59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
1
Evaluation Of Genotoxicity Nanocelulose Línter
Mariana de Abreu Barga¹, João Paulo Saraiva de Morais2, Maria Cléa Brito de Fiqueirêdo2, Morsyleide de Freitas
Rosa2, Leonardo Fernandes Fraceto3, Renata de Lima1,4,5
Departamento de Biotecnologia, Universidade de Sorocaba (UNISO), Rodovia Raposo Tavares km 92; 2 Embrapa
Algodão, Campina Grande – PB; 3 Embrapa Agroindústria Tropical, Fortaleza – CE; 4 Universidade Estadual Paulista “Julio
de Mesquita Filho” – UNESP, Campus Sorocaba, Avenida Três de Março, 511, Alto da Boa Vista; 5 Universidade Federal de
São Carlos – UFSCar, Campus Sorocaba, Rodovia João Leme dos Santos (SP-264), Km 110.
1
[email protected]
Keywords: genotoxicity, nanocelulose linter, in vitro test, Allium cepa and Comet assay.
Nanotechnology is based on the use of nanometer scale of the materials by changing the initial property. Their application
has been used in several products like electronics, biomedical, high performance materials and consumer products
(pharmaceuticals, cosmetics among others). When we referring to Nanomaterials we compared to macroscopic structures
showed differents characteristics such as: high surface area, mechanical properties, optical, magnetic or chemical thereby
unique characteristics to these materials. The nanofibers are an important class of nanomaterials which has attracted
attention over recent years. The continuous development of the nanomaterials and their researchs has generated advances
about its applications in the biomedical and biotechnological industries, as well as in the production of biodegradable
materials aimed at sustainability. Many of these products are marketed and can come into contact with the human body.
But there is no clear about the mechanisms of the interaction of these substances in the molecular reactions and since
these materials have unknown characteristics they may affect the ADN, causing damage directly or indirectly. Thus the
aim of this study was to evaluate the genotoxicity of Nanocelulose linter through the test of Allium cepa and in vitro
Comet assay. The results concluded that tested nanofiber has the capacity to induce changes and DNA damage in the cells
used (plant cells and human lymphocytes), which draws attention to the need for further studies before its application.
Financial Support: Cnpq, EMBRAPA, Rede Agronano e UNISO.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
2
Assessment of genotoxicity cellulose
nanofibers test using Allium Cepa and Comet
Macedo,J ¹;Lima, R1; Corrêa, AC2; Teixeira, EM2; Teodoro, KBR2,3 ;Marconcini, JM2 ; Mattoso, LHC2
1
Universidade de Sorocaba, departamento de Biotecnologia; 2 Laboratório Nacional de Nanotecnologia para o
Agronegócio (LNNA), Embrapa Instrumentação, Rua 15 de novembro, 1452, CEP: 13560-970, São Carlos, SP, Brasil; 3
Universidade Federal de São Carlos (UFSCar), Dep. de Química, Caixa Postal: 676, CEP: 13565-905, São Carlos, SP, Brasil
[email protected]
Keywords: Nanoparticles, cellulose, genotoxic, comet assay and allium cepa.
Nanotechnology is a new science with the number of industrial products growing day by day. Due to this exist
a growing the concern with the with their adverse effects to the environmental consequence and human health.
The small size of nanoparticles allows the penetration into the cells and the interact to the DNA. This study
aimed to analyze the genotoxic potential of cellulose whiskers nanostructures such as Allium cepa and Comet
assay. The nanofiber treatments (surface modified cotton, cotton unmodified surface modified kraft pulp,
unbleached kraft pulp modified) were studied in difeferentes concentrations of Allium cepa (1%) and comet assay
(0.1, 0.25 and 0.50%). The results showed different behaviors regarding to damage to DNA in the treatments
performed, although the differences were not significant there were greater damage to treatments performed
with Kraft Pulp. These results suggest the need for further studies to the user of nanofiber commercially.
Financial Support: CNPq, Finep, Capes, Fapesp (process no 2008/03606-9) and Project MP1 Rede Agronano –
Embrapa.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
3
Evaluation of mutagenicity and
antimutagenicty of Byrsonima verbascifolia
and Byrsonima correifolia
Espanha, LG1; Resende, FA1; Lima Neto, JS2; Vilegas, W2,3; Varanda, EA1.
Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, SP; 2Instituto de Química, UNESP, Araraquara, SP; 3Câmpus
Experimental de São Vicente, UNESP,São Vicente, SP
1
[email protected]
Keywords: Byrsonima, Ames Test, mutagenic, antimutagenic and folk medicine
The Brazilian cerrado contain several native plants used in folk medicine for treating many diseases. The genus
Byrsonima is one of them. There are several reports of its use: the treatment of fungal and bacterial infections,
tuberculosis, chronic wounds, Chagas disease, as a diuretic and antiemetic. The phytochemical profile shows tannins,
flavonoids, triterpenes, which indicates the pharmacological potential of this genus. Although these plants are widely
used by population in folk medicine, there aren’t studies to ensure the safety of their uses. Every day we come into
contact a huge amount of compounds capable of promoting mutation. Thus, it is interesting to identify compounds
that potentially reduce these effects. The aim of this study was to investigate the mutagenicity and antimutagenicty of
standardized ethanolic extracts (70%) of two species of Byrsonima: B. verbascifolia (BV) and B. correifolia (BC) by Ames
Test. The Ames Test was performed according to preincubation assay, in absence and presence of metabolic activation
system, using five concentrations of each sample, in triplicates. For mutagenic assay were used TA98, TA97, TA100
and TA102 strains of Salmonella typhimurium. The concentrations of extracts, in mg/plate, varied 0.2-16.7 for BV and
0.52-16.7 for BC. A sample was considered positive when the mutagenic index was ≥2 for at least one of the tested
doses and when the response was dose-dependent. For antimutagenicity assay were used TA98, TA100 and TA102
and the concentrations of extracts, in mg/plate, varied 0.01-2 for BV and 0.008-4 for BC. The mutagenic agents used
were 4-nitro-o-phenylenediamine (NPD), mitomycin C (MMC), aflatoxin B1(AFL) and benzo(a)pyrene (B[a]P). A
non-antimutagenic effect was considered when a value lower than 25% was obtained, a moderate effect when a value
between 25% and 40% was obtained, and strong antimutagenicity at values greater than 40%. The mutagenicity test
showed that BV and BC did not induce an increase in the number of revertant colonies indicating absence of mutagenic
activity. In the antimutagenicity test, BV has demonstrated different levels of reduction: 48% for NPD, 91% for AFL
and 82% for B[a]P. BC has demonstrated 51% of reduction for NPD, 80% for AFL and 83% for B[a]P. No significant
reduction was found to MMC with both extracts. Both samples has been shown as a strong preventive and potential to
be used as a chemopreventive. These results have demonstrated the continued importance of studies on medicinal plants.
Finnacial Support: FAPESP
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
4
Joannesia princeps: genotoxicity evaluation
of the aqueous extract through the
micronucleus test
de Lima, VM1; Teixeira, JT dos S1; Gomes, JV1; Netto, LKA3; Bastos, LO1; dos Santos, HLC1; Gonçalves, L1; da Silva, F
de A2; Berto, BP1; Borba, HR1.
Instituto de Biologia, UFRRJ, Rio de Janeiro, RJ; 2Instituto de Ciências Exatas, UFRRJ, Rio de Janeiro, RJ; 3Instituto de
Biociências, UNIRIO, Rio de Janeiro, RJ
1
[email protected]
Keywords: citotoxity, genotoxicity, Allium cepa system, micronucleous, Joannesia princeps
Joannesia princeps, also known as “cotieira”, is a huge tree of the Euphorbiaceae family. It’s found in the Northern,
Northeastern and Southeastern Brazilian regions and known for its medicinal effects. It is used in folk medicine as a
purgative and in the treatment of various diseases, such as menstrual disorders, pernicious fever, as an antimicrobial,
syphilis, scrofula and swelling. However, despite the current trend for the use of alternative medicine, vegetal products
used for therapeutic purposes should undergo analysis processes, in order to to ensure that their use is made with
relative safety. Among the analysis performed, there are methods that allow the evaluation of the cytotoxic and
genotoxic potential of these products. Therefore, this study aimed to analyze the cytotoxic and genotoxic potential
of the aqueous extract obtained from the leaves of J. princeps, using for this the micronucleus test. For this test, mice
were divided into three treatment groups of 10 animals each. Group I: received a daily dose of 4g/kg aqueous extract
of leaves of J. princeps for three consecutive days. Group II (negative control) received only water. Group III (positive
control) received a single dose of cyclophosphamide (50mg/kg). The results showed that the aqueous extract of leaves
of J. princeps shows no genotoxic activity; however, a cytotoxic activity of the extract was found on the mice’s bone
marrow cells. These results corroborate those obtained previously by our research group using the Allium cepa test
system, in which it was also observed cytotoxic activity of aqueous extract at 20mg/ml on meristematic cells of A. cepa
without the presence of genotoxic activity. Thus, the aqueous extract of the leaves of J. princeps showed antiproliferative
cytotoxic activity on concentrations used in the two systems, which may be indicative of their therapeutic potential
for the inhibition of cell cycle. Nevertheless, although the extract has not shown mutagenic effects, further studies are
needed to establish the safefy of use of this plant by the population.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
5
Epigenetic activity of valproic acid and
trichostatin A on NIH 3T3 cells
Felisbino, MB1; Gatti, MSV2; Mello, MLS1
1
Department of Structural and Functional Biology, Unicamp, Campinas, SP; 2 Department of Genetic, Evolution and
Bioagents, Unicamp, Campinas, SP
[email protected]
Keywords: NIH 3T3 cells, chromatin remodelling, histone deacetylase inhibitor
Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase (HDAC) inhibitors. In consequence
of their effects on histones, alteration in the chromatin supraorganization and in the nuclear architecture would be
expected. Indeed, it has been found that their epigenetic activity reflects on chromatin supra-organization, nuclear
architecture and cellular proliferation, particularly in tumor cells, as HeLa cells. In this work, chromatin remodeling
with effects extending to condensed chromatin areas, was studied by image analysis in VPA- and TSA-treated
non-transformed mouse fibroblastic NIH 3T3 cells, that are particularly interesting because of the conspicuous
heterochromatin areas these cells contain. Information on HDAC activity and histone acetylation was also assessed.
NIH 3T3 cells were treated with different doses of VPA (0.05; 0.5; 1.0 mM) and TSA (10; 20; 100 ng/mL) for
1 to 24 h. No loss of viability (as indicated by the MTT assay) was found. Image analysis of the Feulgen-stained
cells revealed chromatin decondensation affecting both euchromatin and heterochromatin. HP1-α, a hallmark for
mammalian heterochromatin, identified immunocytochemically, was depleted from the heterochromatic areas with
both VPA and TSA treatment. These observations were concomitant with decrease in HDAC activity and with
hyperacetylation of histone H3. Treatment for 48 h with 5.0 mM VPA or 500 ng/mL TSA induced a significant
reduction in the mitotic index and an increase in the number of micronuclei but no change in cell death ratio.
Chromatin decondensation promoted by VPA and TSA was thus demonstrated in non-transformed NIH 3T3 cells
similar to previous observations in HeLa cells. Additionally, chromatin remodeling induced in the NIH 3T3 cells
by both VPA and TSA also affected the heterochromatic areas supposed to be transcriptionally repressed. Although
low doses of VPA and TSA for relatively short times could induce these effects on chromatin, drastic effects on
cell proliferation and micronucleation required a longer exposure to more concentrated doses of VPA and TSA.
Financial support: FAPESP (2010/50015-6, 2009/11763-0), CNPq (301943/2009-5, 475261/2012-2)
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
6
Mutagenic potential of Punica granatum
L. plant extract using micronucleus test in
peripheral blood from Swiss mice
Guimarães, B.D.¹; Santos, J.T.T.2; Medeiros, A.C.D.3; Lira,W.M. 4
Discente do curso de Farmácia – UEPB; 2 Discente do curso de Enfermagem – UEPB; 3 Departamento de Farmácia –
CCBS/UEPB; 4 Departamento de Biologia – CCBS/UEPB
1
[email protected]
Keywords: Mutagenicity; micronucleus; polychromatic erythrocytes; pomegranate; Punica granatum L.
The mutagenic agents which alter the DNA bases sequence can accelerate or increase the appereance of mutations.
To detect chemical, physical and biological agents that can be mutagenic, the micronucleus test is largely accepted
by international agencies and government institutions. The mundial world population is frequently exposed to
mutagenic substances, and many of them may be of plant origin, as plant extracts. Pomegranate is often used for
medicinal purposes; on strep throat treatment and helminths combat. Many active principles which are present in
the fruit peel can be emphasized, such as alkaloids, gallic tannins, ellagitannins, gallic acid derivatives, flavonoid
glycosides and anthocyanins. The present study was performed to investigate the mutagenic potential of Punica
granatum L. fruit peel plant extract. In the micronucleus test, Swiss albino mice Mus musculus were used (27g weighing
approximately). The animals were divided in 5 groups with 6 mice each (3 males and 3 females): a positive control
group, cyclophosphamide (50mg/kg) applied intraperitoneally; a negative control group, distilled water; and 3 other
groups for three different dosages of Punica granatum L. extract, at concentrations 700 mg/mL, 350 mg/mL and
175mg/mL. The treatments for the extract were inserted by gavage at maximum volume 0,1 mL for each 10 g body
weight. The animals’ blood was collected 30 hours after the treatments by tail pulsation and placed in slides, where
the smears were prepared. The slides were fixed for 10 min on metanol and stained for 25min in Giemsa. Micronuclei
frequency in blood cells quantification was performed by counting 2000 polychromatic erythrocytes per animal. The
results demonstrated that there was no increase in MNPCE (micronucleated polychromatic erythrocytes) frequency
in the animals treated with Punica granatum L. extract. Statistical analysis found that MNPCE frequency of all
three dosages, when compared with negative control, did not presented significant values. That can be explained by
phytochemicals present in the fruit peel, with emphasis on tannins, which are compounds that possess antimutagenic,
antioxidant and antitumor activity. Thus, on the tested experimental conditions, Punica granatum L. extract did
not present mutagenic potential, indicating that the tested substance does not induce chromosome damage.
Financial Support: UEPB, CNPq, PROPESQ.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
7
Evaluation of mutagenic and antimutagenic
activities of Capsicum chinense var. Bode
Silva, GGO1, Costa, BO1, Leal, APF2, Buccini, DF2, Moreno, SE2.
1
Graduação em Ciências Biológicas, Universidade Católica Dom Bosco, Campo Grande, MS; 2Mestrado em
Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS.
[email protected]
Keywords: pepper, micronucleus test, DNA damage, eukaryote cells, erythrocyte
Diet plays an important role in the prevention of some diseases related to DNA damage, such as cancer. The pepper
Capsicum chinense var. bode is widely consumed in Brazil, and is an important source of food antioxidants, that is
correlated with the contents of flavonoids and total phenolic compounds. Unfortunately, there is little data about
mutagenic and antimutagenic activities of these peppers in eukaryote cells. Objective: The aim of this study was
evaluate the mutagenic and antimutagenic property of hexanic and ethanolic extracts of Capsicum chinense var.
bode. Methodology: The experiments were assayed in Swiss mice (Ethic Committee no.004/11). The mutagenic and
antimutagenic activity was determined through micronucleus index in erythrocyte of peripheral blood. The mutagenic
effect was evaluated in 24, 48 and 72 after treatment of mice with different doses (1, 5, 15mg/kg, s.c) of the extracts.
The antimutagenic assay was carried on in mice treated with colchicine and extracts of C. chinense. Results: At all
times evaluated, the hexanic and ethanolic extracts of C. chinense (1, 5 and 15mg/kg, s.c) are not able to induced
increase in micronucleus number, suggesting that these extracts are not provide of mutagenic effect. On the other
hand, the hexanic and ethanolic extracts of C. chinense presented a significant anti-mutagenic effect, inhibiting totally
the micronucleus formation induced by colchicine, suggesting the protection against DNA damage of the C. chinense.
Financial Support: CAPES, CNPq.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
8
Evaluation of themutagenicity ofliquid
effluentsgenerated by gas stations
Toledo, MBF; Camargos, LF; Porto, TP; Rosa, NMO; Santos, FV.
Laboratório de Biologia Celular e Mutagênese, Campus Centro Oeste, Universidade Federal de São João del Rei,
Divinópolis, MG.
[email protected]
Keywords: Ames assay, mutagenicity, gas station, liquideffluents.
The liquid effluents generated in gas stations are important sources of contamination by chemicals, especially thatfrom
petroleum. In general, only physicochemical parameters are used for monitoring these effluents, and biological
parameters are not a legal requirement for this monitoring. Thus, in order to know the possible mutagenic potential
of wastewater from gas stations, mutagenicity tests were performed with samples of residues from these sources. The
samples were collected before and after through wastewater treatment system in periods of drought and rain. For
mutagenic analysis was performed the Salmonella mutageniticy assay using the strains TA98 and TA100, in tests
with and without metabolic activation. Different concentrations of the studied samples (1.17%, 2.22%, 4.34%, 6
38%, 8.33%) were analyzed in triplicate by the method of direct incorporation. The results were expressed as the
mean, standard deviation and by calculating the mutagenicity index (MI). To be considered mutagenic a sample
should present MI>2. Samples that result in revertant frequency statisticallyhigher than the control group and that
presentIM between 1.5 and 1.9 were classified as samples with mutagenicity signs. Complementarily, all samples
were evaluated about some physical and chemical parameters present in Brazilian legislation that regulates the
liquid effluent emissions by gas stations. Only two samples (11 and 12) showed alterations in revertant frequency
when compared with control. The sample 11, in studies performed with TA100 (+S9) showed mutagenicity signs
with RM 1.9 at concentration 1.17%. For other concentrations there was a trend to decrease in the frequency of
revertants, indicating RM around 0.6, an indication of cytotoxicity. The results observed to sample 12 in TA 100 with
metabolic activation, show RM 1.6, also indicating mutagenicity signs. Compounds present in these samples, after
metabolization, become genotoxic. Besides, at higher concentrations they induced a reduction of cell survival. The
analysis of the physical and chemical parameters of the effluents showed that only onesample was in agreementwith
Brazilian legislation. Results presented herein show that the physical and chemical parameters used to consider a waste
sample safe to be released in environment are compatible with the biological safety of these mixtures.However, some
samples showed to be able to induce DNA damage in statically significant levels. In this way, more attention should
be directed to the treatment of these residues and alternative forms of treatment should be developed because the
cumulative effects of these substances (and of the DNA damages induced) may be dangerous in long-term processes.
Financial Support: FAPEMIG and CNPq.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
9
The absence of mutagenic activity of multiwalled, functionalized carbon nanotubes, in
the somatic cells of drosophila melanogaster
Machado, NM1; Orsolin, PC1; Silva-Oliveira, RG1; Oliveira, VC2; Nepomuceno JC1,2.
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, MG; 2 Laboratório de
Citogenética e Mutagênese, Centro Universitário de Patos de Minas, Patos de Minas, MG
1
[email protected]
Keywords: Carbon nanotubes. Genotoxic. Drosophila melanogaster. SMART
Carbon nanotubes (CNTs) are rigid molecules, flexible and resistant to tensions. They are composed of sheets of
carbon (graphene), which, at high temperatures, curl, forming nanometer diameter tubes. Among the different
types of CNTs, the multi-walled carbon nanotubes (MWCNTs) comprise a set of concentric nanotubes with
perfect structures and defect free materials. Several uses for this material have been suggested including biological
applications such as manufacturing of biosensors, carriers of drugs and vaccines and other biomaterials. However,
before these materials can be put on the market, it is necessary to know their genotoxic effects. Thus, this study
aims to evaluate the mutagenicity of carbon nanotubes in somatic cells of Drosophila melanogaster, using the
somatic mutation and recombination Test (SMART). For this purpose we have used 72-hour larvae from standard
(ST) and high bio activation (HB) crosses. The larvae from both crosses were treated with solutions containing
different concentrations of multi-walled carbon nanotubes (MWCNTs) functionalized (0,50, 100, 150, 200, 250
mµ /mL). The trans-heterozygous (MH) descendants, analyzed in both ST and HB crosses, had no significant
effects that could be considered to be due to the nanotube treatment, on the frequency of mutant spots when
compared with negative control (reverse osmosis water). Based on the results and on the experimental conditions
mentioned in this study, it was concluded that MWCNTs were not mutagenic in Drosophila melanogaster.
Financial support: FAPEMIG, CNPQ, UFU e UNIPAM
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
10
Mutagenicity assessment of a synthetic
analogue of marine 3-alkylpyridine alkaloids
with antitumor potential
Barbosa, MC1; Silva, IVG1;Costa, AC²; Gomes, GKA1; Gonçalves, AMMN3; Silva, LM4; Varotti, FP2; Viana, GHR3;
Santos, FV1.
Laboratório de Biologia Celular e Mutagênese, Campus Centro Oeste, Universidade Federal de São João del Rei (UFSJ),
Divinópolis, MG; 2Laboratório de Bioquímica de Parasitos, campus Centro Oeste, Universidade Federal de São João del
Rei (UFSJ), Divinópolis, MG; 3Laboratório de Síntese Orgânica, campus Centro Oeste, Universidade Federal de São João
del Rei (UFSJ), Divinópolis, MG; 4Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG.
1
[email protected]
Keywords: marine alkaloids, mutagenicity, micronucleus test, cytotoxicity, 3-alkylpiridine alkaloids.
