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.

Small de novo CNVs as biomarkers of parental exposure to low doses of ionizing radiation of caesium-137.

The radiological accident in Goiania in 1987 caused a trail of human contamination, animal, plant and environmental by a radionuclide. Exposure to ionizing radiation results in different types of DNA lesions. The mutagenic effects of ionizing radiation on the germline are special concern because they can endures for several generations, leading to an increase in the rate of mutations in children of irradiated parents. Thus, to evaluate the biological mechanisms of ionizing radiation in somatic and germline cells, with consequent determination of the rate mutations, is extremely important for the estimation of genetic risks. Recently it was established that Chromosomal Microarray Analysis is an important tool for detecting wide spectra of gains or losses in the human genome. Here we present the results of the effect of accidental exposure to low doses of ionizing radiation on the formation of CNVs in the progeny of a human population accidentally exposed to Caesium-137 during the radiological accident in Goiânia, Brazil.

In vivo genotoxicity evaluation of efavirenz (EFV) and tenofovir disoproxil fumarate (TDF) alone and in their clinical combinations in Drosophila melanogaster.

This study focuses on the antiretrovirals efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor, and tenofovir disoproxil fumarate (TDF), an oral prodrug of tenofovir analog of adenosine 5'-monophosphate, which belongs to the class of nucleotide reverse transcriptase inhibitors. Both compounds act on the mechanisms of HIV replication, inhibiting the action of reverse transcriptase and thus preventing viral DNA synthesis. The toxic and genotoxic potential of EFV and TDF alone and in combinations {EFV+combivir [zidovudine (AZT)+lamivudine (3TC)] and TDF+3TC} were assessed using the comet assay and the somatic mutation and recombination test (SMART) in Drosophila melanogaster. The results indicate that EFV was toxic at high concentrations and induced genotoxicity using the comet assay, but showed neither mutagenic nor recombinogenic effects using SMART. In combination with combivir, EFV exhibited antagonic genotoxic effects in both tests. Inversely, TDF did not show toxicity but induced genotoxicity at all concentrations tested in both the comet assay and SMART. The prevalence of recombinogenic events in all treatments with TDF alone and in combination with 3TC was detected using SMART. Homologous recombination is an important parameter to be taken into consideration in the evaluation of carcinogenicity of medicines used in antiretroviral therapy regimens, due to the need for lifelong adherence and the unknown effects of long-term treatments.

Genotoxic and mutagenic effects of Atrazine Atanor 50 SC on Dendropsophus minutus Peters, 1872 (Anura: Hylidae) developmental larval stages.

The potential mutagenic and genotoxic effects of the herbicide atrazine were investigated in different developmental stages of Dendropsophus minutus tadpoles. These animals were exposed to 4 nominal concentrations of atrazine (2.25, 4.5, 9, and 18 mg/L) and 40 mg/L of Cyclophosphamide as a positive control, for 96 h. Negative controls were also added to the experiment. The tadpoles were divided into three groups according to Gosner's developmental stages, namely GS 25-33 as premetamorphic, GS 36-39 as prometamorphic, and GS 42-43 as metamorphic climax. Our results showed that the premetamorphic and metamorphic stages were more sensitive than the prometamorphic stage to the herbicide. A comet assay and micronucleus test for the sensitive stages demonstrated DNA damage in a concentration-dependent curve. Although a dose-response effect was not observed for the prometamorphic stage, a statistically significant difference was found between the treatment of 18 mg/L and the negative control. Moreover, the highest concentration of atrazine showed both the largest amount of DNA damage and the highest micronucleus frequency regardless of the developmental stage of D. minutus. In conclusion, atrazine was genotoxic and mutagenic for D. minutus in a dose-sensitive manner, dependent on larval developmental stages. Considering the prometamorphic stages showed no dose-response effect to atrazine, we suggest caution when using this stage in biomonitoring studies in order to avoid false negative results. Amphibians have been proven to be useful bioindicators, and we suggest replicating biomonitoring studies using different species to represent ecosystems' environmental impacts.

DNA damage in peripheral blood lymphocytes and association with polymorphisms in the promoter region of the CYP2E1 gene in alcoholics from Central Brazil.

DNA damage caused by the accumulation of bio-products generated in the biotransformation of ethanol to acetaldehyde mediated by the CYP2E1 enzyme has been studied. To evaluate DNA damage in peripheral blood lymphocytes and the possible association with polymorphisms in the promoter region of the CYP2E1 gene, we performed a case-control study including 75 alcoholics and 59 individuals who consume alcohol socially. Alcoholics were previously diagnosed by the Psychosocial Care Center - Alcohol and Drugs (CAPS A/D) in the city of Goiania, Goias state, Central Brazil. DNA damage was evaluated by comet assay. The analysis of the rs3813867, rs2031920, and rs2031921 polymorphisms in the promoter region of CYP2E1 gene was performed by Sanger sequencing. Men older than 35 years old were the most common alcoholics. We found increased DNA damage in the case group, compared to the control group (p < 0.001). Alcoholics who were heterozygous in the rs3813867, rs2031920, and rs2031921 polymorphisms showed higher DNA damage (tail length and olive tail moment), compared to individuals with the homozygous non-mutated allele. Previous studies have shown that polymorphisms in the promoter region of the CYP2E1 gene could cause higher CYP2E1 transcriptional activity, increasing enzyme activity compared with nondrinkers, indicating that the presence of the mutated allele (heterozygous or homozygous) may be associated with higher alcohol metabolic rates and therefore show increased acetaldehyde levels after alcohol consumption, which then can exert its carcinogenic effect.

Assessment of DNA damage in Brazilian workers occupationally exposed to pesticides: a study from Central Brazil.

We evaluated 41 rural workers occupationally exposed to pesticides and 32 subjects as a control group, using the micronucleus (MN) and the comet assay. For the comet assay, we evaluated the peripheral blood, and for the MN, we sampled cells from the oral epithelium. Damage to DNA was measured by tail length, % DNA in tail (% tail), olive tail moment (OTM), and tail moment (TM). The exposed group presented an 8× increase in MN frequency, when compared to the control group (p <0.05). When we contrasted the MN frequencies between the individuals that use and do not use personal protective equipment, we found a mean of 7.5 MN (57 % variance) and 12.1 MN (130 % variance), respectively. The binucleated cells were 0.04 and 0.005, in the exposed and control groups, respectively, indicating 8× increase in the number of binucleated cells, when comparing the groups (p <0.05). In the comet assay, we demonstrated statistically significant differences in three parameters (% DNA, OTM, and TM) indicating that the rural workers presented high levels of genomic damages. Our results indicate that occupational exposure to pesticides could cause genome damage in somatic cells, representing a potential health risk to Brazilian rural workers that deal constantly with agrochemicals without adequate personal protection equipment.