ICCB 2012
Medimond - Monduzzi Editore International Proceedings Division
proceedings of the
10th International
Congress on Cell Biology
July 25-28, 2012 - Rio de Janeiro (Brazil)
Editors
Hernandes F. Carvalho, Patricia Gama
MEDIMOND
International Proceedings
© Copyright 2013 by MEDIMOND s.r.l.
Via G. Verdi 15/1, 40065 Pianoro (Bologna), Italy
www.medimond.com • [email protected]
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Printed in April 2013 by Editografica • Bologna (Italy)
ISBN 978-88-7587-672-2
Index
Apoptosis
Differentially Expressed Apoptosis-Related Genes Profile of T47D Breast Cancer
Cells Treated With Angiotensin-(1-7)
Alecrim C., Ribeiro de Noronha S.M., Shimuta S.I., Nakaie C.R., Correa-Noronha S.A.A., Guerreiro da Silva I.D.C. .. 1
Cancer
Characterization of the expression of Na+, K+-ATPase subunits in human breast
cancer cells
Almeida Dias M., Marques Capella M.A., Lopes A.G. .........................................................................................................7
human Chorionic Gonadotropin and Deriveted-Angiotensin Peptides Effects on
Cell Viability and on Apoptosis in Tumoral (MCF-7) and in Normal (MCF10A)
Epithelial Breast Cells
Alves Correa-Noronha S.A., Ribeiro de Noronha S.M., Shimuta S.I., Nakaie C.R., Cotrim Guerreiro da Silva I.D. ......11
Human chorionic gonadotrifin (hCG) and Angiotensin-(1-7) decrease the
tumorigenic capacity of the undifferentiated Breast Cancer Cell Line SKBR3
Binda Neto I., Ribeiro de Noronha S.M., Bernardo W., Kede J., da Silva Freitas E.H.; Alves Corrêa de Noronha S.A.,
Cotrim Guerreiro da Silva I.D. ...............................................................................................................................................17
Cell Biology Education
Description of a pedagogical experiment on building undergraduate students’
clinical thinking coupled up with cellular processes and biochemistry understanding
Vargas de Mesquita L., Abbud Hanna Roque C.E., Ávila de Almeida C., Alves Moreira R.F., Rocha C.B. .....................23
Education with technology: the use of an adaptive interface facilitates the teaching
and learning
Lustosa de Oliveira M., Carvalho H.F. ..................................................................................................................................27
Teaching at Distance: Interactive Multimedia of the Cell Biology of Trypanosoma
cruzi
Benchimol M., Teixeira D.E., Crepaldi P.H., de Souza W. ....................................................................................................33
©2013 by MEDIMOND s.r.l.
III
IV
Index
Cell Signalling
Eph/ephrin- mediated interactions govern many aspects of thymus biology
Zapata A.G., Alfaro D., Garcia-Ceca J., Cejalvo T., Tobajas E., Montero S., Muñoz J.J. ....................................................39
Developmental Biology
Morphological changes in the placenta of alloxan induced diabetic rats
Farias P.S., dos Santos Souza K., Marçal A.C., dos Santos M.R.V., Fioretto E.T., Aires M.B. ..............................................43
Retinoic Acid and the Development of Neurotransmitter Systems
Zieger E., Schubert M. ............................................................................................................................................................49
Inflammation
Effects of the Peripheral Sympathetic-CRH-Histamine Axis on the Immune
Function of Macrophages
Renck Nunes P., Homem de Bittencourt Jr. P.I. ....................................................................................................................55
Role of matrix metalloproteinases and inflammasome pathway in the development
of airway inflammation and fibrosis
Lagente V., Gicquel T., Victoni T., Robert S., Viel R., Fautrel A., Valença S., Porto L.C., Boichot E. ...............................61
Natural compounds and toxicants
Evaluation of the cellular response hematologic of Cebus apella species exposed to
carcinogen N-methyl-N-nitrosourea (MNU) and treated with CANOVA®
Azevedo Feio D.C., Pereira Carneiro Muniz J.A., Burbano R.M.R., Cardoso De Brito Junior L., Lima de Lima P.D. ......67
Evaluation of genotoxicity of Solanum lycocarpum aqueous extract utilizing Allium
cepa test-system
Moreira de Lima V., dos Santos J.T., Vieira Gomes J., da Silva de Mello M., Fampa P., Resende Borba H. .....................73
Genotoxic activity of extracts of marine algae from the coast of Alagoas - Brazil
da Silva B.H., Alvelino E.X., Guedes É.A.C., Sant’Ana E.G., Rodarte R.S. ..........................................................................79
Evaluation of genotoxic effects in lymphocytes of mice from extracts Ziziphus
joazeiro Martius (Rhamnaceae)
da Silva B.H., Rocha Brito I.R., Alvelino E.X., de Matos Rodarte C.C., Sant’Ana A.E.G., Rodarte R.S. ...........................85
Does nitric oxide activate antioxidant enzymes in plants exposed to arsenic?
Monteiro de Andrade H., de Oliveira J.A., Neto J.L., Cambraia J., dos Santos Farnese F. ...............................................91
Activation of the ascorbate-glutathione cycle by nitric oxide: signaling under stress
conditions induced by arsenic
Farnese F.S., de Oliveira J.A., da Silveira N.M., Gusman G.S., Siman L.I. ...........................................................................95
Role of nitric oxide molecule in the tolerance to arsenic in plants: signal or
antioxidant?
Farnese F.S., de Oliveira J.A., Gusman G.S., Leão G.A., Canatto R.A., Silva C.J. ...............................................................99
Index
V
Evaluation of Joannesia princeps aqueous extract anthelmintic activity in naturally
infected mice by Syphacia obvelata, Aspiculuris tetraptera and Vampirolepis nana
Borba H.R., Gomes J.V., dos Santos Teixeira J.T., da Silva de Mello M., Fampa P., Gonçalves L., de Assis da Silva F.,
Moreira de Lima V. .................................................................................................................................................................103
Organelles
The Hydrogenosome: an unconventional organelle
Benchimol M. ..........................................................................................................................................................................109
Protein regulation and structure
Analysis of genetic and structural similarity of transferrin receptor in
trypanosomatids and their hosts
Ávila R.A., Lucas J.Z., Lopes I.F., Rivaroli L. ..........................................................................................................................115
Chaperonopathies: Impact on protein folding and beyond
Macario A.J.L., de Macario E.C. .............................................................................................................................................119
Tissue biology and cell - cell interactions
Gene Expression Assessment of the Polycomb & Trithorax Complexes in the Brain
of Fat-Tissue-Implanted Polycystic-Ovarian-Sindrome Mice
da Silva Freitas E.H., de Noronha S.M.R., Baptista C.F., Gamboa Ritto M.N., Kede J., Neto I.B.,
Alves Correa-Noronha S.A., Guerreiro da Silva I.D.C. ........................................................................................................125
Influence of pancreatic acinar cell necrosis on stellate cell proliferation in vitro
Geissler K., Krüger B. ..............................................................................................................................................................131
In vitro interaction of Aeromonas spp. strains with HEp-2 and Caco-2 cell lines
Fonseca Ferreira A., Azevedo dos Santos P., Corrêa de Freitas Almeida A. .....................................................................137
In vitro decellularization and recellularization of the kidneys
Martins A.B., Silva-Mendes B.J., Nascimento J.S., Fonseca R.N., Silva J.R., Moraes J., De Barros C.M.,
Campos de Carvalho A.C., Souza-Menezes J., Goldenberg R.C.S. ....................................................................................143
Brain cAMP/Ca(2+) Signaling Genes in Fat Tissue Implanted Polycystic Ovarian
Syndrome Mouse
Kede J., da Silva Freitas E.H., Batista C.F., Gamboa Ritto M.N., Binda Neto I., Ribeiro de Noroña S.M.,
Alves Corrêa de Noroña S.A., Guerreiro da Silva I.D.C. .....................................................................................................147
Gain control in the outer retina
Joselevitch C., Kamermans M. ...............................................................................................................................................153
Cell death and in vivo melanoma tumor growth remission determined by a snake
toxin with specificity for actively proliferating cells
Sancey L., Márcia Neiva, Nascimento F.D., Costa B.A., Pereira A., Oliveira E.B., Tersariol I.L.S., Coll J.L., Kerkis I.,
Hayashi M.A.F. ........................................................................................................................................................................159
Author Index ...........................................................................................................................................................................167
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Differentially Expressed Apoptosis-Related Genes
Profile of T47D Breast Cancer Cells Treated With
Angiotensin-(1-7)
Alecrim C.1, Ribeiro de Noronha S.M.1, Shimuta S.I.2, Nakaie C.R.2,
Correa-Noronha S.A.A.1, Guerreiro da Silva I.D.C.1
1
Ginecologia Molecular/ Ginecologia, UNIFESP-R. Pedro de Toledo, 791- 4o. andar- V. ClementinoCEP04039032 -Sao Paulo/SP (BRAZIL)
2
Dep. Biofisica - R. Botucatu, 840- 7o. Andar- V. Clementino-Sao Paulo/SP (BRAZIL)
[email protected], [email protected], [email protected], [email protected],
[email protected], [email protected].
Abstract
Angiotensin-(1-7) [Ang-(1-7)] is an endogenous seven-aminoacid peptide hormone of the reninangiotensin system that has antiproliferative properties. Recent studies suggest that Ang-(1-7) inhibits growth of
lung cancer cells through reduction in cellular proliferation and induction of apoptosis. The aim of this work is to
assess the pro-apoptotic effects of Ang-(1-7) in breast cancer cells (T47D). Methods: T47D cells were treated
with angiotensin-(1-7) (10-6M) for 48 hours. The percentage of apoptotic cells was measured by flow cytometry
(Millipore guava). In addition, we used the SA Biosciences Human Apoptosis RT² Profiler PCR Array profiles to
analyze the expression of 84 key genes involved in programmed cell death. Network analyses of differentially
expressed genes were performed through MetaCore database (GeneGo). Ang-(1-7) caused a significantly higher
rate of apoptosis than the control group. More than 60% of the apoptosis-related-genes were differentially
expressed, of which approximately 50% genes were up-regulated, such as TNF, BCL2, BAX and BIK, which
have shown to be nineteen fifteen, nine and four times more expressed, respectively, than control. Furthermore,
seventeen regulatory gene networks were derived from MetaCore analysis, suggesting the molecular
mechanisms underlying of apoptotic-driven T47D cells. Conclusions: In summary, it is clear that Ang-(1-7)
induces apoptosis in T47D breast cancer cells and it probably does so through activation of BCL2 gene family
and TNF related genes.
Keywords: Angiotensin II; Angiotensin 1-7; Angiotensin II Type 1 Receptor (AT1R); Breast Cancer;
apoptosis; T47D cells.
Introduction
Breast cancer is the most common type of cancer and the second leading cause of death among women
[1]. Therefore, the search for novel chemotherapeutic approaches to human breast cancer and the molecular
mechanism underlying the action of these compounds is particularly relevant. Recent studies have shown that at
a local tissue level, the components of the renin-angiotensin system (RAS) influence tumor growth [2, 3].
Angiotensin-(1-7) [Ang-(1-7)], a component of the RAS, is an endogenous peptide hormone that functions as a
vasodilator agent [4] with antihypertensive [5], antiproliferative [6-8] and antiangiogenic properties [9]. This
peptide is synthesized from Ang I, from Ang II or directly from Ang-(1-9) bypassing the synthesis of Ang II.
The formation of Ang-(1-7) from Ang I requires the action of three tissue endopeptidases: prolyl endopeptidase,
neutral endopeptidase and thimet oligopeptidase [10-12]. Ang-(1-7) may also be synthesized from Ang II by the
action of ACE2 (Angiotensin converting enzyme 2) [13, 14] or from Angiotensin-(1-9) [15]. Ang-(1-7) may be
hydrolyzed by ACE forming Ang-(1-5) and angiotensin-(1-3) [16, 17]. The existence of a receptor for Ang-(1-7)
is controversial, however many studies have shown that this Ang-(1-7) exerts its physiologic effects probably
through activation of a unique G protein-coupled Ang-(1-7) [AT(1-7)] receptor encoded by the mas oncogene
(mas1) The site of the receptor for Ang-(1-7) is controversial [18].
It has been clearly demonstrated by in vitro assays that Ang-(1-7) inhibits proliferation of lung cancer
cells [19]. Furthermore, Ang-(1-7) treatment also decreased microvessel density accompanied by a reduction in
vascular endothelial growth factor (VEGF) and in placental growth factor (PlGF) in lung and in breast tumor
xenografts [20, 21]. These molecular changes suggest that Ang-(1-7) may reduce proangiogenic factors and
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
therefore inhibits tumor growth. Moreover Ang-(1-7) inhibits tumoral fibrosis through activation of the mas
receptor [22], which reinforces the role played by Ang-(1-7) as a general cell growth regulator.
Apoptosis is considered an innate defense mechanism that drives the cell to become antineoplastic; and
many chemotherapeutic agents exert their effect by inducing this type of cell death [23]. Therefore, the aim of
this work is to assess the capacity of Ang-(1-7) to induce apoptosis in T47D breast cancer cells.
Methods
2.1 Cell culture
T47D (Human ductal breast epithelial tumor cell line) cells were grown in Dulbecco’s Modified Eagle
Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (100 U/mL),
human insulin (2UI/mL), glutamine (4mM). Cells were kept at 37ºC in a humidified atmosphere of 5% CO2 and
95% room-air.
2.2 Proliferation Assay
T47D cells were plated in 24-well cluster dishes (1x103 cells/well). Control group was treated with 10%
FBS supplemented medium and the cell treatment groups were treated with FBS and Ang II or with FBS and
Ang-(1-7), to a final concentration of 10-6M. The peptides were replaced daily, due to its rapid degradation [24]
After 2, 6, 9 and 15 days, cells were removed from triplicate wells using trypsin/EDTA and the total number of
cells per well was determined using a hemocytometer.
2.3 Apoptosis Assay
T47D cells were seeded into 24-well plates. Control group was treated with 10% FBS supplemented
medium and the cell treatment groups were treated with FBS and Ang II or with FBS and Ang-(1-7), to a final
concentration of 10-6M. The peptides were replaced daily After 2, 6, 9 and 15 days of treatment, apoptotic cells
were detected via annexin-V staining (Guava Nexin kit - Millipore), following the manufacturer's instructions.
Cells from each well were resuspended in 100µl of supplemented DMEM (1x105 cells/well) and 100µl of Guava
Nexin Reagent. Samples were incubated in the dark at room temperature for 20 min and then submitted to the
cytometer Guava EasyCyteTM (Millipore) analysis.
2.4 RNA Extraction
T47D cells were treated with DMEM supplemented in the absence (control) and in the presence of Ang(1-7) and Ang II (10-6M) for 48 hours. Total RNA from these cells was extracted using the TRIzol reagent
(Qiagen) according to the manufacturer’s instructions and followed by DNase treatment for 10 min using an
RNase-free DNase set (Qiagen).
2.5 Quantitative real-time PCR Array
The Human Apoptosis RT2 Profiler PCR Array kit (PAHS-012) was used to assess the expression of 84
key genes involved in programmed cell death. The RT2 first strand kit, which includes a proprietary buffer to
eliminate any residual genomic DNA contamination in the cell line RNA samples; through this kit 2 μg of total
RNA were converted to cDNA via a reverse transcriptase reaction, according to the manufacturer’s instructions.
A cocktail containing cDNA and all the optimized reagents and buffers needed for gene amplification and
SYBR® Green detection was prepared based upon real-time polymerase chain reactions. Twenty-five microliters
of this experimental cocktail were added to each well on the 96-well plate, which was then loaded into a 7500
Fast Real-Time PCR System (Applied Biosystems) and run in a two-step cycling program, with one initial cycle
of 95°C for 10 min and 40 cycles of 15 s at 95°C and 1 min at 60°C.
2.6 Analysis of Relevant Biological Processes and Networks by MetaCore.
The MetaCore software (GeneGo, St. Joseph,MI) is a computational resource that uses logic operations
for identifying altered biological processes based upon gene expression changes. Genes with altered expression
were mapped to Gene Ontol’ogy (GO) using MetaCore algorithm. GO annotations were used as indicators of
biological functions. GO describes gene products in terms of their associated biological processes, cellular
components, and molecular functions. The GO entries are hierarchically linked, thus allowing construction of
cluster genes of crossed pathways.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
2.7 Statistical analysis
The results for cell proliferation and apoptosis assays were analyzed by descriptive statistics (means and
standard deviation) and inferential statistics through the Student’s t-test, with significance level of 5% (p <0.05).
Real-time PCR array reactions were processed through the online software RT2 Profiler™ PCR Array Data
Analysis (SABiosciences).
Results
3.1 Ang-(1-7) inhibits proliferation of T47D cells
Ang-(1-7) has an antiproliferative effect in T47D breast cancer cells, as shown in Figure 1. Regular
medium treatment (control) did not inhibited cell growth and Ang II increased cell proliferation, which grew
more than the control group after 9 days (p=0.04) and 15 days (p=0.05) of treatment. On the other hand Ang-(17) a significantly reduced cell proliferation when compared to the control group after six, nine and fifteen days
(p=0.01) of treatment.
3.2 Ang-(1-7) induces apoptosis in T47D cells
The ratio of apoptotic breast carcinoma cells was measured by flow cytometry. Therefore a full
apoptotic study (early and late stage apoptosis) could be conducted. We only considered the percentage of cells
stained by annexin-V.
In Figure 2, we can see that the 2-day treated cells exhibit a higher rate than than other time-points.
Furthermore, all groups exhibited similar apoptotic rates, however, Ang-(1-7) treated cells exhibited a
significantly higher apoptotic rate than the control group after six (p=0.04), nine (p=0.02) and fifteen days
(p=0.05) of treatment, whereas cells treated with Ang II had an apoptotic rate similar to that of the control group.
The results indicate that Ang-(1-7) is able to induce apoptosis in T47D cells.
3.3. Profile of differentially expressed genes in cells treated with Ang-(1-7)
The results obtained from the cell proliferation and apoptosis assay clearly demonstrate the significant
effects of Ang-(1-7) in breast carcinoma cells. Therefore in order to verify whether the observed effects were
associated with the expression of genes involved in apoptosis regulation, we performed a qPCR-array assay to
simultaneously determine the expression profile of 84 genes involved in programmed cell death. Table 1 shows
the differential expression of apoptosis-related genes in breast cancer cells treated with Ang-(1-7) calibrated by
the control group samples. This heptapeptide markedly modulates the expression of many apoptosis-related
genes – forty-seven were up-regulated (55.9%) and thirteen were down-regulated (15.7%). In contrast, cells
treated with Ang II exhibited seven up-regulated genes (8.3%) and fifteen down-regulated genes (17.8%)
(Figure 3).
Moreover Ang-(1-7) significantly increased the expression of the caspase family genes and of BCL2
and TNF family genes (Figure 3). Interestingly, all figures show that most of the effects caused by Ang-(1-7) are
opposite to those caused by Ang II. p values were calculated based on Student’s t-test of the replicate 2^ (- Delta
Ct) values for each gene in the control group and treatment groups, and p values less than 0.05.
Fig. 1 Apoptotic effect induced by Ang-(1-7) in T47D breast cancer cells. * P < 0.05.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Fig. 2 Major differential-gene expressions caused by Ang-(1-7) in T47D cells.
3.4 Analysis of transcription regulation networks
Differentially expressed genes detected by the qPCR array analysis were used as the input list for the
generation of biological networks using the Transcription Regulation algorithm with default settings. Analysis of
the gene expression data in MetaCore with the transcription regulation algorithm resulted in multiple apoptotic
signaling pathways (Figure 3) Differentially expressed genes due to angiotensin-(1-7) treatment resulted in
signaling pathways composed by members of the BCL2 gene family or genes related to TNF, classical
intracellular pathways altered in breast cancer.
Fig. 3 Representative biological network based on differentially expressed genes obtained by qPCR using MetaCore. Most of
the components are related to BCL2 gene family and to TNF. Thick cyan lines indicate the fragments of canonical pathways.
Upregulated genes are marked with red circles; downregulated ones with blue circles. The 'checkerboard' color indicates
mixed expression for the gene between files or between multiple tags for the same gene.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Conclusions
There are few works correlating Ang-(1-7) to breast cancer. The initial works considered the Ang-(1-7)
as a therapeutic compound employed it as a hematopoietic agent that stimulates the proliferation of
multipotential and differentiated progenitor cells in cultured bone marrow and in human cord blood [23].
Our results strikingly demonstrate that Ang-(1-7) acts as an anti-proliferative and pro-apoptotic
hormone in breast cancer cells. Cell proliferation is markedly decreased in a dose-dependent manner in late timepoints (six, nine and fifteen-day treatments) and the apoptotic effect is sustained throughout the treatment.
Moreover in order to better understand the molecular mechanism underlying these pro-apoptotic effects of Ang(1-7) in T47D cells, the results obtained herein suggest that Ang-(1-7) drives these tumoric cell line to apoptosis
by activating intracellular pathways composed by TNF and BCL2 gene family related genes.
In summary, there has been abundant evidence from the assays described herein that the hormone Ang(1-7) drives the ER-positive cell line T47D to apoptosis and that this hormone performs alterations in
intracellular pathways associated with cell survival. Therefore, the use of Ang-(1-7) as a therapeutic or
preventive tool, by driving these breast cancer cells to apoptosis, should be considered.
Acknowledgments: The work was supported by Grant number 2011/10516-0 and 2008/54383-0 from the Sao
Paulo Research Foundation (FAPESP)-Brazil.
References
[1]
Cancer
Among
Women.
(Published
United
States
Cancer
Statistics,
2010),
http://www.cdc.gov/cancer/dcpc/data/women.htm. Accessed 20 Dec 2010.
[2] Ager EI, Neo J, Christophi C. The renin–angiotensin system and malignancy. Carcinogenesis.2008; 29(9):
1675-1684.
[3] Deshayes F, Nahmias C. Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab. 2005; 16(7):
293-9.
[4] Santos RA, Campagnole-Santos MJ, Andrade SP. Angiotensin-(1-7): an update. Regul Pept. 2000; 91(13):45-62.
[5] Benter IF, Diz DI, Ferrari CM. Pressor and reflex sensitivity is altered in spontaneously hypertensive rats
treated with angiotensin-(1-7). Hypertension. 1995; 26:1138-1144.
[6] Strawn WB, Ferrario CM, Tallant EA. Angiotensin (1-7) reduces smooth muscle growth after vascular
injury. Hypertension. 1999; 33: 207-11.
[7] Freeman EJ, Chisolm GM, Ferrario CM, Tallant EA. Angiotensin-(1-7) inhibits vascular smooth muscle cell
growth. Hypertension. 1996; 28(1): 104-8.
[8] Tallant EA, Ferrario CM, Gallagher PE. Angiotensin-(1–7) inhibits growth of cardiac myocytes through
activation of the mas receptor. Am J Physiol Heart Circ Physiol. 2005; 289(4): H1560-H1566.
[9] Machado RD, Santos RA, Andrade SP. Opposing actions of angiotensins on angiogenesis. Life Sci. 2000;
66(1):67-76.
[10] Santos RA, Brosnihan KB, Jacobsen DW, DiCorletto PE, Ferrario CM. Production of angiotensin-(1-7) by
human vascular endothelium. Hypertension. 1992; 19(2):56-61.
[11] Yamamoto K, Chappell MC, Broshinan KB, Ferrario CM. In vivo metabolism of angiotensin I by neutral
endopeptidase (EC 3.4.24.11) in spontaneously hypertensive rats. Hypertension. 1992; 19(6): 692-6.
[12] Chappell MC, Tallant EA, Brosnihan KB, Ferrario CM. Conversion of angiotensin I to angiotensin-(1-7) by
thimet oligopeptidase (E.C.3.4.24.15) in vascular smooth muscle cells. J Vasc Biol Med. 1994; 5:129-137.
[13] Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A human homolog of angiotensinconverting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol
Chem. 2000; 275(43): 33238-33243.
[14] Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, et al. Angiotensin-converting
enzyme 2 is an essential regulator of heart function. Nature. 2002; 417(6891):822-8.
[15] Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, et al. A novel angiotensinconverting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res.
2000; 87(5): E1-E9.
[16] Chappell (MC), Pirro NT, Sykes A, Ferrario CM. Metabolism of angiotensin-(1-7) by angiotensinconverting enzyme. Hypertension. 1998; 31: 362-7.
[17] Yamada K, Iyer SN, Chappell MC, Ganten D, Ferrario CM. Converting enzyme determines the plasma
clearance of angiotensin-(1-7). Hypertension.1998; 32(3): 496-502.
© Medimond . P726C0005
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[18] Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I, et al. Angiotensin-(1-7) is an
endogenous ligand for the G protein-coupled receptor mas. Proc. Natl Acad. Sci. USA. 2003; 100(14):
8258-63.
[19] Gallagher PE, Tallant EA. Inhibition of human lung cancer cell growth by angiotensin-(1-7).
Carcinogenesis. 2004; 25(11): 2045-52.
[20] Soto-Pantoja DR, Menon J, Gallagher PE, Tallant EA. Angiotensin-(1-7) inhibits tumor angiogenesis in
human lung cancer xenografts with a reduction in vascular endothelial growth factor. Mol Cancer Ther.
2009; 8(6): 1676-83.
[21] Menon J, Soto-Pantoja DR, Callahan MF, Cline JM, Ferrario CM, Tallant EA, Gallagher PE. Angiotensin(1-7) Inhibits Growth of Human Lung Adenocarcinoma Xenografts in Nude Mice through a Reduction in
Cyclooxygenase-2. Cancer Res. 2007; 67(6): 2809-15 .
[22] Cook KL, Metheny-Barlow LJ, Tallant EA, Gallagher PE. Angiotensin-(1-7) reduces fibrosis in orthotopic
breast tumors. Cancer Res 70, 8319–8328 (2010).
[23] Rodgers KE, Oliver J, diZerega GS. Phase I/II dose escalation study of angiotensin 1-7 [A(1-7)]
administered before and after chemotherapy in patients with newly diagnosed breast cancer. Cancer
Chemother Pharmacol. 2006;57(5):559-68.
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Characterization of the expression of Na+, K+-ATPase
subunits in human breast cancer cells
Almeida Dias M., Marques Capella M.A., Lopes A.G.
Instituto de Biofísica Carlos Chagas Filho (Brazil)
[email protected], [email protected], [email protected]
Abstract
Background: It is well established that the known Na+, K+-ATPase has cell adhesion molecule role in
adherens junctions [16]. In so, β subunit acts on maturation and membrane targeting of α catalytic subunit.
Studies have demonstrated the importance of the sodium permeability in tumorigenesis, but there’s no study of
Na+, K+-ATPase α and β subunit’s expression. The aim of the present study is to characterize the expression of
Na+, K+-ATPase subunits in human breast cancer cells.
Methods and Results: To observe the expression of α1 and β1 in MCF-7, MDA-231 and MDCK cells
were seeded in 6 well plates for 24, 48 and 72 hours. The measurement of of α1 and β1 expression by Western
Blotting showed that tumor cell lines do not express β1 subunit.
Conclusion: Our results indicate that there’s a distinct adhesion mechanism in tumor cells, which should
explain metastasis.
Keywords: Na+,K+-ATPase, breast cancer, MCF7, MDA-MB-231
Introduction
The maintenance of an ionic gradient is necessary to homeostasis of various cells types. In so, Na+,K+ATPase has a fundamental role on building a ionic gradient, acting in a wide range of physiologic and pathologic
cell functions, as it is in cancer. It is well established that there’s a variation of sodium permeability in
tumorigenesis [10], so regulation of activity and expression of Na+,K+-ATPase are relevant to the understanding
of this processes.
Recently, the hypothesis of a lower threshold of tumour cells to Na+,K+-ATPase inhibitors has been put
forward by H. Weidemann. It implies that tumour and normal cells have a similar pattern toward Na+,K+-ATPase
inhibitors, in which tumour cells respond with reduction of cell viability in physiologic concentrations of
digitalis-like compounds and normal cells respond with proliferation, while in high plasma concentrations,
normal cells respond with apoptosis and tumour cells with inhibition of proliferation[15]. Until now, there’s no
data about the effect of sub-physiologic digitalis-like compounds in malignant cells. It has been suggested that
this different subunits expression of Na+,K+-ATPase subunits in the membrane can be the reason to this lower
response threshold to digitalis-like compounds.
In the same context, Na+,K+-ATPase subunits expression can be crucial to tumours development, as we
know that β subunit act as a cell adhesion molecule [14], and inhibition of Na+,K+-ATPase can sensitize cancer
cells toward anoikis[9].
The objective of this study was to see the effects of picomolar concentrations of ouabain and
characterize the expression of Na+,K+-ATPase subunits in MCF-7 and MDA-MB-231 human breast cancer cells.
Methodology
1.1.1 Cell counting
Cell viability was assessed by light microscopy and trypan blue dye exclusion. Cell numbers were
evaluated by direct counting, using a Neubauer chamber. All experiments were performed in duplicate.
1.1.2 Western Blotting
Slab SDS/PAGE was used, according to the method of Laemmli [3],
polyacrylamide gel.
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by using 7,5% SDS-
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After SDS-PAGE, the protein was transferred to 0.2m m pore-sized nitrocellulose at 350 mA for 2 h by
using a Loccus Biotecnologia electrophoresis unit. The nitrocellulose was incubated with monoclonal antibody
against human Na+,K+-ATPase subunits at concentration 1 : 200 in the invitrogen WesternBreeze kit blocking
solution. In order to analyze Na+,K+-ATPase subunits, second antibody-alkaline phosphatase conjugated antimouse and anti-rabbit IgG was added and incubated for 60 min under gentle shaking. Then nitrocellulose was
washed with kit’s washing solution four times for 5 minutes and submitted to chromogenic reagent.
1.1.3 Statistical Analyses
The results were analysed by Analysis of Variance (ANOVA) and Dunnett's Multiple Comparison Test
was done to check statistical significance. P<0.05 was considered significant.
Results
1.2
Cell counting
Measurement of the effect of picomolar concentrations of ouabain showed different patterns of response
in MCF-7 and MDA-MB- 231 cell lineages. While MCF7, a positive estrogen receptor cell, do not show any
significant proliferation, MDA-MB-231, that does not express estrogen receptor respond with significant
proliferation at concentrations of 10-11 and 10-12M of ouabain. This point to the lower threshold hypothesis, in
which sub-physiologic concentrations of digitalis-like compounds stimulates proliferation in some malignant
cells.
Figure 1: MDA-MB-231 and MCF7 cell counting in trypan blue. Cells were seeded in 24 wells plate for 24h with picomolar
concentrations of ouabain
1.3
Na+,K+-ATPase subunits expression
Western Blotting analysis showed a impairment of alpha-1 and beta-1 (most common isoforms in
epithelial tissues) Na+,K+-ATPase subunits expression in tumor cells. In so, there’s no participation of alpha-3
and beta-3 subunits (figure 3). These results point to a distinct mechanism of cell adhesion, in which there’s no
participation of beta-1 or beta 3 subunits. Later studies will be performed to analyze alpha-2 and beta-2
expression in both cell lines.
Figure 2: Expression of α1 and β1 Na+/K+ ATPase subunits. To observe the expression of α1 and β1 subunits in MCF-7,
MDA-231 and MDCK cells were seeded into 6 well plates for 24, 48 and 72 hours. The MDCK cell line was used as a
positive control for the expression of both. The measurement of α1 and β1 expression by Western Blotting showed that both
tumor cell lines do not express β1 subunit (n=6).
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Figure 3: Expression of α3 and β3 Na+,K+-ATPase subunits. To observe the expression of α3 and β3 in MCF-7 and MDA231 were seeded onto 6 well plates for 24, 48 and 72 hours. Rat brain extract was used as a positive control for the
expression of α3 subunit, while rat liver extract was used for β3 subunit. (Lane 1). The measurement of α3 and β3 expression
by Western Blotting showed that both tumor cell lines do not express neither α3 nor β3 subunits (n=4).
Conclusions
The role of Na+,K+-ATPase in malignant cell’s metabolism remains to be clarified, but our preliminary
results points to a potencial therapeutic target in the treatment of breast cancer, as previous works showed [7].
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Hartsock A and Nelson WJ. (2008) Adherens and tight junctions: structure, function and connections to
the actin cytoskeleton. Biochim Biophys Acta 1778: 660-669.
Ino Y, Gotoh M, Sakamoto M, Tsukagoshi K, and Hirohashi S. (2002). Dysadherin, a cancer associated
cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis. Proc Natl Acad Sci U
S A 99: 365-370.
Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 15;227(5259):680-5.
Landon J. Inge, Sigrid A. Rajasekaran, Koji Yoshimoto, Paul S. Mischel, William McBride, Elliot
Landaw, and Ayyappan K. Rajasekaran.(2008). Evidence for a potential tumor suppressor role for the
Na,KATPase ß1-subunit. Histol Histopathol. 23(4): 459–467.
Largent, JA; Bernstein, L; Horn-Ross, PL; Marshall, SF; Neuhausen, S; Reynolds, P; Ursin, G; Zell, JA;
Ziogas, A; Anton-Culver, H. (2010). Hypertension, antihypertensive medication use, and breast
cancerrisk in the California Teachers Study cohort. Cancer Causes Control DOI 10.1007/s10552-0109590-x.
Larre I, Lazaro A, Contreras RG, Balda MS, Matter K, Flores-Maldonado C, Ponce A, Flores-Benitez
D, Rincon-Heredia R, Padilla-Benavides T, Castillo A, Shoshani L, and Cereijido M. (2010). Ouabain
modulates epithelial cell tight junction. Proc Natl Acad Sci U S A 107: 11387-11392.
Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, Kiss R. (2007). Cardiotonic steroids on
the road to anti-cancer therapy. Biochim Biophys Acta. 1776(1):32-57.
Rajasekaran SA and Rajasekaran AK. (2009). Na,K-ATPase and epithelial tight junctions. Front Biosci
14: 2130-2148.
Simpson, CD. (2009) Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes
Cancer Cells to Anoikis and Prevents Distant Tumor Formation. Cancer Res;69:2739-2747.
Shen SS, Hamamoto ST, Bern HA, Steinhardt RA.(1978) Alteration of sodium transport in mouse
mammary epithelium associated with neoplastic transformation. Cancer Res. 1356-61.
Shoshani L, Contreras RG, Roldan ML, Moreno J, Lazaro A, Balda MS, Matter K, and Cereijido M.
(2005). The polarized expression of Na+,K+-ATPase in epithelia depends on the association between βsubunits located in neighboring cells. Mol Biol Cell 16: 1071-1081.
Tokhtaeva, E. (2012) Identification of the amino-acid region involved in the intercellular interaction
between the Na,K-ATPase β1 subunits. Jour of Cell Scie.
Tokhtaeva E, Sachs G, and Vagin O. (2009). Assembly with the Na,K-ATPase alpha(1) subunit
isrequired for export of β(1) and β(2) subunits from the endoplasmic reticulum. Biochemistry 48:
11421-11431.
Vagin, O; Dada,LA; Tokhtaeva, E; and Sachs, G. (2012). The Na,K-ATPase α1β1 heterodimer as a cell
adhesion molecule in epithelia Am J Physiol Cell Physiol.
Weidemann H. (2012) "The Lower Threshold" phenomenon in tumor cells toward endogenous
digitalis-like compounds: Responsible for tumorigenesis? J Carcinog.11:2.
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[16]
[17]
Winnicka, K; Bielawski, K; Bielawska, A; Suraz˙y´nski, A. (2008). Antiproliferative Activity of
Derivatives of Ouabain, Digoxin and Proscillaridin A in Human MCF-7 and MDA-MB-231 Breast
Cancer Cells. Biol. Pharm. Bull. 31(6) 1131—1140
Zhao H, Liang Y, Xu Z, Wang L, Zhou F, Li Z, Jin J, Yang Y, Fang Z, Hu Y, Zhang L, Su J, and Zha
X. (2008) N-glycosylation affects the adhesive function of E-Cadherin through modifying the
composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-Journal of
cellular biochemistry 104: 162-175.
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human Chorionic Gonadotropin and DerivetedAngiotensin Peptides Effects on Cell Viability and on
Apoptosis in Tumoral (MCF-7) and in Normal (MCF10A)
Epithelial Breast Cells
Alves Correa-Noronha S.A.1, Ribeiro de Noronha S.M.1, Shimuta S.I.2,
Nakaie C.R.2, Cotrim Guerreiro da Silva I.D.1
1
Ginecologia Molecular/ Ginecologia, UNIFESP-R. Pedro de Toledo, 791- 4o. andar- V. ClementinoCEP04039032 -Sao Paulo/SP (BRAZIL)
2
Dep. Biofisica - R. Botucatu, 840- 7o. Andar- V. Clementino-Sao Paulo/SP (BRAZIL)
[email protected], [email protected], [email protected], [email protected], [email protected].
Abstract
Angiotensin-(1-7) [Ang-(1-7)] is an endogenous heptapeptide hormone of the renin-angiotensin system
that has antiproliferative properties. The aim of this work was to evaluate the anti-proliferative and pro-apoptotic
properties of Ang-(1-7) and of Ang-(1-7)- substituents 9-fluorenylmethyloxycarbonyl (Fmoc) e Ang IIderivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) in normal
(MCF10A) and in tumoral (MCF7) epithelial mammary cell lines. Both cell lines received an hCG and
angiotensin peptides 24-hour treatment, in combination or alone followed by cell viability, apoptosis and cell
cycle assays performed by flow cytometer (GUAVA). After hCG, Ang-(1-7), hCG+Ang-(1-7) and hCG+Ang(1-7)-Fmoc treatments, MCF7 displayed cell viability decrease and mid-apoptosis increase. We also observed
cell viability decrease in MCF10A after Ang-(1-7), Ang-(1-7) Fmoc and hCG+AngII Toac treatments. These
cells had an increase in late apoptosis and necrosis after AngII Toac, hCG + Ang-(1-7) and hCG+Ang-(1-7)Fmoc treatments. Regarding the cell cycle analysis, we did not observed any changes in cell cycle phases. In
summary, cell viability was decreased and apoptosis (initial, mid and late) was increased after hCG and/or Ang(1-7) peptides treatments. These results point out hCG and Ang-(1-7) as effective compounds to inhibit cell
proliferation, since they decrease cell viability and increase apoptosis in both normal and in tumoral breast cells,
being the effect more pronounced in the tumoral cell line. Our results support the idea of investigating more
closely the putative use of these compounds as novel therapeutic agents for breast cancer.
Keywords: Angiotensin II; Angiotensin 1-7; Angiotensin II Type 1 Receptor (AT1R); Breast Cancer;
apoptosis; human Chorionic Gonadotropin.
Introduction
Breast cancer is the most common cancer among American women, except for skin cancers. About one
in eight (12%) women in the US will develop invasive breast cancer throughout her lifetime. The American
Cancer Society's estimates that 226,870 new cases of breast cancer will be diagnosed in 2012 in the United
States; about 63,300 new cases of carcinoma in situ (CIS) will be diagnosed and about 39,510 women will die
from breast cancer (http://www.cancer.org).
The renin angiotensin system (RAS) is a hormone widely known for regulating blood pressure. The
proteolytic cascade of the RAS begins with the release of renin (REN), an aspartyl protease which cleaves
angiotensinogen (AGT) produces angiotensin I (Ang I), which is hydrolyzed by angiotensin converting enzyme
(ACE) and releases angiotensin II (Ang II); this octapeptide exerts its functions through its specific membrane
receptors, angiotensin II receptor type 1 and type 2 (AGTR1 and AGTR2, respectively) [1]. Most of the
physiological effects are attributed to the signaling pathways activated by the AGTR1. However, other
components of the renin-angiotensin system (RAS) have been described as having mitogenic and angiogenic
activities [2], [3]. Since angiogenesis and proliferative processes are related to the development, progression and
metastasis of cancer, we believe that there might be an association between these other RAS components with
cancer [4]. Recent studies have shown that at a local tissue level, the components of the RAS influence tumor
growth by changing its microenvironment [5], [6]. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide
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hormone that functions as a vasodilator [7] with antihypertensive [8], antiproliferative [9-11] and antiangiogenic
properties [12]. The formation of Ang-(1-7) from Ang I requires the action of three tissue endopeptidases: prolyl
endopeptidase, neutral endopeptidase and thimet oligopeptidase [13-15]. Ang-(1-7) may also be synthesized
from Ang II by the action of the ACE2 (Angiotensin converting enzyme 2) [16], [17] or from Angiotensin-(1-9)
[18]. Ang-(1-7) may be hydrolyzed by ACE forming Ang-(1-5) and angiotensin-(1-3) [19], [20]. The existence
of a receptor for Ang-(1-7) is controversial. Most studies have shown that this Ang-(1-7) exerts its physiologic
effects probably through activation of a unique G protein-coupled Ang-(1-7) [AT(1-7)] receptor encoded by the
mas oncogene (mas1) [21].
The aim of this work was to evaluate the anti-proliferative and pro-apoptotic properties of Ang-(1-7), of
Ang (1-7)-FMOC and of Ang II-TOAC analogues in normal (MCF10A) and in tumoral (MCF7) epithelial
mammary cells. These cell lines were also treated with Human Chorionic Gonadotropin (hCG), a hormone that
elicits life-long refractoriness to carcinogenesis by differentiation of the breast epithelium in order to assess any
possible synergistic effect of these compounds.
Matrials and Methods
2.1 Peptides
Ang II-TOAC, an Ang II analogue containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4amino-4-carboxylic acid) spin label synthesized by solid phase methodology [22] and Ang-(1-7)-FMOC, an
Ang-(1-7) analogue containing the FMOC (9-fluorenylmethyloxycarbonyl) group was used for protection of the
amine function [23]. These peptides were supplied by dr. C.R. Nakaie (department of Biophysics/UNIFESPBRAZIL). hCG was obtained from Ovidrel®.
2.2 Cell Culture and Treatments
MCF7 cells (HTB-22, ATCC) were grown in Dulbecco’s Modified Eagle Medium (DMEM), 4.5 g/l of
glucose, supplemented with 5% fetal calf serum, 100 U/mL of penicillin (PAA), and 100 mg/mL of streptomycin
(PAA). MCF10A cells (CRL- 10317, ATCC) were cultured in DMEM/F-12 medium (PAA, Carlsbad, CA)
supplemented with 10 mg/mL of human insulin (Sigma, St. Louis, MO), 20 ng/mL of epidermal growth factor
(Sigma, St. Louis, MO), 0.5 mg/mL of hydrocortisone (Sigma, St. Louis, MO), 5% horse serum (Invitrogen),
100 U/mL of penicillin (PAA) and 100 mg/mL of streptomycin (PAA). All the cells lines employed in this work
were cultured at 37°C in a humidified atmosphere and 5% CO2. The following treatments were performed on
these cells: control (1); hCG (2); Ang II (3); Ang-(1-7) (4); Ang-(1-7)-FMOC (5); Ang II-TOAC (6); hCG+Ang
II (7); hCG+Ang-(1-7) (8); hCG+Ang-(1-7)-FMOC (9); hCG+Ang II-TOAC (10).
2.3 Flow Cytometer (GUAVATM) Assays
MCF7 and MCF10A cells were seeded into 24-well plates with DMEM supplemented with 10% FBS in
the absence (control) or presence of hCG. The cells were treated with 20 μM of peptide, the most active
concentration value [25], and Angiotensin-deriveted peptides [Ang-(1-7)-Fmoc and Ang II-Toac] in the
concentration of 10-6M. After 24 hours of treatment, it was possible to determine viability index, and percentage
of apoptosis, and cell cycle by means of biochemical assays in flow cytometer (Guava EasyCyteTM-Millipore)
using kits ViaCount ® Guava, Guava Nexin ® and cell cycle assay ®, respectively.
2.4 Statistical analysis
The results for cell proliferation, apoptosis, and cell cycle assays were analyzed by descriptive statistics
(means and standard deviation) and inferential statistics through the Student’s t-test with significance level of
5% (p <0.05).
Results
3.1 Cell Viability Assays
MCF-7 cells showed cell viability decrease while the mid-apoptosis increased after Ang-(1-7), Ang-(17)-FMOC and hCG+Ang II-TOAC treatments (Fig. 1A). On the other hand MCF10A cells showed cell viability
decrease, while the mid-apoptosis increased after Ang-(1-7) and hCG+Ang-(1-7)-FMOC treatments (Fig. 1B).
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3.2 Apoptosis Assays
Late apoptosis and dead cells ratio increase in MCF-7 cells after hCG, Ang-(1-7), and hCG+Ang-(1-7)FMOC treatments (Fig. 2A). Late apoptosis or dead cells increase after hCG treatment while early apoptosis
increases in MCF10A cells after hCG+Ang-(1-7)-FMOC treatment (Fig. 2B).
3.3 Cell Cycle Assays
MCF7 and MCF10A cells did not evidenced alterations for G0/G1 or S cell cycle after treatments.
Except for hCG that increases G2/M in MCF-7 (Fig. 3A) and for Ang II-TOAC and hCG+Ang-(1-7)-FMOC that
increase G2/M in MCF-10A (Fig. 3B).
Fig. 1 Cell viability analysis after peptides, analogues and hCG treatments in MCF-7 (A) or MCF10A (B) cells. control (1);
hCG (2); Ang II (3); Ang-(1-7) (4); Ang-(1-7)-FMOC (5); Ang II-TOAC (6); hCG+Ang II (7); hCG+Ang-(1-7) (8);
hCG+Ang-(1-7)-FMOC (9); hCG+Ang II-TOAC (10). *, p<0.05 (compared to controls).
Fig. 2 Apoptosis analysis after peptides, analogues and hCG treatments in MCF-7 (A) and in MCF10A (B) cells. control (1);
hCG (2); Ang II (3); Ang-(1-7) (4); Ang-(1-7)-FMOC (5); Ang II-TOAC (6); hCG+Ang II (7); hCG+Ang-(1-7) (8);
hCG+Ang-(1-7)-FMOC (9); hCG+Ang II-TOAC (10). *, p<0.05 (compared to controls).
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Fig. 3 Cell cycle analysis after peptides, analogues and hCG treatments in MCF-7 (A) and in MCF10A (B) cells. control (1);
hCG (2); Ang II (3); Ang-(1-7) (4); Ang-(1-7)-FMOC (5); Ang II-TOAC (6); hCG+Ang II (7); hCG+Ang-(1-7) (8);
hCG+Ang-(1-7)-FMOC (9); hCG+Ang II-TOAC (10). *, p<0.05 (compared to controls).
Discussion
Ang II stimulates proliferation of AT1R-positive breast cancer cells through PI3-kinase/Akt pathway
activation (26) and Ang-(1--7) has a clinical and pre-clinical activity in vascular sarcomas by reducing HIF1alpha and PlGF genes expression [27]. In the present study, cell viability was decreased and mid-apoptosis was
increased in both tumoral and in normal cells (MCF-7 and MCF10A) after Ang-(1-7) and analogs containing
TOAC or FMOC treatments. Also, we observed that Ang II containing the TOAC substituent became an antiproliferative hormone. Late apoptosis and cell death were increased after hCG, Ang-(1-7), and Ang-(1-7)-FMOC
trreatments in MCF-7 cells. Early apoptosis was increased after stimulation with Ang-(1-7) and the effects of the
peptides containing an FMOC group were powered by hCG in MCF10A cells.
Furthermore, cell cycle changes were not observed after peptide treatments, only hCG increased the
stationary phases of the cell cycle (G2/M) in MCF-7 cells. Moreover in MCF10A cells, only the peptides
containing the groups FMOC and TOAC triggered this same type of change. Therefore it is possible to say that
the TOAC and FMOC substituent groups potentiate the anti-proliferative or pro-apoptotic effect in both cell
types. Moreover we could also observe that Ang II becomes anti-proliferative in these mammary cells when the
TOAC group is added to the hormone molecule and finally that hCG enhances the effects of the peptides.
In summary, cell viability was decreased and apoptosis (initial, mid and late) was increased after hCG
and/or Ang-(1-7) peptides treatments. These results point out hCG and Ang-(1-7) as effective compounds to
inhibit cell proliferation, since they decrease cell viability and increase apoptosis in both normal and tumoral
breast cells, being the effect more pronounced in the tumoral cell line. Our results support the idea of
investigating more closely the putative use of these compounds as novel therapeutic agents for breast cancer.
References
[1] Frossard PM, Malloy MJ, Lestringant GG and Kane JP. (2001) Haplotypes of the human renin gene
associated with essential hypertension and stroke. J Hum Hypertens; 151: 49–55.
[2] Fujita M, Hayashi I, Yamashina S, Itoman M, Majima M.(2002) Biochem Biophys Res Commun.; 294:441447.
[3] Greco S, Muscella A, Elia MG, et al.( 2003)Angiotensin II activates extracellular signal regulated kinases via
protein kinase C and epidermal growth factor receptor in breast cancer cells.J Cell Physiol ; 196: 370–377.
[4] Deshayes F and Nahmias C. Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab 2005;
16: 293–299.
© Medimond . P726C0010
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[5] Ager EI, Neo J, Christophi C. (2008) The renin–angiotensin system and malignancy. Carcinogenesis; 29(9):
1675-1684.
[6] Deshayes F, Nahmias C. (2005) Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab;
16(7): 293-9.
[7] Santos RA, Campagnole-Santos MJ, Andrade SP. (2000) Angiotensin-(1-7): an update. Regul Pept; 91(13):45-62.
[8] Benter IF, Diz DI, Ferrari CM. (1995) Pressor and reflex sensitivity is altered in spontaneously hypertensive
rats treated with angiotensin-(1-7). Hypertension; 26:1138-1144.
[9] Strawn WB, Ferrario CM, Tallant EA. (1999) Angiotensin (1-7) reduces smooth muscle growth after
vascular injury. Hypertension; 33: 207-11.
[10] Freeman EJ, Chisolm GM, Ferrario CM, Tallant EA. (1996) Angiotensin-(1-7) inhibits vascular smooth
muscle cell growth. Hypertension; 28(1): 104-8.
[11] Tallant EA, Ferrario CM, Gallagher PE. (2005) Angiotensin-(1–7) inhibits growth of cardiac myocytes
through activation of the mas receptor. Am J Physiol Heart Circ Physiol; 289(4): H1560-H1566.
[12] 9. Machado RD, Santos RA, Andrade SP.(2000) Opposing actions of angiotensins on angiogenesis. Life
Sci; 66(1):67-76.
[13] Santos RA, Brosnihan KB, Jacobsen DW, DiCorletto PE, Ferrario CM. (1992) Production of angiotensin(1-7) by human vascular endothelium. Hypertension; 19(2):56-61.
[14] Yamamoto K, Chappell MC, Broshinan KB, Ferrario CM. (1992) In vivo metabolism of angiotensin I by
neutral endopeptidase (EC 3.4.24.11) in spontaneously hypertensive rats. Hypertension; 19(6): 692-6.
[15] Chappell MC, Tallant EA, Brosnihan KB, Ferrario CM. (1994) Conversion of angiotensin I to angiotensin(1-7) by thimet oligopeptidase (E.C.3.4.24.15) in vascular smooth muscle cells. J Vasc Biol Med; 5:129137.
[16] Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. (2000) A human homolog of
angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive
carboxypeptidase. J Biol Chem; 275(43): 33238-33243.
[17] Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, et al. (2002) Angiotensinconverting enzyme 2 is an essential regulator of heart function. Nature; 417(6891):822-8.
[18] Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, et al. (2000) A novel angiotensinconverting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res;
87(5): E1-E9.
[19] Chappell (MC), Pirro NT, Sykes A, Ferrario CM. (1998) Metabolism of angiotensin-(1-7) by angiotensinconverting enzyme. Hypertension; 31: 362-7.
[20] Yamada K, Iyer SN, Chappell MC, Ganten D, Ferrario CM. (1998) Converting enzyme determines the
plasma clearance of angiotensin-(1-7). Hypertension; 32(3): 496-502.
[21] Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I, et al. (2003) Angiotensin-(17) is an endogenous ligand for the G protein-coupled receptor mas. Proc. Natl Acad. Sci. USA; 100(14):
8258-63.
[22] Nakaie CR, Silva EG, Cilli EM, Marchetto R, Schreier S, Paiva TB, Paiva AC. (2002) Peptides.
Jan;23(1):65-70.Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin
analogs.
[23] Jubilut GN, Cilli EM, Crusca E, Silva EH, Okada Y, Nakaie CR.Chem Pharm Bull (Tokyo). (2007)
Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for
Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis. Mar;55(3):468-70.
[24] Noronha, Samuel M.R.; Correa-Noronha, Silvana A.A.; Russo, Irma H.; de Cicco, Ricardo
López; Santucci-Pereira, Julia; Russo, José.(2011)Human chorionic gonadotropin and a 15 amino acid
hCG fragment of the hormone induce downregulation of the cytokine IL-8 receptor in normal breast
epithelial cells. Hormone Molecular Biology and Clinical Investigation. Volume 6, Issue 3, Pages 241–
245
[25] Morbeck DE, Roche PC, Keutmann HT, McCormick DJ. (1993) A receptor binding site identified in the
region 81-95 of the beta-subunit of human luteinizing hormone (LH) and chorionic gonadotropin (hCG).
Mol Cell Endocrinol. Nov;97(1-2):173-81.
[26] Zhao Y, Chen X, Cai L, Yang Y, Sui G, Fu S.(2010) Angiotensin II/angiotensin II type I receptor (AT1R)
signaling
promotes
MCF-7 breast
cancer cellssurvival via
PI3-kinase/Akt
pathway.
J Cell Physiol. Oct;225(1):168-73.
[27] Petty WJ, Aklilu M, Varela VA, Lovato J, Savage PD, Miller AA. (2012) Reverse translation of phase I
biomarker findings links the activity of angiotensin-(1--7) to repression of hypoxia inducible factor-1alpha
in vascular sarcomas. BMC Cancer. Sep 11;12(1):404.
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Brazil)
il)
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Human chorionic gonadotrifin (hCG) and
Angiotensin-(1-7) decrease the tumorigenic capacity
of the undifferentiated Breast Cancer Cell Line
SKBR3
Binda Neto I.1,3, Ribeiro de Noronha S.M.3, Bernardo W.3, Kede J.1,2,3,
da Silva Freitas E.H.1,3; Alves Corrêa de Noronha S.A.3,
Cotrim Guerreiro da Silva3 I.D.
1
Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro – RJ (BRAZIL);
Laboratório de Ginecologia Molecular, Universidade Federal de São Paulo, São Paulo – SP (BRAZIL);
3
Fundação Universitária Serra dos Órgãos, Teresópolis – RJ (BRAZIL).
[email protected], [email protected], [email protected], [email protected],
[email protected], [email protected], [email protected].
2
Abstract
Breast cancer is the most common cancer among women. Angiotensin-(1-7) [Ang-(1-7)] has been
correlated with cancer antiproliferative and apoptotic effects, similar properties of the human Chorionic
Gonadotrofin (hCG). Estrogens (E2) are related to tumor development and progression. The aims of this work
are to evaluate the role of Ang-(1-7), of estradiol (E2) and of hCG in modulating the expression of Nuclear
Receptors and Coregulators related genes in the tumorigenic breast cell line SK-BR3. Three experimental
groups were created: control; hCG, hCG+Ang-(1-7). Cells were treated for 11 days and then had their RNA
extracted. Samples were loaded into PCR Array plates containing 84 genes relate to Nuclear Receptors and
Coregulators pathways. Gene expression data were used to construct canonical pathways (MetacoreTM). hCG
and hCG+Ang-(1-7) treatments markedly modulate the expression of Nuclear Receptors and Coregulators
related genes. hCG diferentially expressed 17% of the genes, being 29% upregulated and 71% downregulated.
Meanwhile, hCG+Ang-(1-7) changed the expression of 30% of the genes on the plate, among these genes 56%
were upregulated and 44% downregulated. Among these differentially expressed genes, we highlight Esr1,
Nr2f2, and Nr2f1, Esr1, Hdac5, and Nr4A1 (>4 fold). Finally MetaCore analysis based on Gene Ontology (GO)
generated six networks for hCG and ten networks for the combined treatment. All generated networks are related
to regulation of apoptosis or to Programmed Cell Death processes. In summary, our results herein demonstrate
that the modulation of sexual hormones and of other nuclear factor genes expression might underlie the
tumorigenic protection effect and the induction of cell differentiation caused by the hormones hCG and Ang-(17) [20], especially in CSCs
Keywords: Breast Stem cancer cells; SK-BR3; hCG; Angiotensin-(1-7); estradiol.
Introduction
Breast cancer is the most common cancer among women, accounting for thousands of deaths annually.
In 2012 the estimated number of new breast cancer cases is above two-hundred and twenty-nine thousand [1].
Among the various mediators that act in the carcinogenic process, the components of the renin-angiotensin
system (RAS) have assumed an important role [2-5]. Angiotensin II (Ang II), better known peptide obtained
from the cascade of events of RAS, has vasoconstrictive, angiogenic, hyperplastic, proliferative and metastatic
properties [6, 7].
Moreover, it has also been demonstrated an association between genetic polymorphisms of some RAS
components with breast cancer [8-10]. Many are the evidences that the RAS is related to neoplasia of the breast
tissue and also that its disruption may be involved in one or more steps that lead to carcinogenesis [11].
On the other hand, angiotensin-(1-7) [Ang-(1-7)] another peptide component of the RAS, has been
extensively studied lately, for its vasodilator, antiproliferative and apoptotic effects, opposite effects generated
by Ang II [12, 13].
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
New breast cancer approaches have identified a small population of highly tumorigenic cells with stem
cell properties in the human breast and in other solid tumors. These cells have been considered the source of
tumor initiation and of its maintenance. These highly proliferative cells are referred to as cancer stem cells
(CSCs) [14].
Altogether, the aims of this work are to evaluate the role of Ang-(1-7), of estradiol (E2) and of hCG in
modulating the expression of Nuclear Receptors and Coregulators related genes in the tumorigenic breast cell
line SK-BR3 in order to better understand the molecular mechanisms underlying the effects triggered by these
compounds in CSCs.
Methods
2.1 Cell Culture and treatments
The SKBR3 cell line was grown in DMEM supplemented with 10% FBS, 2mM glutamine, 100 U/ml
penicillin and 100 μg/ml streptomycin. Threee experimental groups were created: control, hCG and hCG+Ang(1-7). Cells were treated for 11 days.
2.2 RNA Extraction
Pelleted cells were homogenized in Trizol reagent (Invitrogen) according to the manufacturer’s
protocol. Total RNA was purified with Qiagen RNeasy Mini Kit and subjected to treatment with DNase A. The
quantity and quality of extracted RNA were measured by espectrophotometer (Nanodrop Technologies Inc.,
Rockland, DE).
2.3 Real Time PCR Array
According to the manufacturer’s (Qiagen) methodology, reverse transcriptase (RT) was carried out for
the synthesis of cDNA. For each sample we used as a template a PCR array plate containing 84 different pairs
of primers for studying genes related to Nuclear Receptors and Coregulators pathways (RT ² Profiler ™ PCR
Array; SABiosciences).
2.4 Analysis of Relevant Biological Processes and Networks by MetaCore.
The MetaCore software (GeneGo, St. Joseph,MI) is a computational resource that uses logic operations
for identifying altered biological processes based upon gene expression changes. Genes with altered expression
were mapped to Gene Ontol’ogy (GO) using MetaCore algorithm. GO annotations were used as indicators of
biological functions. GO describes gene products in terms of their associated biological processes, cellular
components, and molecular functions. The GO entries are hierarchically linked, thus allowing construction of
cluster genes of crossed pathways.
2.5 Statistical analysis
These results were analyzed by descriptive statistics (means and standard deviation) and inferential
statistics through the Student’s t-test, with significance level of 5% (p <0.05). Real-time PCR array reactions
were processed through the online software RT2 Profiler™ PCR Array Data Analysis (SABiosciences).
Results
hCG and hCG+Ang-(1-7) treatments markedly modulates the expression of Nuclear Receptors and
Coregulators related genes (Figures 1 and 2). hCG diferentially expressed 17% of the genes, being 29%
upregulated and 71% downregulated. Meanwhile, hCG+Ang-(1-7) changed the expression of 30% of the genes
on the plate, among these 56% were upregulated and 44% downregulated. In general, the combined treatment
generates a more downregulated expression profile of these genes than hCG itself (Figs. 1 and 2).
Among these differentially expressed genes, we highlight Esr1, Nr2f2, and Nr2f1, Esr1, Hdac5, and
Nr4A1 (>4 fold) (Fig. 3). Finally MetaCore analysis based on Gene Ontology (GO) generated six networks for
hCG treatment and ten networks for the combined treatment. All generated networks are related to regulation of
apoptosis or to Programmed Cell Death processes (Fig. 4).
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Fig. 1 Heat Map of SKBR3 cells gene expression after treatment.
Fig. 2 Gene expression analysis presented by Scatter Plot graphs.
Fig. 3 Most differentially expressed genes caused by both treatments in SKBR3 cells.