3-alkylpiridine alkaloids (3-APAs) are molecules that in most cases have a long aliphatic chain containing nitrogen
atoms in the terminal portion. Studies show that this group of compounds is normally obtained from sponges
of the Haplosclerida Order and generally are linear monomers. An important member of this molecular class is
the Theonelladin C, which is isolated from marine sponge Theonella swinhoei. This monomer has demonstrated
antimicrobial activity against fungi and some bacteria and cytotoxic activity in vitro against mouse lymphoma cells
(L1210) and human KB carcinoma cells. However, the full potential of any substance has to be carefully evaluated
and a correlation between its therapeutic benefits and its collateral effects should be performed. In this way, the
aim of the present study was to assess the cytotoxicity and the mutagenicity of a synthetic analogue of marine
3-alkylpiridine alkaloids in the human tumor cell lines RKO AS-45-1 (human colon carcinoma) and Hela (human
cervix carcinoma). The non-tumor cell line WI 26VA4 (human lung fibroblasts) was used to assess the selectivity
of the compound studied. The cell viability assay was performed employing the colorimetric MTT method. The
cytokinesis-block micronucleus assay was carried out in RKO cell line to assess the potential of the synthetic alkaloid
to induce chromosomal mutations. Three different concentrations of the synthetic analogue of marine alkaloids
were evaluated and the treatments were performed in culture medium without serum for three hours. The results
found show that the synthetic alkaloid reduced the cell viability after treatments and the IC50 values obtained in
RKO, Hela and WI cells were 19.1, 8.5 and 99.1 µM, respectively. These results show that the tumor cell lines
used present higher sensitivity to the synthetic alkaloid than non-tumor cell line WI. In mutagenicity assays, the
higher concentration employed (17.8 µM) was mutagenic inducing chromosomal damages in RKO cell line. Results
presented herein demonstrate that the synthetic analogue of marine 3-alkylpiridine alkaloid present potential to be
an antitumor agent once it has a selective cytotoxic effect and act inducing DNA damages. Complementary studies
are necessary to assess the potential of these chromosomal damages induces the triggering of apoptotic processes.
Financial Support: FAPEMIG
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
11
Mutagenicity of standardized extracts of
plants from Cerrado used in popular medicine
Resende, FA1; De Grandis, RA1; Weiss, MB1; Espanha, LG1; Boldrin, PK1; Nogueira, CH1; Lima Neto, JS2; Rocha, CQ2;
Souza, LP2; Saldanha, LL3; Dokkedal, AL3; Vilegas, W4; Varanda, EA1
1
Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, SP; 2 Instituto de Química, UNESP, Araraquara, SP; 3 Instituto de
Biociências de Botucatu, UNESP, Botucatu, SP; 4 Campus do Litoral Paulista, Unidade São Vicente, UNESP, São Vicente, SP
[email protected]
Keywords: Brazilian Cerrado, popular medicine, medicinal plants, Ames test, mutagenicity
The Brazilian Cerrado (neotropical savanna) is one of the major biogeographic regions of the world with more than
7000 native species of vascular plants. Many of these plants are commonly used as natural remedies by people living
in this area to treat several illnesses. Thus, the evaluation of the possible mutagenic effect is required to allow the
safe use in humans. In this study, the mutagenicity of standardized extracts (70% ethanol) of species widely used
in the region of the Cerrado was assessed using the Ames test. The Ames test was performed by the preincubation
methodology using TA98, TA100, TA97 and TA102 strains of Salmonella typhimurium in the absence (-S9)
and presence (+S9) of metabolic activation system in five concentrations, varying from 24.0 to 0.62 mg/ plate.
According to obtained results, the species of the genus Astronium (A. fraxinifolium, A. graveolens and A. urundeuva),
used to treat allergies, inflammation, diarrhea and ulcers, were not mutagenic either in the presence or absence of
metabolic activation. Machaerium hirtum, used in the treatment of diarrhea, cough and cancer, the ethanol extracts
(70%) of leaves and stalk also no showed mutagenic potential. Species of the genus Byrsonima (B. fagifolia and
B. intermedia) were not mutagenic and are commonly used for its antiseptic, antimicrobial, anti-inflammatory
properties and in the treatment of diarrhea and dysentery. On the other hand, the ethanol extract (70%) of roots
of Arrabidaea brachypoda, widely used in traditional medicine in Southeastern and Northeastern Brazil for kidney
stones and painful joints was mutagenic in the TA98 strain (-S9). Another species highly mutagenic was Myrcia
bella, in all strains tested, except TA102. Studies demonstrated that this specie has antihyperglycemic potencial.
This study demonstrate the risk which the population is subjected due to indiscriminate use of non-standardized
extracts. Although many compounds derived from plants have considerable pharmacological activities, some
undesirable properties such as mutagenicity, carcinogenicity and toxicity may restrict their use as therapeutic agent.
Financial Support: FAPESP and CNPq (Brazil).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
12
Cytotoxic and genotoxic effects of Styrax
camporum hydroalcoholic extract on V79 cells
Oliveira, PF; Nicolella, HD; Damasceno, JL; Pauletti, PM; Tavares, DC.
Universidade de Franca, UNIFRAN, São Paulo, SP
[email protected]
Keywords: Styrax camporum, Styracaceae, cytotoxic, genotoxic, V79 cells
The genus Styrax is the most important representative of Styracaceae family and differs from other genera of the
family by producing a resinous material secreted from injuries incurred in the stem. The specie S. camporum Pohl
is popularly found in Brazil, occurs in the Cerrado of Minas Gerais, São Paulo, Mato Grosso do Sul and semideciduous forest of the Paraná basin. The ethnopharmacological reports the use of S. camporum in the treatment
of gastric disorders. Regarding the therapeutic interest, the present study investigated the cytotoxic and genotoxic
potential of S. camporum hydroalcoholic extract in Chinese hamster lung fibroblasts (V79 cells). The cytotoxicity was
evaluated by the colony forming assay, which the cultures were treated with concentrations ranging from 2.5 to 5000
µg/mL. The results revealed that concentrations up to 80 µg/mL showed significant cytotoxicity. The cytotoxicity
also was evaluated by the nuclear division index (IDN) and the cell viability by Trypan blue exclusion method.
The genotoxic potential was assessed by the micronucleus and comet assays. The V9 cells cultures were treated
with four concentrations of extract (5, 10, 20, 40 and 60 µg/mL) for three hours. The methyl methanesulfonate
(MMS) was used as a positive control at concentration of 44 μg/mL and 22 μg/mL for the micronucleus and
comet assays, respectively. A statistically significant increase in the DNA damage and micronucleus frequencies
was observed in the culture treated with the highest concentration of the S. camporum extract tested (60 µg/mL)
in comparison to the control group. The cultures treated with this concentration of the S. camporum extract also
showed IDN values significantly lower in relation to negative control cultures. All the concentrations tested showed
cell viability up to 85% by the Trypan blue exclusion method. Thus, under the present experimental conditions,
the S. campoum demonstrated cytotoxicity and genotoxicity in the highest concentration tested. This effect
can be attributed to the presence of benzofuran lignans, egonol and homoegonol, in the hydroalcoholic extract.
Financial support: São Paulo Research Foundation (FAPESP, Brazil, grant number 2011/21310-2).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
13
Using the onion (Allium cepa) to evaluate the
cytotoxic, genotoxic, and mutagenic effects of
artificial powdered juice
Pereira, LLV1; Itoyama, MM1; Castiglioni, L2
UNESP - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, Departamento de Biologia,
São José do Rio Preto, SP, Brasil; 2UNIRP - Centro Universitário de Rio Preto, São José do Rio Preto, SP, Brasil.
1
[email protected]
Keywords: Mutagenesis, artificial juice, cytotoxicity, mitosis.
Genetic Toxicology investigates toxic or mutagenic agents that are capable of inducing changes in DNA. In this study,
we tested two different flavors of three different artificial powdered juice brands, six samples in total, with the objective
of evaluating the potential cytotoxic, genotoxic, and mutagenic effects of the artificial juices. The solutions were
prepared at the concentration levels suggested on the packet label and compared in positive (Trifluralina, a pesticide
with Toxicity Class III: moderately toxic) and negative (Milli-Q water) controls in a bioassay conducted with the Allium
cepa test system. Analysis showed that after three days of incubation in the Milli-Q water, the roots of the onion grew
an average of 1.8 cm. After seven days of incubation in Trifluralin and the artificial juice, the roots grew 0.3 cm and 0.5
cm, respectively. With respect to mitotic division, different phases of mitosis were found to be unaltered in the Milli-Q
water. On the other hand, the roots of the onion treated with the six artificial juices and trifluralin showed: a) the majority
of cells in interphase; b) prophases with amoeba nuclei and chromosome loss; c) polyploid metaphases; d) anaphases
and telophases with chromatin bridges; and e) C-metaphases. As previously mentioned, the roots treated with artificial
juice showed the majority of cells in interphase and therefore lacking in growth. Moreover, the cells in other phases of
division showed several changes, indicating that substances in the artificial juice directly affected the DNA. These results
were similar to those obtained with the positive control group, Trifluralina. In conclusion, it is important to study
mammalian test systems in addition to this study in order to determine whether a correlation exists between the two.
Financial Support: FAPESP and FUNDUNESP
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
14
Evaluation of the genotoxicity of a digoxin
synthetic derivative
Oliveira, JT1; Silva, IVG1; Gomes, GKA1; Barbosa, LAO2; Villar, JAFP3; Santos, FV1.
1
Laboratório de Biologia Celular e Mutagênese, Campus Centro Oeste, Universidade Federal de São João del Rei, Divinópolis,
MG; 2Laboratório de Bioquímica Celular, Campus Centro Oeste, Universidade Federal de São João del Rei, Divinópolis, MG;
3
Laboratório de Síntese Orgânica Campus Centro Oeste, Universidade Federal de São João del Rei, Divinópolis, MG
[email protected]
Keywords: Digoxin, Comet Assay, Micronucleus Assay
The identification of compounds potentially noxious to the structure and function of genetic material is a constant
preoccupation. The induction of mutations has been linked to effects caused by many substances used as medicines.
Digoxin is a drug used to treat congestive cardiac insufficiency. Complementarily, digoxin has been correlated as a
potential antitumor, which could be observed in studies that evaluated their cytotoxicity in cell strains derived from
malignant tumors. However, this cytotoxicity for most of the cells tested, only been detected in high concentrations.
In this way, an interesting and promising strategy is realize structural modifications in the molecule of digoxin in
order to obtain derivatives with potential significant cytotoxic against tumor cells in relatively low concentrations
and do not cause excessive changes in non-tumor cells. The present study aimed to evaluate the potential of a digoxin
synthetic derivative in reduces cell viability and cause DNA damage in non-tumor cell line CHO-k1. The MTT assay
is a colorimetric method based on the reduction of MTT (3 - [(4,5 dimethylthiazol-2-yl) -2,5 diphenyltetrazolium
bromide]) into formazan, which allows inference of cell viability after exposure to compound. The Comet assay is
used to identify genotoxic agents, which based on the lysis of cells exposed to treatment followed by electrophoresis,
which allows identification of the levels of DNA fragmentation. The Micronucleus assay is a method used to identify
mutagenic compounds that cause chromosomal damages. Independent experiments were realized for the MTT assay
(24 and 48 hours exposure) and the synthetic compound was evaluated in concentrations of 2.5, 5, 7.5, 10, 12.5, 15,
20, 25, 37.5 and 50 μM. The Comet assay and Micronucleus assay were performed in triplicate with 3 hours of drug
exposure at concentrations of 20, 35 and 50 μM, and 1.8 nM, 5.0 and 10 μM, respectively. For statistical analysis was
realized ANOVA followed by Student-Newman-Keuls post-test. It was observed that the digoxin derivative only affect
cell viability in both higher concentrations and 48 hours of treatment (37.5 and 50 μM). In Comet assay, the substance
was genotoxic at the highest concentration tested in the MTT. The compound was mutagenic in the Micronucleus
assay, only the highest dose tested in this test (10 μM). Results presented herein show that the compound assessed is
genotoxic at high concentrations. The correlation between results observed in comet assay and micronucleus assay may
indicate that this substance acts as aneugenic compound once concentrations that induced micronucleus formation did
not cause DNA fragmentation in Comet assay. Complementary studies are necessary to identify the action mechanism
of this compound and its correlation with the cytotoxicity observed against tumoral cell lines in previous studies.
Financial Support: FAPEMIG and CNPq
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
15
Genotoxic and antigenotoxic effects of manool,
diterpene of Salvia officinalis, in HepG2 cells
Nicolella, HD; Oliveira, PF; Costa, GFD; Veneziani, RCS; Tavares, DC
Universidade de Franca, Franca, SP
[email protected]
Keywords: Salvia officinalis, manool, genotoxicity, antigenotoxicity, HepG2 cells
Savia officinalis is an herb and medicinal plant, widely used in food, pharmaceutical, and cosmetic industries. Sage
enjoys the reputation of being a panacea because of its wide range of medical effects. The diterpene manool is a
chemical marker of S. officinalis and shows potent antimicrobial activity. In order to better understand the effects
of S. officinalis, the present study was carried out to evaluate the genotoxicity and antigenotoxicity of manool in
HepG2 cells (hepatocellular carcinoma) by micronucleus test. The cell cultures were treated during 24 h with different
concentrations of manool, 0.5, 1.0, 2.0, 4.0 and 8.0 µg/mL. These concentrations were chosen using the cytotoxicity
as criterion. For antigenotoxicity assessment, four different concentrations of manool (0.5, 1.0, 2.0 and 4.0 µg/
mL) were associated with the mutagen methyl methanesulfonate (MMS; 44 µg/mL). The results showed that the
cultures treated with the highest concentration of manool (8.0 µg/mL) demonstrated a statistically significant increase
when compared to the negative control cultures. The simultaneous treatment with manool (0.5 and 2.0 µg/mL)
and MMS showed a significant reduction in the frequency of micronuclei in relation to MMS group alone. The
IDN values no showed significant differences when compared to the negative control group. Therefore, the present
results indicate that manool shows the characteristic of a “Janus” compound, i.e., manool is genotoxic at higher
concentrations, while at lower concentrations it display a chemopreventive effect on MMS-induced genotoxicity.
Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp, São Paulo, Brazil) and Conselho
Nacional de Desenvolvimento Científico e Tecnológico (PIBIC/CNPq, Brasília, Brazil).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
16
Antimutagenicity and gene expression
alterations on breast cancer cells induced by
nemorosone, isolated from Clusia rosea resin
De Grandis, R.A1; Camargo, MS1; Resende, FA1; Oliveira, MT2; Cuesta-Rubio, O3; Vilegas, W4; Varanda, EA1.
Faculdade de Ciências Farmacêuticas de Araraquara, UNESP, São Paulo, Brasil; 2Universidade Estadual de Londrina,
UEL, Paraná, Brasil; 3Departamento de Ciencias Farmacéuticas, Universidad de La Habana, Havana, Cuba; 4Instituto de
Química de Araraquara, UNESP, São Paulo, Brasil
1
[email protected]
Keywords: nemorosone, antimutagenicity, gene expression, breast cancer
Nemorosone is the major constituent of Clusia rosea floral resins and brown cuban propolis. The antitumoral activity
of nemorosone has been reported to be a potent cytotoxic agent particularly in aggressive cancer models characteristic
for their highly chemoresistance. Studies have demonstrated that nemorosone exerted an antiproliferative activity
in breast cancer cells, among other types of cancer. It is known that chemical substances that present the capacity
to interact with mutagenic compounds or their metabolites and reduce their effects, are a possible alternative for
preventing cancer. In view of this, the aim of this study was to evaluate the antimutagenicity of nemorosone and
their influence on breast cancer cell line (MCF-7 BUS) gene expression. To performing the procedures, floral resin of
Clusia rosea was submitted to extraction in absolute ethanol. Nemorosone was crystallized in ethanol-water solution
and purificated. The antimutagenic effect was evaluated using mutagens with direct action (mitomycin C, aflatoxin
and 4-nitro-o-phenylenediamine). This study was based on Salmonella typhimurium reversion assay using TA98,
TA100 and TA102 strains. According to the methodology of preincubation in plates, developed by Maron and
Ames, different doses of nemorosone were mixed with bacterial culture and the mutagenic agent and then incubated.
After incubation, top agar was added and the content of each tube was poured on a plate with minimum glucose
agar. The plates were incubated 48h and the number of revertant colonies per plate was counted. To perfoming the
RT2PCR Array (Qiagen, USA), the cells were incubated with nemorosone during 24 h. After treatment the cells
RNA was isolated, cDNA was synthesized, mixed with the SYBR green master mix and loaded onto the 96-well
array. Real-time PCR was performed heating the plate to 95 °C for 10 min, followed by 40 cycles of 95ºC for 15s
and 60ºC for 1 min, followed by dissociation curve. The polymerase chain reaction (PCR) array technology is a
96-well plate containing primers for a set of 84 genes involved in different cellular processes. In this study the genes
are involved in the breast cancer, and encode important enzymes that contribute in apoptosis, hormone receptors,
cell cycle, metabolism, DNA repair, growth factors and transcription factors. Nemorosone showed antimutagenicity
inhibiting the mutagenc activity of mitomycin C in 53% to TA102 strain and in 31% the aflatoxin activity to TA100.
Gene expression analysis revealed significant changes of 20, out of the 84 genes examined. In conclusion, our study
demonstrates that nemorosone is an antimutagenic compound and causes gene alterations in breast cancer cells.
These differential expressions are related with the apoptosis increase, decrease of hormone receptors expression and
alterations on cell cycle, leading to cell death. It can indicate that this compound may be a therapeutic target to cancer.
Financial Support: FAPESP, Processo: 2009/03057-8
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
17
Biomonitoring the genotoxicity of raw and
anaerobically digested slurry using the
Tradescantia Micronucleus Test
Campos, CF1,2; Dayrell, DM1,2; Oliveira, SR1,2; Miranda Filho, R¹; Morais, CR1,2; Marques, JGB3; Rodrigues, FFS1,2; De
Campos Júnior, EO2; Pereira, BB1,2; Morelli, S2.
Fundação Carmelitana Mário Palmério, FUCAMP, Monte Carmelo, MG; 2Instituto de Genética e Bioquímica, UFU,
Uberlândia, MG; 3Departamento Municipal de Água e Esgoto, DMAE, Monte Carmelo, MG.
1
[email protected]
Keywords: Ecotoxicology, Genotoxic, Sanitary landfill, Leachate, Sustainability
Urban solid waste production in Brazil reaches 228,413 tons daily. The common allocation, of the most of these residues
is in sanitary (or controlled) landfills, contradicting green viable possibilities as the use of matter for composting and/
or recycling. The residues in degradation allocated in the landfills, in the occurrence of precipitation, are percolated
and form a dense liquid and with complex mixture, known as slurry. The ecotoxicological/genotoxic potential of
this leachate is very high, including contamination of watercourses, groundwater and cellular damages in somatic
and germinative cells of species that live in the aquatic environment and in species that use contaminated water. The
present study aimed to evaluate the genotoxic potential of raw and treated slurry from the controlled landfill of the city
of Monte Carmelo, Minas Gerais, Brazil, using the Tradescantia Micronucleus Test (Trad-MCN). Physico-chemical
analyzes were performed. The plants were cut and led to flasks with distilled water for acclimatization. Then 15
plants in each type of chorume were exposed, including the positive control (As2O3) and negative control (Hoagland
solution) for 24 hours. The plants after this time were harvested and placed in distilled water for 24 hours to recover.
Following the recovery period, the inflorescences were collected and fixed in Carnoy solution (ethanol/acetic acid 3:1
ratio) for 24 hours at 4°C. The inflorescences were stored in 70% ethanol at 4°C until the analysis time. Five slides
were prepared for each analysis. The cells were stained with 2% acetic carmine. The slides were examined in optical
microscope on 400x magnification. Three hundred tetrads were analyzed per slide. The frequency of micronuclei is
expressed per 100 tetrads, using statistical analysis. The results from the bioassays conducted elucidate to the frequency
of micronuclei that remains high even after treatment of the slurry (ANOVA, Tukey; p<0.05). This allows us to infer
that in this case, slurry treatment with anaerobic and aerobic microorganisms must be complemented by chemical
treatment, considering that after treated the slurry is discarded in watercourses that are used by the local population.
Financial Support: FAPEMIG, UFU , FUCAMP, CAPES and CNPQ.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
18
Assessment of Banisteriopsis anisandra
genotoxic activity in Wistar rats
Mendes-Costa, M.C¹; Oliveira, G.K¹; Marcelino, DP¹; Alves, M.C¹; Faria Júnior, E¹; Messora, MR1,2.
¹ Centro Universitário de Lavras- Núcleo de Pesquisa em Ciências Biológicas-NPCBio; ² Universidade de São Paulo –
FORP
[email protected]
Keywords: polychromatic erythrocytes, hallucinogenic alkaloids, micronucleus test.
The indiscriminate use of medicinal plants as a therapeutic resource is an ancient practice. However, studies have
reported that some of these plants contain chemical constituents that are able to play mutagenic and genotoxic
activity, in prokaryotic and eukaryotic cells. The therapeutic use of medicinal plants is wide; it is an alternative of
easy access and low cost. Studies performed using infusions of the plant Banisteriopsis anisandra on the growth of
Allium cepa L. confirmed genotoxic and cytotoxic behavior. To detect the genotoxic potential of many substances
that are used by the population, it is necessary to employ tests that indicate the mutations occurring in cells, such
as the Micronucleus Test. The MN is also analyzed in polychromatic erythrocytes (young) bone marrow of mice
or rats. The in vivo mouse bone marrow micronucleus test is a mutagenicity test system for the detection of agents
that induce chromosome fragments and/or aneuploidy. Therefore, data obtained with this assay represent a step
further to the appropriate evaluation on the genotoxicity and cytotoxicity of B. anisandra infusion. The leaves of B.
anisandra were dried at 40º C in a forced ventilation stove and ground in a fraction mill to a dry powder that was
submitted to the hot aqueous extraction process. Doses of the infusion (1, 2.5 and 4%) were orally administered
via needle gavage once daily to groups of five Wistar rats (with approximate 250g) for each treatment. A negative
control group (sterile distilled water) was also included. The administration of the test substance was made for 30
days. After the sacrifice of the animal, a sample of the material of the bone marrow was collected from femurs,
in a small quantity (3mL) of fetal bovine serum. After centrifugation and discard the supernatant, two smears
were prepared for each animal. The cells were stained 24 h after the preparation of the blades, with the purpose
of differentiating PCE (non-nucleated polychromatic erythrocytes) of NCE (normochromatic erythrocytes), using
Leishman eosin-blue. The frequency of micronuclei in 1000 cells per repetition (5 repetitions) of polychromatic
erythrocytes, increased to the extent that the concentrations were diminishing. The statistical analysis (ANOVA
and Regression) showed significant results. The results for all tested doses indicate significant increase in MNPCE
frequencies when compared with the negative control group (P>0.05), leading to the conclusion that B. anisandra
harbor a genotoxic effect. Compared with the negative control, the concentration of 1% was the highest amount of
micronuclei. We also observed cells with two and three micronuclei. The results indicate, therefore, that infusions of
Banisteriopsis anisandra is genotoxic in the concentrations tested not being recommended for medicinal purposes.
