Abstract
Background: Pesticides play an important role in controlling the pests on agricultural crops and thereby to increase the yield of agricultural produce. Farmers occupationally exposed to pesticides during spraying activities are more prone to genotoxicity than unexposed. Aim: To assess the genotoxicity in farmers, engaged in spraying complex mixture of pesticides in the cultivation of cotton crops. Material and methods: A total number of 152 male subjects were selected randomly from Guntur district of Andhra Pradesh (AP), South India. The demographic particulars viz., personal habits, duration of exposure to pesticides, types of pesticides used were collected from the study subjects using an interview schedule. Among them 76 subjects were farmers and the remaining individuals served as unexposed or controls. Blood samples from these subjects were collected for assessing the genetic damage by chromosomal aberrations (CAs) test and micronucleus test (MNT). Results: The results of the study indicated that CA was significantly higher with 2.8% in farmers who were exposed to pesticides when compared to unexposed (0.72%). However, there was a minor difference in MN with 0.13% and 0.12% between exposed and unexposed which was not statistically significant (p < 0.05). Conclusion: A correlation between CA frequency and exposure to benzene hexachloride (BHC) pesticide residue was observed.
Introduction
Agriculture is one of the major occupations of rural India. Pesticides play an important role in controlling agricultural pests. 1 At present, there are more than 1000 chemicals classified as pesticides and some of them have been found to be potential genotoxic agents. The most frequently employed pesticides are those containing organophosphates which are known to induce acute toxic effects on health in different experimental systems.2–4 Though there are many reports available on the toxicity of these chemicals, information regarding their long-term effects on genotoxicity of the farmers cultivating cotton crops are scanty in India.5–11
Exposure to pesticides has been the subject of concern as several pesticides are known to exhibit mutagenic effects both in vitro and in vivo test systems in view of their possible role in the induction of short/long-term effects, such as cancer and congenital malformations.5,8,12 Although million cases of pesticide poisonings are documented every year around the World, there are only limited data available on cytogenetic effects in the individuals occupationally exposed to pesticides. 12 Genotoxicity in individuals exposed to pesticides showed that a number of biomarker studies using cytogenetic end points, viz., chromosomal aberrations (CAs), micronuclei (MN) frequency, Comet assay, and sister chromatid exchanges (SCE) can give more insight into the damage of individuals exposed to pesticides.6,13–18
The objective of the present study was to biomonitor the genotoxic effects in agricultural farmers cultivating cotton crops using CA test, and MNT in peripheral blood lymphocytes due to exposure to complex mixture of pesticides. In Guntur district of Andhra Pradesh in South India, the use of technical grade pesticides for agricultural farming was maximum (130–515 metric tons) when compared with other districts.
Methods
Study population
A stratified proportionate random sampling procedure was adopted for the selection of exposed and unexposed subjects to assess the genotoxic effects associated with pesticide exposure.
A total of 152 subjects participated in this study (comprising 76 exposed—agricultural farmers spraying complex mixture of pesticides for a minimum period of 10 years and 76 unexposed human volunteers from the same district but were not occupationally exposed to pesticides at any point of time and were engaged in other professional activities such as hotel vending, electrical work, tailoring, and carpentry work). The subjects were matched for age, gender, and socio-economic status with the exposed group, and so on.
A pretested questionnaire was given to the subjects, which consisted of the information on demographic particulars and their personal habits, types of pesticides applied to cotton crops (exposed), and duration of exposure to pesticides. Blood samples from 152 subjects were collected from both exposed and unexposed subjects. Ethical clearance was obtained from the institutional review board for the study protocol. Further, an informed consent was also obtained from subjects selected for study before collecting the samples.
Collection of samples
A 5-ml aliquot of venous blood was collected in heparin vials from 152 individuals and were transported to the study area in gel packs and processed within 24 h. Samples were divided into three aliquots: one each for chromosomal aberration analysis, lymphocyte micronucleus test, and pesticide residue analysis.
Cytogenetic analysis
The lymphocyte cultures were processed in duplicates separately for assessing chromosomal aberrations and for micronucleus analysis using the modified method of Moorhead et al. 19 and Fenech. 20 The serum was separated from third aliquot for assessing the pesticide residues.
Chromosomal aberrations
The cultures were arrested at metaphase by adding 50 μl of colchicine (40 μg/ml) at the 70th hour of culture initiation. The cells were harvested by centrifugation, resuspended in a prewarmed hypotonic solution (0.56% KCl) for 20 min at 37°C and fixed in Carnoys fixative (methanol: acetic acid 3:1 v/v). The slides were prepared by flame drying and stained with 4% Giemsa. They were analyzed at ×100 magnification using a light microscope. One hundred metaphase plates were screened for each sample.