Control cells were used as the calibrator sample
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Fig. 4 Top scored (by number of pathways) network generated by the active experiments. Thick cyan lines indicate the
fragments of canonical pathways. Up-regulated genes are marked with red circles; down-regulated with blue circles. The
'checkerboard' color indicates mixed expression for the gene between files or between multiple tags for the same gene.
Discussion
The search for possible new molecular targets to treat or to early detect breast cancer is of paramount
importance. Nowadays many researchers are focusing on the actions elicited by the hormone hCG, which has
been shown to decrease proliferation of mammary tumor cells [15].
The results here presented clearly demonstrate that hCG alters the expression profile of many genes
encoding for proteins that act as nuclear transcription factors or as nuclear receptors.
Surprisingly both treatments restored the expression of the estrogen receptor 1 (ESR1) gene in this
originally estrogen receptor negative cell line. The ESR1 is a ligand-activated transcription factor composed of
several domains important for hormone binding, DNA binding, and activation of transcription. Estrogen
receptors are also directly involved in different pathological processes including breast cancer, endometrial
cancer, and osteoporosis (Pubmed).
Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer.
Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms
DNA-damaging species. In SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the
lack of ESR1 and the low levels of ESR2 expression suggest that hCG and Ang-(1-7) modulate the expression of
sexual hormone genes [16].
Nuclear Receptor Subfamily 2 (NR2F2) encodes a member of the steroid thyroid hormone superfamily
of nuclear receptors. The encoded protein is a ligand inducible transcription factor involved in regulation of
many different genes (pubmed). Members of this family inhibit cell differentiation and increase cell growth.
Inhibition of COUP-TFII (Nr2f2) may offer a novel therapeutic approach to breast cancer [17]. In the present
study hCG downregulated COUP-TFII, which might partially explain the breast cancer protection brought about
by hCG. Besides that, hCG restores ESR1 gene expression, which might be beneficial when considering the
antineoplastic drugs available to treat breast cancer. At the same time, NR2F2 downregulation indicates that
hCG seems to induce cell differentiation in SKBR3 cells[17].
hCG also caused downregulation of the Nuclear Receptor Subfamily 2 (NR2F1), which may partially
explain the anti-proliferative effects of this hormone [18].
Another important action of hCG was to increase the expression of the NR4A1 gene, which has an
antimigration effect on normal cells.
Histone Deacetylase 5 (HDAC5) is an enzyme responsible for maintenance/assembly of the
heterochromatin structure. As previously demonstrated its specific inhibition might contribute to increase the
efficacy of DNA alteration-based cancer therapies in clinic [19]. hCG inhibits expression of HDAC5, reducing
cancer progression and cell survival.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
In summary, our results herein demonstrate that the modulation of sexual hormones and of other nuclear
factor genes expression might underlie the tumorigenic protection effect and the induction of cell differentiation
caused by the hormones hCG and Ang-(1-7) [20], especially in CSCs [15,21].
Acknowledgments: The work was supported by Grant number 2011/10516-0 and 2008/54383-0 from the Sao
Paulo Research Foundation (FAPESP)-Brazil.
References
[1] American Cancer Society. Cancer Facts & Figures 2012. Atlanta: American Cancer Society; 2012.
[2] Kosaka T, Miyajima A, Shirotake S, Kikuchi E, Oya M. . Phosphorylated Akt up-regulates angiotensin II
type-1 receptor expression in castration resistant prostate cancer. Prostate. 2011 Feb 14.
[3] Queisser N, Oteiza PI, Stopper H, Oli RG, Schupp N. Aldosterone induces oxidative stress, oxidative DNA
damage and NF-κB-activation in kidney tubule cells. Mol Carcinog. 2011 Feb;50(2):123-35.
[4] Hoshino K, Ishiguro H, Teranishi JI, Yoshida SI, Umemura S, Kubota Y, Uemura H. Regulation of androgen
receptor expression through angiotensin II type 1 receptor in prostate cancer cells. Prostate. 2010 Dec 28.
[5] Okamoto K, Tajima H, Ohta T, Nakanuma S, Hayashi H, Nakagawara H, Ohnishi I, Takamura H, Kitagawa
H, Fushida S, Tani T, Fujimura T, Kayahara M. The role of renin-angiotensin system independent
angiotensin II production in progression and fibrosis of intrahepatic cholangiocarcinoma. Gan To Kagaku
Ryoho. 2010 Nov;37(12):2231-3.
[6] Fujita M, Hayashi I, Yamashina S, Itoman M, Majima M. Biochem Biophys Res Commun. 2002; 294:441447.
[7] Greco S, Muscella A, Elia MG, e cols.. Angiotensin II activates extracellular signal regulated kinases via
protein kinase C and epidermal growth factor receptor in breast cancer cells.J Cell Physiol 2003; 196: 370–
377.
[8] Alves Corrêa SA, Ribeiro de Noronha SM, Nogueira-de-Souza NC, Valleta de Carvalho C, Massad Costa
AM, Juvenal Linhares J, Vieira Gomes MT, Guerreiro da Silva ID. Association between the angiotensinconverting enzyme (insertion/deletion) and angiotensin II type 1 receptor (A1166C) polymorphisms and
breast cancer among Brazilian women. J Renin Angiotensin Aldosterone Syst. 2009 Mar;10(1):51-8.
[9] Mendizábal-Ruiz AP, Morales JA, Castro Marti Nez X, Gutierrez Rubio SA, Valdez L, Vásquez-Camacho
JG, Sanchez Corona J, Moran Moguel MC. RAS polymorphisms in cancerous and benign breast tissue. J
Renin Angiotensin Aldosterone Syst. 2010 Nov 25.
[10] Namazi S, Monabati A, Ardeshir-Rouhani-Fard S, Azarpira N. Association of angiotensin I converting
enzyme (insertion/deletion) and angiotensin II type 1 receptor (A1166C) polymorphisms with breast cancer
prognostic factors in Iranian population. Mol Carcinog. 2010 Dec;49(12):1022-30.
[11] del Pilar Carrera M, Ramírez-Expósito MJ, Mayas MD, García MJ, Martínez-Martos JM. Mammary reninangiotensin system-regulating aminopeptidase activities are modified in rats with breast cancer. Tumour
Biol. 2010 Dec;31(6):583-8.
[12] Krishnan B, Smith TL, Dubey P, Zapadka ME, Torti FM, Willingham MC, Tallant EA, Gallagher PE.
Angiotensin-(1-7) attenuates metastatic prostate cancer and reduces osteoclastogenesis. Prostate. 2012 May
29.
[13] Soto-Pantoja DR, Menon J, Gallagher PE, Tallant EAAngiotensin-(1-7) inhibits tumor angiogenesis in
human lung cancer xenografts with a reduction in vascular endothelial growth factor..
Mol Cancer Ther. 2009 Jun;8(6):1676-83.
[14] Economopoulou P, Kaklamani VG, Siziopikou K. The Role of Cancer Stem Cells in Breast Cancer
in Breast Cancer Initiation and Progression: Potential Cancer Stem Cell-Directed Therapies.
Oncologist. 2012
Aug 31
[15] Noronha, Samuel M.R.; Correa-Noronha, Silvana A.A.; Russo, Irma H.; de Cicco, Ricardo
López; Santucci-Pereira, Julia; Russo, José. Human chorionic gonadotropin and a 15 amino acid hCG
fragment of the hormone induce downregulation of the cytokine IL-8 receptor in normal breast epithelial
cells. Hormone Molecular Biology and Clinical Investigation. Volume 6, Issue 3, Pages 241–245
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[16] Hevir N, Trošt N, Debeljak N, Rižner TL.Expression of estrogen and progesterone receptors and estrogen
metabolizing enzymes in different breast cancer cell lines. Chem Biol Interact. 2011 May 30;191(1-3):20616.
[17] Qin J, Chen X, Xie X, Tsai MJ, Tsai SY. COUP-TFII regulates tumor growth and metastasis by modulating
tumor angiogenesis. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3687-92.
[18] Moré E, Fellner T, Doppelmayr H, Hauser-Kronberger C, Dandachi N, Obrist P, Sandhofer F, Paulweber B.
Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor
II (COUP-TFII) expression in human breast cancer cell lines. J Endocrinol. 2003 Jan;176(1):83-94.
[19] Peixoto P, Castronovo V, Matheus N, Polese C, Peulen O, Gonzalez A, Boxus M, Verdin E, Thiry
M, Dequiedt F, Mottet D. HDAC5 is required for maintenance of pericentric heterochromatin, and
controls cell-cycle progression and survival of human cancer cells. Cell Death Differ. 2012 Jul;19(7):123952.
[20] Kocdor H, Kocdor MA, Russo J, Snider KE, Vanegas JE, Russo IH, Fernandez SV. Human chorionic
gonadotropin (hCG) prevents the transformed phenotypes induced by 17 beta-estradiol in
human breast epithelial cells. Cell Biol Int. 2009 Nov;33(11):1135-43
[21] Katherine L. Cook, Linda J. Metheny-Barlow, E. Ann Tallant, et al. Angiotensin-(1-7) Reduces Fibrosis in
Orthotopic Breast Tumors. Cancer Res 2010;70:8319-8328
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) Description of a pedagogical experiment on building
undergraduate students’ clinical thinking coupled up
with cellular processes and biochemistry
understanding
Vargas de Mesquita L.1, Abbud Hanna Roque C.E.2, Ávila de Almeida C.3,
Alves Moreira R.F.4, Rocha C.B.5
1
Undergraduate Medical Student at Universidade Federal do Estado do Rio de Janeiro – UNIRIO (Brazil)
Undergraduate Medical Student at UNIRIO, Fellow of the Extension Department of UNIRIO (Brazil)
3
Undergraduate Medical Student at UNIRIO (Brazil)
4
Ph.D. Associate Professor in the Public Health Department, UNIRIO (Brazil)
5
Ph.D. Associate Professor in the Biochemistry Department, UNIRIO (Brazil)
[email protected], [email protected]
2
Abstract
Introduction: Traditional teaching methods have been criticized for under stimulating students’
autonomy. “Problem Based Learning” (PBL) is pointed as a promising tool to improve the development of
students’ decision making ability as well as research skills. It is useful to help students with hard sciences-related
subjects.
Aims: To trigger undergraduate health sciences students’ interest in cell biology, biochemistry and
research.
Methods: UNIRIO students from second semester and above elaborate case reports by means of
analysis of selected Gaffrée e Guinle University Hospital’s (HUGG) medical records. They receive orientation
from tutors, and must present the case in classroom to first semester students, showing the cellular and molecular
processes involved in the illnesses. The case selection contemplates patients with listed metabolic diseases.
Results: Classroom participation increased. A crescent amount of students have volunteered to enter the
project after they pass the course, including a few who have previously failed, indicating that the method
influenced student-course relation.
Conclusion: The case report elaboration and presentation have enriched classroom experience,
modifying the traditional teacher-centred method into a dynamic collaboration between the parts. Students are
exposed to tangible connections among Biochemistry, Cell biology and Clinical Practice, which is valuable to
courses that usually bring the students to question the “real life” use of the taught information.
This project was approved by the HUGG´s Research Ethical Committee and has funding support of the
UNIRIO´s Extension Department.
Keywords: case report, biochemistry, cell biology, health sciences.
Introduction
Traditional teaching methods are usually based on the model of content exposure made by a professor
or tutor, followed by individual student efforts at home/library, in order to memorize the subjects and become
prepared for further inquiry. This technique is considered non-challenging, lacking proper stimulus for students’
autonomy and ability to find their own way of learning [1],[2].
Over the past years, educators have been concerned about applying teaching methodologies that
emphasize the role of students on their own knowledge acquisition. This matter resulted in worldwide
curriculum changes [3].
When it comes to medical training, case reports have long been suggested as an efficient technique to
unite practice and theory [4], inspiring also the discussions of Problem Based Learning (PBL) [5]. The method
itself was created in the environment of a medical school, as an important tool to teach students how to make the
bridge between classroom and clinical practice, through constant analysis and discussions of hypothetical
situations.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) On the context of PBL, case reports elaboration offers students an opportunity to direct their own
learning path, whilst exploring scientific processes implicated on complex clinical settings. Case reports are
commonly used to train decision making abilities, whereas PBL original idea is mainly to promote scientific
subjects acquisition.
Health sciences graduation students must germinate clinical reasoning, a foundation element of
diagnosis hypothesis skills, and its consequent differential and definitive diagnosis making.
By elaborating and discussing case reports, students are able to develop an interactive way of
connecting practice and theoretical content. The relevance of this method is precisely the linkage made between
basic science and medical praxis [6], thus proving to be an important learning tool all over medical training
rather used on most of the curriculum, especially on courses that bring hard sciences contents, such as
biochemistry and cell biology.
Medical Biochemistry is a branch of clinical specialties that assists the interpretation of signs and
symptoms presented by the patients, providing instruments to diagnose and solve health issues. These
investigations are involved on a large spectrum from prevention to prognostics, including diagnosis, monitoring
amid others. Biochemical tests may comprehend over a third of all laboratory investigations of a hospital.
Best clinical and therapeutic practice is a result of constant training and knowledge of every available
tool, and the academic environment is a privileged place moment to start producing these actions. This is the
reason to carry out this project of conjugating theory and case report elaboration in the medical biochemistry
course of Universidade Federal do Estado do Rio de Janeiro (UNIRIO).
Aims


Guide groups of students on the preparation of case reports and lectures based on real medical cases
(assisted inwards the University Hospital Gaffrée e Guinle, HUGG) exploring their clinical biochemistry
and cell biology knowledge.
Take advantage of the hypothetical deductive method in order to encourage autonomy, creativity and selfeducation skills.

Trigger undergraduate health sciences students’ interest in cell biology, biochemistry and research; also
enrich the ability to discuss and criticize case reports of hypothetical and real patients, stimulating their
clinical thinking; research accomplishment.

Accumulate reports to create a bank of biochemical disorders treated on HUGG through analysis of
medical records.
Methodology
Groups of senior UNIRIO’s students who have already taken the course “Medical Biochemistry” are
selected to voluntarily participate of this program, while two students receive a scholarship. They have weekly
meetings with the professors in order to plan their actions, study better practices and discuss possible case
reports.
On an attempt to integrate pupil monitoring and extension, these students from second semester and
above elaborate case reports by means of analysis of selected HUGG’s medical records. They receive periodic
orientation from tutors, and must present the case in classroom to first semester students, showing the cellular
and molecular processes involved on a previously chosen illness. The case selection contemplates patients with
listed metabolic diseases, and is made by the students after they visit and explore the medical records archives.
Once the case is selected, they show it to the professor who will advise and direct their studies. They will then
elaborate a case report presentation, with the task of give junior students a view not only of the disease but also
of its metabolic etiology and biochemical components behind it. It must be included on the course content.
The project is being developed on the premises of the department of biochemistry and Gaffrée e Guinle
University Hospital. Students analyze medical records of patients seen and hospitalized in this hospital over
recent years. Case reports are being developed based on data obtained from HUGG’s medical records,
emphasizing biochemical aspects contained on the results of specific and complementary tests, combined with
findings described on scientific literature. All documentation is accompanied by appropriate clinical laboratory
results. Every data obtained from medical records is collected respecting patients’ privacy, and their identities
remain confidential, following the guidelines of the Ethics Committee.
Case reports produced during this extension project are used in classroom. Tutored students involved in
the project send in advance the cases to freshman year students (enrolled in this course), with questions they
must try to answer. The questions usually demand a diagnostic hypothesis, laboratory tests they could ask to help
them to test their hypothesis, and typical findings and parameters of the named disease. They have a week to
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) prepare the answers. After this period, the tutored students deliver lectures about the disease described on the
cases accentuating the cellular and biochemical processes of it, and check with audience possible doubts and
different hypothesis, stimulating them to rethink alternatives and differential diagnostic ways.
As a matter of illustration, a group of students delivered a lecture based on a rare case of paroxysmal
nocturnal hemoglobinuria (PNH) treated on HUGG. To show the connections between the illness itself and its
biological etiology, this group not only described the symptoms and course of the disease but emphasized the
cellular and molecular systems that allowed such medical condition. They explained how its etiology is related to
a surface protein malformation, and showed the cellular paths that lead to this characteristic and its consequent
symptomatology. The subject was part of the protein metabolism content, which was being taught on previous
classes by the professor. This way they could make a link between the topic and a possible clinical finding.
This project was approved by the HUGG´s Research Ethics Committee (n.08/10; CNS196/96) and has
funding support of the UNIRIO´s Extension Department.
Results
This method appears to benefit the students’ comprehension of the subjects on a broader spectrum, for it
offers them a bridge connecting what can be perceived by a freshman as “harsh content” into a pleasant
discovery of the practical side of the theme.
Classroom participation increased, signaled by the number of questions and comments after the lectures.
Students often brought up some of the discussed content on the following classes, what can be perceived as an
indicative of an appropriation of what was taught.
We have also observed that an increasing amount of students have volunteered to enter the project after
they are approved on the course, including a few who have previously failed on medical biochemistry, indicating
that this method influenced student-course relation. The project started on the first semester of 2010 with three
invited students, chosen because of their merits (grades and interest).On the first semester of 2011, we had one
more student join the team, he also volunteered to participate. On 2011, second semester, 15 new students
volunteered. On the first semester of 2012 we had a total of 21 students, which are still part of the team. Because
of a national universities strike, we had to adjust the calendar and weren’t able to accept new volunteers for the
second semester of 2012, although the work was maintained. This numbers support our hypothesis that the
students became prone to join the team after taking the course and observing the effects of their colleagues’
efforts on their own learning processes, what leaded to an interest to volunteer and also become part of the team.
Although this result has been shown through assessments and class observation, the evaluation of this
study is still incipient, once the tools to quantify these data are still being developed. At the moment
questionnaires are being tested to make a quantitative record of students’ perception of the subject before and
after taking this course.
Conclusion
Case report elaboration and presentation have enriched classroom experience, modifying the traditional
teacher-centered method into a dynamic collaboration between the parts. Students are exposed to tangible
connections among Biochemistry, Cell Biology and Clinical Practice, which is valuable to courses that usually
bring the students to question the “real life” use of the taught information. Both senior and junior students are
harvesting the benefits of PBL: senior by learning research skills, case report elaboration, practicing their oratory
and lecture delivering, preparing themselves for peers inquiry and more. Juniors benefit from being early
exposed to practical content, as well as being pushed to start building their own problem solving expertise. The
interaction between students from different levels proved to be an extra gain, for they have an opportunity to
exchange their college experience and learn how to do teamwork.
Further investigations must be made to quantify these achievements, although this practice appears to be
successful on its purposes.
References
[1]
[2]
[3]
[4]
[5]
Headrick, K.L.; J. Chem. Educ. 78, 1281, 2001.
Lowe, J.P.; J. Chem. Educ. 78, 1185, 2001.
Cajén,S.G.; Castiñeiras,J.M.D.; Fernandez,E. G. R. Enseñanza de Las Ciencias, 20,17, 2002.
Breathnach, C.S.; Med Educ. 34(12): 974-5, 2000.
Andrade, M.A.B.S.; Campos, L M L.; Atas do V Encontro Nacional de Pesquisa em Educação de Ciências,
Bauru, Brasil, 2005.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [6] Abensur, S.I., Abensur, H., Malheiros, D.M.A., Zatz, R. and Barro, R.T.; Revista Brasileira de Educação
Médica, 31 (3) : 291 – 295, 2007.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Education with technology: the use of an adaptive
interface facilitates the teaching and learning
Lustosa de Oliveira M.1, Carvalho H.F.2
1,2
Dept. of Structural and Functional Biology - Institute of Biology, State University of Campinas (BRAZIL).
E-mails: [email protected], [email protected].
Abstract
The intense transformation in the goals of science education at all levels requires a change in the
methodologies used to teach Cell Biology. This research presents the construction and application of an adaptive
interface to follow these changes facilitating and motivating the students in the teaching-learning process. The
content of the interface is related to cell cycle and proliferation. The interface was applied to two groups of 3rd
year high school students, while other two groups received conventional lecture based. Prior to application, a
pre-test was given to the students in order to assess their previous knowledge, and after implementation, a posttest was given. They were also asked to produce concept maps in order to do an integrative synthesis of concepts
allowing the assessment of acquired. To compare the groups, ANOVA and T test were performed. In the pre-test
that there was no significant difference (p = 0.197). It is assumed that all students had the same level of prior
knowledge. However, when comparing the results of the post-tests, a statistically significant difference was
obtained between students who received the methodology and those who did not (p = 0.001). In addition, the
concept maps constructed by those who received classes with the interface presented more appropriate
connections and theoretical associations. So, it could be inferred that students who had classes with the interface
showed greater acquisition of knowledge about the content taught. The research was approved by the ethics
committee of the institution.
Keywords: adaptive interface, cell biology, teaching and learning.
Introduction
The cells are highly dynamic structures. Every day, thousands of reactions are processed inside them.
Everything, from reproduction to infections or even how broken bones are recovered, everything happens by the
action of the cells. Although the study of the cell is fantastic, the teaching of the cell biology, reduced to a static
lists of names, cycles and biochemical reactions [1]. Students learn concepts without understanding the dynamic
of biological systems; this is not the goal of science education. In this sense, to achieve the goals of the science
education at all levels are necessary changes in the methodologies used to teach. This research presents the
construction and application of an adaptive interface to follow these changes, facilitating the teaching-learning
process, motivating the students and making the cell biology more interactive and dynamic.
According Langley [2] an adaptive interface teaching of is a virtual space that improves the ability to
interact by constructing a "user model" based on a partial experience with that user. This definition makes clear
that an adaptive interface does not exist in isolation, but rather is designed to interact with a specific user. The
content of the interface described in this study is related to proliferation and cell cycle
(http://biocelunicamp.wix.com/ppg) and it is characterized as “adaptive” because interacts with three users
model, each one with a specific page in the interface, on which the content taught is adapted to the students level
of knowledge, i.e., despite having the same theme for all audiences, each group has a tab with language and an
in-depth approach suited to a specific educational level.
The page intended to elementary school is called "Basic", the one prepared for high school is called
"Intermediate" and the page destined to undergraduate is called "Advanced" (Fig. 1). Even though having a
definite direction by the tabs (basic, intermediate, advanced), the users can also play a quiz called "level test", in
order to check which tab is best suited to their knowledge. Thereby, students can do perform a self-evaluation, in
a playful way, and then access the virtual environment more suited to their level of knowledge about cell cycle
and proliferation. It is noteworthy that, the users are not limited to their tabs, if there are doubts or objections,
they can change to other tab more appropriate, advancing or returning whenever they consider it necessary.
Thus, the interface is adaptive, but at the same time, can be adapted by the users, because they can play a game
to check their knowledge, being directed to a the tabs according to the answers given to the questions of the
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game, or choose one of the three tabs (Basic, Intermediate or Advanced) according to their level in school, their
affinity or their understanding about the content.
Although a variety of research efforts have shown the potential of adaptive user interfaces, there
remains a considerable room for extending their flexibility and their interaction style in the education. Moreover,
in the bibliography, no information was found regarding the use of adaptive interfaces in the science education
or cell biology, which represented the incentive for the effort of this research. Thus, the objective of this study
was to extend the potential of adaptive interfaces, applying it to four high school classes. Tests, interviews and
concept maps were used to evaluate the efficiency of the interface as a facilitator resource of the teachinglearning of cell biology.
Figure 1. Tabs created to specific public. A) Elementary school or basic level of knowledge, B) high school or intermediate
level of knowledge, and C) Undergraduate or advanced level of knowledge. All the tabs contain the same subject:
proliferation and cell cycle.
Methodology
The activity with the interface for high school was based in the tab of the intermediate level. This tab
was built in five pages, whose titles and content are summarized in the table below:
TITLE
Vision
Cell Cycle
Proliferation
Quiz
Experience
SYNTHESIS OF THE CONTENT
A cartoon and a short text mention the limitations of our visual capacity and software
explains the origin and operation of the microscopes.
Four softwares are grouped in this tab. The terms taught in each one are: transcription,
translation, replication and mitosis.
A short text and two videos are displayed; one of them shows cellular proliferation and
other the relation between cell proliferation and cancer.
On this stage users are directed to review and then to answer a Quiz game.
This tab proposes a group activity, in which students should complete a science fiction
story answering some questions related to the content.
To evaluate this part of the adaptive interface, four classes of high school, senior year, from the
“Technical School of Campinas”, totaling 131 students were engaged. Two were in the control group (3º M and
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3º T), and had conventional teaching about the subject; and two on the experimental group (3º P and 3º E), had
classes with the interface about the same subject. Before applying the interface, all students responded a pre-test
to assess their prior knowledge. As previously mentioned, after the pre-test, two classes had the explanation of
the content using the adaptive interface and other two classes were taught in a conventional way. Thereafter, all
classes performed a post-test to estimate the acquired knowledge and to compare the results with and without the
use of the interface. The comparison of the pre-tests scores were held using ANOVA, and for analyze the effect
of learning with and without the interface t-test was used.
Students who had conventional classes had access to the interface after the post-test. Finally, the
students were required to construct concept maps to assess the learning and the terms and connections
established about the content. The maps are graphical tools for organizing and representing knowledge. They
include concepts, usually enclosed in circles or boxes of some type, and relationships between concepts indicated
by a connecting line linking two or more concepts. Three criteria were used to evaluate the maps constructed by
students: the presence of key-words, links between these words and the organization of the conceptual map. The
students also received a questionnaire to evaluate several aspects of the adaptive interface.
Results and Discussion
When comparing the scores of the pre-tests of all groups, it was observed that no significant difference
between them (p = 0.197), i.e., all students had the same level of knowledge before classes (Fig. 2). This analysis
is important to check the presence of inhomogeneity in the population. The pre-tests also can help students to
focus on the key topics that will be covered. Moreover, pre-tests help measure true learning. By comparing pre
and post-tests, teachers can see what students actually learned [3], [4]. In this case, when comparing the results
of the post-tests (Fig. 3), using the t-test, a significant difference between students who received the
methodology and those who did not, was verified (p = 0.003). The mean score of the students, who used the site,
with the activities set and tools provided in this resource, was higher (0.74) than the mean of the students who
have not used it (0.64). Thus, it can be inferred that students who had classes with the interface obtained greater
acquisition of concepts and knowledge about the content taught than students who had conventional classes.
Figure 2. Mean in the pre-test. It was conclude that there wasn’t significant difference between them (p > 0.01) then, all
students had the same level of knowledge.
According to Sutherland et al [5] high levels of student engagement are normally associated with the
use of technologies, whether in school or at home, because the students can work for extended periods of time
investigating their own questions and experimenting with ideas in an interactive way. The adaptive interface
allows interactivity by the presence of videos, cartoons, image gallery, softwares and games that allow students
to learn by playing. During interviews students mentioned that some concepts of Cell Biology are considered
abstract because belong to the “microscopic world”, but in the interface, these concepts became clearer and more
tangible. Furthermore, in the experimental group the students did not write during the class, they just listened the
teacher and interacted with the interface. Most of the students who take notes during a lecture tend to process
information listening and simply recording what they hear. However, students who process information visually,
imagining what they hear, usually outperform students who process information differently, in terms of their
recall ability [6]. Visualization in the form of creating mental images is a fundamental cognitive process that has
been shown to improve student learning, particularly in science [7]. But in some cases, students may have
misconceptions and imagine wrong ways the biologic event. Images, videos and softwares that show what is
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being described by the teacher are important, because they improve the student conceptual understanding and,
ultimately, student performance on assessments.
Figure 3. Mean of post-test. The experimental group scores were higher than control group (p< 0.01).
Another assessment tool used was the conceptual map. When constructed by the students, these maps
present in a highly concise manner the key concepts and principles learned [8], [9]. Comparing the maps of the
two groups (as shown in table 1), it is possible to verify that the students in the experimental group showed
greater acquisition of key words, more connections between the terms and maps more organized, relative to the
control group. Furthermore, this fact demonstrates the development of a meaningful learning by students in the
experimental group; since the fundamental characteristic of the meaningful learning is integration of new
knowledge with the learners’ previous concept [8] and propositional frameworks, proceeding from the more
general to more specific concepts structured in conceptual maps serves to encourage and enhance meaningful
learning. Thus, there is no question that the use of adaptive interface deepened students' understanding about the
cell biology.
Table 2. Different criteria was used to evaluate the conceptual maps. The percentage of each criterion indicates the amount
of students who done correct relation of subject using the criteria. The mean in the experimental group was higher than the
control group.
Conclusions and perspectives
Our results suggest that the "adaptive interface" presented is an efficient alternative to facilitate the
teaching and learning of cell biology to students in high school.
The next stage of the research will be the application and evaluation of the interface to other two public:
elementary school and undergraduates. We hope that the new approach to teaching and learning of Cell Biology,
as well as the suggestions expressed in this research, help the teachers to clarify the content for the students, and
its efficient implementation provides new avenues to be explored by the academic community in the near future.
References
[1] Hillel J. Chiel, Jeffrey P. Gill, Jeffrey M. McManus, Kendrick M. Shaw. (2012). Learning Biology by
Recreating and Extending Mathematical Models. Science 336 (6084) pp. 993-994.
[2] Langley, P. (1999). User modeling in adaptive interfaces. Proceedings of the Seventh International
Conference on User Modeling. Banff, Alberta: Springer pp. 357-370.
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[3] Bonnie J.M. Swoger. (2011). Closing the Assessment Loop Using Pre- and Post-Assessment. Reference
Services Review 39 pp. 244-59.
[4] Hufford, J.R. (2010). What are they learning? Pre- and post-assessment surveys for LIBR 1100, Introduction
to Library Research. College & Research Libraries 71(2), pp. 139-59.
[5] Sutherland, R.; Robertson, R., & John, P. (2004). Interactive education: teaching and learning in the
information age. Journal of Computer Assisted Learning. 20(6) pp. 410-412.
[6] Revak MA, Porter DB. (2001). The toothless bathing beauty and the t-test. Teach Stat. pp. 23:22–23.
[7] Wu H-K, Shah P. (2004). Exploring visuospatial thinking in chemistry learning. Sci Educ. 88, pp. 465–492.
[8] Novak, J. D. & A. J. Cañas. (2006). The Theory Underlying Concept Maps and How to Construct and Use
Them. Technical Report IHMC CmapTools. Florida Institute for Human and Machine Cognition.
http://cmap.ihmc.us/Publications/ ResearchPapers/TheoryUnderlyingConceptMaps.pdf.
[9] Derbentseva, N., Safayeni, F., & Cañas, A. J. (2007). Concept maps: Experiments on dynamic thinking.
Journal of Research in Science Teaching 44(3) pp. 448–465.
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Brazil)
il)
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Teaching at Distance: Interactive Multimedia of the
Cell Biology of Trypanosoma cruzi
Benchimol M.1,2, Teixeira D.E.2, Crepaldi P.H.1,4, de Souza W.3,4
1
Universidade Santa Úrsula, Rio de Janeiro, RJ, Brasil
Fundação CECIERJ, Rio de Janeiro, RJ, Brasil
3
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil
4
INMETRO, Rio de Janeiro, RJ, Brasil
E-mails [email protected], [email protected], [email protected], [email protected]
2
Abstract
CECIERJ is a state public foundation focused in education. It includes CEDERJ arm, which is
specialized in teaching at distance as university. Our group has developed an intense work producing multimedia
material to undergraduate students in Biology. The aim of this work was to develop educational materials in the
graphical version using three-dimensional (3D) animations, to visualize the morphology, dynamic processes and
basic knowledge of the Cell Biology as a whole and here, in special of Trypanosoma cruzi, the causative agent of
Chagas disease. Parasitic protozoa are important agents of human and veterinary diseases not only in Brazil but
also all over the world. The life cycle of this protozoan is presented in different levels of education, from
fundamental school to graduation level. Videos and animations include: cell division, endocytosis and flagellar
beating; the interaction of the parasite with a vertebrate host cell and the behaviour of this protozoan in the
digestive tract of the invertebrate host. Thus, this material could: (1) facilitate the learning and teaching of cell
biology of parasites, (2) provide good material that can be used by several people at different levels, such as
lectures, classes, research, thesis, etc.
Keywords: Biology and Parasitology education, protozoan, Trypanosoma cruzi, life cycle, Chagas
disease, 3D animation
Introduction
One of the biggest challenges of the contemporary world is to provide quality in higher education,
democratizing access to education and encouraging lifelong learning. Distance Education (DE) has been
presented as an alternative modality for training individuals who, for some reason, cannot or are unable to attend
an ordinary graduate classroom and, increasingly, by students who identify themselves with the methodology of
distance learning because they are autonomous. This mode uses new information technologies and
communication technologies (ICTs) with possibilities of interaction in the virtual environment. The multiple
possibilities of relationships in the network potentiate a "new relationship with knowledge" [1]. Follow this
thought, CECIERJ is a state public foundation focused in education. It includes CEDERJ arm, which is
specialized in teaching at distance as university.
1.1
Distance Education in CECIERJ Foundation and CEDERJ Consortium
In the State of Rio de Janeiro, CEDERJ consortium is linked to the Fundação Centro de Ciências e
Educação a Distância do Estado do Rio de Janeiro (CECIERJ) and seven public institutions based in the state:
Universidade do Estado do Rio de Janeiro (UERJ), Universidade Estadual do Norte Fluminense Darcy Ribeiro
(UENF), Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Universidade Federal do Rio de Janeiro
(UFRJ), Universidade Federal Fluminense (UFF), Universidade Federal Rural do Rio de Janeiro (UFRRJ) and,
recently, the CEFET/RJ (Centro Federal de Educação Tecnológica Celso Suckow da Fonseca). Currently, the
following 12 courses are offered in distance mode (Administration, Public Administration, Biological Sciences,
Physics, History, Linguistics, Mathematics, Pedagogy, Chemistry, Tourism, Computer Systems Technology and
Tourism Technology) with support on 33 study centers.
The CEDERJ consortium, working in partnership with municipal governments that are responsible for
regional study centers, aims to contribute to an increased access opportunities to public higher education and free
and quality education in the State of Rio de Janeiro, leading public universities into the periphery and densely
populated towns in the metropolitan area. The decision to offer courses drawing on the modality of distance
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
education (DE) allows the entry of people hitherto excluded from the process of public higher education for
different reasons. Among them, geographical access (because they live far from the university campuses) or the
unavailability of time to attend the traditional classroom courses have prevented people from taking courses.
In the CEDERJ consortium, the responsibility of academic courses lies with the public universities
(institutions that confer diplomas to students). This task includes to be responsible for the design of educationallearning courses, the contents of teaching materials and in the conduction of the courses (including learning
assessment and evaluation and training of tutors). In addition, CECIERJ Foundation takes care for the logistics
and management's own mode of distance education, including the design and production of didactic material,
construction and management of a virtual learning environment, assembly and management of regional study
centers, managing the entry process and institutional assessment.
Our group has developed an intense work producing of learning multimedia material for undergraduate
students in Biology courses that was described in Benchimol et al. [2]. In the current report, we present a new
instructional material with modern schemes and dynamic models, which includes 3D animations. We aim to give
this material to students in order to improve the understanding of the life cycle of a parasitic protozoan.
As an instructional tool, the animations are more effective than the static graphics for teaching dynamic
events ([3], [4]). Rieber [5] demonstrated that the animations reduce the abstractions associated with the
temporal transitions of the process of teaching. The dual-coding model, developed by Paivio [6], suggests that
long-term memory retention is facilitated by a combination of verbal and visual cues. Animations are a valuable
resource in supporting the visual aspects of long-term memory. Previous research suggests that when animations
are simultaneously presented with a narration, the dual-coding model is further supported [7]. Studies in biology
courses have shown that 3D animations lead to increased student understanding and retention of cell biology
information [8]. A systematic review of 17 studies multimedia resources has shown that multimedia learning was
more effective than many traditional educational methods in tertiary level life science education [9].
The aim of this work was to develop educational material in the graphical version using threedimensional (3D) animations, to visualize the morphology, dynamic processes and basic knowledge of the Cell
Biology as a whole and here, in special of Trypanosoma cruzi, the causative agent of Chagas disease. Videos and
animations include: cell division, endocytosis and flagellar beating; the interaction of the parasite with a
vertebrate host cell and the behaviour of this protozoan in the digestive tract of the invertebrate host. Thus, this
material could: (1) facilitate the cell biology of parasites learning and teaching, (2) provide good material which
can be used by several people at different levels, such as lectures, classes, research, thesis, etc.
The depth of this subject correlates to the level of education. The subject matter becomes more in-depth
as the student advances from high school to an undergraduate or graduate institution. Similarly, the subject
matter becomes more involved as the student advances from basic science to biomedical science or specializes in
parasitology. The protozoa of the Trypanosomatidae family are a popular experimental model to study basic
biological processes, including RNA editing and compartmentalization of the glycolytic pathway in glycosomes
([10], [11]). Therefore, our multimedia materials will be useful for researchers that work with the protozoa of the
Trypanosomatidae family. Several researchers can take advantage of our accessible materials for their own
presentations and classes.
Methods
The 3D models and animations were produced by designers working at the CECIERJ
Foundation/CEDERJ Consortium. This material is derived from scientific basic research and experience of the
authors, including light and electron microscopy. Our analysis also used information obtained by different
research groups. The multimedia materials will be useful for a broad audience, which includes students, teachers,
and any member of the general public that may be interested in parasites. All animations and images were
produced using software such as 3ds Max, Maya, Poser, Blender, Hexagon and Flash.
Results and Discussion
1.2
Life Cycle
During its life cycle, T. cruzi infects both invertebrate and vertebrate hosts. Fig. 1 shows a general view
of its life cycle. Previous published figures typically showed only the various developmental stages, as depicted
in
a
figure
from
the
Center
of
Disease
Control
(CDC)
site
(http://www.dpd.cdc.gov/dpdx/html/trypanosomiasisamerican.htm). In our recent publication [12], we
incorporated the general shape of the various developmental stages (see numbers 1 to 5 in the upper portion) and
details of the intracellular life cycle in the vertebrate (see numbers 6 to 15 in the lower portion) into Fig. 1. In the
central portion of the figure, we added the most important animal reservoirs involved in the maintenance of the
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parasite in the domestic and peridomestic environment. Therefore, we believe that the schematic in Fig. 1 is the
most comprehensive view of the life cycle of T. cruzi.
Fig. 1. The life cycle of Trypanosoma cruzi. 1. The insect vector (female or male) bites a mammalian host and ingests
trypomastigotes located in the blood. 2. Metacyclic trypomastigotes. 3. Trypomastigotes transform into epimastigotes and
some spheromastigotes. 4. Epimastigotes multiply in the midgut. 5. Epimastigotes transform into metacyclic trypomastigotes
in the hindgut. 6. The insect vector passes the metacyclic trypomastigotes in feces near a bite site after feeding on a
mammalian host. 7. Metacyclic trypomastigotes form. 8. Metacyclic trypomastigote infects macrophages. 9. Metacyclic
trypomastigote transforms into amastigote. 10. Amastigote is released from the parasitophorous vacuole. 11. Amastigotes
multiply in the cytoplasm. 12. Amastigotes transform into trypomastigotes. 13. Trypomastigotes burst out of the cell. 14.
Amastigotes and trypomastigotes form. 15. (a) Trypomastigotes and (b) amastigotes infect macrophages. In the central
portion of the figure, we added the most important animal reservoirs involved in the maintenance of the parasite in the
domestic and peridomestic environment. After Teixeira et al. [12].
The parasite can also penetrate the human body in other ways: by blood transfusion (now almost
eliminated from Brazil), during pregnancy and even by eating insect accidentally crushed along with sugar cane
or fruit while beverage preparation (oral contamination) [13].
We have reported a video with these basic aspects of the life cycle of T. cruzi in the human host
and
in
the
triatomine
insect
(http://www.imbebb.org.br/conteudo.asp?idsecao=242)
( http://www.imbebb.org.br/conteudo.asp?idsecao=243) in our recent publication [12].
The trypanosome life cycle begins with a triatomine insect, which is infected with T. cruzi, biting a
human host. During the bite, the insect injects saliva that prevents the blood from clotting. Following the
ingestion of blood, the metacyclic trypomastigotes are released with the feces from the insect near the surface of
the bite. Trypomastigotes enter the host through the bite site when the host has a scratching reaction.
Trypomastigotes invade the mammalian’s blood and invade macrophages and other cell types. Parasitic
attachment to the macrophage surface is followed by the recruitment and fusion of host cell lysosomes, which
form a parasitophorous vacuole. Inside the vacuole, the trypomastigote transforms into an amastigote. This
maturation is accompanied by the disruption of the parasitophorous vacuole membrane. The amastigotes are
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released into the cytoplasm of the host cell and divide multiple times. Following division, the amastigotes
transform into trypomastigotes that burst out of the cell and enter the bloodstream.
Trypomastigotes in the bloodstream pass back to the insect once the insect ingests blood from an
infected host. In the insect stomach, trypomastigotes transform into epimastigotes and spheromastigotes.
Epimastigotes pass through the stomach to the intestine, divide by binary fission and attach to the perimicrovillar
membranes of the intestinal cells. Epimastigotes travel to the rectum and transform into metacyclic
trypomastigotes, which are eliminated with the feces. During subsequent insect bites, metacyclic trypomastigotes
infect the vertebrate host, restarting the cycle.
1.2.1 The behavior of T. cruzi in the invertebrate host
The life cycle of T. cruzi in the invertebrate host, begins with the ingestion of trypomastigotes present in
the blood of the vertebrate host during a blood meal. In the invertebrate stomach, some of the trypomastigotes
are lysed and others become rounded. These rounded trypomastigotes possess a long flagellum and are known as
spheromastigotes. Epimastigotes are also formed and migrate to the midgut, which is the only site for the
epimastigotes to proliferate in the intestinal tract. At the most posterior region, many of the epimastigotes attach
to the intestinal surface. Subsequently, the epimastigotes transform into trypomastigotes, which are known as
metacyclic trypomastigotes that migrate to the rectum and are released in the feces and urine during the insect’s
blood meal.
1.2.2 The interaction of T. cruzi with vertebrate host cells
As part of the life cycle, the infective trypomastigote and amastigote forms of T. cruzi interact with
different types of cells in the mammalian hosts. This interaction has been studied in some detail in cell culture
(both phagocytic and non-professional phagocytic cells). The first step of this interaction involves adhesion and
recognition, which is followed by signaling and invasion ([14], [15]). The parasites first attach to the
macrophage surface and are then internalized into a vacuole, which is known as the parasitophorous vacuole
(PV) [16]. During the formation of the PV, the host cell lysosomes fuse with the nascent PV [17]. For
epimastigotes, the destruction of the intravacuolar parasite occurs. In trypomastigotes, the fusion of the
lysosomes and the PV occurs during the intermediate stage when the trypomastigotes are gradually transforming
into amastigotes. Simultaneously, there is a process of disintegration of the PV membrane, which allows the
amastigotes to directly contact the cytoplasmic components of the host cell. The low pH environment of
lysosomes facilitates parasite egress from the vacuole and delivery into the host cytosol [18]. Once in the host
cytoplasm, the amastigotes divide several times to occupy most of this area. Next, they undergo an almost
synchronous transformation into trypomastigotes, via an intermediate stage. The trypomastigotes are highly
motile and once the cell ruptures, hundreds of infective trypomastigotes are released into the intercellular space.
These forms are able to infect other cells and this lytic cycle can be repeated several times. A similar process
occurs when amastigotes infect host cells. The process of infection in heart muscle cells, is similar to that
intracellular cycle already described in macrophages.
The multimedia materials described herein will present a comprehensive view of the protozoan life
cycle to students. These materials also offer dynamic models improve the understanding of some important
biological processes.
Acknowledgments: This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do
Estado do Rio de Janeiro (FAPERJ). The authors would like to thank Celso Sant´Anna, Gustavo Rocha, Kildare
Miranda, Danielle Cavalcanti, Tecia Maria Ulisses de Carvalho, Marina Verjovsky, Rodrigo Leite, Marcelo
Xavier and Ricardo Amaral for their support during the development of this work.
References
[1]
[2]
[3]
[4]
[5]
Lévy, P. (1999). Cibercultura. São Paulo, SP: Ed. 34.
Benchimol, M., Outeiro, M.A.F., Carvalho, R.A., Teixeira, D.E. (2010). Desenvolvimento de material
multimídia no ensino de biologia. EAD em Foco - Revista Científica em Educação a Distância, 1(1), pp.
99-158.
Pollock, E., Chandler, P., and Sweller, J. (2002). Assimilating complex information. Learning Instruction
12(1), pp. 61–86.
Tversky, B., and Morrison, J.B. (2002). Animation: can it facilitate? Int. J. Human Comput. Stud. 57(4),
pp. 247–262.
Rieber, L. P. (1994). Computers, graphics, and learning. Madison, WI: Brown & Benchmark.
© Medimond . P726R9106
36
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Paivio, A. (1991). Dual coding theory: retrospect and current status. Can J Psychol 45(3), pp. 255–287.
Mayer, R.E., and Anderson, R.B. (1991). Animations need narrations: an experimental test of a dualcoding hypothesis. J Educ Psychol 83(4), pp. 484-490.
McClean P, Johnson C, Rogers R, Daniels L, Reber J, et al. (2005). Molecular and cellular biology
animations: development and impact on student learning. Cell Biol Educ 4(2), pp. 169–179.
Rolfe, V. and Gray, D.T. (2011). Are multimedia resources effective in life science Education? A metaanalysis. Bioscience Education, 18(3).
De Souza, W. (2002). Special organelles of some pathogenic protozoa. Parasitol Res. 88(12), pp. 10131025.
Parsons, M. (2004). Glycosomes: parasites and the divergence of peroxisomal purpose. Mol. Microbiol.
53(3), pp. 717-724.
Teixeira, D.E. ; Benchimol, M. ; Crepaldi, P.H. ; De Souza, W. (2012). Interactive Multimedia to Teach
the Life Cycle of Trypanosoma cruzi, the Causative Agent of Chagas Disease. PLoS Neglected Tropical
Diseases 6(8), pp. e1749.
Yoshida, N. (2008). Trypanosoma cruzi infection by oral route: how the interplay between parasite and
host components modulates infectivity. Parasitol Int. 57(2), pp. 105-109.
Burleigh, B.A., and Woolsey, A.M. (2002). Cell signaling and Trypanosoma cruzi invasion. Cell
Microbiol 4(11), pp. 701-711.
De Souza, W., De Carvalho, T.M., and Barrias, E.S. (2010). Review on Trypanosoma cruzi: Host Cell
Interaction. Int J Cell Biol 29, pp. 295394–295412.
De Souza, W. (2005). Microscopy and cytochemistry of the biogenesis of the parasitophorous vacuole.
Histochem Cell Biol. 123(1), pp. 1-18.
Andrade, L.O., and Andrews, N.W. (2005). The Trypanosoma cruzi-host-cell interplay: location,
invasion, retention. Nat Rev Microbiol. 3(10), pp. 819-823
Mott, G.A., and Burleigh, B.A. (2008). The role of host cell lysosomes in Trypanosoma cruzi invasion.
Subcell Biochem. 47, pp. 165-173.
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Eph/ephrin- mediated interactions govern many
aspects of thymus biology
Zapata A.G.1, Alfaro D.1, Garcia-Ceca J.1, Cejalvo T.2, Tobajas E.1, Montero S.1,
Muñoz J.J.2
(1) Department of Cell Biology, Faculty of Biology,
(2) Centre for Cytometry and Fluorescence Microscopy, Complutense University, 28040 Madrid, Spain
The thymus is a primary lymphoid organ in which a 3D epithelial network supports the functional
maturation of lymphoid progenitor cells into T lymphocytes. This complex process is highly dependent on the
migration of developing thymocytes to the adequate thymic niches in which thymic epithelial cells (TECs) and
thymocytes establish critical interactions. Eph receptors and their ligands, ephrins, partially mediate these
interactions and are, therefore, involved in numerous processes occurring in the thymus. In the present article,
available information supporting this assumption is reviewed.
Eph is the largest family of tyrosine kinase receptors of animal cells. Their ligands, ephrins, are also
extensively represented in numerous cell types. Both Eph and ephrins are divided into two families A and B,
based on gene sequence similarities and ligand binding preferences. Although there are some exceptions, some
EphA can interact with ephrin B and vice versa, EphA (10 members) bind GPI-anchored ligands, ephrins A (6
members), whereas EphB (6 members) bind transmembrane proteins, ephrins B (3 members). Each Eph kinase
can bind with different affinities, several ephrins and vice versa and both receptors and ligands transmit signals,
forward and reverse, respectively to the expressing cells. Accordingly, this plastic system allows many different
cell interactions that result in a wide spectrum of cellular functions, including cellular attachment/detachment,
cell shape and cell migration, cell fate, etc…1. Both subfamilies of Eph and ephrins are largely expressed in
thymus although it is difficult to establish a specific pattern of expression restricted to the two thymic
compartments, cortex and medulla. Importantly, both thymocytes and TECs express Eph and ephrins and a same
cell can express several Eph and/or ephrins2.
Several years ago we determined that the in vitro blockade of Eph/ephrin A interactions partially
blocked T-cell differentiation3. In that study, the supply of either soluble EphA-Fc or ephrinA-FC fusion proteins
to fetal thymus organ cultures (FTOCs) resulted in a significant reduction of the total yielded cells that affected
all the thymic subsets, but largely the DP cell compartment and was accompanied by increased proportions of
apoptotic thymocytes. Although these first results suggested certain non-specificity of the Eph/ephrin effects and
the possibility that in the absence of one Eph or ephrin any other member of the same family could substitute its
lack, further in vivo results demonstrated that observed effects were specific to the distinct Eph analyzed.
Thus, whereas EphA4-deficient mice showed decreased numbers of thymocytes together with an important
blockade of T-cell maturation with decreased proportions of DP cells4, EphB2- and/or EphB3-deficient thymuses
also showed decreased cell numbers and high proportions of apoptotic thymocytes but few changes in the
proportions of T-cell subsets5. However, the thymic epithelium showed profound alterations, specific for each
mutant type but exhibiting some common features: occurrence of K5+K8+MTS-10+ immature medullary
epithelial cells, abundant K5-K8-MTS-20+ cells, K5+K8+ cells in the cortex and large K5-K8- areas devoid of
epithelial cell markers6. Remarkably, other authors have failed to observed phenotypical changes in the thymus
of EphB2- or EphB6-deficient mice7, 8.
Because, as mentioned above, both thymocytes and TECs express Eph and ephrins, a second important
issue was to determine which thymic cell type is responsible for the observed phenotypes. We, therefore,
analyzed the thymic phenotype when only thymocytes or TECs were deficient in Eph or ephrins B. These studies
demonstrated that TEC-thymocyte interactions are critical for the Eph/ephrin-mediated effects on thymus.
Whereas EphA4 KO bone marrow (BM) progenitors differentiate normally in SCID thymuses with a normal
Eph/ephrin expression, WT progenitors fail to differentiate in EphA4 KO fetal thymus lobes grafted under the
kidney capsule of WT mice, showing decreased proportions of DP thymocytes4. In correlation with these results,
an immunohistochemical study demonstrated the collapse undergone by the EphA4-deficient cortical epithelium
where DP cells mature. In similar experiments, SCID mice that received BM progenitors deficient in different
Eph B showed blockade of T-cell differentiation at distinct stages of maturation confirming the specificity of
these molecules. Chimaeras established with EphB2-/- or EphB2/B3-/- progenitor cells show blockade of
thymocyte development at DN stage. SCID mice receiving precursor cells that express a truncated EphB2
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molecule, EphB2LacZ, that can transmits reverse signals to ephrinB-expressing neighboring cells, but not forward
signals, reach the DP stage but do not generate SP thymocytes. Finally, EphB3-/- precursor cells injected in SCID
mice show a partial blockade of T-cell development at DN stage resulting in decreased proportions of DP
thymocytes. Together these results indicate that both EphB2 and EphB3 exert an autonomous control on
thymocyte development but the lack of EphB2 results in a more severe phenotype. In addition, the epithelial
phenotype of these chimaeric thymuses was similar to that observed in the EphB KO mice demonstrating that
the thymic epithelial organization requires a non-autonomous EphB-dependent induction by developing
thymocytes9. Thymuses whose epithelium was deficient in ephrinB1 and/or B2 showed a smaller size than WT
counterparts and profound alterations in the thymic epithelial network but few changes in the thymocyte
subsets10. These changes were restricted to the double mutants in which a relationship could be established
between altered T-cell development and severity of epithelial changes. The expression of ephrinB1 and
ephrinB2 in TECs is necessary for a correct epithelial organization and T-cell maturation. In the thymuses
in which ephrins B were selectively deleted in thymocytes, there was a partial blockade of T-cell maturation with
increased proportions of DN3 cells10. Nevertheless, the phenotype severity was highly dependent on the mouse
strain, being more evident in those of mixed background, a phenomenon described for other Ephs and ephrins.
The thymic epithelial component also showed thymic medulla fragmentation and numerous, big epithelial cysts.
Taken together these results support that: Eph and ephrins affect thymus phenotype through the modulation
of thymocyte-TEC interactions; EphB2 and EphB3 and their ligands ephrinB1 and ephrinB2 play
autonomous and non-autonomous roles in T-cell differentiation and thymic epithelial network
organization, and the different reported thymic phenotypes are the result of the balance of combined
Eph/ephrin transmitted signals.
The relevance of Eph/ephrin-mediated thymocyte-TEC interactions for thymus functioning was
confirmed in in vitro experiments. EphrinB1-Fc fusion protein supplied to reaggregate thymus organ cultures
formed by fetal TECs and DP thymocytes disorganized the three-dimensional epithelial network of thymus.
Furthermore, ephrinB1-Fc proteins prevented thymocyte-TEC associations and altered TcR signaling11. More
recently, we demonstrated that the numbers of thymocyte-TEC conjugates formed decreased when using EphB2or ephrinB-deficient DP thymocytes, but increased when thymocytes expressed a truncated EphB2 molecule, the
unique SCID chimaeras that, as described above, exhibited normal proportions of DP thymocytes9. Analysis of
thymus development in several Eph/ephrinB-deficient mice has confirmed that the effects of Eph/ephrinB are
not restricted to adult thymus. The thymic primordium is a pharyngeal endoderm derivate which progresses as an
outgrowth with a central lumen. As early as E11.5 this outgrowth is formed by polarized pseudo- or bi-stratified
epithelium that immediately folds in a branching morphogenesis pattern. When this early branching process is
examined in EphB2-deficient mice, the lack of EphB2 does not affect its general pattern but alters the
development of epithelial branches that appear collapsed.
The arrival of lymphoid progenitors to E12 thymic primordium determines changes in the TEC shape
and the 3D-organization of thymic epithelium. Studies performed on in vitro colonization of FTOCs have
demonstrated that EphB2 also plays a role in this process12. We found decreased immigration into the mutant
thymus of all BM progenitors studied, including the WT ones. EphB2-/- , but not EphB2LacZ, progenitors had
reduced capacity to colonize WT FTOCs in comparison with WT progenitors. On the other hand, FTOCs from
EphB2-/- or EphB2LacZ mice, which received EphB2-/- BM progenitors showed significantly lower capacities for
homing than those receiving EphB2LacZ or WT precursors. When WT and mutant progenitors competed to
colonize FTOCs, EphB2-/- cells showed reduced capacities. Furthermore, mutant progenitors had a lower
capacity to reach the centre of lobes, remaining on the periphery, than WT ones, particularly when lobes were
derived from mutant mice12.
On the other hand, very early EphB-deficient thymuses (E12.5-13.5) exhibit small size, delayed
maturation of TEC progenitors, loose cortical epithelium and changes in the phenotype of medullary areas.
These early changes result in a reduced cortical epithelial network and alter position and size of medullary areas
(E15.5) to finally induce (E17.5, 2PN) a more severe phenotype in later stages6.
Recently, we also analyzed the role of ephrinB1 and ephrinB2 in cortical and medullary epithelium
development10. In the adult thymic cortex, two Ly51hi and Ly51lo cell subsets, can be identified. Ly51hi TECs
appeared in the subcapsulary cortex and also formed a discontinuous layer in the cortico-medullary border. In
these two areas some Ly51hi cells are arranged longitudinally and radially to the medulla whereas Ly51lo cells
appeared between them. In thymuses with ephrinB1-deficient TECs, the occurrence of two Ly51 subsets is
difficult to evidence: cTECs are retracted and form a homogeneous and dense thymic cortex. On the contrary, in
thymuses in which TECs do not express ephrinB2 there is a clearly defined Ly51lo outer cortex and a Ly51hi
region in the inner cortex. In thymuses with double ephrinB-deficient TECs, the thymic cortex shows
homogeneous Ly51 expression although it is possible to identify some Ly51hi cells in the inner cortex and Ly51lo
in the outer area. In these double mutants, flow cytometry analysis demonstrated that the lack of both ephrins
results in a delayed appearance of Ly51hi cells during thymus ontogeny with accumulation of Ly51lo cells
at E14.5 and decreased numbers of Ly51med and Ly51hi cells in the later developmental stages. On the other
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hand, ephrinB1 expression on TECs was necessary for the stratification and differentiation of thymic
medullary epithelium, ephrinB2 for the longitudinal growth of epithelial branches and both ephrins are
involved in the expansion and 3D-organization of the thymic epithelial network10.
Acknowledgements: This work was supported by grants BFU 2010-18250 from the Spanish Ministry of
Education and Science and RD06/0010/0003 from the Spanish Ministry of Health and Consumption
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
PASQUALE EB. Eph-ephrin bidirectional signaling in physiology and disease. Cell 133, 38-52, 2008
MENDES-DA-CRUZ DA, STIMAMIGLIO MA, MUÑOZ JJ, ALFARO D, TERRA-GRANADO E,
GARCIA-CECA J, ALONSO-COLMENAR LM, SAVINO W, ZAPATA A. Developing T-cell migration:
role of semaphorins and ephrins. FASEB J July 19, Epub ahead, 2012
MUÑOZ JJ, ALONSO CL, SACEDON R, CROMPTON T, VICENTE A, JIMENEZ E, VARAS A,
ZAPATA A. Expression and function of the EphaA receptors and their ligands ephrins A in the rat thymus.
J Immunol 169, 177- 184, 2002
MUÑOZ JJ, ALFARO D, GARCIA-CECA J, ALONSO LM, JIMÉNEZ E, ZAPATA A. Thymic
alterations in EphA4-deficient mice. J Immunol 177, 804-813, 2006
ALFARO D, MUÑOZ JJ, GARCIA-CECA J, CEJALVO T, JIMÉNEZ E, ZAPATA A. Alterations in the
thymocyte phenotype of EphB-deficient mice largely affect the double negative cell compartment.
Immunology 125, 131-143, 2008
GARCIA-CECA J, JIMÉNEZ E, ALFARO D, CEJALVO T, MUÑOZ JJ, ZAPATA A. On the role of Eph
signalling in thymus histogenesis. Int J Dev Biol 53, 971-982, 2009
SHIMOYAMA M, MATSUOKA H, NAGATA A, IWATA N, TAMEKANE A, OKAMURA A, GOMYO
H, ITO M, JISHAGE K, KAMADA N, SIZUKI H, TETSUO NODA T, MATSUI T. Developmental
expression of EphB6 in the thymus: lessons from EphB6 knockout mice. Biochem Biophys Res Commun
298, 87-94, 2002
COLES MC, ADAMS R, ADAMS S, RODERICK K, NORTON T, WILKINSON D, KIOUSSIS D. The
role of Eph receptors and ephrin ligands in T cell development in the thymus.Clin Invest Med 56D, 2004
ALFARO D, MUNOZ JJ, GARCIA-CECA J, CEJALVO T, JIMÉNEZ E, ZAPATA A. The Eph/ephrin B
signal balance determines the pattern of T-cell maturation in the thymus. Immunol Cell Biol 89, 844-852,
2011
CEJALVO T. Role of ephrinB1 and ephrinB2 in the development and function of thymus (in Spanish).
PhD thesis, Complutense University, Madrid, 2011
ALFARO D, GARCIA-CECA J, CEJALVO T, JIMÉNEZ E, JENKINSON EJ, ANDERSON G, MUÑOZ
JJ, ZAPATA A. EphrinB1-EphB signalling regulates thymocyte-epithelium interactions involved in
functional T cell development. Eur J Immunol 37, 2596-2605, 2007
STIMAMIGLIO MA, JIMÉNEZ E, SILVA-BARBOSA SD, ALFARO D, GARCIA-CECA J, MUÑOZ JJ,
CEJALVO T, SAVINO W, ZAPATA A. EphB2-mediated interactions are essential for proper migration of
T cell progenitors during fetal thymus colonization. J Leuk Biol 88, 483-494, 2010
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Morphological changes in the placenta of alloxan
induced diabetic rats
Farias P.S.1, dos Santos Souza K.1, Marçal A.C.1, dos Santos M.R.V.2,
Fioretto E.T.1, Aires M.B.1
1
Department of Morphology, 2Department of Physiology. Federal University of Sergipe, Sao Cristovao, Sergipe,
Brazil. E-mail: [email protected].
