|Year : 2020 | Volume
| Issue : 3 | Page : 123-130
Biomonitoring of biomarkers among pesticide sprayers and nonsprayers across cropping seasons in Punjab, India
JS Thakur1, Soundappan Kathirvel1, Samir Malhotra2, Nusrat Shafiq2, Jatinder Paul Singh Gill3, Kulbhushan Tikoo4, Shyam Biswal5
1 Department of Community Medicine and School of Public Health, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Pharmacology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3 School of Public Health and Zoonosis, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India
4 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Chandigarh, India
5 Department of Environmental Health Sciences, Division of Toxicology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA
|Date of Submission||21-Jul-2020|
|Date of Decision||04-Aug-2020|
|Date of Acceptance||24-Aug-2020|
|Date of Web Publication||30-Sep-2020|
Prof. J S Thakur
Department of Community Medicine and School of Public Health, Post Graduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
Introduction: Pesticide exposure causes acute and chronic adverse health effects on humans affecting the Kelch ECH-associated protein (Keap1)- nuclear factor erythroid 2-related factor 2 (Nrf2) and other oxidative pathways. We assessed the Keap1-Nrf2 pathway and other oxidative stress biomarker levels among a cohort of agricultural pesticide sprayers (SP) and nonspraying (NSP) farmers of a rural community during the cotton and wheat cropping seasons in India.
Methodology: We randomly selected eight villages from the four blocks of Bathinda district, Punjab. Using a cohort study design, we collected the socio-demographic characteristics and biological samples (blood and urine) from 68 SP and 71 NSP at baseline, during the flowering seasons of cotton and wheat crops at scheduled time points. The United States Environment Protection Agency methods and standards were used to detect pesticide residues. Standard and validated enzyme-linked immunosorbent assay-based kits and colorimetric method-based kits were used to detect the biomarkers of Keap1-Nrf2 and oxidative stress pathways.
Results: In Keap1-Nrf2 pathway, cotton season glutathione peroxidase (P = 0.004) and baseline heme oxygenase-1 (P < 0.001) levels were significantly different between SP and NSP. However, glutathione reductase and glutathione s-transferase levels were not significantly different. Among oxidative stress and inflammatory biomarkers, a significant difference (P < 0.05) was observed between the groups during the cotton season in malondialdehyde, isoprostane, protein carbonyl, C-reactive protein, and interleukin-6.
Conclusion: The Keap1-Nrf2 and other oxidative stress biomarker levels between the group were not consistently different all times across the seasons. The biomarker levels of SP and NSP need to be compared with the nonagricultural population.
Keywords: Biomarkers, biomonitoring, farmers, occupational-exposure, pesticide residues
|How to cite this article:|
Thakur J S, Kathirvel S, Malhotra S, Shafiq N, Singh Gill JP, Tikoo K, Biswal S. Biomonitoring of biomarkers among pesticide sprayers and nonsprayers across cropping seasons in Punjab, India. Int J Non-Commun Dis 2020;5:123-30
|How to cite this URL:|
Thakur J S, Kathirvel S, Malhotra S, Shafiq N, Singh Gill JP, Tikoo K, Biswal S. Biomonitoring of biomarkers among pesticide sprayers and nonsprayers across cropping seasons in Punjab, India. Int J Non-Commun Dis [serial online] 2020 [cited 2021 Mar 7];5:123-30. Available from: https://www.ijncd.org/text.asp?2020/5/3/123/296795
| Introduction|| |
Pesticides are the substances or a mixture of elements intended for preventing, destroying, repelling, or mitigating any pests to improve the yield of the crops. Majority of the pesticides are chemicals. Of these, organophosphates, carbamates, and pyrethroids are the main group of pesticides currently in use. Along with controlling pests on crops, chemical pesticides also represent a potential source of occupational and nonoccupational risk to human and the ecological system., Cells have evolved adaptive mechanisms to endure both acute and chronic exposure to a wide array of stressors, especially through Kelch ECH-associated protein (Keap1)-Nrf2 (nuclear factor erythroid 2-related factor2) pathway and other oxidative pathways. The nuclear factor erythroid 2-related factor2 (Nrf2)-antioxidant reducing element (ARE) is one of the major transcription factors that up-regulate cellular defences and play an essential role in diminishing the toxic effects of exogenous (environmental) xenobiotics and endogenous stressors through enzymes.,,, The Nrf2-ARE signaling plays a pivotal role in the transcriptional activation of cytoprotective genes, which facilitates the detoxification of carcinogens and polyaromatic hydrocarbons. It is regarded as a promising strategy in cancer prevention. If these pathways are unchecked, exposure to the pesticide can cause respiratory diseases, neurodegeneration, renal and cardiac diseases, cancer, and premature aging.