Financial support: FAPEMIG
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
19
Antimutagenicity of digoxin, a drug used to
treatheart disease
Silva, IVG; Kéfrem, GAG; Oliveira, JT; Barbosa, MCS; Barbosa, LAO; Santos, FV.
Laboratório de Biologia Celular e Mutagênese, Campus Centro Oeste, Universidade Federal de São João del Rei (UFSJ),
Divinópolis, MG.
[email protected]
Keywords: Digoxin, Micronucleus Assay, Antimutagenicity, Mutagenicity, MTT Assay
Digoxin is a drug used to treat congestive cardiac insufficiency to several years. Despite the use of this drug in longterm treatments, there is no conclusive information concerning their effects on genetic material (possible genotoxic
or protectiveeffects). In the present study, CHO-k1 (Chinese Hamster Ovary), RKO (Human Colon Carcinoma)
and Hela (Human Cervice Carcinoma) cell lines were used to assess the mutagenicity and antimutagenicity of
digoxin employing the Cytokinesis-Block Micronucleus assay.The selection of test concentrations was based on MTT
colorimetric assay. This assay shows that the toxicity of digoxin was extremely variable between the different cell
types. In this way,to assess the mutagenicity of digoxin, high concentrations and low concentrations (near to the
digoxin concentrations observed in plasma of human users, that is around 1.8 nM) were used.CHO-k1 cells were
treated with 1.8 nM and 5, 10, 20, 35 and 50 µM of digoxin during three hours. In Hela cells the concentrations
evaluated were 0.7, 1.8, 3, 25, 50 and 100 nM. In RKO, Hela was used at 0.7, 1.8, 3, 100, 200 and 500 nM.
To evaluate the antimutagenicity of digoxin the micronucleus assay was performed only concentrations near to
the human therapeutic plasmatic level of this drug were assessed. Methyl methanesulfonate (MMS – 400 µM)
was used as positive control in mutagenicity analysis and as DNA damage inductor in antimutagenicity evaluation
(simultaneous treatment with digoxin). Two thousands binucleated cells were analyzed in each treatment and three
independent experiments were performed in each condition. For statistical analysis, the mean of binucleated cells
with micronucleus were calculated for each treatment and an ANOVA followed by Student-Newman-Keuls Multiple
Comparisons Test were performed. Digoxin was mutagenic in CHO-k1 at the higher concentrations employed
(35 and 50 µM) and in Hela (50 and 100 nM) inducing numerical and/or structural chromosomal mutations.
However, digoxin was not mutagenic at the concentrations near that observed in plasma of human users of this
drug. In RKO cell line digoxin was not. The antimutagenicity evaluation shows that digoxin did not reduced the
DNA damages induced by MMSin simultaneous treatmentin CHO-k1. However, in RKO cell line the protection
observed was very high, with a reduction of DNA damage around 90%. In Hela the reduction of DNA damage
was around 20%.Hela and RKO are human tumoral cell lines and an important difference between them is the
reduced expression of p53 in Hela. In this way, results presented herein may indicatesome modulation process of the
p53 expression or activity. Nevertheless, more studies are necessary, employing other treatment regimens and other
clastogenic/aneugenic agents to obtain more information about the mechanisms of the anticlastogenicity of digoxin.
Financial Support: FAPEMIG
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
20
Evaluation of the genotoxicity of
Vochysia divergens by citocinese-block
micronucleus assay
Mendes, SA; Oliveira, PF; Acésio, NO; Silva, MR; Soares, MA; Pimenta, LP, Januário, AH; Tavares, DC
Universidade de Franca, Franca, São Paulo, Brazil
[email protected]
Keywords: Vochysia divergens, genotoxicity, cytotoxicity , micronucleus, V79 cells.
The Vochysia divergens is better known as “cambará” belongs to the genus Vochysia and the family Vochysiaceae. It is
a tree commonly found in moist soils of the Pantanal of Mato Grosso, Brazil. This species is used in folk medicine
against respiratory infections, digestive disorders and asthma. Considering the use of this plant as phytochemical, the
aim of this work was to study the genotoxicity of V. divergens extract in Chinese hamster lung fibroblasts (V79 cells) by
citocinese-block micronucleus assay. For the evaluation of genotoxicity, the cultures were treated during three hours with
different concentrations of V. divergens extract (20, 40, 80, 160 and 320 µg/mL). These concentrations was previously
determined by the XTT colorimetric assay. The negative (without treatment), positive (methyl methanesulfonate,
MMS; 44 µg/mL) and solvent (dimethyl sulfoxide, DMSO; 0.5%) controls were included. The cytotoxicity of the
treatments also was evaluated by the nuclear division index (NDI). The results showed that the treatment with the highest
concentration of V. divergens tested (320 µg/mL) increased the micronuclei frequencies, being statistically different to
the negative control group. The NDI values obtained in the cultures treated with the highest concentration of extract
were significantly lower in compared to negative control cultures. Therefore, the V. divergens extract showed a genotoxic
and cytotoxic effect in the highest concentration evaluated, under the experimental conditions used in this study.
Financial support: São Paulo Research Foundation (Fapesp, São Paulo, Brazil)
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
21
In vitro study of cytotoxic, genotoxic and
antigenotoxic potential of Solanum cernuum
hydroalcoholic extract
Damasceno, JL1; Oliveira, PF1; Lima, M1; Bastos, JK2; Tavares, DC1.
Universidade de Franca, UNIFRAN, Franca, SP; 2Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP, Ribeirão
Preto, SP
1
[email protected]
Keywords: Solanum cernuun, citotoxicity, genotoxicity, antigenotoxicity, micronucleus
The genus Solanum L. is the largest and most complex of the Solanaceae family, among them is found Solanum cernuum
Vell, small Brazilian tree with medicinal potential that is restricted to the southwestern states of Brazil. This species
has been widely used to treat many diseases, being observed antimicrobial and antifungal activities. The present study
aimed to evaluate the cytotoxic, genotoxic and antigenotoxic potential of S. cernuum hydroalcoholic extract (SC) in
Chinese hamster fibroblasts (V79 cells). The cytotoxicity was evaluated by the clonogenic efficiency assay, which SC
concentrations ranging from 9.7 to 5000 μg/mL. The results showed that SC was cytotoxic at concentrations up to 160
µg/mL. Genotoxic and antigenotoxic potential were evaluated by micronucleus test. Four concentrations of SC (40, 80,
160 and 320 µg/mL) were used for the evaluation of its genotoxic potential. The DNA damage-inducing agent methyl
methanesulfonate (MMS, 44 µg/mL) was utilized in combination with extract to evaluate a possible protective effect.
A statistically significant increase in the micronucleus frequencies was observed in the cultures treated with the highest
concentrations of SC (160 and 320 µg/mL). The simultaneous treatment of cells with 20, 40 or 80 µg/mL of SC plus
MMS no showed significant differences of micronucleus frequencies when compared to treatment with MMS only. In
conclusion, under the experimental conditions used in this study, the results showed that SC exerted genotoxic effect in
the highest concentrations tested and no showed protective activity against in relation to MMS-induced genotoxicity.
Financial support: São Paulo Research Foundation (Fapesp, Brazil, grant number 2012/07946-4)
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
22
Influence of Shefflera vinosa extract
on genotoxicity induced by methyl
methanesulfonate in swiss mice
Ferreira NH, Alves JM, Leandro LF, Pimenta LM, Andrade KJB, Cintra LS, Januário AH, Tavares DC.
University of Franca, Franca, São Paulo, Brazil
[email protected]
Keywords: Schefflera vinosa; antigenotoxicity; potentiating effect; comet assay; micronucleus test.
Schefflera is the largest genus of Araliaceae, with 650-900 species distributed mainly in tropical regions, especially
in mountainous formations. The extract of S. vinosa and its compounds isolated (betulin and quercetin-3-O-β-Drhamnoside) revealed antiparasitic and antioxidant effects. This present study evaluated the genotoxic potential of S.
vinosa extract and its influence on genotoxicity induced by methyl methanesulfonate (MMS) by the micronucleus
and comet assays. The cytotoxicity was evaluated by the nuclear division index (NDI) and Trypan Blue exclusion
method. The animals were treated with four different doses of S. vinosa extract (125, 250, 500 and 1000 mg/kg
b.w.) for the genotoxicity assessment. In the study of association, the animals received simultaneous treatments with
S. vinosa extract and MMS (40 mg/kg b.w.). The S. vinosa extract did not showed genotoxic by the bone marrow
micronucleus assay, since the frequency of micronucleus observed after treatments of animals with different doses
of extract was not statistically different than that negative control. However, a statistically significant increase in
the DNA damage frequencies was observed in hepatocytes of the animals treated with the highest dose tested of
the S. vinosa extract (1000 mg/kg b.w.) in comparison to the control group. The data also showed that the animals
treated with the highest dose tested of extract plus MMS demonstrated a statistically significant increase in the
frequency of micronuclei when compared to the MMS group alone. On the other hand, S. vinosa extract at dose
of 500 mg/kg b.w. significantly reduced the DNA damage frequency in Swiss mice hepatocytes induced by MMS.
NDI values obtained in the treatments showed no statistically significant differences when compared to the negative
control group. In addition, the treatments demonstrated cell viability above 90% by Trypan Blue exclusion method.
Therefore, under the experimental conditions used, S. vinosa extract revealed genotoxic activity at the highest dose
tested by comet assay, however no genotoxic effect was observed by micronucleus test. S. vinosa extract exerts different
effects on MMS-induced genotoxicity. The modulatory activity of extract depends on the system test and doses used.
Financial support: São Paulo Research Foundation (FAPESP, São Paulo, Brazil, grant number 2012/24427-0).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
23
Absence of carcinogenic effect of aspirin
(acetylsalicylic acid), assessed by testing
for clones of epithelial tumors in Drosophila
melanogaster
Oliveira, VC2; Orsolin, PC1; Silva-Oliveira, RG1; Machado, NM1; Nepomuceno, JC1,2.
Instituto de Genética e Bioquímica da Universidade Federal de Uberlândia – UFU, Uberlândia – MG; 2Laboratório de
Citogenética e Mutagênese do Centro Universitário de Patos de Minas – UNIPAM, Patos de Minas – MG.
1
[email protected]
Keywords: Cancer, Acetylsalicylic acid, Drosophila melanogaster.
Acetylsalicylic acid, commercially known as aspirin, is a drug widely consumed by the population due to its antiinflammatory proprieties (NSAIDs), also used as antipyretic, analgesic and antithrombotic. Some studies involving
aspirin have been conducted as an attempt to prove its activity in inhibiting the growth of endometrial, esophageal, gastric,
lung, and colon and rectal tumors, and this effect is probably related to the inhibition of prostaglandin production via
the enzyme cyclooxygenase (COX). However, there are still doubts as to the real effects of this drug in relation to cancer.
Therefore, this study aimed to evaluate the carcinogenic effect of aspirin, through testing for the detection of clones of
epithelial tumors in Drosophila melanogaster, heterozygous for the tumor suppressor gene wts. For this, a treatment was
carried out with 72-hour larvae resulting from the crossing between wts/TM3, Sb1 virgin females and mwh/mwh males,
with three different concentrations of aspirin (10 mg/mL, 20 mg/mL and 40 mg/ml) alone. As a positive control, we
used mitomycin C to 0.1 mM, and as a negative control, reverse osmosis water. All larvae descending from the crossover
were treated with the concentrations of aspirin. However, only adult flies carrying the balancer chromosome (TM3,
Sb1) were analyzed since they have the gene under study. After the analysis of the flies were evaluated the statistical
differences between the frequency of tumor in the tested concentrations and controls (positive and negative), calculated
according to the nonparametric Mann-Whitney U test, using the significance level < 0.05. The results obtained in the
evaluation of possible carcinogenic effects of aspirin in the three concentrations tested showed no statistically significant
increase in tumors when compared to the negative control (water). The results indicate that the aspirin (acetylsalicylic
acid), in the present experimental conditions, did not induce the occurrence of tumors in Drosophila melanogaster.
Financial Support: UNIPAM and UFU.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
24
Modulatory effect of Betulinic Acid on the
genotoxicity induced by different mutagens
Acésio, NO; Mastrocola, DFP; Lima, IMS; Munari, CC; Sato, VLFL; Souza, APS; Cunha, WR; Tavares, DC.
Universidade de Franca, Franca, São Paulo, Brazil
[email protected]
Keywords: Betulinic Acid, V79 cells, micronucleus test, genotoxicity, modulatory effect
Betulinic acid (BA) is a naturally occurring triterpene isolated from various plants, largely distributed in tropical
regions. The BA has shown to possess antitumor activity by inducing apoptosis, while normal cells are not affected.
This study was carried out to evaluate the genotoxicity of BA and its influence on the genotoxicity induced by
different mutagens inV79 cells by micronucleus test. The cytotoxicity was evaluated by the clonogenic efficiency
assay. The results obtained showed that the cultures treated with concentrations above 10 μg/mL were cytotoxic.
The cultures of V79 cells were treated with different concentrations of BA (1.25, 2.5, 5.0 and 10.0 µg/mL). For
modulatory effect assessment, the concentrations of BA were associated with methyl methanesulfonate (MMS, 44
μg/mL), doxorubicin (DXR, 1.5 μg/mL), camptothecin (CPT, 43 μg/mL) and etoposide (VP16, 1 μg/mL). BA did
not show genotoxic activity in V79 cells, since the frequency of micronucleus observed after treatments of cells with
different concentrations of BA was not statistically different than that negative control. In addition, the simultaneous
treatment of cells with BA (2.5 µg/mL) and MMS resulted in lower micronucleus frequencies than those observed for
cultures treated with MMS alone. On the other hand, the cultures treated with BA (2.5 and 10 μg/mL) plus DXR
or BA (5 µg/mL) plus VP16 demonstrated a significant increase in the micronucleus frequencies when compared to
mutagens alone. With respect to CPT, the results showed that BA no exerts influence on the genotoxicity induced by
CPT. Therefore, in the experimental conditions used, BA exerted no genotoxic activity. However, BA exerts different
effects on mutagens-induced genotoxicity and modulation depends on the type of mutagen and concentrations used.
Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
25
Effect of dilapiol in the chromosomes of Aedes
(Stegomyia) albopictus Skuse, 1894 from
amazon, Manaus, state of Amazonas
Meireles, S.F1; Domingos, P.R.C.2; Rafael, M.S.2
Universidade do Estado do Amazonas, UEA, Manaus, AM; 2Laboratório de Vetores da Malária e Dengue/CPCS/Instituto
Nacional de Pesquisas da Amazônia, INPA, Manaus, AM.
1
[email protected]
Keywords: Cytogenetics; Genotoxicity; Biological Control, Bioassay, Dengue.
Aedes albopictus is a mosquito of epidemiological importance, due to its vector competence for about 26 arboviruses
such as Dengue, Chikungunya and Yellow Fever found in Brazil. In this country, there are no reports of transmission
of dengue by A. albopictus, but it is an important vector in Asia and Africa, affecting over 50 million people per year.
Natural compounds of herbal, essential oils or their derivatives have been effective as new alternatives to control vectors
of medical importance with known resistance to synthetic insecticides. The dilapiol is a kind of essential oil extracted
of Piper aduncum, and represents a successful example of extract of plants used in control of insect vectors, and also
serving as the basis for the synthesis of semisynthetic potential insecticides. The A. albopictus has 2n = 6 chromosomes,
whose molecular structure can occur mutagenic effects caused by plant extracts, such as dilapiol. Classic cytogenetics
analysis has been a useful tool to characterize micronuclei and other chromosomal malformations in several species of
mosquitoes. We used two compounds dilapiol semisynthetic derivatives, ethyl ether dilapiol (1KL39-B) n-butyl ether
and dilapiol (1KL43-C), in order to assess their potential ovicidal, larvicidal and genotoxic on eggs, larvae and adults of
A. albopictus, as a potential control of this species. Immature samples of Aedes albopictus were collected in Puraquequara
District, Manaus, State of Amazonas, Brazil. Eggs and 3rd-instar larvae obtained of generated colonies of A. albopictus
in insectary, CSAS, INPA, Manaus, were exposed at different concentrations of 1KL39-B and 1KL43-C for 24 hours
(toxicological testing) and 4 hours (genotoxicity assay). The cytological preparations were made according to Imai et
al. (1988). The toxicological assay showed 100% mortality of eggs exposed to concentrations of 70 and 80 mg/ml
1KL39-B and 20 and 30 mg/ml 1KL43-C. Mortality of larvae 76 and 88% for 70 and 80 mg/ml 1KL39, and 54 and
100% for 20 and 30 mg/ml 1KL43-C, respectively, was registered. Increase in the frequency of nuclear abnormalities
in larvae and adults was proportional to the increase of the 1KL39-B and 1KL43-C concentrations. Added to
the increased frequency of abnormalities, there was a reduction in the average number of eggs of couples exposed
compared to control couples. These preliminary results indicate that the more nuclear abnormalities may occur in
succeeding generations, and justify a possible application of these compounds in the alternative control of A. albopictus.
Financial Support: Universal Project, process, nº 1036/2011/FAPEAM_CNPq. Universal Project, process. nº 4809262011/CNPq.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
26
Modulating effect of Rosuvastatin on the DNA
damage induced by Doxorubicin in somatic
cells of Drosophila melanogaster
Orsolin, PC1; Silva-Oliveira, RG1; Machado, NM1; Oliveira, VC2; Nepomuceno, JC1,2.
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, MG, Brasil; 2 Laboratório de Citogenética e
Mutagênese, Centro Universitário de Patos de Minas, Patos de Minas, MG, Brasil.
1
[email protected]
Keywords: Rosuvastatin, modulating effect, SMART, Drosophila melanogaster.
Statins are the most commonly used drugs in the treatment of hyperlipidemia, in order to reduce levels of cholesterolrich lipoproteins. These effects result from the inhibitory activity on the enzyme HMG-CoA reductase, with the
property of blocking the conversion of the substrate HMG-CoA to mevalonate, inhibiting the initial steps of
biosynthesis of cholesterol. In addition to its effects in lowering cholesterol, statins present recognized additional
pharmacological properties. Such pleiotropic effects result mainly from their capacity to inhibit the synthesis of
isoprenoids, which are essential to the functioning of proteins responsible for intracellular signaling. Some recent
researches suggest an important antineoplastic role associated with the use of rosuvastatin, a synthetic statin widely
used today. Thus, this study was carried out with the aim of evaluating the possible protecting effects of Rosuvastatin
against damage induced by doxorubicin, by means of the somatic mutation and recombination test (SMART) in
Drosophila melanogaster. For this, larvae descendants from the standard cross (ST) and the high bioactivation cross
(HB) were treated chronically with a negative control (ethanol 5%), a Doxorubicin positive control (DXR 0.125 mg/
mL), and with five different concentrations of rosuvastatin (18.75, 37.5, 75.0, 150 and 300 µM/mL) separately or
in association with DXR. The results showed no toxic effect (survival rates above 92% at all concentrations tested)
and no mutagenic effect, since there was no significant difference in the total number of spots of subjects treated with
Rosuvastatin and the negative control. The analysis of the progeny co-treated with doxorubicin and the different
concentrations of rosuvastatin showed a reduction in the total number of spots mutants (compared to positive
control), which shows that this compound has an activity for modulating the effects induced by DXR. This effect
was observed at all concentrations tested in the offspring of crossing ST and HB, with no dose dependent relation.
We conclude, therefore, that under the experimental conditions outlined in this study, Rosuvastatin reduces damage
induced by DXR in somatic cells of D. melanogaster, and this effect is independent of metabolic biotransformation.
Financial support: CAPES, UFU, UNIPAM.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
27
Evaluation of toxicity, cytotoxicity and
genotoxicity of aqueous extract of Malpighia
glabra on Allium cepa test system
Silva, FGS da1; Costa, D de AF1; Sousa, DKC de1; Barros, TL de1; Maia Filho, ALM1; Baltatu, OC2.
Faculdade Integral Diferencial, FACID, Teresina, PI; 2Universidade Camilo Castelo Branco, São Paulo, SP
1
[email protected]
Keywords: Malpighia glaba, Allium cepa, genotoxicity
Barbados cherry (Malpighia glaba) is a fruit prized for its high content of vitamin C, besides being a source of
carotenoids and anthocyanins. The composition of the fruit depends on factors such as weather conditions, stage of
maturity, among others, and may also be modified by processing and storage. The system test plant of Allium cepa
presents itself as an ideal biomarker for the first screening of genotoxicity infusions due to its low cost, reliability and
agreement with other genotoxicity tests, aiding studies of prevention of harm to human health. Therefore, this study
aimed to evaluate the toxicity, genotoxicity and cytotoxicity of the aqueous extract of Malpighia glaba system Allium
cepa test. The aqueous extracts Malpighia glabra at concentrations of 5 mg / ml, 10 mg / ml and 15 mg / ml, plus the
negative control and positive control groups formed the five tests, and for each of these groups were used six onion
bulbs. The bulbs were kept for 72 hours in the dark (with exchange of water) at 25 ± 1 º C, the roots of approximately
1-2 cm were selected for the experiment and fixed in a Carnoy solution for 24 hours. The roots were hydrolyzed with
1N HCl at 60 ° C for 11 min, dried and transferred into dark bottles (amber) containing Schiff. The meristematic
region was separated which was added a drop of acetic carmine 2%. The slides were then analyzed. With respect to
the toxicity observed that as the concentration increased, decreased size of the roots, indicating growth inhibition.