Abstract
In Brazil, gestational diabetes could occur on 7.6% of women pregnancies, in which placental function
is altered by carrying problems on nutrients and oxygen transfer to fetus. The objective of this study was to
evaluate the effect of hyperglycemia on placental morphology and organization from alloxan-induced diabetic
female rats. Diabetes was induced by a single injection of alloxan in saline solution (37 mg/kg i.v.) on
gestational day (gd) 8 in Wistar rats. Control group, received only saline solution (i.v.). Positive diabetes was
defined by glycemia ≥200 mg/dL 48 hours after the induction. On 17gd, after female laparotomy, all viable
fetuses and placentas were weighted to determine the placental index (PI). Placentas were sampled and
processed for paraffin embedding and histological analysis. In diabetic group, PI was significantly higher
(0.87±0,02 vs 0.69±0,06) and placentas demonstrated morphological differences in all three layers compared to
control group. Junctional zone was apparently larger presenting predominance of glycogen cells surrounded by
spongiotrophoblast cells and increased number of trophoblast giant cells. The labyrinth zone demonstrated
vascular and cellular disorganization, which includes irregular arrangement of labyrinthic trophoblast cells and
straight arrangement of maternal sinusoids. The results could be correlated to placental dysfunction which affects
the exchange of substances between mother and fetus during gestational diabetes.
Keywords: diabetes, placenta, histological analysis.
Introduction
Diabetes mellitus (DM) is a worldwide public health problem and it is increasing due to the high
prevalence of obesity and sedentarism. A study[1] estimated global prevalence of DM, including all age groups,
at 2.8% in 2000 and 4.4% in 2030, and projected that the number of diabetic individuals could rise from 171
million in 2000 to 366 million in 2030. Diabetes in pregnant women is associated with an increased risk of
maternal and neonatal morbidity and remains a significant medical challenge. Diabetes during pregnancy may be
divided into clinical diabetes (in cases previously diagnosed with type 1 or type 2 diabetes) and gestational
diabetes (GD)[2]. The prevalence of GD varies in a given population or ethnic group. In the Brazilian health
public system, 7.6% of pregnant women older than 20 years are diabetic[3].
Maternal diabetes constitutes an unfavorable environment for embryonic and fetoplacental
development[2]. From both human and rodent diabetic experimental models, it has been suggested that placenta is
a compromised target which largely suffers the impact of maternal diabetes causing problems in maternal and
fetus exchange that increases abnormal fetal development and perinatal morbidity rates. In that way, the study of
the uterine environment during placenta development on an animal model for diabetes could be useful for the
better understanding of gestational diabetes physiopathology in humans.
Methodology
2.1 Animals
Twenty-five female and 05 male adult Wistar rats (Rattus norvegicus) weighing 200-250g were used in
the study. All rats were allowed free access to standard rat laboratory diet and tap water and were maintained on
a 12:12h light/dark cycle. Five females and one male were kept overnight in one cage and the day of spermpositive vaginal smear was considered as gestational day 1 (1gd). Diabetes was induced in the diabetic group on
8gd by a single injection of alloxan monohydrate (37mg/kg, i.v) in saline solution, after 12 hours of starvation.
Animals in the control group received identical volume of saline solution. Blood glucose concentration was
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measured at 8gd, 10gd and at 17gd. Animals exhibiting blood glucose level higher than 200mg/dl at 10gd (AccuChek Performa test strips, Roche Diagnostic) were considered to be diabetic. Maternal weight was taken on 1gd,
8gd and 17gd. On 17gd, control and diabetic mothers were anesthetized with xylazine (20mg/kg) and ketamine
(80mg/kg), exsanguinated and laparotomized to remove the uterine horns for weighing of fetus and placentas.
Placental index was determined by the weight of the placenta divided by weight of the fetus. The experimental
protocols were approved by CEPA 86/2011.
2.2 Tissue processing
Placental samples were fixed in 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4 for 24 hours.
After dehydration in ethanol, tissues were cleared in xylene and embedded in paraffin wax. Subsequently, serial
5 µm sections were cut and mounted on gelatin coated slides. For morphological analysis, sections were stained
with haematoxylin and eosin (H&E).
2.3 Statistical analysis
All data are expressed as means±SD. Comparisons between control and diabetic groups for fetus
weight, placental weight and index were performed by Student’s t-test. Comparisons between maternal weight
and glycemia on gestational days 8 and 17 were performed by on way ANOVA followed by Tukey test. A value
of P<0.05 was considered significant.
Results and discussion
3.1 Blood glucose levels and body weight of diabetic group.
Blood glucose levels and body weight gain of the pregnant rats at 8gd and 17gd are shown in Table 1.
The body weight was not different between groups; however, glycemia in the diabetic group was significantly
higher on 17gd compared to control group. Studies have applied different doses of alloxan and streptozotocin in
rats to induce diabetes during pregnancy reaching glycemic levels between 275 to 353 mg/dl[4,5,6]. In our results,
the mean blood glucose concentration of diabetic rats on 17 gd was 329.3±13.38 mg/dl which is considered
severe diabetes by some authors[7, 8].
Table 1: Body weight (g) and blood glucose levels (mg/dl) on 8gd and 17gd of control and diabetic groups.
Group
Gestational day
Control (n=7)
Diabetic (n=7)
8
17
8
17
Weight
181.1 ± 11.82
209.4 ± 11.08
187.1 ± 11.16
198.2 ± 8.76
Glycemia
83.71 ± 2.97
86.29 ± 2.79
84.43 ± 4.45
329.3 ± 13.38***
Values are means ± SD; n= no. of animals. ***p<0.0001.
3.2 Placental weight, placental index and fetal weight
Diabetic group placental weight was not different from control at 17gd (Table 2). Abnormally increased
placental weight has already been described on diabetic rat at the end of pregnancy[9,10,11]. Probably, we couldn’t
observe, at 17gd, this difference since significant placental increase happens at late gestation (day 20 or 21 of
pregnancy) like was reported by some authors[12,13,14] and is related with the continuous cell proliferation on
placental tissues on diabetic placenta[13,14]. The placentomegaly frequency is associated with moderate diabetes
described with glycemia between 120-300mg/dl[8] which contrasts with our results of severe diabetes.
Placental index was higher (P<0.001) on diabetic group and occurred due to the decrease of fetal weight
(Table 2). In many species, fetal weight near term is positively correlated to placental weight, as a proxy measure
of the surface area for materno-fetal transport of nutrients [15,16]. The PI is usually affected on many pathological
conditions such as gestational hypertension and diabetes[17] suggesting placental dysfunction.
The reduction of fetal weight observed (P<0.0001) (Table 2) is correlated with glycemia above
200mg/dl[18,10,14]. Toxic effects of hyperglycemia on embryonic and fetal growth are related to activation of Baxdependent apoptosis via downregulation of SLC2A1 glucose transporter expression[19], impaired PI 3-kinase
activation and reduced Akt activity in response to increased O-GlcNAcylation[20] which affects the regulation of
embryonic cellular proliferation and survival. Moreover, PI 3-kinase regulates glucose transporter recycling at
the plasma membrane, allowing the embryo to coordinate nutrient transport and protein synthetic activities with
the modified metabolic requirements of stimulated proliferation[21]. Another explanation for the reduced fetal
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weight is the deficit on placental nutrient transport (for example: amino acids, vitamins and minerals) due to
altered placental morphophysiology[22,23]. Indeed, Canavan e Goldspink[24] found reduced fetal protein mass and
synthesis on diabetic pregnancies that correlates with intrauterine growth restriction on severe diabetes.
Table 2: Placental weight (g), placental index and fetal weight (g) of control and diabetic groups at 17gd.
Group
Control (n = 7)
Diabetic (n = 7)
Placental weight
0.43 ± 0.01
0.41 ± 0.01
Placental index
0.81 ± 0.02
0.91 ± 0.02**
Fetal weight
0.54 ± 0.01
0.46 ± 0.01***
Values are means ± SD; n= no. of animals. ***p<0.0001, **p<0.001.
3.3 Histological analysis
Mature rat placenta at 17gd consists of three compartments: decidua, junctional and labyrinth zone (Fig. 1A).
Placentas from diabetic group have a reduced area of the labyrinth zone (Fig. 1B) and a straight arrangement of
fetal capillaries and maternal sinusoides (Fig. 1D) instead of the sinuous pattern of the control group (Fig. 1C). It
was observed an enlargement of junctional zone (Fig. 1B), primarily because of the increased number of
glycogen cells identified as vacuolated cells on conventional H&E sections (Fig. 1F). Trophoblast giant cells
distribution on diabetic group was changed as they were bigger and irregularly interdigitated with
spongiotrophoblast and glycogen cells (Fig. 1F).
Figure 1: Placentas from control and diabetic rats at 17º gd. A and B) Three compartments of mature placenta: decidua (D),
junctional (JZ) and labyrinth zone (LZ), note the enlargement of JZ and the reduced area of LZ on diabetic group. C and D)
LZ with sinuous pattern on control and straight arrangement of fetal capillaries (FC) and maternal sinusoids (MS) on
diabetic group. E) JZ with trophoblast giant cells layer (TGC) delimited by yellow line and spongiotrophoblast with
basophilic cytoplasm (ST, arrow) and glycogen cells with vacuolated cytoplasm (GC, arrowhead). F) Enlargement of JZ
because of the increased number of GC, note large TGC (delimited by superior green line) irregularly interdigitated with ST
and GC (detail, arrow and arrowhead). HE stain, Bars=[A] and [B]: 400 μm, [C] and [D]: 30 μm, [E] and [F] : 100 μm
and 30μm (E, F on detailed view).
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The enlargement of junctional zone on diabetic rat placenta was described as the increased number of
glycogen and giant trophoblast cells[9,11]. In addition, we found further glycogen cells on decidua, which indicate
greater glycogen storage on diabetic placenta. Placental glycogen accumulation in diabetes occurs in marked
contrast to other tissues, such as maternal liver, from which glycogen disappears[25]. Liver and muscle
glycogenesis and glycogenolysis are under insulin control, by regulation of the activities of glycogen synthase
and phosphorylase. However, in the placenta these enzymes are not meaningfully influenced by insulin in in vivo
and in vitro studies[25]. Accordingly, glycogen accumulation in the placenta of diabetic rats is related to the
extent of maternal hyperglycemia and is independent of insulin.
Another way for the increased capacity of glucose uptake by placental cells is the expression of
transporter isoforms of the classic glucose carrier (GLUTs) family. GLUT1 is highly expressed in both the
junctional and labyrinth zones of the rat chorionallantoic placenta[26, 27]. Junctional zone GLUT1 expression is
highest at midgestation and actually decreases in concentration by the end of pregnancy[28,29]. GLUT1 in the
junctional zone likely is associated with metabolic requirements for rapid placental growth at midgestation rather
than transplacental glucose transport[30]. Thus, GLUT1 could be up regulated on junctional zone diabetic
placenta enabling increased uptake of glucose by glycogen cells.
Some studies have described an increase number of spongiotrophoblast cells positive for proliferative
markers at gestational day 17 to 20, when this zone should be shrinking at the end of pregnancy[13].
Spongiotrophoblast cells are known precursors of glycogen cells[31] and thus it could collaborate for the
increment of those cells. Moreover, spongiotrophoblast and trophoblast giant cells produce many proteins with
endocrine activity, e.g., prolactin-like hormones, lactogens and cytokines[32,33] consequently their increase could
change the placental metabolism and affect fetal-maternal exchange of substances.
Placental development depends on careful coordination of trophoblast proliferation and differentiation.
Zorn et al.[13] showed that diabetes promotes increased cell proliferation rate especially in the spongiotrophoblast
on 14gd and in the labyrinth, spongiotrophoblast and giant trophoblast cell regions on 17gd, which may explain
the placentomegalia observed in diabetic placentas on 20gd. Also, a reduced expression of p57, a cell cycle
inhibitor, on rat diabetic placenta on days 17 and 21 could explain the high proliferative activity at the end of
pregnancy[14]. Some researchers hypothesize that hyperglycemia leads to a relative immaturity of rat placentas by
providing a stimulus for continuous growth and cell division-delayed maturation[9]. As a result of this
immaturity, disturbances in placental blood flow and an abnormal nutrient transport could occur from the mother
to fetus leading to fetal death or perinatal mortality.
Conclusion
In conclusion, on our study of severe diabetes, despite of no change on placental weight, marked
morphological alterations on labyrinth and junctional zone may indicate impaired placental function resulting on
reduced fetal weight. More detailed studies are required to explain the cellular and molecular mechanisms
underlying the placentation in diabetic pregnancy.
References
[1] Wild, S., Roglic, G., Green, A., Sicree, R., King, H. (2004). Global prevalence of diabetes: estimates for the
year 2000 and projections for 2030. Diabetes Care 27, pp.1047-53.
[2] Vambergue, A., Fajardy, I. (2011).Consequences of gestational and pregestational diabetes on placental
function and birth weight. World Journal of Diabetes 2(11), pp.196-203.
[3] Ministério da Saúde (2000). Secretaria de Políticas de Saúde. Gestação de alto risco. 3ª ed. Brasília: SPS.
[4] Oh, W., Gelardi, N.L., Cha, C.J. (1988). Maternal hyperglycemia in pregnant rats: its effect on growth and
carbohydrate metabolism in the offspring. Metabolism 37, pp.1146-1151.
[5] Oh, W., Gelardi, N,L., Cha, C.J. (1991). The cross-generation effect of neonatal macrosomia in rat pups of
streptozotocin-induced diabetes. Pediatric Research 29, pp.606-610.
[6] Palomar-Morales, M., Baiza, L. A., Verdín, L. T., Ramos, R. R., Lozano, M. A., Méndez, J. D. (1998). Fetal
Development in Alloxan-Treated Rats. Reproductive Toxicology 12(6), pp. 659-665.
[7] Damasceno, D. C., Volpato, G.T., Calderon, I.M.P., Cunha, R.M.V. (2002). Oxidative stress and diabetes in
pregnant rats. Animal Reproductive Science 72, pp.235-244.
[8] Iessi, I. L., Bueno, A., Sinzato, Y. K., Taylor, K. N., Rudge, M. V. C., Damasceno, D. C.(2010). Evaluation
of neonatally-induced mild diabetes in rats: Maternal and fetal repercussions. Diabetology & Metabolic
Syndrome 2(37), pp.1-8.
[9] Gewolb, I.H., Merdian, W., Warshaw, J.B., Enders, A.C. (1986). Fine structural abnormalities of the placenta
in diabetic rats. Diabetes 35, pp.1254-61.
© Medimond . P726C0996
46
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[10] Calderon, I. M. P., Rudge, M. V. C., Ramos, M. D., Peraçoli, J. C. (1999). Estudo Longitudinal,
Bioquímico, e Histoquímico, de Placentas de Ratas Diabéticas: Relação com a Macrossomia e com o
Retardo de Crescimento Intra-uterino. Revista Brasileira de Ginecologia e Obstetrícia 21(2), pp.91-98.
[11] Padmanabhan, R., Shafiullah, M. (2001). Intrauterine growth retardation in experimental diabetes: possible
role of the placenta. Archives of Physiology and Biochemistry 109, pp.260-71.
[12] Robinson J., Canavan J. P., el Haj A. J., Goldspink D. F. (1988). Maternal diabetes in rats I. Effects on fetal
growth and protein turnover. Diabetes 37, pp.1665-1670.
[13] Zorn, T.M.T, Zúñiga, M., Madrid, E., Tostes, R., Fortes, Z., Giachini, F., San-Martín, S. (2011). Maternal
diabetes affects cell proliferation in developing rat placenta. Histology and Histopathology 26, pp.10491056.
[14] Acar, N., Korgun, E. T., Ustunel, I. (2012). Cell cycle inhibitor p57 expression in normal and diabetic rat
placentas during some stages of pregnancy. Histology and Histopathology, 27(1), pp. 59-68.
[15] Baur, R. (1977). Morphometry of the placental exchange area. Advances in Anatomy, Embryology and cell
biology 53(1), pp.3-65.
[16] Mellor, D.J. (1983). Nutritional and placental determinants of foetal growth rate in sheep and consequences
for the newborn lamb. Brazilian Veterinary Journal 139, pp.307–324.
[17] Barcroft, J. (1947). Researches on prenatal life. 1rst Edition. Springfield: Charles C. Thomas, pp. 42-66.
[18] Rudge, M. V. C., Calderon, I. M. P., Maestá, I., Ramos, M. D., Peraçoli, J. C., Curi, P. (1998).
Insulinoterapia na Prenhez de Ratas Diabéticas: Repercussões Fetais e Placentárias. Revista Brasileira de
Ginecologia e Obstetrícia 20(1), pp. 19-24.
[19] Chi, M.M., Pingsterhaus, J., Carayannopoulos, M., Moley, K.H. (2000). Decreased glucose transporter
expression triggers BAX-dependent apoptosis in the murine blastocyst. The Journal of Biological
Chemistry 275, pp.40252–40257.
[20] Pantaleon, M., Tan, H.Y., Kafer, G.R., Kaye, P.L. (2009). Toxic effects of hyperglycemia are mediated by
the hexosamine signaling pathway and o-linked glycosylation in early mouse embryos. Biology of
Reproduction 82(4) pp.751-758.
[21] Pantaleon, M., Kaye, P.L. (1996). IGF-I and insulin regulate glucose transport in mouse blastocysts via
IGF-I receptor. Molecular Reproduction and Development 44, pp.71–76.
[22] Sandovici, I., Hoelle, K., Angiolini, E., Constância, M. (2012). Placental adaptations to the maternal-fetal
environment: implications for fetal growth and development programming. Reproductive BioMedicine
Online 25, pp. 68-89.
[23] Gauster, M., Desoye, G., Tötsch, M., Hiden, U. (2012). The placenta and Gestational Diabetes Mellitus.
Current Diabetes Reports 12, pp. 16-23.
[24] Canavan J.P., Goldspink D.F. Maternal diabetes in rats II. (1988). Effects on fetal growth and protein
turnover. Diabetes 37(12), pp.1671–7.
[25] Shafrir, E., Barash, V. (1991). Placental glycogen metabolism in diabetic pregnancy. Israel journal of
medical sciences 27 (8-9), pp.449-61.
[26] Korgun, E. T., Acar, N., Sati, L., Kipmen-Korgun, D., Ozen, A., Unek, G., Ustunel, I., Demir, R. (2011).
Expression of glucocorticoid receptor and glucose transporter-1 during placental development in the
diabetic rat. Folia Histochemica et Cytobiologica 49(2), pp. 325–334.
[27] Das, U.G., Sadiq, H.F., Soares, M.J., Hay, W. W., Devaskar, S.U. (1998). Time-dependent physiological
regulation of rodent and ovine placental glucose transporter (GLUT-1) protein. American Journal of
Physiology: Regulatory, Integrative and Comparative Physiology 274, pp. R339-R347.
[28] Desoye, G., Shafrir, E. (1996). The human placenta in diabetic pregnancy. Diabetes Review 4, pp.70-89.
[29] Zhou, J., Bondy, C. A. (1993). Placental glucose transporter gene expression and metabolism in the rat.
Journal of Clinical Investigation 91, pp. 845–852.
[30] Knipp, G.T., Audus, K.L., Soares, M.J. (1999). Nutrient transport across the placenta. Advanced Drug
Delivery Reviews 38, pp.41–58.
[31] Coan, P. M., Conroy, N., Burton, G. J., Ferguson-Smith, A. C. (2006). Origin and characteristics of
glycogen cells in the developing murine placenta. Developmental Dynamics 235(12), pp. 3280-3294.
[32] Soares, M. J. (2004). The prolactin and growth hormone families: pregnancy specific hormones/cytokines at
the maternal-fetal interface. Reproductive Biology and Endocrinology 2(51), pp.1-15.
[33] Hu, D., Cross, J. C. (2010). Development and function of trophoblast giant cells in the rodent placenta. The
International Journal of Developmental Biology 54(2-3), pp. 341-354.
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Retinoic Acid and the Development of
Neurotransmitter Systems
Zieger E., Schubert M.
Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR 7009 – CNRS/UPMC) (FRANCE)
[email protected], [email protected]
Abstract
Retinoic acid (RA) is an important morphogen synthesized from vitamin A (retinol) that is involved in
axial patterning, organ formation, neural development and the regulation of homeostasis of adult tissues and
organs. RA is equally required during all three phases of neurogenesis (induction, determination and
differentiation) to produce neurons from embryonic stem cells. While the general role of RA in inducing and
regulating neuronal differentiation has been well studied, the influence of RA on neurotransmitter determination
remains largely unexplored. Here, we review the existing literature on RA functions in controlling the
establishment of neurotransmitter identities.
Keywords: adrenergic, cholinergic, dopaminergic, GABAergic, neuropeptide Y, serotonergic neurons
Retinoic Acid as a Morphogen
Retinoic acid (RA) is an important morphogen, which is synthesized endogenously from vitamin A [1].
In the body, RA levels must always be balanced, especially during pregnancy, to avoid massive teratogenic
effects. RA binds to two nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR),
that together form heterodimers. RXR/RAR heterodimers bind to specific DNA sequences called retinoic acid
response elements (RAREs), most of which consist of two direct repeats (DRs) with the canonical nucleotide
sequence (A/G)G(G/T)TCA separated by a variable number of nucleotide spacers (usually either 1, 2 or 5
nucleotides). Binding of RA to RAR activates the ligand-dependent transcription factor function of the
RXR/RAR heterodimer, which ultimately leads to the initiation of target gene transcription [2]. Although
different isomeric forms of RA have been identified in vivo (including all-trans, 9-cis and 13-cis RA), the
physiologically active isomer is all-trans RA with biological functions of other isomers still being debated [2,3].
During normal development, RA is involved in axial patterning and organ formation. Furthermore, it is
important for neuronal development and for maintaining homeostasis of adult tissues and organs. RA further
controls cell survival, proliferation and differentiation [2-5]. Because of these properties, RA and its synthetic
analogs (chiefly called retinoids) have great potential as anti-carcinogenic agents and may also be useful for the
treatment of skin, autoimmune and neurodegenerative diseases [6]. Accordingly, it is very important to fully
understand this highly complex signaling network.
Retinoic Acid as a Key Differentiation Factor in Neurogenesis
RA regulates different stages of neurogenesis. Major functions include the induction of neural crest
formation and the patterning of neural plate and nervous system along both the anterior-posterior and dorsalventral axes, particularly in the posterior hindbrain and anterior spinal cord [7-12]. The morphogen exerts its socalled pro-neural and anti-mesodermal activities by up-regulating neural genes, such as wnt1, mash1,
neurofilaments and genes encoding neurotransmitter-synthesizing enzymes, while blocking mesodermal genes
like brachyury, cardiac actin, and -globin [13]. Many studies have focused on the regulation of neuronal cell
determination and specification by RA [14-17], but relatively little is known about the involvement of RA in the
acquisition of specific neurotransmitter phenotypes by neuronal precursor cells. It has previously been suggested
that RA has neurotrophic effects that are confined to certain neuronal subpopulations and can, for instance,
promote the survival of cholinergic neurons without affecting GABAergic neurons [18]. Moreover, RA controls
the expression of several components involved in neurotransmitter signaling and thus might influence neuronal
functions within the adult nervous system, where RA has also been linked to affective disorders [19]. Here, we
provide an overview of the functions of RA in the regulation of cellular neurotransmitter identities.
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2.1
Adrenergic Cells
Norepinephrine (also known as noradrenaline) is the main chemical messenger of central noradrenergic
and peripheral sympathetic pathways and serves a multitude of functions in the brain, including arousal,
attention, mood, learning, memory and stress responses [20,21]. Epinephrine (also known as adrenaline) is
considered a site-selective metabolite of physiological and neuronal importance that functions primarily as a cotransmitter with norepinephrine in the forebrain [22].
In quail neural crest cultures, treatments with RA promote the proliferation of cells expressing tyrosine
hydroxylase (TH), an enzyme necessary for the synthesis of the catecholamine neurotransmitters dopamine,
norepinephrine and epinephrine. Accordingly, a dose-dependent increase in the number of adrenergic cells in
response to elevated RA levels can be observed [23]. Moreover, it was demonstrated that in some adrenergic
cells of newborn rats RA induces expression of a sodium-dependent norepinephrine transporter but decreases
levels of TH [24]. The physiological relevance of this differential regulation by RA remains to be explored.
In the zebrafish hindbrain, noradrenergic precursor neurons were shown to require the activity of the
transcription factor AP-2α to adopt their neurotransmitter phenotype. Given that RA acts upstream of AP-2α, the
differentiation of noradrenergic neurons in the medulla oblongata and the area postrema of zebrafish embryos is
thus regulated by RA signaling through AP-2α [25].
2.2
Dopaminergic Cells
Dopamine (DA) is a catecholamine neurotransmitter controlling a variety of processes in the central
nervous system (CNS), including locomotor activity, cognition, emotion, positive reinforcement, food intake and
endocrine regulation. In the periphery, DA also regulates cardiovascular function, catecholamine release,
hormone secretion, vascular tone, renal function and gastrointestinal motility. The DA receptors (called D2Rs)
are the somatodendritic, impulse-regulating autoreceptors of dopaminergic neurons [26]. D2R is considered a
direct target of RA signaling, given that a functional RARE has been identified in the regulatory region of the
mouse D2R gene and that mice with impaired RXR/RAR show reduced expression of D2R, which in turn leads
to a Parkinsonian phenotype [27-29]. Additionally, high doses of exogenous RA significantly increase D2R
expression levels in rat striatal neurons, while mutations of the RARE reduce D2R expression [27-29]. RA can
thus be considered a key factor in regulating the ontogenetic onset of D2R in dopaminergic neurons [30].
2.3
Cholinergic Cells
Acetylcholine is a classical neuromodulator that alters neuronal excitability, influences synaptic
transmission, induces synaptic plasticity and coordinates firing of groups of neurons. The diverse effects of
acetylcholine have been shown to shape the functions of the nervous system and to underlie complex behavior
[31].
In postmitotic rat sympathetic neurons, RA induces a cholinergic phenotype by regulating
catecholaminergic and cholinergic enzyme activities. After long-term exposure to RA, an increase in the specific
activity of choline acetyltransferase (ChAT) and acetylcholine levels can be observed, whereas the specific
activity of the catecholamine synthetic enzymes, TH and dopamine β-hydroxylase (DBH), as well as the level of
norepinephrine (NE) are reduced [32]. ChAT, TH and DBH activities seem to be regulated by the transcription
factor AP-2, which is induced by RA [33]. RA further induces expression of specific neuronal nicotinic
acetylcholine receptor (nAChR) subunits and of high affinity [³H]-nicotine-binding receptors [34].
2.4
Serotonergic Cells
The biogenic amine serotonin (5-hydroxytryptamin, 5-HT) is involved in various physiological and
behavioral processes [35]. In the hindbrain of frog larvae, application of low doses of exogenous RA causes the
anterior-posterior domain of 5-HT expression to expand [36]. In these larvae, additional serotonergic neurons
appear mainly in the anterior region of the CNS at the level of the eyes. Higher doses of exogenous RA,
however, result in a dose-dependent decrease of serotonergic neurons and the remaining neurons project
exclusively caudally. In contrast, neurites of untreated embryos extend both anteriorly into the forebrain and
posteriorly into the hindbrain [36]. It can thus be concluded that very precise levels of RA serve as spatial cues
for the differentiation of serotonergic neurons.
In cells derived from rat embryonic raphe nuclei, treatment with RA increases the expression levels of
5-HT1A receptor and 5-HT reuptake transporter (SERT). This increase causes an accumulation of 5-HT within
the somata of serotonergic neurons, which is accompanied by a reduction of neurotransmitter release. This
intricate impairment mechanism might at least partially be responsible for the observed increase in depressionrelated behaviors in mice treated with 13-cis-RA [37,38].
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2.5
Neuropeptide Y Cells
Neuropeptide Y (NPY) is abundant in both central and peripheral neurons and can equally be found in
cells of neural crest origin. NPY is involved in regulation of artery blood pressure, circadian rhythms, feeding
behavior and release of catecholamine and hypothalamic hormones [39].
In human neuroblastoma cells, RA exerts a potent negative control on the expression of NPY, causing a
marked reduction in biosynthesis and secretion of this neurotransmitter [40]. In contrast, the phorbol ester 12-Otetradecanoylphorbol-13-acetate (TPA) can stimulate NPY expression by inducing the activity of AP-1 [41].
However, this effect is completely abolished in the presence of exogenous RA [40]. Since there is no RARE in
the regulatory region of the gene encoding NPY, it has been proposed that activated RA-RAR complexes interact
directly with a transcription factor, such as AP-1, preventing the binding of the transcription factor to a DNAbinding site in the regulatory region of the NPY gene in a DNA-independent manner [40,42]. In addition to
regulation of NPY, RA also reduces the expression of the NPY-Y1 receptor (Y1-R) [43].
Conversely, six days of RA-induced differentiation of neuroblastoma cells into neurons results in RAdependent stimulation of pro-NPY processing. The long exposure probably leads to cell differentiation-related
processes, including the induction of processing enzymes like pro-hormone convertases 1 and 2, which catalyze
the cleavage of peptide pro-hormones at dibasic sites [40].
2.6
GABAergic Cells
The neurotransmitter γ-aminobutyric acid (GABA) has different functions in both central and peripheral
nervous systems as well as in some non-neuronal tissues. Albeit generally considered inhibitory, during
neurogenesis GABA appears to fulfill a dual role with a transient excitatory activity observable in the developing
mammalian brain [44,45]. GABA thus also promotes synapse formation and, more generally, is thought to
function as a trophic factor and chemical guiding cue [44,46,47].
In the subventricular zone of mice basal ganglia, RA is required for the differentiation of GABAergic
neurons [48]. The RA-dependent activation of the GABA-synthesizing enzyme glutamic acid decarboxylase
(GAD) is crucial for the specification of GABAergic interneurons migrating to olfactory bulb and cerebral cortex
and for the commitment of striatal projection neurons to a GABAergic fate [48]. The regulation of GAD
expression by RA might be direct through a RARE located in the regulatory region of the GAD gene [49].
Conclusion
RA regulates the expression of various components that are important for the differentiation of specific
neurotransmitter phenotypes. In conferring its biological functions, RA can either act directly via the binding of
RXR/RAR heterodimers to RAREs in the regulatory regions of target genes (such as D2R or GAD), indirectly
via RXR/RAR-mediated induction of intermediary transcription factors (including AP-2) or non-genomically by
direct binding of ligand-receptor complexes to other proteins (such as AP-1). Even though the ability of RA to
induce and regulate neural differentiation has been studied quite extensively, very little is known about the
specific functions and mechanisms, through which RA signaling controls the establishment of different
neurotransmitter systems. Therefore, it is very important to investigate the effects of RA on the development of
neurons with a specific biochemical identity, especially since there is evidence that RA is equally involved in the
regulation of adult neurotransmission with potential links to several neuronal disorders [50,51].
Acknowledgements: The authors are indebted to João E. Carvalho and Ricardo Lara-Ramírez for critical
reading of the manuscript. This work was supported by funds from ANR (ANR-09-BLAN-0262-02 and ANR11-JSV2-002-01) and CNRS to Michael Schubert.
References
[1]
[2]
[3]
[4]
Kim, C.-I. & Lieber, C. S. (1992). Retinol forms retinoic acid via retinal. Arch Biochem Biophys 294, 388393.
Campo-Paysaa, F. et al. (2008). Retinoic acid signaling in development: tissue-specific functions and
evolutionary origins. Genesis 46, 640-656.
Mic, F. A. et al. (2003). Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient
to rescue lethal defect in retinoic acid synthesis. Proc Natl Acad Sci USA 100, 7135-7140.
Ribes, V. et al. (2008).Combinatorial signalling controls Neurogenin2 expression at the onset of spinal
neurogenesis. Dev Biol 321, 470-481.
© Medimond . P726R9113
51
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
Gonzalez-Quevedo, R. et al. (2010).Neuronal regulation of the spatial patterning of neurogenesis. Dev Cell
18, 136-147.
Tang, X.-H & Gudas, L. J. Retinoids, retinoic acid receptors, and cancer. Annu Rev Pathol 6, 345-364
(2011).
Villanueva, S. et al. (2002).Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest
Induction. Dev Biol 241, 289-301.
Rhinn, M. & Dollé, P. (2012).Retinoic acid signalling during development. Development 139, 843-858.
Wilson, L. et al. (2004).Retinoic acid and the control of dorsoventral patterning in the avian spinal cord.
Dev Biol 269, 433-446.
Niederreither, K. et al. (2000).Retinoic acid synthesis and hindbrain patterning in the mouse embryo.
Development 127, 75-85.
Grandel, H. et al. (2002).Retinoic acid signalling in the zebrafish embryo is necessary during presegmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud.
Development 129, 2851-2865.
Schubert, M. et al. (2006). A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and
neuronal specification in the developing central nervous system of the cephalochordate amphioxus. Dev
Biol 296, 190-202.
Bain, G. et al. (1996). Retinoic acid promotes neural and represses mesodermal gene expression in mouse
embryonic stem cells in culture. Biochem Biophys Res Commun 223, 691-694.
Jones-Villeneuve, E. M. et al. (1982). Retinoic acid induces embryonal carcinoma cells to differentiate into
neurons and glial cells. J Cell Biol 94, 253-262.
Novitch, B. G. et al. (2003). Requirement for retinoic acid-mediated transcriptional activation in ventral
neural patterning and motor neuron specification. Neuron 40, 81-95.
England, S. et al. (2011). Roles of Hedgehog pathway components and retinoic acid signalling in
specifying zebrafish ventral spinal cord neurons. Development 138, 5121-5134.
Sabado, V. et al. (2012).Specification of GnRH-1 neurons by antagonistic FGF and retinoic acid signaling.
Dev Biol 362, 254-262.
Wuarin, L. & Sidell, N. (1991). Differential susceptibilities of spinal cord neurons to retinoic acid-induced
survival and differentiation. Dev Biol 144, 429-435.
Bremner, J. D. & McCaffery, P. (2008).The neurobiology of retinoic acid in affective disorders. Progr
Neuro Psychopharmacol Biol Psychiatr 32, 315-331.
Ordway, G. A. et al. (2007). Brain norepinephrine: neurobiology and therapeutics. Cambridge University
Press.
Sofuoglu, M. & Sewell, R. A. (2009). Norepinephrine and stimulant addiction. Addict Biol 14, 119-129.
Mefford, I. N. (1988). Epinephrine in mammalian brain. Prog Neuropsychopharmacol Biol Psychiatry 12,
365-388.
Dupin, E. & Le Douarin, N. M. (1995).Retinoic acid promotes the differentiation of adrenergic cells and
melanocytes in quail neural crest cultures. Dev Biol 168, 529-548.
Matsuoka, I. et al. (1997). Differential and coordinated regulation of expression of norepinephrine
transporter in catecholaminergic cells in culture. Brain Res 776, 181-188.
Holzschuh, J. et al. (2003). Noradrenergic neurons in the zebrafish hindbrain are induced by retinoic acid
and require tfap2a for expression of the neurotransmitter phenotype. Development 130, 5741-5754.
Mercuri, N. B. et al. (1997). Loss of autoreceptor function in dopaminergic neurons from dopamine D2
receptor deficient mice. Neuroscience 79, 323-327.
Samad, T. A. et al. (1997). Regulation of dopaminergic pathways by retinoids: activation of the D2
receptor promoter by members of the retinoic acid receptor-retinoid X receptor family. Proc Natl Acad Sci
USA 94, 14349-14354.
Baik, J.-H. et al. (1995). Parkinsonian-like locomotor impairment in mice lacking dopamine D2 receptors.
Nature 377, 424-428.
Wolf, G. (1998). Vitamin A functions in the regulation of the dopaminergic system in the brain and
pituitary gland. Nutr Rev 56, 354-355.
Valdenaire, O. et al. (1998). Retinoic acid regulates the developmental expression of dopamine D2 receptor
in rat striatal primary cultures. J Neurochem 71, 929-936.
Picciotto, M. R. et al. (2012). Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous
system function and behavior. Neuron 76, 116-129.
Berrard, S. et al. (2004). Retinoic acid induces cholinergic differentiation of cultured newborn rat
sympathetic neurons. J Neurosci Res 35, 382-389.
Kim, H.-S. et al. (2009). Regulation of the tyrosine hydroxylase and dopamine β-hydroxylase genes by the
transcription factor AP-2. J Neurochem 76, 280-294.
© Medimond . P726R9113
52
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[34] Nilbratt, M. et al. (2007). Retinoic acid and nerve growth factor induce differential regulation of nicotinic
acetylcholine receptor subunit expression in SN56 cells. J Neurosci Res 85, 504-514.
[35] Azmitia, E. C. (2007). Serotonin and brain: evolution, neuroplasticity, and homeostasis. Int Rev Neurobiol
77, 31-56.
[36] Ruiz i Altaba, A. & Jessell, T. M. (1991). Retinoic acid modifies the pattern of cell differentiation in the
central nervous system of neurula stage Xenopus embryos. Development 112, 945-958.
[37] Neumeister, A. et al. (2004). Implications of genetic research on the role of the serotonin in depression:
emphasis on the serotonin type 1A receptor and the serotonin transporter. Psychopharmacology (Berl) 174,
512-524.
[38] O’Reilly, K. C. et al. (2007). 13-cis-retinoic acid alters intracellular serotonin, increases 5-HT1A receptor,
and serotonin reuptake transporter levels in vitro. Exp Biol Med 232, 1195-1203.
[39] Higuchi, H. & Sabol, S. L. (1988). Rat neuropeptide Y precursor gene expression. mRNA structure, tissue
distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester. J Biol Chem 263, 62886295.
[40] Magni, P. et al. (2000). Retinoic acid negatively regulates neuropeptide Y expression in human
neuroblastoma cells. Neuropharm 39, 1628-1636.
[41] Andersson, G. et al. (1994). Activation of the human NPY gene during neuroblastoma cell differentiation:
induced transcriptional activities of AP-1 and AP-2. Cell Growth Differ 5, 27-36.
[42] Schüle, R. et al. (1991). Retinoic acid is a negative regulator of AP-1-responsive genes. Proc Natl Acad Sci
USA 88, 6092-6096.
[43] Mannon, P. J. & Kaiser, L. M. (2002). Retinoic acid is a negative regulator of the neuropeptide Y/peptide
YY Y1 receptor gene in SK-N-MC cells. J Neurochem 68, 20-25.
[44] Cherubini, E. et al. (1991). GABA: an excitatory transmitter in early postnatal life. Trends Neurosci 14,
515-519.
[45] Hosokawa, Y. et al. (1994). Developmental changes in spontaneous GABAA-mediated synaptic events in
rat hippocampal CA3 neurons. Eur J Neurosci 6, 805-813.
[46] Redburn, D. A. & Schousboe, A. (1987). Neurotrophic activity of GABA during development. Alan R.
Liss, Inc.
[47] Behar, T. N. et al. (1994). GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic
spinal cord neurons. J Neurosci 14, 29-38.
[48] Chatzi, C. et al. (2011). Retinoic acid functions as a key GABAergic differentiation signal in the basal
ganglia. PLoS Biol 9, e1000609.
[49] Goodman, A. B. & Kline, N. S. (1996). Retinoid dysregulation may result in abnormal expression of
glutamic acid decarboxylase in Schizophrenia. Arch Gen Psychiatry 53, 653-653.
[50] Bailey, S. J. & McCaffery, P. J. (2009). Retinoic acid signalling in neuropsychiatric disease: possible
markers and treatment agents. In: The handbook of neuropsychiatric biomarkers, endophenotypes and
genes (Ritsner, M. S., editor), Springer Science + Business Media B.V., 171-189.
[51] Bremner, J. D. et al. Retinoic acid and affective disorders. J Clin Psychiatry 73, 37-50 (2012).
© Medimond . P726R9113
53
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Brazil)
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Effects of the Peripheral Sympathetic-CRHHistamine Axis on the Immune Function of
Macrophages
Renck Nunes P.1, Homem de Bittencourt Jr. P.I.2
1
Faculty of Biomedicine, UFRGS, Porto Alegre (BRAZIL)
Laboratory of Cellular Physiology, Department of Physiology, ICBS, UFRGS; National Institute of Science and
Technology of Hormones and Womens’ Health (BRAZIL)
[email protected]; [email protected]
2
Abstract
The components of the sympathetic-peripheral CRH-histamine axis (SCH) can modulate the release and
action of each other and influence the immune system in vivo and in vitro, through different ways, although their
direct effects are not clear. To address the effect of epinephrine, norepinephrine, corticotropin releasing hormone
(CRH) and histamine in the innate immunity in vitro, peritoneal macrophages from male Wistar rats (200g250g; n=8) were isolated and incubated for 30 min with autologous serum-opsonized zymosan particles. We
evaluated the effects of the components of the SCH axis at their physiological concentrations with or without the
co-stimulus of phorbol 12-myristate 13-acetate (PMA) on macrophage phagocytic capacity. The number of
particles phagocytosed by each cell in a total of 50 random cells was counted with differential interference
contrast microscopy (640X magnification). The effect on the expression of the enzyme inducible nitric oxide
synthase (iNOS) was verified after 6 h of culture in the presence of the axis’ components by Western blot. All
the components were able to induce the expression of iNOS, thus suggesting participation of nuclear factor B
(NF-B) in this process. Concerning phagocytosis, epinephrine and norepinephrine showed synergic effects with
PMA, augmenting the phagocytosis by 63% and 72% respectively. Histamine was also found to synergize, but in
a lesser extent (46% augment), while CRH did not alter this parameter. We conclude that the CRH seems to have
only a medium to long-term influence on the immune function, while catecholamines and histamine also show
short-term effects (phagocytosis).
Keywords: phagocytosis, histamine, CRH, catecholamines, nitric oxide synthase, macrophage.
Introduction
The sympathetic-peripheral CRH-histamine axis (SCH) comprises several parallel axes that influence
the activity of each other. These axes have in common the stress as a modulating agent. Stress can act in the
hypothalamic-pituitary-adrenal axis (HPA) and induce the release of the corticotrophin releasing hormone
(CRH) by the hypothalamus. CRH causes the release of cortisol using adrenocorticotropic hormone (ACTH) as
an intermediary hormone [1] However and despite the importance of central HPA during stressful situations, it
has also been described the existence of a similar axis involving CRH and catecholaminergic components but
located peripherally [2, 3]. Beside of this, CRH can also increase plasmatic concentration of catecholamines [4],
which are able to increase histamine levels [5]. Moreover, psychological or physical stress alone may increase
the levels of these substances, which in turn influence by different ways the immune system [6, 7].
Stress represented by physical exercise has direct and indirect influence on the function of the innate
immune system. Exercise increases the circulating levels of the components of the SCH axis and stimulates
chemotaxis [8, 9], adhesion [10] and phagocytosis [11-17] of macrophages in vivo. In order to address the
specific contributions of the SCH axis on the immune function, many in vitro experiments were carried out.
Surprisingly, some studies found that addition of catecholamines, histamine and CRH into the culture media
seems to decrease the phagocytic activity [18, 19] and the production of nitric oxide [20, 21].
The differences in the outcome of these studies may be due to the concentrations used. Some in vitro
experiments use concentrations that exceed a million times the in vivo ones, which may lead to inaccurate
results. Therefore, the aim of our work was to address the effects of the components of the SCH axis on the
immune function of macrophages using their physiological concentrations.
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Methods
1.1
Animals
Eight adult Wistar rats (200-250 g) were obtained from the breeding animal unit of the Federal
University of Rio Grande do Sul (UFRGS) and acclimatized in small cages at an animal room (temperature 22 ±
1 ºC; dark-light cycle of 12h). A standard pellet diet and water were available without restriction. Rats were
starved for 12h prior to the experiments. Animals were killed by decapitation always at 8 a.m. to avoid
interference of normal hormone cycles. All animals were treated according to the Guide for Care and Use of
Experimental Animals published by the National Institutes of Health. All procedures in this study were approved
by the Ethics Committee Research of UFRGS (protocol #19964, April 14, 2011).
1.2
Collection of peritoneal macrophages
After decapitation and collection of blood (6 mL), peritoneal macrophages were obtained as previously
described [22, 23]. Briefly, 20 mL of phosphate saline buffer (PBS) were i.p. injected and a 30-s massage was
performed on the rat abdomen. Laparotomy was followed by collection of the PBS with a plastic pipette. The
PBS with macrophages was transferred to a 50 mL plastic tube. The cells were spun down and resuspended in
RPMI 1640 media supplemented with 10% homologous rat serum.
1.3
Preparation of zymosan particles
Zymosan particles (1 mg in 50 uL) were stained with neutral red and incubated with the same volume of
homologous serum at 37 °C for 30 minutes as described [17]. The serum was obtained after centrifugation of the
rat’s blood at 4,050 x g for 5 minutes. After incubation, the particles were washed 3 times in PBS and
resuspended in RPMI 1640 media.
1.4
Phagocytosis assay
Macrophages (105/well) were seeded in a 24-well dish and left to attach at 37 °C in a 5% (v/v) CO2
incubator for 15 min. Next, physiological concentrations of epinephrine (40 nM [24]), norepinephrine (14 nM
[24]) histamine (4.5 ng/mL [25]) or CRH (30 pg/mL [26]) were individually added to the culture media.
Phorbol-12-myristate-13-acetate (PMA, 200 nM) or ethanol diluent was also added to the specific wells. Cells
were incubated for another 15 min under the same conditions before the addition of opsonized zymosan particle
suspension (10% v/v). Phagocytosis was interrupted after additional 30-min incubation period with the exchange
of culture media for ice-cold PBS. Cells were washed 3 times and left on ice in 0.5 mL of cold PBS. Each
sample was analyzed by differential interference contrast microscopy with 640X magnification. At least 50 cells
were analyzed and the number of zymosan particles in each cell was counted. Results are expressed by the
Hishikawa index [27].
1.5
Expression of inducible nitric oxide synthase
Macrophages (107/well) were seeded in a 6-well dish and left to attach as described above. Afterwards,
epinephrine (40 nM), norepinephrine (14 nM) histamine (4.5 ng/mL) or CRH (30 pg/mL) were added to the
culture media in separate wells. Cells were washed in PBS and collected with a cell scraper after 6 h cultivation
in a 5% (v/v) CO2 incubator at 37 °C. Macrophages were sonicated in the presence of 0.1% sodium dodecyl
sulfate (SDS), 2 g/mL aprotinin, 2 g/mL leupeptin, 0,1 mM phenylmethylsulfonyl fluoride and 20 M
tosyllysine chloromethyl ketone hydrochloride (TLCK). Protein concentration was measured by the Bradford
method [28] and 40 ug of protein were separated in a 10% polyacrylamide gel by electrophoresis (SDS-PAGE).
Proteins were electrotransferred onto a nitrocellulose membrane and detected by Western Blot [29]. The
membrane was blocked with 5% non-fat milk and proteins were detected by anti-iNOS (1:500, Santa Cruz
Biotechnology sc-651) and anti-β-actin (Sigma A3854) antibodies. After the addition of HRP-labelled secondary
anti-mouse monoclonal antibodies and development (ECL Plus, GE), signals were assessed in a
videodocumentation system (GE Image Master 350) and digitalized images were analyzed by Image Quant 7.0
software (GE). Results were normalized by the content of β-actin.
1.6
Statistical analysis
Data related to the response of a specific group to the presence of PMA were analyzed by Student’s-t
test. All other statistical comparisons between groups were performed using two-way ANOVA followed by
Student Newman-Keuls post-hoc test. Differences were considered significant if p<0.05.
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Results
Basal (unstimulated) phagocytosis was found to be unaffected by neither treatment (Fig. 1). However,
in PMA-elecited phagocytosis (which enhances phagocytic activity in controls by approx 67%), all treatments,
except histamine, induced a rise in phagocytic capacity of macrophages: epinephrine (63%), norepinephrine
(72%) and histamine (46%). In spite of this, CRH did not cause an increase in phagocytosis that were higher than
that observed in control groups (Fig. 1), however it is been shown that this hormone may have indirect effects on
the immune system through the modulation of other components of the SCH axis.
Figure 1. Phagocytic capacity expressed by the Hishikawa index of macrophages under the influence of different components
of SCH axis with PMA or not. There was no difference among groups that were not co-treated with PMA. In those where
PMA was present we observed that: “a” is different from “Control”, “Norepinephrine”, “Histamine” and “CRH”
(p<0.05); “b” is different from “Control” and “CRH” (p<0.01). * is different from its own control group without PMA
treatment (p<0.05).
Concerning the expression of the inducible form of NOS (iNOS), whose expression is absolutely
dependent on NF-B activation, all SCH axis components were able to induce a roughly 100%-rise (p<0.05), as
depicted in Fig. 2.
*
*
*
*
Figure 2. Expression of iNOS after 6h of treatment with the indicated SCH axis components. Western blot was performed
according to the Methods section. All treatments were able to increase the intracellular content of iNOS when compared to
the control group. Data are expressed as the mean  S.E.M. A representative gel is given. *P<0.05 relative to controls.
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Discussion
Macrophages are present in several tissues under normal conditions. As part of the innate immune
system, their hole is to perform an unspecific and fast reaction against foreigner particles or cell debris. One of
the most important functions of these cells are the phagocytosis and the production of reactive oxygen species to
defeat foreign cells and bacteria. These two reactions are influenced by local and systemic factors, which may
include SCH axis components. However, little is known about the specific effects of each component of the axis
on the immunological activity of macrophages. To our knowledge, this is the first in vitro study that uses in vivo
concentrations of catecholamines, histamine and CRH to better understand the role of the SCH axis on the innate
immune system.
In this study, we observed that the presence of each individual component of the axis by themselves in
the culture media did not significantly change basal phagocytic activity of macrophages. However, under the
stimulation with PMA, a classical protein kinase C (PKC) activator which mimics physiological activation of
macrophages in vivo, all the SCH axis components tested herein, with the exception of CRH, were able to
increase phagocytic activity to levels above those found for control cells, which suggests a co-stimulatory
activity. Hence it can be argued for a synergism between PMA downstream pathways and these inflammatory
mediators.
PMA is a synthetic inducer of PKC, necessary for several pro-inflammatory processes in macrophages,
and was used in pharmacological concentrations to contribute to the phagocytic response of cells [30]. Among
all components of the SCH axis tested, treatment with histamine was the only one where there was no
observation of increased phagocytic activity in the presence of PMA compared to histamine treatment alone.
One possible explanation is the fact that both histamine and PMA use the same phosphorilation sites in PKC
(Tyr311 and Thr505 [31]) to increase the immune response [31]. On the other hand, histamine can also act by
activation of H1 receptors, which activate phospholipases C (PLC), PLA2 and D (directly or not), thus increasing
intracellular concentrations of IP3, Ca2+ and cAMP [32]. The exact mechanism by which the activity of these
enzymes and the concentration of cAMP are balanced to inhibit or activate macrophage phagocytosis is not clear
yet, but may be the reason why the phagocytosis is increased in groups treated with PMA and histamine in
comparison with the group treated only with PMA.
The greatest synergic effects of components of the SCH axis with PMA were showed by groups treated
with epinephrine (63%) and norepinephrine (72%). These catecholamines use different downstream pathways as
compared to PMA in order to produce their effects. They have receptors coupled to G-proteins that increase
cAMP concentrations, activate proteins kinase A (PKA) and may also activate the nuclear factor-B [17], the
latter being probably the case, since iNOS was found to be activated by such treatments.
CRH did not change the phagocytosis of zymosan in unstimulated cells nor in the presence of PMA .
However, this hormone is able to influence the concentration of all other SCH axis components in vivo and then
may have an indirect effect on the phagocytic activity of macrophages.
Another feature to evaluate the immune function of macrophages is the production of reactive species of
oxygen or nitrogen, such as the nitric oxide (NO). In these cells, NO is produced by the inducible form of the
enzyme nitric oxide synthase (iNOS, encoded by the NOS-2 gene) [33]. The expression of this enzyme was
observed after 6 h of treatment with catecholamines, histamine or CRH. Since all treatments were able to
increase the intracellular concentration of iNOS, they may have not only short-term effects of the immune
system (phagocytosis) but also long-term and lasting effects.
The iNOS gene promoter has 2 regulatory sequences. One of them, the NF-B can be activated directly
by catecholamines [17], by histamine [34] and by CRH [35] due to their signalling pathways. However, the
possibility that the transcription of iNOS was indirectly increased by cytokines cannot be ignored. Transcription
of iNOS can also be triggered by pro-inflammatory cytokines, which also act via NF-B downstream pathways
[33], and whose release may be influenced by the presence of component of the SCH axis, especially in vivo.
Therefore, our data are consistent with in vivo observations that physical exercise may increase serum
levels of these substances and that it is capable of increasing the phagocytic activity of macrophages [17].
Altogether, the data suggest a possible interplay among SCH under physiological concentrations whose activity
was not previously determined in some in vitro studies using pharmacological concentrations of the components
of the SCH axis.
References
1.
2.
Papadimitriou, A. and K.N. Priftis, Regulation of the hypothalamic-pituitary-adrenal axis.
Neuroimmunomodulation, 2009. 16(5): p. 265-71.
Elenkov, I.J., et al., Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/
inflammatory response: acute and chronic effects. Ann N Y Acad Sci, 1999. 876: p. 1-11; discussion 11-3.
© Medimond . P726C0385
58
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Elenkov, I.J., et al., The sympathetic nerve--an integrative interface between two supersystems: the brain
and the immune system. Pharmacol Rev, 2000. 52(4): p. 595-638.
Korte, S.M., G.A. Bouws, and B. Bohus, Central actions of corticotropin-releasing hormone (CRH) on
behavioral, neuroendocrine, and cardiovascular regulation: brain corticoid receptor involvement. Horm
Behav, 1993. 27(2): p. 167-83.
Graham, P., G. Kahlson, and E. Rosengren, Histamine Formation in Physical Exercise, Anoxia and under
the Influence of Adrenaline and Related Substances. J Physiol, 1964. 172: p. 174-88.
Inder, W.J., et al., Prolonged exercise increases peripheral plasma ACTH, CRH, and AVP in male athletes.
J Appl Physiol, 1998. 85(3): p. 835-41.
Berkin, K.E., et al., Circulating adrenaline and noradrenaline concentrations during exercise in patients
with exercise induced asthma and normal subjects. Thorax, 1988. 43(4): p. 295-9.
Forner, M.A., et al., Effect of age on adherence and chemotaxis capacities of peritoneal macrophages.
Influence of physical activity stress. Mech Ageing Dev, 1994. 75(3): p. 179-89.
Ortega, E., M.A. Forner, and C. Barriga, Exercise-induced stimulation of murine macrophage chemotaxis:
role of corticosterone and prolactin as mediators. J Physiol, 1997. 498 ( Pt 3): p. 729-34.
Michna, H., The human macrophage system: activity and functional morphology. Bibl Anat, 1988(31): p.
1-84.
Forner, M.A., C. Barriga, and E. Ortega, Exercise-induced stimulation of murine macrophage phagocytosis
may be mediated by thyroxine. J Appl Physiol, 1996. 80(3): p. 899-903.
de la Fuente, M., M.I. Martin, and E. Ortega, Changes in the phagocytic function of peritoneal
macrophages from old mice after strenuous physical exercise. Comp Immunol Microbiol Infect Dis, 1990.
13(4): p. 189-98.
Fehr, H.G., H. Lotzerich, and H. Michna, The influence of physical exercise on peritoneal macrophage
functions: histochemical and phagocytic studies. Int J Sports Med, 1988. 9(1): p. 77-81.
Fehr, H.G., H. Lotzerich, and H. Michna, Human macrophage function and physical exercise: phagocytic
and histochemical studies. Eur J Appl Physiol Occup Physiol, 1989. 58(6): p. 613-7.
Ortega, E., et al., Stimulation of the phagocytic function in guinea pig peritoneal macrophages by physical
activity stress. Eur J Appl Physiol Occup Physiol, 1992. 64(4): p. 323-7.
Ortega, E., et al., Effect of age and of swimming-induced stress on the phagocytic capacity of peritoneal
macrophages from mice. Mech Ageing Dev, 1993. 70(1-2): p. 53-63.
Silveira, E.M., et al., Acute exercise stimulates macrophage function: possible role of NF-kappaB
pathways. Cell Biochem Funct, 2007. 25(1): p. 63-73.
Azuma, Y., et al., Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the
production of TNFalpha and IL-12 by macrophages via H2-receptors. Int Immunopharmacol, 2001. 1(910): p. 1867-75.
Gosain, A., et al., Norepinephrine suppresses wound macrophage phagocytic efficiency through alpha- and
beta-adrenoreceptor dependent pathways. Surgery, 2007. 142(2): p. 170-9.
Zinyama, R.B., G.J. Bancroft, and L.B. Sigola, Adrenaline suppression of the macrophage nitric oxide
response to lipopolysaccharide is associated with differential regulation of tumour necrosis factor-alpha
and interleukin-10. Immunology, 2001. 104(4): p. 439-46.
Sigola, L.B. and R.B. Zinyama, Adrenaline inhibits macrophage nitric oxide production through beta1 and
beta2 adrenergic receptors. Immunology, 2000. 100(3): p. 359-63.
Homem de Bittencourt Junior, P.I., et al., Glutathione metabolism and glutathione S-conjugate export
ATPase (MRP1/GS-X pump) activity in cancer. II. Cell-to-cell variability, relation with cellular activation
state and functional absence of GS-X pump in lymphocytes. Biochem Mol Biol Int, 1998. 45(6): p. 124354.
Homem de Bittencourt Junior, P.I., R. Curi, and J.F. Williams, Glutathione metabolism and glutathione Sconjugate export ATPase (MRP1/GS-X pump) activity in cancer. I. Differential expression in human
cancer cell lines. Biochem Mol Biol Int, 1998. 45(6): p. 1227-41.
Jin, W.L., et al., Repetitive hypoglycaemia increases serum adrenaline and induces monocyte adhesion to
the endothelium in rat thoracic aorta. Diabetologia, 2011.
Irman-Florjanc, T. and L. Stanovnik, Tricyclic antidepressants change plasma histamine kinetics after its
secretion induced by compound 48/80 in the rat. Inflamm Res, 1998. 47 Suppl 1: p. S26-7.
Nunez, H., et al., Fetal undernutrition induces overexpression of CRH mRNA and CRH protein in
hypothalamus and increases CRH and corticosterone in plasma during postnatal life in the rat. Neurosci
Lett, 2008. 448(1): p. 115-9.
Kurisu, K., et al., Radiotherapy of postoperative residual tumor of bile duct carcinoma. Radiat Med, 1991.
9(2): p. 82-4.
Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal Biochem, 1976. 72: p. 248-54.
© Medimond . P726C0385
59
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
29.
30.
31.
32.
33.
34.
35.
Burnette, W.N., "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and
radioiodinated protein A. Anal Biochem, 1981. 112(2): p. 195-203.
Nishihira, J. and J.T. O'Flaherty, Phorbol myristate acetate receptors in human polymorphonuclear
neutrophils. J Immunol, 1985. 135(5): p. 3439-47.
Mizuguchi, H., et al., Involvement of Protein Kinase C{delta}/Extracellular Signal-regulated
Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation
of Histamine H1 Receptor Gene Expression in HeLa Cells. J Biol Chem, 2011. 286(35): p. 30542-51.
Seifert, R., et al., Histamine increases cytosolic Ca2+ in dibutyryl-cAMP-differentiated HL-60 cells via H1
receptors and is an incomplete secretagogue. Mol Pharmacol, 1992. 42(2): p. 227-34.
Wang, Y. and P.A. Marsden, Nitric oxide synthases: gene structure and regulation. Adv Pharmacol, 1995.
34: p. 71-90.
Minami, T., et al., Histamine amplifies immune response of gingival fibroblasts. J Dent Res, 2007. 86(11):
p. 1083-8.
Zhao, J. and K.P. Karalis, Regulation of nuclear factor-kappaB by corticotropin-releasing hormone in
mouse thymocytes. Mol Endocrinol, 2002. 16(11): p. 2561-70.
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Role of matrix metalloproteinases and
inflammasome pathway in the development of
airway inflammation and fibrosis
Lagente V.1, Gicquel T.1, Victoni T.1, Robert S.1, Viel R.2, Fautrel A.1,2, Valença
S.3, Porto L.C.4, Boichot E.1
1: UMR991 INSERM/Université de Rennes 1, Faculté de Pharmacie, Rennes, France
2: Histopathological platform H2P2, University de Rennes 1 ;, Rennes, France
3: Instituto de Ciências Biomédicas, Universidade Federal de Rio de janeiro, Rio de janeiro, Brazil
4: Universidade do Estado de Rio de Janeiro, Rio de Janeiro, Brazil
E-mail : [email protected]
Summary
Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of
extracellular matrix (ECM) and so they are suggested to be important in tissue remodeling such as pulmonary
fibrosis. Pulmonary fibrosis is associated with deposition of ECM components mainly collagen in the lung
interstitium. The excessive airway remodelling as a result of an imbalance in the equilibrium of the normal
processes of synthesis and degradation of extracellular matrix components. We previously showed a correlation
of the differences in collagen deposition in the lungs of bleomycin-treated mice with a reduced molar pro-MMP9/TIMP-1 ratio. We also suggested that early altered regulation of matrix turnover may be involved in the
development of fibrosis. We then demonstrated that NLRP3-inflammasome pathway associated with the IL1R/MyD88 signaling is required in the increased TIMP-1 level and pulmonary fibrosis in mice. Finally, these
observations emphasize those effective therapies for these disorders must be given early in the natural history of
the disease, prior to the development of tissue remodeling and fibrosis.
