Research Article

Assessing Haemato-Biochemical, and Genotoxic Effects of Pesticide Exposure on Rural Inhabitants in South Punjab, Pakistan: A Biomonitoring Study

Abdul Ghaffar1*, Ayesha Maqsood1, Riaz Hussain2, Ghulam Abbas3, Rabia Tahir4, Habiba Jamil1, Fozia Afzal5, Ahrar Khan6,7, Muhammad Ahmad Chishti1, Shahnaz Rashid3, Aliya Noreen8, Kashfa Akram1 and Maria Niaz1

¹Department of Zoology, The Islamia University of Bahawalpur, 63100, Pakistan; 2Department of Pathology, University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan; 3Centre of Excellence in Marine Biology University of Karachi, 75270, Pakistan; 4College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China; 5Department of Zoology, The Islamia University of Bahawalpur, Rahim Yar Khan Campus, 6400, Pakistan; 6Shandong Vocational Animal Science and Veterinary College, Weifang, 261061, China; 7Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan; 8Biology Centre CAS, Institute of Entomology, Ceske Budějovice, Czechia.

Received | April 02, 2024; Accepted | July 26, 2024; Published | September 17, 2024

*Correspondence | Abdul Ghaffar, Department of Zoology, The Islamia University of Bahawalpur, 63100, Pakistan; Email: dr.abdul.ghaffar@iub.edu.pk

Citation | Ghaffar, A., A. Maqsood, R. Hussain, G. Abbas, R. Tahir, H. Jamil, F. Afzal, A. Khan, M.A. Chishti and S. Rashid, A. Noreen, K. Akram and M. Niaz. 2024. Assessing haemato-biochemical, and genotoxic effects of pesticide exposure on rural inhabitants in south Punjab, Pakistan: A biomonitoring study. Sarhad Journal of Agriculture, 40(3): 1071-1080.

DOI | https://dx.doi.org/10.17582/journal.sja/2024/40.3.1071.1080

Keywords | Blood parameters, Pesticidal exposure, Genotoxicity, Biochemical abnormalities

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Pesticides serve specific purposes or target particular species, but they also produce harmful effects on non-targeted organisms, including animals and humans (Hernández et al., 2011). Pesticides are supposed to be less effective for human beings rather than targeted species but not as the case as considered because these are much toxic for human health even in low quantity (Tsatsakis et al., 2009; Zeliger, 2011). It also causes toxic effects by using free radical method that can also be detected with measurements of oxidative degeneration of lipids in body. During developmental period pesticides exposure cause major damage not only in development but also leave its effects in later life especially effecting the brain and hormonal system (London et al., 2012). Pesticides effects can be ensured by observing the chemical changes in life before its exposure in adverse conditions (Patil et al., 2003). Diseases in human beings as well as crops losses are mostly caused by harmful pests (Kazmi et al., 2023), which are being reduced by using pesticides, but its usage is on such large scale that pests are getting immunity against the pesticide day by day. To avoid this trouble, a large number of pesticides have been introduced into the market, which are classified as fungicide, insecticide, herbicides etc. (Speck-Planche et al., 2012). Uncontrollable way of using pesticides has caused a serious damage to our environment regarding human health and other existing all life on this planet (Agrawal et al., 2010), because it spreads toxicity in natural flora and fauna (Rashid et al., 2010). Low quantity of insecticides containing chlorinated hydrocarbons when added to animal feeds remains in their tissues. Chlorinated hydrocarbons (Lipophilic) are amassed in fat particles of the body because they are fat soluble (Mansouri et al., 2017; Reichelt-Brushett, 2023). Workers that work with pesticides have to face the chemical mixing, equipment loading and cleaning and getting rid of waste bottles. Workers regularly exposed to the pesticides have skin diseases rate higher than respiratory diseases (Jallow et al., 2017; Febriana et al., 2023). Second source of exposure is entering the field after treating it with pesticides because their particles remain in the field even after treatment for a short time (Widowati et al., 2022). The exposure is higher at the time of direct handling as compared to second entry (Damalas and Koutroubas).

Pesticides have produced large positive impact on man life by increasing the agricultural products and disease control but on the other hand, their overuse has also destroyed the man health greatly (Al-Saeed et al., 2023). They affect various organs and systems, such as the reproductive, renal, cardiovascular, immune, and respiratory systems, particularly in individuals with extensive pesticide exposure, leading to chronic diseases such as Parkinson’s, aging-related conditions, Alzheimer’s, and kidney diseases (Abdollahi et al., 2004; De Souza et al., 2011; Mostafalou and Abdollahi, 2012). Study for pesticides shows that steroid hormones like estrogens and androgens affects as reduced with several chemicals, environmental pollutants, drugs and pesticides like methoxychlor and DDT have anti-androgenic effect (Aziz et al., 2022).