Pesticides are exogenous xenobiotics which alter the levels of various biomarkers reflecting the interaction between the biological system and environmental agents. Periodic and repetitive detection of biomarkers called biomonitoring is a useful tool for assessing exposure to pesticides. It is being used in the risk assessment of the ecosystem to identify the effects of pesticide exposure at the subcellular level., Such biomonitoring is necessary among agricultural pesticide sprayers who are at high risk of developing acute and chronic pesticide toxicity due to occupational exposure. The exposure also occurs through the multiple routes such as inhalation, ingestion, and dermal absorption, which varies with the type of pesticide used, mode of application, potential dose, and use of personal protective equipment (PPE)., Poor awareness on the toxic effects of pesticides, poor PPE use, excessive or repetitive (nonjudicious) use of pesticides, and unsafe method of pesticide storage were the identified reasons for high exposure among pesticide sprayers.
In developing countries, pesticide use is increasing. Limited evidence is available on the biomonitoring of pesticides in these countries, though there is quite adequate evidence available on acute poisoning. The same applies to India. The biomonitoring studies conducted in India primarily focused on mango orchards and other specific crops. These studies were limited with small sample size, cross-sectional study design, and single crop season assessment.,,,,, Further, assessment of a limited number of pesticide residues and biomarkers of oxidative stress pathway (acetyl/butyl cholinesterase, glutathione, malondialdehyde [MDA], and others) is another limitation of these studies. However, no study assessed comprehensively the oxidative stress biomarkers related to the Nrf2-ARE pathway and pesticide residues across crop seasons in a year. With this background, we conducted the current study to measure and compare the (a) serum and urinary levels of pesticide/residues, and (b) biomarkers of Nrf2-ARE pathway and other oxidative stress pathways among pesticide sprayers and nonspraying farmers in India across cropping seasons.
| Methodology|| |
We used a cohort study design, i.e., we recruited and followed up a group of pesticide sprayers and nonspraying farmers across cropping seasons in the selected study area.
The operational definition of sprayers and nonsprayers
Sprayers (SP) are farmers or nonfarmers involved in the agricultural application of pesticides in the past 12 months or more. Nonsprayers (NSP) are farmers with no or <1-year involvement in spraying pesticides.
Punjab is one of the prosperous states of India situated in the northern part of the country. The state recorded the highest pesticide use in the country, i.e., >17% of pesticide use is by Punjab though it carries just 1.5% of land area. Malwa region (Cotton Belt) accounts for nearly 75% of pesticide usage in the state, a known part for clustering of cancer cases. There are two agricultural cropping seasons, i.e., Cotton (called as “Kharif”) which is between May to October and Wheat (called as “Rabi”)-which is between December to March. Pesticides are applied once a month (“cycle”) for wheat crop and once weekly (“cycle”) for the cotton crop during the flowering season. Flowering season is two and three months after sowing in the wheat and cotton season, respectively. We selected eight villages randomly, i.e., two villages each from all four community administrative blocks of Bathinda district, Malwa Region of Punjab, India.
Study participants, sampling, and sample size
From the selected villages, we recruited pesticide sprayers and nonspraying farmers using convenience sampling after their informed consent and confirmation of their availability throughout the study period. Participants aged <18 years, who did not provide consent, and with any known cancer were excluded.