Introduction
The increasing family of matrix metalloproteinases (MMPs) has been subject to sustained research and
has been widely demonstrated to be important in various fields of medicine including inflammatory process and
pathology. MMPs were primarily described to be involved in homeostasis and the turnover of the extracellular
matrix (ECM), but there has been numerous evidence suggesting that MMPs act on cytokines, chemokines and
protein mediators to regulate various aspects of inflammation and immunity [1,2]. The matrix metalloproteinases
(MMPs) form a group of structurally related extracellular zinc endopeptidases known for their ability to cleave
one or several constituents of the extracellular matrix [3]. Zymogen forms of the MMPs (pro-MMPs) are
secreted into the extracellular space from a large number of cell types, where the activation of the pro-MMPs in
the local microenvironment can result in discrete alterations in the tissue architecture. MMP synthesis and
functions are regulated by transcriptional activation, post-transcriptional processing (release of pro-domain, cell
surface shedding), and the control of activity by a family of endogenous inhibitors collectively known as tissue
inhibitors of metalloproteinases (TIMP). Upon stimulation, many cell types have been identified as producers of
MMPs and TIMPs in a context of inflammatory process, strongly suggesting the involvement of MMPs in
numerous inflammatory diseases. Based on this property, MMPs are not only put forward as physiological
mediators of the “turnover” of the extracellular matrix but are also considered to be critical factors of the
remodeling processes in pathological conditions [4]. Indeed, a marked increase in their expression is observed
and associated with a variety of inflammatory diseases.
Consequently, MMPs have been speculated to play a critical role in various inflammatory diseases, such
as airway diseases associated with inflammatory process including acute lung injury (ALI) and acute respiratory
distress syndrome (ARDS) [5], chronic obstructive pulmonary disease (COPD) [6], pulmonary fibrosis [7], but
also liver diseases, rheumatoid arthritis [8] and cancer [9]. In these conditions, we have to consider MMPs as
therapeutic targets which can be inhibited by non-selective and/or selective inhibitors as possible novel antiinflammatory compounds.
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Role of TIMPs in the development of pulmonary fibrosis
Tissue inhibitors of the metalloproteinases (TIMPs) are specific endogenous inhibitors that bind to the
active site of MMPs in a stoichiometric 1:1 molar ratio, thereby blocking access to extracellular matrix
substrates. Four TIMPs (TIMP-1, -2, -3 and -4) have been identified in vertebrates, and their expressions are
regulated during development, tissue remodelling but also inflammation [10]. The mammalian TIMP family
presents substantial sequence homology and structural identity on a protein level [11]. Basically, all members of
the TIMP family inhibit MMPs activity. This is accomplished through the coordination of the Zn+2 of the MMP
active site by the amino- and carbonyl- groups of the TIMPs N-terminal cysteine residues. Nevertheless, a
selective inhibition of some members of the MMP has been reported. For example, although TIMP-1 is the most
potent inhibitor for most MMP family members, it is a poor inhibitor of the membrane-type MMPs (MT-MMPs)
and MMP-19 [12,13]. TIMP-3 inhibits members of the A Disintegrin And Metalloproteinase (ADAM) family of
proteases, although the mechanism for this inhibition appears to be different from MMP inhibition [14,15].
TIMP-2 selectively interacts with MT1-MMP to facilitate the cell-surface activation of pro-MMP-2 [12,13,16].
Thus, TIMP-2 can both inhibit MMP activity and promote the cell surface activation of pro-MMP-2 by MT1MMP. TIMPs can be regulated on a transcriptional level by various cytokines and growth factors, resulting in
tissue-specific, constitutive, or inducible expression [17].
Strong evidences imply that TIMPs / MMPs imbalance are an important element in the fibrogenic
process: TIMPs, and especially TIMP-1 are upregulated in cases of human pulmonary fibrosis and in bleomycininduced pulmonary fibrosis. TIMPs, and particularly TIMP-1 induction could lead to a "non collagenolytic
microenvironment", building adequate conditions for a further extracellular matrix deposition to occur. Indeed,
we previously reported that TIMP-1 was markedly increased in mice’s lungs, 24 h after the administration of
bleomycin [18]. During this period, we were not able to observe collagen deposition, but bleomycin induced an
important inflammatory reaction characterized by an influx of neutrophils and probably an increase in
macrophage activity. However, the depletion of mice in neutrophils did not modify the level of the TIMP-1
protein in comparison with control mice [18]. We also reported that the non selective MMPs inhibitor,
batimastat, reduced the development of bleomycin-induced fibrosis in mice, associated with a decrease in TIMP1 levels in bronchoalveolar lavage fluid (BALF) [19]. In another study, we reported the inability of phagocytes
from p47phox-/- KO mice to produce large quantities of ROS via the NADPH oxidase pathway inhibits the
development of bleomycin-induced pulmonary fibrosis [20]. This inhibition is associated with changes in IL-6
production and in the molar MMP-9/TIMP-1 ratio in favor of the production of TIMP-1, both probably key
factors in airway remodeling and fibrosis. We also investigated MMP-9, MMP-2, TIMP-1, TIMP-2 and TIMP-3
in the fibrotic response to bleomycin of “fibrosis prone” C57BL/6J and “fibrosis resistant” BALB/c mice [21].
Fourteen days after bleomycin administration, hydroxyproline assay and histological study revealed that BALB/c
mice developed significantly less fibrosis compared to C57BL/6J mice. At day 1, bleomycin enhanced TIMP-1,
MMP-2 and MMP-9 protein levels in broncholaveolar lavage fluid, and induced corresponding genes in lung
tissue of both strains. The rise of Timp-1, Mmp-9 and Mmp-2 genes levels were significantly stronger in lungs of
C57BL/6J, whereas gelatinase activities of MMP-2 and MMP-9 were similar. At day 14, neither MMP-2 nor
MMP-9 levels exhibited strain-dependent protein level or gene expression although TIMP-1 was markedly
associated with fibrosis. Interestingly, bleomycin induced neither Timp-2 nor Timp-3 in lung tissue at any time
of the study [21]. These studies show that early altered regulation of TIMP-1 following bleomycin administration
may be involved in bleomycin-induced pulmonary fibrosis. This strongly suggests that TIMP-1 may be
considered as an available target for tissue remodelling and fibrosis. However, TIMPs which have affinities with
the picomolar range seem ideal inhibitors but they do not present selectivity and possess other biological
functions, which could lead to side effects [22].
It has also been suggested that the TIMP-1 function is also influenced by the cellular context,
specifically in that MMPs, in particular MMP-9, may reduce the effective concentration of TIMP-1 and compete
with TIMP-1 for binding to the cell surface receptor CD63 [22]. In contrast to TIMP-2, TIMP-1 blood
concentrations are increased in cancer patients, particularly in those with breast or colorectal carcinoma, and this
increase is negatively associated with patient outcome [23-25]. These recent studies have demonstrated the
clinical utility of TIMP-1 as a biomarker and independent prognostic factor in breast, colorectal, and several
hematological cancers.
The characterization of receptors for TIMP family members is a first step to understand the MMPindependent, cytokine-like functions of the TIMPs. Hopefully, this can lead to a starting point for the molecular
dissection of signaling events associated with the various activities of these proteins and their function in both
normal physiologic and pathologic processes. It is clear that the pleotropic activities of the TIMP family
members are complex and depend on interactions with other extracellular components, as well as direct
interactions with cell binding partners.
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Role of Inflammasome pathway in the development of pulmonary fibrosis
Several inflammatory diseases have been reported to be linked with the activation of NLRP3inflammasome pathway including gout [26], Crohn’s disease [27], rheumatoid arthritis [28] and cryopyrinassociated periodic syndrome (CAPS) [29].
The best-characterized inflammasome consists of three main components, the Nod-like receptor (NLR)family protein, NLRP3, pro-caspase-1, and the ASC (Apoptosis speck-like protein containing a CARD) adapter,
which bridge interactions between the former proteins [30]. NLRP3 is expressed by myeloid cells and is
upregulated in response to the stimulation of macrophages with pathogen-associated molecule patterns (PAMPs)
or danger-associated molecule patterns (DAMPs) such as ATP. NLRP3 interacts with ASC and pro-caspase-1 to
become effective. Following autoactivation via inflammasome assembly, caspase-1 cleaves pro-IL-1β, whose
biologically active form IL-1β is then secreted.
IL-1β is a cytokine with major roles in inflammation and innate immune responses. This cytokine is
produced by activated monocytes, macrophages and dendritic cells, inducing the production of chemokines or
cytokines such as TNF and IL-6, or proteases such as matrix metalloproteinases (MMPs) associated with
neutrophil recruitement and proliferation of resident cells mainly fibroblasts [31]. Since mature IL-1β is very
potent, its production is tightly regulated on expression, transcription and secretion [32].
It has also been previously demonstrated that pulmonary fibrosis is closely associated with the
activation of NLRP3-inflammasome pathway, production of IL-1 and TIMP-1 [33,34]. Indeed, several studies
using KO mice for several components of inflammasome pathway including NLRP3, ASC and caspase 1 showed
a reduction of experimental pulmonary fibrosis induced by bleomycin in mice [33-35]. The inhibition of the
collagen deposition and fibrosis is well correlated with the imbalance between MMPs and TIMP-1 in favour to
increased TIMP-1 production. Moreover, production of TIMP-1 and collagen deposition is reduced in IL-1
receptor and MyD88 deficient mice [31]. Similarly, cigarette smoke-induced inflammation and elastase-induced
emphysema in mice depends on inflammasome pathway and IL-1R1/MyD88 signaling [36,37].
Abnormal cell activation may provide signal that alert the immune system to danger, triggering innate
immunity activation leading to inflammatory process and remodelling. In this context, dying cells release danger
signals that may activate the immune system and stimulate innate and adaptive immunity. The danger signals are
recognized by membrane receptors such as TLRs [38] or cytosolic receptors such NLRP3-inflammasome [30]. It
was clearly demonstrated that uric acid is a danger signal activating NLRP3-inflammasome pathway in gout
arthritis [26]. Uric acid is a product of purine catabolism which is produced from injured tissue in vivo after
tumor chemotherapy leading to tumor lysis syndrom characterized by hyperuricemia [39]. At high local
concentration, uric acid precipitates and forms crystals that cause inflammation as observed in gout and activate
the caspase-1-containing NALP3-inflammasome, leading to the production of IL-1. It has been demonstrated
that uric acid locally produced in the lung upon bleomycin-induced DNA damage and degradation induced the
activation of NLRP3-inflammasome pathway. Reduction of uric acid levels using the inhibitor of uric acid
synthesis allopurinol or uricase leads to a decrease in bleomycin-induced IL-1 and TIMP-1 production, lung
inflammation and fibrosis. Finally, local administration of exogenous uric acid crystals recapitulates lung
inflammation and fibrosis, which depend on the NLRP3 inflammasome, MyD88, and IL-1R1 pathways and Tolllike receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor.
Adenosine triphosphate (ATP) has been described also as a danger signal activating NLRP3inflammasome leading to the pro-inflammatory cytokine IL-1β release in lung. Extracellular ATP was shown to
play a major role to trigger synthesis and release of mature IL-1β after prestimulation of macrophages by an
inflammatory signal such as LPS [40]. ATP is described as an agonist of purinergic P2 receptor predominantly
expressed on immune cells [41] and is reported to be involved in the pathophysiology of LPS-induced lung
injury, modulating airway inflammatory process and functional changes [42]. ATP mainly activates the P2X7
purinergic receptor, leading to trigger ASC-caspase-1 complex in a NLRP3-dependent manner, leading to the
production of IL-1β. Fibrotic patients have elevated ATP content in BALF in comparison with control
individuals [34]. It has been shown an early increase in ATP levels in BALF on bleomycin administration in
mice. Modulation of eATP levels with the ATP-degrading enzyme apyrase greatly reduced bleomycin -induced
inflammatory cell recruitment, lung IL-1β, and tissue inhibitor of metalloproteinase (TIMP)-1 production.
P2X(7) receptor-deficient mice presented dramatically reduced lung inflammation, with reduced fibrosis markers
such as lung collagen content, TIMP-1 and MMP-9 [34]. This clearly proposed that ATP released from
bleomycin-injured lung cells constitutes a major endogenous danger signal that engages the P2X(7)
receptor/pannexin-1 axis, leading to IL-1β maturation and lung fibrosis. We recently showed that ATPγS and
BzATP, two analogs of ATP are able to potentiate the release of IL-1β from human monocyte-derived
macrophages induced by low concentration of LPS [43]. In the same conditions no increase in IL-1α and IL-6
was observed. We also observed that P2X7R antagonists, A-438079 and A-740003, were able to reduce the
release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP strongly
suggesting the involvement of the P2X7R/NLRP3 pathway in the secretion of IL-1β from ATP-stimulated human
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macrophages [43].
Conclusions and future directions
It has not yet been clearly established which MMP activity needs to be inhibited in order to have an
impact on inflammatory diseases. Since numerous reported evidences suggest that different MMPs play an
important role in the pathogenesis of tissue remodelling associated with inflammatory processes in several
diseases, broad spectrum MMPs inhibitors may have therapeutic potential, nevertheless, associated with adverse
events [44,45]. It is therefore possible that a selective MMP inhibitor may have reduced side-effects.
One alternative is a gene transfer to overexpress TIMPs which can reduce MMPs activity and modulate
tissue remodelling. Several preclinical studies of various diseases have reported encouraging data. For example,
cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by the gene transfer of TIMP-1
and TIMP-3 [46]. However, expressing wild-type TIMPs could have drawbacks because multiple MMPs may be
inhibited. The best route to success is probably the development of engineered TIMPs with altered specificity, to
enable the targeting of specific MMPs. One alternative of selective MMP inhibitors could be the RNA
interference therapy development.
The recent characterization of the involvement of the NLRP3-inflammasome pathway has opened a
large possibility of new therapeutic targets for the reduction of collagen deposition and fibrosis. However, we
need of selective and available tools to validate the right target. For instance, our goal is to investigate the role of
purinergic receptors. Regarding the recent data, P2X7 receptor would be a good candidate. However, it is not
excluded that blockade of one type of receptors may induce a compensation by others. The screening of
potential drugs effective in preclinical models of fibrosis would be the next challenge.
Acknowledgments: The authors are grateful to the CAPES-COFECUB for funding and bilateral agreement
(Brazil-France). This study is also supported by Agence Nationale de la Recherche (ANR) project (ANR2010
MIDI01202) and INSERM.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
GREENLEE KJ, WERB Z, KHERADMAND F Matrix metalloproteinases in lung: multiple, multifarious,
and multifaceted. Physiol Rev 87: 69-98 (2007)
PARKSWC, WILSON CL, LOPEZ-BOADO YS Matrix metalloproteinases as modulators of
inflammation and innate immunity. Nat Rev Immunol 4: 617-629 (2004)
VU T, AND WERB Z. Gelatinase B: structure, regulation, and function. In Matrix Metalloproteinases. W.
C. Parks and R. P. Mecham, editors. Academic Press, San Diego. 115–148 (1998).
MOTT JD, WERB Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 16:
558–564 (2004).
FLIGIEL SE, STANDIFORD T, FLIGIEL HM, TASHKIN D, STRIETER RM, WARNER RL,
JOHNSON KJ, VARANI J Matrix metalloproteinases and matrix metalloproteinase inhibitors in acute
lung injury. Hum Pathol 37: 422-430 (2006).
OHNISHI K, TAKAGI M, KUROKAWA Y, SATOMI S, KONTTINEN YT Matrix metalloproteinasemediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest 78: 10771087 (1998).
MAISI P, PRIKK K, SEPPER R, PIRILA E, SALO T, HIETANEN J, SORSA T Soluble membrane-type
1 matrix metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) in induced sputum and
bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis. APMIS, 110: 771-782
(2002).
KONTTINEN YT, AINOLA M, VALLEALA H, MA J, IDA H, MANDELIN J, KINNE RW,
SANTAVIRTA S, SORSA T, LOPEZ-OTIN C, TAKAGI M Analysis of 16 different matrix
metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and
rheumatoid arthritis. Ann Rheum Dis 58: 691-697 (1999).
URBANSKI SJ, EDWARDS DR, MAITLAND A, LECO KJ, WATSON A, KOSSAKOWSKA AE
Expression of metalloproteinases and their inhibitors in primary pulmonary carcinomas. Br J Cancer 66:
1188-1194 (1992).
BREW K. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys
Acta 1477: 267–283 (2000).
TUUTTILA A, MORGUNOVA E, BERGMANN U, LINDQVIST Y, MASKOS K, FERNANDEZCATALAN C, BODE W, TRYGGVASON K, SCHNEIDER G. Three-dimensional structure of human
tissue inhibitor ofmetalloproteinases-2 at 2.1 A resolution. J Mol Biol 284: 1133–1140 (1998).
© Medimond . P726R9161
64
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
LAMBERT E, DASSE E, HAYE B, PETITFRERE E. TIMPs as multifacial proteins. Crit Rev Oncol
Hematol 49:187–198 (2004).
BAKER AH, EDWARDS DR, MURPHY G. Metalloproteinase inhibitors: biological actions and
therapeutic opportunities. J Cell Sci 115: 3719–3727 (2002).
BREW K, DINAKARPANDIAN D, NAGASE H. Tissue inhibitors of metalloproteinases: evolution,
structure and function. Biochim Biophys Acta 1477: 267–283 (2000).
AMOUR A, SLOCOMBE PM, WEBSTER A, BUTLER M, KNIGHT CG, SMITH BJ, STEPHENS
PE, SHELLEY C, HUTTON M, KNAUPER V, DOCHERTY AJ, MURPHY G. TNF-alpha converting
enzyme (TACE) is inhibited by TIMP-3. FEBS Letters 435: 39–44 (1998).
STETLER-STEVENSON WG. Matrix metalloproteinases in angiogenesis: a moving target for
therapeutic intervention. J Clin Invest 103: 1237–1241 (1999).
CLARK IM, SWINGLER TE, SAMPIERI CL, EDWARDS DR. The regulation of matrix
metalloproteinases and their inhibitors. Int J Biochem Cell Biol 12 :006 (2007).
MANOURY B, NENAN S, GUÉNON I, LAGENTE V, BOICHOT E. Influence of early neutrophil
depletion on MMPs/TIMP-1 balance in bleomycin-induced lung fibrosis. Int Immunopharmacol 7: 900911. (2007).
CORBEL M, CAULET-MAUGENDRE S, GERMAIN N, LAGENTE V, BOICHOT E. Inhibition of
bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor, batimastat. J
Pathol 193: 538-545 (2001).
MANOURY, B., LECLERC, O., NÉNAN, S., GUÉNON, I., BOICHOT, E., PLANQUOIS, J.M.,
BERTRAND, C. AND LAGENTE, V. The absence in reactive oxygen species production protects against
bleomycin induced pulmonary fibrosis in mice. Respir Res, 6: 11 (2005)
MANOURY B., CAULET-MAUGENDRE, S., GUÉNON, I., LAGENTE, V., BOICHOT, E. TIMP-1
is a key factor of fibrogenic response to bleomycin on lung of mice. Int J Immunopathol Pharmacol, 19:
471-487 (2006).
CHIRCO R, LIU XW, JUNG KK, KIM HR. Novel functions of TIMPs in cell signaling. Cancer
Metastasis Rev 25: 99–113, 2006.
HOLTEN-ANDERSEN MN, FENGER C, NIELSEN HJ, RASMUSSEN AS, CHRISTENSEN IJ,
BRUNNER N, KRONBORG, O. Plasma TIMP-1 in patients with colorectal adenomas: a prospective
study. Eur J Cancer 40: 2159–2164, 2004.
MCCARTHY K, MAGUIRE T, MCGREAL G, MCDERMOTT E, O’HIGGINS N, DUFFY MJ. HIGH
levels of tissue inhibitor of metalloproteinase-1 predict poor outcome in patients with breast cancer. Int J
Cancer 84: 44–48, 1999.
NAKOPOULOU L, GIANNOPOULOU I, STEFANAKI K, PANAYOTOPOULOU E, TSIRMPA I,
ALEXANDROU P, MAVROMMATIS J, KATSAROU S, DAVARIS P. Enhanced mRNA expression of
tissue inhibitor of metalloproteinase-1 (TIMP-1) in breast carcinomas is correlated with adverse prognosis.
J Pathol 197:307–313, 2002.
F. MARTINON, V. PETRILLI, A. MAYOR, A. TARDIVEL, AND J. TSCHOPP, Gout-associated uric
acid crystals activate the NALP3 inflammasome, Nature, 440: 237-241, 2006.
VILLANI AC, LEMIRE M, FORTIN G, LOUIS E, SILVERBERG MS, COLLETTE C, BABA N,
LIBIOULLE C, BELAICHE J, BITTON A, GAUDET D, COHEN A, LANGELIER D, FORTIN PR,
WITHER JE, SARFATI M, RUTGEERTS P, RIOUX JD, VERMEIRE S, HUDSON TJ,
FRANCHIMONT D, Common variants in the NLRP3 region contribute to Crohn's disease susceptibility,
Nature Genetics 41: 71-76, 2009.
ROSENGREN, H.M. HOFFMAN, W. BUGBEE, AND D.L. BOYLE, Expression and regulation of
cryopyrin and related proteins in rheumatoid arthritis synovium, An Rheumatic Dise 64: 708-714, 2005.
AGOSTINI L, MARTINON F, BURNS K, MCDERMOTT MF, HAWKINS PN, AND TSCHOPP J,
NALP3 Forms an IL-1beta–processing inflammasome with increased activity in Muckle-Wells
autoinflammatory disorder, Immunity 20: 319-325, 2004.
MARTINON F, BURNS K AND TSCHOPP J, The inflammasome: a molecular platform triggering
activation of inflammatory caspases and processing of proIL-beta, Mol Cell 10: 417–426, 2002.
GASSE P, MARY C, GUENON I, NOULIN N, CHARRON S, SCHNYDER-CANDRIAN S,
SCHNYDER B, AKIRA S, QUESNIAUX VF, LAGENTE V, RYFFEL B AND COUILLIN I, IL1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in
mice,” J Clin Invest, 117: 3786-3799, 2007.
F. MARTINON F, MAYOR A AND TSCHOPP J, The inflammasomes: guardians of the body, Ann
Rev Immunol, 27: 229-265, 2009.
GASSE G, RITEAU N, CHARRON S, GIRRE S, FICK L, PÉTRILLI V, TSCHOPP J, LAGENTE V,
QUESNIAUX VF, RYFFEL B AND COUILLIN I, Uric acid is a danger signal activating NALP3
inflammasome in lung injury inflammation and fibrosis, Am J Resp Crit Care Med 179: 903-913, 2009.
© Medimond . P726R9161
65
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
RITEAU N, GASSE P, FAUCONNIER L, GOMBAULT A, COUEGNAT M, FICK L,
KANELLOPOULOS J, QUESNIAUX VF, MARCHAND-ADAM S, CRESTANI B, RYFFEL B AND
COUILLIN I, Extracellular ATP is a danger signal activating P2X7 receptor in lung inflammation and
fibrosis. Am J Respir Crit Care Med 182: 774-783, 2010.
MARIATHASAN S, WEISS DS, NEWTON K, MCBRIDE J, O’ROURKE K, ROOSE-GIRMA M,
LEE MP, WEINRAUCH Y, MONACK DM AND DIXIT VM, Cryopyrin activates the inflammasome in
response to toxins and ATP, Nature 440: 228–232, 2006.
DOZ E, NOULIN N, BOICHOT E, GUENON I, FICK L, LE BERT M, LAGENTE V, RYFFEL B,
SCHNYDER B, QUESNIAUX VF, COUILLIN I. Cigarette smoke-induced pulmonary inflammation is
TLR4/MyD88 and IL-1R1/MyD88 signaling dependent. J Immunol 180(2):1169-1178, 2008.
COUILLIN I, VASSEUR V, CHARRON S, GASSE P, TAVERNIER M, GUILLET J, LAGENTE V,
FICK L, JACOBS M, COELHO FR, MOSER R, RYFFEL B. IL-1R1/MyD88 signaling is critical for
elastase-induced lung inflammation and emphysema. J Immunol 183(12): 8195-8202, 2009.
TIAN J, AVALOS AM, MAO SY, CHEN B, SENTHIL K, WU H, PARROCHE P, DRABIC S,
GOLENBOCK D, SIROIS C, HUA J, AN LL, AUDOLY L, LA ROSA G, BIERHAUS A, NAWORTH
P, MARSHAK-ROTHSTEIN A, CROW MK, FITZGERALD KA, LATZ E, KIENER PA, COYLE AJ.
Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1
and RAGE. Nat Immunol 8(5): 487-496, 2007.
CAMMALLERI L, MALAGUARNERA M. Rasburicase represents a new tool for hyperuricemia in
tumor lysis syndrome and in gout. Int J Med Sci 4(2):83-93, 2007.
D. FERRARI, C. PIZZIRANI, E. ADINOLFI, R.M. LEMOLI, A. CURTI, M. IDZKO, E. PANTHER,
AND F. DI VIRGILIO, The P2X7 receptor: A key player in IL-1 processing and release, J Immunol, 176:
3877–3883, 2006.
CODDOU C, YAN Z, OBSIL T, HUIDOBRO-TORO JP AND STOJILKOVIC SS, Activation and
regulation of purinergic P2X receptor channels, Pharmacol Rev 63: 641-683, 2011.
Monção-Ribeiro LC, Cagido VR, Lima-Murad G, Santana PT, Riva DR, Borojevic R, Zin WA,
Cavalcante MC, Riça I, Brando-Lima M, Takiya CM, Faffe DS, and Coutinho-Silva R,
Lipopolysaccharide-induced lung injury: role of P2X7 receptor. Respir Physiol Neurobiol, 179: 314-325,
2011.
Gicquel
DORMAN G, CSEH S, HDJU I, BARNA L, KONYA D, KUPAI K, KOVACS L, FERDINANDY P.
Matrix metalloproteinase Inhibitors. Drugs 70: 949-964, 2010.
PAVLAKI M, ZUCKER S. Matrix metalloproteinase inhibitors (MMPIs): The beginning of phase I or
the termination of phase III clinical trials. Cancer Metastasis Rev 22: 177–203, 2003
VAN DER LAAN WH, QUAX PH, SEEMAYER CA, HUISMAN LG, PIETERMAN EJ,
GRIMBERGEN JM, VERHEIJEN JH, BREEDVELD FC, GAY RE, GAY S, HUIZINGA TW, PAP T.
Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of
TIMP-1 and TIMP-3. Gene Ther 10: 234-242, 2003.
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Evaluation of the cellular response hematologic of
Cebus apella species exposed to carcinogen Nmethyl-N-nitrosourea (MNU) and treated with
CANOVA®
Azevedo Feio D.C.1, Pereira Carneiro Muniz J.A.2, Burbano R.M.R.1,
Cardoso De Brito Junior L.1, Lima de Lima P.D.3
1
Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, (Brazil)
Centro Nacional de Primatas, Ministério da Saúde, Ananindeua, PA, (Brazil)
3
Laboratório de Biologia Molecular, Centro de Ciências Biológicas e da Saúde, Universidade Estadual do
Pará, Belém/PA, (Brazil)
[email protected], [email protected], [email protected], [email protected],
[email protected].
2
Abstract
The Immune Response Modifier Canova ® (CA) is a homeopathic remedy indicated for patients with
depressed immune system, since this drug appears to increase the innate immunity and induce an immune
response against multiple and severe pathological conditions, including cancer. The aim of this study was to
evaluate the pattern of hematopoietic cell response in primates of Cebus apella species exposed to carcinogen NMethyl-N-Nitrosourea (MNU) and subjected to CA treatment. Thirteen animals were divided into 4 groups:
control (negative and positive) and three experimental groups (MNU alone (35 days); MNU (35 days) plus CA
(3 days); CA alone (3 days)). The animals received MNU orally and CA by three intravenous injections.
Evaluation of the cellular immune response was performed by hemogram. In the analysis of hematological
values of leukocytes, were observed an increase in the experimental groups treated with CA, a fact explained by
the action of the drug. Monocytes have shown to be decreased in the group treated with MNU and increased in
the groups receiving the drug probably by the CA action on macrophage activation via stimulation of monocytes.
Although it has been reported that CA can increase the number of neutrophils in the present study we did not
observe this action of the drug, probably due to the short time of treatment. CA downplayed the toxicity of
MNU, then being able to restore some components of the hematopoietic system, and can act as adjuvant
chemotherapy.
Keywords: Canova, MNU, Cebus apella, Immune response modification; Non-human primates,
Cellular response.
Introduction
The use of nonhuman primates as experimental models in vivo is very important in studies of
application to human health, due to their similar anatomical, biochemical and phylogenetic with humans.
Moreover, the size of the animal and it´s organs makes possible to repeat procedures such as diagnostics
collection of blood samples and biopsy of the same animal [1]. Although nonhuman primate models are not
common and are expensive compared to rodent models, the long life span observed in nonhuman primates
allows for long-term carcinogenic studies [2].
Immunomodulators are defined as agents that increase the immunity of an individual to promote a
specific immune response [3]. Canova (CA) is a complex homeopathic active immune response modifier of
natural origin and can be defined as an agent which mimics increases, or requires participation of host cells for a
more effective immune response. It is primarily indicated for patients whose immune system is depressed [4,5].
The treatment with CA seems to act by increasing the immunity of the individual to provoke an immune
response against particular states and various pathological conditions, including cancer [6,7]. Canova activates
macrophages both in vivo and in vitro, indirectly induces lymphocyte proliferation and mononuclear
differentiation induction in bone marrow cells [8,9]. Moreover, it is neither toxic nor mutagenic [10].
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A large variety of chemicals have been shown capable of inducing the development of tumors in
various animal species, since this cause genetic and epigenetic changes that lead to neoplastic transformation
[11]. N-methyl-N-nitrosourea (MNU) is an alkylating agent that induces cellular stress, leading to chromosomal
aberrations, point mutations, cell death, and DNA damage, causing the imbalance in the defense system of the
cell and thus stop mechanisms related to cellular metabolism [12]. The oral administration of MNU is
characterized to induce squamous cell carcinoma of the gastrointestinal surface in monkeys (mouth, pharynx,
larynx and stomach), as well as chronic inflammations generally observed in the esophagus causing increased
cell proliferation [13]. When used in high concentrations, MNU is first target to the lymphoid hematopoietic
system, resulting in a high mortality in animals, limiting the development of long-term experimental studies [14].
The goal of this work is to assess the pattern of hematopoietic cell response in Cebus apella primate
species exposed to carcinogenic N-Methyl-N-Nitrosourea (MNU) and subjected to the treatment with the
Immune response modifier Canova®, through the analysis haematological parameters.
Methods
1.1
Nonhuman Primates
We used 13 adults Cebus apella with a mean weight of 3.750 Kg, kept in captivity in the Centro
Nacional de Primatas (CENP), under the conditions of the same patterns in individual precincts within cages,
sheds, subject to natural photoperiod fed daily from fruit, vegetables, tenébrios (Zophobas morio), pelleted food
and water ad libitum. To identification and control, all animals had a "microchip" implanted in the dorsal
interscapular region. The animals were weighed before and every blood collection throughout the experimental
period. According to a basic veterinary examination, all animals were considered healthy at the time of first
blood sampling. This study was approved by the Ethics Committee of Universidade Federal do Pará.
1.2
Experimental Design
The study group consisted of a total of thirteen (13) animals, divided into two main groups control and
experimental, formed as described below. The animals were assigned randomly by lottery in four groups of three
Cebus apella each:
-
Negative control Group (NC): 3 animals which received saline instead of MNU and / or CA;
-
Positive control Group 1 (PC1): 1 animal that is 3 years receiving treatment with MNU;
-
Positive control Group 2 (PC2) animal of the positive control group 1 treated with CA (3 days);
-
Experimental group 1 (EXP.1): 3 animals received treatment with MNU alone (35days);
-
Experimental group 2 (EXP.2): 3 animals received treatment alone CA (3days);
-
Experimental group 3 (EXP.3): 3 animals receiving treatment MNU (35days) plus CA (3days).
The groups were of similar median age and weight (p = 1, Manne Whitney test). CA and sterile
physiological saline solution were injected by slow infusion in the right or left femoral vein of Cebus apella in
each dose.
1.3
Canova and MNU treatment
‘Canova do Brasil’, a Brazilian company, holds the international patent of this medicine
(www.canovadobrasil.com.br). CA is produced in drops, inhalant and intravenous forms, and sold only in
authorized pharmacies and laboratories. CA is standardized and authorized by competent agencies for medicinal
application. The final product contains Aconitum napellus dH20, Apis mellifica dH19, Arsenicum álbum dH17,
Asa foetida dH20, Baryta carbonica dH20, Bryonia alba dH14, Calcarea carbonica dH20, Conium maculatum
dH16, Ipecacuanha dH13, Lachesis muta dH18, Lycopodium clavatum dH20, Pulsatilla nigricans dH13, Rhus
toxicodendrum dH17, Ricinus communis dH14, Silicea dH18, Thuja occidentalis dH16, Veratrum album dH20
and less than 1% ethanol in distilled water. It is an aqueous, colorless and odorless solution. Experiments were
performed with commercial CA donated by ‘Canova do Brasil’. CA solution was vigorously shaken (succussion)
immediately before all treatments. The CA treatment was by intravenous injections at the dose of 1.67 ml/kg,
administered at 0h, 24h and 48h. This therapeutic model regime was adapted taking into account the weight of
the animal from the therapeutic regimen used in the initial treatment in humans [15,16]. Animals treated with
MNU received orally a solution of 667 µl MNU (Sigma-Aldrich of Brazil) daily, diluted in milk (200 ml) and
prepared by dissolving 1.8 g in 25 ml of distilled water, as experimental protocol to study the tumorigenesis as
described [1,13].
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1.4
Animal evaluation
During the treatment periods, the animals were inspected daily and their clinical symptoms were
recorded. Body weight was determined before every collection peripheral blood from the left femoral vein for all
analyzes. Clinical hematology parameters, included red blood cell count, hemoglobin, hematocrit, platelet count,
white blood cell (WBC) and differential segmented neutrophil, lymphocyte, monocyte, eosinophil and basophile
were analyzed. Methods and reference values for male adult animal were previously described [17-19]. The
hematological analysis was carried out using automated cell counter for veterinary use CELM CC-550 and the
differential leucocyte was done manually, by identifying 100 cells in blood smears stained with Wright, using
100x objective.
Results
The hematologic parameters were analyzed individually, through a comparison between the means of
control and experimental groups using analysis of variance (ANOVA test - parametric). The animals in the
control group had their negative hematological parameters considered normal, not presenting statistically
significant difference to the normal values of Cebus apella kept in Captivity [17-19], except the absolute value
of lymphocyte, the only pattern changed, with significantly lower average value as described in the literature,
although lymphocyte percentage values have appeared normal. The results presented in Table 01 demonstrate
statistically significant differences of haematological parameters when compared to the control groups. The
analysis of the hemoglobin, hematocrit and red blood cells of positive control groups were significantly lower as
compared to the negative control groups. We observed an increase of leucocytes in the experimental groups
treated with the CA, since monocytes were reduced in the group treated with MNU and showed increased in both
groups received CA. There was also an increase in platelet counts in the experimental group compared to
negative control. The following parameters: MCV, MCH, MCHC, basophils, eosinophils, myelocytes and
metamyelocytes were analyzed and showed no significant difference.
Table 01 - Mean and standard deviation obtained from the analysis of hematological parameters.
Haematological
Parameter
NC
PC 1
PC 2
EXP.1
EXP.2
EXP3
MEAN±SD
MEAN±SD
MEAN±SD
MEAN±SD
MEAN±SD
MEAN±SD
Leukocytes
3
(x10 /mm3)
6,90±0,56
15,40±0,69*
12,19±3,66†
7,71±2,92
10,55±1,94†
6,97±2,68
Platelets
3
(x10 /mm3)
109,00±12,17
125,00±14,14*
107,00±26,87
162,73±37,06*
122,56±14,13†
177,94±49,60*
Neutrophils (%)
64,67±2,52
59,50±0,70
58,00±2,83
46,97±14,27*
55,11±7,10
44,50±13,87*
Lymphocytes (%)
32,67±2,08
33,50±2,12
34,50±12,02
49,87±14,29§
42±6,75*
51,67±14,03§
Monocytes (%)
1,33±1,15
1±0
4±5,66*
0,53±0,63*
1,22±0,97
0,78±0,88
The data was subjected to one-way analysis of variance (ANOVA) and differences between samples were determined by T
multiple comparison test using the BioEstat 5.0 program.
* Significantly different from NC, p < 0.05.
† Significantly different from Exp1 and Exp3, p < 0.05.
§ Significantly different from NC and PC1, p < 0.05.
Discussion
Literature shows that blood cells of mammals, are an excellent system to test substances for their
capacity to produce damage in the organism, since samples are optimal for different treatment systems [20,21]
because they widely available and easily collectable, so allow performing several tests with answers that are
usually quick and easy analysis [22].
According to hematological parameters hematocrit, erythrocytes, hemoglobin, and leukocyte counts in
the control group, the animal treated with the carcinogen MNU for more than three years presented a pattern of
anemia. The values of these parameters previously cited were significantly smaller as compared to negative
control group, experimental as well as normal values for the species cited in the literature [17-19]. The anemia is
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a common complication in patients with various types of diseases, including cancer [23]. The increased of white
blood cell in the groups treated with MNU (positive control 1 and 2) was probably due to the inflammatory
response against the animal's likely neoplasic process, which is according to the carcinogenic effects on the
lymphoid and hematopoietic systems [24]. In the groups treated with CA (experimental 2 and 3), was also
observed an increase in these cells, a fact explained by the action of this drug, which acts as an immune response
modifier activating macrophages and inducing the production of white blood cell lineage [9]. Our research group
had previously shown that CA indirectly induces the proliferation of lymphocytes in vitro by macrophage
activation [8].
Although the literature reports that the CA may act indirectly by promoting increased number of
neutrophils [25], this study did not observe this action, probably due to the short period (3 days) by which
animals were treated with the drug.
Monocytes represent a large population of circulatory cells that can differentiate into macrophages or
dendritic cells [26]. In this study the number of monocytes shown to be decreased in the group treated with
MNU (experimental 1) and increased in the groups receiving the CA (positive control 2 and experimental 2),
probably because this drug act in the activation of macrophages via stimulation monocyte differentiation in this
cell type [27].
Although we have observed the increase in the number of platelets in both groups receiving MNU
(positive 1 and experimental1) and in the groups that received CA (experimental 2 and 3), the values of these
groups were considered normal when compared with the usual patterns previously described in the literature for
Cebus apella [17].
Conclusion
We found that CA had minimized the toxicity of the MNU when compared certain hematological
parameters; we had observed their partially ability to modify immune response, as improve white blood cell
count, but without changing the pattern of immunological markers. Then CA is able to restore some
hematopoietic system components and can act as an adjuvant in chemotherapy treatments.
Acknowledgements: This study was supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq - 550885/2007-2), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Centro Nacional de Primatas (CENP).
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Takayama S, Thorgeirsson UP, Adamson RH. (2008). Chemical carcinogenesis studies in non-human
primates. Proc Jpn Acad Ser B 84(6), pp. 176-188.
Borges da Costa JFF, Leal MF, Silva TCR, Andrade Junior EF, Rezende AP, et al. (2011). Experimental
Gastric Carcinogenesis in Cebus apella Nonhuman Primates. PLoS ONE 6(7).
Smit E, Pretorius E, Anderson R, Oommen J, Potjo M. (2008). Differentiation of human monocytes in
vitro following exposure to Canova in the absence of cytokines. Ultrastructural Pathology 32(4), pp.14752.
Coelho Moreira CO, Borges da Costa FFJ, Leal MF, Ferreira de Andrade E, et al. (2012). Lymphocyte
proliferation stimulated by activated Cebus apella macrophages treated with a complex homeopathic
immune response modifiers. Homeopathy 101(1), pp. 74-79.
De Oliveira CC, De Oliveira SM, Godoy LM, Gabardo J, Buchi DF. (2006). Canova, a Brazilian medical
formulation, alters oxidative metabolism of mice macrophages. J Infect. 52(6), pp. 420-432.
Sasaki MGM, Mariano FC, Gurgel LP, Probst S. (2001). Estudo clínico randomizado placebo controlado
para avaliar a eficácia e segurança do Método Canova na terapêutica de pacientes portadores de
HIV/AIDS em uso de anti-retrovirais. Braz J Infect Dis, 5(58).
Cesar B, Abud AP, de Oliveira CC, Cardoso F, et al. (2008). Activation of mononuclear bone marrow
cells treated in vitro with a complex homeopathic medication. Micron 39(4), pp. 461-70.
Burbano RR, Leal MF, Costa JB, Bahia, MO, Lima PDL, et al. (2009). Lymphocyte proliferation
stimulated by activated human macrophages treated with Canova. Homeopathy, 98(1), pp. 45-48.
Abud, AP, Cesar B, Cavazzani LF, de Oliveira CC, Gabardo J, Buchi DF. (2006). Activation of bone
marrow cells treated with Canova in vitro. Cell Biology International 30(10), pp. 808-816.
Seligmann IC, Lima PD, Cardoso PC, Khayat AS, Bahia MO, Buchi DF, Cabral IR, Burbano RR.
(2003).The anticancer homeopathic composite “Canova Method” is not genotoxic for human
lymphocytes in vitro. Genet Mol Res, 2(2), pp. 223-228.
© Medimond . P726C0029
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Tsubura A, Lai YC, MIKI H, SASAKI T, UEHARA N, et al. (2011) Review: Animal Models of NMethyl-N-nitrosourea-induced Mammary Cancer and Retinal Degeneration with Special Emphasis on
Therapeutic Trials. In Vivo 25(1), pp. 11-22.
Sintara K, Thong-Ngam D, Patumraj S, Klaikeaw N. (2012). Curcumin Attenuates Gastric Cancer
Induced by N-Methyl-N-Nitrosourea and Saturated Sodium Chloride in Rats. J Biomed Biotechnol. 2012:
915380.
Thorgeirsson UP, Dalgard DW, Reeves J, Adamsno RH. (1994).Tumor incidence in a chemical
carcinogenesis study of nonhuman primates. Regul Toxicol Pharmacol 19, pp. 130-151.
Da Silva Franchi CA, Bacchi MM, Padovani CR, de Camargo JLV. (2003).Thymic lymphomas in Wistar
rats exposed to N-methyl-N-nitrosourea (MNU). Cancer Science 94(3), pp. 240–243.
Cabral MP, Soffritti EM, Nader JR. (2003). Estudo clínico para avaliação do Imunomodulador Canova®
na terapêutica de pacientes oncológicos considerados FPT – Fora de Possibilidade Terapêutica: Fundação
Amor — Entidade Filantrópica de Combate a Dor do Câncer, Juiz de Fora MG.
http://www.canovadobrasil.com.br/trabalhos/TC_Cabral.pdf.
Castanheira PT, Oliveira AP, Rajão MCS, Krieger COP, Modesto NP, et al. (2001) Avaliação do uso de
imunomodulador
Canova
(medicamento
homeopático)
em
pacientes
com
câncer.
http://www.canovadobrasil.com.br/trabalhos/TC_Castanheira2.pdf .
Naves EA, Ferreira FA, Mundim AV, Guimarães EC. 2006. Valores hematológicos de macaco prego
(Cebus apella - Línnaeus, 1758) em cativeiro. Bioscience journal Uberlândia 22(2), pp. 125-131.
Riviello MC & Wirz A. (2001). Haematology and blood chemistry of Cebus apella in relation to sex and
age. J Med Primatol 30, pp. 308–312.
Wirz A, Truppa V, Riviello MC. (2008). Hematological and plasma biochemical values for captive tufted
capuchin monkeys (Cebus apella). Am J Primatol 70(5), pp. 463-472.
Bases R, Rubinstein A, Kadish A, Mendez F, Wittner D, et al. (1979). Mutagen-induced disturbances in
the DNA of human lymphocytes detected by antinucleoside antibodies. Cancer Res 39(9), pp. 3524-30.
Morales-Ramírez P, Vallarino-Kelly T, Cruz-Vallejo VL, López-Iturbe R, Alvaro-Delgadillo H. (2004).
In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics. Mutagenesis 19(3),
pp. 207-213.
Hoffbrand AV, Moss PAH, Pettit JE. (2007). Fundamentos em Hematologia. 5° ed. Porto Alegre.
Artmed.
Adamson JW. (2008). The anemia of inflammation/malignancy: mechanisms and management.
Hematology Am Soc Hematol Educ Program. pp. 159-165.
Uwagawa S, Tsuda H, Inoue T, Tagawa Y, Aoki T. (1991). Enhancing potential of 6 different
carcinogens on multi-organ tumorigenesis after initial treatment with N-methyl-N-nitrosourea in rats. Jpn
J Cancer Res 82 (12), pp. 1397-1405.
Laslo P, Spooner CJ, Warmflash A, Lancki DW, Lee HJ. (2006). Multilineage transcriptional priming
and determination of alternate hematopoietic cell fates. Cell 126(4), pp. 755-766.
Syme R, Bajwa R, Robertson L, Steward D, Gluck S. (2005). Comparison of CD34 and monocytederived dendritic cells from mobilized peripheral blood from cancer patients. Stem Cells 23(1), pp. 74–
81.
Smit E, Oberholzer HM, Pretorius E. (2009). A review of immunomodulators with reference to Canova.
Homeopathy 98 (3), pp. 169-176.
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Brazil)
il)
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Evaluation of genotoxicity of Solanum lycocarpum
aqueous extract utilizing Allium cepa test-system
Moreira de Lima V.1, dos Santos J.T.2, Vieira Gomes J.2, da Silva de Mello M.3,
Fampa P.1, Resende Borba H.1
1-Departamento de Biologia Animal – Instituto de Biologia – Universidade Federal Rural do Rio de Janeiro Brasil
2- Estudante de Graduação em Ciências Biológicas – Instituto de Biologia – Universidade Federal Rural do Rio
de Janeiro - Brasil
3- Bolsista do Projeto Jovens Talentos – FAPERJ – Brasil
[email protected], [email protected], [email protected], [email protected], [email protected]
Abstract
Evaluation of genotoxicity and cytotoxic effects is an essential step to which products of vegetal source
with potential therapeutic applications must be submitted in order to be safely utilized. Solanum lycocarpum St.
Hil, also known as wolf apple, is applied in local folk medicine for its diuretics, tranquilizer, antispasmodics,
anti-ophidic, antiepileptic and hypoglycemia-inducing actions. S. lycocarpum leaves infusion is indicated against
hemorrhoids and urinary tracts infections. In the present work, our goal is to analyze genotoxicity potential of S.
lycocarpum leaves extract utilizing Allium cepa test-system. A. cepa roots were allowed to be in direct contact
with S. lycocarpum aqueous leaves extract (20mg/ml), ethyl metanosulfonate (EMS) 25mM and distilled water.
Analyses were performed after 24 and 48h of contact with the solutions, and the results were obtained by
counting 3000-5000 cells, respectively, for each treatment. We observed that S. lycocarpum aqueous leaves
extract did not present genotoxic activity (p-value of P< 0,05, when χ2 test was applied). Actually, A. cepa cells
treated with S. lycocarpum leaves extract presented a significant lower number of alterations when compared to
positive and negative controls. Therefore those preliminary analyses strongly suggest that S. lycocarpum leaves
extract present an antimutagenic property. Similar results have been also obtained with S. lycocarpum
hydroalcoholic fruit extracts in V79 cell and mice bone marrow-obtained cells, reinforcing S. lycocarpum
antimutagenic potential evidences and demonstrating A. cepa test-system suits as an efficient tool to test plant
extracts putative mutagenic action.
Keywords: Solanum lycocarpum, genotoxicity, Allium cepa.
Introduction
The safe use of plants and plant products with therapeutic effects is determined by tests in order to
discard side toxic effects as well as the existence of noxious contaminants to health, such as heavy metals,
pesticides, microorganisms, metabolic products, among others. As part of the analysis process, there are tests for
the evaluation of genotoxic and citotoxic potentials of the vegetable material to be used. This kind of analysis is
necessary to assure that the usage of the material is reliable with reduced risks for a therapeutic usage, mainly for
long periods of time (1,2,3).Thus, different methodologies have been developed for evaluation of
substances genotoxicity and cytotoxicity inducing potential. Among those, the test-system performed in Allium
cepa, which involves the evaluation of chromosome alterations of A. cepa root cells after treatment exposure, is
noteworthy. This methodology is validated by the International Program of Safe Chemistry (IPCS, OMS) and by
the Environmental Program of United Nations (UNEP) as an efficient test for analysis and in situ monitoring of
genotoxicity and citotoxicity caused by environmental substances, and has been used in several studies aiming
the detection of genotoxicity of vegetables extracts (1, 4, 5, 6, 7, 8, 9).
The Brasilian flora presents a large potential as purveyor of medicinal plants that can be utilized in
treatment of endemic diseases in Brazil and in other tropical countries. Solanum lycocarpum St. Hil, species for
example, also known as lobeira and fruto-de-lobo is used in popular medicine for its diuretic, sedative,
antispasmodic, anti-venom, antiepileptic among others. The tea obtained from its leaves, for example, by
decoction in internal use, is indicated against hemorrhoids, and other infections on the urinary tract, and the
baked and hot fruits are indicated for direct application upon atrophied organs for their regeneration 10.
Furthermore, previous studies have demonstrated the molluscicidal and anthelmintical activity of S. lycocarpum
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leaves aqueous extracts (11, 12). Some recent analysis demonstrated that alcoholic extract of S. lycocarpum
fruits did not present genotoxic effects in bone marrow cells of mice, but exhibited antimutagenic effects instead
(13, 14). Until the present moment there are no reports of studies that evaluate the citotoxic/genotoxic
potential of extracts produced by other parts of the plant, like the leaves, for example, which tea is also used in
popular medicine. Therefore, this study aims to evaluate the genotoxic and citotoxic potentials of extracts
obtained from S. lycocarpum leaves, using the A. cepa test system.
Materials and methods
1.1
Samples collecting and identification
Solanum lycocarpum St Hil., (Solanaceae) leaves collecting was done at UFRRJ campus, Seropédica,
Rio de Janeiro. The botanic material was identified by the Herbaria technician Thiago Azevedo Amorim at the
Department of Botany of the Universidade Federal Rural do Rio de Janeiro, and the exsiccate was placed in the
Herbaria under the number RBR 14071.
1.2
Obtainment of aqueous extract of Solanum lycocarpum leaves
Immediately after the collecting, S. lycocarpum leaves were transported to the Laboratory of Plant
Genotoxic Activity – LAGeP/UFRRJ, where they were spread over the workbench, wrapped in absorbent paper
at room temperature and protected from sun light. Afterwards the leaves were mashed and kept in amber bottles
until the extracts preparation. The leaves were dried and administered as crude aqueous extract in the
concentration of 20mg/ml, prepared as hot infusion and then filtered in flannel tissue.
1.3
Mutagenicity test
Onion bulbs measuring 2,0cm of diameter were utilized, after removal of external cataphylls without
damaging the root buds. For each treatment, three to five bulbs of onion were used. Initially, the bulbs were
placed in a recipient containing distilled water for 48 hours and then the roots were put in contact with the
vegetable extract under considered high concentration of 20mg/ml. The bulbs used as negative control were kept
in distilled water and others used as positive control, were placed in a solution containing ethyl methanesulfonate
(EMS 2,5mM). The control and test bulbs were incubated at 25°C and the solutions were daily replaced. For
each treatment, the apical meristem of the roots with 2 and 2,5cm were removed from the bulbs after 24 and 48
hours of exposition to the solution. The slides were prepared as previously described [4], with slight
modifications. After the removal, the meristems were washed with distilled water and fixed in a solution of
ethanol: acetic acid glacial in the proportion of 3:1 (V/V), being storage under 4°C. Five slides were prepared per
each bulb, using five meristems per bulb. Meristems were washed with distilled water twice for five minutes and
hydrolyzed in HCL5N for 30 minutes, washed in distilled water twice for five minutes and stained with acetic
orcein 2%. The meristematic regions were fragmented utilizing a scalpel blade and the coverslip was placed over
the material. The slides were blindly observed in optic microscope. The presence of chromosome alterations and
aberrations were analyzed in 1000 cells per bulb, in a total of 3000 to 5000 cells for treatment.
1.4
Antimutagenic test
Antimutagenic effects analyses were performed through simultaneous treatment, where bulbs of A. cepa
were submitted to EMS 25mM diluted in aqueous extract of S. lycocarpum 20mg/ml. Slide preparations of this
group, as well as negative and positive control groups and bulbs submitted solely to the extract of S. lycocarpum
treatment were performed as described above. Mitotic index, which considers the number of cells present in each
cellular division phase relative to to the total number of observed cells, were calculated by cell counting for each
treatment group. The presence of chromosome alterations and aberrations were analyzed in 1000 cells per bulb,
in a total of 3000 cells per treatment.
1.5
Statistic analysis
The statistic analysis of the results were performed through the chi-square test (χ2 ) with probability
<0,05.
Results
Treatment with S. lycocarpum leaves extract on 20mg/mL did not show significant cytotoxic or
genotoxic effects on A. cepa cells after 24 to 48 hours of treatment. Conversely, we observed that extract-treated
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A. cepa cells exhibited significantly lower number of genotoxic alterations when compared to positive and
negative control cells (Table 1), indicating a potential antimutagenic effect. The main changes observed in
meristematic cells submitted to EMS were the presence of nucleolus in huge number and with increased volume
(Fig. 1). Further analysis of antimutagenicity (Table 2) demonstrated that S. lycocarpum aqueous extract
significantly reduces the effects caused by EMS, when meristematic cells from onion roots were treated
simultaneously with either S. lypocarpum leaves aqueous extract and EMS (Fig. 1). Results also demonstrated
(Table 3) that the aqueous extract of S. lycocarpum was able to inhibit the action of the mutagen on the cell
division rate, once again reinforcing its protective effect on meristematic cells of A. cepa.
Table 1: Nucleolus changes found in meristematic root cells of A. cepa submitted to different treatments
Treatment groups
Concentrations
VN
Negative control
Distilled water
361(b)
680
(a)
308
(a)(b)
25 mM
EMS
Aqueous extract
20mg/ml
VNG
VNGB
MET C
238(b)
08(b)
01(b)
5000
(a)
(a)
(a)
5000
(b)
5000
1670
124
35
(a)(b)
01
(a)(b)
15
0
Total number of cells
(a) Significant difference compared to negative control (P< 0,05) according to χ2 test; (b) Significant difference compared to
positive control (P< 0,05) according to χ2 test; VN – more than two nucleoli; VNG – one or more huge nucleoli; VNGB –
one or more huge nucleoli and nuclear bud; MET C – metaphase C.
Table 2: Number of cells with alterations in A. cepa roots submitted to antimutagenicity analysis.
Treatment groups
Concentrations
Negative control
EMS
Aqueous extract
EMS + Aqueous extract
VN
Distilled water
25mM
20mg/ml
25mM – 20mg/ml
(a)
47
116
78(a)
11(a)
VNG
Total number of cells
(a)
246
1403
16(a)
523(a)
3000
3000
3000
3000
(a) Sigificant difference compared to EMS (P< 0,05) according to χ2 test; VN – more than two nucleoli; VNG – one or more
huge nucleoli.
Table 3: Mitotic index in A. cepa roots submitted to different treatments.
Treatment groups
Total number of cells
Negative control
3000
EMS
Aqueous extract
EMS + Aqueous extract
Number of cells in division
542
3000
3000
18.06
(a)
15.60
585
(b)
19.50
545
(b)
18.16
468
3000
Mitotic index(%)
(a) Significant difference compared to negative control (P< 0,05) according to χ2 test; (b) Sigificant difference compared to
EMS (P< 0,05) according to χ2 test.
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Fig. 1: Cells from A. cepa roots and the different treatments: A root cells from negative control; B – root cells from positive
control - notice the presence of nucleolus with altered sizes; C – root cells treated with aqueous extract of S.lycocarpum; D –
root cells submitted to the simultaneous treatment (EMS + extract)
Discussion
The fact that the meristematic cells treated with the extract of S. lycocarpum leaves presented a
significantly lower number of alterations related to the nucleolus, when compared to the controls, is an indicative
of the antimutagenic effect of this extract. Similar results were found in 2007 by Tavares and collaborators, when
they analyzed the effect of hydroalcoholic extract of S. lycocarpum fruit in V79 cells and in bone marrow cells
of mice utilizing micronucleus technique. The authors attributed this effect to possible interactions between
different components present in the hydroalcoholic extract of the S. lycocarpum fruits, which presents important
secondary metabolites such as glycoalcaloid and phenolic compound. S. lycocarpum leaves extracts exhibit
phenols, alkaloids and flavonoids in its constitutions 15. These substances have been pointed as responsible for
the antimutagenic effects found in others vegetable extracts, including another species of Solanum (16, 17,
18, 19).
Conclusion
Our results suggest that the use of leaf tea from S. lycocarpum in folk medicine is a safe procedure, at
least with regard to their mutagenic effects, beyond the fact that their antimutagenic potential may contribute to
anticarcinogenic strategies. These results demonstrated the effectiveness of the A. cepa system and reinforce its
capacity as a tool in the analysis of mutagenic and antimutagenic potential of plant extracts.
References
[1]
[2]
Macêdo MFS, Sisenando HAAACN, Queiroz ACCA, Saturnino ACRD, Coelho LCBB, Medeiros SRB
(2008). Determining the genotoxicity of an aqueous infusion of Bauhinia monandra leaves. Rev. Bras. de
Farmacognosia 18(4), pp. 509-516.
Castro LS, Perazzo FF, Maistro EL (2009). Genotoxicity testing of Ambelania occidentalis
(Apocynaceae) leaf extract in vivo. Genetics and Molecular Research 8(2), pp. 440-447.
© Medimond . P726C0333
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Simões CMO (org.), Petrovick PR (2000). Farmacognosia: da planta ao medicamento. 2ª ed. Porto
Alegre/Florianópolis: Ed. Universidade/UFRGS/ Ed. Da UFSC.
Iganci JRV, Bobrowski VL, Heiden G, Stein VC, Rocha BHG (2006). Efeito do extrato aquoso de
diferentes espécies de boldo sobre a germinação e índice mitótico de Allium cepa L. Arq. Inst. Biol. 73
(1), pp. 79-82.
Bagatini MD, Da Silva ACF, Tedesco SB (2007). Uso do sistema teste de Allium cepa como bioindicador
de genotoxicidade de infusões de plantas medicinais. Revista Brasileira de Farmacognosia 17(3), pp. 444447.
Fachinetto JM, Bagatini MD, Durigon J, Da Silva ACF, Tedesco SB (2007). Efeito anti-proliferativo das
infusões de Achyrocline satureoides DC (Asteraceae) sobre o ciclo celular de Allium cepa. Revista
Brasileira de Farmacognosia 17(1), pp. 49-54.
Stange VS, Gomes TDUH, De Andrade MA, Batitucci MC (2009). Avaliação do efeito mutagênico do
extrato hidroalcoólico bruto, por meio de bioensaios in vivo e prospecção fitoquímica de Cecropia
glaziovii Sneth (embaúba), Cecropiaceae. Revista Brasileira de Farmacognosia 19 (2B), pp. 637- 642.
Celik TA, Aslanturk OS (2010). Evaluation of cytotoxicity and genotoxicity of Inula viscosa leaf extracts
with Allium test. Journal of Biomedicine and Biotechnology, pp. 1-8.
Sabini MC, Escobara FM, Bachetti RA, Sutil SB, Contigiani MS, Zanon SM, Sabini LI (2011).
Evaluation of cytogenotoxic effects of cold aqueous extract from Achyrocline satureoides Allium cepa L.
test. Nat. Prod. Commun. 6 (7), pp. 995-998.
Scarpel R, Cunha WR, Lopes RA, Souza MA, Lopes PEVP, Sala MA, Regalo SCH, Petenusci SO
(2006). Hepatotoxicidade e plantas medicinais. LII. Ação da infusão de Solanum lycocarpum St. Hil. No
rato. Investigação – Revista Científica da Universidade de Franca 6(1), pp. 17-20.
