Kátia Kvitko

Evaluation of genetic damage in a Brazilian population occupationally exposed to pesticides and its correlation with polymorphisms in metabolizing genes.

Cytogenetic damage in individuals occupationally exposed to pesticides has received the attention of investigators in several countries, but no definitive conclusions can yet be made. The present study aimed at assessing if prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage. Vineyard workers exposed to pesticides in Caxias do Sul (Brazil) were evaluated using the micronucleus (MN) test in binucleated lymphocytes and the comet assay in peripheral leukocytes. In order to evaluate if genetically determined individual variations in xenobiotic metabolizing capacity could modify individual susceptibility to the possible genotoxic effects of pesticides, the subjects were genotyped for several genes: GSTT1, GSTM1, GSTP1, CYP1A1, CYP2E1 and PON. The study involved a total number of 173 men: 108 were agricultural workers exposed to pesticides and 65 were controls. The present study showed a high rate of MN and DNA damage in pesticide-exposed individuals (P <or= 0.001; Mann-Whitney U-test). In addition, some effects of genetic polymorphisms in PON in the modulation of MN results were observed in the exposed group, and an association between GSTM1, GSTT1 and CYP2E1 polymorphisms was suggested.

Evaluation of genetic damage in open-cast coal mine workers using the buccal micronucleus cytome assay.

Coal is the largest fossil fuel source used for the generation of energy. However, coal extraction and its use constitute important pollution factors; thus, risk characterization and estimation are extremely important for the safety of coal workers and the environment. Candiota is located to the southeast of the state of Rio Grande do Sul and has the largest coal reserves in Brazil, and the largest thermal power complex in the state. In the open‐cast mines, the coal miners are constantly exposed to coal dust. The human buccal micronucleus cytome (BMCyt) assay has been used widely to investigate biomarkers for DNA damage, cell death, and basal cell frequency in buccal cells. The aim of this study was to assess whether prolonged exposure to coal dust could lead to an increase in genomic instability, cell death, and frequency of basal cells using the BMCyt assay. In the analysis of epithelial cells, the exposed group (n = 41) presented with a significantly higher frequency of basal cells, micronuclei in basal and differentiated cells, and binucleated cells compared to the non‐exposed group (n = 29). The exposed group showed a significantly lower frequency of condensed chromatin cells than the non‐exposed group. However, we found no correlation between DNA damage and metal concentration in the blood of mine workers. DNA damage observed in the mine workers may be a consequence of oxidative damage resulting from exposure to coal residue mixtures. In addition, our findings confirm that the BMCyt assay can be used to identify occupational risk. Mol. Mutagen. 2013.

CYP1A1, CYP2E1, GSTM1, GSTT1, GSTP1, and TP53 polymorphisms : do they indicate susceptibility to chronic obstructive pulmonary disease and non-small-cell lung cancer?

Gene polymorphisms of phase I (CYP1A1 and CYP2E of cytochrome P,) and phase II (GSTM1, GSTT1 and GSTP1 of glutathione-S-transferase,) enzymes and the TP53 tumor suppressor gene were studied as markers in a sample of 262 Brazilians of European descent, the sample consisting of 97 patients with non-small-cell lung cancer (NSCLC), 75 patients with chronic obstructive pulmonary disease (COPD) and 90 control individuals. For NSCLC, we found no significant relationship between any of the markers studied and susceptibility to this disease. With respect to COPD, although the distribution of the CYP1A1, GSTM1, GSTP1, GSTT1 and TP53 genotypes was similar to that of the controls the frequency of the CYP2E1*1A/*5B heterozygote was about 6 times higher in COPD patients than in controls (OR= 6.3; CI = 1.1-35.5 for p = 0.04). Individuals who presented the GSTT1 null phenotype and GSTP1 Ile/Val genotype had a risk about four times higher (OR= 4.0; CI = 1.2–14.6 for p = 0.02) of having COPD than individuals without these genotypes, the same being true for individuals having the GSTT1 null phenotype and CYP1A1*1A/*2A genotype (OR= 3.7; 1.1–14.6 for p = 0.04).These results suggest that the CYP2E1 and GSTT1 + GSTP or GSTT1 + CYP1A1 polymorphisms may be predictive of susceptibility to COPD, at least in this population of European ancestry.

