Comparison of biochemical, haematological and plasmatic butyrylcholinesterase parameters in farmers and non-farmers, Morocco
Hasnaa Sine1, Youssef Bouchriti2, Hayat Sine1, Abderrahmane Achbani3
1 Department Life and Health Sciences, Faculty of Medicine and Pharmacy of Rabat, Mohamed V University of Rabat, Morocco
2 Faculty of Sciences of Agadir, Morocco
3 Department of Biology, Faculty of Sciences, University Ibn Zohr, Agadir, Morocco
|Date of Submission||01-Nov-2022|
|Date of Acceptance||01-Feb-2023|
|Date of Web Publication||20-Jul-2023|
Dr. Hasnaa Sine
Department, Life and Health Science- University Mohamed V/Faculty of Medicine and Pharmacy-Rabat - 10100
Source of Support: None, Conflict of Interest: None
Background: The long-term use of pesticides can cause harmful consequences to both human health and the environment. In the present research, we aimed to compare biochemical, hematological, and plasmatic measurements of butyrylcholinesterase (BChE) between farmers and non-farmers.
Materials and Methods: The study is cross-sectional and included 270 participants, with 135 farmers using pesticides and a control population of 135 non-farmers. The recruitment of the participants was conducted from August 2017 to the end of December 2019. Blood samples from participants were collected for the evaluation of biochemical markers of the function of the liver and determination of BChE activity. A whole blood sample with ethylenediamine tetraacetic anticoagulant (EDTA) was also taken for a complete blood count.
Results: The results showed a statistically significant (P = 0.03) decrease in mean corpuscular hemoglobin (MCH) in the cases (28.45 ± 2.94 pg) as compared with controls (29.17 ± 2.54 pg). The statistical analysis of the renal parameters between the two groups determined that the uremia value was significantly higher in cases (34 ± 12 mg/dL) when compared to the control group (29 ± 8 mg/dL) P < 0.001. The cases recorded a significant increase in aspartate aminotransferase (AST) (26.22 ± 11.59 U/L) and alanine aminotransferase (ALT) (25.63 ± 13.47 U/L) enzyme activities among cases versus controls. The results obtained showed a significantly decreased BChE activity in the group of cases exposed to pesticides (7554.52 ± 2107 U/l) compared to the unexposed control group (10135.58 ± 1909 U/l) (t-test, P < 0.001).
Conclusion: The education of the farmers on correct practices concerning phytosanitary use has the potential of reducing their exposure to these products.
Keywords: Aspartate aminotransferases, biomarkers, butyrylcholinesterase, cross-sectional studies, erythrocyte indices, humans, liver, pesticides, uremia
|How to cite this article:|
Sine H, Bouchriti Y, Sine H, Achbani A. Comparison of biochemical, haematological and plasmatic butyrylcholinesterase parameters in farmers and non-farmers, Morocco. Adv Biomed Res 2023;12:181
|How to cite this URL:|
Sine H, Bouchriti Y, Sine H, Achbani A. Comparison of biochemical, haematological and plasmatic butyrylcholinesterase parameters in farmers and non-farmers, Morocco. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:181. Available from: https://www.advbiores.net/text.asp?2023/12/1/181/382069
| Introduction|| |
The productivity of farming worldwide has significantly increased, principally because of the Green Revolution, which enabled the farmers involved to supply food to the growing world population. Pesticide use was a key contributor to this spectacular increase in food production. The worldwide use of pesticides in 2020 has reached 3.48 million tons. Nevertheless, the widespread and improper utilization of pesticides is a major environmental and public health problem in the world. Consequently, over the past decades, the researchers observed a significantly increased concerns regarding the harmful impacts of the chemical pesticide on the health of the general public, farmers, and the environment. In Morocco, the number of poisoning cases recorded during the period between 2008 and 2016 is 11196. In the same period, there was a crude incidence of acute pesticide poisoning at a rate of 3.57 per 100,000 populations (AntiPoison and Pharmacovigilance Center of Morocco).