Costa DPC, Cruz APS, Aguiar LLF, Oliveira JCS, Fernandes GLT, Vasconcelos SDD, Rodrigues JS,
Diré GF, Borba HR (2008). Effect of Solanum lycocarpum aqueous extracts in helminth parasites of
conventionally maintained laboratory mice. Research Journal Parasitology 3(1), pp. 12-19.
Xavier VB, Borba HR, Brandolini SVP (2010). Atividade biológica de Solanum lycocarpum (Solanaceae)
procedente de regiões fitogeográficas distintas sobre Biomphalaria glabrata (Planorbidae). Revista
Brasileira de Zoociências 12 (1), pp. 43-49.
Vieira PM, Costa PM, Silva CR, Chen-Chen L (2010). Assessment of the genotoxic, antigenotoxic, and
cytotoxic activities of the ethanolic fruit extract of Solanum lycocarpum A. St. Hill. (Solanaceae) by
micronucleus test in Mice. Journal of Medicinal Food 13 (6), pp. 1409-1414.
Tavares DC, Munari CC, Araújo, MGF, Beltrame MC, Furtado MA, Gonçalves CC, Tiossi RFJ, Bastos
JK, Cunha WR, Veneziani RCS 2011. Antimutagenic potencial of Solanum lycocarpum against induction
of chromosomal aberrations in V79 cells, and micronuclei in Mice by doxorubicin. Planta Med. 77: 14891494,
Oliveira SCC (2003). Alelopatia em Solanum lycocarpum St. Hill (Solanaceae). Brasília. 78pp.
Dissertação de Mestrado, Universidade de Brasília.
Sultana S, Perwaiz S, Igbal M, Athar M (1995). Crude extracts of hepatoprotective plants, Solanum
nigrum and Cichorium intybus inhibit free radical-mediated DNA damage. J. Ethnopharmacol 45, pp.
189-192.
Ferreira L, Carvalho JCT, Maistro EL (2003). Standardized Solanum melongena extract presents
protective effects against chromosomal aberrations induced by doxorubicin in wistar rat bone marrow
cells. Cytologia 68, pp. 177-181.
Cai Y, Qiong L, Mei S, Corke H (2004). Antioxidant activity and phenolic compounds of 112 traditional
Chinese medicinal plants associated with anticancer. Life Sciences 74, pp. 2157
Fragoso V, Nascimento NC, Moura DJ, Silva ACR, Richter MF, Saffi J, Fett-Neto AG (2008).
Antioxidant and antimutagenic properties of the monoterpene índole alkaloid psychollatine and the foliar
extract of Psychotria umbellate Vell. Toxicol. In Vitro 22, pp. 559-566.
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Genotoxic activity of extracts of marine algae from
the coast of Alagoas - Brazil
da Silva B.H.1, Alvelino E.X.1, Guedes É.A.C.2, Sant’Ana E.G.3, Rodarte R.S.1
1
Laboratory of Molecular Cell Biology, Institute of Biological and Health Sciences – Federal University of
Alagoas (BRAZIL)
2
Laboratory of Phycology, Institute of Biological and Health Sciences – Federal University of Alagoas (BRAZIL)
3
Laboratory of Natural Resources, Institute of Chemistry – Federal University of Alagoas (BRAZIL)
E-mails: [email protected], [email protected], [email protected]
Abstract
Marine organisms are important and promising resources in cancer research. The group showed that
55.17% of the 48 different extracts of seaweed showed cytotoxic activity in different tumor cell lines (NCIH292, Hep-2 and K562). In this work the objective was to evaluate the genotoxic potential of extracts in cultured
lymphocytes from mice. Cells were incubated in 96 well plates (5 x 105 cells) at 30 µg / ml of each extract in
accordance with NCI Protocol. After 24h the samples were evaluated by micronucleus assay, comet assay and
apoptosis. Data were analyzed using one-way ANOVA test followed by Student-Newman-Keuls post-test (p <
0.05). The methanol extract of p. gymnospora and chloroform fraction obtained from the dichloromethane
extract of P. gymnospora induced genotoxicity high, capable of leading to apoptosis more than 25% of the
lymphocytes. Furthermore doxorubicin led lymphocyte apoptosis (20%), a value similar to chloroform fraction
obtained from the dichloromethane extract of H. musciformis. At concentration of 30 ug / ml, extracts induced
breakage in the genetic material of lymphocytes by alkaline comet assay. However, only HM and DM exhibited
lower indices of genotoxicity in micronucleus tests (5 ± 1) compared to those found in control and doxorubicin
(4 ± 1 and 2 ± 1, respectively). The data showed that the extracts are genotoxic in normal cells at different levels.
At present we are examining the genotoxicity of each extract on cultured tumor cells.
Keywords: Marine algae, Genotoxic activity, Comet assay, Micronucleus, Apoptosis.
Introduction
We know that exposure to genotoxic agents are harmful to health and may lead to the development of
different types of cancer [1]. Moreover, the same can have applicability in anticancer therapy, as described in
scientific literature of extracts, fractions or metabolites of plants, microorganisms, fungi, marine organisms [2, 3,
4]. The National Cancer Institute (NCI) of the United States has conducted a screening of various extracts of
plant samples against numerous cell lines. Of the 92 drugs available before 1983 and approved worldwide
between 1983 and 1994, 62%, approximately, can be related to natural origin [5].
Plants are the target of much research in the screening of potential substances with biological activity.
Although 71% of the globe is covered by water, marine organisms are underrepresented in this regard. The
microflora and microalgae constitute over 90% of marine biomass. This wide flora offers a large number of
natural compounds with unique biological activities that may be used to find potential agents of great efficiency
and specificity to treatment of diseases [6]. The marine flora has been used for medicinal purposes in several
countries, such as India, China and also in Europe. Historically, China and Japan use seaweed for consumption.
However, many of these countries use the algae as anthelmintics, anesthetics, ointments, as well as for the
treatment of cough, cold sores, gout, goiter, among other venereal diseases [6].
Algae are important sources of protein, iodo, vitamins and minerals. Their metabolites have shown
interesting antioxidant, antitumor and immunomodulatory activities. The anticancer activity is one of the most
important activities of drugs of marine origin. Some algae and its metabolites have shown cytotoxic effect and
plays an important role in the search for new pharmaceutical compounds with anticancer activity [6, 7]. A
research team tracked down 39 Chinese coast algae and showed that four species of Rhodophyta and three
species of Phaeophyta showed cytotoxic effects against cancer cell lines (KB and HT29). More than 30
compounds, as bromophenols, carotenes and steroids were isolated and purified. Their effects on cell lines have
been tested separately [7]. Studies with H. musciformis showed that chloroform-methanolic (2:1) extract showed
antigenotoxic and chemo protective effect against mitomycin c and ethyl methanesulfonate in human
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lymphocytes in vitro [8]. Within this context, we evaluate the genotoxic effect of some extracts of three species
of algae from the coast of Alagoas: Padina gymnospora, Hypnea musciformis and Digenea simplex.
Metodology
1.1
Animals
Male and female mice of the Swiss strain with 8 to 10 weeks old (20 to 25 g) were obtained from the
Central Biotery of the Federal University of Alagoas (UFAL). All procedures were submitted for approval by the
Ethics Committee of Animal Use (CEUA, Protocol No 19/2012), both of UFAL.
1.2
Biological material
Algae were collected from the Riacho Doce beach, (9°34'0" S and 35°39'0" W) during low tide period
between October 2007 to July 2009. Three species of algae from two divisions were used for this study: i.
Phaeophyta - Padina gymnospora (Kutzing) Sonder - MAC51235; ii. Rhodophyta - Hypnea musciformis
(Wulfen) J.V. Lamouroux - MAC51234 and Digenea simplex (Wulfen) C. Agardh - MAC51231. Vouchers were
deposited in the Herbarium MAC, Environment Institute of Maceió, Alagoas (Brazil) as internal reference
material.
1.3
Extracts
The seaweed were washed with distilled water, dried in circulating air ovens (Blue Mod1401440SC,
USA) at 45 ° C for five hours and then ground in industrial blender (type TA-2 METVISA model, Brazil). To
obtain the crude extract, 500 g of alga powder were suspended in 1000 ml of dichloromethane (DCM),
chloroform, methanol, ethanol and water. Each suspension was maintained for 72 hours. The organic extracts
were filtered and roto-evaporated (Buchii Rotevaporator Heating Bath-B490, Switzerland) at 25 °C and 40 °C.
The aqueous extract was lyophilized (Lyophilizer Edwards High Vacuum, ModE2MB, Brazil). The DCM
extracts from H. musciformis and P. gymnospora were fractionated because they presented high yields: 11.86 g
(2.58%) and 13.96 g (3.04%), respectively. For fractionation by liquid-liquid partition, extracts of H.
musciformis (2.73 g) and P. gymnospora (1.90 g) was suspended in methanol: water (3:1). The extraction was
made with hexane and chloroform, respectively. All solvents used were VETEC-Quimica Fina (RJ-Brazil).
1.4
Cell culture
Lymphocytes were obtained from mesenteric lymph nodes of mice in sterile buffered saline solution
(BSS). Quantified in Neubauer chamber and cultured in R10 - RPMI 1640 (Gibco® Life Technologies) with 10%
fetal bovine serum (LGC Biotechnology Ltda. São Paulo, Brazil) supplemented with 2 mM L-glutamine, 1 mM
sodium pyruvate and nonessential amino 1X acids (Gibco® Life Technologies). In 96-well flat-bottom microtiter
plates, 5 x 105 cells contained in 100uL of R10 medium were incubated for 16 hours with 5 ug / ml of
concanavalin A (Sigma-Aldrich Co.) at 37 °C in humidified atmosphere of 5%. In quadruplicate 30 ug of each
extract were added in the lymphocyte culture in a final volume of 200 uL. The plates were incubated in the
previous conditions for 24 hours.
1.5
Genotoxicity assays
The DNA damage was evaluated using different methods: i) alkaline comet assay, ii) micronucleus test
(MN) and iii) apoptosis. Each assay was performed as described by Tice et al. (2000) [9], Fenech (2000) [10]
and Singh (2000) and Sestili (2006) [11, 12], respectively. For the controls were utilized, lymphocytes treated
with doxorubicin (0.5 ug / ml), hydrogen peroxide (0.4 and 0.8 mM) and lymphocytes untreated (control). Each
of the assays was performed in triplicate.
1.6
Statistical analysis
Data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc.) using the statistical one-way
ANOVA test followed by Student-Newman-Keuls post-test (p < 0.05).
Results
The alkaline comet assay allows assessing the damage that take place in the single strand of DNA. In
this essay we observe that the culture of lymphocytes treated with dichloromethane extract of P. gymnospora
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(PD) did not induce significant damage to DNA in comparison to untreated lymphocyte culture (control - Fig.1).
The aqueous extract of P. Gymnospora (PA) that showed a lower level of DNA damage (Table 1). Comparing
PD and PA with doxorubicin (DX), both extracts showed less damage than the chemotherapeutic agent and
significantly (p < 0.001 and p < 0.01 respectively - Table 1 and Fig. 1). All other treatments induced significant
damage to the DNA (P < 0.001) comparable to DX.
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The micronucleus test identifies breaks into the genetic material, thereby, allows us to infer the presence
of clastogenic substances in the samples. The methanol extracts of the three species did not show significant
damage to double strand of the DNA of lymphocytes compared to control. Extracts of PD, PDC, PE, PA, HDC,
HA seem to present at their chemical composition clastogens. Whereas PD, PE, PA and HDC extracts were more
significant (p < 0.001) than PDC (p < 0.01) and HA (p < 0.05) as compared to control (Fig. 2).
The assay of apoptosis by DNA gel diffusion [13] showed that both PDC and HDC fractions and PM
extract showed significant rates (> 20%). In this context PM extract induced more apoptosis than DX. Moreover,
PA extract had the same performance as the control (Fig. 3).
Discussion
Genotoxicity assays with extracts of seaweed in the scientific literature are not great. Despite the
increase in studies on biological activities of seaweed, few products have emerged with the potential to be
identified or developed [2, 14]. Therefore, this work is important to the scientific community and, possibly, one
of the first to describe genotoxic activity of extracts of seaweed species.
Among the species studied in this work is known that H. musciformes shows activity anti-HSV (Herpes
Simplex Virus) type 1 and type 2, which can be improved in the presence of phytohormones [15]. Study on the
toxicity of this species in Swiss mice showed no change in hepatic or renal [16]. Our results on the genotoxic
potential of H. musciformes in lymphocyte culture have shown that the fraction HDC, compared to the other two
extracts (HM and HA), caused more damage to DNA and, therefore, increased rate of apoptosis. Only with an
analysis of the components present in this fraction can understand your activity.
The species P. Gymnospora displays anti-inflammatory activity mediated by sulfated polysaccharides
[17]. The genus Padina has shown antioxidant and antimicrobial activities by the presence of polyphenols, hence
with potential anticancer [18]. Among the extracts evaluated of this species we show that PDC and PM were
genotoxic to lymphocytes and PM was more effective than chemotherapeutic agent DX. These findings have led
the group to evaluate the chemical composition of these extracts and study the anticancer potential in tumor cell
lines.
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The only extract Digenea simplex (DM) showed only damage in single stranded DNA, since the data
MN and apoptosis were not significant. Searches in the literature on this species are quite incipient.
It is noteworthy that some extracts (HA, PE, PA and DM) did not induce apoptosis significantly.
However, exhibited significantly breaks in DNA strand according to the alkaline comet assays or MN. Such data
report that possibly there are potential chemotherapeutic substances present in these extracts.
Although preliminary, this work reveals the importance of studying the marine flora, especially algal
biomass. While some extracts have shown low genotoxic potential in cultured murine lymphocytes, the group
data has shown cytotoxic effect on tumor line K562 (data not shown). In summary, it is necessary to continue
evaluating each extract in different tumor cell lines, the testing of chemical prospecting and attempt to isolate
biologically active substances.
Conclusion
Our results suggest that there are bioactive substances in these extracts, which makes it necessary to
investigate the composition of these extracts and their effects on tumor cells. Another interesting point suggests
that the extracts behave in almost three levels of genotoxicity. First, those that significantly induced apoptosis,
being classified as high level (PM, PDC and HDC). Second group were those with negligible apoptosis, in
contrast to significant induction of micronucleus and comet, placing them in an intermediate level of DNA
damage (HA, PD, PA and PE). The third group were those with genetic toxicity in only one of the two trials,
MN or comet, placing them with low genotoxic potential (HM and DM).
In this context, we conclude that the analysis of genotoxicity seems to be more credible with the three
trials analyzed together. As well as study the marine flora appears to be quite promising in finding new bioactive
compounds.
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References
[1] Keshava, N. and Ong, T. (1999). Occupational Exposure to Genotoxic Agents. Mutation Research Reviews
437(2), pp. 175–194.
[2] Si-Yuan Pan, Shan Pan, Zhi-Ling Yu, Dik-Lung Ma, Si-Bao Chen, Wang-Fun Fong, Yi-Fan Han, KamMing Ko. (2010). New Perspectives on Innovative Drug Discovery: An Overview. J Pharm Pharmaceut
Sci. 13(3), pp. 450 – 471.
[3] Almeida, A.; Cunha, A.; Gomes, N.C.M.; Alves, E.; Costa, L.; Faustino, M.A.F. (2009). Phage Therapy and
Photodynamic Therapy: Low Environmental Impact Approaches to Inactivate Microorganisms in Fish
Farming Plants. Marine Drugs 7(3), pp. 268-313.
[4] Nobili, S.; Lippi, D.; Witort, E.; Donnini, M.; Bausic, L.; Mini, E.; Capaccioli, S. (2009). Natural
Compounds For Cancer Treatment And Prevention. Pharmacological Research. 59(6), pp. 365–378.
[5] Cragg, G.M.; Newman, D.J. and Snader, K.M. (1997). Natural Products in Drug Discovery and
Development. Journal of Natural Products 60(1), pp. 52–60.
[6] Boopathy, N.S. and Kathiresan, K. (2010). Anticancer Drugs fromMarine Flora: An Overview. Journal of
Oncology Volume 2010, pp. 1-18.
[7] Zandi, K., Ahmadzadeh, S., Tajbakhsh, S., Rastian, Z., Yousefi, F., Farshadopour, F., Sartavi, K. (2010).
Anticancer Activity of Sargassum oligocystum Water Extract Against Human Cancer Cell Lines.
European Review for Medical and Pharmacological Sciences 14, pp. 669-673.
[8] Vatan, O.; Celikler, S.; Yildiz, G. (2011). In Vitro Antigenotoxic and Anti-oxidative Capacity of Hypnea
musciformis (Wulfen) Lamouroux Extract in Human Lymphocytes. African Journal of Biotechnology
10(4), pp. 484-490.
[9] Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E.,
Ryu, J.-C. and Sasaki, Y.F. (2000). Single Cell Gel/Comet Assay: Guidelines for In Vitro and In Vivo
Genetic Toxicology Testing. Environmental and Molecular Mutagenesis 35, pp. 206-221.
[10] Fenech, M. (2000). The In Vitro Micronucleus Technique. Mutation Research 455, pp. 81–95.
[11] Singh, N.P. (2000). A Simple Method for Accurate Estimation of Apoptotic Cells. Experimental Cell
Research 256, pp.328 –337.
[12] Sestili, P., Martinelli, C., Stocchi, V. (2006). The Fast Halo Assay: An Improved Method to Quantify
Genomic DNA Strand Breakage at the Single-Cell Level. Mutation Research 607, pp. 205–214.
[13] Manoharan, K. and Banerjee, M.R. (1985). β-Carotene Reduces Sister Chromatid Exchange Induce
Chemical Carcinogens in Mouse Mammary Cells in Organ Culture. Cell Biol. Int. Rep. 9, pp. 783–789.
[14] Smit, A.J. (2004). Medicinal and Pharmaceutical Uses of Seaweed Natural Products: A Review. Journal of
Applied Phycology 16, pp. 245–262.
[15] Mendes, G.S., Bravin, I.C., Yoneshigue-Valentin, Y., Yokoya, N.S., Romanos, M.T.V. (2012). Anti-HSV
Activity of Hypnea musciformis Cultured with Different Phytohormones. Brazilian Journal of
Pharmacognosy 22(4), pp. 789-794.
[16] J.A.G. Rodrigues; I.W.F. de Araújo; G.A. de Paula; E.S.O. Vanderlei; I.N.L. de Queiroz; A.L.G. Quinderé;
C.O. Coura; É.F. Bessa; T.B. Lima; N.M.B. Benevides. (2011). Isolation, Fractionation and In Vivo
Toxicological Evaluation of Sulfated Polysaccharides from Hypnea musciformis. Ciência Rural 41(7),
pp.1211-1217.
[17] Marques, C.T., de Azevedo, T.C.G., Nascimento, M.S., Medeiros, V.P., Alves, L.G., Benevides, N.M.B.,
Rocha, H.A.O., Leite, E.L. (2012). Sulfated Fucans Extracted from Algae Padina gymnospora Have AntiInflammatory Effect. Brazilian Journal of Pharmacognosy 22(1), pp. 115-122.
[18] Devi, K.P., Suganthy, N., Kesika P. and Pandian, S.K. (2008). Bioprotective Properties of Seaweeds: In
Vitro Evaluation of Antioxidant Activity and Antimicrobial Activity Against Food Borne Bacteria in
Relation to Polyphenolic Content. BMC Complementary and Alternative Medicine 8, pp. 38-48
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Evaluation of genotoxic effects in lymphocytes of
mice from extracts Ziziphus joazeiro Martius
(Rhamnaceae)
da Silva B.H.1, Rocha Brito I.R.1, Alvelino E.X.1, de Matos Rodarte C.C.2,
Sant’Ana A.E.G.2, Rodarte R.S.1
1
Laboratory of Molecular Cell Biology, Institute of Biological and Health Sciences – Federal University of
Alagoas (BRAZIL)
2
Laboratory of Natural Resources, Institute of Chemistry – Federal University of Alagoas (BRAZIL)
E-mails: [email protected], [email protected]
Abstract
Several activities are described for extracts of Z. joazeiro, however, little is known about the genotoxic
potential and the anticancer effect of these extracts. In this study we aimed to evaluate the genotoxic effect of
four fractionated extracts (hexane, chloroform, ethyl acetate and aqueous). This material was obtained from the
crude ethanolic extract of leaves of Z. joazeiro. Lymphocytes (5 x 105 cells) were incubated with 30 ug / ml of
each extract in a 96 well plate. After 24 hours, the samples were evaluated by micronucleus, comet and
apoptosis. Data were analyzed by one-way ANOVA followed by Student-Newman-Keuls test (p <0.05).
According to the data, extracts of chloroform and hexane levels showed significant DNA damage in all three
assays. However, the chloroform extract appears to have greater genotoxic effect. Moreover, the aqueous extract
induced DNA damage only by the alkaline comet assay (99 ± 6 in arbitrary unit). While the ethyl acetate extract
was found to be genotoxic in the comet assays and apoptosis (89 ± 3, in arbitrary unit and 13 ± 1, in percent,
respectively). We conclude that the extracts have different levels of genotoxicity, suggesting that the aqueous
extract is the mildest, while chloroformic the most effective. At present we are evaluating these extracts in
different tumor cell lines in search of compounds with antitumor activity.
Keywords: Ziziphus joazeiro, genotoxicity, comet assay, micronucleus, apoptosis.
Introduction
Ziziphus joazeiro belongs to the family Rhamnaceae, the genus Ziziphus Mill has about 100 species
widely distributed. The Z. joazeiro Mart, known as juazeiro, juá and orange tree of cowboy, is the most notable
representative of the savanna biome [1]. This plant is used by people for the treatment of skin disorders such as
dermatitis and mycosis [2]. Albuquerque et al. (2007) [3] attributed to the whole plant several medicinal uses:
mouthwash, dermatologic problems (dandruff, scabies, seborrhea dermatitis and itching), respiratory problems
(asthma, cough, pneumonia, tuberculosis, bronchitis, sore throat and flu), digestive system problems
(constipation, stomatitis, gastric ulcers and indigestion. It has also been reported healing activity [4].
The cortex of stems and leaves is rich in saponins and so are used in the manufacture of anti-dandruff
shampoos and hair tonic [4]. The antifungal activity was observed against Candida albicans, Cryptococcus
neoformans and Fonsecaea pedrosoi. It has also been demonstrated antioxidant activity of aqueous extract of the
bark of Z. juazeiro. The antipyretic activity of the genus Ziziphus has been reported. In the bark of the plant has
been reported the presence of betulinic acid, oleanolic acid, and saponin. The epicuticular wax of the leaves has
alkanes and triterpenoid [5, 6].
Silva et al. (2011) [7] did not show the same effect antifungal described by Cruz et al (2007) [2] for the
aqueous extract of the bark of juá against C. albicans. Such discrepancies have been attributed to factors such as:
different extractive methods, different collection sites, and the methods employed to detect antimicrobial
activity.
Studies with the extract of leaves showed an antioxidant activity 400 times greater than the bark extract
[7]. This fact should probably be related to the high content of tannins present in this species. In this context, our
study sought to focus on the genotoxic effects of anticancer and extracts juá.
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Methodology
1.1
Animals
Male and female mice of the Swiss strain with 8 to 10 weeks old (20 to 25 g) were obtained from the
Central Biotery of the Federal University of Alagoas (UFAL). All procedures were submitted for approval by the
Ethics Committee of Animal Use (CEUA, Protocol No 19/2012), both of UFAL.
1.2
Biological material
Leaves of Ziziphus joazeiro were provided by the project IMSEAR (Millennium Institute of SemiArid). Samples were collected by botanist Dr ª Tânia Ribeiro on 09/04/2002 road Urandi, Farm Palmeiras, 6 km
from the city of Urandi, state of Bahia, 14 ° 16'S 42 ° 37'W. The plant is registered in the Herbarium of the State
University of Feira de Santana (HUEFS) under the serial number 65975.
1.3
Extracts
The crude ethanolic extract was obtained from 3.8 kg of leaves Ziziphus joazeiro air dried, crushed into
forage machine and submitted to exhaustive extraction with 95% ethanol in percolator at room temperature (27
°C ± 1 °C). The solvent was removed on a rotary evaporator under reduced pressure at temperatures less than 60
°C. The crude ethanolic extract yielded 465.5 g (12.2% yield) and was mixed in methanol:water (2:3), this
solution was submitted to liquid-liquid partition with solvents of increasing polarity. The following fractions
were obtained: hexane (75.95 g - 16.31%), chloroform (59.81 g - 12.84%) in ethyl acetate (71.18 g - 15.29%)
and aqueous (258, 56 g - 55.54%). The samples were concentrated on a rotary evaporator.
1.4
Cell culture
Lymphocytes were obtained from mesenteric lymph nodes of mice in sterile buffered saline solution
(BSS). Quantified in a Neubauer chamber and cultured in R10 - RPMI 1640 (Gibco® Life Technologies) with
10% fetal bovine serum (LGC Biotechnology Ltda. São Paulo, Brazil) supplemented with 2 mM L-glutamine, 1
mM sodium pyruvate and nonessential amino 1X acids (Gibco® Life Technologies). In 96-well flat-bottom
microtiter plates, 5 x 105 cells contained in 100uL of R10 medium were incubated for 16 hours with 5 ug / ml of
concanavalin A (Sigma-Aldrich Co.) at 37 °C in humidified atmosphere of 5%. In quadruplicate 30 ug of each
extract were added in the lymphocyte culture in a final volume of 200 uL. The plates were incubated in the
previous conditions for 24 hours.
1.5
Genotoxicity assays
The DNA damage was evaluated using different methods: i) alkaline comet assay, ii) micronucleus test
(MN) and iii) apoptosis. Each assay was performed as described by Tice et al. (2000) [8], Fenech (2000) [9] and
Singh (2000) and Sestili (2006) [10, 11], respectively. For the controls were utilized, lymphocytes treated with
doxorubicin (0.5 ug / ml), hydrogen peroxide (0.4 and 0.8 mM) and lymphocytes untreated (control). Treatments
of the lymphocytes with the extracts performed in 96 well plates were done in quadruplicate. To confirm the
results were carried out three replicates of each test.
1.6
Statistical analysis
Data from comet assays, micronucleus and apoptosis were analyzed using GraphPad Prism 5 (GraphPad
Software, Inc.) using the statistical one-way ANOVA test followed by Student-Newman-Keuls post-test with a
confidence interval of 95% (p < 0.05).
Results
The alkaline comet assay (Fig. 1) demonstrated that all extracts caused damage to the DNA of normal
lymphocytes. However, the JH extract showed significant arbitrary unit (45.75 ± 3.37) compared to control and
chemotherapic (24.4 ± 2.27 and 104.1 ± 5.0, respectively). JAE extract also showed significance to both
controls, whose damage were similar to that of hydrogen peroxide (0.4 mM). The other two extracts damaged
the genetic material similarly to DX (Fig. 1 and Table 1).
When evaluating data MN (Fig. 2), the hexane extract was found to induce breaks in DNA similarly to
hydrogen peroxide (0.8 mM) (7.25 ± 1.13 and 7.33 ± 1.23, respectively). While the JC extract induced the
presence of micronuclei such as hydrogen peroxide (0.4 mM) (5.5 ± 1.05 and 5.5 ± 0.63, respectively). The JA
and JAE extracts were not significant compared with the control (Fig. 2).
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In analyzing the data obtained with the assay of apoptosis (Fig 3) we can observe that JA did not cause
cell death significantly. On the other hand, the remaining three extracts induced apoptosis. However, the
chloroform extract was what presented rate higher than the chemotherapic agent when compared to both (JC and
DX) to the untreated control.
Discussion
Research on natural products, especially with plant extracts, have shown the potential for the presence
of bioactive substances. The actions are most variable: antioxidant, antimicrobial, anti-inflammatory, antiviral,
cytotoxic, genotoxic, anti-cancer, among others [13, 14, 15]. Some of these activities are harmful to human
health, thus it is necessary to conduct tests quickly and reliably to detect that such effects.
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The genotoxicity of plant extracts has applicability in anticancer therapy [16]. Z. joazeiro presents not
only the activities mentioned above, in fact has many others. However, the genotoxic activity of leaf extracts is
poorly known compared to those already described for bark [2, 7, 17, 18].
According to the results obtained in this study may suggest that the fractionated extracts of crude
ethanol extract of leaves of Z. joazeiro are potentially genotoxic. The ability to damage DNA molecule and
inducing programmed cell death in normal lymphocytes of mice seems to be different for each one of extracts.
This fact is seen when evaluating the three tests together, which suggest that JC extract has the highest genotoxic
effect, followed by JH and JAE extracts. The aqueous extract even though genotoxic to lymphocytes, it looks
like the repair mechanism operates in behalf of cell survival, since they were not detected significant MN and
apoptosis.
In summary, the data presented here matched with other laboratory findings allow us to suggest that
there is antitumoral action in extracts obtained from crude ethanolic extract of Z. joazeiro (data not shown).
However, further studies should be conducted to ascertaining the chemical components of these extracts. And
also to evaluate the action of the extracts at different concentrations on tumor cell lines in vitro.
Conclusion
Front preliminary tests submitted we may conclude that the extracts are genotoxic, although to varying
degrees. Furthermore our studies seem to very promising regarding for anticancer activity of leaf extract
obtained Z. joazeiro Mart.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Lorenzi, H., Matos, F.J.A. (2002). Plantas Medicinais no Brasil: Nativas e Exóticas. Ed. São Paulo:
Instituto Plantarum de Estudos da Flora.
Cruz, M.C.S., Santos, P.O., Barbosa Jr., A.M., Melo, D.L.F.M., Alviano, C.S., Antoniolli, A.R., Alviano,
D.S., Trindade, R.C. (2007). Antifungal Activity of Brazilian Medicinal Plants Involved in Popular
Treatment of Mycoses. J Ethnopharmacol 111, pp. 409-412.
Albuquerque, U.P., Medeiros, P.M., Almeida, A.L.S., Monteiro, J.M., Lins Neto, E.M.F., Melo, J.G.,
Santos, J.P. (2007). Medicinal plants of the Caatinga (Semi-Arid) Vegetation of NE Brazil: A
Quantitative Approach. J Ethnopharmacol. 114, pp. 325–354.
Kato ETM, Ohara MT, Nishitami M. (1998). Evalution of Antimicrobial Property of Ziziphus joazeiro
Martius. Lecta USF 16(2), pp. 75-85.
Oliveira, A.F., Meirelles, S.T., Salatino, A. (2003). Epicuticular Waxes from Caatinga and Cerrado
Species and Their Efficiency Against Water Loss. Ann Braz Acad Sci. 75(4), pp. 431-439.
Oliveira, A.F.M., Salatino, A. (2000). Major Constituents of the Foliar Apicuticular Waxes of Species
from the Caatinga and Cerrado. Zeitschrift fur Naturforschung C-A J Biosci. 55(9-10), pp. 688-692.
Silva, T.C.L., Almeida, C.C.B.R., Veras Filho, J., Peixoto Sobrinho, T.J.S., Amorim, E.L.C., Costa, E.P.,
Araújo, J.M. (2011). Atividades Antioxidante e Antimicrobiana de Ziziphus joazeiro Mart.
(Rhamnaceae): Avaliação Comparativa Entre Cascas e Folhas. Rev Ciênc Farm Básica Apl. 32(2), pp.
193-199.
Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas,
E., Ryu, J.-C. and Sasaki, Y.F. (2000). Single Cell Gel/Comet Assay: Guidelines for In Vitro and In Vivo
Genetic Toxicology Testing. Environmental and Molecular Mutagenesis 35, pp. 206-221.
Fenech, M. (2000). The In Vitro Micronucleus Technique. Mutation Research 455, pp. 81–95.
Singh, N.P. (2000). A Simple Method for Accurate Estimation of Apoptotic Cells. Experimental Cell
Research 256, pp.328 –337.
Sestili, P., Martinelli, C., Stocchi, V. (2006). The Fast Halo Assay: An Improved Method to Quantify
Genomic DNA Strand Breakage at the Single-Cell Level. Mutation Research 607, pp. 205–214.
Manoharan, K. and Banerjee, M.R. (1985). β-Carotene Reduces Sister Chromatid Exchange Induce
Chemical Carcinogens in Mouse Mammary Cells in Organ Culture. Cell Biol. Int. Rep. 9, pp. 783–789.
Demma, J., Engidawork, E. and Hellman, B. (2009). Potential Genotoxicity of Plant Extracts Used in
Ethiopian Traditional Medicine. Journal of Ethnopharmacology 122, pp. 136–142.
Ali, B.H., Wabel, N.A., and Blunden, G. (2005). Phytochemical, Pharmacological and Toxicological
Aspects of Hibiscus sabdariffa L.: A Review. Phytother. Res. 19, pp. 369–375.
Vargas, V.M.F., Guidobono, R.R. and Henriques, J.A.P. (1991). Genotoxicity of Plant Extracts. Mem.
Inst. Oswaldo Cruz 86(Suppl. II), pp. 67-70.
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[16]
[17]
[18]
Pessoa, C., Costa-Lotufo, L.V., Leyva, A., de Moraes, M.E.A., de Moraes, M.O. (2006). Anticancer
Potential of Northeast Brazilian Plants. Lead Molecules from Natural Products. M.T.H. Khan and A.
Ather (eds.). pp. 197-211.
Baglin, I., Mitaine-Offer, A.-C., Nour, M., Tan, K., Cavé, C. and Lacaille-Dubois, M.-A. (2003). A
Review of Natural and Modified Betulinic, Ursolic and Echinocystic Acid Derivatives as Potential
Antitumor and Anti-HIV Agents. Mini Reviews in Medicinal Chemistry 3, pp. 525-539.
Luna, J. de S., dos Santos, A.F., de Lima, M.R.F., de Omena, M.C., de Mendonça, F.A.C., Bieber, L.W.,
Sant’Ana, A.E.G. (2005). A Study of the Larvicidal and Molluscicidal Activities of Some Medicinal
Plants from Northeast Brazil. Journal of Ethnopharmacology 97, pp. 199–206.
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Does nitric oxide activate antioxidant enzymes in
plants exposed to arsenic?
Monteiro de Andrade H.1, de Oliveira J.A.*1, Neto J.L.1, Cambraia J.1,
dos Santos Farnese F.1
1
Federal University of Vicosa, Department of General Biology (Brazil)
[email protected], *[email protected], [email protected], [email protected], [email protected]
Abstract
Nitric oxide (NO) is an important cell signaling agent in responses to various kinds of abiotic stress
conditions. Under oxidative stress plants produce toxic levels of oxygen reactive intermediates (ROIs), causing
damage to cell metabolism. Experimental data is suggestive of an antioxidant action of the NO in plants
subjected to abiotic stresses, such as exposed to toxic levels of arsenic (As). The objective of this research was to
evaluate the effect of NO, exogenously supplied, on the activity of antioxidant enzymes: superoxide dismutase
(SOD), catalase (CAT) and peroxidase (POX) in water hyacinth (Eichhornia crassipes) exposed to toxic levels
of As. Plants grown in nutrient solution were exposed to the following treatments: T0 (control), T1 (20 µM As),
T2 (20 µM As + 100 µM SNP) and T3 (100 µM SNP). The SNP (sodium nitroprusside) was the exogenous
supplement of NO. After 0, 4, 12 and 24 hr root samples were taken and the antioxidant enzymes activities were
determined. The activities of the antioxidant enzymes increased in the plant exposed to toxic levels of As (T1)
and with time. Addition of SNP (T2) to the nutrient solution, however, did not increase the activities of the
enzymes as expected, probably as a result of a reduction in ROIs content, substrate of the antioxidant enzymes.
Probably, NO may be directly used as antioxidant agent reducing the availability of substrates to the enzymes,
and therefore reducing cell damage.
Keywords: Cell signalling, tolerance, toxicity, aquatic plant.
Introduction
Plants are often exposed to conditions of biotic and abiotic stresses and to prevent or minimize cell
damages, occurs structural changes, biochemical and gene expression. Therefore, the plant needs to detect the
condition early unfavorable and activate signaling cascades that lead to appropriate responses. Several of stress
signaling pathways have been described in the literature as pathways mediated by calcium [1] and those mediated
by cGMP [2]. Recently, studies have demonstrated the role of nitric oxide (NO) as a mediator in many signaling
pathways involved in disease resistance, cell death, senescence, in root development, germination, hormonal
responses in growth and flowering. Regarding the response to abiotic stresses, in addition to signaling role, NO has
antioxidant function as found in rice seedlings [3] exposed to many stressors, including arsenic (As).
Arsenic is a metalloid found naturally in the environment, however, due to various anthropogenic
activities such as mining, for example, there has been accumulation and consequent contamination of soil and
surface water and groundwater. The tolerance mechanisms of the plants are still not completely elucidated, but it
is known that systems antioxidant, enzymatic and non-enzymatic, are of great importance.since the main damage
is due to production of intermediates reactive oxygen (ROI's) with great toxic potential. Therefore, the objective
of this study was to evaluate the role of NO as an agent reduction of the toxic effects caused by As.
Material and Methods
Specimens of water hyacinth (Eichhornia crassipes (Mart.) Solms) collected in non-polluted dams at
the Federal University of Viçosa, Viçosa, Minas Gerais State, Brazil, with similar size (about 10.0 g fresh
weight) were transferred to polyethylene pots with 10 L of Clark’s nutrient solution [4], pH 6.5 and maintained
in a growth room with controlled temperature and irradiance (25 ± 2 ºC; 230 µmol m-2 s-1), under a photoperiod
of 16 hours, for an adaptation period of 3 days. After the adaptation period, plants were transferred to 1.0 L
polyethylene pots containing Clark’s nutrient solution, pH 6.5 and exposed to four treatments: T0 (control), T1
(20 µM As), T2 (20 µM As + 100 µM SNP) and T3 (100 µM SNP). The SNP (sodium nitroprusside) was the
exogenous supplement of NO. After 0, 4, 12 and 24 hr root samples were taken and the antioxidant enzymes
activities were determined.
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1.1
Enzyme extraction and assays
To assess enzyme activities, root fresh weight samples were ground in liquid nitrogen and homogenized
in the following buffer media: a) Superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POX, EC 1.11.1.7) and
catalase (CAT, EC 1.11.1.6): 0.1 M potassium phosphate buffer (pH 6.8), 0.1 mM ethylenediamine tetraacetic
acid (EDTA), 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1% (w/v) polyvinylpolypyrrolidone (PVPP)
[5]. In all cases, after being filtrated through four layers of cheesecloth, the homogenates were centrifuged at
12,000 x g for 15 min at 4ºC, and the supernatant was used as the source of crude enzyme. Enzyme activities
were determined by adding 0.1 mL of the crude enzyme extract to: a) POX: 2.9 mL of a reaction medium,
consisting of 0.1 M potassium phosphate buffer (pH 6.8), 20 mM pyrogallol and 20 mM H2O2 ; b) CAT: 2.9 mL
of a reaction medium, consisting of 50 mM potassium phosphate buffer (pH 7.0) and 12.5 mM H2O2. In all
cases, the mixtures were incubated at 30ºC, and the absorbances were measured during the first minute of the
reaction. Enzyme activities were estimated using the following molar extinction coefficients: POX (420 nm; ε:
2.47 mM-1 cm-1); CAT (240 nm, ε: 36 M-1 cm-1). The activity of SOD was determined by adding the root or leaf
crude enzymatic extract to a reaction mixture, consisting of 50 mM potassium phosphate buffer (pH 7.8), 13 mM
methionine, 0.1 mM EDTA, 75 µM nitroblue tetrazolium (NBT) and 2 µM riboflavin. The reaction was carried
out in a chamber with a 15 W fluorescent lamp at 25ºC. After 5 min of illumination, the blue formazan was
measured at 560 nm. All rates were corrected for non-enzymatic activity. One unit of SOD activity was defined
as the amount of enzyme required to cause a 50% inhibition of the rate of NBT reduction. The determination of
the protein concentration was by Lowry method [6].
Results
The activity of superoxide dismutase (SOD) increased in plants exposed to As and was significantly
higher than in the plants treated with As+SNP (Fig. 1A). Similar to the SOD, CAT enzyme activity significantly
increased, mainly after 12 of exposure(Fig. 1B). However, this increase in the activity was not sufficient to
attenuate celular oxidative damage. The addition of SNP induced continuous increase of enzymatic activities
acting as activator of protective mechanism against oxidative damage in the initial exposure.
The plants exposed to As showed continuous increase in the POX activity, with values higher than those
obtained other treatments (Fig. 1C). This increased activity was not sufficient to removing excess H2O2 generated
during As treatment. Moreover, plants exposed to As+SNP there was no significant increase in the POX activity
relative to control treatment indicating that, in this case, the SNP not act as a promoter of this enzyme activity.
Figura 1 – Enzymatic activity of SOD (A), CAT (B) and POX (C) in roots of E. crassipes: Control (
( ) and SNP ( ).
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), As+SNP
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Discussion
Superoxide dismutase (SOD) is a first line enzyme acting in the remotion of superoxide (O2.-)[9]. In this
study, As stress induced an expressive increase in O2.- concentration and, as a consequence, higher SOD activity.
Nevertheless, the increase in the activity of this enzyme was not enough to overcome the amoount of O2.produced during the treatment with As.
Application of sodium nitroprussiate (SNP) on As-treated plants reduced SOD activity, probably due to
a reduction in O2.- concentration. Probably, the nitric oxide (NO) reacted directly with O2.- reducing the amount
of this substrate to the enzyme. Similar results were found in roots of rice exposed to As and SNP [3]. In this
experiment application of SNP also reduced the activity of SOD and other antioxidative enzyme
The NO supplied as SNP induced CAT activity in water hyacinth plants exposed to As, aiding in
mitigating the damage caused by this toxic element. This action was also observed in other stress conditions, as
reported in potato leaves treated with herbicide [11] and Hydrilla verticillata exposed to ammonia [10].
The POX enzymes also acts to removes H2O2 but the affinity for the substrate is different from CAT
enzymes. The POX enzymes are responsible for the fine modulation of H2O2, removing it in smaller amounts,
while CAT acts in high concentrations of this molecule [12].
The balance in the activities of antioxidant enzymes (SOD, CAT and POX) are important to maintain
the redox state of the cell, avoiding the formation of highly toxic hydroxyl radical [13]. The results of this
research suggest that NO acts directly or indirectly in reducing the concentration of ROI’s.
Conclusion
In this study, the exposure of water hyacinth plants to As increased the activities of antioxidant enzymes
superoxide dismutase (SOD), catalase (CAT) e peroxidase (POX), indicating active participation of these
enzymes as part of the strategy to reduce stress-induced As.
Acknowledgments: The authors are thankful to Conselho Nacional de Desenvolvimento Científico
(CNPq) for fellowships and to Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for their
financial support.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Berridge MJ. Bootman, MD. Roderick, HL. (2003). Calcium Signaling: Dynamics, Homeostasis and
Remodelling. Nature Reviews Molecular Cell Biology 4, pp. 517-529.
Meier, S. Madeo, L. Ederli, L. Donaldson, L. Pasqualini, S. Gehring. C. (2009). Deciphering cGMP
Signatures and cGMP-Dependent Pathways in Plant Defence. Plant Signaling and Behavior 4, pp. 307309.
Singh, HP. Kaur, S. Batish, DR. Sharma, VP. Sharma, N. Kohli, RK. (2009). Nitric Oxide Alleviates
Arsenic Toxicity by Reducing Oxidative Damage in the Roots of Oryza sativa (Rice). Nitric Oxide 20,
pp. 289-297.
Clark, RB. (1975). Characterization of Phosphatase of Intact Maize Roots. Journal of Agricultural
and Food Chemistry 23, pp. 458-460.
Peixoto, PHP. Cambraia, J. Sant’ana, R. Mosquim, PR. Moreira, MA. (1999). Aluminum Effects on
Lipid Peroxidation and on Activities of Enzymes of Oxidative Metabolism in Sorghum. Revista Brasileira
de Fisiologia Vegetal 11, pp. 137-143.
Lowry, OH. Rosebrough, NJ. Farr, AL. Randall, RL. (1951). Protein Measurement with the Folinphenol
Reagent. Journal Biology Chemistry 193, pp. 265-275.
Shri, M. Kumar, S. Chakrabarty, D. Trivedi, K. Mallick, S. Misra, P. Shukla, D. Mishra, S. Srivastava, S.
Tripathi, RD. Rakesh, T. (2009). Effect of Arsenic on Growth, Oxidative Stress, and Antioxidant System
in Rice Seedlings. Ecotoxicology and Environmental Safety 72, pp.1102-1110.
Halliwell, B. (2006). Reactive Species and Antioxidants: Redox Biology is a Fundamental Theme of
Aerobic Life. Plant Physiology 141, pp. 312-322.
Alscher, RG. Erturk, N. Heath, LS. (2002). Role of Superoxide Dismutases (SODs) in Controlling
Oxidative Stress in Plants. Journal of Experimental Botany 53, pp. 1331-1341.
Wang, C. Zhang, SH. Li, W. Wang, PF. Li, L. (2011). Nitric Oxide Supplementation Alleviates
Ammonium Toxicity in the Submerged Macrophyte Hydrilla verticillata (L.f.) Royle. Ecotoxicology and
Environmental Safety 74, pp. 67-73.
Beligni MV, Lamattina L. (2002). Nitric oxide interferes with plant photo-oxidative stress by detoxifying
reactive oxygen species. Plant, Cell and Environment 25:737-748
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[12]
[13]
Mittler, R. (2002). Oxidative Stress, Antioxidants and Stress Tolerance. Trends in Plant Science 7, pp.
405-410.
Apel, K. Hirt, H. (2004). Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal
Transduction. Annual Review of Plant Biology 55, pp. 373-99.
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Activation of the ascorbate-glutathione cycle by nitric
oxide: signaling under stress conditions induced by
arsenic
Farnese F.S.1, de Oliveira J.A.2, da Silveira N.M.1, Gusman G.S.1, Siman L.I.2
1
Federal University of Viçosa, Department of Plant Physiology (Brazil)
Federal University of Viçosa, Department of General Biology (Brazil)
[email protected], [email protected], [email protected], [email protected]
2
Abstract
The study evaluated the effect of nitric oxide (NO) in Pistia stratiotes exposed to arsenic (As), with NO
supplied in the form of SNP. Plants were exposed to four treatments: control, SNP (0.1 mg L-1), As (1.5 mg L-1)
and As + SNP (1.5 and 0.1 mg L-1, respectively), for 24 h. In plants exposed only to As, changes was observed in
the concentration of reduced ascorbate. The addition of SNP altered the concentration of reduced ascorbate,
dehydroascorbate and enzymes APX, DHAR and GR, indicating activation of ascorbate-glutathione cycle.
Keywords: oxidative stress, Pistia stratiotes, antioxidants.
Introduction
Arsenic (As) exposure has likely been a longstanding problem in the world. In many Latin American
countries, for example, soil and groundwater are highly enriched with As due to its high density in the region's
abundant volcanic rock and ash. In some countries, mining operations and copper foundries have unearthed As
and enhanced its release into groundwater sources for the past few centuries [1]. A possible solution for this
problem is the use of plants that are able to accumulate As by removing the metalloid from water and soil
through a process called phytoremediation [2]. Aquatic macrophytes are an interesting tool for phytoremediation
and they can effectively remove As from water [3]. In the present study, Pistia stratiotes L. (Araceae) was
selected because of its fast growth, wide distribution and short life span, which are interesting features for
phytoremediation.
Exposure of plants to As promotes the generation of reactive oxygen intermediates (ROIs), triggering
the oxidative stress, which may result in damage to DNA, proteins and lipids [2]. Antioxidant defense system of
plants falls into two general classes: (1) low molecular weight antioxidants, which consist of the lipid soluble
antioxidants (e.g., b-carotene) and the water soluble reductants (e.g., glutathione and ascorbate); and (2)
enzymatic antioxidants [4]. These antioxidant systems are apparently stimulated by signaling molecules, such as
nitric oxide (NO), a small molecule that participates as a signal in several biochemical and physiological
processes in plants [5]. Considering that ascorbate is involved in buffering oxidative stress in plants and that it is
key factor for heavy metal tolerance, understand how the synthesis of this antioxidant occurs in the presence of
As, as well as the involvement of NO in this process, can be crucial for the application of phytoremediation. In
this way, this study evaluated the effect of NO, supplied as sodium nitroprusside (SNP), on the synthesis of
ascorbate and enzymes related to this antioxidant by P. stratiotes exposed to As.
Material and Methods
Specimens of P. stratiotes L. (Araceae) collected in non-polluted dams at the Federal University of
Viçosa, Viçosa, Minas Gerais State, Brazil, were used in all experiments. Plants of similar size (about 10.0 g fresh
weight) were transferred to polyethylene pots with 10 L of Clark’s nutrient solution (Clark, 1975), pH 6.5 and
maintained in a growth room with controlled temperature and irradiance (25 ± 2 ºC; 230 µmol m-2 s-1), under a
photoperiod of 16 hours, for an adaptation period of 3 days. After the adaptation period, plants were transferred to
1.0 L polyethylene pots containing Clark’s nutrient solution, pH 6.5 and exposed to four treatments: control
(nutrient solution only), SNP (0.1 mg L-1), As (1.5 mg L-1) and As + SNP (1.5 and 0.1 mg L-1, respectively). The
plants were maintained under these conditions for 24 h.
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1.1
Concentration of ascorbate and dehydroascorbate
To determine the concentration of ascorbate and dehydroascorbate, 0.4 g of fresh root and leaves were
homogenized in trichloroacetic acid 6% and centrifuged at 15000 xg for 15 min. The supernatant was used as
extract in determining the content of total ascorbate and reduced ascorbate. The concentration of
dehydroascorbate was calculated by the difference between total ascorbate and reduced ascorbate [6].
1.2
Enzymatic antioxidant system
To evaluate the activity of antioxidant enzymes, 0.3 g of fresh root and leaves were homogenized in
extraction medium and centrifuged at 12000 xg for 15 min [7]. The supernatant was used as extract in
determining the activity of ascorbate peroxidase (APX) [8], dehydroascorbate reductase (DHAR) and glutathione
reductase (GR) [7].
Results
1.3
Effect of As and SNP on the concentration of ascorbate and
dehydroascorbate
The exposure to As increased the concentration of total ascorbate in roots. This increase was due to the
higher content of reduced ascorbate (Fig. 1A), while the concentration of dehydroascorbate remained unchanged
(Fig. 1B). The treatment with SNP alone had no effect compared to values obtained in control. When the As was
supplied together with SNP the concentration of reduced ascorbate and desidroascorbato decreased, which
resulted in decreases in the concentration of total ascorbate.
A
B
Figure 1. Concentration of total ascorbate (A) and dehydroascorbate (B) in leaves (
1.4
) and roots ( ) of P. stratiotes.
Effect of arsenic and SNP on the activity of antioxidant enzymes
No changes were observed in any of the three enzymes analyzed in plants exposed only to As (Fig. 2),
and the same was observed in plants exposed to SNP. The addition of SNP in the plants treated with the
pollutant, in turn, was accompanied by an increase in enzymatic activity of APX (Fig. 2A), DHAR (Fig. 2B) and
GR (Fig. 2C) in leaves and roots.
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B
A
C
Figure 2. Activity of APX (A), DHAR (B) and GR (C) in leaves (
) and roots ( ) of P. stratiotes.
Discussion
Ascorbate is an essential compound in plant tissues and act as antioxidants in either enzymatic or nonenzymatic way. It can react directly by reducing superoxide, hydrogen peroxide and hydroxyl radical or
quenching singlet oxygen [9]. Ascorbate also functions as a co-substrate of plant oxidases, such as the APX,
which produces dehydroascorbate. Dehydroascorbate is reduced to ascorbic acid in a reaction dependent of
glutathione catalysed by DHAR, being the GR important in this process. All these reactions, together, constitute
the ascorbate-glutathione cycle, one of the major mechanisms for elimination of ROIs [10]. The results related to
the concentrations of reduced ascorbate, dehydroascorbate and enzymatic activity in the treatment As+SNP
indicate that, probably, NO is involved in the activation of the ascorbate-glutathione cycle. In fact, in this
treatment the observed decrease in the concentration of reduced ascorbate is probably a result of APX enzyme
activity, generating desidroascorbato. The concentration of desidroascorbato, in turn, would be reduced due to
increased activity of DHAR. The increase in the activity of GR is also indicative of the occurrence of the cycle.
Apparently, the ascorbate-glutathione cycle is not a mechanism that is naturally involved in defense
against oxidative stress induced in P. stratiotes, since no changes were observed in dehydroascorbate
concentration in plants exposed to the pollutant. It is likely, therefore, that increasing the concentration of
reduced ascorbate, in the presence of As, relates to the direct elimination of ROIs by this antioxidant or
regeneration of α-tocopherol, which is involved in the elimination of peroxide radicals and singlet oxygen [11].
Thus, it is possible to conclude that NO, supplied in the form of SNP, is capable of alleviating the damage
caused by As. The beneficial effects of NO to plants of P. stratiotes are related with the signaling to increase in
the activities of enzymatic antioxidants and activation of ascorbate-glutathione cycle.
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References
[1]
McClintock, TR. Chen, Y. Bundschuh, J. Oliver, J.T. Navoni, J. Olmos, V. Lepori, EV. Ahsan, H.
Parvez, F. (2012). Arsenic Exposure in Latin America: Biomarkers, Risk Assessments and Related Health
Effects. Science of the Total Environment 429, pp. 76-91.
[2]
Singh, HP. Kaur, S. Batish, DR. Sharma, VP. Sharma, N. Kohli, RK. (2009). Nitric Oxide Alleviates
Arsenic Toxicity by Reducing Oxidative Damage in the Roots of Oryza sativa (Rice). Nitric Oxide 20, pp.
289-297.
[3]
Gonzaga, MIS. Ma, LQ. Pacheco, EP. Santos, WM. (2012). Predicting Arsenic Bioavailability to
Hyperaccumulator Pteris vittata in Arsenic-Contaminated Soils. International Journal of Phytoremediation
14, pp. 939-949.
[4]
Sudhakar, S. D’Souza, SF. (2010). Effect of Variable Sulphur Supply on Arsenic Tolerance and
Antioxidant Responses in Hydrilla verticillata (L.f.) Royle. Ecotoxicology and Environmental Safety 73,
pp. 1314-1322.
[5]
Leitner, M. Vandelle, E. Gaupels, F. Bellin, D. Delledonne, M. (2009). Nitric Oxide Signaling in Plant
Defence. Plant Biology 12, pp. 451-458.
[6]
Kampfenkel, K. Montagu, MV. Inzé, D. (1995). Extraction and Determination of Ascorbate and
Dehydroascorbate from Plant Tissue. Analytical Biochemistry 225, pp. 165-167.
[7]
Anderson, MD. Prasad, TK. Stewart, C.R. (1995). Changes in Isozyme Profiles of Catalase, Peroxidase,
and Glutathione Reductase During Acclimation to Chilling in Mesocotylos of Maize Seedlings. Plant
Physiology 109, pp. 1247-1257.
[8]
Peixoto, PHP. Cambraia, J. Sant’ana, R. Mosquim, PR. Moreira, MA. (1999). Aluminum Effects on Lipid
Peroxidation and Activies of Enzymes of Oxidative Metabolism in Sorghum. Revista Brasileira de
Fisiologia Vegetal 11, pp. 137-143.
[9]
Arasimowicz, M. Floryszak-Wieczorek, J. 2007. Nitric Oxide as a Bioactive Signalling Molecule in Plant
Stress Responses. Plant Science 172, pp. 876-887.
[10] Xiong, J. Fu, G. Tao, L. Zhu, C. 2010. Roles of Nitric Oxide in Alleviating Heavy Metal Toxicity in
Plants. Archives of Biochemistry and Biophysics 497, pp. 13-20.
[11] Leitner, M. Vandelle, E. Gaupels, F. Bellin, D. Delledonne, M. 2009. Nitric Oxide Signaling in Plant
Defence. Current Opinion in Plant Biology 12, pp. 451-458.
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Role of nitric oxide molecule in the tolerance to
arsenic in plants: signal or antioxidant?
Farnese F.S.1, de Oliveira J.A.2, Gusman G.S.1, Leão G.A.1, Canatto R.A.1,
Silva C.J.1
1
Federal University of Viçosa, Department of Plant Physiology (Brazil)
Federal University of Viçosa, Department of General Biology (Brazil)
[email protected], [email protected], [email protected], [email protected],
[email protected]
2
Abstract
Effect of nitric oxide (NO) in mitigating stress induced by arsenic (As) was assessed in Pistia stratiotes,
with NO supplied in the form of SNP. Plants were exposed to four treatments: control, SNP (0.1 mg L-1), As (1.5
mg L-1) and As + SNP (1.5 and 0.1 mg L-1, respectively), for 24 h. The absorption of As triggered various
changes, as the increased production of reactive oxygen intermediates and the damage to cell membranes. These
effects were attenuated by SNP, which participated directly as an antioxidant, eliminating the superoxide anion,
and as signaling, inducing an increase in the concentration of antioxidant enzymatic.
Keywords: oxidative stress, Pistia stratiotes, antioxidants.
Introduction
Environmental contamination with arsenic (As) is one of the most important public health problems in
the world. Although this element occurs naturally in the Earth’s crust, human activities such as mining, the
burning of fossil fuels and pesticide use are the main causes of toxic As accumulation in water and soil [1]. The
As can be absorbed and accumulated by plants, which enables the use of these organisms in the removal of this
pollutant from the environment through a process called phytoremediation [2]. The species with high potential
for phytoremediation is Pistia stratiotes L. (Araceae), a macrophyte that is capable of absorbing large amounts
of toxic metals and that has a high biomass production [3], characteristics that are essential for the use of plants
to extract pollutants [4].
Typical plant responses to As include the increased production of reactive oxygen intermediates (ROIs).
The increase in the production of ROIs may have deleterious effects on plant metabolism [5]. The plants,
however, possess mechanisms to mitigate these effects, using enzymatic antioxidants, such as superoxide
dismutase (SOD), peroxidase (POX), and catalase (CAT). These antioxidant systems are apparently stimulated
by signaling molecules, such as nitric oxide (NO) [6]. The NO, a highly reactive gas molecule, has been the
focus of research investigating the toxic effects of pollutants and the tolerance of plants to these pollutants [7, 6].
This small molecule participates as a signal in several biochemical and physiological processes in plants. The
exogenous supply of NO has already been shown to reduce the stress caused by pollutants, mainly by inducing
an increase in the concentration of antioxidants capable of eliminating ROIs [8]. In this way, this research
evaluated the effect of NO, supplied as sodium nitroprusside (SNP), on the tolerance of P. stratiotes to As.
Material and Methods
Specimens of P. stratiotes L. (Araceae) collected in non-polluted dams at the Federal University of
Viçosa, Viçosa, Minas Gerais State, Brazil, were used in all experiments. Plants of similar size (about 10.0 g
fresh weight) were transferred to polyethylene pots with 10 L of Clark’s nutrient solution [9], pH 6.5, and
maintained in a growth room with controlled temperature and irradiance (25 ± 2 ºC; 230 µmol m-2 s-1), under a
photoperiod of 16 hours, for an adaptation period of 3 days. After the adaptation period, plants were transferred
to 1.0 L polyethylene pots containing Clark’s nutrient solution, pH 6.5 and exposed to four treatments: control
(nutrient solution only), SNP (0.1 mg L-1), As (1.5 mg L-1) and As + SNP (1.5 and 0.1 mg L-1, respectively). The
plants were maintained under these conditions for 24 h.
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1.1
Concentration of reactive oxygen intermediates and cell damages
To determine the concentration of the superoxide anion (O2.-), leaf and roots samples were incubated in
an extraction medium [10] and the reaction was initiated by adding 100 µL of 25.2 mM epinephrine in 0.1 N
HCl. The samples were incubated at 28°C under stirring for 5 min. The absorbance was read at 480 nm for 5
min. Superoxide anion production was assessed by determining the accumulated adenochrome, using the molar
absorption coefficient of 4.0 x 103 M-1 [11]. The hydrogen peroxide (H2O2) concentration was determined using
leaf and roots samples that were homogenized in an extraction medium [12]. Subsequently, 50 µL aliquots of the
supernatant were added to a reaction medium containing 100 µM FeNH4SO4, 25 mM sulfuric acid, 250 µM
xylenol orange and 100 mM sorbitol [13]. The samples were kept in the dark for 30 min, and the absorbance was
determined at 560 nm. The H2O2 concentrations were estimated based on a calibration curve prepared with H2O2
standards.
The integrity of the cell membranes was assessed by analysis of electrolyte leakage [14]. The
conductivity was measured with a conductivity meter (DM31, Digimed, Santo Amaro, Brazil) and expressed as a
percentage of total conductivity.
1.2
Enzymatic antioxidant system
To evaluate the activity of antioxidant enzymes, 0.3 g of fresh root and leaves were homogenized in
extraction medium and centrifuged at 12000 xg for 15 min [15]. The supernatant was used as extract in
determining the activity of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT).
