Assessing Genotoxicity and Mutagenicity of Three Common Amphibian Species Inhabiting Agroecosystem Environment.

Amphibians are constantly exposed to pollutants and the stress of agricultural activities. We selected three anuran amphibian species Dendropsophus minutus, Boana albopunctata, and Physalaemus cuvieri, totaling 309 individuals. We collected tadpoles in 15 permanent ponds: 5 soybean crops, 3 corn crops, and 7 nonagricultural lands. Our study provides the first comparative data on the genotoxicity and mutagenicity of three common amphibian anurans. Dendropsophus minutus was the most vulnerable species compared with B. albopunctata and P. cuvieri for comet assay and micronuclei test. However, the more significant amount of DNA damage seen in D. minutus does not mean that their populations are threatened once such species adapt well to anthropogenic disturbances. Despite, P. cuvieri was less sensitive than the other two species; the DNA damage was significantly higher in soybean crops. Physalaemus cuvieri is a leptodactylidae species that deposit their eggs in foam nests, which are essential to protect eggs from dehydration. Moreover, the foam reduces the contact of eggs with water; thus, P. cuvieri eggs could be less exposed to contaminants present in pounds, compared with D. minutus and B. albopunctata, which deposit their eggs directly in the water. Therefore, this study was sufficiently sensitive to detect genotoxic and mutagenic effects in tadpoles exposed to agroecosystems. We strongly suggest D. minutus in future biomonitoring studies that involve the comparison of anthropized versus not anthropized environments. Overall, we recommend the comet assay and micronucleus test as effective methods for the detection of genotoxic damage in amphibian anurans to the environmental disturbance, especially in agricultural sites.

The effect of low-dose exposure on germline microsatellite mutation rates in humans accidentally exposed to caesium-137 in Goiânia.

A serious radiological accident occurred in 1987 in Goiânia, Brazil, which lead to extensive human and environmental contamination as a result of ionising radiation (IR) from caesium-137. Among the exposed were those in direct contact with caesium-137, their relatives, neighbours, liquidators and health personnel involved in the handling of the radioactive material and the clean-up of the radioactive sites. The exposed group consisted of 10 two-generation families, totalling 34 people. For each exposed family, at least one of the progenitors was directly exposed to very low doses of γ-IR. The control group consisted of 215 non-irradiated families, composed of a father, mother and child, all of them from Goiânia, Brazil. Genomic DNA was purified using 100 µl of whole blood. The amplification reactions were prepared according to PowerPlex® 16, following the manufacturer's instructions. Genetic profiles were obtained from a single polymerase chain reaction amplification. The exposed group had only one germline mutation of a paternal origin in the 'locus' D8S1179 and the observed mutation presented a gain of only one repeat unit. In the control group, 11 mutations were observed and the mutational events were distributed in five loci D16S539, D3S1358, FGA, Penta E and D21S11. The mutation rates for the exposed and control groups were 0.006 and 0.002, respectively. There was no statistically significant difference (P = 0.09) between the mutation rate of the exposed and control groups. In conclusion, the quantification of mutational events in short tandem repeats can provide a useful system for detecting induced mutations in a relatively small population.

Multi-biomarker responses to pesticides in an agricultural population from Central Brazil.

We evaluated farmworkers exposed to pesticides and individuals with no history of occupational exposure to pesticides. It was performed the comet assay to evaluate DNA damage. The immunophenotyping of TCD4+ lymphocyte subpopulations in peripheral blood was performed by flow cytometry. The single nucleotide polymorphisms (SNPs) in PON1, XRCC1, IL6, IL6R, TNF-α, and MIR137 genes were evaluated by real-time PCR. The exposed group was composed mostly by males (69.44%), with direct exposure to pesticides (56%) and with an average age range of 46 ± 13.89 years, being that 58.3% of farmworkers directly exposed to pesticides and reported the full use of personal protective equipment (PPE). DNA damage was greater in the exposed group (p < 0.05), reinforced by the use of PPE to denote a lower degree of DNA damage (p = 0.002). In this context, in the exposed group, we demonstrated that the use of PPE, age, gender and intoxication events were the variables that most contributed to increase DNA damage (p < 0.0001). Besides, the exposed group showed a significant increase in the subpopulations of T lymphocytes CD3+CD4+ (p < 0.05) and CD3+CD4+CD25+ (p < 0.0001) and a significant decrease in CD3+CD4+CD25-FOXP3+ (p < 0.05). SNPs in the TNF-α (rs361525) gene presented a difference in the genotype distribution between the groups (p = 0.002). The genotype distribution of TNF-α (rs361525) was also positively correlated with the DNA damage of the exposed group (r = 0.19; p = 0.01), demonstrating a higher risk of DNA damage in the farmworkers presenting the A mutated allele. Our findings demonstrate that pesticides can exert various deleterious effects on human health by damaging the DNA as well as by influencing the immune system in the case of both direct or indirect exposure and these issues are associated to age, gender, intoxication and the nonuse of PPE.