Farmers from the developing world are professionally being exposed to increased quantities of the exported pesticides, which are banned in the European Union. Some recent studies indicate an increased risk of physical and mental health problems due to pesticide exposures affecting agricultural workers and their families as well as people living in agricultural areas. The many adverse health effects associated with pesticides include dermatological, gastrointestinal, neurological, carcinogenic, respiratory, and reproductive effects., In previous studies, some associations were found with long-term exposure to pesticides, resulting in changes in biochemical and hematological parameters. A number of supplementary laboratory biochemical and hematological parameters have been used as biomarkers to detect the early effects of pesticides., In previous studies, some associations were found with long-term exposure to pesticides, resulting in changes in biochemical and hematological parameters. Such biomarkers may reflect and predict the early stages of disease progression. For this reason, biomonitoring is considered a valid method to assess occupational exposure to pesticides. This exposure can be assessed by measuring the pesticide, its metabolites present in the urine and blood, or its biological effect, such as cholinesterase inhibition. In this study, we aimed to describe the associations of a long-term exposure to pesticides and their potential adverse health effects for agricultural workers, using hematological biochemical parameters and serum cholinesterase activity as biomarkers of effect. We hypothesized that frequent and long-term exposure to pesticides modifies the biological responses, including hematological biochemical parameters and serum acetylcholinesterase activity in exposed individuals.
| Materials and Methods|| |
Participants in the study are farmers and agricultural workers exposed to pesticides in the Souss Massa region (southern Morocco) who are eligible to participate.
Participants in this study are those meeting the following criteria: (i) subjects between the ages of 18 and 60, of any gender; (ii) employed on a farm and/or directly exposed to pesticides for at least one year; and (iii) signed informed consent.
Aged individuals, children, pregnant women, and individuals with chronic metabolic diseases or neurological disturbances are all excluded from the population under study to avoid interference with the biological parameters being evaluated.
Case and control sample selection
The agricultural and farm worker participants (livestock worker, tree worker, horticultural worker, or greenhouse worker) exposed to pesticides are identified during the period between August 2017 and the end of August 2019. One hundred and thirty-five cases (n = 135) of people consented to participate in this study.
Control subjects, with no previous experience of pesticide use or medical history, were recruited into the study with their consent. The 135 controls were matched to the 135 cases according to the following criteria: age ± 2 years, gender, and locality of residence, to reduce the potential number of confounding factors.
The size was determined by the OpenEpi calculator. According to this formula, the minimum sample size required is 270 participants, divided into 135 cases and 135 controls.
Blood collection and sample preparation
After at least 12 hours of fasting, blood samples are collected by puncturing the vein in the crease of the elbow between 8.00 and 10.00 am. The collected blood is collected in EDTA tubes (hematological parameters) and labeled, numbered dry tubes. After collection, the samples are immediately stored in a cooler and transported to the laboratory for processing after two hours. The dry tubes are then centrifuged at 805 xg for 15 minutes. The serum obtained is used to determine biochemical and enzymatic parameters and can be stored at − 20°C for further analysis.
Analysis of hematological parameters
The blood count is performed in a medical analysis laboratory, using an automated counter (ABX MICROS 60), on a blood sample taken from a tube containing EDTA as an anticoagulant.
Analysis of biochemical and enzymatic parameters
Determination of butyrylcholinesterase activity
The determination of butyrylcholinesterase (BChE) activity is performed by the spectrophotometric method of Ellman et al. (1961). Acetylthiocholine can be used as a substrate for the plasma BChE assay. Acetylthiocholine is subdivided into acetate and thiocholine. The thiol grouping of thiocholine reduces dithiodinitrobenzene to thionitrobenzene, a yellow compound that has a maximum absorbance at 412–415 nm. The increase in coloration over time indicates the formation of thiocholine, which is a reflection of the activity of the enzyme.
Determination of the enzymatic activity of transaminases (AST, ALT)
The enzyme transaminase catalyzes the transfer of the amine group from aspartate aminotransferase (AST) or alanine aminotransferase (ALT) to oxaloglutarate with formation of glutamate and oxaloacetate (for AST) or pyruvate (for ALT). Measurements are carried out using coupled reactions to allow the use of the coenzyme NADH/H+, whose decrease in absorbance is measured. Thus, oxaloacetate is reduced to malate or pyruvate to lactate using dehydrogenases (Malate dehydrogenases or Lactate dehydrogenases) coupled to NADH/H+. The oxidation rate of NADH is proportional to the enzymatic activity of the transaminases. It is determined by measuring the decrease in absorbance at 340 nm.