Sample collection and period
The study collected blood and urine samples from the participants during the flowering season of the crops, i.e., during maximum pesticide application. The samples were collected one month after the start of flowering season of wheat and cotton crops, i.e., 3rd month for the wheat season and 4th month for the cotton season, respectively [Figure 1]. Approximately 10 ml of venous blood was collected in heparinized and ethylenediaminetetraacetic acid vials. Similarly, 50 ml of early morning urine was collected. Samples were transported to the laboratory in an ice-cold box immediately. Blood samples were stored at −60°C, and urine samples were stored at −80°C without any pretreatment until analysis.
|Figure 1: Biological sample collection for assessing the Keap1-Nrf2 pathway and oxidative biomarkers in baseline, wheat, and cotton season among sprayers and nonsprayers|
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Estimation of pesticide residues
The United States Environment Protection Agency (USEPA) method 8081A in gas chromatography (GC) and USEPA method 8141A in GC-capillary column technique were used for the detection of organochlorine and organophosphorus pesticides, respectively. Protocols for determining dialkyl phosphates were standardized using GC-mass spectrometry (MS) equipped with an electron ionization system as described by Ueyama et al. GC-MS confirmed the detected compounds.
Estimation of biomarkers
In the Nrf2-ARE pathway, the study estimated the reduced glutathione (GSH), glutathione s-transferase (GST), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1). GSH estimation was done using the method mentioned by Ellman et al. The activity of GST was measured using 1-Chloro-2,4-dinitrobenzene (CDNB) as a substrate. The amount of CDNB conjugate formed was measured by recording the absorbance at 340 nm by using the enzyme-linked immunosorbent assay (ELISA) kit. Similarly, the GPx activity and HO-1 were determined by measuring the oxidation of nicotinamide adenine dinucleotide phosphate at 340 nm, and ELISA kit, respectively.
Other oxidative stress biomarkers like (a) nitrotyrosine, total antioxidant capacity (TAC) and acetylcholinesterase (AChE) in serum, (b) isoprostane and protein carbonyl in urine, and c) MDA in both serum and urine was estimated. Further, inflammatory biomarkers such as C-reactive protein (CRP) and interleukin-6 (IL-6) were estimated in serum. AChE inhibition and CRP levels were determined using the kits based on the colorimetric method. MDA level was determined using the method described by Ohkawa et al. Isoprostane, protein carbonyl, nitrotyrosine, TAC, and IL-6 were estimated using the ELISA kits.
Data analysis was performed using the Statistical Package for the Social Sciences ® version 16 (SPSS Inc., SPSS for Windows, Chicago, USA). Descriptive statistics such as mean (standard deviation) and proportions were calculated for socio-demographic characteristics and pesticide levels in blood and urine. The Chi-square test and independent t-test were used to compare the socio-demographic characteristics between SPs and NSPs. Similarly, independent sample t-test or Mann–Whitney U-test was used to compare the difference in pesticide residues and oxidative stress biomarker levels between SPs and NSPs across the cropping seasons depending on the distribution (normality) of data. P < 0.05 was considered statistically significant.
Ethical approval was obtained from the Institute Ethics Committee of Postgraduate Institute of Medical Education and Research, Chandigarh, India. A written, informed consent was obtained from all the study participants.
| Results|| |
Out of 139 participants recruited, 68 were SPs and 71 were NSPs. All the study participants were male farmers. The mean age (standard deviation) of the participant was 39.6 (11.3) years. Of the total participants, a total of 30 (21.6%) were illiterate, and 21 (15.1%) were unmarried. [Table 1] describes the socio-demographic characteristics of the study participants. Mean age (standard deviation) of SPs and NSPs was 37.9 (10.8) and 40.8 (11.6) years, respectively. More than 60% of the SPs and NSPs were doing agricultural activities such as ploughing, weeding, replanting, and watering for at least ten years. All participants were engaged in both cropping seasons of the year and spent 5-8 h/day on an average in farm works. There was no statistically significant difference observed in any of the socio-demographic characteristics between SPs and NSPs.
|Table 1: Socio-demographic characteristics of pesticide sprayers and non-sprayers of Punjab, India|
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Among SPs, 12%, 21%, and 67% of them were involved in personally mixing and applying pesticides for 2–5 years, 6–10 years, and more than ten years, respectively. Only 3% of the SPs use PPEs such as air respirator/gas mask, face mask, chemically resistant boots, gloves, and goggles. None of the SPs used any type of chemically resistant cloths while spraying. Endosulfan, chlorpyrifos, and monocrotophos were the most commonly used pesticides. The retail shop owners and the peer farmers were the primary sources of information regarding the prevailing pesticides use practice (results not tabulated).