The importance of understanding the distribution of GSTM1 and GSTT1 genotypes and haplotypes in a region with intense agriculture activity.

Brazil is one of the largest pesticide consumers in the world, mainly due to its intense agricultural activity. The State of Goias, situated in Central Brazil, is a region recognized as an essential producer of soy, corn, beans, sorghum, sugar cane, and cotton. In this study, we evaluated 602 unrelated individuals, distributed in central and southern regions in Goias, presenting combined frequencies (haplotypes) of the GSTT1 and GSTM1 genes. In all municipalities, the frequency of the GSTT1 null genotype was 38.2% and of the GSTM1 null genotype was 50.3%. Goiania, the capital of Goias, presented the highest frequencies of GSTT1 and GSTM1 null genotypes, probably due to a founder effect of non-representative colonizing ancestors. So, the ancestral population adapted to the environment, with the frequencies observed in Goiania. However, nowadays, as there is excessive use of pesticides, the community becomes susceptible to the harmful effects of xenobiotics exposure, mainly due to the high frequency of GSTT1 and GSTM1 null genotypes. As in Goias, the consumption of pesticides has shown considerable growth, haplotypes with null alleles are of high risk for the population. Our results indicated that it is essential to understand the frequencies of the GSTT1 and GSTM1 genes for the monitoring of risk groups, like farmers, who have contact with pesticides, directly or indirectly, as well as assisting in the development of preventive medicine practices.

Increased DNA damage is not associated to polymorphisms in OGGI DNA repair gene, CYP2E1 detoxification gene, and biochemical and hematological findings in soybeans farmers from Central Brazil.

Our study evaluated 163 individuals, being 74 soybean farmers, occupationally exposed to pesticides, and 89 individuals from Goias municipalities, Central Brazil, with similar conditions to the exposed group, comprising the control group. Of the 74 soybean farmers, 43 exposed directly to pesticides and 31 exposed indirectly. The exposed group consisted of individuals aged 19 to 63 years, 21 women and 53 men, and the control group had ages ranging from 18 to 64 years, being 36 women and 53 men. 18.9% of the exposed group were poisoned by pesticides, and the most common symptoms were headache and gastrointestinal problems. The genotype frequencies of the rs2031920 (T>C) polymorphism in the CYP2E1 gene present significant differences between the exposed and control groups (p = 0.02), showing that 24.3% of the exposed group were heterozygotes against 6.7% in the control group. For the OGG1 gene, two SNPs, rs1052133 (G>C) and rs293795 (T>C), were evaluated and the genotype frequencies were not statistically different between the exposed and control groups. The DNA damage was distinct (p < 0.05) in the three analyzed comet parameters (tail length, Olive tail moment, %DNA) between groups. However, there was no influence of age and alcohol consumption between the groups associated with the polymorphisms in the CYP2E1 and OGG1 genes and DNA damage. We also did not find altered hematological and biochemical parameters in the exposed group. Thus, this pioneering study at Goias State carried out an overview of the health of soybean farmers. We evaluated classic laboratory exams, associated with exposure markers (comet assay) and susceptibility markers (genetic polymorphisms), emphasizing the need to expand the Brazilian health assessment protocol. We found, in soybean farmers, increased DNA damage and a higher number of heterozygotes in CYP2E1 gene, compared with the control group, despite the lack of association with age, educational level, smoking, drinking habits, and genetic polymorphisms.

Evaluating genotoxic risks in Brazilian public health agents occupationally exposed to pesticides: a multi-biomarker approach.