Determination of urea levels
Plasma urea is determined by colorimetric and enzymatic methods. The reaction consists of an enzymatic reaction coupled with a color reaction. The urease hydrolyzes the urea, producing ammonium (NH4+). The ammonium ions react in an alkaline medium with salicylate and hypochlorite to form a blue-colored indophenol. The staining is catalyzed by the nitroprusiate, and the reading is taken at 600 nm.
Determination of creatinine levels
Creatinine is determined by the kinetic method in human plasma. The assay is carried out by a colorimetric reaction (Jaffé method) using picric acid in an alkaline medium, with the kinetics of color development being measured at 490 nm.
The data were encoded and analyzed using IBM SPSS version 22 software. Comparisons between groups were made based on percentage numbers, using Student (T) and Chi-square tests. The Chi-square test was used to look for dependency between categorical variables for a population size >5 and the Fisher test for a population size <5, the Student test was used for continuous variables for a population size >30, and the Mann–Whitney test for a population size <30. To compare more than two groups, Kruskal–Wallis and Anova tests were applied.
Because of the unequal sample size, the premise of equality of variances was systematically verified. If the test is significant (unequal variances), the Welsch test is used. Differences are considered significant at P < 0.05 and highly significant at P < 0.01.
| Results|| |
The controls were matched with cases based on age and sex, with a ratio of one control to one case. A total of 270 eligible participants (135 cases and 135 controls) agreed to participate in this study. The general demographic characteristics of cases and controls were compared [Table 1]. There were no differences between cases and controls by age, with a mean age of about 43.51 ± 12.02 years for controls (t = 0.42, P > 0.05). The age distribution was similar in the controls due to matching. There were no significant differences between the case and control groups by gender. The education level was not similar between cases and controls.
|Table 1: Comparison of sociodemographic variables between cases and controls|
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Results of analyses of hematological parameters
The hematological parameters are analyzed in both groups: the pesticide exposed group (cases) and the unexposed group (controls). The data showed that all hematological parameters were within the normal range for the farmers and the control group.
The results in [Figure 1]a show a similar mean red blood cell (RBC) count between cases (4,994 ± 0,566,106/mm3) and controls (4,915 ± 0,619,106/mm3) (P = 0.27). Our results showed no statistically significant difference in white blood cell count between the cases (7,062 ± 1,685,103/mm3) compared to the controls (7,078 ± 1,622,103/mm3) (P = 0.79) [Figure 1]b.
|Figure 1: Distribution of cases and controls based on blood count results (a) mean red blood cell count (b) mean white blood cell count (c) mean haematocrit (d) mean haemoglobin (e) mean platelet count|
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[Figure 1]c shows a statistically non-significant decrease in the mean hematocrit value for cases (41.92 ± 4.33%) compared to controls (42.28 ± 5.34%) (P = 0.54). As for the hemoglobin level, the mean is 14.12 ± 1.48 g/dl in the case group and 14.28 ± 1.47g/dl in the control group. These two averages are similar (P = 0.35) [Figure 1]d.
Platelet levels varied around a mean of 271.17 ± 69.77.103/mm3 in the case group, compared to a mean of 263.32 ± 63.24.103/mm3 in the control group. Comparison of these means yields no statistically significant difference (P = 0.33) [Figure 1]e.
According to the results of the calculation of the RBC constants, there is a slight, non-significant decrease in mean corpuscular volume (MCV) in the case group (84.45 ± 7.97 μm3) compared to the control group (85.84 ± 7.35 μm3) [Figure 2]a. Whereas, [Figure 2]b shows a statistically significant (P = 0.03) decrease in mean corpuscular hemoglobin (MCH) in the cases (28.45 ± 2.94 pg) compared to the controls (29.17 ± 2.54 pg).
|Figure 2: Distribution of cases and controls according to red blood cell count results (a) Mean corpuscular volume (MCV) (b) Mean corpuscular hemoglobin (c) Mean corpuscular hemoglobin concentration rate|
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[Figure 2]c shows a non-significant decrease in MCH concentration (MCHC) in cases (33.72 ± 2.05%) compared to controls (34.03 ± 1.85%).