Results
1.3
Effect of As and SNP on the concentration of reactive oxygen
intermediates and cell damages
The exposure to As increased the concentration of O2.- in leaves and roots, while treatment with SNP
alone had no effect on the concentration of the anion compared to values obtained in control (Fig. 1A). The
concentration of H2O2 also enhanced in leaves and roots of plants after exposure to As, being the increase more
significant in roots (Fig. 1B). The application of SNP to plants treated to As, in turn, attenuated this effect.
A
B
Figure 1. Concentration of superoxide anion (A) and hydrogen peroxide (B) in leaves ( ) and roots ( ) of P. stratiotes.
The treatment of plants with As increased the electrical conductivity, which is indicative of damage to
cell membranes (Fig. 2). There was no difference in damage generated between roots and leaves, despite the
higher As accumulation in roots. The SNP alone had no effect on electrolyte leakage, but when added to plants
treated with As, attenuated the damage triggered by this metalloid.
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Figure 2. Electrolyte leakage in leaves ( ) and roots ( ) of P. stratiotes.
1.4
Effect of arsenic and SNP on the activity of antioxidant enzymes
The activities of SOD, CAT and POX increased significantly in leaves of plants treated with As (Fig.
3). In the roots, however, only SOD activity increased with exposure to As (Fig. 3A). The application of SNP
alone had no effect on the enzymes activity. The addition of SNP in the plants treated with As avoided the
increase in SOD activity induced by As (Fig. 3A). In the case of POX and CAT the addition of SNP resulted in a
further increase in enzyme activity compared to that observed in plants subjected only to As (Fig. 3B and C).
B
A
C
Figure 3. Enzymatic activity of SOD (A), POX (B) and CAT (C) in leaves ( ) and roots ( ) of P. stratiotes.
Discussion
In the specimens of P. stratiotes exposed to As occurred oxidative stress, as evidenced by the increase
in the concentration of ROIs and by loss of membrane integrity. This toxic effect of As, however, was reduced
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by addition of NO, supplied as SNP, which participated both as a direct antioxidant, eliminating O2.-, and as a
signaling molecule, increasing the response of the antioxidant mechanisms.
In some types of abiotic stress SNP acts as a signal to increase the activity of SOD and thus decrease
the concentration of O2.- [16]. In P. stratiotes, however, SOD activity did not change in the presence of SNP and
it is likely that this molecule has acted directly as an antioxidant [17, 18]. In the case of enzymes CAT, POX and
APX the NO release from SNP was active as a signaling agent in the increased activity, which resulted in
significant reduction in the concentration of H2O2.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Kumar, BM. Suzuki, KT. (2002). Arsenic Around the World: A Review. Talanta 58, pp. 201-235.
Tu, S. Ma, LQ. (2003). Interactive Effects of pH, Arsenic and Phosphorus on Uptake of As and P and
Growth of the Arsenic Hyperaccumulator Pteris vittata L. under Hydroponic Conditions. Environmental
and Experimental Botany 50, pp. 243-251.
Prasad, MNV. Greger, M. Smith, B. (2001). Aquatic Macrophytes. In: Prasad MNV, ed. Metals in the
Environment: Analysis by biodiversity. New York: Marcel Dekker. pp. 259-288.
Melo, EEC. Nascimento, CWA. Santos, ACQ. (2006). Solubility, Phytoextraction and Fractionation of
Heavy Metals as a Function of Chelating Agents Applied to Soil. Rev Brasileira de Ciencia do Solo 30, pp.
1051-1060.
Rao, KP. Vani, G. Kumar, K. Wankhede, DP. Misra, M. Gupta, M. Sinha, AK. (2011). Arsenic Stress
Activates MAP Kinase in Rice Roots and Leaves. Archives of Biochemistry and Biophysics 506, pp. 7382.
Leitner, M. Vandelle, E. Gaupels, F. Bellin, D. Delledonne, M. (2009). Nitric Oxide Signaling in Plant
Defence. Plant Biology. 12, pp. 451-458.
Wojtazek, P. (2000). Nitric Oxide in Plants: To NO or not to NO. Phytochemistry 54: 1-4.
Hayat, S. Hasan, SA. Mori, M. Fariduddin, Q. Ahmad, A, (2010). Nitric Oxide: Chemistry, Biosynthesis,
and Physiological Role. In: Hayat S, Mori M, Pichtel J, Ahmad A, eds.
Clark, RB. (1975). Characterization of Phosphatase of Intact Maize Roots. Journal of Agricultural
and Food Chemistry 23, pp. 458-460.
Mohammadi, M. Karr, AL. (2001). Superoxide Anion Generation in Effective and Ineffective Soybean
Root Nodules. Journal of Plant Physiology 158, pp. 1023-1029.
Boveris, A. Alvarez, S. Bustamante, J. Valdez, L. (20002. Measurement of Superoxide Radical and
Hydrogen Peroxide Production in Isolated Cells and Subcellular Organelles. Methods Enzymology 105,
280-287.
Kuo, MC. Kao, CH. (2003). Aluminium Effects on Lipid Peroxidation and Antioxidative Enzyme
Activities in Rice Leaves. Biologia Plantarum 46, 149-152.
Gay, C. Gebicki, JM. (2000). A Critical Evalution of the Effect of Sorbitol on the Ferric-xylenol Orange
Hydroperoxide Assay. Analitical Biochemistry 284, 217-220.
Lima, ALS. DaMatta, FM. Pinheiro, HA. Totola, MR. Loureiro, ME. (2002). Photochemical Responses
and Oxidative Stress in Two Clones of Coffea canephora under Water Deficit Conditions. Environmental
Experimental Botany 47, pp. 239-247.
Peixoto, PHP. Cambraia, J. Sant’ana, R. Mosquim, PR. Moreira, MA. (1999). Aluminum Effects on Lipid
Peroxidation and on Activies of Enzymes of Oxidative Metabolism in Sorghum. Revista Brasileira de
Fisiologia Vegetal 11, pp. 137-143.
Arasimowicz, M. Floryszak-Wieczorek, J. (2007). Nitric Oxide as a Bioactive Signalling Molecule in
Plant Stress Responses. Plant Science. 172, pp. 876-887.
Singh, HP. Kaur, S. Batish, DR. Sharma. VP. Sharma, N. (2009). Nitric Oxide Alleviates Arsenic
Toxicity by Reducing Oxidative Damage in the Roots of Oryza sativa (Rice). Nitric Oxide 20, pp. 289-297.
Xiong, J. Fu, G. Tao, L. Zhu, C. (2010). Roles of Nitric Oxide in Alleviating Heavy Metal Toxicity in
Plants. Archives Biochemistry Biophysics 497, pp. 13-20.
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Evaluation of Joannesia princeps aqueous extract
anthelmintic activity in naturally infected mice by
Syphacia obvelata, Aspiculuris tetraptera and
Vampirolepis nana
Borba H.R.1, Gomes J.V.2, dos Santos Teixeira J.T.2, da Silva de Mello M.3,
Fampa P.1, Gonçalves L.1, de Assis da Silva F.4, Moreira de Lima V.1
1-Departamento de Biologia Animal – Instituto de Biologia – Universidade Federal Rural do Rio de Janeiro Brasil.
2- Estudante de Graduação em Ciências Biológicas – Instituto de Biologia – Universidade Federal Rural do Rio
de Janeiro – Brasil.
3- Bolsista do Projeto Jovens Talentos – FAPERJ- Brasil.
4-Departamento de Química – Instituto de Ciências Exatas – Universidade Federal Rural do Rio de Janeiro –
Brasil.
[email protected], [email protected] , [email protected] , [email protected], [email protected] ,
[email protected], [email protected]
Abstract
Joannesia princeps Vell. (Fam. Euphorbiaceae), known as ‘’cotieira’’ or andá-açú’’ presents medicinal
value like purgative and is indicated for syphilis manifestations. The root peel tea is used as laxative and the seed
tea shows strong anthelmintic activity. In the present work, our goal was to evaluate the therapeutic potential of
J. princeps aqueous extracts as anthelmintic in naturally infected mice by V. nana, A. tetraptera and S. obvelata.
J. princeps leaves were collected at Cabo Frio-RJ and transported to the lab in UFRRJ. Dry leaves were
submitted to infusion followed by filtration. Final concentration of obtained products was expressed in g/100 ml.
J. princeps extracts were applied in young male and female mice weighting around 25g. Applications were
performed by intragastric administration in the final volume of 0,04mL/g during three consecutive days.
Eliminated feces 24h after each application were collected and observed at the stereoscopic microscope. V. nata
proglottids were collected and had their wet weight obtained. Eliminated nematodes were identified and counted.
After 96h, mice were sacrificed and necropsied, evaluating V. nana segments wet weight and the number of
reminiscent nematodes. Identical proceeding was adopted to control (C) group and with pirantel pamoat/
praziquantel reference treatment (protocols approved by CEPEB). Anthelmintic evaluation of the extract was
expressed in percentage of elimination, and the results showed that J. princeps leaves aqueous extracts (10%) did
not reproduce previous reported pattern described for seed extracts. It was not observed significant difference in
percentage of elimination to any of tested helminthes. J. princeps leaves extract as anthelmintic potential
therapeutic use analysis still requires pharmacological and clinical assays for its validation.
Keywords: Joannesia princeps, extract, anthelmintic, medicinal plant
Introduction
Medicinal plants utilization is based on family tradition and became a widespread practice in the
popular medicine, especially in developing countries. Nowadays, many factors contribute for the increasing use
of this resource, among that, the high cost of private medicine, the precariousness of public health services, and
the high costs of industrialized medicaments, as well as the tendency, in present day, to use products of natural
origin ([1], [2]). The Brazilian flora presents a huge potential as supplier of medicinal plants used in the
treatment of endemic diseases of Brazil as well as other tropical countries. Joannesia princeps is an important
example of Brazilin flora specimens. It is a native plant known as ‘’cotieira’’ or ‘’andá-açú’’ and can be found in
Brazil North, Northeast and Southeast geographical regions, mainly in the Atlantic Forest biome. The oil of the
peel is used as laxative and in the popular medicine is indicated for purgative, for disturbs in the menstrual
period, pernicious fever, antimicrobial, syphilis, scrofulosis, and swelling [3]. J. princeps seed extracts exhibited
anthelmintic activity ([3], [4]). Endoparasites represent a huge issue of public health in Brazil, affecting mainly
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the low income population who lives in precarious conditions of sanitation and hygiene [5]. Considering, the
current tedency of using phytotherapies and the fact of many medicaments come from medicinal plants [6], it is
important that the plants with such properties to be tested regarding the bioactivity of the extracts obtained from
the different organs, and therefore better comprehend its therapeutic properties. This work had the goal of
evaluating anthelmintic potential of aqueous extract from J. princeps leaves using naturally infected mice by
Syphacia obvelata, Aspiculuris tetraptera e Vampirolepis nana as experimental model.
Material and Methods
2.1 Extracts collecting, identification and preparation
Young and adult leaves of J. princeps were collected at Região dos Lagos, Jardim Peró, Cabo Frio-RJ
and immediately transported to the Laboratory of Anthelmintic Activity of Plants – LAAP/UFRRJ,
where they were spread over the bench, previously wrapped in absorbent toweling at room temperature and
protected from sunlight. Then the leaves were crushed and stored in amber bottle until the moment of extracts
preparing. The identification of the botanic material was done by the botanical Prof. Pedro Germano Filho,
Department of Botanic/Inst°. of Biology-UFRRJ. The voucher specimen was stored in the Herbaruim RBR at the
registration number 34.630. The leaves conserved in the dry form were administered to the animals in crude
aqueous extract form at 10% p/v, prepared by hot infusion and after filtered in flannel tissue.
2.2
Anthelmintic evaluation tests
Albino mice, male and female obtained from Fundação Oswaldo Cruz – FIOCRUZ, weighing around
25g and naturally infected by V. nana, A. tetraptera and S. obvelata, were utilized at anthelmintic evaluation
tests. The animals were maintained in individual cages of polypropylene (30 x 20 x 13 cm), with a stiff wire
mesh frame in the floor (mesh 7 x 7 mm) on absorbent toweling, aiming to facilitate the daily collecting of
feces([7], [8]). The extracts were applied orally (intragastric), in the volume of 0,04 mL/g, with a flexible thin
probe during three consecutive days. The feces collected 24 hours after the applications, for a total of four fecal
collects, were softened, transferred into a tamis mesh of 125 micrometers and examined at stereoscopic
microscope, with the objective of counting and identifying the eliminated nematodes and collecting the segments
of V. nana, from the second to the fifth day of experiment. On the fifth and last day of tests, mice were sacrificed
by inhalation ethyl ether vapors, examining the content of caecum, colon and small intestine, to evaluate the
number of S. obvelata, A. tetraptera and collect the V. nana proglottid remains [9]. In these tests the aqueous
extract of J. princeps dry leaves, 10% infusions, were used. Additional groups of mice were used as controls,
receiving 70mg/kg/day doses of pyrantel pamoate/ Praziquantel (Strondal®) and submitted to identical procedure
of anthelmintic evaluation as described above. Another control group, without any treatment was used to
estimate the spontaneous elimination of the studied helminths. For calculate the anthelmintic activity a critical
test was used[10]. The anti-cestoid effect was expressed as mean percentage terms of proglottids elimination, by
calculating the humid weight of the eliminated segments on the feces after treatment regarding the total segments
mass. The anti-nematode effect was also expressed as mean percentage of eliminated nematodes, considering the
total number of eliminated nematodes. The procedures were approved by the Comissão de Ética na PesquisaCOMEP/UFRRJ, protocolo no 193/2012.
2.3 Statistic analysis
Obtained results in the anthelmintic tests were submitted to a statistic treatment, applying the Student
‘’t’’ test and being the adopted significance level of p<0,05[11].
Results and Discussion
Table 1 show the percentage data of S. obvelata elimination in mice submitted to administration of J.
princeps extracts. We observed that the aqueous extract of the leaves at 10% was not capable to produce
significant removal of the oxyuridae (p>0,05), in relation to spontaneous elimination percentage registered for
control group. The anthelmintic test using the same concentration, with the purpose of verifying the influence of
the same infusion of ‘’cotieira’’ at the elimination of A. tetraptera and V. nana was negative (p>0,05), as we
observe in table 2 and 3. The data also demonstrate that the prevalence of S. obvelata, A. tetraptera and V.
nanam in tested groups of mice was 100%. This high percentage of naturally infected animals allowed obtaining
significant results of anthelmintic activity of the studied plant.
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TABLE 1: Anthelmintic activity of aqueous extract obtained from J. princeps and of routine chemotherapy in
elimination of S. obvelata (NEMATODA: OXYURIDAE), in naturally infected mice.
Used organ
Leaf
pyrantel
pamoate/
Praziquantel
Administration form
Fecal exam
Necropsy
% of
Elimination
Infusion 10%
14/14a
204
319
39,0
Suspension
10/10a
233
99
70,2
14/14a
335
417
44,5
Control
a
Number of Helmints
Number of
animals
Number of alive animals, regarding the total number of animals per treatment.
TABLE 2: Anthelmintic activity of aqueous extract obtained from J. princeps and of routine chemotherapy in
elimination of A. tetraptera (NEMATODA: HETEROXYNEMATIDAE), in naturally infected mice.
Administration
form
Number of
animals
Leaf
Infusion 10%
pyrantel
pamoate/
Praziquante
Suspension
Used part
Control
a
Number of Helmints
% of
Elimination
Fecal exam
Necropsy
14/14a
09
1003
1,0
09/09a
159
81
66,2
12/12a
0
962
0
Number of alive animals, regarding the total number of animals per treatment.
TABLE 3: Anthelmintic activity of aqueous extract obtained from J. princeps and of routine chemotherapy in
elimination of V. nana (EUCESTODA:HYMENOLEPIDIDAE), in naturally infected mice.
Used part
Leaf
pyrantel
pamoate/
Praziquante
Administration
form
Number of
animals
Infusion 10%
Suspension
Control
a
Weight(mg) of Helmints
Fecal exam
Necropsy
14/14a
25,4
195,9
11,5
10/10a
88,9
0,0
100,0
14/14a
11,0
104,1
9,6
Number of alive animals, regarding the total number of animals per treatment.
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mination Percentage of
S. obvelata
Anthelmintic activities of the aqueous extract from the J. princeps leaves were represented considering
the accumulated percentage of S. obvelata, A. tetraptera elimination and of proglottids from V. nana during 96
hours, compared to the activity exercised by the routine chemotherapy (Pictures 1, 2 e 3 respectively). Antinematode and anti -cestoid routine chemotherapy activity occurred in the first 24 hours of the test, while the
elimination percentages produced by ‘’cotieira’’ infusion, expressed graphically as mean percentages, reveal a
gradual effect along the test, but with values near or lower than the obtained in the control group in the
conditions of the experiment. The aqueous extract of J. princeps leaves did not reproduced the results found with
seed extracts, that exhibited strong anthelmintic activity [3]. The anthelmintic effect found in the ‘’cotieira’’ was
attributed to isolated alkaloids of different parts of the plant [12].
80
70
60
50
40
30
20
10
0
mination Percentage of
A. tetraptera
Fig. 1: Effect of anthelmintic activity of aqueous extract from J. princeps in naturally infected mice by S. obvelata.
70
60
50
40
30
20
10
0
imination Percentage of
V. nana
Fig. 2: Effect of anthelmintic activity of aqueous extract from J. princeps in naturally infected mice by A. tetraptera.
120
100
80
60
40
20
0
Fig. 3: Effect of anthelmintic activity of aqueous extract from J. princeps in naturally infected mice by V. nana.
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Conclusion
In the experiments performed, despite of the high and repeated doses to which animals were submitted,
no toxic effects were observed. The results obtained with aqueous extract of J. princeps leaves does not exclude
the possibility that others extracts or other vegetable parts will reveal an anthelmintic effect.
Reference
[1]
Brasileiro, B.G.; Pizziolo, V.R.; Matos, D.S.; Gemano, A.M.; Jamal, C.M.(2008). Plantas
Medicinais Utilizadas pela População Atendida no “Programa de Saúde da Família”, Governador
Valadares, MG, Brasil. Revista Brasileira de Ciências Farmacêuticas, 44(4), pp. 629-635.
[2] França, I. S. X.; De Souza, J. A.; Baptista, R. S.; Britto, V. R. S.(2008). Medicina Popular: Benefícios e
Malefícios de Plantas Medicinais. Revista Brasileira de Enfermagem, 61(2), pp. 201-208.
[3] Souza, O. V., Fioravante, I. A.; Yamamoto, C. H.; Alves, M. S.; Araújo, A. L. A.(2007). Propriedades
Biológicas das Sementes de Joannesia princeps Vellozo. HURevista,33 (1), pp. 23-27.
[4] Souza, O. V.; Fioravante, I. A.; Del-Vechio-Vieira, G.; Caneschi, C. A.(2009). Investigação das Atividades
Antinociceptiva e Antiedematogênica do Extrato Etanólico das Folhas de Joannesia princeps Vellozo.
Revista de Ciências Farmacêuticas Básica e Aplicada, 30 (1), pp. 91-97.
[5] Andrade, E. C.; Leite, I. C. G.; Rodrigues, V. O.; Cesca, M. G.(2010). Parasitoses Intestinais: Uma Revisão
sobre seus Aspectos Sociais, Epidemiológicos, Clínicos e Terapêuticos. Rev. APS, 13(2), pp. 231-240.
[6] WHO.Traditional
Medicine.
Fact
sheet
nº134,
December
2008
acesso
internet
<http://www.who.int/mediacentre/factsheets/fs134/en/print.html> acesso em 15/06/2012.
[7] Amorim, A.; Borba, H.R.; Silva, W.J.(1987). Ação Anti-Helmíntica de Plantas. Revista Brasileira de
Farmácia, 68, pp. 64-70.
[8] Amorim, A.; Borba, H.R.(1990). Ação Anti-Helmíntica III. Efeito de Extratos Aquosos de Punica
granatum L. (romã) na Eliminação de Vampirolepis nana e de Oxiurídeos em Camundongos. Revista
Brasileira de Farmácia, 71(4), pp. 85-87.
[9] Amorim, A.; Borba, H.R.; Carauta, J.P.P.; Lopes, D.; Kaplan, M.A.C.(1999). Anthelmintic Activity of
Látex of Fícus Species. Journal of Ethnopharmacology, 64(3), pp. 255-258.
[10] Steward, J.S.(1955). Anthelmintic Studies: A Controlled Critical Entero-Nemacidal Test. Parasitology, 45,
pp. 231-241.
[11] Snedecor, G.W. (1946). Statistical Methods. The Collegiate Press, Inc., Ames, Iowa, USA, 486p.
[12] Achenbach, H.; Benirschke, G.(1997). Joannesialactone and other compounds from Joannesia princeps.
Phytochemistry, 45, pp. 149-157.
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il)
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The Hydrogenosome: an unconventional organelle
Benchimol M.
Laboratório de Ultraestrutura Celular, Universidade Santa Úrsula, Rio de Janeiro, Brazil
Summary
Some organisms lack conventional mitochondria and instead contain divergent mitochondrial-related
organelles, called hydrogenosomes, which are double-membrane bound organelles and produce molecular
hydrogen. The hydrogenosome is found in non-mitochondrial organisms such as some Parabasalia and ciliated
protists and chytrid fungi which live in anaerobic or microaerophilic environments. Like mitochondria the
hydrogenosome is surrounded by two closely apposed membranes and presents a granular matrix, produces
ATP, participates in the metabolism of pyruvate formed during glycolysis, incorporates calcium, imports
proteins post-translationally and divides in the same way. However, this organelle differentiates from
mitochondria due of absence of genetic material, at least in trichomonas, lack a respiratory chain and
cytochromes, absence of the F0- F1 ATPase, absence of the tricarboxylic acid cycle, and absence of cristae.
Introduction
The hydrogenosome (Figs. 1-5) is a crucial organelle for some organisms that inhabit oxygen-poor
environments protists such as some free-living ciliates, unicellular parasites, termites flagellates and rumen fungi
and ciliates. These organisms lack conventional mitochondria. Hydrogenosomes are polyphylogenetic and have
arisen independently in several eukaryotic lineages (Embley and Hirt, 1998).
The most-studied hydrogenosomes are those that infect cattle, Tritrichomonas foetus (Figs. 1-2) and the
pathogen of the urogenital tract of humans, Trichomonas vaginalis (Figs. 3), which the complete genome
sequence is available (Carlton et al., 2007). These organisms have been subject to extensive investigation due to
the presence of hydrogenosomes (Figs. 1-5) an organelle that has a special mode of respiration, because it
produces molecular hydrogen and ATP by oxidizing pyruvate or malate under anaerobic conditions (Müller,
1993) (Fig. 3). It has been proposed that in the course of the mitochondria-to hydrogenosome transition, aspects
of typical mitochondrial energy metabolism were lost, including the citric acid cycle and the respiratory chain.
Similarities with Mitochondria
Hydrogenosomes are considered as descend from the same eubacterial endosymbiont as mitochondria,
but they differ from the mitochondria in several aspects. Like mitochondria, both organelles: (a) are enveloped
by two membranes (Figs. 1-2) (Benchimol and De Souza, 1983), (b) divide autonomously by fission (Fig. 4)
(Benchimol et al., 1996b), (c) import proteins post-translationally (Johnson et al., 1993), (d) produce ATP (Fig.
8) (LIndmark and Müller, 1973), (e) present a beta-succinyl-coenzyme A synthetase, a soluble hydrogenosomal
protein with an amino-terminal sequence that resembles mitochondrial presequences (Lahti et al., 1992), (f)
present a membrane targeting pathway, a member of the mitochondrial carrier family (Dyall et al., 2000), (g)
they may divide at any phase of the cell cycle (Benchimol and Engelke, 2003), (h) both present a mitochondrialtype 70 kDa heat shock protein and Cpn60 (Bui et al., 1996); (i) they contain the NADH dehydrogenase module
of mitochondrial complex I (Hrdý et al., 2004); (j) hydrogenosomal proteins are synthesized on free
polyribosomes (Lahti and Johnson, 1991); (k) they incorporate calcium (Fig. 2) (Benchimol et al., 1982), (l)
they are able to utilize oxygen as a terminal electron acceptor (Cerkasov et al., 1978), (m) present a relationship
with the endoplasmic reticulum, (n) both present Frataxin, a conserved mitochondrial protein (Dolezal et al.,
2007). The presence of cardiolipin is still controversial (Andrade et al, 2006; Guschina et al, 2009).
Differences with Mitochondria
The hydrogenosome differs from mitochondria because: (a) it produces molecular hydrogen (b) it has
hydrogenase, (c) lacks cytochromes and members of complexes I–IV, with the exception of NADH
dehydrogenase 51 kDa (Ndh51) and 24 kDa (Ndh24) subunits (Dyall et al., 2004; Hrdý et al., 2004), (d) it does
not present the tricarboxylic acid cycle, (e) lacks the F0- F1 ATPase (f) lacks oxidative phosphorylation (Hrdý et
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al., 2008), (g) the lack of sensitivity to metabolic inhibitors such as rotenone and cyanide (Cerkasov et al., 1978)
(h) lacks a respiratory chain, (i) it lacks a genome (Clemens and Johnson, 2000), with the possible exception of
hydrogenosomes from Nyctotherus ovalis (Akhmanova et al, 1998).
Fig. 1. Thin section of Tritrichomonas foetus as seen in transmission electron microscopy showing several hydrogenosomes
(H). P, pelta; G, Golgi complex; ER, endoplasmic reticulum; N, nucleus; V, vacuoles; Gl, glycogen granules; C, costa, B,
basal bodies. Bar, 500 nm.
Fig.2. Routine preparation of hydrogenosomes (H) from Tritrichomonas foetus. Note that the hydrogenosome is
spherical, enveloped by a double membrane and presents a peripheral vesicle (asterisk) which is a calcium storage. Bars, 60
nm.
Materials and methods
Transmission Electron Microscopy
Cells were fixed overnight at room temperature in 2.5% glutaraldehyde in 0.1 M cacodylate buffer.
Afterwards, the cells were washed in PBS and post-fixed for 15 min in 1% OsO4 in 0.1 M cacodylate buffer
containing 5 mM CaCl2 and 0.8% potassium ferricyanide. The cells were dehydrated in ethanol and embedded in
Epon. Ultra-thin sections were harvested on 300 mesh copper grids, stained with 5% uranyl acetate and 1% lead
citrate and then observed with a JEOL 1210 transmission electron microscope.
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Fig. 3. Metabolic pyruvate metabolism of Trichomonas hydrogenosomes. The process starts in the cytosol, where
glycolysis takes place. The glycolysis originates intermediate products, such as pyruvate and malate which enters in the
hydrogenosome. Pyruvate is oxidatively decarboxylated and the electrons are transferred primarily to protons with H2
formation. Oxidative pyruvate decarboxylation in T. vaginalis, differently from the other eukaryotic cells, is catalyzed by a
different enzyme, pyruvate:ferredoxin oxidoreductase, an enzyme found in several bacteria and in a number of
microorganisms.
Fig. 4. Dividing hydrogenosomes. Views of the process of dividing hydrogenosomes which are similar to mitochondria. (a)
Segmentation. The organelle in process of segmentation where it is elongated showing a constriction in the central region.
(b) Partition. The hydrogenosome becomes larger and an invagination of the inner hydrogenosomal membrane is observed,
gradually dividing the hydrogenosomal matrix in two compartments. (c) Heart-form. The hydrogenosme take a heart-shape
and divides in two new organelles. Bars (a) 80 nm, (b) 50 nm, (c) 100 nm.
Results
The Hydrogenosome Morphology
In trichomonads (Figs. 1-5) (Benchimol et al., 1996a) hydrogenosomes are spherical or slightly
elongated organelles (Figs. 1-4) and possess a peripheral vesicle (Figs. 1-2) that is a distinct compartment within
the organelle (Díaz and De Souza 1997).
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Fig. 5. Abnormal hydrogenosomes. The hydrogenosomes present very abnormal shapes when they are submitted to stress
conditions, such as incubation with drugs as metronidazole, colchicine or cytochalasins. Their sizes which can reach 2 µm.
Note that the organelles are not spherical as in the routine preparations and can present internal membranes and abnormally
enlarged peripheral vesicles. Note in Fig. d a dense nucleoid which is formed when the hydrogenosome is out of order. Bars,
Fig. a, 400 nm; Fig. b, 300 nm; c, 200 nm; d, 100; e, 300; f, 80 nm.
Hydrogenosome Size
Hydrogenosome sizes vary according to the species or if the cell is submitted to stress conditions (Fig.
5) (Benchimol, 1999, 2001, Madeiro and Benchimol, 2004). In routine and normal conditions, hydrogenosomes
of trichomonads (Figs. 1-2) present an average diameter of 300 nm, but they may reach 2 µm when under stress
conditions (Fig. 5), such as drug treatment. Hydrogenosomes are enveloped by two very thin and very closely
apposed unit membranes in all species studied (Fig. 2). As a general rule no space is observed between the two
membranes. Each membrane has a thickness of 6 nm and may present a certain undulation (Benchimol and De
Souza, 1983).
The Peripheral Vesicle
The hydrogenosome peripheral vesicle is completely surrounded by two closely apposed unit
membranes and this compartment contains calcium (Fig. 2) (Benchimol and De Souza, 1983; Benchimol et al.,
1996a).
The Matrix of the Hydrogenosome
The hydrogenosome matrix is homogeneous (Figs. 1-2), with a granular appearance and differing from
the cytoplasmic matrix and occasional calcium deposits can been found (Benchimol et al., 1982).
Hydrogenosomes Biogenesis
Hydrogenosomes, as almost all other organelles, grow by proliferation of preexisting organelles (Fig.
4). Hydrogenosomes, like mitochondria, may divide by three distinct processes: (1) segmentation (Fig. 4a), (2)
partition (Fig. 4b), and (3) heart-form (Fig. 4c). In the segmentation process (Fig. 4a) the hydrogenosome grows,
becoming elongated with the appearance of a constriction in the central portion (Benchimol et al. 1996b).
Microfibrillar ou membranous structures (Fig. 4a) appear to help the furrowing process, ending with a total
fission of the organelle. In the partition process Fig. 4b), rounded hydrogenosomes, in a bulky form, are further
separated by a membranous internal transversal septum separating the organelle matrix into two compartments
(Benchimol et al. 1996b). A necklace of intramembranous particles delimiting the outer hydrogenosomal
membrane in the region of organelle division was observed by freeze-etching (Benchimol and Engelke, 2003).
Figure 4c shows the heart form which also occurs in mitochondria.
Hydrogenosome Metabolism
The trichomonad metabolism starts in the cytosol, where glycolysis takes place; various intermediates
of the glycolysis give rise to intermediate products, such as the pyruvate, which corresponds to the classical
glycolysis pathway observed in other eukaryotic cells. Malate can also be produced and like pyruvate, enters into
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the hydrogenosome (Fig. x). Within the hydrogenosome, pyruvate is oxidatively decarboxylated to acetyl
coenzyme A and CO2. During pyruvate decarboxylation electrons are transferred primarily to protons with H2
formation and in this reaction CO2 is liberated and the acyl moiety is transferred to coenzyme A (CoA) to form
acetyl–CoA;this latter product is converted to acetate and the energy of the thioesther bond is conserved in two
successive steps, resulting in substrate level phosphorylation. Oxidative pyruvate decarboxylation in T.
vaginalis, differently from the other eukaryotic cells, is catalyzed by pyruvate:ferredoxin oxidoreductase, an
enzyme found in several bacteria and in a number of microorganisms. Electrons released from pyruvate are
transferred to ferredoxin, a low molecular weight electron carrier protein. The protons are terminal electrons
acceptors through the action of a hydrogenase and thus producing molecular hydrogen, and this process is
coupled to ATP synthesis (Muller, 1990).
Hydrogenosomes Under Drug Treatments
When trichomonads were treated with drugs, such as metronidazole, colchicines, cytochalasins, among
others, important alterations are observed in hydrogenosome morphology: giant organelles, invaginations of the
hydrogenosome membrane delimitating inner compartments, abnormal sizes and shapes and distinct electron
density in the hydrogenosomal matrix (Fig. 5).
Effect of zinc on hydrogenosomes
The hydrogenosome constitutes the main site of the initial effect of zinc, where the hydrogenosomal
vesicle increases in electron density and size. Electron spectroscopy imaging and the electron energy loss
spectrum showed the presence of zinc, calcium and oxygen in the electron-dense areas of the hydrogenosome
(Benchimol et al., 1993). Zinc has been described in prostatic fluid and it has been demonstrated that the normal
concentration found in men is lethal for most isolates of T. vaginalis.
Conclusions
The hydrogenosome is a very an unusual organelle because produces ATP and molecular hydrogen. It
is found in Parabasalia protozoa, certain chytrid fungi and certain ciliates. Hydrogenosomes are not present in
multicellular animals or plants or in other anaerobic protists, such as amoebas and giardias. The most extensive
studies of this organelle have been carried out in the trichomonad species such as Trichomonas vaginalis and
Tritrichomonas foetus. This organelle is a good target for drugs treatment since it presents differences with
mitochondria and thus, it is not present in higher eukayotic cells.
Acknowledgements: This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico), PRONEX (Programa de Núcleo de Excelência), FAPERJ (Fundação Carlos Chagas Filho de
Amparo à Pesquisa do Estado do Rio de Janeiro), and AUSU (Associação Universitária Santa Úrsula).
References
AKHMANOVA A, VONCKEN F, VAN ALEN T, VAN HOEK A, BOXMA B, VOGELS G, VEENHUIS M,
HACKSTEIN JH A hydrogenosome with a genome. Nature 396: 527-528, 1998.
ANDRADE RI, EINICKER-LAMAS M, BERNARDO RR, PREVIATTO LM, MOHANA-BORGES R,
MORGADO-DIAZ J, BENCHIMOL M Cardiolipin in hydrogenosomes: evidence of symbiotic origin.
Eukaryot Cell 5: 784-787, 2006.
BENCHIMOL M Hydrogenosome autophagy in Tritrichomonas foetus: an ultrastructural and cytochemical
study. Biol Cell 91: 165-174, 1999.
BENCHIMOL M Hydrogenosome morphological variation induced by fibronectin and other drugs in
Tritrichomonas foetus and Trichomonas vaginalis. Parasitol Res 87: 215-222, 2001.
BENCHIMOL M, ALMEIDA JCA, DE SOUZA W Further studies on the organization of the hydrogenosome in
Tritrichomonas foetus. Tiss Cell 28: 287-299, 1996a.
BENCHIMOL M, ALMEIDA JC, LINS U, GONÇALVES NR, DE SOUZA W Electron microscopic study of
the effect of zinc on Tritrichomonas foetus. Antimicrob Agents Chemother 37: 2722-2726, 1993.
BENCHIMOL M, DE SOUZA W Fine structure and cytochemistry of the hydrogenosome of Tritrichomonas
foetus. J Protozool 30: 422-425, 1983.
BENCHIMOL M, ELIAS CA, DE SOUZA W Ultrastructural localization of calcium in the plasma membrane
© Medimond . P726R9107
113
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
and in the hydrogenosome of Tritrichomonas foetus. Exp Parasitol 54: 277-284, 1982.
BENCHIMOL M, ENGELKE F Hydrogenosome behavior during the cell cycle in Tritrichomonas foetus. Biol
Cell 95: 283-293, 2003.
BENCHIMOL M, JOHNSON PJ, DE SOUZA W Morphogenesis of the hydrogenosome: an ultrastructural
study. Biol Cell 87: 197-205, 1996b
BUI ET, BRADLEY PJ, JOHNSON PJ A common evolutionary origin for mitochondria and hydrogenosomes.
Proc Natl Acad Sci USA 93: 9651-9656, 1996.
CARLTON JM, HIRT RP, SILVA JC, DELCHER AL, SCHATZ M, ET AL. Draft genome sequence of the
sexually transmitted pathogen Trichomonas vaginalis. Science 315: 207–212, 2007.
CERKASOV J CERKASOVOVÁ A, KULDA J, VILHELMOVÁ D Respiration of hydrogenosomes of
Tritrichomonas foetus. I. ADP-dependent oxidation of malate and pyruvate. J Biol Chem 253: 1207-1214,
1978.
CLEMENS DL, JOHNSON PJ Failure to detect DNA in hydrogenosomes of Trichomonas vaginalis by nick
translation and immunomicroscopy. Mol Biochem Parasitol 106: 307-313, 2000.
DÍAZ JAM, DE SOUZA W Purification and biochemical characterization of the hydrogenosomes of the
flagellate protist Tritrichomonas foetus. Eur J Cell Biol 74: 85-91, 1997.
DOLEZAL P, DANCIS A, LESUISSE E, SUTAK R, HRDÝ I, EMBLEY TM, TACHEZY J Frataxina
conserved mitochondrial protein,in the hydrogenosome of Trichomonas vaginalis. Eukaryot Cell 6: 1431 –
1438, 2007.
DYALL SD, KOEHLER CM, DELGADILLO-CORREA MG, BRADLEY PJ, PLÜMPER E,
LEUERNBERGER D, TURCK CW, JOHNSON PJ Presence of a member of the mitochondrial carrier
family in hydrogenosomes: conservation of membrane targeting pathways between hydrogenosomes and
mitochondria. Mol Cell Biol 20: 2488-2497, 2000.
DYALL SD, YAN W, DELGADILLO-CORREA MG, LUNCEFORD A, LOO JA, CLARKE CF, JOHNSON
PJ. Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex. Nature 431:1103
–1107, 2004.
EMBLEY TM, HIRT RP. Early branching eukaryotes? Curr Opin Genet Dev 8, 624–629, 1998.
GUSCHINA AI, HARRIS KM, MASKREY B, GOLDELBERG B, LLOYD D, HARWOOD JL. The
microaerophilic flagellate, Trichomonas vaginalis, contains unusual acyl lipids but no detectable
cardiolipin. J Eukaryot. Microbiol 56: 52–57, 2009.
HRDÝ I, HIRT RP, DOLEZAL P, BARDONOVA L, FOSTER PG, TACHEZY J, EMBLEY TM. Trichomonas
hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I. Nature 432: 618
–622, 2004.
HRDÝ I, TACHEZY J, MÜLLER M. Metabolism of trichomonad hydrogenosomes. In: Tachezy J, ed.
Hydrogenosomes and Mitosomes: Mitochondria of Anaerobic Eukaryotes. pp 113–145. Springer-Verlag,
Berlin, Heidelberg, 2008.
JOHNSON PJ, LAHTI CJ, BRADLEY PJ. Biogenesis of the hydrogenosome: an unusual organelle in the
anaerobic protist Trichomonas vaginalis. J Parasitol 79: 664-670, 1993.
LAHTI CJ, D'OLIVEIRA CE, JOHNSON PJ. Beta-succinyl-coenzyme A synthetase from Trichomonas
vaginalis is a soluble hydrogenosomal protein with an amino-terminal sequence that resembles
mitochondrial presequences. J Bacteriol 174: 6822-6830, 1992.
LAHTI CJ, JOHNSON PJ. Trichomonas vaginalis hydrogenosomal proteins are synthesized on free polyribosomes
and may undergo processing upon maturation. Mol Biochem Parasitol 46, 307-310, 1991.
LINDMARK DG, MÜLLER M. Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate,
Tritrichomonas foetus, and its role in pyruvate metabolism. J Biol Chem 248: 7724-7728, 1973.
MADEIRO RF, BENCHIMOL M. The effect of drugs in cell structure of Tritrichimonas foetus. Parasitol. Res
92: 159-170, 2004.
MÜLLER M . Biochemistry In: Honigberg BM (ed) Trichomonads Parasitic in Humans. pp 36-83. SpringerVerlag. New York, 1990.
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Analysis of genetic and structural similarity of
transferrin receptor in trypanosomatids and their
hosts
Ávila R.A.1, Lucas J.Z.1, Lopes I.F.2, Rivaroli L.1
1
Natural Sciences Department, Federal University of São João del Rei (Brazil)
Zootechny Department, Federal University of São João del Rei (Brazil)
E-mails: [email protected], [email protected], [email protected],
[email protected]
2
Abstract
Iron (Fe) is a very important element for parasites and hosts. Vital events as oxidative metabolism,
oxygen transport, DNA synthesis and other, require iron to occur. Mammals evolved an Iron delivery system
that involves serum iron chelator proteins. The unavailability of Iron on mammalian blood prevents access of
parasites to it. The most common iron chelating protein is transferrin (Tf), which is internalized into the target
cell after linked to the transferrin receptor (TfR). This system is not fully efficient to avoid parasites of the genus
Trypanosoma, which also express a Transferrin Receptor. The objective of this study was to compare, in silico,
the primary structure similarity between transferrin receptor of human (mammalian host) and of the
Trypanosoma brucei parasite.
Keywords:
Reconstrucion
Transferrin
Receptor,
Transferrin,
Iron, Tripanosoma,
Alignment,
Phylogenetic
Introduction
Iron is an essential element for metabolic and developmental processes of most living being. Iron is the
fourth most common element in the earth's crust and is the most abundant transition metal in the human body
[1]. In the mammalian extracellular environment, iron is associated with transferrin (Tf), a serum protein that
acts as a chelator. Only cells expressing the transferrin receptor (TfR) has access to iron. Once in contact with
the receptor, occurs formation of complex Tf-TfR which is endocyted [2]. The Homo sapiens transferrin receptor
is a glycoprotein composed by two chains of 760 amino acids both located on the plasma membrane [3].
Among the strategies of the Trypanosoma sp for obtaining iron is the use of proteins similar to the
hosts, such as the transferrin receptor. Understanding how the Tf-TfR interaction takes place in parasites and
hosts is crucial to comprise how occurred the emergence of this protein in parasites. Trypanosoma brucei is a
human parasite that causes sleeping sickness and obtains the iron necessary for its survival through a transferrin
receptor composed of a membrane glycoprotein heterodimer. This protein is encoded by two expression-siteassociated genes, ESAG6 and ESAG7 [4]. To comprise the parasite protein structure, identifying their
similarities and key differences in relation to human receptor is critical for understanding how the interaction
between TfR trypanosomatids and transferrin occurs.
Material and methods
1.1 Amino Acid Sequences
The amino acid sequences of the Transferrin Receptor of Trypanosoma brucei and Homo sapiens were
obtained in FASTA format (Fig. 1) from the protein database of the NCBI (National Center for Biotechnology
Information).
1.2 Structural Data Analysis
To verify the similarity between amino acid sequences of T. brucei and human receptors, multiple alignments
were performed in T-COFFE application (www.ebi.ac.uk/Tools/msa/tcoffee) implemented in Expasy, the site of
European Bioinformatics Institute (EBI) [5]. The multiple alignment is a precondition for further analysis of
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homology, modeling, phylogenetic reconstruction and verification of conserved and variable sites within a
family protein. The application T-COFFEE aligns the two sequences and constructs a series of local alignments
and subsequently a global alignment of proteins.
>gi|339516|gb|AAA61153.1| transferrin receptor [Homo sapiens] !
MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGT !
IAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDF !
TSTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLV !
YLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVL !
IYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNME !
GDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSG !
VGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLG !
TSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCED !
TDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRA !
DIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHV !
FWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF!
!
Fig. 1. Primary structure of human TfR. > Represents the beginning of the sequence, followed by the code of the protein
(gi|339516|), access code in GenBank (gb | AAA61153.1 |) and protein identification. From the second line, we have the
sequence itself, with the amino acids represented by letters.
The result of the alignment can be observed in the application Jalview (editor alignments in Java) on
page of EBI. The application shows the quality of alignment and consensus between the sequences.
1.3
Phylogenetic reconstruction
Phylogenetic reconstruction of the proteins was performed on the platform Phylogeny.fr
(www.phylogeny.fr/), a free online platform that analyzes and establishes phylogenetic relationships between the
amino acid sequences. The analysis was performed in "A la Carte" mode, which consists on Muscle alignment
and phylogenetic tree constructed on the TreeDyn model, ideal for sequences with more than 200 amino acids.
Results
The alignment of sequences ESAG 6 and 7 with the sequence of the human TfR revealed little
similarity between the primary structures of these proteins, including on the main sites of interaction with human
transferrin (Fig.2).
Fig. 2. Partial view of the multiple alignment in Jalview application, in the region of the main sites of Tf-TfR human
interaction. From top to bottom, we have the sequences of TfR ESAG 6 and 7 of T. brucei and TfR of Homo sapiens. The
black bars indicate the consensus sequences and yellow bars represent the amino acid conservation and quality of alignment.
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For TfR human, has been described that the amino acids L (leucine) and Y (tyrosine) at positions 619
and 643, respectively, are essential for interaction with transferrin [3]. In the parasite, these positions are
occupied by different amino acids that exhibit different physicochemical characteristics. In both TfR subunits are
found Y (tyrosine) at position 619 and the analysis suggests a gap at position 643.
In Fig. 3 are presented the phylogenetic reconstruction of Homo sapiens and Trypanosoma brucei TfR.
Can be noticed a fairly sharp evolutionary distance, suggesting marked differences in tertiary and quaternary
structure among proteins. It is unlikely that there is a common ancestral receptor protein from Homo sapiens and
trypanosomes and if there is rather primitive and does not provide identifiable phylogenetic relationship between
these proteins.
Fig.3 Phylogenetic reconstruction of T. brucei, T. evansi and Homo sapiens TfR. The number at the bottom indicates the
scale of the represented branch. The size and position of the branch relative to the source node are proportional to the rate
of evolutionary distance between the proteins.
Conclusions
Phylogenetic analysis suggests no close evolutionary relationships among Transferrin Receptors of
humans and T. brucei. Alignments revealed poor similarity between the primary structures of these organisms
TfR, including major sites of Tf-TfR interaction. Further studies are required to understand how differences
between receptors correlate with affinity for transferrin. Furthermore, understanding the interaction of TfR T.
brucei and transferrin may indicate a treatment way for sleeping sickness.
References
[1]
[2]
[3]
[4]
[5]
Halliwell, B. E; Gutteridge, J. M. C. (2007). Free Radicals in Biology and Medicine. 4a Ed. Oxford
University Press, Oxford, UK.
Muñoz, M.; Villar, I.; García-Erce, J. A. (2009). An update on iron physiology. World J. Gastroenterol.
15(37), pp. 4617-4626.
Aisen, P. (2004). Transferrin receptor 1. The International Journal of Biochemistry & Cell Biology. 36,
pp. 2137–2143
Fast, B.; Kremps,K.; Boshart, M; Sterverding, D. (1999). Iron-depedent regulation of transferring
receptor expression in Trypanosoma brucei. Biochemistry Journal. 342, pp. 691-696
Notredame, C.; Higgins, D.G.; Heringa, J. (2000). T-Coffee: A Novel Method for Fast and Accurate
Multiple Sequence Alignment. Journal of Molecular Biology. 302, pp. 205-217
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il)
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Chaperonopathies: Impact on protein folding and
beyond
Macario A.J.L.1,2, de Macario E.C.1
1
Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore, and
IMET, Baltimore, MD, USA;
2
Istituto Euro-Mediterraneo di Scienza et Tecnologia (IEMEST), Palermo, ITALY.
e-mails: [email protected], [email protected]
Abstract
Chaperonology encompasses the study of molecular chaperones and heat-shock proteins in all their
aspects, normal and abnormal; physiological and pathological, including medico-clinical; biochemical;
molecular biological; genetic; and biological. A subfield of Chaperonology deals with the chaperonopathies, i.e.,
diseases in which chaperones play a pathogenic role, participating in the mechanism of disease as etiologicpathogenic factors. These diseases can be classified as any other in the Medical textbooks, considering genetic
features, molecular mechanism, age of onset, clinical manifestations and course, response to treatment, and so
on. Some are genetic and hereditary while others are acquired and not transmissible, the former are due, for
example, to mutations in a chaperone gene, whereas the latter are due, for example, to post-translational
modifications of the chaperone protein molecule. Since this is a new field, only recently defined within Medicine
and that is not treated in most textbooks of Medicine or Pathology, this article will consist of an introductory
overview. Various types of genetic and acquired chaperonopathies will be listed, considering that malfunctioning
chaperones affect not only protein homeostasis (i.e., the canonical role of chaperones) but also other unrelated
cellular functions, pertaining for example, to cancer and autoimmune diseases. Some of these are classified as
chaperonopathies by mistake or collaborationism. In them, one or more molecular chaperone, even if normal,
actively favors disease, e.g., certain types of cancer require chaperones for cell growth and dissemination.
Keywords: genetic chaperonopathies, acquired chaperonopathies, chaperonopathies by defect,
chaperonopathies by excess, chaperonopathies by mistake, cancer, autoimmunity.
Introduction
In order to understand chaperonopathies it is necessary to recall the classification of chaperones, which
for practical purposes are grouped considering molecular weight, Table 1.
Table 1. Subpopulations of Hsp-chaperones classified according to apparent molecular
weighta
a
MW (kDa)
range
Classical family in this MW
range
Other members in this range implicated in the
causation of disease (chaperonopathies)
200 or
higher
None
Sacsin
100-199
Hsp100-110
None
81-99
Hsp90
Paraplegin [SPG7]
65-80
Hsp70/DnaK
Spastin [SPG4]; LARP7
55-64
Hsp60 (chaperonins Groups I
and II, e.g., Cpn60 and CCT,
respectively)
Myocilin; protein disulphide-isomerases (PDI).
35-54
Hsp40/DnaJ
AIP; AIPL1; torsin A; clusterin; DNAJC19 (TIM14)
34 or less
sHsp (crystallins)
Hsp10 (Cpn10); Alpha Hemoglobin-Stabilizing Protein
(AHSP); cyclophilin type peptidyl-prolyl cis-trans
isomerases (PPI); alpha-synuclein; HSPB11.
Source: References [1, 2].
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Another important basic concept is that chaperones contrary to what was believed until not long ago can
occur not only inside but also outside cells, in all intra- and extracellular compartments, Table 2.
Table 2. Residences of chaperones in eukaryotesa
a
Location
Compartment
Intracellular
Nucleus; Cytosol; Mitochondria; Endoplasmic reticulum;
Lysosomes; Vesicles; Membrane on the inside, and
transmembrane; Chloroplasts (in plants)
Pericellular
Membrane on the outside
Extracellular
Intercellular space; Blood (plasma, serum): soluble or in
vesicles (e.g., exosomes); Lymph; Cerebrospinal fluid;
Intersynovial space (joint cavity); Secretions (e.g., saliva,
urine)
Source: references [2,3].
Lastly, understanding the concept and field of chaperonopathies is facilitated by assuming that the
whole set of chaperones, co-chaperones, and co-factors of an organism constitute a physiological system. A
schematic representation of the forms a chaperone can assume, the locales at which it may work, and its
movements in cellular and extracellular compartment is shown in Fig. 1.
Fig.1. The chaperoning system. Circled C, molecular chaperone; 1, mobile chaperone in the cytosol; 2, chaperone inside an
organelle, such as the nucleus or mitochondrion; 3, sessile chaperone anchored to a particle (e.g., ribosome) in the cytosol;
4 and 5, sessile chaperone anchored to the cell membrane on the cytosolic side (4) or on the outside in the extracellular
space (5) - chaperones can also be located in the plasma membrane (i.e., transmembrane protein); 6, mobile chaperone in
the intercellular space; 7, mobile chaperone in circulation inside a vessel (blood or lymph) in suspension or, 7a, on the
surface of circulating erythrocytes, lymphocytes, granulocytes, or platelets; 8, sessile chaperone anchored to the vessel wall
on the inside; 8a, chaperone inside a biological space, such as the intersynovial space in the cavity of many joints, and the
space between the pia and the arachnoid maters in the central nervous system (cerebrum ventricles, cisterns, and sulci, and
spinal cord central canal); 9, mobile chaperone in the cytosol like that shown in 1, but imported from another cell. Molecular
chaperones can be found also in other locations such as cerebrospinal fluid (8a) and secretions (e.g., saliva and urine), the
latter two not shown in this figure (see Table 2); 10, mobile or sessile chaperone that originated in the blood or on a nearby
cell (same as 6 if mobile) and is now in the intercellular space. Arrows indicate the various directions of movement of a
chaperone molecule from inside a cell to the extracellular space or vessel lumen and vice versa. A chaperone can gain the
extracellular space from inside a cell or from inside a vessel and it can go into a vessel directly from a cell or from the
extracellular space. Source: References [2,4].
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The chaperonopathies
Chaperonopathies are pathological conditions in which one or more chaperones are abnormal and
contribute to disease rather than protect against it as one might expect, considering the cytoprotective roles
traditionally attributed to chaperones. The abnormality of the pathogenic chaperone molecule may be the
consequence of a gene mutation or of a post-translational modification, for example. Thus, the chaperonopathies
may be classified into genetic or acquired. If we consider the type of chaperone abnormality the
chaperonopathies may be classified as by excess, defect, or mistake, Table 3.
Table 3. Classification of chaperonopathies according to pathogenic mechanisma
Chaperonopathies by:
Mechanism, features
Excess
Quantitative, e.g., due to gene dysregulation and overexpression
Qualitative, e.g., gain of function
Defect
Quantitative, e.g., gene downregulation
Qualitative, e.g., due to structural defect genetic or acquired
Mistake
a
Normal chaperones can contribute to disease, e.g., some tumors that
need chaperones to grow, and autoimmune conditions in which a
chaperone is the autoantigen
Source: references [5,6].
At the molecular level, structural abnormalities whether genetic or acquired that characterize
chaperonopathies may affect any of their functional domains, Fig. 2.
Fig. 2. Key for the structural domains, from left to right in top left scheme: ATP-binding-ATPase, substrate binding,
chaperone or cofactor-chaperone interaction (needed for the assembly of the chaperoning networks, i.e., interaction with
other chaperones and chaperoning teams), oligomerization (needed for the formation of oligomers, i.e., the chaperoning
complex or team such as the homo-heptamer formed by Hsp60 or the hetero-octamer characteristic of CCT); hinge (needed
for allowing the allosteric changes accompanying all functions of the chaperone molecule - several of these domains are
usually present); ubiquitin-proteasome interaction (needed for interaction with the ubiquitin-proteasome system for protein
degradation). Filled forms represent structurally altered domains due to mutation or post-translational modification. Source:
reference [7].
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Many diseases today labeled variously can in fact be chaperonopathies. Examples of genetic and
acquired chaperonopathies are displayed in Table 4.
Table 4. A few illustrative examples of pathologic conditions, in which an abnormality in one
or more Hsp-chaperone is pathogenic in some cases that can be considered
chaperonopahiesa
Chaperonopathy
type
Abnormal Hspchaperone
Disease, syndrome
Genetic,
hereditary,
structural
(mutation)
Small-size
Cataracts, distal hereditary motor neuropathies,
Charcot-Marie-Tooth
Chaperonin Group I
(Hsp60)
Hereditary spastic paraplegia (SPG13); MitCHAP-60
Disease (Pelizaeus-Merzbacher-like)
Chaperonin Group
II (CCT)
Hereditary sensory neuropathy; McKusick-Kaufman
(MKKS) and Bardet-Biedl (BBS)
Hsp40(DnaJ)
Distal hereditary motor neuropathy
Hsp70(DnaK)
Stomach cancer
Super heavy
Sacsinopathies
Dedicated
Autosomal dominant spastic paraplegia-4 (SPG4),
dilated cardiomyopathy with ataxia (DCMA), autosomal
recessive spastic paraplegia 7 (SPG7), recessive
osteogenesis imperfect
Gene
dysregulation
Various
Beta-Thalassemia, some neurodegenerative, e.g.,
Alzheimer’s; Huntington’s; Parkinson’s
Gene
polymorphism
Hsp70 and others
Localized in the regulatory (promoter) and in coding
regions
Substrate
mutation
Alpha-Crystallin,
AHSP, PPI, CCT
Cataract, Beta-thalassemia, cystic fibrosis, Von HippelLindau disease
Alpha-A crystallin,
alpha-B crystallin
Cataracts, retinopathy, inclusion body myositis
Autoimmunity
inflammation
Hsp60
Vasculitis, atherosclerosis, rheumatoid arthritis,
diabetes, ulcerative colitis, Hashimoto’s thyroiditis,
myasthenia gravis
Cancer
Hsp60, Hsp70,
Hsp90
Large bowel, breast, prostate, glioblastoma, uterine
cervix
Acquired
Ageing
individuals
By mistake
a
Source: references [8, 9].
It has to be borne in mind that the diseases listed can be considered chaperonopathies if it is
demonstrated that one or more chaperone is abnormal and has an impact on pathogenesis, namely, it contributes
to pathogenic pathways leading to disease. Many diseases that are clinically apparent with very similar signs and
symptoms are usually grouped under a single name although they may not have the same etiology or pathogenic
mechanism. For instance, a diagnosis of Bardet-Biedl syndrome (BBS) in a patient does not mean that this
particular case is a chaperonopathy because proteins-genes other than chaperones may be affected. Only some
cases of BBS are associated with mutations in a chaperone gene and these are chaperonopathies. The same line
of thought applies to virtually all condition listed in Table 4.
Well characterized examples of genetic chaperonopathies are hereditary spastic paraplegia SPG14 and
MitCHAP-60 disease, both affecting the mitochondrial chaperone Hsp60 (also called Cpn60) [10, 11] and some
types of Charcot-Marie-Tooth disease with abnormalities in the sHsp HSPB1 [12].
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Likewise, examples of common acquired chaperonopathies are several autoimmune disorders in which
a chaperone becomes auto-antigenic and reacts with auto-antibobies causing disease [4, 13], and various types of
cancer that need chaperones to grow and metastasize [3, 5].
Perspectives
The future will bring advances at least in three directions: 1) the identification of other
chaperonopathies not yet described as such; 2) the characterization of chaperonopathies in more detail at all
levels, clinical, pathological, and mechanistic. In this regard it will be necessary to elucidate the molecular
mechanisms involved in the causation of the cellular, histological, and organismal lesions and abnormalities
observed in chaperonopathies. This will require the development and use of experimental models with
prokaryotes (e.g., archaea, which have chaperones more similar to those of humans than bacteria do) and
eukaryotes (e.g., mouse, rat, and others); and 3) the expansion of chaperonotherapy, currently in its beginnings.
This will include positive chaperonotherapy, namely the use of chaperone genes and/or proteins to replace
defective or absent chaperones, and negative chaperonotherapy [3, 15]. The latter consists in the use of
antichaperone agents to block or eliminate chaperones that participate in pathogenesis, favouring development of
disease, as it occurs in some cancers and autoimmune disorders.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Macario, A.J.L. (1995). Heat-shock proteins and molecular chaperones: Implications for pathogenesis,
diagnostics, and therapeutics. Intl. J. Clin. Lab. Res. 25:59-70.
Macario, A.J.L., Conway de Macario, E. (2009). The chaperoning system: Physiology and pathology.
Exp
Med
Rev
Vol.
2-3:
Years
2008/09,
pp.
9-21.
Available
on-line
at:
http://www.unipa.it/giovanni.zummo .
Cappello, F., Conway de Macario, E., Marasa, L., Zummo, G., Macario, A.J.L. (2008). Hsp60: new
locations, functions, and perspectives for cancer diagnosis and therapy. Cancer Biol. Ther. 7:801-809.
Macario, A.J.L., Cappello, F., Zummo, G., Conway de Macario, E. (2010). Chaperonopathies of
senescence and the scrambling of the interactions between the chaperoning and the immune systems. Ann
New York Acad Sci 1197: 85-93. http://www.ncbi.nlm.nih.gov/pubmed/20536837
Macario, A.J.L., Conway de Macario, E. (2007). Chaperonopathies by defect, excess, or mistake. Ann N
Y Acad Sci 1113:178-191. http://onlinelibrary.wiley.com/doi/10.1196/annals.1391.009/abstract
Cappello, F, Macario, A.J.L. (2012). Mitochondrial chaperonin Hsp60: locations, functions and
pathology. In Houry, W.A. (ed.), Protein Homeostasis: The Biomedical & Life Sciences Collection,
Henry Stewart Talks Ltd, London (online at http://hstalks.com/bio). Direct talk access links:
http://hstalks.com/lib.php?t=HST142.3142&c=252
Macario, A.J.L., Conway de Macario, E. (2007). Molecular chaperones: Multiple functions, pathologies,
and potential applications. Front Biosci 12: 2588-2600. To see Table of Contents, Abstract, Figures, and
Tables go to http://www.bioscience.org/2007/v12/af/2257/fulltext.htm
Macario, A.J.L., Conway de Macario, E. (2005). Sick chaperones, cellular stress and disease. New Eng. J.
Med. 353: 1489-1501.
Macario, A.J.L., Grippo, T.M., Conway de Macario, E. (2005). Genetic disorders involving molecularchaperone genes: A perspective. Genet. Med. 7: 3-12.
Hansen, J.J., Durr, A., Cournu-Rebeix, I., Georgopoulos, C., Ang, D., Davoine, C.S., Brice, A., Fontaine,
B., Gregersen, N., Bross, P. (2002). Hereditary spastic paraplegia SPG13 is associated with a mutation in
the gene encoding the mitochondrial chaperonin Hsp60. Am. J. Hum Genet. 70:1328-1332.
Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E.,
Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., Mandel, H. (2008). Mitochondrial
hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain
hypomyelination and leukodystrophy. Am. J. Hum. Genet. 83: 30-42.
Almeida-Souza, L., Goethals, S., de Winter, V., Dierick, I., Gallardo, R., Van Durme, J., Irobi, J.,
Gettemans, J., Rousseau, F., Schymkowitz, J., Timmerman, V., Janssens. S. (2010). Increased
monomerization of mutant HSPB1 leads to protein hyperactivity in Charcot-Marie-Tooth neuropathy. J.
Biol. Chem. 285: 12778-12786.
Cappello, F., Conway de Macario, E., Di Felice, V., Zummo, G., Macario, A.J.L. (2009). Chlamydia
trachomatis infection and anti-Hsp60 immunity: The two sides of the coin. PLoS Pathogens, 5(8):
e1000552.
doi:10.1371/journal.ppat.1000552,
2009.
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[14]
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000552;jsessionid=D8B
6EB8FE00D7C5051888FE9EBC98271
Macario, A.J.L., Conway de Macario, E. (2007). Chaperonopathies and chaperonotherapy. FEBS Letters.
581: 3681-3688.
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Gene Expression Assessment of the Polycomb &
Trithorax Complexes in the Brain of Fat-TissueImplanted Polycystic-Ovarian-Sindrome Mice
da Silva Freitas E.H.¹,², de Noronha S.M.R.², Baptista C.F.¹,²,
Gamboa Ritto M.N.¹,², Kede J.¹,²,³, Neto I.B.¹,², Alves Correa-Noronha S.A.²,
Guerreiro da Silva I.D.C.²
1
Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro – RJ BRAZIL;
Laboratório de Ginecologia Molecular, Universidade Federal de São Paulo, São Paulo – SP BRAZIL; 3
Fundação Universitária Serra dos Órgãos, Teresópolis – RJ BRAZIL.
[email protected], [email protected], [email protected],
[email protected], [email protected], [email protected], [email protected], [email protected].
2
Abstract
The Polycystic Ovary Syndrome (PCOS) is the most common androgenic disorder in women during
reproductive life. PCOS may also be accompanied by metabolic syndrome and recent studies point to leptin as
playing a role in disrupting infertility and in changing the energy balance in obese mice through its action on the
hypothalamus. To assess the expression of the Polycomb & Trithorax Complexes genes in brain of mice
transplanted with fat tissue from normal mice, in order to better understand the neuronal mechanisms underlying
the reversion of PCOS. Three B6 V-Lepob/J mouse groups: normal weight, obese and seven-day-treatment
obese had their brain RNA extracted and submitted to an 84 Polycomb & Trithorax Complexes genes PCR
Array plate and MetacoreTM pathways localization. Genomic profiles obtained were compared to the ones of the
normal-weight-mice group. Differentially expressed genes were 13% and 26% respectively to control and
treatment. Major changes were in genes: Snai1/31; Smarca1/-17; Dnmt3b/4.7; Ezh1/15. Altered genes were
associated to canonical pathways and provided 3 networks related to epigenetics. Underlying neuronal changes
caused by leptin in obese mice brain, there is an important role being played by the histone code. Here there is
evidence that leptin drives the chromatin packing to a more condensed pattern. Upregulation of
methyltransferase genes, like Ezh1, favors this thought. In summary the Polycomb & Trithorax complexes might
answer for the silencing of some downregulated genes in the obese mice brain when exposed to leptin, like Neu4
and Scg2.
Keywords: Polycistic Ovarian Sindrome; Polycomb and Trithorax Complexes; leptin; fat mouse
Introduction
The Polycystic Ovary Syndrome (PCOS) is the most common androgenic disorder in women during
reproductive life [1]. PCOS may also be accompanied by metabolic syndrome and recent studies point to leptin
as playing a role in disrupting infertility and in changing the energy balance in obese mice through its action on
the hypothalamus [2, 3].
DNA methylation is involved in the regulation of many genes expressed in a tissue, playing a crucial
role in determining cells fate [4,5] and it is closely related to chromatin structure [6, 7]. Cellular differentiation
involves various genome-wide histone (H) modifications that have been directly linked to active and inactive
structures of the chromatin [8, 9]. Acetylation of lysine (K) residues is a prevalent and reversible
posttranslational modification and diminishes the interaction between histone and DNA molecules facilitating
the binding of non-histone proteins to targeted regions of the DNA [10]. It is also known that nonhistone
methylation may function as a fingerprint that establishes repressive and active chromatin configuration at target
loci causing heritable changes in gene expression [11]. The histone code tightly controls DNA packaging and
chromatin remodeling, which leads to repression or activation of gene transcription [12].
Proteins of the polycomb group exert their function by forming three multi-protein complexes that act
as transcriptional repressors, PRC1, PRC2 and PhoRC [11]. The PRC2 complex trimethylates H3K27, a wellknown mark for silenced chromatin associated with promoters and regulatory elements of PcG target genes [13-
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15]. Furthermore the PhoRC interacts with H3K27 upstream of PcG target genes and PRC1 complex maintains
the chromatin in a silenced state by binding to H3K27me3 [16].
Trithorax group (TrxG) proteins form multi-subunit complexes to exert their functions as well. Three
complexes have been purified in mammalian cells so far, SET1-like, BRM and the MLL supercomplex [11]. The
SET1-like complex contains HMTase activities and also methylates lysine 4 of histone 3, which is highly
associated with promoter regions of transcription active loci [17-19]. The BRM complex contains the SWI/SNF
chromatin-remodeling ATPase BRM [20] and the MLL supercomplex has got HMTase and chromatin
remodeling activities [21].
The objective of this study is to assess the expression of the Polycomb & Trithorax Complexes genes in
the brain of mice transplanted with fat tissue from normal mice, in order to better understand the neuronal
mechanisms underlying the reversion of PCOS.
Methods
2.1 Experimental Groups
B6.V-Lepob / J mice with 2 and 3 months of age were divided into four groups: normal weight control,
obesity control, obese and obese 7 days 45 days. After each treatment, these mice were sacrificed and their
brains were processed for RNA extraction.
2.2 RNA Extraction
After using liquid nitrogen for cryogenic soaking, tissues were homogenized in Trizol reagent
(Invitrogen) according to the manufacturer’s protocol. Total RNA was purified with Qiagen RNeasy Mini Kit
and treated with DNase A. The quantity and quality of extracted RNA were measured by espectrophotometer
(Nanodrop Technologies Inc., Rockland, DE).
2.3 Real Time PCR Array
According to the manufacturer’s (Qiagen) methodology, reverse transcriptase (RT) was carried out for
the synthesis of cDNA. For each sample we used a PCR array plate containing 84 different pairs of primers as a
template in order to study the expression of genes related to the Polycomb & Trithorax Complexes (RT ²
Profiler ™ PCR Array; SABiosciences).
2.4 Analysis of Relevant Biological Processes and Networks by MetaCore.
The MetaCore software (GeneGo, St. Joseph,MI) is a computational resource that uses logic operations
for identifying biological processes that are altered because of changes in gene expression. Genes with altered
expression were mapped to Gene Ontol’ogy (GO) using MetaCore algorithm. GO annotations were used as
indicators of the biological functions. GO describes gene products in terms of their associated biological
processes, cellular components, and molecular functions. The GO entries are hierarchically linked, thus allowing
construction of cluster genes of crossed pathways.
2.5 Statistical analysis
These results were analyzed by descriptive statistics (means and standard deviation) and inferential
statistics through the Student’s t-test, with significance level of 5% (p <0.05). Real-time PCR array reactions
were processed through the online software RT2 Profiler™ PCR Array Data Analysis (SABiosciences).
Results
Differentially expressed genes were 13% and 26% respectively to control and treatment (Figures 2 and
3). Major changes were in genes: Snai, Smarca, Dnmt3b, Ezh1 (Figure 3). Altered genes were associated to
canonical pathways and provided 3 networks related to epigenetic mechanisms of gene expression regulation
(Figure 4).
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Fig. 1 Gene expression change Heat Map. groups: obese vs. control; 7-day treatment vs. control;. 45-day treatment vs.
control.
Fig. 2 Gene expression changes scatter plot. Group 1, obese, Group 2, 7-day treatment and Group 3 to 45-day treatment.
Fig. 3 Fold regulation of genes that presented an important differential expression after leptin treatment of the experimental
groups.
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Fig. 4 Metacore Analysis: Control vs 7-day treatment: 03 Networks Main 1: EZH2, Rb protein, p21, Histone H3, SUZ12.
GO Processes: regulation of gene expression (87.5%).
Conclusions
Polycomb (PcG) proteins were originally identified as part of an epigenetic cellular memory system that
controls gene silencing via chromatin structure. However, recent reports suggest that they are also involved in
controlling dynamics and plasticity of gene regulation, particularly during differentiation, by interacting with
other components of the transcriptional apparatus [22].
Enhancer of zeste homolog 1 (Ezh1) is a gene that encodes a PcG protein that initiates repression in
certain chromatin domains and regulates developmental genes expression, which is closely related to
cell proliferation [23]. This gene is up-regulated after 7 days of treatment
Others genes related to transcription factors, DNA methyltransferases and chromatin remodeling
proteins member were also studied. Snail homolog 1 (SNAl1) proteins are zinc-finger that play important roles
in determining cell-fate [24]. This gene is up-regulated after 7 days of treatment.
DNA methyltransferase 3B (Dnmt3b) dynamically regulate chromatin remodeling and gene expression.
DNA methyltransferases contribute to the establishment and maturation of cell fate during retinal development
[25]. This gene is also upregulated after 7 days of treatment.
SWI/SNF related, matrix associated, actin dependent regulator of chromatin (Smarca1) is a component
of the chromatin remodeling complex. This gene is related to neuron differentiation, brain development and
chromatin remodeling. In the brain, SMARCA1 is prevalent in proliferating cell populations whereas, it is
predominantly expressed in terminally differentiated neurons after birth and in adult animals. These results
suggest that SMARCA1 complexes have distinct functions associated with cell maturation or differentiation
[26]. This gene is downregulated in this study.
In summary, exogenous leptin drives the phisyological variables related to POS to a normal status
(glycemia, ovulation and fertility, body weight and food intake) only after a 45-day treatment period. Exogenous
leptin reestablishes a normal pattern of ovarian cycle controling hormones release after a 7-day treatment period.
Underlying neuronal changes caused by leptin in obese mice brain, there is an important role being played by the
histone code. Here there is evidence that leptin drives the chromatin packing to a more condensed pattern.
Upregulation of methyltransferase genes, like Ezh1, favors this thought. In summary the Polycomb & Trithorax
complexes might answer for the silencing of some downregulated genes in the obese mice brain when exposed to
leptin, like Neu4 and Scg2.
References
[1] Goldzieher JW, Green JA. The polycystic ovary I Clinical and histological features. J Clin Endocrinol Metab.
1962; 22:325-31.
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[2] Hahn S, Bering van Halteren W, Roesler S, Schmidt M, Kimmig R, Tan S, Mann K, Janssen OE. The
combination of increased ovarian volume and follicle number is associated with more severe hyperandr
ogenism in German women with polycystic ovary syndrome. Exp Clin Endocrinol Diabetes. 2006;
114:175- 81.
[3] Premoli AC, Santana LF, Ferriani RA, Moura MD, DeSa MF, Reis RM. Growth hormone secretion and
insulin-like gr owth factor-1 are related to hyperandrogenism in non obese patients with polycystic ovary
syndrome. Fertil Steril. 2005; 83: 1852- 5.
[4] SHIOTA, K. (2004). DNA methylation profiles of CpG islands for cellular differentiation and development
in mammals. Cytogenet Genome Res 105: 325-334.
[5] YAGI, S., HIRABAYASHI, K., SATO, S., LI, W., TAKAHASHI, Y., HIRAKAWA, T., WU, G.,
HATTORI, N., OHGANE, J., TANAKA, S. et al. (2008). DNA methylation profile of tissue-dependent
and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific
gene expression. Genome Res 18: 1969-78.
[6] HATTORI, N., NISHINO, K., KO, Y.G., OHGANE, J., TANAKA, S. and SHIOTA, K. (2004). Epigenetic
control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J Biol Chem
279: 17063-17069.
[7] IKEGAMI, K., OHGANE, J., TANAKA, S., YAGI, S. and SHIOTA, K. (2009). Interplay between DNA
methylation, histone modification and chromatin remodeling in stem cells and during development. Int J
Dev Bio 53: 203-214.
[8] OHGANE, J., HATTORI, N., ODA, M., TANAKA, S. and SHIOTA, K. (2002). Differentiation of
trophoblast lineage is associated with DNA methylation and demethylation. Biochem Biophys Res
Commun 290: 701-706 [9]Shiota et al., 2002
[10] SHOGREN-KNAAK, M., ISHII, H., SUN, J.M., PAZIN, M.J., DAVIE, J.R. and PETERSON, C.L. (2006).
Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311: 844-847.
[11] BASKIND, H.A., NA, L., MA, Q., PATEL, M.P, GEENEN, D.L. and WANG, Q.T (2009). Functional
conservation of Asxl2, a murine homolog for the Drosophila enhancer of Trithorax and Polycomb gene
Asx. PLoS ONE 4(3): e4750.
[12] ESCARGUEIL, A.E., SOARES, D.G., SALVADOR, M., LAERSEN, A.K. AND HENRIQUES, J.A.
(2008). What histone code for DNA repair? Mutat Res 658(3): 259-270.
[13] CZERMIN, B., MELFI, R., McABE, D., SEITZ, V., IMHOF, A., et al. (2002). Drosophila enhancer of
Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb
sites. Cell 111(2): 185–196.
[14] KUZMICHEV, A., NISHIOKA, K., ERDJUMENT-BROMAGE, H., TEMPST, P. and REINBERG, D.
(2002). Histone methyltransferase activity associated with a human multiprotein complex containing the
Enhancer of Zeste protein. Genes Dev 16(22): 2893–2905.
[15] IKEGAMI, K., OHGANE, J., TANAKA, S., YAGI, S. and SHIOTA, K. (2009). Interplay between DNA
methylation, histone modification and chromatin remodeling in stem cells and during development. Int J
Dev Bio 53: 203-214.
[16] SCHUETTENGRUBER, B., CHOURROUT, D., VERVOORT, M., LEBLANC, B. and CAVALLI, G.
(2007). Genome regulation by polycomb and trithorax proteins. Cell 128: 735-745.
[17] Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, et al. (2004) Leukemia proto-oncoprotein MLL
forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol
Cell Biol 24(13): 5639–49.
[18] Bernstein BE, Kamal M, Lindblad-Toh K, Bekiranov S, Bailey DK, et al. (2005) Genomic maps and
comparative analysis of histone modifications in human and mouse. Cell 120: 169–181.
[19] Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane-Robinson C, et al. (2004) Histone H3 lysine 4
methylation patterns in higher eukaryotic genes. Nat Cell Biol 6: 73–77.
[20] Papoulas O, Beek SJ, Moseley SL, McCallum CM, Sarte M, et al. (1998) The Drosophila trithorax group
proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes. Development 125(20): 3955–
66.
[21] Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T, et al. (2002) ALL-1 is a histone
methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol
Cell 10(5): 1119–1128.
[22] Prezioso C, Orlando V. Polycomb proteins in mammalian cell differentiation and plasticity. FEBS
Lett. 2011 Jul 7;585(13):2067-77.
[23] Margueron R, Li G, Sarma K, Blais A, Zavadil J, Woodcock CL, Dynlacht BD, Reinberg D. Ezh1 and Ezh2
maintain repressive chromatin through different mechanisms. Mol Cell. 2008 Nov 21;32(4):503-18.
[24] Zhuge X, Kataoka H, Tanaka M, Murayama T, Kawamoto T, Sano H, Togi K, Yamauchi R, Ueda Y, Xu
Y, Nishikawa S, Kita T, Yokode M. Expression of the novel Snai-related zinc-finger transcription factor
gene Smuc during mouse development. Int J Mol Med. 2005 Jun;15(6):945-8.
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[25] Nasonkin IO, Lazo K, Hambright D, Brooks M, Fariss R, Swaroop A. Distinct nuclear localization patterns
of DNA methyltransferases in developing and mature mammalian retina. J Comp Neurol. 2011 Jul
1;519(10):1914-30.
[26] Lazzaro MA, Picketts DJ. Cloning and characterization of the murine Imitation Switch (ISWI) genes:
differential expression patterns suggest distinct developmental roles for Snf2h and Snf2l. J
Neurochem. 2001 May;77(4):1145-56.
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Influence of pancreatic acinar cell necrosis on
stellate cell proliferation in vitro
Geissler K.1, Krüger B.2
1
Dept. of Gastroenterology, University of Rostock, Rostock, Germany
Dept. of Medical Biology, University of Rostock, Rostock, Germany
[email protected] , [email protected]
2
Abstract
Pancreatic stellate cells (PSC) are coupled to acinar cells morphologically, but nothing is known about
their functional relationships, especially in acute pancreatitis. To shed some light on this issue we compared both
cell types in pure and co-cultures. After the first day of cultivation 90% of acini were damaged. Cell debris and
digestive proteases were released into medium, mimicking conditions of pancreatic necrosis. Under these
conditions PSC adhesion was inhibited, but division rate stimulated. As shown by RT-PCR and immune-staining
cultured PSC express acinar specific proteins, in a time-dependent manner but independent from any acinar state.
These data correspond with results of CCK-induced calcium release. Thus our data indicate that pancreatitisrelated PSC transformation is affected by cultivation time and by the state of co-cultivated acinar cells in vitro.
These cellular interactions are probably an important part of regeneration of pancreatic tissue.
Keywords: pancreas, stellate cells, acinar cells, co-culture, cellular interactions
Introduction
Pancreatic stellate cells (PSC) play a key role in pathobiology of major disorders of the exocrine
pancreas. PSC are localized in the periacinar space encircling the base of acinar cells. Nevertheless nothing is
known about functional relationships between PSC and acini, especially regarding tissue repair capabilities after
acinar cell necrosis. Comparing both cell types in pure and co-cultures we analyzed the influence of acinar cell
state on PSC proliferation in vitro.
Materials and Methods
Pancreatic acini (physiological units of acinar cells) were isolated by collagenase digestion according to
Williams et al. [1], and PSC were separated by density gradient centrifugation according to Apte et al. [2], both
from male Wistar rats. PSC were co-cultivated with pancreatic acini at equicellular concentrations (each with a
biovolume of 1 mm³/ml) in the same culture medium and at the same conditions described for acini alone [3].
Co-cultivation was carried out in 24 well microtiter plate Boyden chambers (Greiner Bio-one), separating both
cell types by an insert with filter pore size of 0.45 µm. Results of co-cultivation were compared with pure
cultures of each cell type.
Cell number and biovolume concentration were determined with the Cell Analyzing System CASY TT
(Schärfe GmbH, Germany), for PSC at a diameter range between 11-23 µm and for acini between 24-80 µm [4].
Gene expression of trypsinogen and CCK-A receptor in PSC was quantified by RT-PCR.
Paraformaldehyde fixed PSC were immune-stained with primary anti-trypsin antibody (Biogenesis) or
anti-CCK-A receptor antibody (Progen), linked with fluorescent Alexa 488 secondary antibody (Invitrogen) [5].
CCK-A receptor dependent calcium release induced with 10 nM caerulein, an analogue of cholecystokinin
(CCK), was monitored with FURA2-AM (2 µM) by ratio imaging (TILL Photonics, Germany) [3].
Values in figures 1-3 and 6 denote means +/- SEM of triplicates, representative of at least three
experiments.
Results
After the first day of cultivation almost all acini were necrotized, whether in pure or co-culture (fig. 1).
Acinar cell debris and digestive proteases were released into culture medium, mimicking conditions of pancreas
necrosis. As a result, the division rate of co-cultured PSC was stimulated (fig. 2), but PSC adhesion to plate
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bottom was inhibited (fig. 3). PSC proliferation was accompanied by expression of acinar specific proteins. As
shown by RT-PCR (fig. 4) and immune-staining (fig. 5), PSC are able to synthesize trypsinogen and CCK-A
receptor, mostly in a time-dependent manner, but independent from any state of co-cultivated acini. While in
freshly isolated PSC almost no immune-staining was observed (data not shown), more than 50% of PSC were
immune-positive five days after beginning of cultivation. These data correspond with time dependence of CCKA receptor induced calcium release of PSC, stimulated by the CCK analogue caerulein (fig. 6).
Figure 1: Time course of disintegration and necrosis of acinar cells in vitro. Acinar biovolume concentration was measured
with the Cell Analyzing System CASY in mono- (blue line) and co-culture with PSC (red line). Dead cells were quantified
with the trypan blue method (black line). No differences in necrosis between pure and co-cultured acini were found.
Figure 2: Time course of PSC proliferation in pure and co-cultures with necrotic pancreatic acini. PSC, co-cultured with
necrotic acini, have higher division rates (inset), leading to smaller sized single cells (main graph).
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Figure 3: Time course of PSC adherence. Acinar cell necrosis reduced the number of adherent PSC and increased PSC
mobilization.
Figure 4: Expression of trypsinogen and CCK-A receptor mRNA in freshly isolated pancreatic acini (A) and in pure PSC
cultures (day 0, 2, 4, 6, and 8). No expression differences from co-cultivated PSC were detected (data not shown). Results of
RT-PCR.
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Figure 5: Immune-staining for trypsinogen (A) and CCK-A receptor (B) in pure, five days old PSC cultures (Ex 488 nm/Em
530 nm). No significant differences from co-cultured PSC were determined.
Figure 6: Intracellular calcium release in PSC induced with caerulein (10 nM). The percentage of caerulein-positive cells
increased during the first five days of cultivation. Except on day 7 (refer to corresponding bar chart), no differences were
observed in calcium release between pure (P) and co-cultivated (C) PSC.
Conclusions
Our data provide evidence that damaged pancreatic acinar cells are involved in, and possibly are
required for the acceleration of cell division of co-localized PSC, the mobilization of adherent PSC, and
consequently the faster transmigration of PSC into epithelial tissue during acute pancreatitis. The results
strengthen the suspicion of a high differentiation potency of PSC. It is concluded, that PSC are capable of typical
activities of exocrine acinar cells and may even contribute to regeneration after acute pancreatitis.
Acknowledgements: We thank Uta Naumann and Simone Rackow for their expert technical assistance.
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References
[1] WILLIAMS JA, KORC M, DORMER RL. Action of secretagogues on a new preparation of functionally
intact, isolated pancreatic acini. Am J Physiol. 235(5): 517-524, 1978.
[2] APTE MV, HABER PS, APPLEGATE TL, NORTON ID, McCAUGHAN GW, KORSTEN MA, PIROLA
RC, WILSON JS. Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut
43(1): 128-133, 1998.
[3] KRÜGER B, ALBRECHT E, LERCH MM. The role of intracellular calcium signaling in premature protease
activation and the onset of pancreatitis. Am J Pathol. 157(1): 43-50, 2000.
[4] SCHNEKENBURGER J, MAYERLE J, KRÜGER B, BUCHWALOW I, WEISS FU, ALBRECHT E,
SAMOILOVA VE, DOMSCHKE W, LERCH MM. Protein tyrosine phosphatase kappa and SHP-1 are
involved in the regulation of cell-cell contacts at adherens junctions in the exocrine pancreas. Gut 54(10):
1445-1455, 2005.
[5] MAYERLE J, SCHNEKENBURGER J, KRÜGER B, KELLERMANN J, RUTHENBÜRGER M, WEISS
FU, NALLI A, DOMSCHKE W, LERCH MM. Extracellular cleavage of E-cadherin by leukocyte elastase
during acute experimental pancreatitis in rats. Gastroenterology 129(4): 1251-1267, 2005.
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Brazil)
il)
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In vitro interaction of Aeromonas spp. strains with
HEp-2 and Caco-2 cell lines
Fonseca Ferreira A., Azevedo dos Santos P., Corrêa de Freitas Almeida A.
Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Ciências Médicas, Universidade do
Estado do Rio de Janeiro, RJ, Brazil
E-mails: [email protected], [email protected]
Abstract
The genus Aeromonas is a significant human pathogen. Studies have shown that the pathology caused
by these bacteria involves numerous virulence factors, such as the ability to produce toxins, adhesion and
invasion. The properties conferred by these factors have been extensively studied in experiments of interaction
between bacterial strains and cell culture. We investigated the interaction of Aeromonas strains with HEp-2 and
Caco-2 cell lines as the action of cytotoxic enterotoxin, the pattern of adhesion and invasive capacity. Ten strains
of Aeromonas isolated from fish sold in open markets were used, 9 of A. caviae and one of A. hydrophila. For
the cytotoxicity assay, aliquots of the sterile filtrate from bacterial culture were added to the cells cultures with
incubation period of 2, 24 and 48 hours. In adhesion and invasion tests, aliquots of bacterial grown culture were
added to cell cultures. Adhesion test had incubation period of 90 minutes and 6 hours. The ability of 6 of the 10
strains to invade HEp-2 with 3 hours of incubation was evaluated. Among 10 strains, 90% produced cytotoxic
effect on HEp-2 cells, while 50% of the strains produced the same effect on Caco-2 cells. Displaying the
adherence patterns diffuse and aggregative, the strains were adherent to both cell lines. The strains tested showed
low rates of invasion and are considered non-invasive. HEp-2 cells showed to be superior for the tests, while the
Caco-2 cells are not suitable for the testing of cytotoxicity.
Keywords: Aeromonas, virulence, cell lines.
Introduction
Aeromonads are Gram-negative bacilli or coco-bacilli, facultative anaerobic, non-sporulating and
generally mobile. The genus Aeromonas is characteristically oxidase and catalase positive, glucose fermentor
and is included in the family Aeromonadaceae [1], [2]. These bacteria are essentially ubiquitous, being widely
distributed in aquatic environments. Members of the genus are also found in contaminated environments and
food, such as fish and seafood, raw vegetables, raw meat, pasteurized milk and its derivatives [3], [4], [5].
Aeromonas species have been known by causing several diseases in animals, especially those live in
aquatic environments. Recently, Aeromonas has been gaining importance due its increasing occurrence in
medicine. Among the species, three are major in cases of gastroenteritis, sepsis and infections of the skin and
other tissues in humans: A. caviae, A. hydrophila and A. veronii bv sobria [2], [6]. Gastroenteritis is the most
commonly reported infection associated with Aeromonas disease, ranging from mild, self-limited watery
diarrhoea to a more severe and invasive dysenteric form [2], [7].
The genus owns a set of virulence factors considered responsible for intestinal and extra-intestinal
infections. The properties conferred by these factors have been extensively studied in experiments of interaction
between bacterial strains and cell culture. However, it’s still unclear the way these factors combine to establish a
specific disease and how they are distributed among species of the genus [8], [9], [10]. Toxins and adhesion
capacity are the most studied virulence factors in the genus and have been shown to be the major determinant of
pathogenicity for several enteropathogenic bacteria, including the aeromonads [11]. The ability of Aeromonas
strains to invade epithelium surfaces needs to be studied further to explain the evolution of infection, especially
in severe gastroenteritis.
In this study, we investigated the interaction of Aeromonas strains with HEp-2 and Caco-2 cell lines as
the action of cytotoxic enterotoxins, the pattern of adhesion and invasive capacity.
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Material and methods
1.1
Bacterial strains
Ten Aeromonas spp. strains previously isolated from fish sold in open markets, nine A. caviae and one
A. hydrophila, were selecetd for study [5]. The strains were maintained in refrigerated stock agar TSA (Difco
Lab, Detroit, MI, USA) and at -70°C in skim milk (Difco) containing 20% (v/v) glicerol, being subcultured on
TSB without glucose (tryptic soy broth; Difco) and incubated at 37°C overnight for the experiments.
1.2
Cell lines and culture conditions
The cell line HEp-2 (ATCC CCL23), originary from human laryngeal carcinoma, was used in
cytotoxicity, adherence and invasion assays. The cell line Caco-2 (ATCC HTB37), originary from human
intestine adenocarcinoma (colon), was used in the cytotoxicity and adherence assays.
HEp-2 and Caco-2 cells were cultivated in plastic bottles containing, respectively, 5ml of MEM
(Minimal Essential Medium, Gibco, NY, USA) or DMEM (Dulbecco’s Modified Eagle Medium, Gibco)
maintenance medium (supplemented with antibiotics [gentamicin 50 µg.ml-1 and fungizone 2.5 µg.ml-1, Gibco],
5% FCS [fetal calf serum, Gibco] and 0.5% glutamine 2.5 mM) in 5% CO2 atmosphere at 37°C until confluence
(after 2-3 days for HEp-2 and 3-4 days for Caco-2), when subculture was made.
1.3
Cytotoxic assay
HEp-2 and Caco-2 cells were cultivated in 96-well tissue culture plates containing, respectively, MEM
or DMEM maintenance medium in 5% CO2 atmosphere at 37°C, until reach confluence. Overnight Aeromonas
grown cultures were inoculated into 5 ml of double-strength TSB without glucose and incubated at 37°C
overnight with shaking (200 rev.min-1). Sterile filtrates were prepared by centrifugation (10000g for 2 min) with
subsequent filtration of the supernatants through a 0.22 µm pore size filter (Millipore, São Paulo, Brasil). The
filtrates were immediately used or stored at –20°C for 3 months. Aliquots of sterile filtrates were added in
duplicate to confluent monolayer of HEp-2 or Caco-2 cells in well containing 200 µl of MEM or DMEM usage
medium (supplemented with 2% FCS, 1% glutamine 2.5 mM and 1% D-manose) without antibiotics to reach 1:5
and 1:10 final dilutions. The tissue-culture plates were incubated in 5% CO2 atmosphere at 37°C. Monolayers
were examined after 2, 24, and 48 hours of incubation using inverted light microscopy for cytotoxic activity. In
each well, cytotoxic positive result was the beginning of cell death observation comparing to cells untreated by
filtrate. Negative and positive controls consisted, respectively, of A. caviae ATCC 15468 and Aeromonas
hydrophila ATCC 7966 [12]. Experiments were performed at least twice.
1.4
Adherence assay
HEp-2 and Caco-2 cells were cultivated on 13-mm-diameter glass coverslips placed in 24-well tissue
culture plates containing, respectively, MEM or DMEM maintenance medium in 5% CO2 atmosphere at 37°C,
until reach halfconfluence. Bacterial strains were inoculated in TSB without glucose overnight at 37°C and
aliquots of 35 µl bacterial suspensions were added in duplicate to halfconfluent monolayers of HEp-2 or Caco-2
cells in well containing 1 ml of MEM or DMEM usage medium without antibiotics. The tissue-culture plates
were incubated in 5% CO2 atmosphere at 37°C for 90 minutes and 6 hours. After the incubation, cells
monolayers were washed with PBS-D (Dulbecco’s phosphate-buffered saline) to remove nonadherent bacteria.
The monolayers were fixed with methanol for 20 minutes and stained with 20% Giemsa solution (Merck, Rio de
Janeiro, Brasil) for 30 min at room temperature. The coverslips were mounted on glass slides and examined by
optical microscopy. Nonadherent Escherichia coli K12 (DH5α) and enteroaggregative E. coli 17-2 (EAEC 042)
were also included respectively as negative and positive controls [13], [14]. Experiments were performed at least
twice.
1.5
Invasion assay
HEp-2 cells were cultivated in 24-well tissue culture plates containing, MEM maintenance medium in
5% CO2 atmosphere at 37°C, until reach confluence. Six bacterial strains of both species were cultivated as
described before for adherence assay. Aliquots of bacterial grown culture were added to PBS-D for optical
density measurement (λ=680nm, OD =0,14) to obtain 108 CFU.ml-1. From standard bacterial suspension, 100µl
(107 CFU.ml-1) were added to HEp-2 monolayers in wells containing 1ml of MEM usage medium without
antibiotics. The infected monolayers were incubated for 3 hours and then washed with PBS-D. In some wells,
DMEM usage medium supplemented with 250 µg.ml-1 of gentamicin was added to the monolayers and
incubated for 1 h at 37°C in 5% CO2 atmosphere. After incubation, the cells were washed with PBS-D (10-1 to
10-5) and lysed with 1% Triton X-100 (BioRad, Richmond, CA, USA). Aliquots of cell lysates were diluted in
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PBS-D , plated in TSA Petri dishes and incubated at 37°C for 18 hours to quantify the CFU of viable
intracellular bacteria. The invasive ability of Aeromonas strains was compared with positive and negative
controls, respectively Salmonella Typhimurium (C20) and non-invasive E. coli (DH5α) [12], [14]. Invasion rates
were calculated from the ratio of the number of total and intracellular bacteria recovered from HEp-2
monolayers. Experiments were performed at least twice.
2 Results
2.1 Cytotoxic effect
In assays which HEp-2 cells were used, nine strains (90%) of both A. caviae and A. hydrophila
produced cytotoxic effect, occurring lysis of cell monolayers in wells containing 1:5 concentration of filtrate
after 48 hours of incubation (Table 1). Only one strain showed no cytotoxic activity on this cell line. On assays
which Caco-2 cells were used, four A.caviae strains (40%) produced cytotoxic effect, in the same concentration
as observed with HEp-2 cells after 48 hours of incubation. The A. hydrophila strain produced cytotoxic effect
after 24 hours of incubation (Table 2).
Table 1: Cytotoxic effect observed in HEp-2 cells after 2, 24 and 48 hours of incubation with sterile filtrate from cultivation
of Aeromonas spp. strains
Strains (nº)
2 hours
24 hours
48 hours
Non-cytotoxic
A. caviae (9)
0
0
8 (89%)
1 (11%)
A. hydrophila (1)
0
0
1 (100%)
0
TOTAL
0
0
9 (90%)
1 (10%)
Table 2: Cytotoxic effect observed in Caco-2 cells after 2, 24 and 48 hours of incubation with sterile filtrate from cultivation
of Aeromonas spp. strains
2.2
Strains (nº)
2 hours
24 hours
48 hours
Non-cytotoxic
A. caviae (9)
0
0
4 (44%)
5 (56%)
A. hydrophila (1)
0
1 (100%)
0
0
TOTAL
0
1 (10%)
4 (40%)
5 (50%)
Adherence pattern
Aeromonas strains tested showed aggregative and diffuse adherence to both cell lines used after 90
minutes and aggregative adherence after 6 hours (Table 3 and Table 4). The aggregative adherence was similar
to the pattern observed in positive control (EAEC 042), especially on the cell border and on glass surfaces.
Diffuse adherence was characterized by a uniform distribution of bacteria on the cell surface, but generally
containing a low number of bacteria per cell. Some A. caviae strains were adherent to Caco-2 cells, but had not
defined pattern. One A. caviae strain was non-adherent to HEp-2 and Caco-2 cells after 90 minutes of incubation
and no cell destruction was observed (Fig. 1), unlike the partial monolayer destruction and cell lysis observed
when the most of strains exhibited aggregative adherence after 6 hours of incubation (Fig. 2).
Table 3: Relation between the 10 Aeromonas strains studied and the adherence patterns expressed on HEp-2 after 90
minutes and 6 hours of incubation
Strains
(nº)
A. caviae
(9)
A. hydrophila
(1)
TOTAL
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90 minutes
6 hours
Aggregative
Diffuse
Non-adherent
Aggregative
Nonadherent
6 (67%)
2 (22%)
1 (11%)
9 (100%)
0
1 (100%)
0
0
1 (100%)
0
7 (70%)
2 (20%)
1 (10%)
10 (100%)
0
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Table 4: Relation between the 10 Aeromonas strains studied and the adherence patterns expressed on Caco-2 after 90
minutes and 6 hours of incubation
Strains
(nº)
A. caviae
(9)
A. hydrophila
(1)
TOTAL
90 minutes
6 hours
Aggregative
Diffuse
Nonadherent
UP
Aggregative
UP
5 (56%)
2 (22%)
1 (11%)
1 (11%)
7 (78%)
2 (22%)
1 (100%)
0
0
0
1 (100%)
0
6 (60%)
2 (20%)
1 (10%)
1 (10%)
8 (80%)
2 (20%)
UP – undefined pattern
Figure 1: Non-adherent strain and HEp-2 cells
after 90 minutes of incubation.
2.3
Figure 2: Strain showing aggregative adherence
to HEp-2 cells after 6 hours of incubation.
Invasive activity
In invasion assays, approximately 107 CFU.ml-1 of six strains (A. caviae and A. hydrophila) were added
to HEp-2 cells. The invasion rates of these strains were very low when compared to the values of the positive
strain Salmonella Typhimurium after 3 hours of incubation. The 6 strains tested were considered non-invasive.
Discussion
The genus Aeromonas is an important pathogen for both human and other animals, being responsible
for the etiology of intestinal and extraintestinal diseases [2]. Several virulence factors have been detected in
Aeromonas strains isolated from several sources and the lack of information about how these characteristics are
combined in the pathogenesis justifies the interest of studying the mechanisms of cytotoxicity, adhesion and
invasion. HEp-2 and Caco-2 cell lines have been used as a model for detection of virulence factors on in vitro
interaction studies with Aeromonas. [4], [8], [9], [12], [15].
Aeromonas species produce two types of cytotoxins: the cytopathic, which does not cause destruction of
crypts and villi in the intestines, and the cytotoxic, which causes extensive destruction of intestinal epithelium
[10]. Vero cells are indicated as the most sensitive cell line to these toxins [12]. In purpose of ensure the
realization of experiments in prolonged periods without destroying HEp-2 and Caco-2 monolayers, ten cytotoxic
strains after 48 hours or that did not exhibit cytotoxic activity on Vero cells were selected (data not shown), and
tests were reproduced with cell lines used in this study. The strains showed cytotoxic activity on HEp-2 cells
similar to that detected on Vero cells, whereas Caco-2 cells were less sensitive to the cytotoxicity test, showing
greatest variation in tests.
The adhesion to mucosal cells promotes colonization, where the bacteria produce toxins and may invade
the tissues being protected from host defenses [2], [11]. In a comparative study with Caco-2 cells, it was found
that HEp-2 cell line, although not originating from the intestine, is a good model for studies of adherence [11]. In
this study, strains tested were adherent to both cell lines, tending to aggregative adherence pattern, especially
after 6 hours of interaction. Studies have shown that ability of adhesion to cell lines is connected to presence of
polar and lateral flagella, demonstrating the importance of motility to this characteristic [15], [16]. The strains
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studied were previously characterized for the presence of lafA and flaA genes, lateral and polar flagella,
respectively. Most of strains were positive for one or both genes and motility positive (data not shown) and
adherent to cell lines. Caco-2 cell line polarizes in vitro and studies show that Aeromonas strains are more
adherent to non-polarized monolayers [12], [17]. The present study also found that all strains were adherent to
non-polarized Caco-2 monolayers, and the same occurs in HEp-2 cells. According to previous studies and
present results, HEp-2 cells were considered superior for adherence and invasion assays by its characteristics.
Cell invasion is another strong feature linked to the pathogenicity of bacteria, which gives access to an
environment protected from the host immune system and enables the survival and propagation of bacterial cell
by cell [18]. It was observed that Aeromonas strains isolated from diarrheal feces were able to invade HEp-2
cells, whereas strains from other sources were mostly non-invasive, suggesting that this characteristic is more
related to strains obtained from clinical cases [19], [20].
The detection of Aeromonas spp. isolated from fishes freely traded expressing virulence factors such as
cytotoxic effect and adherence highlights the role of food as a potential vehicle of transmission of this
microorganism to humans.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Abbott, S.L.; Cheung, W.K.W.; Janda, J.M. (2003). The genus Aeromonas: Biochemical characteristics,
atypical reactions and phenotypic identification schemes. J. Clin. Microbiol. 41: 2348-2357.
Janda, J.M. & Abbott, S.L. (2010). The Genus Aeromonas: Taxonomy, Pathogenicity, and Infection. Clin.
Microbiol. Rev. 23: 35-7.
Freitas, A.C.; Nunes, M.P.; Milhomem, A.M.; Ricciardi, I.D. (1993). Occurrence and characterization of
Aeromonas species in pasteurized milk and white cheese in Rio de Janeiro, Brazil. J. Food Prot. 56: 6265.
Pereira, C.S.; Possas, C.A.; Viana, C.M.; Rodrigues, D.P. (2004). Aeromonas spp. e Plesiomonas
shigelloides isoladas a partir de mexilhões (Perna perna) in natura e pré-cozidos no Rio de Janeiro, RJ.
Ciênc. Tecnol. Aliment. 24: 562-566.
Vieira, R.C.; Peçanha, R.C.; Oliveira, S.S.; Freitas-Almeida, A.C. (2007). Resistência antimicrobiana e
perfil plasmidial de Aeromonas spp. isoladas de peixes. In: 24º Congresso Brasileiro de Microbiologia,
Brasília, Brasil. Anais do 24º CBM, CD-rom.
Botarelli, E. & Ossiprandi, M.C. (1999). Aeromonas Infections: An update. Online available at
<http://www.unipr.it/arpa/facvet/annali/1999/bottarelli2/bottarelli.htm>. Accessed on 01/17/2012.
Holmes, P. & Nicolls, L.M. (1995). Aeromonads in drinking-water supplies: their occurrence and
significance. J. Chart. Inst. Water Environ. Manage. 9: 464-469.
Kirov, S.M. (1997). Aeromonas and Plesiomonas species. In: Food Microbiology Fundamentals and
Frontiers. Ed: Doyle, M. P.; Beuchat, L. R.; Monteville, T. J. Washington DC, ASM Press.
Ágarwal, R.K.; Kapoor, K.N.; Kumar, A. (1998). Virulence factors of Aeromonas - an emerging food
borne pathogen problem. J. Commun. Dis. 30: 71-78.
Chopra, A.K. & Houston, C.W. (1999). Enterotoxins in Aeromonas-associated gastroenteritis. Microb.
Infect. 1: 1129-1137.
Kirov, S.M.; Hayward, L.J.; Nerrie, M.A. (1995). Adhesion of Aeromonas sp. to cell lines used as model
for intestinal adhesion. Epidemiol Infect 115, 465–473.
Couto, C.R.A.; Oliveira, S.S.; Queiroz, M.L.P.; Freitas-Almeida, A.C. (2007). Interactions of clinical and
environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells. Lett. Appl.
Microbiol., 45: 405-410.
Nataro, J.P.; Deng, Y.; Cooksno, S.; Cravioto, A.; Savarino, S.J.; Guers, L.D.; Levine, M.M.; Tacket, O.
(1995). Heterogeneity of Enteroaggregative Escherichia coli virulence demonstrated in volunteers. J.
Infect. Dis. 171: 465-468.
Rosa, A.C.P.; Vieira, M.A.M.; Tibana, A.; Gomes, T.A.T.; Andrade, J.R.C. (2001). Interactions of
Escherichia coli strains of non-EPEC serogroups that carry eae and lack the EAF and stx gene sequences
with undifferentiated and differentiated intestinal human Caco-2 cells. FEMS Micriobiol. Lett. 20:117–
22.
Santos, P.G.; Santos, P.A.; Bello, A.R.; Freitas-Almeida, A.C. (2010). Association of Aeromonas caviae
polar and lateral flagella with biofilm formation. Lett. Appl. Microbiol. 52: 49–55.
Gavín, R.; Merino, S.; Altarriba, M.; Canals, R.; Shaw, J.G.; Tomás, J.M. (2003). Lateral flagella are
required for increased cell adherence, invasion and biofilm formation by Aeromonas spp. FEMS
Microbiol. Lett. 224: 77-83.
Rocha-De-Souza, C.M.; Mattos-Guaraldi, A.L.; Hirata, R.J.; Moreira, L.O.; Monteiro-Leal, L.H.; FreitasAlmeida, A.C.; Previato, L.M.; Previato, J.O; Andrade, A.F.B. (2003). Influence of polarization and
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[18]
[19]
[20]
differentiation on interaction of 43-kDa outer-membrane protein of Aeromonas caviae with human
enterocyte-like Caco-2 cell line. Intern. J. Mol. Med. 11: 661-667.
Cossart, P. & Sansonetti, P.J. (2004). Bacterial invasion: the paradigms of enteroinvasive pathogens.
Science, 304: 242-248.
Lawson, M.A.; Burke, V.; Chang, B.J. (1985). Invasion of HEp-2 cells by fecal isolates of Aeromonas
hydrophila. Infect. Immun. 47:680-683.
Rall, V.L.M.; Iaria, S.T.; Heidtmann, S; Pimenta, F.C.; Gamba, R.C.; Pedroso, D.M.M. (1998).
Aeromonas species isolated from pintado fish (Pseudoplastystoma sp): virulence factors and drug
susceptibility. Rev Microbiol. 29: 222-227.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) In vitro decellularization and recellularization of the
kidneys
Martins A.B.1, 2, Silva-Mendes B.J.2, Nascimento J.S.3, Fonseca R.N.1,
Silva J.R.1, Moraes J.1, De Barros C.M.3, Campos de Carvalho A.C.2,
Souza-Menezes J.1, Goldenberg R.C.S.2
1 – Laboratório Integrado de Bioquímica Hatisaburo Masuda – Núcleo de Pesquisas em Ecologia e
Desenvolvimento Sócio-Ambiental de Macaé – UFRJ-Macaé.
2 – Laboratório de Cardiologia Celular e Molecular – Instituto de Biofísica Carlos Chagas Filho - UFRJ
3 – Laboratório Integrado de Morfologia - Núcleo de Pesquisas em Ecologia e Desenvolvimento SócioAmbiental de Macaé – UFRJ-Macaé.
[email protected] - [email protected]
Abstract
The extracellular matrix (ECM) is custom designed and manufactured by the resident cells of each
tissue and organ and it is in dynamic equilibrium with its surrounding microenvironment.
The aim of this study was to develop a decellularized kidney scaffold feasible for recellularization.
The kidney harvesting process was performed to minimize vasoconstriction and clotting. After excision
the kidney was rinsed with phosphate buffered saline (PBS) and securely attached through the hilo in an ex-vivo
perfusion system. The decellularization was performed with 1% sodium dodecyl sulfate (SDS). After 15h of
perfusion the tissue became translucid. Decellularized kidney scaffold was gently washed with PBS and fixed in
4% PF and embedded in paraffin, or stored in antibiotic-antimicotic solution at 4oC.
The detergent-based perfusion protocol successfully produced acellular kidney ECM that retaining the
web-like appearance of the basement membrane. Hematoxylin and eosin and DAPI staining confirmed the
removal of the cells. After 3 days LLC-PK1 recellularization procedure cell adhesion on renal ECM was
observed particularly at the outer epithelial layer.
These findings demonstrated that decellularized kidney sections retain critical structural and functional
properties necessary for the use as a three-dimensional scaffold and promote cellular repopulation. Further, this
study provides the initial steps in developing new regenerative medicine strategies for renal tissue engineering
and repair
Introduction
The kidney is one of the most important life-sustaining organs displaying complex structures and
multiple functions (Atala, 2006). The current available treatments for kidney disease are dialysis and renal
transplantation, which can only be used to replace kidney function during end-stage renal disease. However,
these treatments are limited in scope. Dialysis, although life-preserving, replaces only a small fraction of normal
kidney function (Humes, 1999; Tarantal et al, 2010). In contrast, renal allotransplantation can restore all renal
functions; however, it is limited by donor shortage, allograft failure, and long-term immunosuppression (Tarantal
et al, 2010).
Tissue engineering is an extension of cell therapy combining biologic and engineering techniques to
construct devices to replace tissue or organ functions (Langer and Vacanti, 1993; Tarantal et al, 2010).
Extracellular matrix (ECM) scaffolds obtained from biological decellularized tissues would be
interesting because they have the histoarchitecture that best represents in vivo organization and are in theory
more capable to facilitate tissue regeneration (Wang C.Y, 2011). The ECMs retain the innate matrix components
necessary for promoting cell adhesion, migration, proliferation, differentiation, and regulation of intercellular
signaling (Badylak, 2010).
For tissue engineering, the kidney is an interesting model because of the complexity of cell composition
and structures (Atala, 2006).
The goal of this study was to develop a decellularized kidney scaffold feasible for recellularization.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) Material and methods
Perfusion and decellularization of rat kidney
All animal work was approved by the Institutional Animal Care and Use Committee (Universidade
Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil). The rats were sedated and anesthetized with 10 mg/kg
xylasine and 100 mg/kg ketamine solution. Following sedation and anesthesia, 200 UI of heparin solution was
intraperitoneally administrated to avoid blood clotting. After five minutes a laparotomy was performed for
kidney excision. The kidneys were washed twice with PBS followed by decellularization with 1% sodium
dodecyl sulfate (SDS) (Vetec, Brazil) for 15 hours followed by perfusion for 15 minutes with phosphate buffered
saline solution (in mM, 138.9 NaCl; 2.7 KCl; 0.9 KH2PO4; 6.4 Na2HPO4). The kidney extracellular matrix
(ECM) was fixed in 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 3 days and embedded in
paraffin or stored in antibiotic-antimicotic solution (100 U/ml penicillin, 100 U/ml streptomycin and 2.5μg/mL
amphotericin B, Gibco) in PBS to use as scaffolds for culture.
Microscopic and Histochemical Evaluation of decellularization
Decellularized kidneys were fixed in 4% paraformaldehyde (Sigma-Aldrich) and imbedded in paraffin.
Sections (5-μm) were stained with hematoxylin- eosin (HE) and 4’, 6’ Diamidino-2-Phenylindole (DAPI Sigma) to determine if residual nuclear structures could be identified after decellularization. Picrosirius was used
to visualize ECM components such as collagen.
Recellularization
The porcine proximal tubule cell line (LLC-PK1) was expanded in 25cm2 flasks using culture medium
Dubelco’s Modified Eagle Medium – 5.5mM of glucose (DMEM) (Cultilab) supplemented with 5% fetal bovine
serum (FBS) (Gibco), 100 U/ml penicillin, 100 U/ml streptomycin (Sigma) at 37°C in 5% CO2 atmosphere. The
LLC-PK1 cells were plated in ECM recovered from decellularized kidney, after antibiotic-antimicotic treatment,
using the medium described above. Three days after culture initiation, partially recellularized ECM were fixed in
4% paraformaldehyde (Sigma-Aldrich) and embedded in paraffin for histochemical analyses with H&E and
periodic acid-Schiff (PAS). DAPI staining was also performed to confirm cell adhesion in ECM.
Results
After 15h of decellularization process, the kidneys became translucid (figure 1 – A e B). Qualitative
analysis of decellularized tissues with 1% SDS at room temperature revealed well-preserved architecture and
demonstrated the absence of cell nuclei, confirming removal of intact cells. The detergent-based perfusion
protocol successfully produced acellular kidneys that retained the web-like basement membrane. HE (Figure 1 –
C e D), Picrosirius (Figure 1 – E) and DAPI (Figure 1 –F) staining confirmed the removal of cellular material. After LLC-PK1 recellularization procedure cell adhesion on renal ECM was observed. PAS (figure 2 -A and B)
and DAPI staining (C and D) staining showed cell adhesion to the decellularized kidney ECM.
Discussion and conclusion
Tissue engineering focuses on growing tissue replacements by combining cells and matrices under
defined laboratory conditions to replace, repair, or enhance the biological function of damaged cells (Langer R
and Vacanti JP, 1993; Tarantal, et al, 2010). Currently, cartilage, bone, skin, tendon, and bladder have been
successfully replaced by tissue engineering techniques. Cartilage and skin products have been commercialized
(Willers et al., 2007; Clark et al., 2007). However, in these studies, only simple structural tissues were generated.
Tissue engineering of vital organs, such as kidneys, remains a difficult task because of the kidney’s complex
structures and functions.
Our findings demonstrate that decellularized kidney sections retain critical structural properties
necessary for their use as a three-dimensional scaffold for recellularization.
Recellularization of renal ECM with a differentiated cell line (LLC-PK1) was used in this study to
confirm the adequacy of this ECM for cell adhesion. To access if the renal ECM is able to induce cell
differentiation, recellularization using undifferentiated cells, such as pluripotent or multipotent stem cells, will be
necessary.
This study provides the initial steps to develop strategies for renal tissue engineering and repair.
Financial Support: FAPERJ, CNPq, Brazilian Health Ministry, CAPES and FUNEMAC.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) Figure 1: Decellularization SDS protocol: (A) Photograph of translucent acellular kidney after 15 hours in the protocol (B)
Renal extracellular matrix viewed under stereoscopic microscope. Note that vessel structures have been preserved. (C and
D) H&E staining showing preserved cortical renal extracellular matrix microstructure with no evidence of residual nuclei or
intact cells. (E) Picrosirius staining of the basement membrane proteins (collagen in red). (F) DAPI staining showing the
absence of cell nuclei.
Figure 2: Recellularization protocol using LCCPK-1 cells for 3 days in culture on ECM from decellularized
kidneys: (A and B) PAS staining showing cell adhesion to the ECM (black arrows) particularly at the outer
epithelial layer. (C and D) DAPI staining showing LLCPK-1 cell adhesion to the renal ECM (scale bar, 50 µm
and 100 µm respectively).
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) References
Atala, A. 2006 Recent applications of regenerative medicine to urologic structures and related tissues.
Regenerative medicine and urologic structures Current Opinion in Urology 2006, 16:305–309
Humes HD, Buffington DA, MacKay SM, et al. Replacement of renal function in uremic animals with a tissue
engineered kidney. Nat Biotechnol 1999;17:451–455.
Tarantal, A.F; Lee, I.C; Batchelder,A.C. , 2010 Decellularized Rhesus Monkey Kidney as a Three-Dimensional
Scaffold for Renal Tissue Engineering. Tissue engineering.16: 7
Langer, R., and Vacanti, J. P. 1993. Tissue engineering. Science 260, 920–932.
Wang, C.Y; Wu, J; Du, Z et al, 2011. Self-assembly of renal cells into engineered renal tissues in
collagen/Matrigel scaffold in vitro. Journal Of Tissue Engineering And Regenerative Medicine
Research Article. J Tissue Eng Regen Med. 10.1002/term.484.
Badylak, F.S; D.G Ratner; Castner D.B. 2010. Surface characterization of extracellular matrix scaffolds.
Biomaterials 31: 428–437
Willers C, Partsalis T, Zheng MH, et al. 2007; Articular cartilage repair: procedures versus products. Expert Rev
Med Devices 4:373–392.
Clark RA, Ghosh K, Tonnesen MG. 2007; Tissue engineering for cutaneous wounds. J Invest Dermatol 127:
1018–1029.
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ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
Brain cAMP/Ca(2+) Signaling Genes in Fat Tissue
Implanted Polycystic Ovarian Syndrome Mouse
Kede J.1,2,3, da Silva Freitas E.H.1,2, Batista C.F.1,2, Gamboa Ritto M.N.1,3,
Binda Neto I.1,2, Ribeiro de Noroña S.M.2, Alves Corrêa de Noroña S.A.2,
Guerreiro da Silva I.D.C.2
1
Universidade Federal do Estado do Rio de Janeiro (BRAZIL)
Universidade Federal de São Paulo (BRAZIL)
3
Unifeso, Faculdade de Medicina de Teresópolis (BRAZIL)
[email protected], [email protected],[email protected], [email protected],
[email protected] , [email protected], [email protected], [email protected].
2
Abstract
Background: A relationship among cAMP, Ca(2+) and pulsatile GnRH secretion exists. Mice with
polycystic ovarian syndrome (PCOS) show leptin deficiency in hypothalamic neurons; cAMP responses in gene
transcription in hypothalamic neurons are leptin regulated. In reaching leptin sufficient level, there is restoration
of gonadotrophin secretion and reproductive function. Altogether these facts point to a promising research field.
Aims: Analysis of cAMP/Ca2+ gene`s expression in PCOS obese mouse brain, transplanted with leptin rich
adipose tissue fragments from normal mice, intending to identify possible changes in neuronal
behavior/environment. Methods: Four B6. V-Lepob/J mice in each of the following groups: normal weight
(control), obese (control), seven-day-treatment obese and forty-five-day-treatment obese had their brain RNA
extracted and submitted to an 84 cAMP/Ca2+ pathway genes PCR Array plate and MetacoreTM pathways
localization. Results: Mice’s differentially expressed genes were 04%, 65% and 18% respectively to obese
control, seven-day-treatment and forty-five-day-treatment. A specific major expression changes were found in
the following genes: Cnn1/308; Scg2/-53; Thbs1/48; Vip1/-17. Canonical pathways generated 15 and 2 networks
together with 51% and 80% mice cAMP/Ca2+ gene hyper-regulation, respectively in seven and forty-five-daytreatment. Conclusions: Our results provide a general view of the molecular mechanisms underlying neuronal
plasticity, cytoskeletal organization, apoptosis, and dendrite proliferation in the treated mice brain. Ferhat’s work
(2003) demonstrates Cnn1 gene brain expression causing dendritic spine plasticity. In addition, Scg2 modulates
neuronal plasticity when its expression decreases. These data confirm our findings: leptin influences neuronal
plasticity.
Keywords: Polycystic Ovary Syndrome, Cyclic AMP, Calcium-Binding Proteins, Leptin, neuronal
plasticity, Hypothalamus.
Introduction
1.1 Considerations about the Hypothalamic-Pituitary-Ovarian Biomolecular Cycle
Hypothalamic neurons through a standard pulsatile secretion of gonadotropin releasing hormone
(GnRH) controls the reproductive system of mammals. GnRH acts on anterior pituitary gonadotropic cells,
through their membrane surface receptor (GnRH-R), a member of the of G-protein coupled receptors family,
producing both luteinizing hormone (LH) and follicle stimulating hormone (FSH), both being responsible for the
production of gonadal steroids. GnRH activates adenylyl cyclase, producing 3', 5'-cyclic adenosine
monophosphate (cAMP), whose action on signaling molecules mobilize intracellular Ca(2+) stocks activating C
and A kinase proteins, propagating the signaling cascade (1). However, prolonged exposure of cells to the GnRH
results in marked desensitization of gonadotropin release due to reduction in GnRH-R, Gq/11 levels in αT3-1
cells and also the downregulation and subsequent reduction of signaling related to C kinase protein (PKC),
cAMP and Ca(2+) [1].
In women the pulse rate of GnRH secretion gradually rises during the follicular phase of the menstrual
cycle, and high levels of LH and FSH are kept during ovulation. In the luteal phase there is a reduction of the
secretory pulse because of the regulatory effect of estradiol and of progesterone through actions trigered by
hypothalamic opioid peptides [2].
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Concerning the molecular structure, both LH and FSH share the same alpha subunit and they are
distinguished by different beta subunits, encoded by three distinct genes. Increase of GnRH pulse rate secretion
promotes LH beta unit transcription, thereby increasing the LH /FSH ratio. Conversely, reduction of GnRH pulse
rate secretion induces FSH beta unit transcription, generating higher secretion of FSH by the adenohypophysis [2].
1.2 The Polycystic Ovary Syndrome
Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in women during the
reproductive period, with prevalence ranging between 6.5% and 7.0% [3, 4, 5, 6, 7]. A characteristic of this
syndrome are hypothalamic-pituitary-ovarian cycles associated with elevated levels of LH and decreased levels
of FSH, causing increased androgen production by ovarian theca cells, whose conversion to estrogens is the
substrate for chronic anovulation [8,9,10 ]. This syndrome is usually associated with obesity, hirsutism, insulin
resistance, metabolic syndrome and cardiovascular disorders [2,8,9,11].
The PCOS pathogenesis remains unclear, but hypotheses considering the synergy of environmental
impact on genetic predisposition modulating hormonal and metabolic processes are being considered
[11,12,13,14,15]. A polymorphism of the LH beta subunit revealed that the carriers of this gene variant, although
predominantly healthy, are predisposed to infertility, PCOS, breast cancer and prostate cancer [16]. Other lines
of evidence suggest the prenatal development as the origin of the syndrome [17,18]. Studies in monkeys based
on excessive prenatal exposure to androgens favors the development of a human phenotype of PCOS in these
adult animals (19,20,21,22), reinforcing the fetal origin hypothesis for the syndrome [23,24]. These studies are
coincident with the notion of being the communication between neurons susceptible to constant changes, even in
the adult brain. This ability of neuronal circuits to strengthen or weaken their specific synaptic interactions (a
phenomenon known as synaptic plasticity) may occur due to different environmental demands, which favors the
idea that dynamic changes in communication between neurons underlie the behavioral flexibility, i.e. processes
of learning and memory [25].
1.3 The hypothalamus, cAMP, Ca(2+) and leptin
The rhythmic activity of hypothalamic neurons related to pulses of GnRH release, presents, as
modulating factor, the control of Ca(2+) cationic channels, dependent of intracellular levels of cAMP, as the key
mechanism of reproductive cycle control [26,27,28]. In animal models endocrine reproductive phenotype similar
to PCOS when the leptin gene is silenced may be observed [9]. Genetic mutations in the leptin molecule or in its
receptors located on hypothalamic neurons are associated with physiological abnormalities, such as obesity, and
infertility [29,30]. Cannabinoid receptors and neurons related to LH are involved in motivational aspects of food
intake; leptin inhibits the entry of Ca(2+) in these neurons, decreasing the synthesis of endocannabinoids,
consistent with the possibility of regulating the signaling circuits related to appetite [31].
