Investigation of pesticide exposure by genotoxicological, biochemical, genetic polymorphic and in silico analysis.

Soybean farmers are exposed to various types of pesticides that contain in their formulations a combination of chemicals with genotoxic and mutagenic potential. Therefore, the objective of this paper was to evaluate the genetic damages caused by this pesticide exposure to soybean producers in the state of Mato Grosso (Brazil), regarding biochemical, genetic polymorphic and in silico analyses. A total of 148 individuals were evaluated, 76 of which were occupationally exposed and 72 were not exposed at all. The buccal micronucleus cytome assay (BMCyt) detected in the exposed group an increase on DNA damage and cell death. No inhibition of butyrylcholinesterase (BchE) was observed within the exposed group. The detection of inorganic elements was made through the particle-induced X-ray emission technique (PIXE), which revealed higher concentrations of Bromine (Br), Rubidium (Rb) and Lead (Pb) in rural workers. A molecular model using in silico analysis suggests how metal ions can cause both DNA damage and apoptosis in the exposed cells. Analysis of the compared effect of X-ray Repair Cross-complement Protein 1 (XRCC1) and Paraoxonase 1 (PON1) genotypes in the groups demonstrated an increase of binucleated cells (exposed group) and nuclear bud (non-exposed group) in individuals with the XRCC1 Trip/- and PON1 Arg/- genes. There was no significant difference in the telomere (TL) mean value in the exposed group in contrast to the non-exposed group. Our results showed that soybean producers showed genotoxic effect and cell death, which may have been induced by exposure to complex mixtures of agrochemicals and fertilizers. In addition, XRCC1 Arg/Arg could, in some respects, provide protection to individuals.

DNA damage and epigenetic alteration in soybean farmers exposed to complex mixture of pesticides.

Exposure to pesticides can trigger genotoxic and mutagenic processes through different pathways. However, epidemiological studies are scarce, and further work is needed to find biomarkers sensitive to the health of exposed populations. Considering that there are few evaluations of soybean farmers, the aim of this study was to assess the effects of human exposure to complex mixtures of pesticides. The alkaline comet assay modified with restriction enzyme (hOGG1: human 8-oxoguanine DNA glycosylase) was used to detect oxidised guanine, and compared with the buccal micronucleus cytome assay, global methylation, haematological parameters, biochemical analyses (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, gamma-glutamyl-transferase and butyrylcholinesterase), and particle-induced X-ray emission (PIXE) for the analysis of inorganic elements. Farm workers (n = 137) exposed to different types of pesticides were compared with a non-exposed reference group (control; n = 83). Results of the enzyme-modified comet assay suggest oxidation of guanine in DNA generated by pesticides exposure. It was observed that DNA damage (comet assay and micronucleus test) was significantly increased in exposed individuals compared to the unexposed group. The micronucleus test demonstrated elimination of nuclear material by budding, defective cytokinesis and dead cells. Occupationally exposed individuals also showed genomic hypermethylation of DNA, which correlated with micronucleus frequency. No differences were detected regarding the haematological and biochemical parameters. Finally, significantly higher concentrations of Al and P were observed in the urine of the soybean farmers. DNA damage could be a consequence of the ability of the complex mixture, including Al and P, to cause oxidative damage. These data indicate that persistent genetic instability associated with hypermethylation of DNA in soybean workers after long-term exposure to a low-level to pesticides mixtures may be critical for the development of adverse health effects such as cancer.

Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international inter-laboratory scoring exercise.