Regarding the significance of the mitotic index was negative control in relation to concentrations of 5mg/ml (p <0.05),
and 15mg/ml (p <0.05), and there is no significance when comparing the concentrations of 5mg/ml, 10mg/ml and
15mg/ml between themselves. In the aspect of chromosomal aberrations was not significant when comparing the
groups. When analyzing the number of micronuclei found no significant difference was observed between the groups
analyzed, however there was an increasing number of micronuclei with increasing concentration. It can be concluded
that the aqueous extract of Malpighia glabra showed toxicity at concentrations of 10 mg / ml and 15 mg / ml and
showed no cytotoxicity and genotoxicity at any of the concentrations when measured by the test system Allium cepa.
Financial Support: Scientific initiation program voluntary- PIVICFACID
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
28
Genotoxicity effects of Banisteriopsis
anisandra alkaloids on polychromatic
erythrocytes in the bone marrow of Wistar rats
Oliveira, GK¹; Oliveira, AR¹; Mendes-Costa,MC¹.
¹Centro Universitário de Lavras- Núcleo de Pesquisa em Ciências Biológicas- NPCBio
[email protected]
Keywords: medicinal plants, micronucleus test, alucinógenos
The lack of more detailed information about the content of toxicity and genotoxicity of the active principles of plants of
the genus Banisteriopsis in particular of alkaloids, makes it attractive for studies. Large part of Brazilian native plants still
have no scientific studies to allow its safe and effective use, require greater quality control, since the scientific literature
indicates that many of these may present toxic substances or variable chemical composition. The genus Banisteriopsis
is known for its diversity of psychoactive alkaloids, such as the indole- b-carboline harmina and harmalina found in
B. caapi, the latter being used in religious rituals in Brazil, Bolivia, Ecuador and Peru. The aim of this study was to
assess the genotoxic activities of the alkaloids Harmina and Harmalina using the micronucleus test in polychromatic
erythrocytes in bone marrow of rats in vivo.The alkaloids were obtained from Sigma Aldrich. The substance was
administered via needle gavage once daily in 8 young Wistar rats during two consecutive days. The animals were
sacrificed after 48 h of the last administration.The dosages used were 1, 2 and 3 mg/kg, the negative control was done
with distilled water and the positive control with cyclophosphamide. After the sacrifice of the animal, a sample of the
material of the bone marrow was collected from femurs, in 3 mL of fetal bovine serum. After centrifugation and discard
the supernatant, two smears were prepared for each animal. The cells were stained 24 h after the preparation of the
blades, with the purpose of differentiating PCE (non-nucleated polychromatic erythrocytes) of NCE (normochromatic
erythrocytes), using Leishman eosin-blue. Our results showed that neither Harmaline nor Harmine presented an
increase in the micronucleated polychromatic erythrocytes (P>0.05) using the micronucleus test, suggesting absence
of genotoxicity in both tests. We also observed some cells with more than one micronucleus. In view of these results it
can be concluded that the alkaloids Harmina and Harmalina had no genotoxic action on bone marrow cells of rodents
in the concentrations tested, but it is not recommended the use of this kind of plants in folk medicinal purposes.
Financial support: FAPEMIG
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
29
Effect of leachate from Sanitary landfills
on water quality in Perdizes river, monte
Carmelo, MG, Brazil
Dayrell,DM1,2; Campos, CF1,2; de Campos Junior,EO2; Pereira,BB1,2; Rodrigues, FFS1,2 Morais, CR1; Oliveira, SR1,2;
Marques, JGB1; Morelli, S2
FUCAMP, Fundação Carmelitana Mário Palmério, Monte Carmelo – MG, Brazil; 2 Laboratório de Citogenética. Instituto
de Genética e Bioquímica, UFU, Uberlândia – MG, Brazil.
1
[email protected]
Keywords: Micronucleus. Cytotoxicity. Biomonitoring, Genotoxicity, Allium cepa
Chemical compounds, originating from industrial and agricultural activities and domestic residues are major concerns
with regard to contamination and pollution of water resources. The practices employed for the control of water quality
and effluent treatment is not always effective. The environmental biomonitoring consists in employing organisms to
identify factors that influence in the environmental quality. The objective of the present study was to evaluate the
influence of treated leachate by sanitary landfill discarded in Perdizes River, Monte Carmelo, Minas Gerais, Brazil,
using the Allium test. Water samples for bioassays and chemical testing on three stretches of the river (S2: before
to discard of the leachate; S3: location discard of the leachate and S4: location after discard of the leachate) were
collected. Tests for referential site (distilled water) and positive control (cyclophosphamide) were performed. For
cytogenetic analysis were used eight bulbs of Allium cepa which were exposed for 72hs, with subsequent excision of
meristematic cells of their roots, which were removed and fixed in Carnoy solution for 12 hours. Then the roots were
washed and submitted to hydrolysis in 1N HCl solution for 10 minutes at 60°C. Subsequently, the roots were stained
with Schiff and 2% acetic carmine. Seven slides were prepared for each point, including the control groups. 2000
cells per slide with an optical microscope under the magnification of 400x were analyzed. Analysis of root growth
rate and mitotic index (MI) were performed in meristematic root cells treated with water samples from the five sites,
including the Reference site, characterized as S1 and positive control. The MI analysis results were significant at all
tested sites (S2, S3 and S4) compared to S1. Similarly the root growth rate has demonstrated that the sites in the test
group showed significant retardation of growth compared to the control group, especially in site 3. The Micronucleus
test (MN) in accordance with the physical and chemical parameters of the water showed that sites S3 and S4 differed
significantly (p<0.05) from site 1, according to Mann-Whitney test. It’s possible infer that the discard of leachate, even
after treatment at sanitary landfill, directly affects the quality of water in some sites of the river, directly interfering
in the organisms that is found in such sites, and for the people who make use of this water without due treatment.
Financial support: Fapemig, Capes, Cnpq, UFu, FUCAMP.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
30
Evaluation of chromosomal damage
frequency induced by Persea americana pulp
oil in in vitro mammalian cells
Corrêa, MB; Oliveira, PF; Damasceno JL; Silva DC; Cunha WR; Tavares, DC
Universidade de Franca, Franca, São Paulo, Brazil
[email protected]
Keywords: Persea americana, genotoxicity, micronucleus test, V79 cells
Persea americana Miller is commonly known by “avocado”. This plant belonging to Lauraceae family and is
considered a tropical fruit, where the pulp oil is known to be rich in vitamins, proteins and phytosterols. Among
its biological activities can cite the prevention of prostate hyperplasia and cholesterol reduction. Considering the
pharmacological potential of this plant, this study aimed to evaluate the genotoxicity of P. americana pulp oil in V79
cells by the micronucleus test. The cell cultures were treated during three hours with the P. americana pulp oil in
different concentrations: 400, 600, 800 1000 and 1200 µg/mL. These concentrations were previously determined
by the XTT colorimetric assay using the cytotoxicity as criterion. The negative (without treatment), solvent (Tween
80; 0.5%) positive (methyl methanesulfonate, MMS; 44 µg/mL) controls were included. The cytotoxicity of the
treatments also was evaluated by the nuclear division index (NDI). The results showed a significant increase in the
micronuclei frequencies in the cell cultures treated with the highest concentration of P. americana oil (1200 µg/
mL) when compared to negative control cultures. The NDI values were significantly lower in the cultures treated
com 1200 µg/mL of P. americana oil in relation to negative control group. Therefore, the P. americana pulp oil
showed genotoxic and cytotoxic effects in the highest concentration tested, under the experimental conditions used.
Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
31
Cytotoxic and genotoxic effects of crude
metanolic extract of Banisteriopsis malifolia
(Malpighiaceae) on Allium cepa L. root tip cells
Xavier,LB1; Oliveira,AR1; Frias, UA1; Mendes-Costa, MC1
1
Centro Universitário de Lavras, Lavras, MG; Núcleo de Pesquisas em Ciências Biológicas-NPCBio
[email protected]
Keywords: Micronucleus test, medicinal plants, chromosome aberrations
The use of plants with phytochemical properties is without doubt one of the most widespread habits in humanity.
Brazil as a rich country in biodiversity has a large number of species that already has medicinal uses by local population
and this reason has led to the need to assess their genotoxicity. The plants of the genus Banisteriopsis present significant
importance since not only their medicinal activity but also their psychotropic use were already known for long ago.
Some species of the family Malpighiaceae are known as hallucinogenic, as is the case of Banisteriopsis caapi, used in
religious practice. The genus Banisteriopsis has 66 species, and is one of the genera with the highest number of species
in Malpighiaceae. Among many pharmacologically active compounds, which were found in B. malifolia alkaloids,
flavonoids and tannins seem to be the most noteworthy, corroborating with literature data. Due to the scarcity of
data in the literature, the aim of this work was to evaluate both the cytotoxicity and the genotoxicity of metanolic
extract of B. malifolia using Allium cepa L. root tip cell (onion). Plant samples were collected in Reserva Particular do
Patrimônio Natural do Clube de Caça e Pesca Itororó de Uberlândia. The bioassays for the assessment of phytotoxicity
were carried out through four different dilutions (1:1; 1:2; 1:4 e 1:10 (p/v)) of the extract. Sterile water was used as a
negative control, with 4 replications per dilution with 50 onion seeds purchased in local trade, for each replication.
The cytotoxicity evaluation was performed through the inhibition of growth, taking into account the average size of
the root and stem, as well as the percentage of germination. The genotoxic potential of the extract was carried out
by counting 4000 cells per dilution extract by the technique of scanning, and then by calculating the mitotic index
(MI), micronucleus and chromosomal aberrations. Statistical analyzes were performed using ANOVA and Regression
Analysis. The results of the IM was statistically significant at dilutions of 1:1 and 1:2 being observed positive correlation
with chromosomal aberrations that were also significant at this same dilutions. The cytotoxic action of the extract was
shown at dilutions of 1:1, 1:2 and 1:4, with thickening of roots, dark color and lower growth. These results suggest
inhibitory and genotoxic activity of the metanolic extract of the plant Banisteriopsis malifolia at dilutions 1:1; 1:2 and 1:4.
Financial Support: CNPq
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
32
Evaluation of the angiogenic potential and
genotoxic activity of Hancornia speciosa latex
Ribeiro, T.P.1; Silva, J.F.2; Sousa, T.R. 1; Peixoto, N.1; Melo-Reis, P.R.3; Graeff, C.F.O.2; Gonçalves P.J.4; Almeida, L.M.1
1
Universidade Estadual de Goiás – UnU Ipameri, Brazil; 2 Faculdade de Ciência de Bauru – Universidade Estadual
Paulista (UNESP), Bauru, SP; 3 Departamento de Biomedicina da Pontifícia Universidade Católica de Goiás – PUCGo; 4
Instituto Física–Universidade Federal de Goiás (UFG), Goiânia, GO.
[email protected]
Keywords: CAM assay; biomaterials, cometa test, mutagenesis, mangabeira.
Plants are promising sources of new bioactive compounds, especially to medicinal field. Hancornia speciosa, popularly
known as “mangabeira”, is a fruit plant with wide distribution in Brazil. The latex of this plant is used in folk medicine
on the treatment of tuberculosis, liver dysfunction, ulcers, warts and dermatitis. In the present study, the angiogenic
activity of Hancornia speciosa latex was evaluated using the chick embryo chorioallantoic membrane (CAM) assay and
the genotoxic activity by comet assay. Analysis and quantification of the neoformed vascular net in CAM assay were made
using 20 eggs for 5 treatments: 1) Dexamethasome (negative control); 2) water; 3) Regerderme (positive control); 4)
mangabeira latex diluted in water and 5) Mangabeira latex with 0.01% ammonium. The images were captured images
by camera and the percentage area of each assay was determined using the programs Gimp for Windows and Image J. The
results showed significant increase of the vascular net (P<0,005) in latex samples when compared as to the negative control
(dexametasona and water), but there was not identify significant difference between mangabeira latex and Regederme.
For comet assay, the fibroblast cells treated with mangabeira latex were submitted to electrophoresis on the slides.
Three conditions were tested: negative control, mangabeira latex and mangabeira latex with 0.1% ammonium. The
percent of DNA in tail was assessed by Comet II image analysis system. The results showed that mangabeira latex is not
genotoxic when compared with negative control, however the latex with ammonium present DNA damage (P<0,05). In
summary, the mangabeira latex has angiogenic potential and is not genotoxic and can be a good biomaterial for medical
applications. This study contributes to understanding of the potentialities of mangabeira latex as sources of new drugs.
Financial Support: CNPq and FAPEG
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
33
Comparison of the effects of methanolic
extracts of leaves Chrysobalanus icaco in lung
cell lines
Cruz, LO1; Fróes, BB1,2; Nascimento, LB1,2; Gagliardi, RF3; Oliveira, MBN1; De Mattos, JCP1; Caldeira-de-Araujo, A1;
Dantas, FJS1.
Laboratório de Radio e Fotobiologia, UERJ, Rio de Janeiro, RJ; 2Famath- Faculdades Integradas Maria Thereza, Niterói,
RJ; 3Núcleo de Biotecnologia Vegetal, UERJ, Rio de Janeiro, RJ
1
[email protected]
Keywords: Cytotoxicity, Medicinal plants, Methanolic extract.
Plants have been used in folk medicine for the treatment of several diseases, despite the little scientific knowledge
about their potential toxicity. Studies have been developed to ensure safe and effective use of medicinal plants. The
aqueous extract of leaves Abajeru (Chrysobalanus icaco L.) is used popularly as a diuretic and hypoglycemic. The
aim of this study was to evaluate the biological effects of this extract in a neoplastic cell line and its corresponding
wild. Cuttings of C. icaco were collected at the Parque das Dunas in Cabo Frio (22 ° 54’26, 4’’ S, 42 ° 02’05, 5’’ W),
RJ and leaves separated into two groups: (i) intact and (ii ) preyed, and both were maintained in methanol (50g /
L). Following, the extracts were submitted to rotary evaporator. Cytotoxic effect was assayed in lung carcinoma cells
(A549 cells) and its wild line lung (MRC5). The cell cultures were distributed in 24-well plates with F12 medium
and incubated at 37 ° C and 5% CO2 for 24h. After this, the culture medium was discarded, cells were washing
with phosphate buffered saline (PBS), the fresh growth medium was added to each well, plus different plant extract
concentrations (6.25 and 12.5 µg/ml). The cells were incubated for 24 hours and then, treated with trypsin-EDTA
solution (0.01% and 0.1). After centrifugation, cells were stained with Trypan Blue and counted in a Neubauer
chamber for determining the number of viable and non-viable cells. Cell viability was also evaluated for its ability to
multiply. After treatment, aliquots containing 300 cells were incubated at 37°C in a 5% CO2 atmosphere for ten days.
Following, cells were washed with PBS, fixing solution was added and stained with Giemsa solution. After drying at
room temperature, cell clusters were counting. Genotoxic potential of the plant extract was determined by the comet
assay, under alkaline conditions. After treatment, cells were homogenized with low melting point agarose and arranged
in glass slides containing normal melt point agarose and then kept in ice-cold lysis solution for 48 hours. After this
period, the slides were subjected to horizontal electrophoresis in alkaline buffer. After this, slides were neutralized,
stained with silver solution and analyzed under a light microscope and assessed damage according to the intensity tails
of comets, in 4 different classes. Higher cytotoxic effect was measured by trypan blue assay when cells were exposed
to the extract concentrations of 6.25 and 12.5 µg/ml. The survival rate was lower in A549 cells, when compared
to the MRC5 cell line. The neoplasic cells showed decreasing in mitogenic capacity and the comet assay showed
increasing damage in DNA of treated A549 cells. In a general manner, cancer cells were more sensible than wild type.
Financial support: Faperj, CNPq e UERJ
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
34
Evaluation of guine ethanolic extract
cytotoxicity in a549 line cells
Nascimento, LB1,2 ;Fróes, BB1,2; Cruz, LO1; Soares, BO3; Gagliardi, RF3; Oliveira, MBN1; De Mattos, JCP1; Caldeirade-Araujo, A1; Dantas, FJS1
1
Laboratório de Radio e Fotobiologia, UERJ, Rio de Janeiro, RJ; 2Famath- Faculdades Integradas Maria Thereza, Niterói,
RJ; 3Núcleo de Biotecnologia Vegetal, UERJ, Rio de Janeiro, RJ
[email protected]
Keywords: Petiveria alliacea L., Medicinal Plants, trypan blue, cancer cells, cell death
The great biological diversity has been used as a resource for extractive activities in the natural environment, among
which stands out the collection of plant species used in medicinal practices, which have attracted interest due to the
pharmacological, economic and cultural activities. Several studies have been conducted to ensure safe and effective use
of medicinal plants. The leaf ethanol extract of Petiveria alliacea, known as guine, in Rio de Janeiro, is recommended
for external use in the treatment of rheumatism, while the root one is used against toothache. The dibenzil trisulfide
(DTS) is the main lipophilic compound of P. alliacea and has been identified as an immunomodulatory component,
which also has neurotoxic and cytostatic properties. The aim of this study was to evaluate the cytotoxicity of the
ethanol extract of P. alliacea in A549 cancer cell line. Samples of P.alliacea L. were collected in Niterói, Rio de
Janeiro (22 ° 53’55 .95 “S and 43º 05’09 .37”), the leaves were washed, dried in an oven at 45°C, macerated and
kept in an incubator. Ethanol solution (100%) was added and the vials were sealed and placed under stirring and
then they were subjected to a rotary evaporation. The A549 cell cultures were digested at room temperature with
0.01% Trypsin-EDTA and centrifuged. Following, an aliquot (10 µL) was stained with 0.4% Trypan Blue dye and
the viable cells was determined in a Neubauer chamber. The cultures were plated and incubated for 24h (37oC, 5%
CO2). In the next day, they were treated with the P. alliacea extracts (2,5 – 100 ug/ml) and incubated for 24h (37oC,
5% CO2) Viability was assessed once more by means of trypan blue exclusion. A549 cells treated with ethanol extract
of P. alliacea had a lower survival rate when compared to control. The highest difference (p<0.05) was observed
after incubation with a concentration of 10mg/ml, occurring cell death of more than 90% of the population.
Financial support: UERJ, FAPERJ, CNPq.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
35
Citotoxicity of Petiveria alliacea in vitro
ethanolic extract in A549 cells
Fróes, BB1,2; Nascimento, LB1,2; Cruz, LO1; Soares, BO3; Gagliardi, RF3; Oliveira, MBN1; De Mattos, JCP1; Caldeirade-Araujo, A1; Dantas, FJS1.
1
Laboratório de Radio e Fotobiologia, UERJ, Rio de Janeiro, RJ; 2Famath- Faculdades Integradas Maria Thereza, Niterói,
RJ; 3Núcleo de Biotecnologia Vegetal, UERJ, Rio de Janeiro, RJ
[email protected]
Keywords: Medicinal plants, Antineoplastic effect, Cell death, Guiné, Trypan blue
The vegetable active principles are the most source to provide raw material to chemical industries in the achievement
of drugs. Consequently, several studies have been developed to ensure safe and effective use of medicinal plants. Many
biological properties of Petiveria alliacea have been described in extract of field plant, including the inflammatory
activities, attributed to polysulfides derived from secondary metabolism. The aim of this study was to evaluate the
cytotoxicity of the P.alliacea extract produced in vitro in neoplastic A549 cell line. P.alliacea leaves produced and
maintained in vitro culture for 24 months were macerated and incubated in ethanol (100%) and then the extract
was subjected to a rotary evaporation. The lung cancer cells (A549 line) were distributed into 24 wells plates and
incubated in F12 medium at 37 °C in 5% CO2 atmosphere for 24h. After this period, F12 medium was discarded
and plates washed with phosphate buffered saline (PBS) then treated in F12 medium with P.alliacea extract. The cells
were once more incubated in the same conditions for 24 hours and subjected to trypsin-EDTA solutions (0.01 and
0.1%). Subsequently, they were stained with Trypan Blue and counted in a Neubauer chamber for evaluation of the
cell viability. Cells treated with different concentrations of plant extract showed a reduction in the survival rate. Thus,
compared to the control, the concentration of 50mg/ml promoted a significant difference (p<0.05), in the survival rate
of the cells. The concentration of 50mg/ml showed the greatest potential antitumor effect under the tested conditions.
Financial support: Faperj, CNPq e UERJ
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
36
Micronucleus Test in peripheral blood of
rats treated with hyperbaric oxygen after
splenectomy with inferior pole preservation
Souza, IF1; Silva, KB1; Wandekoken, KG1; Freitas, JV2; Paulo MSL1; Paulo DNS1; Errera, FIV1.
Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória (EMESCAM), ES; 2Universidade Federal do Espírito
Santo (UFES)
1
[email protected]
Keywords: Esplenectomy, Hyperbaric oxygen treatment, Micronucleus Test, Mutagenic potential, Wistar rats.
After the verification of the advantages of total or partial preservation of the spleen, due to it’s importance and functional
diversity, conservative surgery of this organ began to be more valued. It was demonstrated that hyperbaric oxygen
treatment (HBO) in rats submitted to conservative surgery like partial splenectomy with inferior pole preservation
(ESTPI), led to cellular and vascular proliferation, among other benefits. However, HBO has some collateral effects
such as pulmonary and neurological toxicities, and it could also be related to carcinogenic events and the cellular
aging. The micronucleus (MN) test is an easy, fast and with low cost method to identify chromosomal alterations in
vivo. It is a small mass of chromatin separated from the main nucleus formed during telophase of mitosis or meiosis,
which results from chromosome loss or malsegregation of chromosomes (non-disjunction). To evaluate the mutagenic
potential of HBO, the MN test was performed in peripheral blood of male Wistar rats submitted to ESTPI, followed
by HBO. It were used 12 animals, with 2-3 months of age, submitted to ESTPI, randomly distributed into 2 groups
of 6 animals: group 1 - not submitted to HBO and group 2 - submitted to HBO. Blood samples were collected
from the tail of all rats before the surgical procedure (T0) and immediately after the 10th session of HBO (Ti). The
animals were euthanized after the last collect. The number and the frequency of micronucleated normochromatic
erythrocytes (MNNE) were determined by the blindfold count of 2000 normochromatic erythrocytes per animal,
totaling 12000 cells, for each group. The chi-square test was used (x2) and p< 0,05 was considered significant. The
variables studied were the presence or absence of micronucleus, as well as the quantity of them. After the analysis, were
counted, in the group 1, at T0, 3 MNNE and the frequency of MNNE/NCE was 2,5 x 10-4. At Ti, 1 MNNE was
counted and the frequency of MNNE/NCE was 8,3 x 10-5. In group 2, at T0, 2 MNNE were seen and the frequency
of MNNE/NCE was 1,7 x 10-4. At Ti, in the same group, none MN was found. It was verified that the treatment
protocol used by the present study did not induce MN formation, since the analysis of the obtained results did not
show significant increase of the MNNE frequency in the group submitted to HBO when compared to the negative
control group. These negative results might be associated to a DNA repair mechanism suggested by other studies.