Pesticide residues detected in blood and urine of the study population are given in [Table 2]. All these samples belonged to the cotton season. Wheat season sample results were discarded due to the operational issues and problems in standardizing the testing protocols. Of the organochlorine pesticides, dichloro, diphenyl, dichloro-ethylene (p, p 1-DDE) and endosulfan sulfate were detected in 14 (10.1%) and 6 (4.3%) of participants, respectively. β-hexachlorohexane (β-HCH) was detected from a NSP (0.01 ppm). In the organophosphorus group, parathion was detected from 8 (5.8%) of the study population. Ethion (0.014 ppm) was detected from a SP and monocrotophos was detected from two participants, one each from SP (0.027 ppm) and NSP (0.021 ppm). In urine, diethyl phosphate (DEP) and diethyl thiophosphate (DETP) were detected in 43 (30.9%) and 10 (7.2%), respectively. There was no statistically significant difference observed between SPs and NSPs in any of the detected pesticide residues.
|Table 2: Blood and urine levels of pesticide residues in cotton season among sprayers and non-sprayers of Punjab, India|
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The results for all oxidative stress biomarkers are available for baseline and cotton crop season. The total number of samples tested for different biomarkers was not same due to various operational issues, namely nontraceability of participants across the season, difficulty in transporting and storing the samples, and the problems with the protocol standardization (initially). No biomarkers results except GSH and MDA (serum and urine) for the wheat season are available due to the above-mentioned operational issues. Among the Nrf2-ARE pathway biomarkers, a statistically significant difference was observed in cotton season Gpx (P = 0.004) and baseline HO-1 (P< 0.001) activity between SPs and NSPs [Table 3].
|Table 3: Comparison of biomarkers of nuclear factor erythroid 2-related factor 2- antioxidant reducing element pathway between sprayers and non-sprayers across the cropping season in Punjab, India|
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Among other oxidative and inflammatory biomarkers [Table 4] and [Table 5], serum MDA (P = 0.017), isoprostane (P = 0.049), CRP (P = 0.012), and IL-6 (P< 0.001) levels were significantly different between the groups in the cotton season. A statistically significant difference was also observed in AChE (P = 0.043) and protein carbonyl (P = 0.034) levels at baseline and in urinary MDA level (P< 0.001) at the wheat season.
|Table 4: Comparison of levels of other oxidative stress biomarkers between sprayers and nonsprayers across cropping season in Punjab, India|
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|Table 5: Comparison of levels of neurotransmitters and inflammatory biomarkers between sprayers and non-sprayers across cropping season in Punjab, India|
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| Discussion|| |
The current study is the first comprehensive study (at the author's knowledge) assessed the biomarkers of pesticide exposure, including pesticide residues among humans from India. We assessed the Nrf2-ARE pathway and other oxidative stress biomarkers, including inflammatory biomarkers among 68 SPs and 71 NSPs. The study found a significant difference between the group at baseline for HO-1, AChE, and protein carbonyl levels, at the cotton season with Gpx, serum MDA, isoprostane, CRP, and IL-6 levels. In addition, the wheat season urine MDA level was also significantly different between the SPs and NSPs. Both organophosphorus (parathion, ethion, and monocrotophos) and organochlorine (p, p 1-DDE, endosulfan sulfate, and β-HCH) were detected in the biological samples of the study participants.
The current study confirms the use of endosulfan, chlorpyrifos, and monocrotophos, similar to other studies conducted in this region and other states of India.,, Among these, endosulfan and chlorpyrifos are moderately hazardous, and monocrotophos is a highly hazardous pesticide. Although organochlorine pesticide use is banned in many countries, India is still manufacturing and using DDT for indoor residual spraying under the National Vector Borne Disease Control Programme. Although DDT was banned for agricultural use, it cannot be ruled out due to the poor implementation of regulations and surveillance in India.,
The presence of DDT residue in the current study could be due to the exposure to persistent DDT in the environment and illegal use either stand-alone or as part of cocktail use. Studies confirm the possibility of above explanation since 28% of the farmers were not aware of instruction written on the container, 64.5% were not aware of the recommended dose of input and >50% did not know how to store it safely., In case of pesticide exposure, the levels of urinary dialkyl phosphate (DAP) like DEP and DETP level will be increased. Although we have found a higher level of DETP among SPs, the DEP level was high among NSPs.