This is the first study demonstrating genotoxic effects and whole transcriptome analysis on community health agents (CHAs) occupationally exposed to pesticides in Central Brazil. For the transcriptome analysis, we found some genes related to Alzheimer's disease (LRP1), an insulin-like growth factor receptor (IGF2R), immunity genes (IGL family and IGJ), two genes related to inflammatory reaction (CXCL5 and CCL3), one gene related to maintenance of cellular morphology (NHS), one gene considered to be a strong apoptosis inductor (LGALS14), and several transcripts of the neuroblastoma breakpoint family (NBPF). Related to comet assay, we demonstrated a significant increase in DNA damage, measured by the olive tail moment (OTM), in the exposed group compared to the control group. Moreover, we also observed a statistically significant difference in OTM values depending on GSTM1 genotypes. Therefore, Brazilian epidemiological surveillance, an organization responsible for the assessment and management of health risks associated to pesticide exposure to CHA, needs to be more proactive and considers the implications of pesticide exposure for CHA procedures and processes.

Do GSTT1 and GSTM1 polymorphisms influence intoxication events in individuals occupationally exposed to pesticides?

This study evaluated the variability of GSTM1 and GSTT1 polymorphisms in individuals occupationally exposed to pesticides in ten Goias municipalities that present intense agricultural activity. We evaluated blood samples of 235 individuals, which 120 were rural workers occupationally exposed to pesticides and 115 formed the control group, analyzing GST polymorphisms by quantitative polymerase chain reaction (qPCR).The exposed group consisted of 111 men and nine women only getting an average of 39 ± 9 years. These workers were from ten 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 (42 %), use of Personal Protection Equipment (PPE; 52 %) and if the worker prepared the pesticide (7 %), or if just applied the pesticide (22 %) or if the worker prepared 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. Finally, we could not associate a null GSTT1 or null GSTM1 polymorphisms or both to intoxication events caused by pesticides, but instead we presented the importance to use PPE to prevent such harm, once we found a statistically significant association between the use of PPE and events of intoxication (p ≤ 0.001).

Verificação genotípica do polimorfismo rs1143634 do gene il1b em indivíduos com a doença periodontal crônica / Genotypic Verification rs1143634 polymorphism IL1B gene in Individuals with Chronic Periodontal Disease

O objetivo desta pesquisa foi verificar a frequência do polimorfismo rs1143634 do gene IL1B em indivíduos com a doença periodontal crônica (DPC) e a relação do mesmo com o risco de afecção. Foram analisadas 39 amostras de um grupo de indivíduos diagnosticados com DPC, sendo 77% com nível leve, 21% com o nível moderado e 3% com o nível severo, apresentando uma média de idade de 43,26. Durante o estudo foram utilizadas as técnicas de PCR e RFLP para o rastreamento do SNP (Do Inglês, Single nucleotide polymorphism - Polimorfismo de Núcleotídeo Único) rs1143634, verificando-se diferenças significativas (p<0,0001) e uma redução absoluta de risco de 53,8% referente à presença do alelo C, indicando o alelo T como um fator de risco. No entanto, este resultado também sugere a possibilidade da participação de outros fatores, uma vez que a redução obtida foi pouco acima de 50%, e deste modo, poderia apontar para o envolvimento de elementos relacionados aos hábitos de vida (higiene bucal, tabagismo e etilismo) e/ou outros aspectos genéticos, considerando que o gene IL1B entre outros mediadores implicados com a patogênese da DPC possuem várias regiões polimórficas.(AU)

The current work aimed to determine the allelic frequency regarding the SNP rs1143634 in the IL1B gene of individuals with chronic periodontal disease (CPD) and the potential to predict the relative risk for the condition. Thus, 39 patients, with a mean age of 43.26, diagnosed with CPD were clinically distributed according the level of disease in low level (77%), moderate (21%), and severe (3%). In order to genotype the SNP, PCR and RFLP methodologies were used. Allele C in rs1143634 was related to an absolute relative risk reduction of 53.8%, showing statistically significant difference (p<0,0001) On the other hand, the presence of T in rs1143634 can be considered a risk factor for CPD. Additional to the results from the current study, the participation of other factors, since reduction obtained was slightly above 50%, suggested to involvement others elements including and life style (oral hygiene, smoking, and alcoholism) and the genetic risk when considering the roll of IL1B gene in the pathogenesis of CPD.(AU)