Financial Support: Foundation for Research Support of the State of Espírito Santo (FAPES)
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
37
DNA repair in bacterial cultures and plasmid
DNA exposed to infrared laser for treatment
of pain
Canuto, KS1, Sergio, LPS 1, Marciano, RS1, Guimarães, OR 1, Polignano, GAC 1, Geller, M 1,2, Paoli, F3 and Fonseca, AS 1,4,5
Centro de Ciências da Saúde, Centro Universitário Serra dos Órgãos, Teresópolis, RJ, Brazil; 2Setor de Facomatoses do
Serviço de Genética Clínica do IPPMG, Universidade Federal do Rio de Janeiro, RJ, Brazil; 3Departamento de Morfologia,
Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Campus Universitário, São Pedro, Juiz de Fora,
MG, Brazil; 4Departamento de Ciências Fisiológicas, Instituto Biomédico, Universidade Federal do Estado do Rio de
Janeiro, RJ, Brazil; 5Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcântara Gomes, Universidade
do Estado do Rio de Janeiro, RJ, Brazil
1
[email protected]
Keywords: infrared laser; Escherichia coli; DNA repair; pain; treatment
Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols
are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of
this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in
Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures of AB1157 (wild type),
BW9091 (xthA-) and BH20 (fpg-), and plasmids exposed to infrared laser (830 nm) at fluences used to treat pain (60
and 120 J/cm2), bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic
profile. As controls, E. coli cultures and plasmids not exposed to laser. In 24h of stationary growth phase, data from
survival assay of AB1157 (control and 120 J/cm2) were (media±standard deviation): 1.0±0.19 and 1.9±0.27; to
BW9091, 1.0±0.18 and 1.0±0.23, to BH20, 1.0±0.14 and 1.0±0.19. Data from filamentation assay to AB1157 were:
2.2±0.38 and 8.3±1.53; to BW9091, 18.7±3.21 and 33.0±2.10; to BH20, 1.3±0.58 and 4.3±0.56. Percentages of
supercoiled form were: 88.7±0.38 and 83.1±4.09. Percentages of supercoiled form of plasmids incubated with DNA
repair enzymes were: 92.1±1.01 and 92.0±1.14 (exonuclease III); 75.6±1.27 and 72.3±3.34 (fpg). Low intensity
infrared laser at the therapeutic fluences used to treat pain significantly (p<0.05) increases survival of E. coli wild type
and induces filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA
lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III
or formamidopyrimidine DNA glycosylase.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
38
The effect of the captopril against the
mutagenicity and recombinogenicity of
doxorubicin in the Drosophila wing spot test
Silva-Oliveira RG1, Orsolin PC1, Machado NM1, Oliveira VC2 And Nepomuceno JC1,2
Instituto de Genética e Bioquímica da Universidade Federal de Uberlândia – UFU, Uberlândia – MG; 2Laboratório de
Citogenética e Mutagênese do Centro Universitário de Patos de Minas – UNIPAM, Patos de Minas – MG.
1
[email protected]
Keywords: Angiotensins. SMART. Antihypertensive.
Captopril is an antihypertensive class of inhibitors of angiotensin converting enzyme (ACE) inhibitors whose
mechanism of action occurs by blocking ACE, reducing the formation of angiotensin II. Apart from its effect on blood
pressure regulation, the captopril is being further evaluated as a potential cancer chemopreventive agent. Thus, this
study aims to evaluate the mutagenic and antimutagenic potential of Captopril through the SMART test (Somatic
Mutation and Recombination Test) in somatic cells of Drosophila melanogaster. For the treatment, were used larvae
trans-heterozygous descendants of the high- bioactivation crossing (HB) and standard (ST). In this procedure were
used five concentrations of captopril (500, 250, 125, 62.5 and 31.25 µg/mL), a positive control (doxorubicin - DXR
125 µg/mL) and a negative control (water). All concentrations of captopril associated with chemotherapy DXR
125 µg/mL were also used. In the ST crossing, the results show that there were no significant increases in mutant
frequencies of spots when the individuals were treated only with captopril isolated concentrations, when compared
to the negative control, except for the concentration of 250 µg /mL whose diagnosis was statistically significant
(α> 0.05) for total spots (TM), whereas at associated concentrations, the diagnosis was significant only at 62.5 µg /
mL + DXR compared to the positive control. At the crossing HB we observed that in both isolated concentrations
and associated concentrations, the captopril showed no significant diagnostic when compared to their respective
controls. Considering the above information we can conclude that in individuals MH of the crossing ST the captopril
presented a mutagenic and / or recombinogenic potential as well as an ability to modulate the effect of DXR, but these
effects are not dose dependents. However in the individuals descendants from the crossing HB, where there is high
expression of enzyme cytochrome P450, the captopril did not present mutagenic and / or recombinogenic activity.
Financial Support: CAPES, UFU, UNIPAM.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
39
Biological monitoring of the Mumbuca
stream in Monte Carmelo/MG, Brazil in Bagre
(Rhamdia quelen)
de Campos Junior EO1; Pereira BB1,2; Dayrell DM2; Campos, CF2; Morais, CR1,2; Oliveira, SR1,2; Rodrigues, FFS1,2,
Morelli, S1
Laboratório de Citogenética. Instituto de Genética e Bioquímica, UFU, Uberlândia – MG, Brazil; 2 Facihus, Fundação
Carmelitana Mário Palmério, FUCAMP, Monte Carmelo – MG, Brazil.
1
[email protected]
Keywords: Biomonitoring, Rhamdia quelen, Daphinia, Genotoxicity, Citotoxicity
Chemical compounds that are foreign to local environments have been discovered and are influencing the dynamic
of the ecosystems, modifying the development of indigenous organisms and their interrelations. Environmental
toxicology characterizes the effects of chemical substances present in some species and their biological implications.
The induction of mutations in germ cells can result in an increased frequency of genetic damages. The biological
monitoring is used to identify the situation of the environment evaluating the potential contaminated organisms,
besides value the relation of the strange compounds and the physical factors that the organisms are subject. The study
aimed to evaluate the genotoxic potential by cytogenetic methods and its possible interference of 60 individuals of a
population of Rhamdia quelen, using the piscine micronucleus test, besides the use of bioassays of chronic toxicity with
Daphinia magna. The collections were carried out in four sites (areas were characterized by different standards of water
quality) in the Mumbuca stream, County of Monte Carmelo – Minas Gerais, Brazil. From each data site 12 individuals
were analyzed including the positive control. The analysis involved 2,000 cells from each fish for determination of
the frequency of micronuclei. The water quality index (WQI) showed decreasing quality of site 1(referential site) to
site 4. The analysis of the site 3 pointed to the presence of Aluminum and lead compounds, derivatives of effluent
from human activities, while the last site showed the presence of industrial and agricultural compounds. Duncan test
showed a variation in reproduction (p < 0.05) by the presence of chronic toxicity, indicating the presence of doses
of environmental aggressors in toxicity test. The frequency of micronucleus (MN) in the sites 3 and 4 represented
higher rate than the referential site, the same thing was observed by the number of micronucleus cells (CMN).
The statistical test of Mann-Whitney, it showed that there was significant difference between the collected Sites (F
= 44.08). The increase in micronucleus rates, as well as the chemical variables, showed the fact that sites 3 and 4
(located in an area with high genotoxic potential) present greater interference for the fish of the site in question. Thus,
Rhamdia quelen was demonstrated as a good indicator of genotoxicity, combined with chemical and toxicity tests.
Financial Support: Capes, CNPq, Fapemig e UFU.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
40
Biomonitoring air genotoxicity during and
after a public transportation strike in the
center of Uberlândia, Minas Gerais, Brazil
Pereira, BB1,2; De Campos Júnior, EO1; Morelli, S1.
Instituto de Genética e Bioquímica, UFU, Uberlândia, MG; 2Fundação Carmelitana Mário Palmério, FUCAMP, Monte
Carmelo, MG.
1
[email protected]
Keywords: Genotoxicity, Biomonitoring, Vehicle pollution, Micronuclei, Tradescantia pallida
The economic development, increasing rates of urbanization, political and fiscal incentives for the production and
purchase of vehicles and, particularly, the lack of public policies encouraging and modernizing public transportation
have all been cited as causal factors for the significant increase in individual motorization in Brazil. In Uberlândia,
as in any other large urban center, increasing numbers of vehicles have intensified traffic, causing congestion and
inconvenience. Additionally, the combustion of fuel for automotive engines contaminates the urban atmosphere
with ozone, sulfur and nitrogen oxides, heavy metals, organic compounds and particulate matter, causing damage to
the environment and, consequently, to human health. On May 17th, 18th and 19th in 2010, the public transport
workers of Uberlândia went on strike, reducing the bus traffic to approximately 50% of the normal amount observed
on working days. As there is a lack of information concerning the air quality in the city, and the occurrence of the
strike presented an opportunity to conduct a natural experiment, we attempted to biomonitor air genotoxicity in the
city center during and after the public transportation strike using a Tradescantia micronucleus test. The aim of this
study was to address the lack of information concerning the air quality in the city of Uberlândia, Minas Gerais, Brazil.
In this study, we conducted an unprecedented experiment involving the in situ biomonitoring of air genotoxicity
in the city center during and after a public transportation strike using the Tradescantia micronucleus test. Twenty
inflorescences were exposed daily from May 17th through May 23rd in 2010 in the city center and at the reference site.
The inflorescences were exposed each day during the same daily public transportation circulation period in the city at
each site between 06:00 am and 11:59 pm, totaling 18 hours of exposure per day during the three-day transportation
stoppage and for four days after the stoppage. The frequency of micronuclei was significantly higher in the city center
compared with the reference site (Mann-Whitney test, p<0.05), with the highest MN levels being observed during
public transport stoppage (Kruskal-Wallis, Dunn p<0.01). In addition, the multiple linear regression analyses revealed
that the low circulation of buses during public transport stoppage and the increase in the concentration of particulate
matter from the increased flow of vehicles in the city center during the strike positively influenced the MN frequency.
Financial Support: CAPES, FAPEMIG, UFU
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
41
Anti-/-genotoxicity of Cinnamomum camphora
in propylene glycol and essential oil solution,
in vitro
Santos, JC1; Bellini MF1.
1
Universidade Sagrado Coração, USC, Bauru, SP, Brasil
[email protected]; [email protected]
Keywords: Cinnamomum camphora, micronucleus, propylene glycol, essential oil, genotoxic
Plants have been used for medicinal purposes for centuries, improving life quality. Cinnamomum camphora (Camphor)
is a terpenoid ketone and represents a class of bioactive compounds synthesized by plants. It is widely prescribed for
treatment of inflammation diseases, but some high doses of camphor volatile oils are neurotoxicity. According to the
Technical Opinion No6, December 21st, 2010, the ANVISA, highlight the toxicity risk of camphor preparations,
because it can cause hepatotoxicity. Genetic-toxicological studies on Camphor are scarce and some publications
of Cinnamomum sp have shown contradictory results. The aim of this study was to evaluate the anti-/genotoxic
activities induced by 1% of camphor in propylene glycol and essential oil solution, in cultured of human lymphocytes
(approved by the Institutional Ethics Committee), by cytokinesis-block micronucleus (CBMN) assay. To perform
CBMN assay was used cell suspension of human peripheral blood, of 4 people (2 males and 2 females), that was
incubated in a glasshouse, for 6 hours, and then added the following treatments: negative control (C-), positive
control (methyl methane sulfonate - 4 x 10-²M (MMS)), 1% of Camphor in propylene glycol solution (PG), 1%
of Camphor in essential oil solution (EO), 1% of Camphor in propylene glycol solution + MMS (PG + MMS)
and 1% of Camphor in essential oil solution + MMS (EO + MMS). After 44 hours exposed to the treatments,
cytochalasin-B (6µg/mL) was added to the culture that was interrupted by completing 72 hours. The slides were
produced by drip and Giemsa stain for 5 minutes. Slides analysis was performed by counting 2000 binucleated
cells per treatment, on optical light microscopy. Also, cell viability was determined ≥ 50% in all treatments, by
trypan blue exclusion: 82,57% ± 0,014 (C-), 60,33% ± 0,0075 (MMS), 62,06% ± 0,1625 (PG), 81,83% ± 0,1035
(EO), 63,53% ± 0,5655 (PG+MMS) and 68,48% ± 0,6145 (EO+MMS), enabling the assay. The CBMN assay we
observed a increasing of micronucleus formation in camphor treatments, 8,78% ± 3,211 (PG), 10,72% ± 0,7538
(PG+MMS), 13,48% ± 2,386 (EO), and 7,21% ± 1,803 (EO+MMS),when compared to C- (6,15% ± 1,820),
however, these results are close to MMS (14,95% ± 1,834). Test t-student comparing the average of viable / nonviable cells, showing no cytotoxic activity of the treatments. By the micronucleus presence, the average of camphor
treatments were higher than the average of the C-, but they are no not significantly different (p > 0,05). According to
these preliminary data, statistically we cannot verify genotoxic activity, meanwhile the biologic analysis implying it.
Financial Support: CNPq
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
42
Evaluation of the mutagenic activity of
ethanolic extract of Caesalphinia ferrea Mart.
(Caesalpinioideae) by Salmonella/Microsome
assay (Ames test)
De Grandis, RA1; Müller, MM1; Wyrepkowski, CC2,3; Resende, FA1; Sinhorin, AP4; Rocha, CQ2; Santos, LC2; Varanda,
EA1.
Faculdade de Ciências Farmacêuticas de Araraquara, UNESP, São Paulo, Brasil; 2Instituto de Química de Araraquara,
UNESP, São Paulo, Brasil; 3Instituto Federal do Mato Grosso, IFMT, Mato Grosso, Brasil; 4Universidade Federal do Mato
Grosso, Mato Grosso, Brasil
1
[email protected]
Keywords: mutagenicity, medicinal plants, Ames test, Caesalpinia ferrea, Brazilian Cerrado
In recent decades, many studies have aimed to identify natural products with therapeutic properties. Caesalpinia
ferrea has traditionally been used in Brazil for many medicinal purposes, such as the treatment of bronchitis, diabetes
and wounds. Despite its use as a medicinal plant, there are no studies to ensure the safety of their use. Thus, the
aim of this study was to investigate the mutagenicity of the ethanolic extract of stem bark of C. ferrea by Ames
test, since the balance between the therapeutic vs. the toxicological effects is an important parameter in assessing its
applicability in relation to phytotherapeutic potential. A preliminary phytochemical study of the stem bark of C.
ferrea revealed the presence of flavonoids, saponins, tannins, coumarins, steroids and phenolic compounds. The Ames
test was performed using TA98, TA100, TA97 and TA102 strains of Salmonella typhimurium, as sensitive indicators
of DNA damage, in the absence (-S9) and presence (+S9) of metabolic activation system in five concentrations,
varying from 2.08 to 0.26 mg/ plate. The results showed that none of the concentrations induced any increase in
the number of revertant colonies on the strains tested, either in the presence or absence of metabolic activation,
indicating the absence of any mutagenic activity. The absence of such an effect by C. ferrea extract against S.
typhimurium bacterial strains in the Ames assay is a positive step towards determining its safe use in traditional
medicine. Although many compounds derived from plants have considerable pharmacological activities, some
undesirable properties such as mutagenicity, carcinogenicity and toxicity may restrict their use as therapeutic agent.
Thus, it is extremely important that does a detailed assessment about the safety of medicinal plants used popularly.
Financial Support: FAPESP, CAPES and CNPq.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
43
Comparative analysis of genotoxicity test
Allium cepa applied in natural environment
and laboratory
Leite, AK ¹ ² ³; Fernandes, FMC ²
¹Programa de Pós-Graduação em Genética e Biodiversidade, UFBa, Salvador, BA; ²Laboratório de Genética de
Populações e Evolução Molecular, UFBa, Salvador, BA; ³Centro de Ecologia e Consevação Animal, UCSal, Salvador- BA
[email protected]
Keywords: Genotoxicity, Allium cepa, natural environment, laboratory, Pituaçu lake.
Water is a natural resource of great importance for all living organisms. With the increasing use of water resources,
these natural ecosystems have become vulnerable to the process of degradation, with the emission of pollution agents.
Many of these substances can change the genetic material of living organisms, they are call genotoxic, which can be
induced by chemical, physical or biological agents. Because of this, new methodologies have been developed to assess
the potential of these genotoxic substances to inducing changes in the genetic material of organisms exposed to them.
One of those is the Allium cepa test which is frequently used to evaluation genotoxic in this ecosystem. This tests are,
in most studies, realize in artificial environment. Despite tests in artificial environments are more controlled and easier
to handle, the application of this methodology in natural environment to evaluate genotoxicity allows a result closer
to the real context, considering that organisms are exposed to environrmental variables which influence the mutagenic
processes in the field. This work aims to evaluate the efficiency of Allium cepa test conducted in natural environment
and compare with the same procedures applied in the laboratory. It was cultivated 12 organisms in each environment,
6 exposed to the freshwater from Pituaçu lake and 6 exposed to distilled water (negative control) in both cases.
Genotoxic experiments were performed from the protocol suggested by Guerra & Souza, 2002. We analyzed 1000
root cells (from apical meristem) per bulb and quantified the cells in mitosis, which has made it possible to calculate
the mitotic index (MI). In each treatment, MI from freshwater was compared with the negative controls, using the
statistical test ANOVA one way, Turkey on BioEstat 5.0. The organisms exposed in in natural environment did not
detect genotoxicity (p= 0.8663), different from the result obtained in the laboratory, which presented significant
difference when compared with the control (p 0.0073), shows the presence of some mutagen substance in the
environment. According to the results obtained, the present study showed that the treatment performed in natural
environment has a potential of discoverability for mutagens lower than treatment in the laboratory environment, it
is suggested that this was due to the fact that the bulbs were submitted to different condition, submerged to a much
greater volume of water than in the laboratory, in addition to having been exposed to rains in two periods which may
provide in greater dilution of genotoxic agents, thus decreasing the mutagenic effect on organisms exposed to them.
Financial Support: Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB)
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
44
Evaluation of the cytotoxicity and
mutagenicity of water contaminated with
tannery effluents
Rocha, CAM1; Costa, MHP2; Paixão, KCG2; Pinheiro, RHS3; Pessoa, CMF2; Rodrigues, CAC1,4
Coordenação de Recursos Pesqueiros e Agronegócio, Instituto Federal de Educação, Ciência e Tecnologia do Pará;
Coordenação de Biologia, Instituto Federal de Educação, Ciência e Tecnologia do Pará; 3Universidade Federal do Oeste
do Pará; 4Curso de Engenharia de Pesca da Universidade Federal Rural da Amazônia.
1
2
Keywords: Allium cepa, Astronotus ocellatus, chromium, micronucleus, mitotic index.
Aquatic ecosystems are those which have suffered the greatest impacts of pollution, since the water ends up being
the final destination of almost any pollutant. Chemical pollution of water by heavy metals is one of the most serious
environmental problems caused by residential and industrial sewages. Cytotoxicity of metals is important because
some metals are potential mutagens able to induce tumors in humans and other animals. Hexavalent chromium is
biologically reactive, highly toxic, genotoxic and carcinogenic, and can damage DNA in several ways. In our study,
water samples from the Piraíba River from a chromium-containing tannery effluent discharge site, in the Icoaraci
District, Belém City, State of Pará were subjected to the two bioassay: the Allium cepa test system was used to assess the
mitotic index, micronuclei and rate of mitotic abnormalities; the mutagenicity of the water samples also was assessed
using micronucleus test in erythrocytes from young of the fish Astronotus ocellatus (average length of 11.3 ± 1.17 cm and
weight of 22.3 ± 4.61 g). As positive control was used potassium dichromate (12mg/L). ANOVA one-way was used for
statistical analysis of the date among the groups. The mitotic index showed an increase in treatments, but differences were
not significant; the frequency of micronuclei and mitotic abnormalities increased significantly in the groups treated.
On the other hand, the micronucleus frequency in the fish blood was low and the statistical analyses demonstrated
that its frequency was not divergent in 5% probability, although nuclear changes, included blebbed, notched and
lobed nuclei, have increased significantly in treatments. Results in this work indicate that onion root-tips were more
sensible to environmental genotoxic agents. These are partial results and more analyses are needed to reinforce them.
Support: IFPA/PIBEX.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
45
Cytogenetic and molecular biomonitoring
of individuals occupationally exposed to
pesticides
Batista, MP1,2; Godoy, F.R1., Melo, C.A.O1,2., Carvalho; W.F1.; Cunha, D.M.C.C1, Ribeiro, C.L1., Pinto, G.S1., da Cruz,
A.D1,3., Silva, D.M.1,2
Núcleo de Pesquisas Replicon, Pontifícia Universidade Católica de Goiás; 2Programa de Pós-Graduação em Genética
e Biologia Molecular, Universidade Federal de Goiás; 3Laboratório de Citogenética Humana e Genética Molecular,
Laboratório de Saúde Pública Dr. Giovanni Cysneiros, Secretaria do Estado da Saúde de Goiás.
1
Keywords: translocation 14;18, pesticide, intoxicação
The impact of pesticide use on farm workers is an issue that has received attention from the scientific community
worldwide. Occupational exposure of agricultural workers occurs due to lack of information or lack of skilled technical
resources. These workers are exposed to pesticides in crops using, and this exposure may be responsible for genetic
damage causing a health risk. A problem with the use of pesticides is genotoxicity, which may lead to the onset of
disease. A major evaluation of individuals exposed to pesticides is the research of chromosomal translocation t (14;
18) (q31, q21), which can be investigated at the molecular level. Thus, this study aimed to evaluate the presence of
the t (14; 18) (q31, q21) by Fluorescent in situ Hybridization (FISH), in 30 farm workers exposed to pesticides for
more than 15 years, in the municipalities of Silvânia and Itapuranga, both at Goias State. We collected blood samples
of these individuals, and soon after it was made a culture of T lymphocytes for 47 hours. We used the manufacturer’s
suggested protocol for the hybridization of the probes of the samples already determined. The fluorescence images
were captured with a motorized epifluorescence microscopy, using specific filters and analyzed with the aid of Isis ®
software. 50 Cells were analyzed 50 (metaphases and nuclei) for each patient. In this study, we did not observe any cells
with translocation 14, 18, which can be explained by the low incidence in the population / number of cells analyzed.
