To link Geno toxicity with various human blood parameters, blood samples were analyzed for total blood count. The test results were analyzed in accordance with Indian recommendations and normal ranges for all parameters. Table 1 shows data on blood sugar, white blood cells, hemoglobin, and blood urea. With increasing exposure time, Blood sugar, WBC, Hemoglobin, and blood urea levels were all greater in the exposed group than in the control group. Changes in the biochemical profile of blood sugar, white blood cell count, hemoglobin, and blood urea level as a result of environmental exposure are shown in Figure 1. The median result and a p-value of blood sugar, white blood cell count, hemoglobin, and blood urea are 0.000 it is statistically significant.
DNA damage analysis was done for selected groups that were monitored in the comet assays for the level of DNA damage. There was no obvious DNA damage in controls. Pesticide exposed groups Cells were graded into four categories depending on DNA damage and DNA Migration levels. Intact Cells (IC) or control (A), B Slightly damaged cells (SDC), C damaged cells (DC), and D Highly Damaged cells (HDC). This is shown in Fig: 2 Based above factors, DNA Damages were analyzed. One of the causes is Geno-toxic, and we discovered considerable DNA damage in pesticide-exposed people as a function of exposure time in our study. This demonstrates that the length of exposure has an impact on DNA damage The tail length of DNA migration was chosen from the photos for the estimation of Mean Head and Tail DNA damage which is shown in Fig: 3
The research results show clearly who had been exposed to pesticides for over 20 years. Their karyotyping results Case no 1-7 and 10,11,12,14 their chromosomal abnormalities were not found it represents normal karyotype. In cases 8, 9, and 13 were shown 14pstk+, 15pstk+ Satellite and 1qh+ Heterochromatin region enlargements were discovered to be the exposed groups and there were no obvious chromosomal abnormalities in controls. It was observed that those with longer exposures had higher aberrations. Figure 4 shows the meta-phase chromosomal abnormalities.
Gene toxicity analysis was done by a group of pesticides and the PBR322 DNA was docked. Three screened compounds were subjected to docking of pesticide and DNA interaction analysis. Initially, based on the scoring function, all the selected compounds were screened. The results of glide docking of the molecules with the DNA (5'-D (*AP*CP*CP*GP*GP*CP*GP*CP*CP*AP*CP*A) -3') for A chain and DNA (5'-D (*TP*GP*TP*GP*GP*CP*GP*CP*CP*GP*GP*T) -3') for the B chain length 12 are presented in Fig. 1. A Glide docking demonstrates that the best pose of compound 1(Atrazine) has the better glide score in comparison to the other 2 compounds. The glide scores in their best poses of compound 1 is -5.936 kcal/Mol. The best docking pose of the compound atrazine showed that the model and glide energies are a -28.690 kcal / Mol respectively and Fig.5. This demonstrates that the compound atrazine recognizes both strands of the DNA and binds within the grooves of the helix. Hydrogen bonding indicates that the compound atrazine involves the interaction in DGA: 7, DCA: 8, DGB: 19 bases of the DNA. Molecular docking results showed that this compound recognizes both the strands of the DNA (5'-D(*AP*CP*CP*GP*GP*CP*GP*CP*CP*AP*CP*A) -3') for A chain and DNA (5'-D (*TP*GP*TP*GP*GP*CP*GP*CP*CP*GP*GP*T) -3') for the B chain within the minor groove. Docking results reveal that compound 1 has the best binding capability with the DNA duplex. The results are concluded that the binding of the drug molecules to DNA is sequence-dependent and the specific sequence of the DNA may be playing a key role in the binding process. These results may enhance future prospects for future drug development and toxicity assessment. The glide score of Atrazine was -5.936 and Glide Energy (-28.690) Atrazine compound has a good glide score and also potentially interacted with PBR322 DNA. That formed 2 hydrogen bonds interacting with DGA: 7, DCA: 8, DGB: 19 with the distance of 1.68, 2.15, and 2.98. 3IXN glide score of Atrazine was -2.858 and glide energy (-20.976) also formed 2 hydrogen bonds (Pi-Pi Stacking) interacting with DAB:6, DCB:7 in the distance of 2.40 and 2.48. Atrazine has a top racking and good glide score. DICHLORVAS (PubChem ID 3039) Glide score of dichlorvos was -3.749 and Glide Energy (-20.764) dichlorvos compound has good glide score and also potentially interacted with PBR322 (Fig.6) That formed 2 hydrogen bonds interacting with DGA:7 and, DGB: 19 with the distance of 2.43 and 2.64. ENDOSULPHAN (PubChem ID 3224) Glide score of Endosulphan was -4.218 and Glide Energy (-28.011) endosulfan compound has good glide score and also potentially interacted with PBR322 (Fig.7) that formed 1 hydrogen bonds interacting with DGA: 7 with the distance of 1.94.
