Abstract
The main aim of this study was to evaluate genotoxic effects of pesticides in association with glutathione S-transferase (GST) polymorphism. To achieve this aim, DNA damage and the genotypes of the GSTM1 and GSTT1 genes were studied from blood lymphocytes of pesticide-exposed and unexposed (control) agricultural workers of the Punjab region of northwestern India. The blood samples were collected from 40 exposed and 27 unexposed subjects from the Kakrala and Sanour villages of Patiala district. DNA damage was evaluated by using an alkaline comet assay. The analysis of the comets was done through visual scoring and image analysis software (Tritek's CometScore™). Damage Index (DI), Damage Frequency (DF) (calculated by visual scoring method), and % DNA in tail (measured by image analysis software) were considered for assessing DNA damage. The DNA extraction from blood cells was done using proteinase K and the phenol-chloroform method, and genotyping of GSTM1 and GSTT1 was done using multiplex PCR. It was found that all the pesticide-exposed subjects showed higher DI, DF, and % DNA in tail in comparison to the controls. The statistical comparison of DNA damage between the exposed group and unexposed group revealed highly significant differences (p < 0.05; Mann–Whitney U-test). In addition, the GSTT1 gene deletion and simultaneous deletions of GSTM1 and GSTT1 genes in increasing DNA damage were observed in the exposed group.
Introduction
A human detoxification enzyme like glutathione S-transferase (GST) is a family of isoenzymes that use glutathione in reactions contributing to the transformation of a wide range of compounds, including carcinogens, therapeutic drugs, and products of oxidative stress; these enzymes have provided evidence that individual susceptibility to DNA damage is mediated by individual genome organization. The GST family comprises of at least four generic classes: alpha, mu, pi, and theta. The GSTs act by catalyzing the reaction of glutathione with an acceptor molecule to form an S-substituted glutathione (S indicates sulfur). At least two enzyme steps are involved in the processing of toxicants in the liver of humans—cytochrome p450 enzyme and GST. The enzymes of the liver first deactivate the toxic substances and then convert the toxin into water-soluble forms, which are eliminated through urine, bile, feces, sweat, etc. GSTM1 and GSTT1 have been of special interest in molecular epidemiological studies because it has been reported that individuals having gene deletions of GSTM1 and GSTT1 are at a greater risk when exposed to carcinogens because of reduced detoxification ability. Polymorphism at GSTM1 locus genes encoding the mu class of enzymes are organized in a gene cluster on chromosome 1, 3 and GSTT1 locus genes encoding the theta class of enzymes are organized in a gene cluster on chromosome 22. 4
Materials and Methods
The present study was undertaken in Kakrala (Samana) and Sanour villages of the Patiala district, Punjab (northwestern border of India). A group of 40 workers exposed to various pesticides along with a group of 27 unexposed control workers having an average age of 36.1 ± 10.64 years (range, 20–61years) and 38.41 ± 14.54 years (range, 20–75 years), respectively, were studied.
Comet assay
The alkaline SCGE assay was carried out using the technique of Singh et al. 1 with slight modification by Ahuja and Saran. 5 A silver staining method was used to stain the cells for analysis. A total of 100 cells were examined per subject using a Zeiss microscope. Undamaged cells have intact nuclei without a tail and appear as a “halo,” whereas damaged cells have the appearance of “comets.” Through visual scoring, each cell was assigned a visual score. Two parameters, Damage Index (DI) and Damage Frequency (DF), were calculated by summing up the visual score of 100 cells of each individual and by finding the percentage of cells showing migration, respectively. Another comet parameter, i.e., the % DNA in tail was also considered and measured with the help of computerized image analysis software (TriTek CometScore™).
DNA extraction and genotyping
Genomic DNA was isolated from whole blood collected in EDTA by employing the standard proteinase K and phenol-chloroform extraction method described by Blin and Stafford. 6 Two polymorphic genes, GSTM1 and GSTT, were determined by genotyping using multiplex PCR described by Arand et al. 7 The 215-bp GSTM1 and the 480-bp GSTT1 fragments were co-amplified with 350-bp albumin (positive control) fragments in the same reaction. The genes were genotyped using 40–50 ng of genomic DNA. The amplification protocol consisted of initial denaturation at 95°C for 5 min, final denaturation of 1 min at 95°C, followed by annealing for 1 min at 56°C and an extension of 1 min at 72°C for 35 cycles and at last a final extension of 7 min at 72°C. The absence of either GSTM1 or GSTT1 fragments indicated the corresponding null genotype. PCR products were electrophoresed on 2% agarose gel.
Statistical analysis
Results are expressed as mean ± standard deviation (SD). The chi-squared test and t-test were used to compare the demographic data. The statistical analysis of differences in DI, DF, and % DNA in tail between exposed and unexposed groups were carried out using the t-test and Mann–Whitney U nonparametric test.