The research of chromosomal translocations is of paramount importance for the understanding of the processes
involved in mutagenesis and carcinogenesis, helping to minimize the risks to individuals susceptible to pesticides.
Financial support: CNPq e FAPEG
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
46
Evaluation of the genotoxic potential of the
crude extract of Inga laurina by comet assay in
HepG2 cell line
Navegante, G1, Falcoski, TORS1; Serafim, RB1; Agustoni, D1; Marqui2, SR; Silva, DHS2; Valente, V1; Soares, CP1.
School of Pharmaceutical Sciences of Araraquara – UNESP; 2Institute of Chemistry of Araraquara - UNESP
1
Keywords: Inga laurina, Genotoxicity, Comet Assay
Since ancient times, medicinal plants are used to treat diseases and have become an important source of chemotherapeutic
agents. Therefore metabolites of plants have been studied to assess their ability to modify the activity of mutagens and
carcinogens. The plant Inga laurina belongs to the family Fabaceae and is among the species that are widely studied
phytochemically, presenting numerous biological activities. These species are one of the largest and most important
botanical families worldwide distributed specially in tropical and subtropical regions. The fruit-shaped pods are edible
and the seeds are surrounded by a flaky white flesh sweet. Many tribes of the Amazon used it to treat diseases, such as
flatulence, skin bruises, earaches among others. Previous studies have shown that phenolic compounds present in the
extract may be responsible for the antioxidant activity. This study aims to analyze the genotoxic potential of the crude
extract of Inga laurina through the comet assay. The leaves and branches of Inga laurina were collected in the city of
Assis, state of São Paulo, and the processes of extraction and purification were performed in Nubbe (Core of Bioassays,
Biosynthesis and Ecophysiology of Natural Products), Chemistry Institute - UNESP Araraquara. The evaluation of
genotoxicity was performed in a cell line of human hepatocellular carcinoma (HepG2). HepG2 cells were treated at
concentrations of 7.40, 22.22, 66.67, and 200.0 mg / mL. The parameter used in this study was the percentage of
DNA in the tail of comet images, which allows us to estimate the amount of fragmented DNA. Data were subjected
to ANOVA, Kruskal-Wallis with Dunnett’s post-test in comparisons with the vehicle control. Although the extract
evaluated did not show cytotoxic activity in previous studies, screening of genotoxicity of crude extracts of Inga laurina
demonstrated genotoxic activity statistically significant (p <0.0001) compared to vehicle control at all concentrations
tested, with higher concentrations showing larger damage. Therefore, we can say that the crude extract from Inga
laurina can induce genotoxic damage in HepG2 cells.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
47
Mutagenic and genotoxic potential of
wastewater from wet processing of coffee
Aguiar, LL1, David, JAO1
1
Departamento de Biologia, Centro de Ciências Agrárias, Universidade Federal do Espírito Santo.
[email protected]
Keywords: Oreochromis niloticus, micronucleus test, nuclear abnormalities, mutagenicity, biomonitoring.
The coffee bean is one of the most traded agricultural product in the world. The wet processing of the coffee bean
produces a better quality drink, but generates effluents with high concentrations of organic matter and agrochemicals.
Considering the importance of coffee production to the national economy, the concern with the quality of coffee
produced and the volume of waste generated in the wet processing of the grains, this study sought to evaluate the
mutagenic and genotoxic potential of wastewater from the wet processing of coffee, using as bioindicator the fish
Oreochromis niloticus. The fishes were divided into 4 groups with 11 individuals each and were allocated in tanks with
constant aeration. The wastewater was collected in a property in which it is done the wet processing of coffee, located
in the municipality of Muniz Freire, in the south of the state of Espírito Santo. There were four treatments: negative
control group (dechlorinated tap water); Group 1:80 (wastewater: dechlorinated tap water); Group 1:60 (wastewater:
dechlorinated tap water); Group 1:40 (wastewater: dechlorinated tap water). After 8 days of exposure, a blood sample
was withdrawn and used to make two slides with blood smears, which were stained with quick panoptic staining dye
and examined under light microscope. The slides were coded, allowing a blind test without prior identification of
treatments. To determinate the micronucleus frequency, 3000 erythrocytes were analyzed per slide (6000 per fish). Cells
carrying other nuclear alterations were also analyzed. The data were subjected to statistical analysis using the BioStat 5.0
software and the nonparametric Mann-Whitney test for pairwise comparison of the experimental groups. The average
frequency of micronuclei was equal to 0.2‰ (±0.2) in fishes from Group 1:80 and 1:60, and 0.2‰ (±0.4) in fishes
from Group 1:40. There were no micronucleated cells in fishes from negative control. All treatments, with the exception
of 1:40, presented a significant increase in micronucleated cells in relation to the negative control. In the group 1:40,
45.5% of individuals died during the experiment, probably due to a toxic effect of the tested effluent, which may have
influenced the statistical analyzes. The average frequency of nuclear abnormalities was equal to 0.1‰ (±0.3) for the fishes
of negative control and 19.9‰ (±15.5), 22.8‰ (±9.8) and 19,4‰ (±9.5) for fishes of Groups 1:80, 1:60 and 1:40,
respectively. All treatments showed significant increase compared to the negative control. The results obtained showed
that the wastewater from the wet processing of coffee presents mutagenic and genotoxic potential to fishes exposed
for 8 days, even in higher dilutions of the effluent. The authors recommend greater care in its disposal and/or reuse.
Financial Support: UFES (scholarship)
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
48
Genotoxic effects on Tradescantia pallida
(Rose) D.R Hunt. by vehicle traffic pollution in
Dourados, Mato Grosso do Sul/Brasil
Spósito, JCV¹; Crispim, BA¹; Grisolia, AB¹, Mussury, RM¹.
Faculdade de Ciências Biológicas e Ambientais, UFGD, Dourados, Mato Grosso do Sul, MS
1
[email protected]
Keywords: micronucleus, air pollution, genotoxicity, mutations e flow of vehicles.
The atmosphere in urban areas now contain complex mixtures of pollutants, including mutagenic substances, being
a consequence of the increasing number of automobiles, industries and other polluting activities that cause harm to
all living beings. Thus, biomonitoring is being increasingly used because of its advantages such as reduced costs and
efficiency for monitoring large areas. Among the bioindicator plant species, which may show chromosomal aberrations
as chromosomal breaks, causing fragments called micronuclei (MCN), one can cite Tradescantia pallida (Rose) DR
Hunt cv. ‘Purpurea’ Boom. In this study, the micronucleus test in Tradescantia (Trad-MCN) was used to evaluate the
mutagenic potential of air in three places with different vehicle traffic intensity in the town of Dourados, Mato Grosso
do Sul, Brazil. Standardized methods were used to analyze Trad-MCN. The experiment was analyzed as randomized
design and the treatments arranged in plots split in time, the plots being the sampling sites, and the splits being the
periods, with five repetitions each. Data were evaluated by ANOVA and significance, when found, subjected to Tukey
5% test. There were no significant interaction between sites and sampling times, showing statistical differences for
the factors alone. The greater occurrence of MCN (36,13) was observed in the samples during the month of May
coinciding with the great drought in the region capable of altering the air pollutants suspension, coincidentally the
lowest incidence (14,60) occurred in July when the relative humidity index increased. At the same time, the highest
incidence of MCN (28,64) was observed in the Hayel Bon Faker Street sample group, an area with higher vehicular
flow, and lower incidence (15,32) at UFGD. We conclude that the higher frequencies of MCN observed can be
attributed to air pollution. Considering that the bioindicator plant presents an integrated response to abiotic factors,
such as pollutants and climatic conditions, this may come to be used as an additional tool that can point to synergic
effects of environmental variables on organisms.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
49
Identification of the chromosomes that
carry doxorubicin-induced chromosomal
aberrations in brazilian brown brocket deer
Tomazella, IM1,2; Abril, VV1,2; Duarte, JMB1
Núcleo de Pesquisa e Conservação de Cervídeos (NUPECCE), Faculdade de Ciências Agrárias e Veterinárias (FCAV),
Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Jaboticabal – SP; 2Programa de Pós-graduação em
Genética e Melhoramento Animal, Faculdade de Ciências Agrárias e Veterinárias (FCAV), Universidade Estadual Paulista
“Júlio de Mesquita Filho” (UNESP), Jaboticabal – SP
1
[email protected]
Keywords: Mazama gouazoubira, Cervidae; chromosome evolution, chromosomal fragility, G-banging
The process of karyotype evolution of deer descending from a hypothetical ancestor with diploid and fundamental
numbers equal to 70 was characterized by complex chromosomal rearrangements. This ancestral karyotype, which
was retained, is standard for Mazama gouazoubira. Several animals of this species presented karyotype variation that
can be explained by chromosomal fragility.Among the Brazilian deer species, M. gouazoubira presented the highest
rates of chromosomal aberrations (CA) in cell cultures subjected to doxorubicin. Thus, this paper aims to identify the
chromosomes carrying doxorubicin-induced aberrations and to locate the regions of chromosome breaks (proximal,
middle or distal). In order to do so, we have conducted a cell culture of lymphocytes from six individuals, both treated
and untreated, aiming to induce CA through the incorporation of DXR (0,25 µg/mL). For each animal, 300 metaphase
preparations treated with DXR (treated culture – TC) and 300 untreated metaphase preparations (control culture –
CC) were analyzed by conventional staining in order to identify and quantify nine different types of chromosomal
aberrations (ring, dicentric, chromatid gap, chromosomal gap, chromatid break, chromosomal break, triradials form,
quadriradials form and rearrangements); 100 metaphase preparations of TC and 100 metaphase preparations of CC
were analyzed by G-banding aiming the precise identification of the chromosomes that carry aberrations and the
regions where the breaks occur. Moreover, the differences were statistically evaluated with regard to the total number
of CA and the number of metaphases that presented CA. The mitotic index (MI) was determined by the ratio between
the number of metaphases found in 6000 cells, expressed in percentage. The results of the types of CA showed that the
chromatid break is the most frequently found type of CA in both CC and TC. Statistical analysis showed a statistically
significant difference (P < 0,05) between CC and TC in the total number of CA and in the number of metaphases
that presented CA, but there was no significantly difference between males and females. The MI values obtained
showed that the TC did not show statistically significant differences compared to the CC, indicating the absence of
citotoxicity. Chromosome pairs 1, 2, 4, 5, 6, 7, 15 and 16 and the X chromosome showed chromosomal aberrations in
all analyzed animals. Among all the chromosomes that presented chromosomal aberrations, most of them were located
in the middle and distal regions. Based on these data, we can conclude that the doxorubicin did not lead to random
CA in Mazama gouazoubira chromosomes, pointing out that the CA might be spotted at specific areas of some
chromosomes. The results also suggest that the middle and distal regions of the chromosome may be more susceptible
to chromosomal fragility and, consequently, could be involved in the karyotype differentiation of Mazama species.
Financial support: FAPESP (2013/06100-7) and FCAV/UNESP
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
50
Preliminary analysis of Água Boa Stream
water mutagenicity potential in Dourados/MS,
Brazil
Silva, BV1, Dourado, PLR2; Rocha, MP2, Crispim, BM1; Oliveira, KMP1,2. Grisolia, AB1,
Faculdade de Ciências Biológicas e Ambientais, UFGD, Dourados, MS; 2Faculdade de Ciências Exatas e Tecnologias,
UFGD, Dourados, MS.
1
[email protected]
Keywords: Biomonitoring, micronucleus, Astyanax sp., industrial effluents, streams
The Água Boa Stream has its source in the urban area and is located in the city of Dourados/MS, presenting various
anthropogenic influences along its route: deforestation, uncontrolled occupation of the area, inappropriate land use,
waste disposal, domestic and industrial sewage. The aim of this study was to evaluate the mutagenic potential of water
samples from the Água Boa Stream through the micronucleus test using Astyanax sp. Samples were collected in August
and December of 2012 at three sampling points which differentiate into P1 (upstream), P2 (middle), P3 (downstream)
of the tannery wastewater disposal and as a negative control it was used water samples from UFGD tank. The water
was collected in 20 liters gallons and placed in aerated aquariums for 24 hours. After this period, the fish from the
Douradense pisciculture were acclimated and continued in aquariums for 96 hours. At the end of this period, the fish
were anesthetized and euthanized, and soon after, a cut was made on the caudal fin for blood collection. The collected
material was used for blood smears on slides. The samples were fixed with absolute ethanol, stained with fast Panoptic,
being made 2 blades for fish of each point and counted 2000 cells per slide. For the statistical analysis we used ANOVA
and, when significant, we used Tukey’s test (<0,05). It was observed that there was significant interaction (p<0,05)
between sites and sampling times for samples collected in August. During the month of December, the highest
micronuclei incidence (5,40) occurred among individuals in the P2 sample group and the lowest (0,20) for P1. In
general, larger amounts of MCN observed occurred during the month of December for all sampling points, compared
with the month of August. Because the month of December has the largest rain rate, the quantity of micronucleus
suggests that there is a greater disposal of waste in this period, which can also be related to the possibility of xenobiotics
presence in the water, as the amount of these products is higher during the rainy season. Thus, from the results obtained
by the micronucleus rate, it is suggested that there was mutagenic damage at the observed points during the two periods,
increasing as approaching the place of tannery discharge P2>P3>P1. Thus, it can be concluded that the micronucleus
test is a powerful tool to verify mutagenic damage found in fish cells, contributing to examine the damage to the
natural environment. Furthermore, there is a need for more collections in different periods of the year so there is a
detailed monitoring of the site, particularly in relation to the effects of tannery disposal in the fish mutagenicity process.
Financial support:UFGD
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
51
Biomonitoring through toxicity and
cytotoxicity tests of Água Boa Stream/MS
water, Brazil
Dourado, PLR1; Rocha, MP1; Silva, BV2; Crispim,BA1, Oliveira, KMP1,2. Grisolia, AB1,2
Faculdade de Ciências Exatas e Tecnologias, UFGD, Dourados, MS; 2Faculdade de Ciências Biológicas e Ambientais,
UFGD, Dourados, MS.
1
[email protected]
Keywords: Água Boa, cytotoxicity, Allium cepa, biomonitoring, streams
The Água Boa Stream is located in the city of Dourados/MS and traverses the southern outskirts of the city, agricultural
areas, receives effluent from the Industrial District and flows into the Dourados River, with great economic importance
for the municipality. The aim of this study was to evaluate the toxic and cytotoxic potential of the Água Boa Stream
water. The samples collection were conducted in the months of December 2012 and February 2013 at three sampling
points (P1, P2, P3). Water samples were collected in sterile vials, being P1 (200 meters above the disposal site), P2
(in the vicinity of the disposal site) and P3 (10km after the disposal site). Five bioassays were mounted, a negative
and a positive control and the other (3) with water samples to be analyzed. The cytotoxicity assays containing Allium
cepa seeds were performed on plates and the toxicity ones were performed using bulbs cultured with water samples
from collection points for 3-4 days. After this period the seeds were collected, fixed in Carnoy, subjected to acid
hydrolysis and stained with Schiff reactive. For each sampling point 5000 cells were counted. Statistical analyzes were
performed using the ANOVA method and, when significant, the Tukey test (p <0,05). The results showed inhibition
of root growth in P2 and P3, which grew less than 2 cm compared with the negative control, with growth greater
than 3 cm in the December collect. In the samples collected during the month of February, the root growth was also
inhibited, not reaching 0,5 cm long. The cytotoxicity analysis showed that there was an increase in the number of
aberrant cells, with the exception of P3, however, for those that were collected in February it was possible to found
chromosomal alterations in all collection points. Moreover, the possible action of Chrome on Allium cepa cells was
detected due to the presence of c-metaphase cells during the analysis. Thus, it is suggested that Chrome may be a
potential contaminant, showing that tannery wastewater can damage the DNA of cells and jeopardize the water
quality. There was no inhibition of the mitotic index in any of the samples collected and the interaction between site
and sampling date was not significant, statistical difference was found for the factors evaluated separately, with the
highest mutagenic incidence (5,21) occurring during the month February. This way, it becomes extremely important
that further studies be conducted to encourage the development of research related to environmental pollution,
not only of this stream, but many others receiving effluents from different industrial hubs, including tanneries.
Financial support: CAPES and UFGD
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
52
Analysis of exons 6 and 8 of the TP53 gene in
canine mammary tumors
Faro, TAS¹; Pinheiro, DR¹; Ferreira, WAS²; Sousa, RM¹; Aguirra, LRVM¹; Monger, SGB2; Pereira, WLA¹; Harada, ML²;
Borges, BN¹
Universidade Federal Rural da Amazônia, Belém, PA; ²Universidade Federal do Pará, Belém, PA
1
[email protected]
Keywords: Cancer, dogs, mutations
Cancer is a disease with multiple stages, originated due to changes in the control and activity of genes responsible for
regulating cell growth and differentiation, resulting in uncontrolled proliferation. It is considered primarily a genetic
disease caused by a sequential accumulation of mutations in genes that control the cell cycle. Among the various
tumor types, breast tumors are the most commonly observed in female dogs. Studies suggest that the TP53 gene is one
of the main mutated during mammary carcinogenesis. Nevertheless, there are no records, at the moment, of molecular
studies involving the TP53 gene in breast tumors of dogs from the Northern region of Brazil. So the aim of this work
was to assess the mutational status of exons 6 and 8 of the TP53 gene in samples of breast tumors in female dogs
from Belém, Pará (Brazil). The samples of neoplastic and non-neoplastic tissue were collected during mastectomy at
the UFRA’s Veterinary Hospital (HOVET) and subjected to DNA extraction followed by polymerase chain reaction
(PCR) and automatic sequencing. The obtained fragments were aligned and compared with a reference sequence using
the BioEdit software. We were able to identify several mutational events, such as indels, transversions and transitions.
Many of the observed alterations were shared by both non-neoplastic and tumoral samples. Comparing tumoral and
non-tumoral samples, we did not observed any difference in the statistical analysis related to the presence of molecular
alterations and race, histological type of tumor or age at diagnosis. In conclusion we can suggest that alterations in exons
6 and 8 of the TP53 gene are common in canine mammary cancer. However, an increased in our sample size would
probably help us to confirm this result and the potential use of these markers in the diagnosis of this cancer type. As
far as we know, this is the first study using these molecular markers in canine mammary tumors in the Northern Brazil.
Financial Support: CNPq, UFRA
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
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53
Evaluation of the mutagenic activity of water
from the Arara Stream in the city of Rio
Brilhante (MS – Brazil) using micronuclei test
Maceda, EB1; Vaini, JO2; Santos, JCG3; Pinto, JVC3; Lima, LAM1; Grisolia, AB1.
Faculdade de Ciências Biológicas e Ambientais, UFGD, Dourados, MS; 2Faculdade de Ciências Exatas e Tecnologia,
UFGD, Dourados, MS; 3Faculdade de Ciências Agrárias, UFGD, Dourados, MS
1
[email protected]
Keywords: Aquatic contamination, Astyanax sp., bioassay, bioindicator, conservation
Some chemical compounds are capable of change the characteristics of the water. They are able to induce mutations
and genetic alterations, both in aquatic organisms and human beings, which make this environment a subject of
several studies in order to evaluate its mutagenic potential. The objective of this study was to evaluate the activity
mutagenicity of the water Arara Stream, at the city of Rio Brilhante/MS. The bioassay was conducted in April 2013,
therefore, water was collected at three different sites of Arara Stream. The site 1 of the stream suffers with human
disturbance, the site 2 is located right after the Sewage Treatment Station of the city and it is surrounded by farms
and pig breeding and the site 3 is characterized by surrounding plantations. The collected water was transported
to the Laboratory of Mutagenesis, Faculdade de Ciências Biológicas e Ambientais/UFGD and placed in different
aquariums, being kept under aeration during 24 hours. After this period, 5 specimens of Astyanax sp. (from the
tanks of Douradense Piscicultura, Dourados-MS) were added to each aquarium. The specimens were kept in the
aquariums during 72 hours and then anesthetized and sacrificed. The peripheral blood of the fish was taken, followed
by blood smear procedure. The smears were fixed in 96% ethanol and then stained with Panótico Rápido. We
analyzed 2000 cells from each fish at the optical microscope at the 400X total increase. The results found were
submitted to a variance analysis (ANOVA) and then also submitted to Tukey test 5%. There was no statistical
difference between the incidences of micronuclei in the different sampling points. The following micronuclei
averages were observed on the bioindicator subject at water Arara Stream for site 1 (3,10), site 2 (2,70) and site 3
(1,88). However, we were able to identify that the place suffers with aquatic contamination by possible mutagens
that damaged the DNA of the fish cells. This research serves as a base to future plans of conservation steps of
water Arara Stream – Rio Brilhante/MS, since until the present moment no research of this nature was ever made.
Financial Support: Universidade Federal da Grande Dourados (UFGD).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
54
Arrabidaea chica mutagenic potential
measure, through in vivo bioassays
Souza, K.H.S1,3 ;Leitão, W.S.1; Sousa, F.C.1; Monteiro Neto, M.A.B2.
¹ Acadêmicos do curso de Biomedicina da Faculdade Estácio/Seama-AP; ² Orientador da pesquisa, Doutor, pesquisador
do Instituto de pesquisas Científicas e tecnológicas do Estado do Amapá (IEPA), Professor da Universidade Federal do
Amapá (UNIFAP) e Estácio/SEAMA-AP; 3 Estagiária do Instituto de pesquisas Cientificas e tecnológicas do Estado do
Amapá (IEPA)
[email protected]; [email protected]
Keywords: Erythrocytes, Genotoxicity, micronucleus, Traditional medicine.