Compared to various biomarker levels at non/low exposure condition, the levels of GSH, GPx, and GST were low, and HO-1 was high in this study. The decrease in GSH, GPx, and GST was more in SPs during the cotton or wheat season than baseline. These observations were correlating with the previous studies, which reported that these levels might increase or decrease.,,
Acute or chronic exposure to pesticide is usually measured and managed by estimating the cholinesterase levels. Organophosphate and carbamate pesticides inhibit the AChE activity., The inhibition of AChE observed in this study was not significantly different between SPs and NSPs at the cotton season. It could be due to near equal exposure among NSPs due to their involvement in farm works, especially immediately after pesticide application. Lipid peroxidation, an oxidative stress reaction, is indicated by an increased level of serum or urinary MDA. In our study, the cotton season serum MDA and wheat season urine MDA were significantly high in SPs compared to NSPs.,
The detected biomarker levels are not found consistently different between SPs and NSPs for all biomarkers. Similarly, some of the differences were found statistically significant at baseline. It could be due to variation in the duration of exposure, antioxidant levels (enzymatic and nonenzymatic), presence of any morbidity, use medication or tobacco/alcohol use, type of pesticides and its mixtures, use of PPE (and its type), and other factors among the study participants. The similar level of biomarkers in NSPs compared to SPs could be due to the exposure of NSP during and immediately after pesticide spraying and for a longer duration than SPs.
Periodic biomonitoring of biomarkers, provision of PPE, and enforcement of regulatory actions on violators need to be recommended for the risk reduction among the high-risk population for pesticide exposure. Low level of PPE use needs further investigation, especially exploring the user's perspective, including its availability and affordability. Activation of Nrf2-ARE pathway antioxidant enzymes for the prevention of oxidative damage at the cellular level and further prevention of acute and chronic diseases through various interventions are the potential area of research for the future. The present study did not evaluate the second-line antioxidant defence system, i.e., the nonenzymatic system (Vitamin A, C, E, and β-carotene). Both enzymatic and nonenzymatic systems are interlinked, and it is indeed to study both the systems because its complex tackles reactive oxygen species in a complex way.
Our study result needs cautious interpretation considering the following strengths and limitations. The present study assessed the oxidative stress and inflammatory biomarkers of pesticide exposure comprehensively, including the pesticide residue assessment. The assessment was at the different cross-section covering both the cropping seasons, i.e., baseline, cotton season, and wheat season using the same study participants. However, the varying sample size for different biomarkers and nonavailability of all biomarker results except glutathione (reduced) and MDA (serum and urine) for the wheat season were the important limitations of the current study. Both blood and urine were tested using the standardized techniques/methods, and the sample size was higher than other reported study from India. However, the study participants may not represent the study area completely since we used nonprobability sampling. Recall bias could be one of the limitations while assessing the exposure, i.e., SP or NSP. Inclusion of participants with diseases other than cancer and on medications which affect the biomarker levels and noncomparison of biomarker level with non-agricultural population are the other limitations.
| Conclusion|| |
The study detected various organochlorine and organophosphorus pesticide residues among SPs and NSPs. The Keap1-Nrf2 and other oxidative stress biomarkers, including the inflammatory biomarker levels were not consistently different between the groups all times across seasons. In the cotton season, the levels of GPx, MDA, isoprostane, protein carbonyl, C-reactive protein, and interleukin-6 were significantly different between the pesticide sprayers and nonsprayers. The biomarker levels of SP and NSP need to be compared with the nonagricultural population following standardized protocols.
The authors acknowledge the support provided by Dr. RS Dhaliwal, Scientist E, Non Communicable Diseases (NCD) Division, ICMR, New Delhi to this study.
Financial support and sponsorship
The authors are grateful to the Indian Council of Medical Research (ICMR), New Delhi, for their financial assistance to conduct this study.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]