Pesticides used on a large scale includes organochlorines (OC), organophosphates (OP), and carbamates (CB). The action of OB and CB inhibits the activity of the acetylcholinesterase enzyme (Kwong, 2002). The poisoning effect of CB is short-term compared to OP. Organochlorines, which are readily lipid-soluble, neurotoxic, chemically stable, and have endless half-lives in the environment, are known to cause continuous lethal effects by opening sodium gates in neurons, leading to decreased KB absorption and reduced Ca, K, Na-ATPase. Pesticides which cause 50% poisoning are organophosphates and also used as dangerous nerve gas (Soltaninejad et al., 2007; Karami-Mohajeri and Abdollahi, 2011; Jayaraj et al., 2016). The current study was therefore devised to detect hematological and biochemical alterations in rural inhabitants exposed to pesticides and to monitor genotoxic potential using single-cell gel electrophoresis in blood samples from rural pesticide-exposed workers.

Materials and Methods

Collection of samples

Survey was done for collection of blood samples of pesticides exposed workers from district Bahawalnagar, Punjab, Pakistan. The total 5ml blood was collected of which 2.5ml added in EDTA tubes for the study of DNA damage, hematology and biochemical analysis and 2.5ml in Eppendorf tubes for the study of serum. The total 100 samples were collected included 50 of pesticides exposed workers and 50 of shopkeepers working with pesticides. The workers of different age groups were selected ranging from 15 to 70 years. The samples were collected and handled with great care. Additionally, a survey was conducted on 100 pesticide-exposed and 100 unexposed individuals to gather information on various diseases, including diabetes, flu, fever, hepatitis, skin allergy, throat infection, uric acid, sneezing, nasal allergy, kidney problems, eye irritation, chest tightness, blood pressure, and cardiac problems.

Hematological analysis

For hematological analysis, blood samples were inspected to study different parameters, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin (Hb), platelets (PLT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), lymphocytes (LYM), hematocrit (HCT), monocytes and eosinophils.

Serum analysis

Different parameters of freshly collected blood have been analyzed with available kits in the market with the use of spectrophotometer. The blood centrifugation was done at first for 10-15 minutes at 1600 rpm for the separation of serum and plasma and collected the serum in Eppendorf tubes. The parameters selected for analysis are bilirubin, alanine aminotransferase (ALT), Urea, creatinine, lactate dehydrogenase (LDH), aspartate aminotransferase (AST) Alkaline Phosphatase (ALP), creatinine phosphokinase (CPK), sodium (Na), phosphorus (P), potassium (K), triglycerides, calcium (Ca) and cholesterol. Alkaline Comet assay was performed with protocols described by Singh et al. (1988) with small changes.

Statistical analysis

The data collected in this study underwent statistical analysis using SPSS 15 software. Results are presented as means for hematological and serum analyses, comparing exposed and unexposed workers through multiple comparisons.

Results and Discussion

Clinical abnormalities

The survey conducted in this study revealed a higher prevalence of diseases among pesticide-exposed workers compared to those who were not exposed. Table 1 illustrates the percentages of clinical abnormalities observed in both groups, highlighting significantly higher values in pesticide-exposed individuals across various conditions such as throat infection, nasal allergy, skin allergy, eye irritation, and others. Previous research, as noted by Srivirojana et al. (2005), has indicated a substantial prevalence of pesticide exposure among agricultural workers in Pakistan, particularly in crops like cotton and sugarcane. This exposure has led to a significant number of health impacts on the affected workers, motivating the current investigation and the implementation of safety measures (Aroonvilairat et al., 2015).

 

Table 1: Percentage of clinical damage within exposed and unexposed rural dwellers of pesticides.

Parameters	Exposed	Unexposed
Diabetes	09%	07%
Throat infection	46%	42%
Blood pressure	22%	20%
Cardiac problems	21%	17%
Cough	78%	70%
Kidney problems	11%	08%
Hepatitis	11%	09%
Skin allergy	38%	31%
Asthma	52%	48%
Fever	35%	30%

 

The study examined demographic and socioeconomic factors, including age, education level, marital status, work experience with pesticides, and training received. Additional factors such as monthly household income, social class, household size, and number of children were also considered. These parameters are detailed in the accompanying Table 2. The age range of pesticide users in this study varied from 18 to 59 years, with exposure durations ranging from 3 to 35 years, consistent with findings reported by Chuisseu et al. (2015). The study further confirmed that the observed clinical abnormalities in pesticide-exposed individuals were likely attributable to pesticide exposure compromising their immune systems (Henneberger et al., 2014).