The Arrabidaea chica belongs the Bignoniaceae family, which contains around 120 genus and 800 species, distributed
in tropical regions (South America and Africa). In Brazil, those family’s plants occur from Amazon till Rio Grande
do Sul (a Brazilian state, located in the south of the country), and don’t have unique habitat. Depending on the
region , the A. chica receives various names by the folk medicine, being known as: Crajiru, Pariri, Carajuru, Puncapanga. Researches conducted to assess the safe use of medicinal plants in Brazil are still incipient, as well as control of
commercialization. For that, it is extremely important to do cytogenetical tests to measure those so called medicinal
plants’ genotoxic potential. The A. chica extract is being used to treat various diseases, and has various therapeutic
indications: anti-inflammatory, anemia, antioxidant, amongst other. Facing those various biological activities and
increasing therapeutic interest, the present paper measured the A. chica’s genotoxic potential surrounding the
micronucleus frequency on Swiss mice. The animals were treated with different concentrations of A. chica, these being
40.80 and 160mg/kg b.w. Were included negative controls groups (water) and positive (duxorrubicin, DXR; 15mg/kg
b.w.). The A. chica concentrations were given to the animals during 48h through gavage. Samples were collected from
peripheral blood 48h after the treatment. The frequency of micronucleated polychromatic erythrocytes was achieved
from the analysis of 3000 polychromatic erythrocytes anucleated per animal. The results reveal that the treatment
applied didn’t show statistically significant differences due to the negative control group, revealing the genotoxicity’s
absence. Based on the applied methodology, this paper concludes that A. chica didn’t present genotoxic effect.
Financial support: Institute of Scientific Research and Technology of the State of Amapá (IEPA) and College Estácio
/ Seama.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
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55
Genotoxic effect of Spilanthes oleracea L.
polychromatic cells of swiss mice
Costa, I.Q1 ; Carmo, K.L.T1; Santos, G.N1 ; Monteiro Neto, M.A. B.
¹Estagiários do Instituto de pesquisas Cientificas e tecnológicas do Estado do Amapá (IEPA) e Acadêmicos do curso de
Biomedicina da Faculdade Estácio/Seama-AP; ²Orientador da pesquisa, Doutor, pesquisador do Instituto de pesquisas
Científicas e tecnológicas do Estado do Amapá (IEPA), Professor da Universidade Federal do Amapá (UNIFAP) e Estácio/
SEAMA-AP.
[email protected]
Keywords: Erythrocytes, genotoxicity, micronucleus,Traditional medicine, Swiss Mice.
Spilanthes oleracea L. Asteraceae is a native of the Amazon region, herbaceous perennial vegetable, semi-erect and
decumbent branches. The blossoms are small and yellow, arranged in chapters. It is also known as watercress Pará, Brazil
cress, watercress North jabuaçu, wacky weed, jaburama, Buttercup, among others. Besides widely used in cooking, S.
oleracea L., has medicinal importance, because it has active ingredients such as essential oils, saponins, espilantinas,
afinina, filoesterina Hill triterpenoids and especially the espilantol, which had the structure discovered by Yasuda. This
study identified the genotoxic potential of S. oleracea L.. on the frequency of micronucleus present in Swiss mouse.
The animals were treated with different concentrations of S. oleracea L.. being respectively 60. 30 and 15 mg / kg
w.c. Control groups were included negative (water) and positive (doxorubicin, DXR, 15 mg / kg w.c). S. oleracea L.
concentrations during 48 hours were administered by gavage to the animals. Samples were collected from peripheral
blood 48 hours after treatment. The frequency of micronucleated polychromatic erythrocytes was obtained from the
analysis anucleated 3.000 polychromatic erythrocytes per animal. The results show that animals treated with different
concentrations of S. oleracea L.. had micronucleus frequencies similar to those of the negative control. From these
data was concluded that S. oleracea L. showed no genotoxic or cytotoxic effects in the conditions used in this work.
Financial support: Institute of Scientific Research and Technology of the State of Amapá (IEPA) and College Estácio
/ Seama.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
56
Evaluation of the influence of GSTT1 and
GSTM1 polymorphism in individuals
occupationally exposed to pesticides
Godoy,FR1,3; Melo, COA1; Minasi,LB1,5; Costa,EOA1,3; Carvalho, WF1,3; de Melo, AV1; Cruz,AS1,5; Cunha,DMC1; da
Cruz,AD1,2,3,5; Silva,DM1,3,4
Núcleo de Pesquisa Replicon, Departamento de Biologia PUC-GO; 2Laboratório de Citogenética Humana e Genética
Molecular/ La Gene - LACEN, Laboratório de Saúde Pública Dr. Giovanni Cysneiros/ SES; 3Mestrado em Genética,
Programa de Pós Graduação e Pesquisa - PROPE/ PUC-GO; 4Universidade Federal de Goiás, Instituto de Ciências
Biológicas; 5Programa de Pós-Graduação em Biologia, Instituto de Ciências Biológicas, Universidade Federal de Goiás.
1
[email protected]
Keywords: Polymorphism, GSTT1, GSTM1, Pesticides; Intoxication.
The impact of pesticide on agricultural workers is an issue that has received attention from the scientific community
worldwide. These workers are exposed to pesticides in crops and this exposure may be responsible for genetic damage
causing a health issue. A problem with the use of pesticides is the genotoxicity, which may lead to the onset of diseases.
Little is known about the relationship between genotoxicity and genetic polymorphisms xenobiotics metabolism that
can modify individual susceptibility. Therefore, there is a need to study genes as glutathione-S-transferase mu (GSTM1)
and glutathione-S-transferase theta (GSTT1) encoding detoxification enzymes. In this context, this study aimed to
evaluate the variability of GSTM1 and GSTT1 polymorphisms in individuals occupationally exposed to pesticides
in Goias municipalities that present intense agricultural activity. We evaluated 235 individuals which 120 were rural
workers occupationally exposed to pesticides and 115 individuals were not exposed to pesticides, forming the control
group. The exposed group consisted of 111 men and 9 women only getting an average of 39 + 9 years old. These
workers were form 12 rural municipalities situated at Goias state. It was found that 18% of the exposed individuals
had the GSTT1 null genotype and 49% had the GSTM1 null genotype, and 10% had both null genotypes. Data as
intoxication (41%), use of Personal Protection Equipment (52%) and if the worker handled the pesticide (7%), or if
just applied the pesticide (22%) or if the worker manipulated and applied (71%) have all been correlated with genetic
polymorphisms. There were no statistically significant differences between the GSTM1 and GSTT1 polymorphisms
between control and exposed groups. Thus, the study of genetic polymorphisms as biomarkers of susceptibility is of
fundamental importance in understanding the processes involved in mutagenesis and carcinogenesis and could help
minimize the risk to susceptible individuals who are exposed to pesticides.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
57
Biomonitoring of genotoxic capacity of
the Santa Maria river, Monte Carmelo - MG
through the micronucleus test in Geophagus
brasiliensis
MORAIS, CR¹,²; PEREIRA, BB¹,²; VIEIRA, TC¹,²; DE CAMPOS JUNIOR, EO²; CAMPOS, CF¹,²; DAYRELL, DM¹,²; OLIVEIRA,
SR¹,²; RODRIGUES, FFS¹,²; MORELLI, S².
Fundação Carmelitana Mário Palmério, FUCAMP, Monte Carmelo, MG; 2Instituto de Genética e Bioquímica, UFU,
Uberlândia, MG.
1
[email protected]
Keywords: Geophagus brasiliensis, genotoxic, micronuclei, toxicology, cytotoxic
The existing water resources for the mains human activities suffer intense anthropogenic degradation processes.
Used as a biological tool for toxicology, fish Geophagus brasiliensis have tolerant sensitivity to chemical compounds,
derived from effluents of various activities (household residues, industrial and agricultural), representing an
effective bioindicator. The present research aimed evaluate the genotoxic potential of the Santa Maria river, Monte
Carmelo, MG, Brazil, using the micronucleus test in Geophagus brasiliensis. Then, 40 Geophagus brasiliensis samples
at 4 points of the Santa Maria river (Monte Carmelo – MG) were collected, samples were stained with Giemsa
and tested with micronuclei (MN), statistical analysis was later made (p>0,05). In this context, samples with a
positive (ciclofosfamida) and a negative control were compared. After collecting and analyzing water chemistry,
the indices of water quality with aid of IQADATA software were obtained and classified in accordance with
Resolution 357 of 2005 of the National Council of Environment (CONAMA) relative to the conditions of use.
The results of the micronuclei frequency corroborate with the chemical analysis of the water, showing an increase of
micronuclei in the points with bad water quality, which have in their qualitative parameters cytotoxic and genotoxic
products. The degradative process intensifies as it following the course of the river along the points 1, 2, 3 and 4.
Finacial Support: CAPES, CNPQ, FAPEMIG, UFU, FUCAMP.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
58
Quantitative exfoliative cytology of oral
mucosa and micronucleus test in smokers
Galego, L.G.C1; Moraes, M.I.2
1
Universidade Federal do Triângulo Mineiro (UFTM), Uberaba (MG); 2Universidade Paulista (UNIP), São José do Rio Preto
(SP)
[email protected]
Keywords: Smoking, cell alterations, micronuclei test, Papanicolaou staining
Smoking is a serious health problem worldwide. The habit of smoking is related to various cellular benign and
malignant lesions of the oral cavity and oropharynx. One of the most common ways to diagnose changes in the oral
mucosa is the analysis of cell morphology which is a criterion for the evaluation of tissue changes, but if the goal of
the analysis is to diagnose cell and tissue alterations that suggest the neoplasia´s formation, the micronucleus test is
appropriate. In both cases, oral mucosal smears are materials for obtaining simple, non-invasive, low-cost oral cytology
for analysis and subsequent staining, for example, the Papanicolaou staining, which can also be used for micronuclei
visualization. In this context, the present study evaluated the prevalence of micronuclei and alterations of oral mucosa
cell morphology in the smokers and non-smokers of sexes and the suitability of Papanicolaou technique for this
purpose. Samples of oral mucosa and tongue of a total of 120 subjects, divided into 4 groups (A, B, C and D)of 30
people were collected with wooden spatulas. The groups A and B was composed for men and women, respectively,
who never smoked. The groups C and D was composed for men and women, respectively, who smoke for at least five
years, more than 15 cigarettes for day. The collected material of men and women oral mucosa was distributed on glass
slides from the spreading technique and properly fixed with 95% ethanol and then stained using the Papanicolaou
techniques. All individuals who agreed to participate in the collections provided information relating to smoking and
signed the informed consent protocol, which received a copy. The altered cells number was counted and compared
between groups by ANOVA (p<0,05) and the micronuclei frequencies each sample group were compared with the
chi-square test. All comparisons indicated significant differences in the cell distribution pattern, whereas smokers
showed an increase in the basal cells amount and in the cellular alteratiuons. Furthermore, the presence of micronuclei
in cells from smokers (6.83 / 1 000 cells) was much higher than in non-smokers (<1/1000 cells). The cytology
analysis of oral mucosa from smokers and non-smokers allow to conclude that the tobacco use increases the likelihood
that individuals showed cell alterations and of the presence of micronuclei in their cells and the Papanicolaou
technique can be used as a tool to identify changes these cells and may contribute significantly to preventive actions.
Financial Support: UNIP
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
59
In vivo Evaluation of antimutagenic
potencial of extract Spondias purpurea L by
micronucleus teste in peripheral blood of mice
Santo, JTT²; Tavares, AV¹; Guimarães, BD3; Santos, VL4; Lira, WM5.
¹Discente de Biologia UEPB, Campina Grande, PB; ²Discente de Enfermagem UEPB, Campina Grande, PB; 3Discente de
Farmácia UEPB, Campina Grande, PB; 4,5Centro de Ciência Biológicas e da Saúde UEPB, Campina Grande, PB
[email protected]
Keywords: Spondias purpurea L, antimutagenic, micronucleus
Due to the wide use of medicinal plants in folk medicine to treat various diseases, plant research has been intensified
by the existence of important sources of biologically active natural products. In many opportunities though it is not
known how far their effects can be beneficial, for a medicinal plant, considering the toxicological point of view, does
not only present immediate effects, easily correlated with their intake, but also long-term ones in an assymptomatic
way. This study had the objective to evaluate the antimutagenic potential of Spondias purpurea L, popularly known
as cirigueleira or serigueleira, which produces red mombin fruit, Spanish prune or plum. A phytochemical study
showed it to be rich in secondary metabolites, possessing moderate values of phenolic compounds, ascorbic acid
and anthocyanins. In addition to these compounds it also presents, in a greater amount in the shell, flavonoids
such as quercetin. Studies have shown therapeutic effects such as anti-inflammatory activity. in vivo micronucleus
test in peripheral blood of mice was used to evaluate the antimutagenic potential. Swins albinos Mus musculus
mice (approximately 28g) were used. The animals were divided in groups of 6, 3 males and 3 females. Each group
received different doses established by previous studies of the plant extract in a maximum volume of 0.1 ml per 10g
body weight, along with cyclophosphamide at a dose of 50mg/kg by intraperitoneally. Starting from the maximum
dose the concentrations used were 2000 mg/kg 1000 mg/kg and 500 mg/kg by gavage. Cyclophosphamide was
used as a positive control (50 mg/Kg p.c) and distilled water as negative control (0.3 mL). Blood was collected
30 hours after administration by means of pulse flow. The slides were stained with Giemsa for further analysis.
The frequency of micronuclei in blood cells was obtained by counting 2000 polychromatic erythrocytes. Results
were evaluated by Student’s t-test. Under the conditions tested Spondias purpurea L presented no antimutagenic
activity. There were no statistically significant differences for the groups undergoing the treatments at 500mg/kg
bw and 1000/kg pc, compared with the positive control, and there was cytotoxicity at a dose of 2000mg/kg pc.
Financial Support: CNPq; UEPB.
59º Congresso Brasileiro de Genética
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www.sbg.org.br - ISBN 978-85-89109-06-2
60
Genotoxic activity of Momordica charantia L.
trought the test system in vivo
Fernandes, WA1;Castilho, L.L1; Santos,G.N1; Souza, K.H.S1;Monteiro Neto, M.A.B2
¹Estagiários do Instituto de pesquisas Cientificas e tecnológicas do Estado do Amapá (IEPA) e Acadêmicos do curso de
Biomedicina da Faculdade Estácio/Seama-A; ²Orientador da pesquisa, Doutor, pesquisador do Instituto de pesquisas
Científicas e tecnológicas do Estado do Amapá (IEPA), Professor da Universidade Federal do Amapá (UNIFAP) e Estácio/
SEAMA-AP.
[email protected]; [email protected]
Keywords: Erythrocytes, genotoxicity, micronucleus,Traditional medicine, Swiss Mice.
Momordica charantia L. has origin from Asian, brought from Africa by slaves, is a species belonging to the Cucurbitaceae
family,the genre Cucurbita, is a vine. In tradicional medicine, it is used in the treatment of skin lesions, healing and
anti-rheumatic, beingused by the public mainly against diabetes. In face of the great demand for products based on
medicinal plants found in the present,it is necessary to investigate how these products being offered to the consumer,
in accordance with the law and specific normas scientifically established. This study identified the genotoxic potential
of Momordica charantia L. on the frequency of micronucleus present in Swiss mouse. The animals were treated
with different concentrations of Momordica charantia L. being respectively 1.0, 2.0 and 4.0 mg / kg w.c. Control
groups were included negative (water) and positive (doxorubicin, DXR, 15 mg / kg w.c).Momordica charantia L.
concentrations during 48 hours were administered by gavage to the animals. Samples were collected from peripheral
blood 48 hours after treatment. The frequency of micronucleated polychromatic erythrocytes was obtained from the
analysis anucleated 3.000 polychromatic erythrocytes per animal. The results show that animals treated with different
concentrations of M. charantia L. had micronucleus frequencies similar to those of the negative control. From these
data was concluded that M.charantia L. showed no genotoxic or cytotoxic effects in the conditions used in this work.
Financial support: Institute of Scientific Research and Technology of the State of Amapá (IEPA) and College
Estácio / Seama.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
61
Genotoxic activity of rawhide extract of the
Licania macrophylla Benth in swiss mice
Bezerra, E.C¹; Carmo, K.L.T¹; Fernandes, W.A1; Sales, P.F1; Monteiro Neto,M.A.B¹,².
¹Estagiários do Instituto de pesquisas Cientificas e tecnológicas do Estado do Amapá (IEPA) e Acadêmicos do curso de
Biomedicina da Faculdade Estácio/Seama-AP; ²Orientador da pesquisa, Doutor, pesquisador do Instituto de pesquisas
Científicas e tecnológicas do Estado do Amapá (IEPA), Professor da Universidade Federal do Amapá (UNIFAP) e Estácio/
SEAMA-AP.
[email protected]; [email protected]
Keywords: Cytotoxicity, genotoxicity, micronucleus.
Licania macrophylla Benth owns the Chrysobalanaceae family, containing 18 genres and 531 species distributed in
both the tropics. Are formed by trees or shrubs. The L. macrophylla B. is popularly known as “anauerá” or “anuera”,
being found mainly in the margins of the lowland regions of the lower Amazon. The extract of L. macrophylla B.
has been used for the treatment of various diseases, which has a diverse therapeutic indications: diseases generally
caused by protozoa such as giardiasis and amoebic, dysentery, toothache, venereal diseases, malaria, and intestinal
disorders in general. In face of these various biological activities and the increasing therapeutic interest, this study
evaluated the genotoxic potential of L. macrophylla B. on the frequency of micronucleus present in Swiss mice. The
animals were treated with different concentrations of L.macrophylla B. being respectively 60, 30 and 15 mg / kg w.c.
Control groups were included negative (water) and positive (doxorubicin, DXR, 15 mg / kg w.c) . L.macrophylla B.
concentrations during 48 hours were administered by gavage to the animals. Samples were collected from peripheral
blood 48 hours after treatment. The frequency of micronucleated polychromatic erythrocytes was obtained from the
analysis of 3.000 anucleated polychromatic erythrocytes per animal. In order to evaluate the cytotoxicity of different
treatments were analyzed 600 erythrocytes / animal for obtaining the index of nuclear division (IDN). The results
demonstrated that animals treated with different concentrations of L.macrophylla B. had micronucleus frequencies
similar to those of the negative control. The IDN values obtained showed that the treatments did not show statistically
significant differences, compared to control (water), do not demonstrating effect of cytotoxicity. Therefore, with this
data was concluded that L. macrophylla B. did not show genotoxic nor cytotoxic effects with the concentrations used.
Financial support: Institute of Scientific Research and Technology of the State of Amapá (IEPA) and Faculty Estácio
/ Seama.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
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www.sbg.org.br - ISBN 978-85-89109-06-2
62
Micronucleus and binucleated cells in the oral
epithelium of rural workers occupationally
exposed to pesticides: a case-control study
from Central Brazil
Queiroz, F.J1; Carvalho, W.F1., Gonçalves, M.W2., Melo, C.A.O2., Godoy, F.R1., Cunha, D.M.da C1., de Melo, A.V1., da
Cruz, A.S3., da Cruz, A.D1,3., Silva, D.M2.
Núcleo de Pesquisas Replicon, Departamento de Biologia, Pontifícia Universidade Católica de Goiás; 2Programa de PósGraduação em Genética e Biologia Molecular, Universidade Federal de Goiás; 3Programa de Pós-Gradução em Biologia
Celular e Molecular, Universidade Federal de Goiás
1
Indiscriminate use of pesticides results in a number of consequences for the environment and one of the problems
with the use of these products is the genotoxicity. The micronucleus assay is a cytogenetic analysis that can assess
the potential mutagenicity and genotoxicity of compounds, being simple, rapid, and sensitive. In this context, we
evaluated 71 rural workers from 10 municipalities in Goiás state and 68 individuals with no history of occupational
exposure. The collection of oral mucosa cells was performed using a tongue depressor at sites with no visible ulcers
and other injuries. Hydrolysis was performed on the slides title 1:10 (10%). After the hydrolysis, the slides were
dipped in basic fuchsin solution for 15 minutes under light and soon after, lightly rinsed with water to remove
excess dye. Then, the slides were stained with Fast Green (10%). The analysis was carried out by optical microscopy,
and it consisted of an evaluation of at least 1,000 cells of each sample. The average age of individuals who
participated in the study for the control group was 41,98 ±16,11 and 39.90 ± 13,46 for the exposed group. About
50% of workers reported exposure for more than 15 years. Glyphosate was responsible for 56.34% of use among
workers. Of all workers who reported signs of intoxication, 53.52% did not use ppE’s. This study demonstrated
a statistically significant difference in the micronucleus assay, found an average of 5:42 + 7:32 micronucleus in
the exposed group that did not use ppE’s (p <0.001). There were statistically significant differences between the
frequency of micronucleus and binucleated cells in the case and control groups (p <0.05). Therefore, pesticides
are capable of causing mutations in individuals occupationally exposed, especially those who do not use ppE’s.
Financial support: CNPq; FAPEG-Ministério Público; CAPES.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
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63
Hydroalcoholic extract obtainment from
Dalbergia subcymosa ducke and evaluation of
its genotoxic in cells of mammals
Kiryu,A.C.F.C.1, Vale,E.P.1, Costa,I.R.Q.1, Monteiro Neto,M.A.B.1.2
Faculdade Estácio Seama, Macapá, AP; 2 Instituto de Pesquisa Científica e Tecnológica do Estado do Amapá (IEPA)
Macapá, AP
1
[email protected]
Keywords : Dalbergia subcymosa Ducke. Micronucleus. Genotoxicity.