Genotoxic biomonitoring of tobacco farmers: Biomarkers of exposure, of early biological effects and of susceptibility

Tobacco farming presents several hazards to those who cultivate and harvest the plant. The genotoxic and mutagenic effects in tobacco farmers were investigated. In order to verify the relationship between genetic susceptibility and biomarkers GSTT1, GSTM1, GSTP1, CYP2A6, PON, OGG1, RAD51, XRCC1, and XRCC4 genes polymorphism were evaluated. Oxidative stress markers and trace elements content were determined. Peripheral blood cells samples were collected from 111 agricultural workers during pesticides application and leaf harvest, and 56 non-exposed subjects. Results show that farmers are exposed to mixture of substances with genotoxic and cytotoxic potential. Only GSTM1 null and CYP2A6*9 showed significant associations with cytokinesis-blocked micronuclei assay results. In pesticide application an increase in trace elements content was observed. The results indicated that exposure to pesticides and nicotine can influence antioxidant enzymes activity. Our study drives the attention once more to the need for occupational training on safe work environment for farm workers.

BER gene polymorphisms (OGG1 Ser326Cys and XRCC1 Arg194Trp) and modulation of DNA damage due to pesticides exposure.

The susceptibility of individuals to the genotoxic effect of pesticides can be modulated by genetic variations in the xenobiotic detoxification and DNA repair processes. This study evaluates if the two BER polymorphisms (XRCC1Arg194Trp and OGG1Ser326Cys) or the combined genotypes of these polymorphisms with PON1Gln192Arg could modify individual susceptibility to pesticide exposure in vineyard workers, as measured by micronucleus formation and DNA damage induction in peripheral leukocytes. The study population comprised 108 agricultural workers exposed to pesticides and 65 nonexposed. Our results demonstrate that individuals with the variant allele (OGG1Cys) showed higher DNA damage, detected by the comet assay, in relation to individuals carrying the wild‐type OGG1Ser allele. Considering the combined influence of metabolizing PON1 and the DNA repair OGG1 genes, we observed significantly higher DNA damage in the comet assay in the exposed group when a less efficient OGG1Cys allele was acting independently of the PON1 genotype, reinforcing the importance of the OGG1 repair enzyme in the response to DNA damage by pesticide exposure. The association of the PONGln/Gln genotype with higher MN frequency suggests that the PON1 genotype is a major determinant of genotoxic risk in individuals exposed to pesticides. Analysis of the compared effect of XRCC1 and PON1 genotypes in the exposed group suggested that, among the poorly metabolizing PON1Gln/Gln individuals, the XRCC1Arg/Trp genotype has a protective effect with respect to MN formation. These results indicate that enhanced XRCC1 function may provide some protection from the enhanced genotoxic risk associated with inefficient xenobiotic detoxification in the studied population. Environ. Mol. Mutagen. 52:20–27, 2011.

Genetic and oxidative damage of peripheral blood lymphocytes in workers with occupational exposure to coal

Coal is an important fossil fuel used to generate energy. Coal dust is constituted primarily of hydrocarbons and metals. During coal extraction, large quantities of coal dust particles are emitted, contributing to environmental pollution. Coal miners are constantly exposed to coal dust and its derivatives. The goal of this study was to evaluate the potential genotoxic effects of coal and oxidative stress in individuals from Candiota who were exposed to coal as part of their occupation. The comet assay and micronucleus (MN) test were used to assess these effects. This study involved 128 male participants of whom 71 reported work that included exposure to coal (exposed group) and 57 reported working at different jobs (unexposed group). The exposed group had a significantly increased damage index and damage frequency, as assessed using the comet assay, and increased MN and nucleoplasmic bridge frequencies, as assessed using the MN assay, compared with unexposed individuals. Significant and positive correlations between MN frequencies in the lymphocytes and buccal cells of control and exposed individuals were observed. The exposed individuals presented lower average levels of thiobarbituric acid reactive substances (TBARS) and catalase activity (CAT), while the mean superoxide dismutase activity (SOD) levels were higher in this group. The exposed group also had higher hematocrit levels. No correlation between DNA damage and inorganic elements, as identified using PIXE, was found; however, there was a correlation between the damage index and zinc. The evidence that exposure to coal and its derivatives presents a genetic hazard demonstrates the need for protective measures and educational programs for coal miners.