Results
The results are shown in Table 1. The analysis of comet assay values (mean ± SD) indicated a significant increase in DI and DF (p < 0.05; Mann–Whitney U-test) for the exposed group compared to the control group. Similarly, the % DNA in tail was significantly higher in exposed group compared to the control group (p < 0.05; t-test; Table 1). No significant differences in DI, DF, and % DNA in tail were detected between the smokers and the nonsmokers (data not shown). Apart from this, the other confounding factors like age, tobacco chewing, and exposure time showed a significant difference in the DF values only (p < 0.05; Mann–Whitney U-test) (data not shown).
Significant difference (Mann–Whitney U-test). For Z-value: at 0.05% > 1.95, significant; 0.05% < 1.95, nonsignificant.
Highly significant (p < 0.05).
No result for one sample.
SD, Standard deviation; N, number of individuals; DI, damage index; DF, damage frequency.
The effects of the GSTM1 and GSTT1null genotypes were examined for association with DNA damage risk. The DI and DF frequencies were greater in exposed workers having the GSTT1 null genotype (189.33 ± 49.22 and 93.5 ± 5.21, respectively) compared with the non-null genotype (143.39 ±61.60 and 85.70 ± 10.88, respectively). The DI showed a statistically significant difference (p < 0.05; 2.41; Mann–Whitney U-test). The GSTT1 null genotype showed more % DNA in tail (14.43 ± 4.05) compared to the GSTT1 non-null genotype (9.82 ± 3.17) with a statistically significant difference (p < 0.05, t = 1.90).
The DI and DF values were lower in subjects having the GSTM1 null genotype (145.29 ± 42.46 and 86.64 ± 13.21, respectively) compared with the non-null genotype (153.36 ±70.79 and 87.04 ± 9.03, respectively). The DF values showed a statistically significant difference (p < 0.05; Mann–Whitney U-test). Similarly, % DNA in tail of the GSTM1 null subjects (10.28 ± 2.88) showed a decrease compared to the GSTM1 non-null genotype (10.67 ± 4.09) and the difference was statistically nonsignificant (p > 0.05).
A complete deletion of both GSTT1 and GSTM1 genotypes showed a high % DNA in tail (14.09 ± 2.40) compared with non-null types of these genotypes in the exposed group (9.92 ± 3.55) and the difference was significant (p < 0.05). Similarly, there were increases in DI and DF values among both the complete null genotypes of exposed subjects (186.5 ± 28.99 and 95.00 ± 7.07, respectively) compared to both the non-null genotypes (146.24 ± 71.54 and 85.95 ± 9.29, respectively) but the differences were nonsignificant (p > 0.05).
Discussion
During the last decade, the comet assay has become a popular technique for the determination of genotoxicity. DNA damage is influenced by various exogenous and endogenous factors, and the individual difference in the susceptibility to damage depends on various detoxifying genes. The results of the present study demonstrate that there is an increase in DNA damage in pesticide-exposed workers as compared to the unexposed workers (Table 1; p < 0.05). Similar findings have been reported in many earlier studies. 8
The confounding factors, including age, duration of exposure, and tobacco chewing, were found to influence the DNA damage because all of the values of Z (Mann–Whitney U-test) indicated significant differences in the DF parameter. Similar findings were reported earlier by Bolognesi et al. 9 On the other hand, smokers and nonsmokers did not show such a difference as also reported by Ulka et al. 10
Recent studies have explored the influence of single genotypes and the interaction of genotypes on genotoxic exposure to biomarker levels. 11 The determination of polymorphisms has become an increasingly important aspect that may increase the sensitivity and the specificity of assays. 12 GST is an isoenzyme involved in the conjugation of reactive chemical intermediates and plays an important role in detoxification. GSTM1 and GSTT1 polymorphisms owing to gene deletions result in null alleles, and individuals homozygous for deletions lack enzyme activity, which increases the risk of DNA damage and may lead to cancer development. 13
The present study and an earlier study by Norppa 14 showed a significant increase in DNA damage (% DNA in tail) in GSTT1 null as compared to non-null subjects. GSTM1 null subjects showed a nonsignificant decrease in DNA damage. This could be due to the fact that GST isoenzymes exhibit overlapping substrate specificities and the deficiencies of these GST isoenzymes may be compensated by other isoforms and utilization of alternative metabolic pathways. 15 A similar finding was reported by da Silva et al. 8 and Bolognesi, 9 where no significant increase in DNA damage in relation to GSTM1 was observed. Only 2 subjects (5%) were found to have complete deletions of both the GSTM1 and GSTT1 genes and showed a significantly elevated level of % DNA in tail when compared with the subject who did not show deletion of both these genes.
Conclusion
The analysis of comet parameters has indicated a significant increase in DNA damage in the pesticide-exposed subjects compared to control subjects. The DI and % DNA in tail were not found to be influenced by any of the confounding factors, whereas the DF was seen to be influenced by the confounding factors except smoking. Only the GSTT1 gene deletion was found to be associated with the susceptibility to DNA damage in the exposed group.
Footnotes
Acknowledgments
The authors express their gratitude to all the individuals who volunteered to participate in this study and gratefully acknowledge financial support from Punjab State Council for Science & Technology, MGSIPA Complex, Near Sacred Heart School, Sector 26, Chandigarh.