The Dalbergia subcymosa Ducke also known as “Verônica”, is a native plant of Brasil, French Guiana, Guiana and
Venezuela. In Brazil it is found mainly in the regions of Pará, Amazonas, Amapá and Parintins. It natural habitat is dry
land forests and clay, having an ascending shrubby habit, but is commonly grown in gardens, by presenting flowers with
colors blue, pink and white from months May to July. It has antianemic and antiinflammatory medicinal properties
and is used by population in the treatment of genital disorders, anemia and inflammations. The D.subcymosa Ducke’s
tea has large amount of soluble iron in its composition and is used in the treatment of iron deficiency anemia. Due to
studies showing the anti-inflammatory effects and antianemics Dalbergia subcymosa Ducke, the present study through
the test system in vivo was performed assessment of genotoxic hydroalcoholic of the plant in Swiss mice using the
micronucleus test for its advantages such as speed of analysis, low cost, plus the ability to assess chromosomal damage,
and the possibility to be reproduced in different species. For the animals treatment of this study, were used three
different concentrations of D. subcymosa Ducke (76mg, 38mg and 19mg /Kg b.w.). Were included the negative control
group, where the animals were treated with water, and the positive control group treated with doxorubicin (DXR= 15
mg/kg b.w.). The different concentrations of Dalbergia subcymosa Ducke were administered to the animals by gavage
method in a volume of 0.5 mL / kg b.w. Made treatment, after 48 hours, samples were collected from peripheral
blood which were analyzed 3000 polychromatic erythrocytes per animal to obtaining the frequency of micronucleous
formation. The results showed that the groups treated with the Dalbergia subcymosa Ducke were not statistically
significant relative frequency of micronuclei in the control group and in the concentrations used. Thus, it is concluded,
with the use of the concentrations of Dalbergia subcymosa Ducke in this work, it does not present risk of genotoxicity.
Financial support: Institute of Scientific Research and Technology of the State of Amapá (IEPA) and Faculty Estácio
/ Seama.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
64
TP53 codon 72 polymorphism and oral cancer:
pilot study
Vanessa Christine Martins, Marcus Vinicius de Matos Gomes, Roberta Losi Guembarovisk, Regina Célia PoliFrederico
Keywords: p53 codon 72 polymorphism, oral cancer, genetic polymorphism
The TP53 codon 72 polymorphism has been investigated extensively for its association with various cancers around
the world. It is still unclear whether the TP53 codon 72 polymorphism is associated with oral cancer risk. The p53
tumor suppressor gene contributes to the maintenance of genomic stability by controlling cell cycle and facilitating
DNA repair in response to DNA damage. It is a gatekeeper or guardian gene of cell division. The p53 gene was found
to be polymorphic with a single nucleotide polymorphism in the exon 4 in codon 72. The substitution of G to C
changes the amino acid from arginine to proline. It gives rise to three genotypes, Arg/Arg, Arg/Pro and Pro/Pro. More
recently, the p53Arg and p53Pro proteins are reported to be biologically and biochemically different from each other.
The aim of our study was to evaluate the association between the TP53 codon 72 polymorphism and the oral cancer
risk. A hospital-based case–control study with 20 patients with oral cancer and 20 matched controls was conducted.
Genomic DNA was isolated from peripheral blood, and gene polymorphisms were analyzed by polymerase chain
reaction–restriction fragment length polymorphism. The initial results of this pilot study showed that the frequencies
of Arg / Arg and Pro / Pro genotypes were 43% and 57% in patients and frequency of Pro / Pro genotypes were100%
in controls. The studies may help to elucidate the role of TP53 polymorphism in oral cancer.
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
65
Evaluation of genotoxicity and other nuclear
alterations through the micronucleous assay
of buccal mucosa of diabetic patients with
chronic kidney disease in hemodialysis
José Ricardo M. Ceroni, Melissa N. V. de Freitas, Vinicius A. S. e Santos, Júlio Boschini Filho, Debora Ap.
Rodrigueiro, Marta Wey Vieira
OBJECTIVE: Evaluate the possible genomic damage of the hemodialysis in diabetic chronic kidney disease patients
through the micronucleus assay of buccal mucosa. METHODS: After the acceptance of the Ethical committee of the
Faculdade de Ciências Médicas e da Saúde PUC-SP, 20 adult Diabetics mellitus type2 patients with chronic kidney
disease in hemodialysis were compared to 20 diabetic patients with chronic kidney disease not in hemodialysis and
20 asymptomatic patients through the micronucleus assay of buccal mucosa. The collection and preparation of the
samples were done according to the HUMN protocol. The genomic damage was evaluated by the determination
of the frequency of micronucleation and other nuclear alterations in 1000 cells of the casuistry buccal mucosa.
Cellular types were classified according to the following criteria: indicative cells of proliferative index, of genotoxic
and cytotoxic actions, and cellular death. For result analysis it was used Kruskal-Wallis test, chi-squared test and
Mann-Whitney test. The significance level was fixed in 0,05. RESULTS: The results suggest the diabetic chronic
kidney disease patients in hemodialysis and diabetic chronic kidney disease patients not in hemodialysis showed a
higher frequency of micronucleous when compared to the control group. When compared between the first two,
diabetic patient with chronic kidney disease in hemodialysis showed a higher frequency of micronucleous then the
group not in hemodialysis. CONCLUSIONS: The micronucleus assay of buccal mucosa was effective identifying
important nuclear alterations, observing proliferation taxes, genotoxicity and cytotoxicity markers, as they reflect
the cellular aging in the control group and the genotoxical and clastogenic found in diabetic patients with chronic
kidney disease in hemodialysis and not in hemodialysis. The result of the micronucleation frequency and other cellular
alterations in buccal mucosa suggest the hemodialysis can aggravate the genomic damage of the diabetic type2 patients
in consequence of the oxidative stress produced by the hyperglycemia in these patients. Worsen of the genomic
damage caused by the hemodialysis could be explained by the interaction of the blood and biocompatible membranes,
in which circulating neutrophils use oxygen and generate reactive oxygen species and inflammatory complications.
FINANCIAL SUPPORT: CNPq
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
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66
Electrophoresis tank for slides of the comet
assay
Prof. Dr. Cristiano José da Silva1 and Profa. Dra. Catarina Satie Takahashi2
1
Federal Institute of Education, Science and Technology of Goiás – Brazil; 2Department of Genetics, Faculty of Medicine
of Ribeirão Preto, São Paulo University, Brazil
[email protected]
Keywords: Eletrophoresis Tank, Comet Assay, DNA Damage
The Comet assay or single-cell gel electrophoresis assay (SCGE) is accepted by inspection/regulators worldwide; the
method is promising in toxicology, because it allows the detection of DNA damage in a population of eukaryotic
cells. Tools that optimize the performance of this assay allows to make it an important test for routine laboratory.
The aim of this work was develop an opaque electrophoresis tank for lysis, neutralization and run electrophoresis
vertically in agarose slides to realize the Comet assay and other purposes. It may be performed Comet assay with low
amounts of solutions, with a drainage system that allows to change the required solution at various stages of the assay,
without the need to manipulate the slides. It is made of opaque material eliminating the need for manual coating. It
allows run electrophoresis in several slides simultaneously. Intended for laboratory (medical and academic researchers)
for research in health, environment and biotechnology. To conduct this study we used doxorubicin, epirubicin
hydrochloride and etoposide, three well-known genotoxic agents.We used the cell line V79 (hamster fibroblasts). The
culture medium used in the experiment was HAM-F10 + D-MEN, 1:1, supplemented with 10% fetal bovine serum.
Doxorubicin was tested at concentrations of 0.15 mg, 0.30 mg and 0.75 mg / mL of culture medium, etoposide 0.15
mg and 0.30 mg / ml medium and epirubicin hydrochloride 0.03 mg / mL. As a negative control we used the proper
medium. The cells are exposed to genotoxic agents for two hours, after this period takes place the comet assay; 500
cells were analyzed for each concentration. We did not observe differences in the mean damage scores in the DNA
between electrophoresis tank experimental (built by us) and conventional Tank to agents and concentrations tested,
except for epirubicin hydrochloride at 0.30 mg / mL (experimental tank at constant current 300mA and voltage 4V)
that the average of damage score in experimental tank was significantly large than the conventional tank in statistical
terms (Student`s t-test).We can conclude that the experimental tank has the same effectiveness in detecting DNA
damage in V79 cells that conventional tank, so we suggest continuity tests to introduce it in the routine laboratory.
Support: CNPq, CAPES, Department of Genetics – FMRP, IFG
59º Congresso Brasileiro de Genética
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Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
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67
Absence of genotoxicity induced by Copaifera
duckei oilresin in swiss mice by micronucleus test
Ozelin SD¹, Oliveira PF¹, Leandro LF¹, Oliveira WL¹, Ambrósio SR¹, Bastos JK² and Tavares DC¹.
¹Universidade de Franca, Franca, SP; ²Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Ribeirão Preto, SP.
[email protected]
Keywords: Copaifera duckei, Copaifera oilresin, genotoxicity test, Swiss mice and micronucleus test.
The Copaifera duckei, an abundant plant in northern Brazil, is widely used in folk medicine in the region. Copaifera
oilresin has been used to treat various diseases, which have diverse therapeutic indications: anti-inflammatory,
anthelmintic, antimicrobial, anticancer, gastroprotective and trypanocidae. The aim of the study was to evaluate
the genotoxicity of C. duckei oilresin in Swiss mice bone marrow cells using the micronucleus test. The different
doses of C. duckei oilresin (500, 1000, 1500 and 2000 mg/kg body weight [b.w.]) were administered by gavage. The
negative (water), solvent (Tween 80 5%) and positive (methyl methanesulfonate, MMS; 40 mg/kg b.w.) controls
groups were included. The samples were collected 24 hours after the treatments. Two thousand polychromatic
erythrocytes were analyzed to determine the micronucleus frequency. The nuclear division index (NDI) was
used for the evaluation of the cytotoxicity. For the NDI values, it was analyzed four hundred erythrocytes. The
results demonstrated that the animals treated with different doses of C. duckei oilresin no shown micronucleus
frequencies significantly different when compared to the negative control group. As expected, the animals treated
with MMS showed a significant increase of micronuclei frequencies in relation to negative control. The NDI
values no shown significant statistically differences between different treatments and negative control groups.
Thus, under these experimental conditions used, the C. duckei oilresin no showed genotoxic and cytotoxic effects.
Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil, grant number 2011/136307) and Apis Flora Industrial e Comercial Ltda (Ribeirão Preto, São Paulo, Brazil).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
68
Lemon and acerola juices: comparative
assessment of modulation of genotoxicity by
micronucleus test in mice.
Bernardes, LF; Melo, AS; Figueiredo, VB; Arrais, LML, Cecchi, AO
Universidade de Franca, UNIFRAN, Franca, SP
[email protected]
Keywords: antimutagenesis, lemon, acerola, mice, micronucleus test
Oxidative stress is the term used to describe the condition of oxidative damage that results when the critical balance
between free radical generation and antioxidant defenses are unfavorable. Free radicals can originate endogenously from
normal metabolic reactions or exogenously as components of tobacco smoke and air pollutants and indirectly through
the metabolism of certain solvents, drugs, and pesticides as well as through exposure to radiation. On the other hand,
it is known that balanced diets rich in antioxidants have the potential to neutralize and reduce the reactive action of
our organism. The high consumption of fruits has been associated with low incidence of chronic-degenerative diseases,
probably due to the presence of bioactive compounds in these foods, like vitamin C. Many fruits are considered a
good source of vitamin C that has antioxidant properties to protect against oxidative damage caused by free radicals
preventing cell damages caused by oxidation. Therefore, their consumption should be encouraged. Based on these data,
this study aims to investigate and compare the antimutagenic potential of lemon and acerola juices in mice treated with
cyclophosphamide (CPA) using the micronucleus test in peripheral blood cells. The juices, prepared from fresh fruits
(100%), were administered by gavage (10 mL kg-1 on body weight - b.w.) and CPA, intraperitoneally (100 mg kg-1 b.w.),
24 h before. It was used a total of 30 male Swiss mice weighing approximately 30 g. Blood samples were collected 24 h
after treatment and were analyzed in light microscopy common, where the frequency of micronucleated polychromatic
erythrocytes (PCEMNs) were recorded. Our results showed that lemon and acerola juices showed no genotoxic potential
to damage DNA and inhibited the genotoxicity induced by CPA. These results showed positive effects on human health
and may be applied to better explore the characteristics of complex mixture of lemon and acerola as a health promoter.
Financial support: FAPESP (2013/10321-9) e Universidade de Franca
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
69
Assessment of micronuclei frequency in civil
construction workers
Cury, FP¹; Ramos, MCAS¹ and Silveira, HCS¹
¹Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP, Brazil
[email protected]
Keywords: civil construction workers; micronuclei; buccal cells; occupational factors; biomarker.
Nowadays, the civil construction sector plays a major role in Brazilian economy, totaling 15.6% of the country’s
GNP (Gross National Product). Workers have to work long hours and are exposed to several agents in their work
environment that can lead to diseases, such as cancer. The exposure to materials containing asbestos, silica and metals,
as well as the exposure to solar radiation, can interfere with the stability of human genome, causing breaks to DNA
which can lead to genetic alterations that might be critical to the normal functions of cellular cycle and survival. Due
to the great number of jobs in the construction area, it is essential to be concerned about quality of life and the impacts
on the health of the workers who are exposed to hazardous agents. Hence, using an exposure biomarker, such as
micronuclei, we can assess genotoxic damages to these subjects and compare them to the control group. Furthermore,
a questionnaire to evaluate socio-demographic data, life style, medical and occupational history of all volunteers
was carried out. Our study comprises two groups: civil construction workers and control (administrative personnel
from Barretos Cancer Hospital). To assess micronuclei, exfoliated buccal cells were collected using (Cytobrush – BD
Biosciences) and (Surepath – BD Biosciences). The slides were stained using Papanicolaou, through liquid based
cytology platform (BD Surepath – PrepStain). After that, 1000 cells were counted for each subject in the study. For
statistical analysis, the non-parametrical test Mann-Whitney was used to assess if there were differences between
the groups. According to partial results, it could be seen that the frequency of micronuclei in the study group was
statistically different from the control group (p-value <0.001). Thus, results suggest a mutagenic potential of the
materials that workers are exposed to on a daily basis, which may lead to a possible increase of the risk of cancer.
Financial Support: CNPq and Barretos Cancer Hospital
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
70
Toxicogenetic evaluation of Annona muricata
extract in swiss mice
Carrijo, GS; Batista, TH; Acésio, NO; Tozatti, MG; Cunha, WR; Tavares, DC.
University of Franca, Franca, São Paulo, Brazil
[email protected]
Keywords: Annona muricata, Swiss mice, micronucleus test, genotoxicity, toxicogenetic
Annona muricata L (Annonaceae), commonly known as “graviola”, it is a plant found in almost all countries of tropical
America. Although not native to Brazil, this species is grown extensively in the northern and northeastern regions of the
country. A. muricata is employed for a number of medicinal purposes and studies have demonstrated its antitumor action.
Thus, the aim of this study was to evaluate the genotoxic potencial of A. muricata hydroalcoholic extract in Swiss mice bone
marrow by micronucleus test. The cytotoxicity was evaluated by the polychromatic erythrocytes (PCE)/normochromatic
erythrocytes (NCE) ration. The animals were treated by gavage with the different doses of A. muricata extract, 500,
1000, 1500 and 2000 mg/kg b.w.. The negative (water), solvent (Tween 80 5%) and positive (methyl methanesulfonate;
MMS 40 mg/kg b.w.) controls groups were also included. The bone marrow samples were collected 24 hours after the
treatments. The animals treated with the highest dose of A. muricata extract (2000 mg/kg b.w.) showed signs of toxicity
and died within 24 hours. The micronuclei frequencies observed in animals treated with 500, 1000 or 1500 mg/kg
b.w. of extract were not significantly different from the negative control group. The PCE/NCE rations obtained in the
animals submitted to treatments with the A. muricata extract no shown significantly differences in relation to negative
control animals. Therefore, A. muricata extract revealed absence of genotoxicity and cytotoxicity in the evaluated doses.
Financial support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
71
Nanotechnology in the use of nitric oxide for
tumors treatment
Coelho, Bruna Oliveira1; Haddad, Paula2; Seabra, Amedea B.2; Lima, Renata1
1
Universidade de Sorocaba, UNISO, Sorocaba, SP; 2 Universidade Federal de São Paulo (UNIFESP), São Paulo, SP
[email protected]
Keywords: Iron nanoparticles, Nitric Oxide and Nanotoxicity.
After being seen as a mere environmental pollutant for several years, nitric oxide (NO) is currently one of the most
studied molecules in biomedical sciences. The chemical nature of NO makes it the key molecule in bioregulation
of important physiological processes such as vasodilatation and communication between cells. NO is a free radical,
devoid of charge, with a small size, lipophilic and it diffuses freely in biological, crossing most of the physiological
barriers. Currently, it is known that NO is involved in diverse biological processes, however, at high concentrations
can be extremely toxic to cells. This toxicity can be used in order to destroy tumor cells, so there is a great interest in
developing shippers and releasing of NO materials, which can be used as conveyers. Recently, a promising strategy
that has been used is the iron nanoparticles, which have excellent magnetic properties, and when coated with DMSA
(dimercaptosuccinic acid) have low agglomeration capacity and a decrease of possible cellular damage, since DMSA
increases its biocompatibility. This project aimed to perform genotoxic analysis of functionalized magnetic iron
nanoparticles to use in combating tumor cells. The ability of magnetic nanoparticles coupled with the release of NO
showed excellent results, since the functionalized nanoparticles did not show genotoxicity when NO was absent, unlike
the condition of transport of NO, which have proved to be extremely toxic. These results creates expectations to the use of
new strategies for controlled release of NO with great clinical potential, so it can be used without harm to human health.
Financial Support: UNISO e UNIFESP
59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
72
Mutagenic effects of petrochemical waste of
Terminal Admiral Soares Dutra (Osorio-RSBrazil) through the Allium cepa test
Machado. AT.1; Cardoso, V V.2; Casali, EA1,3
Laboratórios de Estudos Sobre as Alterações Celulares e Teciduais, Departamento de Ciências Morfológicas, ICBS,
UFRGS; 2 Laboratório de Mutagênese e Toxicologia, Centro Universitário Metodista- IPA; 3 Centro de Estudos em Estresse
Oxidativo, Departamento de Bioquímica, ICBS, UFRGS.
1
Water resources activities associated with the petrochemical industry has received attention due to possible mutagenic
and carcinogenic potential of chemicals contained in the effluents of these industries. This study aimed to evaluate
the mutagenic potential of the effluent from petrochemical terminal Almirante Soares Dutra (TEDUT), located in
the city of Osório, RS, through the Allium cepa test doses resulting from exposure to residual hydrocarbons. Samples
of the effluent were collected at the exit of Water Separator and Oil Terminal and 12 copies of Allium cepa were used
for the experiment followed the method proposed by FISKESJÖ, 1985. Samples were collected during three shifts
(morning, afternoon and evening), with an interval of 8 h between samples, making a total of 3 samples, representing
one day. Chemical analysis of total petroleum hydrocarbons were performed for the samples collected, in order to
support the biological investigations. Frequencies (mean ± SEM) for interphase changes were significant in all samples
when compared to the control group (15.00 ± 1.00, 19.00 ± 1.00, 14.50 ± 1.50, 5, 00 ± 2.00 respectively). As a
parameter of cytotoxicity was estimated mitotic rates in these analyzes of the mitotic index, referring to rootlets treated
effluent collected showed a significant increase in the rate of cell division, for samples collected in the morning and
afternoon, when compared to the control test negative. The physico-chemical analysis of the effluent indicated that
among the parameters analyzed in May 2013 there are present at levels above CONSEMA tolerated by law 128/2006.
The chemical analysis of the effluent showed that the concentration of Polycyclic Totals (TPH) in the sample collected
on the same date of sampling for the mutagenicity test is less than 0.2 mg / L. These results may be related to the high
sensitivity of the Allium cepa test determines that contamination does not go unnoticed, even when complex mixtures
are tested. The results of this study indicated that the effluent from the Water Separator and Oil TEDUT presents
indicative mutagenic. This effect can be attributed to its ability to promote changes in meristematic cells of Allium
cepa, such as cells carrying nuclear buds, micronuclei and binucleate cells. However, the results presented represent the
study of raw effluent, not considering their dilution in the receiving water body. Additionally, representing the effluent
released in a single day by Petrochemical Terminal, not serving as a parameter environmental monitoring. Therefore
further studies monitoring mutagenicity of the effluent in combination with testing samples Lagoa do Armazém.
59º Congresso Brasileiro de Genética
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Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
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73
Estimating Mutation Rates in 21 STR
Autossomal Loci in Goiânia-GO
Vieira, TC1,2,3,4; Gigonzac, MAD4; Melo, COA1,3; Rodovalho, RG5; Silva, DM1,3; Da Silva, CC 2,3,4; Da Cruz, AD1,2,3.
Universidade Federal de Goiás, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia; 2LaGene
- Laboratório de Citogenética Humana e Genética Molecular, Lacen – Laboratório de Saúde Pública Dr. Giovanni
Cysneiros, SES-GO; 3Pontifícia Universidade Católica de Goiás, Departamento de Biologia, Núcleo de Pesquisas
Replicon; 4Universidade Estadual de Goiás, Eseffego/Unidade Goiânia; 5Laboratório Biochroma, Laboratório de Análise
de DNA.
1
[email protected]
Keywords: mutation rates, STR, paternity testing
The present study estimated the mutation rate of 21 autosomal STR loci in the population of Goiânia, Goiás, with the
aim of creating a database for statistical calculations of interest, both in testing genetic link, as in studies of induced
mutations. We studied 3,353 paternity tests, all performed in Goiânia-GO, from August 2012 to February 2013. It
was sampled a total of 147,532 alleles. The biological material was collected by venipuncture, DNA was extracted and
amplified by conventional PCR and all samples were subjected to analysis on an automatic sequencer 3500 (Applied
Biosystems), to obtain the allelic profiles of each marker. We identified 74 mutations in 19 loci. The loci that presented
more mutations were FGA, with a total of 13 mutations and mutation rate of 0.8 x 10-4. The results showed mutational
rates ranging from 0.8 to 1.3 x 10-4 mutations/ site/region and the overall mutational rate was 5 × 10-4, which are
within the expected values for the STR markers. We found a higher rate of mutations of paternal origin, indicating that
changes in male gametes are more frequent, probably due to a peculiar feature of spermatogenesis, in which embryonic
germ cells undergo multiple rounds of replication and mitotic division. This result points to the need to confirm the
mutations by sequencing, considering the importance of using STR markers in DNA typing for paternity testing.
Financial Support: CAPES; FAPEG.