Micronucleus test
Bi-nucleate cells were obtained by adding 5 μg/ml cytochalasin B (Sigma, USA) to the cultures at 44th hour and were subsequently harvested at 72nd hour. 21 The cells were obtained after gentle centrifugation at 800 rpm for 10 min, fixed in Carnoys fixative (methanol: acetic acid 3:1 v/v). The slides were then prepared, stained with 4% Giemsa and analyzed at ×40 magnification using a light microscope. One thousand bi-nucleated cells were scored for each sample.
Analysis of pesticide residues
One milliliter of serum sample from each subject was processed for the analysis of persistent organochlorine pesticide residues using solid phase extraction (SPE) cartridges. The serum samples were loaded into the SPE cartridges after preconditioning with methanol and then eluted the residues as per the standard procedures. To determine the quality of the methodology, a recovery study was performed on 10 spiked replicates of blank human blood sera, which presented contamination levels below the detection limits. The spike studies were conducted for standard compounds of α, β, γ, and δ benzene hexachloride (BHC), dichloro diphenyl trichloroethane (DDT), dichloro diphenyl dichloroethane (DDD), dichloro diphenyl trichloro ethylene (DDE), α and β endosulfan and endosulfan sulphate at the level of 100, 50, 25, 10 ng/ml. After extraction from the serum 1 μl was injected into gas chromatography (GC, Varian CP-3800) apparatus. It revealed the mean recovery values ranged from 90.5% to 93.8%. To confirm the determination of compounds that corresponded to organochlorine pesticide residue peaks, a gas chromatograph–mass spectrometer (GC-MS, Perkin Elmer and Turbomass with auto system XL-GC) was used. The peaks eluted were confirmed by comparing the results of mass spectra of substances from blood sera to those of standard compounds selecting specific ions obtained from an ion trap detector. The linearity curves were drawn for each compound. The minimum detection limit for the organochlorine pesticides studied was 1.0 ng/ml.
GC-operating conditions
Detector: electron capture detector; column: 5% phenyl 95% methyl polysiloxone column was used with a diameter of 0.53 mm, thickness of 0.50 μm and with 30 m length with split less system. Iolar-1 nitrogen was used as a carrier gas with a flow rate of 1.5 ml/min.
Temp: (°C): 100 (2 min); 190 at 5°C per min (5 min); 25 min 250° (4 min) 35 min: injector port: 270°; detector; 300°.
Statistical analysis
Descriptive statistics were used for all the parameters studied. Pearson correlation and Spearman’s non-parametric correlation were calculated between the duration of spraying activity, total number of CAs/cell, mean MN/cell, smoking history, tobacco chewing, alcohol consumption, and pesticide residues.
Results
Demographic particulars and personal habits
The data analysis showed that about 77% of the farmers were engaged in agriculture for the cultivation of cotton crops and about 23% were agricultural laborers. The mean age of all the subjects was similar in both the groups, that is, exposed and unexposed with 37.8 ± 9.51 years and 37.3 ± 12.2 years, respectively. The prevalence of smoking in the pesticide-exposed group was 35% and 34% in unexposed individuals. Similarly, non-alcohol consumers in unexposed and exposed groups were 45% and 47%, respectively. These differences were not statistically significant (p > 0.05) indicating that both the groups were homogeneous (Table 1 ).
Personal habits of the subjects included in the study
The farmers (80%) used a mixture of organophosphorus and organochlorine pesticides. The most widely used pesticides were acephate (O,S-dimethyl acetylphosphoramidothioate, C4H10NO3PS), chlorpyriphos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl, monocrotophos (36% E.C.) (dimethyl (E)-1-methyl-2-(methylcarbamoyl) vinyl phosphate, C7H14NO5P) (supplied by|| M/s. Bayer India Pvt Ltd) and synthetic pyrethroids, such as cypermethrin (RS) -α-cyano-3-phenoxybenzyl (1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl) -2,2-dimethyl cyclopropanecarboxylate (C22H19Cl2NO3), and an organochlorine viz., endosulfan (supplied by M/s. Rallis India Pvt Ltd) for their pest control operations. However, 20% of the farmers used combination of commercially available pesticides. Further, it was observed that the exposed subjects were occupationally involved in spraying complex mixture of pesticides for 16.3 ± 8.41 years with an average of 5.5 ± 1.74 hour’s exposure per day for a period of about 4 months in a year.
Genotoxic effects on the subjects—chromosomal aberrations
In the present study, out of the 7600 metaphase plates scored, in each group, about 213 cells (2.8%) exhibited CAs in the exposed group (Figure 1a and b ) and 55 (0.72%) in the unexposed. This showed a significant difference (p < 0.05) between both the groups (Table 2 ).
Chromosomal aberrations indicated with arrows in pesticide-exposed individuals (a) Plate showing break and (b) Plate showing gap.
Frequency of chromosomal aberrations in the pesticide-exposed and unexposed individuals
CA: chromosomal aberrations.
a p < 0.05.