Results of analyses of biochemical parameters
Analysis of renal exploration parameters
Biochemical analysis of the sera revealed that the averages of the main indicators of kidney function, urea and creatinine, were within biological norms. Statistical analysis of these parameters between the two groups determined that the uremia value was significantly higher in the cases (34 ± 12 mg/dL) compared to the control group (29 ± 8 mg/dL) P < 0.001 [Figure 3]a. Serum creatinine levels were 10.4 ± 6.07 mg/l and 10.19 ± 2.67 mg/l in the cases and controls, respectively. This difference in creatinine concentration is not statistically significant (t-test, P = 0.71) [Figure 3]b.
|Figure 3: Analysis of renal parameters in both groups (Case/Control) (a) Average uremia rate (b) Average creatinine rate|
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Analysis of hepatic enzymes
The results in [Figure 4] show that the cases recorded a significant increase in AST enzyme activity (26.22 ± 11.59 U/L) (P < 0.001), compared to controls where the enzyme activity did not exceed 21.86 ± 9.10 U/L.
|Figure 4: Analysis of hepatic enzymes (AST/ALT) in both groups (Case/Control)|
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In addition, a significant increase in enzyme activity was noted for ALT (25.63 ± 13.47 U/L) (t-test, P < 0.01) compared to controls, which was (22.12 ± 9.39 U/L).
Analysis of the enzymatic activity of plasma BChE
The results obtained showed a significantly decreased BChE activity in the group of cases exposed to pesticides (7554.52 ± 2107 U/l) compared to the unexposed control group (10135.58 ± 1909 U/l) (t-test, P < 0.001) [Figure 5]. A significant decrease of 26% was recorded in the case group.
| Discussion|| |
The present study was conducted to evaluate the adverse effects of pesticides on certain hematological and biochemical parameters in farmers chronically exposed to pesticides, in the Souss Massa region, in Morocco. The study recruited 270 participants (135 farmers and 135 controls) with an average age of 42.96 + 11.32 years. The educational level of the farmers interviewed in this study was low; 44% had no schooling at all, and 43% had no more than primary schooling. These results are similar to other regions in Morocco., Several studies, have associated low levels of education with the abuse and inappropriate application of pesticides and difficulty in deciphering pictograms and reading instructions and hazard warnings on pesticide container labels.
The results of this study concerning the use of pesticides and hematological parameters are consistent with other studies that have not found a constant association between pesticide exposure and hematological parameters., A further Indian study found only an increase in the number of leukocytes in the farmers compared to the control group, while RBCs, platelets, and hemoglobin showed no significant difference between the two groups.
On the other hand, another study showed an increase in the MCV, hematocrit, and hemoglobin in the exposed group than in the reference group, while measurements of leukocytes, lymphocytes, platelets, and MCHC were significantly higher in the exposed group than in the reference group. In our study, the average MCH was lower among farmers compared to controls. The low MCH reflects a moderate anemia. According to the study conducted on animals, the exposure of pesticides, in particular to organophosphate, can induce anemia.
The serum enzymes analyzed, which reflect hepatic damage (ALT, AST), were significantly increased in the farmers compared to the controls. These data could be explained by the hepatotoxic effects of pesticides. These findings are consistent with the results of experimental studies supporting the elevation of such enzymes after exposure to pesticides., Also, the level of urea was significantly higher in farmers than in controls. This may be due in part to the disturbance of urea synthesis because of impaired hepatic function or to a decrease in the filtration of the kidney.
Serum cholinesterase activities (BChE) were significantly reduced in the farmers compared to the control group (P < 0.001). Similar results were found in other studies,,, that suggested that occupational exposure to organophosphate and carbamate pesticides induces the inhibition of serum cholinesterase activity.
| Conclusion|| |
The present study indicated that pesticide exposure can seriously alter various parameters of biological systems. Our data revealed the importance of biomonitoring in farmers as well as studying early pathophysiological alterations, to prevent long-term illness and improve the quality of life of farm workers. Raising farmers' awareness of good practices related to pesticide use and the correct wearing of personal protective equipment could also reduce pesticide exposure.
Special thanks to the biologists and laboratory technicians of SAADA and MOUHDI Laboratories for their pleasant assistance and collaboration.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]