Blood parameters

Results of blood parameters expressed in Table 4 as results revealed significant hematological changes in pesticide-exposed workers compared to unexposed individuals. Specifically, hemoglobin level, WBCs, RBCs, HCT, MCV, MCH, and MCHC were markedly decreased in pesticide-exposed workers with clinical abnormalities, such as blood pressure, skin allergy, cardiac problems, and diabetes, as detailed in Table 3. Conversely, lymphocyte and monocyte counts were significantly higher in pesticide-exposed workers compared to their unexposed counterparts. This finding aligns with similar research where decreased levels of total leukocytes and monocytes were also reported (Aroonvilairat et al., 2015). Another study investigating the health impacts of pesticide exposure during peak agricultural periods documented a significant decrease in monocytes and platelets, coupled with an increase in total leukocytes (Saraiva, 2009). Interestingly, contrasting results were observed in another study, which noted an increase in total leukocytes, platelets, and monocytes among pesticide-exposed individuals (Wafa et al., 2013). These findings collectively suggest that pesticides may induce changes in leukocytic activity and potentially

 

Table 2: Demographic appearances of pesticide exposed rural inhabitants in surroundings of Bahawalnagar district.

Characteristics	No. (%)
Age
≤ 25	26%
26- 36	24%
37-46	30%
≥ 47	20%
Highest level of education
None	82%
1-7th grade	15%
8th- 10th	3%
Marital status
Married	72%
Uumarried	28%
Number of years working in pesticides
≥1	15%
2-10	75%
≤11	10%
Received information or training about pesticides
Received	1%
Not received	99%
Monthly household income
≤5000	55%
6000-15000	35%
≥16000	10%
Social classes
Upper	1%
Middle	15%
Poor	84%
Number of households members
≤3	30%
4-6	60%
≥6	10%
Number of children in household
None	29%
≥1	71%

 

activate immune defense mechanisms (Cattelan et al., 2018). These observations emphasize the complex impact of pesticide exposure on blood parameters and suggest potential health consequences that require further investigation. Understanding these hematological alterations is crucial for developing effective strategies to mitigate the health risks associated with pesticide use in agricultural settings.

Biochemical parameters

In our study, pesticide-exposed workers showed high levels of phosphorus and potassium, but low levels of sodium and calcium. While Serum parameters such as glucose, urea, creatinine, bilirubin, ALT, and ALP in exposed individuals, indicative of greater prevalence of kidney problems, asthma, and skin allergies (Table 4). The group that was exposed to pesticides showed lower mean total cholesterol in relation to the group that does not use, a result consistent with a similar study involving exposed agricultural populations (García-García et al., 2016; Cattelan et al., 2018). However, other studies (Remor et al., 2009; Wafa et al., 2013; Adad et al., 2015) presented divergent results for this laboratory data. The effects of pesticides differ according to their toxicological classification (Karami-Mohajer and Abdollahi, 2011) , present different mechanisms of action and exert distinct toxicological effects. In the early stages of exposure, carbohydrates are used to provide energy and address stressful conditions, and later lipids and proteins serve as the main energy sources. This may explain the divergent results among various studies involving agricultural populations, given that research encompasses several classes and mixtures of pesticides and different occupational exposure conditions. All liver determinations were within reference values for both groups, but alkaline phosphatase and albumin concentrations were significantly lower in the pesticide-using group. Similar findings were reported in other studies (Aroonvilairat et al., 2015), suggesting that while other liver enzymes are within reference values, reduced alkaline phosphatase and albumin levels may indicate alterations in hepatic function without hepatocellular necrosis (García-García et al., 2016; Cattelan et al., 2018). These findings highlight the need for further research to understand the mechanisms behind these biochemical changes and to develop strategies to mitigate the health risks of pesticide exposure, considering the effects of different pesticide classes and occupational exposure conditions.

 

 

Genotoxicity

In the results and discussion section, the study reveals significant differences in DNA damage between pesticide-exposed and unexposed individuals across various age groups. The comet assay categorizes DNA damage into highly damaged, slightly damaged, and undamaged cells. Among pesticide-exposed workers, there is a notable increase in the proportion of damaged DNA cells with advancing age, contrasting with unexposed individuals who show a higher prevalence of undamaged DNA.

