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] All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form, or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission, in writing, from the publisher. 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 © Medimond . P726C0005 1 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. © Medimond . P726C0005 2 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. © Medimond . P726C0005 3 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. © Medimond . P726C0005 4 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 5 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. © Medimond . P726C0005 6 ICCB 2012. Proceedings of the 10th International Congreso on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0007 7 by using 7,5% SDS- ICCB 2012. Proceedings of the 10th International Congreso on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0007 8 ICCB 2012. Proceedings of the 10th International Congreso on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0007 9 ICCB 2012. Proceedings of the 10th International Congreso on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C0007 10 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0010 11 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0010 12 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0010 13 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 14 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. © Medimond . P726C0010 15 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C0010 16 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726C0818 17 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). © Medimond . P726C0818 18 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 © Medimond . P726C0818 19 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. © Medimond . P726C0818 20 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 © Medimond . P726C0818 21 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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 © Medimond . P726C0818 22 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. © Medimond . P726C0911 23 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 © Medimond . P726C0911 24 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. © Medimond . P726C0911 25 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. © Medimond . P726C0911 26 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 © Medimond . P726C1008 27 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C1008 28 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C1008 29 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1008 30 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C1008 31 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C1008 32 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9106 33 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 © Medimond . P726R9106 34 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9106 35 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9106 37 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726R9106 38 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9015 39 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9015 40 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9015 41 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726R9015 42 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0996 43 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0996 44 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0996 45 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0996 47 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C0996 48 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9113 49 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726R9113 50 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. 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[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 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726R9113 54 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. © Medimond . P726C0385 55 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0385 56 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0385 57 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0385 60 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9161 61 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9161 62 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9161 63 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). 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Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726C0029 67 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726C0029 68 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0029 69 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). 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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). 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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. © Medimond . P726C0029 71 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C0029 72 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0333 73 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0333 74 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0333 75 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 76 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. © Medimond . P726C0333 77 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C0333 78 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0400 79 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0400 80 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) (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. © Medimond . P726C0400 81 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0400 82 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0400 83 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0400 84 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0401 85 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0401 86 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0401 87 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) © Medimond . P726C0401 88 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0401 89 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C0401 90 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0862 91 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 ( ). © Medimond . P726C0862 92 ), As ( ), As+SNP ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0862 93 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C0862 94 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0939 95 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0939 96 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0939 97 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0939 98 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1102 99 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1102 100 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C1102 101 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1102 102 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C1194 103 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1194 104 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1194 % of Elimination 105 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1194 106 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1194 107 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C1194 108 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9107 109 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9107 110 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726R9107 111 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9107 112 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9107 114 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C1107 115 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C1107 116 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C1107 117 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C1107 118 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726R9006 119 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726R9006 120 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726R9006 121 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726R9006 122 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726R9006 123 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726R9006 124 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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- © Medimond . P726C0706 125 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0706 126 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0706 127 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0706 128 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C0706 129 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C0706 130 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0265 131 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0265 132 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0265 133 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0265 134 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0265 135 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C0265 136 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0561 137 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0561 138 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0561 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%) 0 139 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0561 140 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726C0561 141 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) [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. © Medimond . P726C0561 142 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. © Medimond . P726C0564 143 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. © Medimond . P726C0564 144 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). © Medimond . P726C0564 145 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. © Medimond . P726C0564 146 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]. © Medimond . P726C0840 147 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0840 148 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0840 149 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0840 150 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. 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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. 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Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9026 153 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 © Medimond . P726R9026 154 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) (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 © Medimond . P726R9026 155 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. 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(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. © Medimond . P726R9026 157 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726R9026 158 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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]. © Medimond . P726C0246 159 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 37C 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 37C, followed by washing with PBS) was performed using the red channel (e.g. excitation 595 nm © Medimond . P726C0246 160 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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 -80C (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 © Medimond . P726C0246 161 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0246 162 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. © Medimond . P726C0246 163 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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). © Medimond . P726C0246 164 ICCB 2012. Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Brazil) 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. 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Proceedings of the 10th International Congress on Cell Biology (July 25th- 28th, 2012 - Rio de Janeiro, Braz Brazil) il) © Medimond . P726C0246 166 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