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. Environ. Mol. Mutagen., 2012.

Genotoxic assessment in tobacco farmers at different crop times

Agricultural workers engaged in tobacco cultivation are constantly exposed to large amounts of pesticides as well as to the nicotine present in raw tobacco leaves. Pesticides have been considered potential chemical mutagens: experimental data revealed that various agrochemicals possess mutagenic properties. Studies have affirmed that nicotine absorbed through the skin results in the characteristic green tobacco sickness (GTS), an occupational illness reported by tobacco workers. This study sought to determine genotoxic effects in farmers occupationally exposed to agrochemicals and nicotine. Peripheral blood samples were collected from 30 agricultural workers, at different crop times (off-season, during pesticides application and leaf harvest), and 30 were non-exposed. We obtained data on DNA damage detected by the Comet assay and Micronucleus test as biomarker of occupational exposure and effect. The serum cholinesterase level, which in general present relation with exposition to organophosphates and carbamates, as well as serum cotinine level, which is a metabolite of nicotine, were also evaluated. The results showed a significant increase in Damage index and frequency in tobacco farmers compared to the non-exposed group, for all different crop times; and a significant increase in micronucleated cells in the off-season group. No correlation was found between age and exposure time in relation to biomarker tests. The DNA damage was greater in males than in females, but with a significant difference only in off-season group. No difference, in cholinesterase activity, was seen among the group of farmers and non-exposed group. Elevated level of cotinine was observed in leaf harvest group. This investigation suggests increased DNA damage in all tobacco crop stages, calling attention to the significant increase during the off-season and tobacco leaf harvest.

Susceptibility to DNA damage in workers occupationally exposed to pesticides, to tannery chemicals and to coal dust during mining

Our mutagenesis group has been studying with important economic drivers of our state, such as agriculture, the foot-wear and leather industry and open-cast coal mining. Working conditions in these sectors have potentially harmful to humans. The aim of these studies is to determine the health risk of workers by biomonitoring subjects exposed to genotoxic agents. The main results of our studies with vineyard farmers we observed a high rate of MN and DNA damage in individuals exposed to pesticides (p < 0.001). In addition, some effects of genetic polymorphisms in the modulation of MN results were observed in this group. Tobacco farmers were also evaluated at different crop times. The results showed a significant increase in the Damage index and frequency in tobacco farmers compared to the non-exposed group, for all crop times. The results for footwear and tannery workers showed a significant increase in the mean ID for the solvent-based adhesive (p < 0.001) group in comparison to the water-based adhesive group and control (p < 0.05). For open-cast coal mine workers, the EBCyt indicated a significant increase in nuclear bud frequency and cytokinetic defects in the exposed group compared to the non-exposed group (p < 0.0001). We were able to associate specific genetic susceptibility with each type of exposure and with the non-use or improper use of personal protection equipment and diet adequacy. These results show how important the continuous education of exposed workers is to minimizing the effect of the occupational exposure and the risk of disease associated with the work.

Investigation of potential biomarkers for the early diagnosis of cellular stability after the exposure of agricultural workers to pesticides

Agricultural workers involved in the harvest of tobacco crops are regularly exposed to large quantities of pesticides. In order to determine how this exposure to pesticides induces genetic alterations in these workers, blood samples were obtained from 77 exposed individuals, as well as from 60 unexposed subjects. DNA damage was analyzed by the Comet assay and by the micronucleus (MN) test. The antioxidant profile was evaluated by activity of superoxide dismutase (SOD), and the polymorphism of gene PON1 was used as a susceptibility biomarker. The content of inorganic elements in the blood samples was determined by PIXE analysis. Our results demonstrated that the damage frequency, damage index, the MN frequency, and the SOD activity were significantly elevated in the exposed relative to the unexposed group. A modulation of the MN results for the PON1 gene was observed in the exposed group. The concentrations of inorganic elements in the exposed group were higher compared to those of the unexposed group. In this study, we observed that genetic damage, and change in oxidative balance were induced by the exposure of workers to complex mixtures of pesticides in the presence of inorganic compounds, whereby an influence of the genotype was evident.