 

 

 

Specifically, highly damaged cells, characterized by greater tail lengths in the comet assay, are predominantly observed in pesticide-exposed workers, whereas unexposed individuals exhibit predominantly undamaged cells. These findings are detailed in Table 5 and Figures 1, 2, 3, 4, 5, illustrating the varying extents of DNA damage observed. This study’s comet assay results underscore significant DNA damage among pesticide-exposed workers, with damage severity correlating with increasing age. The prevalence of highly damaged cells with elongated tails further supports the notion that pesticide exposure contributes to genetic alterations. This aligns with previous research by Cook and Brazell (1975), reinforcing the heightened genetic risk faced by agricultural workers exposed to pesticides.

 

Table 5: Pesticides exposed and unexposed individuals showing different levels of DNA damaged with reference to age groups.

Age groups (years)	Grades of DNA damages
	Undamaged		Slightly damaged		Damaged		Highly damaged
	No.	%	No.	%	No.	%	No.	%
Less than 20
Exposed	2900	82.9	300	8.6	250	7.1	50	1.4
Unexposed	3100	88.6	250	7.1	130	3.7	20	0.6
20-29
Exposed	2500	71.4	635	18.1	280	8	85	2.4
Unexposed	2980	85.1	315	09	165	4.7	40	1.1
30-39
Exposed	2200	62.9	800	22.9	365	10.4	135	3.9
Unexposed	2844	81.2	417	11.9	182	5.2	57	1.6
40-49
Exposed	1990	56.9	937	26.8	415	11.9	158	4.5
Unexposed	2680	76.6	526	15.0	217	6.2	77	2.2
Greater than 50
Exposed	1540	44	1060	30.3	615	17.6	285	8.1
Unexposed	2330	66.6	772	22.0	308	8.8	90	2.6

 

 

Genetic biomonitoring serves as an essential tool in assessing the potential carcinogenic risks associated with pesticide exposure. While all populations face some risk from pesticide exposure, workers directly involved in pesticide production and agricultural application are particularly vulnerable to its genetic effects (Sailaja et al., 2006). This underscores the importance of continuous genotoxic monitoring in these populations to mitigate long-term health impacts. The comet assay’s utility in evaluating DNA damage in lymphocytes of farmworkers exposed to pesticide mixtures has been demonstrated in previous studies (Garaj-Vrhovac and Zeljezic, 2000; Muniz et al., 2008). Consistently, our findings corroborate increased DNA damage in farmworkers, indicating exposure to genotoxic components of pesticides.

Conclusions and Recommendations

It is concluded that pesticides have potential for causing clinical and hematological abnormalities plus serum biochemical alterations and genotoxic effects in pesticides exposed workers. There is a requirement to form training institutes that teach the farmers about safety measures while using pesticides. Non-targeted organisms such as local inhabitants are found to get affected by the application of pesticides. Engineering measures should be taken to get rid of these chemicals and aware the occupational workers about the hazardous impact of these pesticides. There is also needed to start a treatment program to treat the pesticide exposed workers who indicate clinical abnormalities in their bodies.

Acknowledgements

We are grateful to NRPU Project # 3392 entitled “Monitoring of Adverse Effects of Pesticide Exposure on Health of Agriculture and Industrial Workers and their amelioration with vitamin C and E” for providing the funds to conduct this research, and thankful to the staff of Aquaculture, Genetic Toxicity, and Molecular Biology Laboratory, Department of Zoology, IUB.

Novelty Statement

This research presents a pioneering comprehensive investigation into the effects of pesticides in a structured and systematic manner, providing valuable insights to the rural inhabitants.

Author’s Contribution

Abdul Ghaffar: Supervision, conceptualization, formal analysis.

Ayesha Maqsood: Executed sampling and data collection and laboratory work.

Riaz Hussain: Formal analysis, data curation.

Ghulam Abbasand Rabia Tahir: Writing review and editing.

Habiba Jamil: Data curation.

Fozia Afzaland Ahrar Khan: Edited and formatted the manuscript.

Muhammad Ahmad Chishti Shahnaz, Aliya Noreen: Helped data curation and proofreading of the MS.

Kashfa Akram and Maria Niaz: Helped in sampling, data collection, and laboratory work.

Conflict of interest

The authors have declared no conflict of interest.

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