Micronuclei analysis
In the present investigation, of the 76,000 bi-nucleated cells analyzed from the exposed and unexposed groups (each) 102 cells (0.13%) and 89 cells (0.12%) were found to be positive for micronuclei. However, this was not significant (Table 3 ).
Frequency of micronuclei in the pesticide-exposed and unexposed individuals
Results of pesticide residue analysis
Of the 10 pesticide residues analyzed in the blood samples from both exposed and unexposed groups, there was a correlation between the parameters (CA test and MNT). However, this was not statistically significant with respect to DDT and its isomers including α and β endosulfan and endosulfan sulphate. It was observed that there exists significant correlation between CAs/cell and BHC group of compounds when analyzed by Spearman’s non-parametric test (r = 0.264; p < 0.05; Table 4 ).
Range of BHC (pesticide residue) group of compounds in pesticide-exposed and unexposed individuals
BHC: benzene hexachloride.
Discussion
Review of literature indicated that some of the pesticides are clastogenic agents causing DNA damage resulting in chromosomal breaks. These effects seem to be cumulative for chronic/sub-chronic exposure to low levels of complex mixture of pesticides. Increased CAs has also been associated with increased risk of cancer and further, the cytogenetic damage induced by pesticides appears to depend on the degree and type of exposure. 6 A large number of studies were carried out in different populations in the past. However, studies on the genotoxic effects of pesticides to agricultural farmers cultivating cotton crops have not been studied so far. Therefore, the present study was carried out in Guntur district of AP, wherein the cultivation of cotton crop is more and the use of pesticides is very high when compared to the other districts of South India.
For biomonitoring purpose, the selection of genotoxic parameters should be simple, reliable, and cost effective. In order to assess the suitable indicators of genotoxicity in the agricultural farmers, occupationally exposed to complex mixture of pesticides (for an average period of 16.3 years with a minimum of 5 h exposure per day for a period of 4–5 months in a year during the cropping season), CA and MN parameters were chosen. The exposed individuals showed significant increase in CA when compared to unexposed subjects. Previous reports from all over the world have shown that commonly used pesticides have similar undesirable genotoxic properties. 21
In contrast to the evaluation of CA, the scoring of MN in lymphocytes is simple rapid, cost effective test. Therefore, it is considered as an important biomarker which allows the evaluation of both clastogenic and aneuploidogenic effects in a wide range of cells, since they are detected in interphase. The formation of MN may be due to the condensation of acentric chromosomal fragments arising from chromosomal breaks or by whole chromosomal lagging during the cell division (Figure 2). However, in the present study, there was no significant variation observed between the exposed and unexposed groups. This shows that the endpoints may vary based on the mechanism of induction of genotoxicity by different chemicals which is in conformity with earlier observations made by other workers. 12
In the present study the influence of personal habits such as smoking, which is one of the major confounding factors on the genotoxic effects in agricultural farmers exposed occupationally to a complex mixture of pesticides, was not significant (p > 0.05) between exposed and unexposed. Hence, the observations made in the present study reveal that the chronic/sub-chronic occupational exposure was the main contributing factor in the induction of CA. A number of genotoxic studies have been performed on individuals exposed to a variety of pesticides during spraying.5,15,22,23 It has also been shown that pesticide sprayers using proper safety measures had significantly reduced genetic damage. 24 Genotoxicity observed in the agricultural farmers of the present study may be due to lack of proper protective measures while spraying or storing pesticides. Since the agricultural farmers are frequently exposed to mixture of pesticides, it is difficult to attribute the genotoxic damage to any particular chemical class or compound.
Though farmers were using a mixture of organophosphate compounds, but the persistent and toxic organochlorine pesticide residues viz., BHC groups of compounds (α, β, δ, and γ-BHC) were found along with DDT and its isomers in the present study. A significant association was observed with respect to BHC groups of compounds in the exposed subjects with increased CA and not with MN. However, previous studies reported similar effect of δ-BHC as MN frequencies in cell lines. 25
Micronucleus indicated with arrow in pesticide-exposed individuals.
The genotoxicity revealed by exposure to pesticides may be taken as an early warning signal for future development of diseases such as cancer and congenital malformations. Further, it is suggested that the exposed workers are made aware about the potential harmful effects of pesticides and the concerned authorities should ensure that protective measures are used by farmers while working in agricultural fields.
Footnotes
Acknowledgements
The helpful cooperation provided by State Department of Agriculture, Guntur and Dr KRS Sambasivarao, Professor and Head, Department of Biotechnology, Nagarjuna University, Guntur District, AP, India, are gratefully acknowledged.
Funding
The author Padmaja R Jonnalagadda thanks the Department of Science and Technology, (DST – Grant No SR/WOS- A/LS-683/2003.), New Delhi, India for providing the financial support for the study.