Materials and Methods
2.2 Experimental Groups (samples)
We selected fifteen transgenic leptin deficient mice (B6.V-Lepob / J, designated as ob/ob mice) and five
lean (wild) littermates, obtained from the Centre for the Development of Experimental Models (CEDEME),
Federal University of Sao Paulo (UNIFESP). All animals were maintained in a temperature-controlled
environment at ±24°C under a 12/12-h light-dark cycle and handled at least one time per week.Ten ob/ob mice
received adipose tissue transplant as described in Gavrilova and Marcus-Samuels et al. (2000) and Pereira et al.
(2011). The adipose gonad tissue obtained from wild mice after euthanasia by cervical dislocation was placed in
phosphate buffered solution (PBS) and fragmented into small pieces. The grafts were implanted in the
subcutaneous tissue through small shaved skin incisions on the dorsal region of the animal anesthetized with
isoflurane. All the animals had their brain removed by the reported procedure, kept in liquid nitrogen until use
and divided into groups as follows:
2.2.1 - Control group (CG): Five normal weight B6.V-Lepob / J mice with two to three months of life and
ovulatory cycles.
2.2.2 - Obese group (OG): Five ob/ob mice with two to three months of life and anovulatory cycles.
2.2.3 - Obese group transplanted (OGT): Ten ob/ob mice with two to three months of life and anovulatory cycles
implanted with adipose gonad tissue of mice with ovulatory cycles. Five of these animals were
sacrificed in 7 days of the procedure and the remainder after 45 days of transplanting.
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2.3 Ribonucleic acid (RNA) extraction
The brains retrieved from liquid nitrogen were macerated and the tissue homogenized in Trizol reagent
(Invitrogen). After the complete dissociation of nucleoprotein complexes, the phase separation was achieved with
chloroform and centrifugation. The precipitated and RNA obtained in the aqueous phase was washed with 75%
ethanol. The RNA was dried and dissolved in RNase-free water. Total RNA was purified with Qiagen RNeasy
Mini Kit (Qiagen) and subjected to treatment with DNase A (Qiagen). The quantity and quality of the extracted
RNA was assessed by spectrophotometry using Nanodrop (Nanodrop Technologies Inc., Rockland, DE).
2.4 Real Time PCR Array
According to the manufacturer’s (Qiagen) methodology, reverse transcriptase (RT) was carried out for
the synthesis of cDNA. For each sample we used as a template a PCR array plate containing 84 different pairs
of primers for studying genes related to Mouse cAMP / Ca (2 +) Signaling Pathway Finder PCR Array [32] (RT
² Profiler ™ PCR Array; SABiosciences).
2.6 Statistical Analysis
The fold change ratio for each gene was compared between treatment and control groups. Statistical
differences for quantitative analysis of gene expression by Real Time were evaluated by Student's t test or chisquare test. In all tests genes are reported as differently expressed between groups when the fold change was at
least three-fold up-regulated or down-regulated and the p value (analysis of variance and posttest) was lower
than or equal to 0.05.. The program GraphPad Prism 5 software was used to assist in statistical data analysis and
production of graphics. For PCR analysis it was used an array specific program available on the SA Biosciences,
the "RT2 PCR Array Data Analysis ProfilerTM."
Results
Mice’s differentially expressed genes were 04%, 65% and 18% respectively to obese control, sevenday-treatment and forty-five-day-treatment (Figures 1 and 2). A specific major change expression was found in
the following gene folds: Cnn1/308; Scg2/-53; Thbs1/48; Vip1/-17. Canonical pathways (Figure 3) showed 15
and 2 networks together with 51% and 80% mice cAMP/Ca2+ gene hyper-regulation, respectively in seven and
forty-five-day-treatment. According to Gene Ontology the cellular process was 89% of the analyzed processes.
Fig. 1 Gene expression change heat Map. groups: obese vs. control group; 7-day obese group transplanted vs. control
group;. 45-day obese group transplanted vs. control group.
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Fig. 2 Scatter plot of gene expression changes of the experimental groups: obese vs. control group; 7-day obese group
transplanted vs. control group;. 45-day obese group transplanted vs. control group.
Fig. 3 The twelfth scored (by the number of pathways) network from active experiments. Thick cyan lines indicate the
fragments of canonical pathways. Up-regulated genes are marked with red circles; down-regulated with blue circles. The
'checkerboard' color indicates mixed expression for the gene between files or between multiple tags for the same gene.
Discussion
Exogenous leptin drives the physiological variables related to POS to a normal status (glycemia,
ovulation and fertility, body weight and food intake), reestablishing a normal pattern of ovarian cycle controlling
hormones, after a 7-day treatment period. It seems that leptin acts in the cAMP/Ca2+ in the brain of obese mice
causing drastic changes in gene expression mainly after 7 days of leptin treatment. In 45 days treatment we
noticed a tendency towards the pre-treatment status, suggesting a tolerance effect related to a persistent leptin
stimulus on hypothalamic neurons. Our results correlate with neuronal plasticity, cytoskeleton organization,
apoptosis, and proliferation. In the brain, increased Cnn1 causes dendrite spine plasticity [33]. Furthermore,
Scg2 modulates neuronal plasticity and other neuronal mechanisms when its expression decreases [34).
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Conclusion
Further studies will be necessary to establish the pathways leading to neuronal tolerance under a
prolonged leptin stimulus on hypothalamic neurons. These data show the potential effects on neuronal behavior
in many cerebral areas, since the hypothalamus maintain widespread connections in the brain. We consider the
hypothesis of the relationship among hypothalamic neurons, cAMP, Ca(2+) and leptin receptors to be a common
feature in PCOS syndrome. For these studies, we consider the insertion of PCOS as part of various syndromes
and each of them defining multifactorial aspects.
References
[1] Liu F, Austin DA, Webster NJG. Gonadotropin-Releasing Hormone-Desensitized LβT2 Gonadotrope Cells
Are Refractory to Acute Protein Kinase C, Cyclic AMP, and Calcium-Dependent Signaling.
Endocrinology. 2003, 144(10):4354–4365.
[2] Marshall JC, Dalkin AC, Haisenleder DJ, Griffin ML, Kelch RP. GnRH pulses – The Regulators of Human
Reproduction. Trans Am Clin Climatol Assoc. 1993; 104: 31–46.
[3] Diamanti-Kandarakis E, Kouli CR, Bergiele AT, Filandra FA, Tsianateli TC, Spina GG, Zapanti ED, Bartizis
MI. A Survey of the Polycystic Ovary Syndrome in the Greek Island of Lesbos: Hormonal and Metabolic
Profile. The Journal of Clinical Endocrinology & Metabolism.1999; 84(11): 4006-11.
[4] Tehrani FR, Simbar M, Tohidi M, Hosseinpanah F, Azizi F. The prevalence of polycystic ovary syndrome in
a community sample of Iranian population: Iranian PCOS prevalence study. Reproductive Biology and
Endocrinology. 2011; 9:39. http://www.rbej.com/content/9/1/39 (Access in 09/17/2012).
[5] Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz B. The Prevalence and Features of the
Polycystic Ovary Syndrome in an Unselected Population. The Journal of Clinical Endocrinology &
Metabolism. 2004; 89(6):2745–2749.
[6] Asunción M, Calvo RM, Millán JLS, Sancho J, Avila S, Escobar-Morreale HF. A Prospective Study of the
Prevalence of the Polycystic Ovary Syndrome in Unselected Caucasian Women from Spain. The Journal of
Clinical Endocrinology & Metabolism. 2000; 85(7): 2434-8.
[7] Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence of the Polycystic
Ovary Syndrome in Unselected Black and White Women of the Southeastern United States: A Prospective
Study. Journal of Clinical Endocrinology and Metabolism. 1998; 83(9): 3078- 82.
[8] Ciccone NA, Kaiser UB. The biology of gonadotroph regulation. Current Opinion in Endocrinology,
Diabetes & Obesity. 2009; 16:321–327.
[9] Pereira Júnior M; Vidotti DB; Borra RC; Simões Mde J; Da Silva ID; Haidar MA. Involvement of GDF-9,
leptin, and IGF1 receptors associated with adipose tissue transplantation on fertility restoration in obese
anovulatory mice. Gynecol Endocrinol. 2011;27(10): 759-66.
[10] Blank SK, Helm KD, McCartney CR, Marshall JC. Polycystic ovary syndrome in adolescence. Ann N Y
Acad Sci. 2008;1135:76-84.
[11] Barontini M, García-Rudaz MC, Veldhuis JD. Mechanisms of Hypothalamic-Pituitary-Gonadal Disruption
in Polycystic Ovarian Syndrome. Archives of Medical Research. 2001; 32:544–552 .
[12] Ehrmann DA. Polycystic Ovary Syndrome. N Engl J Med. 2005; 352:1223-1236.
[13] Legro RS, Driscoll D, Strauss III JF, Fox J, Dunaif A. Evidence for a genetic basis for hyperandrogenemia
in polycystic ovary syndrome. Proc. Natl. Acad. Sci. USA. 1998; 95: 14956–14960 .
[14] Xita N, Georgiou I, Tsatsoulis A. The genetic basis of polycystic ovary syndrome. European Journal of
Endocrinology. 2002;147:717–725.
[15] Nardo LG, Patchava S, Laing I. Polycystic ovary syndrome: pathophysiology, molecular aspects and
clinical implications. Panminerva Med. 2008; 50(4):267-78.
[16] Huhtaniemi IT, Pettersson KSI. Alterations in gonadal steroidogenesis in individuals expressing a common
genetic variant of luteinizing hormone. Journal of Steroid Biochemistry and Molecular Biology.1999;
69:281-285.
[17] Abbott DH, Dumesic DA, Franks S. Developmental origin of polycystic ovary syndrome – a hypothesis.
Journal of Endocrinology.2002;174:1–5.
[18] Franks S, Mccarthy MI, Hardy K. Development of polycystic ovary syndrome: involvement of genetic and
environmental factors. International Journal of Andrology. 2006; 29: 278–285.
[19] Eisner JR, Dumesic DA, Kemnitz JW, Abbott DH. Timing of Prenatal Androgen Excess Determines
Differential Impairment in Insulin Secretion and Action in Adult Female Rhesus Monkeys. The Journal of
Clinical Endocrinology & Metabolism. 2000; 85(3): 1206- 10.
© Medimond . P726C0840
151
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[20] Abbott DH, Barnett DK, Levine JE, Padmanabhan V, Dumesic DA, Jacoris S, Tarantal AF. Endocrine
Antecedents of Polycystic Ovary Syndrome in Fetal and Infant Prenatally Androgenized Female Rhesus
Monkeys. BIOLOGY OF REPRODUCTION. 2008;79:154–163.
[21] Abbott DH, Zhou R, Bird IM, Dumesic DA, Conley AJ. Fetal programming of adrenal androgen excess:
lessons from a nonhuman primate model of polycystic ovary syndrome. Endocr Dev. 2008 ; 13: 145–158.
[22] Abbott DH, Tarantal AF, Dumesic DA. Fetal, infant, adolescent and adult phenotypes of polycystic ovary
syndrome in prenatally androgenized female rhesus monkeys. Am J Primatol. 2009; 71(9): 776–784.
[23] Barker DJP, Clark PM. Fetal undernutrition and disease in later life. Reviews of Reproduction.1997; 2;
105–112.
[24] Barker DJ. Maternal nutrition, fetal nutrition, and disease in later life. Nutrition. 1997; 13(9):807-13.
[25] Ruggiero RN, Bueno-Júnior LS, Ross JB, Fachim HA, Padovan-Neto FE, Merlo S, Rohner CJS, Ikeda ET,
Brusco J, Moreira JE. Neurotransmissão glutamatérgica e plasticidade sináptica: aspectos moleculares,
clínicos e filogenéticos. Medicina (Ribeirão Preto). 2011;44(2): 143-56.
[26] Charles A, Weiner R, Costantin J. cAMP Modulates the Excitability of Immortalized Hypothalamic (GT1)
Neurons via a Cyclic Nucleotide- Gated Channel. Molecular Endocrinology. 2001; 15(6): 997–1009.
[27] Chen Q, Weiner RI, Blackman BE. Decreased Expression of A-Kinase Anchoring Protein 150 in GT1
Neurons Decreases Neuron Excitability and Frequency of Intrinsic Gonadotropin-Releasing Hormone
Pulses. Endocrinology. 2010; 151(1):281–290 .
[28] Krsmanovic LZ, Mores N, Navarro CE, Tomic M , Catt KJ. Regulation of Ca2+-Sensitive Adenylyl Cyclase
in Gonadotropin- Releasing Hormone Neurons. Molecular Endocrinology. 2001; 15(3): 429–440.
[29] Qiu J, Fang Y, Rønnekleiv OK, Kelly MJ. Leptin excites POMC neurons via activation of TRPC channels.
J Neurosci. 2010; 27; 30(4): 1560–1565.
[30] Hsuchou H, He Y, Kastin AJ, Tu H, Markadakis EN, Rogers RC, Fossier PB, Pan W. Obesity induces
functional astrocytic leptin receptors in hypothalamus. Brain. 2009; 132: 889–902.
[31] Jo Y-H, Chen Y-JJ, Chua Jr. SC, Talmage DA, Role LW. Integration of Endocannabinoid and Leptin
Signaling in an Appetite-Related Neural Circuit. Neuron. 2005; 48(6): 1055–1066.
[32] http://www.sabiosciences.com/rt_pcr_product/HTML/PAMM-066A.html (Accessed in 11/04/2012).
[33] Ferhat L, Esclapez M, Represa A, Fattoum A, Shirao T, Ben-Ari Y. Increased levels of
acidic calponin during dendritic spine plasticity after pilocarpine-induced seizures. Hippocampus
2003;13(7):845-58.
[34] Paulussen M, Van Brussel L, Arckens L. Monocular enucleation profoundly reduces secretogranin
II expression in adult mouse visual cortex. Neurochem Int. 2011;59(7):1082-94.
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Gain control in the outer retina
Joselevitch C.1, Kamermans M.2,3
1
Instituto de Psicologia, Universidade de São Paulo (BRAZIL)
Retinal Signal Processing, Netherlands Institute for Neuroscience (THE NETHERLANDS)
3
Department of Neurogenetics, AMC, University of Amsterdam (THE NETHERLANDS)
[email protected]; [email protected]
2
Abstract
Gain control mechanisms are present at all retinal layers. They are especially important for cells that
receive mixed-input from rods and cones, in order to avoid premature saturation as light levels increase. Here we
describe some of the mechanisms at work in the outer plexiform layer that modulate the effectiveness of the
photoreceptor-bipolar cell synapse.
Keywords: vision, retina, gain control, photoreceptors, bipolar cells.
What is gain control and what is it good for?
Gain can be defined as the amplification factor of a physiological response in relation to the stimulus
[1]. In the context of a synapse, gain control is any mechanism that influences synaptic communication, be it by
stabilizing or by modifying this amplification factor. Such adaptive changes are of paramount importance in any
neural circuit: they consolidate synapses, optimize information transfer and spare energy resources. There are
several mechanisms that can modulate synaptic gain, each one most suitable for dealing with particular kinds of
signals. A single synapse may display thus various types of adaptive behavior, depending on the strength, spatial
extent and duration of the stimulus.
Gain control in vision
As in any neural structure, the visual system needs to adapt to the stimulus it receives in order to
optimally use the limited dynamic range of its neurons [2;3]. This is not a trivial task, because neuronal
responses to light stimuli saturate after only two log units, while visual scenes vary widely in absolute luminance
(which could be freely translated into the concept of “brightness”), local luminance (“contrast”) and spectral
distribution (“color”). In addition, the fact that our eyes are in constant movement complicates the task of visual
coding even further. Eye movements imply in abrupt changes in contrast, which could potentially lead to
neuronal saturation [3;4] .
At all levels of the visual system, therefore, one will find several gain control mechanisms at work,
many concomitantly, such as to enable optimal coding of the ever changing visual world [1;4]. In this article, we
concentrate on the gain control mechanisms found at the first retinal synapse, namely the triad formed between
photoreceptors, horizontal cells (HCs) and bipolar cells (BCs). We will further narrow down the scope of this
work by describing post-synaptic mechanisms intrinsic to BCs, since other mechanisms have been more
extensively studied (comprehensive reviews, see [5-7]).
Gain control mechanisms in the outer retina
Because photoreceptors themselves also have limited dynamic range, the visual system uses two kind of
photosensitive neurons with different absolute sensitivity, rods and cones, to bridge the 10 to 12-log-unit light
intensity range one encounters in the course of a day. Each particular type of photoreceptor has a dynamic range
of about 2.5 log in a given adaptive state. In addition, it displays intrinsic adaptation such as to extend its
working range to 6 log unit as light levels change. This way, rods are able to bridge the 6 log-unit range one
encounters from low scotopic to mesopic levels, and cones are able to function for the remaining 6 log units and
up [8;9].
In some species, both mammalian and non-mammalian, there are post-synaptic neurons that connect to
rods and cones and are potentially able to function over a very broad range of light intensities. For those neurons,
controlling the gain of the rod synapse is of paramount importance to allow good signal-to-noise ratio at low
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light levels and at the same time prevent saturation as light levels increase. It is not surprising, therefore, to find
several mechanisms at BC dendrites that modulate the strength of their response to light.
1.1
Post-synaptic mechanisms at BC dendrites
There are two major classes of bipolar cells in the retina, which stratify at different levels of the inner
plexiform layer (Fig. 1): OFF BCs (Fig. 1B) follow the voltage of the photoreceptor, while ON BCs (Fig. 1A)
invert this voltage, transforming the light-induced hyperpolarisation of the photoreceptor into a depolarising
response. A third type of BC is able to both hyperpolarise or depolarise to light stimulation of their receptive
field centre, depending on stimulus wavelength [10] and/or intensity [11].
Fig. 1: BC types. A) Top: Fluorescence micrograph of an ON BC of the goldfish retina filled with Lucifer Yellow (LY) and
still attached to the patch pipette. Bottom: Light responses of the same cell to a 250 μm slit and to a full field at 550 nm and
similar intensities (-0.44 and -0.68 log photons*mm2*s-1, respectively). B) Top: Fluorescence micrograph of an OFF BC
filled with LY and still attached to the recording electrode. Bottom: Light responses of the same cell to a 250 μm slit and to a
full field at 550 nm and similar intensities (-0.11 log and -0.01 log photons*mm2*s-1, respectively). Horizontal bars indicate
stimulus timing (500 ms), vertical bar = 2 mV for (A) and 4 mV for (B). Note that in both cases there are gain control
mechanisms working: BC light responses decrease in amplitude over time, even though the light stimulus is a square pulse.
ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion
cell layer.
The kind of light-induced response at the BC level is determined by the glutamate receptor these cells
express at their dendrites: OFF BCs connect to photoreceptors via AMPA/Kainate ionotropic receptors [12;13],
while ON BCs use mGluR6, a metabotropic receptor [14] coupled to a G protein, Go [15], which modulates the
activity of TRPM1 channels located at the dendritic tips [16-20]. An unusual type of receptor, supposedly
responsible for cone-driven ON BC responses, was described in the fish retina [21]. Its pharmacology is
consistent with that of a glutamate transporter [22], most likely EAAT5 [23], which is associated with a Clconductance. Activation of the glutamate transporter opens the CL-conductance. Although this
transporter/receptor has been classically linked to light-induced depolarisations, it is interesting to note here that
the response polarity depends on the equilibrium potential of Cl-, and can, in principle, go both ways.
It has long been postulated that the division into ON- and OFF channels might subserve the codification
of positive and negative contrasts, respectively (for a discussion, see [2]). Although this might hold true at the
ganglion cell level, ON and OFF BC responses perform equally well for positive and negative contrasts,
responding with much higher gain for small contrasts than for large ones [24]. This could be explained by the
fact that in both kinds of neurons the gain at threshold is maximal [25], despite their differences in receptor
makeup, although the latter renders amplification within ON BCs larger [26].
Nonetheless, because of the varying nature of the light response in these BCs, it is only logical that
different gain control mechanisms are used by each kind of neuron. The effect is however similar: a modulation
of response amplitude in time and at different adaptive states, which changes both amplification and kinetics
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(Fig.1). This way, the retina generates at the first synapse several channels aimed at amplifying different parts of
the photoreceptor dynamic range and at transmitting information at different speeds, effectively covering
complementary parts of the visual continuum [27].
1.1.1
Voltage-independent voltage control
Rod-cone convergence is of special interest here because it can also act as a gain control mechanism
[25]. The effect of one input onto the gain of the other greatly depends on BC receptor makeup, even when rods
and cones drive the same kind of glutamate receptor. For instance, in mixed-input OFF BCs in which both
photoreceptors drove AMPA/Kainate receptors, there would be a push-pull mechanism between rod and conedriven synapses. Despite a mutual shunt in darkness, dim backgrounds that only hyperpolarise rods slightly
would increase the gain of cone synapses, because the closure of rod-driven channels would augment the driving
force for the cone conductance. For mixed-input ON BCs in which both receptors drove mGluR6, however, the
effect would be different. In addition to the shunting effect of the few cone-driven channels that are open in
darkness, dim backgrounds would only worsen the situation, because the rod-driven depolarisation would
decrease the gain of both rod and cone synapses.
In this sense, the glutamate transporter found in cone synapses of fish mixed-input ON BCs presents an
interesting case. Because in these cells the transporter current is inward in darkness and has a negative reversal
potential [22;28], cone convergence would keep these neurons hyperpolarised at light levels to which cones are
not sensitive, effectively increasing the gain of rod-driven, mGluR6-mediated responses. With increasing
backgrounds, the gain of rod-driven responses would decrease due to the opening of rod-driven channels; at the
same time, the gain of cone-driven synapses would increase, since the open rod-driven channels would
depolarise the cell and augment the driving force for the cone conductance.
While mGluR6 allows for great response amplification due to the intracellular cascade it activates, its
use necessarily implies in response delays due to the time needed to activate and deactivate each cascade step
[29]. In ON BCs, therefore, one finds several mechanisms that aim at speeding up response times. One of them
is the direct closure of the membrane channels modulated by mGluR6 by Ca2+ ions [30], which permeate the cell
when TRPM1 channels are open [31]. The gain decrease caused by Ca2+ entry serves a dual purpose: at low light
levels, it transiently repolarises the cells, effectively speeding up signal transmission [32;33]; as mean light
levels increase, it decreases the size of the BC response to light and reduces the gain of the photoreceptor-BC
synapse by keeping part of the TRPM1 channel pool tonically closed [30], an effect analogous to response
compression in photoreceptors [5].
The amplification yielded by the mGluR6 cascade in ON BCs can also be modulated intracellularly by
cGMP levels (for a review, see [34]). An increase in intracellular cGMP augments responses near threshold,
boosting ON BC responses to dim light stimulation, but not for more intense stimulation [35]. This increase in
gain at low light levels would increase light detection at low scotopic levels. As a result of amplification, the
latency of ON BC responses is reduced when cGMP levels are high. It remains to be determined which the
intracellular targets of cGMP are and how the concentration of this nucleotide is modulated at different light
levels.
1.1.2
Voltage-dependent voltage control
Voltage-dependent currents interact with BC light responses in two ways: they can either boost voltage
responses for larger neurotransmitter release, or they can dampen light responses for faster signal transmission.
Action potentials do both things due to the interplay between their several underlying voltage-gated
conductances. BCs were long thought as non spiking neurons, but it turns out that some ON BCs express
voltage-gated Na+ channels [36] and are able of generating classical action potentials [37]. This feature might be
especially useful for cells with very thin axons, which could potentially filter small, graded voltage changes. In
addition, mixed-input ON BCs of the fish retina were shown to generate Ca2+- dependent spikes at their axon
terminals under some circumstances [38;39]. The significance of spiking for those cells is less clear, since their
axons are fairly thick and do not seem to act as a filter [40]. However, the fact that light-driven Ca2+ spikes are
more frequently observed in younger larvae [41] raises the possibility that this feature might play a bigger role in
development.
Response dampening due to the action of voltage-gated conductances is also a powerful gain control
mechanism. Outwardly rectifying K+ channels were shown to determine BC response characteristics in ON and
OFF BCs [42;43]. Injecting currents into these cells lead to a decrease in static gain, defined as the voltage
response to a sustained input increment, and to increased response speed. Both effects can be abolished by agents
known to block voltage-gated K+ channels. Driving BCs away from their resting membrane potential, therefore,
leads to the activation of voltage-gated currents that change significantly their response properties. Since these
effects can be observed in isolated cells, they nicely demonstrate that BC voltage-dependent currents play a key
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role in part of their adaptive response to light stimulation, preventing premature saturation and/or regulating the
gain of individual rod and cone synapses according to mean light levels for BCs that receive mixed input.
Conclusions
Already at the first retinal synapse, a plethora of gain control mechanisms coexist in order to enable the
visual system to respond in a constant way to the constantly changing image that falls onto the retina. Together,
they shape BC output and determine which information is filtered out and which is sent to ganglion cells – and
ultimately to the brain – for further processing.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Shapley,R.M. & Enroth-Cugell,C. (1984) Visual adaptation and retinal gain controls. Progress in Retinal
Research 3, pp. 263-343.
Barlow,H.B. (1981) The Ferrier Lecture, 1980. Critical limiting factors in the design of the eye and visual
cortex. Proceedings of the Royal Society of London. Series B, Containing Papers of Biological Character
212, pp. 1-34.
Rieke,F. & Rudd,M.E. (2009) The challenges natural images pose for visual adaptation. Neuron 64, pp.
605-616.
Demb,J.B. (2008) Functional circuitry of visual adaptation in the retina. The Journal of Physiology 586,
pp. 4377-4384.
Burns,M.E. & Lamb,T.D. (2003) Visual transduction by rod and cone photoreceptors. In: The visual
neurosciences (Chalupa,L.M. & Werner,J.S., eds), Cambdridge, MIT Press, pp. 215-233.
Klaassen,L., Fahrenfort,I., & Kamermans,M. (2012) Connexin hemichannel mediated ephaptic inhibition
in the retina. Brain Research . In press.
Thoreson,W.B. & Mangel,S.C. (2012) Lateral interactions in the outer retina. Progress in Retinal and Eye
Research 31, pp. 407-441.
Burkhardt,D.A. (1994) Light adaptation and photopigment bleaching in cone photoreceptors in situ in the
retina of the turtle. The Journal of Neuroscience 14, pp. 1091-1105.
Rodieck,R.W. (1998) The first steps in seeing., Sunderland Sinauer Associates,
Kaneko,A. (1973) Receptive field organization of bipolar and amacrine cells in the goldfish retina. The
Journal of Physiology 235, pp. 133-153.
Joselevitch,C. & Kamermans,M. (2007) Interaction between rod and cone inputs in mixed-input bipolar
cells in goldfish retina. Journal of Neuroscience Research 85, pp. 1579-1591.
DeVries,S.H. & Schwartz,E.A. (1999) Kainate receptors mediate synaptic transmission between cones
and 'Off' bipolar cells in a mammalian retina. Nature 397, pp. 157-160.
DeVries,S.H. (2000) Bipolar cells use kainate and AMPA receptors to filter visual information into
separate channels. Neuron 28, pp. 847-856.
Nakajima,Y., Iwakabe,H., Akazawa,C., Nawa,H., Shigemoto,R., Mizuno,N., & Nakanishi,S. (1993)
Molecular characterization of a novel retinal metabotropic glutamate receptor mglur6 with a high agonist
selectivity for l-2- amino-4-phosphonobutyrate. The Journal of Biological Chemistry 268, pp. 1186811873.
Dhingra,A., Lyubarsky,A., Jiang,M., Pugh,E.N., Jr., Birnbaumer,L., Sterling,P., & Vardi,N. (2000) The
light response of ON bipolar neurons requires G[alpha]o. The Journal of Neuroscience 20, pp. 90539058.
Li,Z., Sergouniotis,P.I., Michaelides,M., Mackay,D.S., Wright,G.A., Devery,S., Moore,A.T.,
Holder,G.E., Robson,A.G., & Webster,A.R. (2009) Recessive mutations of the gene TRPM1 abrogate
ON bipolar cell function and cause complete congenital stationary night blindness in humans. American
Journal of Human Genetics 85, pp. 711-719.
Morgans,C.W., Zhang,J., Jeffrey,B.G., Nelson,S.M., Burke,N.S., Duvoisin,R.M., & Brown,R.L. (2009)
TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells. Proc. Natl. Acad. Sci.
U. S. A 106, pp. 19174-19178.
Shen,Y., Heimel,J.A., Kamermans,M., Peachey,N.S., Gregg,R.G., & Nawy,S. (2009) A transient receptor
potential-like channel mediates synaptic transmission in rod bipolar cells. The Journal of Neuroscience
29, pp. 6088-6093.
van Genderen,M.M., Bijveld,M.M., Claassen,Y.B., Florijn,R.J., Pearring,J.N., Meire,F.M., McCall,M.A.,
Riemslag,F.C., Gregg,R.G., Bergen,A.A., & Kamermans,M. (2009) Mutations in TRPM1 are a common
© Medimond . P726R9026
156
ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil)
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
cause of complete congenital stationary night blindness. American Journal of Human Genetics 85, pp.
730-736.
Koike,C., Obara,T., Uriu,Y., Numata,T., Sanuki,R., Miyata,K., Koyasu,T., Ueno,S., Funabiki,K., Tani,A.,
Ueda,H., Kondo,M., Mori,Y., Tachibana,M., & Furukawa,T. (2010) TRPM1 is a component of the retinal
ON bipolar cell transduction channel in the mGluR6 cascade. Proc. Natl. Acad. Sci. U. S. A 107, pp. 332337.
Saito,T., Kondo,H., & Toyoda,J.I. (1978) Rod and cone signals in the On-center bipolar cell: their
different ionic mechanisms. Vision Research 18, pp. 591-595.
Grant,G.B. & Dowling,J.E. (1995) A glutamate-activated chloride current in cone-driven ON bipolar cells
of the white perch retina. The Journal of Neuroscience 15, pp. 3852-3862.
Arriza,J.L., Eliasof,S., Kavanaugh,M.P., & Amara,S.G. (1997) Excitatory amino acid transporter 5, a
retinal glutamate transporter coupled to a chloride conductance. Proceedings of the National Academy of
Sciences of the United States of America 94, pp. 4155-4160.
Burkhardt,D.A. (2011) Contrast processing by ON and OFF bipolar cells. Visual Neuroscience 28, pp.
69-75.
Falk,G. (1988) Signal transmission from rods to bipolar and horizontal cells: a synthesis. Progress in
Retinal Research 8, pp. 255-279.
Ashmore,J.F. & Falk,G. (1980) Response of rod bipolar cells in the dark-adapted retina of the dogfish,
Scyliorhinus canicula. The Journal of Physiology 300, pp. 115-150.
Sterling,P. (2004) How retinal circuits optimize the transfer of visual information. In: The Visual
Neurosciences (Chalupa,L.M. & Werner,J.S., eds), Cambridge, MIT Press, pp. 234-259.
Saito,T., Kondo,H., & Toyoda,J.I. (1979) Ionic mechanisms of two types of On-center bipolar cells in the
carp retina. I. The responses to central illumination. The Journal of General Physiology 73, pp. 73-90.
Kim,H.G. & Miller,R.F. (1993) Properties of synaptic transmission from photoreceptors to bipolar cells
in the mudpuppy retina. Journal of Neurophysiology 69(2), pp. 352-360.
Shiells,R.A. & Falk,G. (1999) A rise in intracellular Ca2+ underlies light adaptation in dogfish retinal 'on'
bipolar cells. The Journal of Physiology 514 ( Pt 2), pp. 343-350.
Nawy,S. (2000) Regulation of the on bipolar cell mGluR6 pathway by Ca2+. The Journal of
Neuroscience 20, pp. 4471-4479.
Berntson,A., Smith,R.G., & Taylor,W.R. (2004) Postsynaptic calcium feedback between rods and rod
bipolar cells in the mouse retina. Visual Neuroscience 21, pp. 913-924.
Nawy,S. (2004) Desensitization of the mGluR6 transduction current in tiger salamander On bipolar cells.
The Journal of Physiology 558, pp. 137-146.
Snellman,J., Kaur,T., Shen,Y., & Nawy,S. (2008) Regulation of ON bipolar cell activity. Progress in
Retinal and Eye Research 27, pp. 450-463.
Shiells,R.A. & Falk,G. (2002) Potentiation of 'on' bipolar cell flash responses by dim background light
and cGMP in dogfish retinal slices. The Journal of Physiology 542, pp. 211-220.
Zenisek,D., Henry,D., Studholme,K., Yazulla,S., & Matthews,G. (2001) Voltage-dependent sodium
channels are expressed in nonspiking retinal bipolar neurons. The Journal of Neuroscience 21, pp. 45434550.
Saszik,S. & DeVries,S.H. (2012) A mammalian retinal bipolar cell uses both graded changes in
membrane voltage and all-or-nothing Na+ spikes to encode light. The Journal of Neuroscience 32, pp.
297-307.
Zenisek,D. & Matthews,G. (1997) Calcium action potentials in retinal bipolar neurons. Visual
Neuroscience 15, pp. 69-75.
Protti,D.A., Flores-Herr,N., & Von Gersdorff,H. (2000) Light evokes Ca2+ spikes in the axon terminal of
a retinal bipolar cell. Neuron 25, pp. 215-227.
Arai,I., Tanaka,M., & Tachibana,M. (2010) Active roles of electrically coupled bipolar cell network in
the adult retina. The Journal of Neuroscience 30, pp. 9260-9270.
Dreosti,E., Esposti,F., Baden,T., & Lagnado,L. (2011) In vivo evidence that retinal bipolar cells generate
spikes modulated by light. Nature Neuroscience 14, pp. 951-952.
Mao,B.Q., MacLeish,P.R., & Victor,J.D. (1998) The intrinsic dynamics of retinal bipolar cells isolated
from tiger salamander. Visual Neuroscience 15, pp. 425-438.
Mao,B.Q., MacLeish,P.R., & Victor,J.D. (2002) Relation between potassium-channel kinetics and the
intrinsic dynamics in isolated retinal bipolar cells. Journal of Computational Neuroscience 12, pp. 147163.
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Cell death and in vivo melanoma tumor growth
remission determined by a snake toxin with
specificity for actively proliferating cells
Sancey L.1, Márcia Neiva2, Nascimento F.D.3, Costa B.A.2, Pereira A.4,
Oliveira E.B.5, Tersariol I.L.S.6, Coll J.L.1, Kerkis I.4, Hayashi M.A.F.2
1
INSERM U823, Institut Albert Bonniot, Grenoble, France;
Depto. Farmacologia, Universidade Federal de São Paulo (UNIFESP-EPM), São Paulo, SP, Brazil;
3
Grupo de Estudos em Odontologia, Universidade Bandeirante de São Paulo (UNIBAN), São Paulo, SP, Brazil;
4
Lab. Genetics, Butantan Institute, São Paulo, SP, Brazil;
5
Depto. Bioquímica e Imunologia, Universidade de São Paulo (USP), Ribeirão Preto, Brazil;
6
Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes, Mogi das
Cruzes, SP, Brazil;
2
Abstract
Crotamine is a toxin from the venom of a South American rattlesnake with cell-penetrating property, a
dose-dependent cytotoxic activity, and an interesting specificity for actively proliferating cells, demonstrated by
our group both in vitro and in vivo. Evidences showing that the cytotoxic effect of crotamine is dependent on the
cell internalization of the toxin, followed by endocytic vesicle lysis and subsequent release and activation of
proteases in the cytoplasm were previously demonstrated [Hayashi et al., Toxicon 2008]. More recently, we also
showed the involvement of mitochondrial depolarization followed by rapid intracellular calcium transients in the
cell death triggered by crotamine. Use of selective inhibitors of different calcium mobilization pathways, also
indicated the involvement of mitochondria, lysosomes and endoplasmic reticulum, as well as of extracellular
calcium entry for the calcium-mobilizing effect of crotamine. Considering that we have previously shown that
crotamine also carries and delivers nucleic acid molecules into cells, we suggest that crotamine can be used for a
dual purpose: to target and detect growing tumor tissues as well as to selectively trigger cell death of tumor cells,
potentially carrying therapeutic genes. Moreover, the ability of crotamine to target proliferating tumor cells in
vitro as well as in vivo, demonstrated by intraperitoneal injection of labeled crotamine allowing observe an
efficient targeting of remote subcutaneous tumors engrafted in the mice hind limb flank, stimulated us to
evaluate the anticancer cells toxicity both in vitro and in vivo and its efficacy to inhibit the tumor growth in
mouse model of melanoma was shown. Thus we believe that crotamine belongs to a class of natural peptides
with a potential interest as theranostic compound with potential application for both cancer diagnosis and
treatment.
Introduction
Crotamine is a polypeptide present in the venom of a South American rattlesnake Crotalus durissus
terrificus. It is a shot molecule composed by 42 amino acid residues and the main structural characteristics of
this molecule are the high content of basic amino acid residues, mainly lysine and arginine, and the compact 3Dstructure folded by three intramolecular disulfide bonds. The first biological activity of crotamine described was
the paralysis of the hind limb in rodents that led to classify crotamine as a myotoxin [1].
Posterior investigations of the crotamine biological properties showed that this toxin is a natural cell
penetrating peptide (CPP). The first CPPs described were found within a domain of larger proteins, for instance
the Antennapedia homeobox protein and the TAT protein from HIV-1 [2]. Crotamine is the first CPP with
defined 3D-structure described in the literature, and it is ability to penetrate mammalian cells with a particular
preference to actively proliferating (AP) cells, both in vitro and in vivo, was demonstrated by our group
[3][4][5].
Studies of the mechanisms of internalization of crotamine indicated the involvement of endocytosis and
the essential participation of cell membrane heparan sulfate proteoglycans [4]. The interaction of crotamine with
nucleic acid molecules and its ability to carry and deliver them into living cells was also demonstrated [4].
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The cytotoxicity of crotamine was evaluated in vitro and in vivo and the toxic effect was found to be
cell and dose-dependent. The cytotoxicity depends on the cell internalization of the toxin, which triggers
endocytic vesicle lysis and a subsequent activation of proteases in the cytoplasm [5]. It was observed that the
toxin also induces mitochondrial depolarization and rapid intracellular calcium transients [6]. The use of
selective inhibitors of different calcium mobilization pathways indicated the involvement of mitochondria,
lysosomes and endoplasmic reticulum, as well as of extracellular calcium entry [6]. Together, these events
triggered by crotamine induce the cell death, probably converging to apoptosis pathway.
The search for specific therapeutic drugs that act exclusively on tumor tissues and that are not harmful
to normal cells is one of the main goals of researches in the field. To achieve this purpose, the main strategies
currently employed are based on the use of antibodies against surface markers or ligands for receptors and
proteins preferentially expressed on the surface of tumor cells in the target tissue [7][8]. Although these therapy
antibodies can successfully target tumors [9], their use is hindered by limitations as genetic instability of the
tumor cells and lack of amplification, since each target molecule binds at most one single probe [10]. Due to
these limitations, the use of nanoparticles was proposed, but clinical investigations about its efficacy and toxicity
are still required [11]. Another strategy that has been explored is the use of small specific ligands like RGD
peptides as delivery drug system [12][13].
The recently demonstrated in vitro and in vivo specific cancer cells cytotoxicity of crotamine
[5][14][15] and also its ability to target highly proliferating tumor cells confirmed by its efficient targeting of
remote subcutaneous tumors engrafted in the flank of nude mice [6] allowed us to confirm that this natural
peptide owns all features expected for a potential application for cancer diagnosis and treatment [6]. This could
be further confirmed by the effective anti-tumor activity observed for crotamine in mice animal model bearing
melanoma tumor cells, which was able to significantly inhibit tumor growth evaluated by measuring the tumor
mass weight [14].
In this context, supported by our data, we suggest the use of crotamine for a dual purpose: to target and
detect growing tumor tissues, as well as to selectively trigger cell death of tumor cells, potentially carrying
therapeutic genes.
Methodology
2.1. Materials
Cell culture medium and supplements were from Invitrogen (Gaithersburg, MD, USA). Crotalus
durissus terrificus venom was extracted from snakes maintained at the Faculdade de Medicina de Ribeirão Preto
(FMRP) serpentarium, São Paulo University and crotamine was obtained essentially as previously described
[16]. All chemicals, when not specified, were from Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Cell culture
B16-F10 murine melanoma cells were cultured in RPMI (Invitrogen Corporation, Carlsbad, CA, USA),
supplemented with 10% fetal bovine serum (FBS, LGC, Biotecnologia, São Paulo, Brazil) without antibiotics.
Culture medium was changed every 3 days. The cell cultures were maintained at 37C in a humidified
atmosphere with 5% CO2.
2.3. Cell viability assay
Cell viability after exposure to crotamine was examined using the MTT assay, whereby metabolically
active mitochondrial dehydrogenases convert the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, MO, USA) to insoluble purple formazan crystals
at 298 nm, which was proportional to cell viability. Primary culture of mice astrocyte cells, rat
pheochromocytoma PC12 cells, Chinese hamster ovary CHO-K1 cells, and tumoral mice melanoma B16F10
cells maintained in appropriate culture media supplemented with 10% of FBS were used for cytotoxicity analysis
of crotamine, as previously described [5]. Percent of cytotoxicity was calculated as 100  (1-[optical density at
570-620 nm with toxin]/[optical density at 570-620 nm without toxin]). Results are expressed as mean values ±
SD of three independent experiments.
2.4. Mitochondrial membrane potential analysis
Mitochondrial activity was determined by using both the fluorescent probe tetramethylrhodamine,
methyl ester, perchlorate (TMRM) (Molecular Probes®, Life Technologies, Grand Island, NY, USA) and the
mitochondria potential sensor JC-1 (#T3168; Invitrogen, Carlsbad, CA, USA). Detection of the loss of orangered fluorescence after treatment with 5 µM crotamine, in TMRM stained B16F10 cells (100 nM of TMRM for
30 min at 37C, followed by washing with PBS) was performed using the red channel (e.g. excitation 595 nm
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and emission: band-pass filter 610-635 nM). Quantification of the mitochondrial membrane potential was
determined by measuring the cellular uptake of the fluorescent dye JC-1 after treatment with 1 µM crotamine.
For quantitative analysis, at least 10 regions of interest (ROIs) were selected to quantify changes at 488 and 543
nm. All ROIs comprised cells chosen by the criteria of well-defined cellular limits, clear identification of the
nucleus, and absence of intersection with neighboring cells. The size, number of pixels, and ratios between
fluorescence intensity in the red (high membrane potential) and green (low membrane potential) channels in each
ROI were calculated. Data are expressed as mean values of three independent experiments ± SD. Pictures were
taken using an inverted LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a 40.0  1.25
numerical aperture (N.A.) oil immersion objective, essentially as previously described [6].
2.5. Labeling of crotamine with fluorescent dye
Fluorescent crotamine derivatives were prepared using the Alexa-700 Fluor dye (Invitrogen, Carlsbad,
CA, USA) following the instructions of the manufacturer.
2.6. In vivo intratumoral localization of crotamine
All animals were caged and handled ethically according to international rules of animal care, stated by
the International Animal Welfare Recommendations, in accordance with the Guidelines for the Use of Animals
in Biochemical Research [17], and were approved by the Institutional Ethics Committee (CEP No. 0649/11; Sep
4th, 2012). B16F10 cells (105 cells/200 μL) were subcutaneously injected into 5-weeks old C57/Bl/6 males
weighting about 16-18 g. Crotamine-Alexa 700 was intraperitoneally (IP) injected into mice (2.5 μg per animal)
bearing tumor, which were euthanized and dissected 24 h after injection. Fluorescence reflectance imaging (2DFRI) was carried out in mice anesthetized with 3.5/4% isoflurane/oxygen for induction and 1.5/2% thereafter
(CSP, Cournon, France). A crotamine-Alexa 700 solution (10 μg/2 nmol in 100 μL/animal) was injected IP at the
beginning of the experiment. Mice were illuminated with 660 nm light-emitting diodes equipped with
interference filters, and both fluorescence images and black & white pictures were acquired by a back-thinned
CCD camera at -80C (ORCAII-BT-512G, Hamamatsu, Massy, France) fitted with a high-pass RG 9 filter
(Schott, Clichy, France). At the end of the experiment, mice were euthanized and dissected to better visualize the
co-localization of crotamine-Alexa 700 with the bioluminescent tumor cells in the tumor tissue.
2.7. Histological analysis of mouse tissues
To assess the localization of crotamine in tumor tissue formed by SC injection of TS/A tumor cells,
crotamine-Alexa 700 IP injected mice were sacrificed and the tumor was removed and fixed. Frozen sections
from excised tumor nodules were obtained using a cryo-microtome (Model CM 1100, Leica, Germany) and were
analyzed by LSM 510 confocal microscopy.
2.8. In vivo tumor growth arrest in response to crotamine treatment
Groups of 5 mice (male), 4-weeks old and weighing between 14 to 16 g were used. Each animal
received 105 B16F10 cells (200 μL PBS) by subcutaneous injection. The crotamine-treated group was treated
with 1 μg of crotamine in 200 μL of saline solution per animal (by IP injection) during 15 days, starting 24 h
after cells inoculation. The second group (control) received only saline solution (200 μL). After last injection, the
animals were euthanized and the tumor tissues samples were evaluated, measuring the size and weight. Before
the inoculations, the groups were weighted and their body mass were daily measured during 7 days.
Results
3.1 In vitro cytotoxic effect of crotamine on several cultured cells
The toxic concentration of crotamine for several cell lines was determined after incubation with 1 to 50
μg/ml concentrations of native toxin during 24 hours, followed by addition of MTT solution and subsequent
reading after 4 hours. The more pronounced cytotoxic effect of crotamine to highly proliferating cells as the
Chinese hamster ovary CHO-K1 and tumoral mice melanoma B16F10 cells cultured in the presence of 10% of
FBS compared to several normal cell lines namely mice astrocyte primary culture cells, and rat
pheochromocytoma PC12 cells, is demonstrated (Figure 1).
3.2 Assessment of crotamine effect on the mitochondrial potential
Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the
respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent
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death. Several fluorescent probes (JC-1 and TMRM,) can be used to determine Δψm in a variety of cell types.
The change in fluorescence intensity reflects the change in relative levels of ΔΨm.
Figure 1. Cytotoxic effect of crotamine. Primary culture of mice astrocyte cells, rat pheochromocytoma PC12 cells, Chinese
hamster ovary CHO-K1 cells and tumoral mice melanoma B16F10 cells were incubated with 0, 10, 20 and 50 µM of
crotamine for 24 h, and the cell survival rate was measured by the MTT assay. The percentage of cell growth in the control
group was designated as 100%. Results are expressed as mean values ± SD of three independent experiments.
Tetramethylrhodamine methyl ester (TMRM) is a potentiometric, cell-permeable fluorescent indicator that
accumulates in the highly negatively charged interior of mitochondria. Membrane potential-driven accumulation of
TMRM within the inner membrane region of healthy functioning mitochondria results in a dramatic increase in
TMRM-associated orange fluorescence. Detection of the loss of orange-red fluorescence in TMRM stained cells is
a reliable method for assessing apoptosis induction or oxidative stress-induced mitochondrial depolarization in
experimental cell populations [18]. The lower levels of TMRM fluorescence resulting from 5 µM crotamine
treatment reflect the depolarization of mitochondrial membrane potential (Figure 2).
Figure 2. Mitochondrial membrane potential depolarization evaluated by fluorescence microscopy. Mitochondrial
potential of B16F10 cells after treatment with 5 µM crotamine was monitored by the detection of the loss of orange-red
fluorescence in TMRM stained cells. This reliable method for assessing apoptosis induction was performed using the red
channel (e.g. excitation 595 nm and emission: band-pass filter 610-635 nM) in an inverted LSM 510 confocal microscope
(Carl Zeiss, Jena, Germany) equipped with a 40.0  1.25 numerical aperture (N.A.) oil immersion objective. Mitochondrial
depolarization could be monitored by the time-course decrease of the red fluorescence after the treatment with crotamine, as
shown by the quantification represented in the graphic below.
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JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a
fluorescence emission shift from green (500 to 550 nm) to red (565 to 615 nm). At low membrane potentials, JC1 produces green fluorescence; at high membrane potentials, it forms ‘J-aggregates’ with red fluorescence
correlated with mitochondrial activity. A decrease after crotamine treatment in the red/green fluorescence
intensity ratio of about 3.35-fold is shown (Figure 3).
Figure 3. Mitochondrial potential determined by biphotonic fluorescence microscopy. Mitochondrial potential of B16F10
cells before (A) and after (B) treatment with 1 µM crotamine evaluated with JC-1 dye. Mitochondrial polarization could be
monitored by the red (functional) or green (depolarized) fluorescence. Red/green ratios before and after treatment with
crotamine are shown below. Results are expressed as means ± SD.
3.3 In vivo crotamine uptake into tumors
Non-invasive real-time optical imaging was used for tracking the specific localization of fluorescently
labeled crotamine to tumors. The accumulation of crotamine into both B16F10 and TS/A-pc SC tumors after IP
injection of crotamine-Alexa 700 was shown [6]. High crotamine uptake was observed macroscopically in TS/Apc tumors by 2D-FRI in living animal, the best tumor-to-background fluorescence ratio was observed 3 h after
crotamine-Alexa 700 injection (Figure 4A-B). Cryostat sections analysis of the excised tumor tissue in confocal
microscopy also confirmed the presence of crotamine-Alexa 700 preferentially in the proliferating cells localized
at the peripheral region of the tumor mass (Figure 4C-E).
Figure 4. Uptake of IP injected crotamine-Alexa 700 by subcutaneously injected murine breast TS/A-pc tumors in live
animal. 2D-FRI optical imaging of TS/A-pc tumors-bearing mice was obtained about 3 h after fluorescent crotamine IP
injection (10 mol/100 L/mouse), showing a major tumor uptake of the crotamine. (A) B/W: Black and white imaging of the
mice; (B) 2D-FRI imaging of tumor-bearing mice; (C-E) crotamine-Alexa 700 accumulated in this same TS/A-pc tumors.
Following removal of TS/A-pc tumors, frozen slices of this tissue were analyzed under fluorescent microscopy. (E) Merged
images of C & D obtained by (D) differential interference contrast (DIC) and (C) fluorescent confocal microscopy (Fm).
Scale bars: C-E = 20 µm.
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Mice with tumors engrafted by IP injection of bioluminescent TS/A-pc-pGL3 cells were also monitored
2D-FRI imaging after IP injection of crotamine-Alexa 700. Overlapping bioluminescence (green) and crotamineAlexa 700 fluorescence (red) was observed in tumor tissues, either in living animals lying on their sides or lying
on their back, and after sacrifice and organs exposure or in dissected tumors (Figure 5).
Figure 5. Optical imaging of IP TS/A-pc-pGL3 tumor-bearing mice showing a specific accumulation of crotamine in
tumor tissues. Mice with IP TS/A-pc-pGL3 tumors were IP injected with luciferin (green, A) and 10 mol crotamine-Alexa
700/mice (red, B) 5 min and 3 h and before optical imaging, respectively. Co-localization (yellow, C) of bioluminescence and
crotamine fluorescence in tumors could be observed in live animals. Pictures were taken with the animals lying on their sides
or lying on their back and, after sacrifice, of the internal organs exposed and respective dissected tumors.
Chronic treatment (daily IP injection of 1 µg of crotamine per animal) started at the first day after 105
B16F10 melanoma cells subcutaneous injection allowed observing that crotamine significantly inhibits the tumor
growth, as evaluated by measuring the tumor mass weight, and also prolongs the lifespan of these mice bearing
B16F10 tumor (Figure 6). All together these data indicate that crotamine could potentially be used for a dual
purpose: to target and detect growing tumor tissues and also to selectively trigger tumor cell death due to its
selective cytotoxic effect [6].
Figure 6. In vivo crotamine anti-tumor activity in mouse animal model. A) Mouse bearing subcutaneous melanoma tumor
15 days after injection of 105 B16F10 cells. The tumor nodules were dissected to evaluate their size without (control, B) and
with (C) crotamine treatment by daily IP injection of 1 µg/mice. Mice were sacrificed and the tumors size determined by
weight determination (D).
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Acknoledgement: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We are grateful to
Marcela B. Nering and Francisco R. dos Santos for the technical assistance.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Gonçalves, J.M., Arantes, E.G. (1956). Estudos sobre venenos de serpentes brasileiras. III. Determinação
quantitativa de crotamina no veneno de cascavel brasileira. An Acad Bras Cien. 28, pp. 369-371.
Kerkis A, Hayashi MA, Yamane T, Kerkis I. (2006). Properties of cell penetrating peptides (CPPs).
IUBMB Life. 58(1), pp. 7-13.
Kerkis, A., Kerkis, I., Rádis-Baptista, G., Oliveira, E.B., Vianna-Morgante, A. M., Pereira, L.V.,
Yamane, T. (2004). Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus
durissus terrificus. FASEB J. 18, pp. 1407-1409.
Nascimento, F.D., Hayashi, M.A., Kerkis, A., Oliveira, V., Oliveira, E.B., Rádis-Baptista,G., Nader,
H.B., Yamane, T., Tersariol, I.L., Kerkis, I. (2007). Crotamine mediates gene delivery into cells through
the binding to heparan sulfate proteoglycans. J. Biol. Chem. 282, pp. 21349-21360.
Hayashi, M.A., Nascimento, F.D., Kerkis, A., Oliveira, V., Oliveira, E.B., Pereira, A., Rádis-Baptista, G.,
Nader, H.B., Yamane, T., Kerkis, I., Tersariol, I.L. (2008). Cytotoxic effects of crotamine are mediated
through lysosomal membrane permeabilization. Toxicon 52, pp. 508-517.
Nascimento FD, Sancey L, Pereira A, Rome C, Oliveira V, Oliveira EB, Nader HB, Yamane T, Kerkis I,
Tersariol IL, Coll JL, Hayashi MA. (2012). The natural cell-penetrating peptide crotamine targets tumor
tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells. Mol Pharm. 9(2), pp.
211-21.
Albrecht, H., Radosevich, J.A., Babich, M. (2009). Fundamentals of antibody-related therapy and
diagnostics. Drugs Today 45, pp. 199-211.
Eberle, A.N., Mild, G. (2009). Receptor-mediated tumor targeting with radiopeptides. Part 1. General
principles and methods. J. Recept. Signal Transduct. Res. 29, pp. 1-37.
Griggs, J., Zinkewich-Peotti, K. (2009). The state of the art: immune-mediated mechanisms of
monoclonal antibodies in cancer therapy. Br. J. Cancer 101, pp. 1807-1812.
Ko, E.C., Wang, X., Ferrone, S. (2003). Immunotherapy of malignant diseases. Challenges and strategies.
Int. Arch. Allergy Immunol. 132, pp. 294-309.
Ting, G., Chang, C.H., Wang, H.E. (2009). Cancer nanotargeted radiopharmaceuticals for tumor imaging
and therapy. Anticancer Res. 29, pp. 4107-4118.
Jin, Z.H., Josserand, V., Foillard, S., Boturyn, D.,Dumy, P., Favrot, M.C., Coll, J.L. (2007). In vivo
optical imaging of integrin avb3 in mice using multivalent or monovalent cRGD targeting vectors. Mol.
Cancer 6, pp. 41.
Foillard, S., Dumy, P., Boturyn, D. (2009). Highly efficient cell adhesion on beads functionalized with
clustered peptide ligands. Org. Biomol. Chem. 7, pp. 4159-4162.
Pereira, A. Kerkis, A., Hayashi, M.A.F, Pereira, A.S.P., Silva, F.S., Oliveira, E.B, Prieto da Silva, A.R.B.,
Yamane, T., Rádis-Baptista, G., Kerkis, I. (2011). Crotamine toxicity and efficacy in mouse models of
melanoma. Expert Opin. Investig. Drugs 20(9), pp. 1189-1200.
Yamane ES, Bizerra FC, Oliveira EB, Moreira JT, Rajabi M, Nunes GL, de Souza AO, da Silva ID,
Yamane T, Karpel RL, Silva PI Jr, Hayashi MA. (2012). Unraveling the antifungal activity of a South
American rattlesnake toxin crotamine. Biochimie. [Epub ahead of print].
Hayashi MA, Oliveira EB, Kerkis I, Karpel RL. (2012). Crotamine: a novel cell-penetrating polypeptide
nanocarrier with potential anti-cancer and biotechnological applications. Methods Mol Biol. 906, pp. 33752.
Giles, A. R. Guidelines for the use of animals in biomedical research. (1987). Thromb. Haemostasis 58,
pp. 1078−1084.
Wlodkowic, D., J. Skommer, and J. Pelkonen. (2006). Multiparametric analysis of HA14-1 induced
apoptosis in follicular lymphoma cells. Leukemia Res. 30, pp. 1187-1192.
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Author Index
Abbud Hanna Roque C.E., 23
Aires M.B., 43
Alecrim C., 1
Alfaro D., 39
Almeida Dias M., 7
Alvelino E.X., 79, 85
Alves Corrêa de Noroña S.A., 17, 147
Alves Correa-Noronha S.A., 11, 125
Alves Moreira R.F., 23
Ávila de Almeida C., 23
Ávila R.A., 115
Azevedo dos Santos P., 137
Azevedo Feio D.C., 67
Baptista C.F., 125
Batista C.F., 147
Benchimol M., 33, 109
Bernardo W., 17
Binda Neto I., 17, 147
Boichot E., 61
Borba H.R., 103
Burbano R.M.R., 67
Cambraia J., 91
Campos de Carvalho A.C., 143
Canatto R.A., 99
Cardoso De Brito Junior L., 67
Carvalho H.F., 27
Cejalvo T., 39
Coll J.L., 159
Corrêa de Freitas Almeida A., 137
Correa-Noronha S.A.A., 1
Costa B.A., 159
Cotrim Guerreiro da Silva I.D., 11, 17
Crepaldi P.H., 33
da Silva B.H., 79, 85
da Silva de Mello M., 73, 103
da Silva Freitas E.H., 17, 125, 147
da Silveira N.M., 95
de Assis da Silva F., 103
De Barros C.M., 143
de Macario E.C., 119
de Matos Rodarte C.C., 85
© MEDIMOND s.r.l.
de Noronha S.M.R., 125
de Oliveira J.A., 91, 95, 99
de Souza W., 33
dos Santos Farnese F., 91
dos Santos J.T., 73
dos Santos M.R.V., 43
dos Santos Souza K., 43
dos Santos Teixeira J.T., 103
Fampa P., 73, 103
Farias P.S., 43
Farnese F.S., 95, 99
Fautrel A., 61
Fioretto E.T., 43
Fonseca Ferreira A., 137
Fonseca R.N., 143
Gamboa Ritto M.N., 125, 147
Garcia-Ceca J., 39
Geissler K., 131
Gicquel T., 61
Goldenberg R.C.S., 143
Gomes J.V., 103
Gonçalves L., 103
Guedes É.A.C., 79
Guerreiro da Silva I.D.C., 1, 125, 147
Gusman G.S., 95, 99
Hayashi M.A.F., 159
Homem de Bittencourt Jr. P.I., 55
Joselevitch C., 153
Kamermans M., 153
Kede J., 17, 125, 147
Kerkis I., 159
Krüger B., 131
Lagente V., 61
Leão G.A., 99
Lima de Lima P.D., 67
Lopes A.G., 7
Lopes I.F., 115
Lucas J.Z., 115
168
Lustosa de Oliveira M., 27
Macario A.J.L., 119
Marçal A.C., 43
Márcia Neiva, 159
Marques Capella M.A., 7
Martins A.B., 143
Monteiro de Andrade H., 91
Montero S., 39
Moraes J., 143
Moreira de Lima V., 73, 103
Muñoz J.J., 39
Nakaie C.R., 1, 11
Nascimento F.D., 159
Nascimento J.S., 143
Neto I.B., 125
Neto J.L., 91
Oliveira E.B., 159
Pereira A., 159
Pereira Carneiro Muniz J.A., 67
Porto L.C., 61
Renck Nunes P., 55
Resende Borba H., 73
Ribeiro de Noroña S.M., 147
Ribeiro de Noronha S.M., 1, 11, 17
Author Index
Rivaroli L., 115
Robert S., 61
Rocha Brito I.R., 85
Rocha C.B., 23
Rodarte R.S., 79, 85
Sancey L., 159
Sant’Ana A.E.G., 85
Sant’Ana E.G., 79
Schubert M., 49
Shimuta S.I., 1, 11
Silva C.J., 99
Silva J.R., 143
Silva-Mendes B.J., 143
Siman L.I., 95
Souza-Menezes J., 143
Teixeira D.E., 33
Tersariol I.L.S., 159
Tobajas E., 39
Valença S., 61
Vargas de Mesquita L., 23
Victoni T., 61
Vieira Gomes J., 73
Viel R., 61
Zapata A.G., 39
Zieger E., 49