The buccal micronucleus cytome (BMNcyt) assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and follow-up of precancerous oral lesions. The main limitation of the assay is the large variability observed in the baseline values of micronuclei (MNi) and other nuclear anomalies mainly related to different scoring criteria. The aim of this international collaborative study, involving laboratories with different level of experience, was to evaluate the inter- and intra-laboratory variations in the BMNcyt parameters, using recently implemented guidelines, in scoring cells from the same pooled samples obtained from healthy subjects (control group) and from cancer patients undergoing radiotherapy (treated group). The results indicate that all laboratories correctly discriminated samples from the two groups by a significant increase of micronucleus (MN) and nuclear bud (NBUD) frequencies and differentiated binucleated (BN) cells, associated with the exposure to ionizing radiation. The experience of the laboratories was shown to play an important role in the identification of the different cell types and nuclear anomalies. MN frequency in differentiated mononucleated (MONO) and BN cells showed the greatest consistency among the laboratories and low variability was also detected in the frequencies of MONO and BN cells. A larger variability was observed in classifying the different cell types, indicating the subjectivity in the interpretation of some of the scoring criteria while reproducibility of the results between scoring sessions was very good. An inter-laboratory calibration exercise is strongly recommended before starting studies with BMNcyt assay involving multiple research centers.

Genetic damage in soybean workers exposed to pesticides: evaluation with the comet and buccal micronucleus cytome assays.

Soybean cultivation is widespread in the State of Rio Grande do Sul (RS, Brazil), especially in the city of Espumoso. Soybean workers in this region are increasingly exposed to a wide combination of chemical agents present in formulations of fungicides, herbicides, and insecticides. In the present study, the comet assay in peripheral leukocytes and the buccal micronucleus (MN) cytome assay (BMCyt) in exfoliated buccal cells were used to assess the effects of exposures to pesticides in soybean farm workers from Espumoso. A total of 127 individuals, 81 exposed and 46 non-exposed controls, were evaluated. Comet assay and BMCyt (micronuclei and nuclear buds) data revealed DNA damage in soybean workers. Cell death was also observed (condensed chromatin, karyorhectic, and karyolitic cells). Inhibition of non-specific choline esterase (BchE) was not observed in the workers. The trace element contents of buccal samples were analyzed by Particle-Induced X-ray Emission (PIXE). Higher concentrations of Mg, Al, Si, P, S, and Cl were observed in cells from workers. No associations with use of personal protective equipment, gender, or mode of application of pesticides were observed. Our findings indicate the advisability of monitoring genetic toxicity in soybean farm workers exposed to pesticides.

DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death.

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.

Application of the buccal micronucleus cytome assay and analysis of PON1Gln192Arg and CYP2A6*9(-48T>G) polymorphisms in tobacco farmers.

Tobacco is a major Brazilian cash crop. Tobacco farmers apply large amounts of pesticides to control insect growth. Workers come into contact with green tobacco leaves during the tobacco harvest and absorb nicotine through the skin. In the present study, micronucleus frequency, cell death, and the frequency of basal cells were measured in tobacco farmers using the buccal micronucleus cytome assay (BMCyt), in parallel with measurement of blood butyrylcholinesterase (BChE) and nicotine levels. Polymorphisms in PONIGln192Arg and CYP2A6*9(-48T>G) were evaluated to verify the relationship between genetic susceptibility and the measured biomarkers. Peripheral blood and buccal cell samples were collected from 106 agricultural workers, at two different crop times (during pesticide application and leaf harvest), as well as 53 unexposed controls. BMCyt showed statistically significant increases in micronuclei, nuclear buds, and binucleated cells among exposed subjects in differentiated cells, and in micronuclei in basal cells. In addition, the exposed group showed higher values for condensed chromatin, karyorrhectic, pyknotic, and karyolitic cells, indicative of cell death, and an increase in the frequency of basal cells compared to the unexposed control group. A slight difference in mutagenicity using the BMCyt assay was found between the two different sampling times (pesticide application and leaf harvest), with higher micronucleus frequencies during pesticide application. Elevated cotinine levels were observed during the leaf harvest compared to the unexposed controls, while BChE level was similar among the farmers and controls. PONIGln192Arg and CYP2A6*9(-48T>G) polymorphisms were associated with DNA damage induced by pesticides and cell death.