DNA Cleavage: The cleavage activity was demonstrated by gel-electrophoresis experiments using super-coiled (SC) plasmid pBR322 DNA in a medium TAE buffer. These experiments were monitored by the addition of varying concentrations of the pesticides (10–50ng). When DNA was incubated with increasing concentrations of the pesticides, SC DNA was degraded to nicked circular form. The cleavage activities of pesticides 1 were depicted in Figure 8. The activity of 1 starts at a concentration as low as 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, and 50ng/ml a complete conversion of SC plasmid DNA into the NC form was observed, and the DNA completely smeared (lanes 1, 2, and 3). In contrast, only 63% cleavage was achieved and (lanes 4, 5, and 6) 37% cleavage was observed. To ensure that the pesticide was solely responsible for the cleavage, several control experiments were performed under identical conditions. When circular plasmid DNA is subjected to electrophoresis, relatively fast migration will be observed in the intact supercoiled form (Form-I). If one strand is scissored (nicked), the supercoiled will relax and produce a slow-moving open circular form (Form- II). If all strands are cleaved, a linear form (Form-III) is produced that migrates between Forms I and II. The cleavage effect upon irradiation of the plasmid pBR322 DNA in the presence of different concentrations of pesticide has been tested. Rest of two compounds, DNA cleavage is shown in (Fig.9) Form-II increases and Form-I decreases gradually as the concentration of pesticides Increases. The findings point to single-strand cleavage of supercoiled Form-I to nicked Form-II in a concentration-dependent manner. Under comparable experimental conditions, pesticide 1 exhibits more effective DNA cleavage activity than the other 2 pesticide compounds.The different cleaving efficiency may be ascribed to the different binding affinities of Pesticide and PBR322 DNA.
Cell Viability Assay: Cell viability of the Atrazine, Endosulfan and Dichlorvos treated PBLC (Peripheral Blood Lymphocytes Cells) sample with 10 ng, 20 ng, 30 ng, 40ng and 50 mg were observed in 24 hour, 48 hour and 72 hours. The result shows a gradual decrease in the cell viability as the time and concentration of the pesticide increase. The cell viability is less on the cell treated with 50 mg concentrations of the pesticide after 72 hours of treatment. In case of sample treated with 30 ng concentrations of pesticides, 75 percentage of cells were viable after 72 hours of incubation. When it’s come in the case of 10 mg concentrations of pesticide treatment, 84 percent of the cell are alive. In case of 40ng it gradually decreases of viability after 72 hours of treatment, it showed (Fig 10). This trend shows that cell viability decreases with increase in concentration of pesticide and time of exposure.
General, lifestyle and Environmental factors associated breast cancer risk
The findings reveal how the lifestyle patterns of women affected among different age groups due to the exposure of pesticides that increases their chances of getting breast cancer in women. The results showed that the age group 45-55 had the highest risk, followed by the age group 55-65, When comparing the risk variables for married and unmarried women, married women had a high risk towards causing BC. By classifying the risk of BC among educated and uneducated women, exposed to pesticides in farmlands. Women who are mostly uneducated and are not aware of the harmful effects of pesticides. Because of their ignorance on pesticide exposure while using pesticides on agricultural lands, uneducated women were more likely to expose to pesticides than educated women. Table 2 shows data on general factors associated with breast cancer risk. The study on the Pesticide Exposure is classified into eight different ways of pesticide exposure among women working farmlands and as other skilled works in farmlands. The pesticide exposure levels are of , which are defined as follows - Working in '8' hours of pesticide sprayed farms, - Living nearby pesticide sprayed farm areas, -women handling harmful pesticides , -Pesticides stored in home, - Consuming more amounts of pesticides sprayed food , - Washing of pesticide sprayed clothes with bare hands, - Farmers working with bare hands in pesticide sprayed farms and - Without pesticide exposures. The results obtained based on the pesticide exposure shows that being higher when compared to other pesticide exposure levels, people living near the pesticide sprayed farm areas had the highest risk of having BC. Table 3 shows data of pesticide exposure and breast cancer risk. Our data show that occupational status of women working in farmlands may be linked to an elevated risk of breast cancer.