Assessment of acetylcholinesterase and butyrylcholinesterase activities in blood plasma of agriculture workers

Abstract

Background:

Cholinesterase determination indicates whether the person has been under pesticide exposure is not. It is recommended that the worker′s cholinesterase level should be assessed for workers at a pesticide applied region. Hence, cholinesterase activities in blood samples of agricultural workers exposed to vegetables and grape cultivation with age matched, unexposed workers, who never had any exposure to pesticides, were estimated.

Methods:

The detailed occupational history and lifestyle characters were obtained by questionnaire. Cholinesterase activity was determined by the method of Ellman as modified by Chambers and Chambers.

Results:

AChE was ranging from 1.65 to 3.54μmoles/min/ml in exposed subjects where as it was ranged from 2.22 to 3.51μmoles/min/ml in control subjects. BChE activity was ranging from 0.16 to 5.2μmoles/min/ml among exposed subjects, where as it was ranged from 2.19 to 5.06μmoles/min/ml in control subjects. The results showed statistically significant reduction in enzyme activities (AChE 14%; BChE 56%) among exposed subjects.

Conclusion:

It was concluded that the reduction in cholinesterase activity may lead to varieties of effects. Hence it is compulsory to use protective gadgets during pesticide spray. Further a continuous biomonitoring study is recommended to assess pesticide exposure.

INTRODUCTION

In India, more than 1 billion people are engaged in agricultural activities and a large quantity of pesticides is used to protect their crop against pests to get more yields. India is the largest producer of pesticides in Asia and the third largest consumer of pesticides in the world.[1] Pesticide consumption has been increasing steadily in the past few years and there has been a distinct shift from organochlorine to organophosphorous (OP) and carbamate pesticides. These pesticides interfere with or inhibit the activity of cholinesterase (ChE) enzymes in nerves and muscle tissue,[2,3] which results in accumulation of the neurotransmitter acetylcholine (ACh) in the nervous system. Acute toxicological effects of OP pesticides are a result of the inhibition of acetylcholinesterase (AChE) in the nervous system, which can cause respiratory, myocardial, and neuromuscular transmission impairment. Chronic effects of OP exposures are not well documented; however, several recent reports indicate that certain birth outcomes (e.g., decreased gestational age, decreased birth length) and abnormal reflex functions in infants may be associated with low level environmental exposures to OP pesticides.[4–6]

AChE inhibition causes clinical features due to overstimulation of cholinergic synapses in the parasympathetic system, neuromuscular junction, and central nervous system. Decrease in ChE activity by 15–25%, 25–35%, and 35–50% is caused by low, moderate, and severe intoxication with pesticides, respectively.[7] People are directly exposed to these pesticides through dermal contact and inhalation, and indirectly through the food chain. Annually, about 3 million people worldwide are intoxicated with organophosphates; out of this, 300,000 either die or are severely injured.[8] A most economical blood test for the monitoring of farm workers who are exposed to OP insecticide is measurement of plasma butyrylcholinesterase (BChE) activity. Monitoring of plasma BChE has been recommended in the OP-exposed population, as this could be a useful biomarker to predict and prevent health hazards of pesticides.[9]

ChE determination indicates whether the person has been under pesticide exposure or not. It is recommended that the worker′s ChE level should be assessed before they start working at a pesticide applied region. In view of this, the present study was aimed to evaluate the AChE and BChE activities among agriculture workers occupationally exposed to pesticide.

MATERIALS AND METHODS

Study area and subjects

The study was conducted in the neighboring villages of Chikkaballapur town, rural Bangalore, South India, from December 2010 to March 2011. This study included 28 rural people who were agriculture workers, engaged in floriculture, and cultivation of cabbage, potato, and grape. A control group consisting of 13 unexposed workers, who never had any exposure to OP pesticides, was taken as the reference group. A detailed history, including the personal and occupational details, was recorded through a questionnaire. The list of pesticides used and the frequency of usages were also investigated.

Blood sample collection

Ethical clearance was obtained from Institute Ethical Committee. A written informed consent was taken from all study subjects after explaining the importance of the study in their local language. Five milliliters of venous blood was collected in dried heparinized tubes and transported in ice box to the laboratory. Blood samples of voluntarily participated agriculture workers (n = 28) who have been involved mainly in pesticide spraying activities in vegetable and grape gardens were collected. A control group consisting of 13 male subjects who belonged to a similar age group and socioeconomic status and were not exposed to any kind of pesticides was selected for the study from the same localities.

Sample processing and enzyme assay

Blood was centrifuged at 4000 rpm for 10 min at 4°C to separate the plasma. ChE activity was determined by the method of Ellman et al.[10] as modified by Chambers and Chambers.[11] Three milliliters of 0.25 mM of 5′,5′-dithiobis (2-nitrobenzoic acid) (DTNB) prepared in 0.05 M phosphate buffer was pipetted out into a cuvette, in which 20 μl of thoroughly mixed plasma sample and 100 μl of 1 mM substrate (acetylthiocholine iodide for AChE assay) were added. The contents were mixed gently and allowed to stabilize for 60 sec. The sample was placed on a UV-VIS spectrophotometer set at a wavelength of 410 nm. The change in absorbance with a light path of 1 cm width was recorded following time drive kinetic spectrophotometric method for 5 min to ensure that the linear phase of the reaction was measured at a time lag of 30 sec. All samples were assayed in duplicate. A nonenzymatic blank was included to assess the background levels of hydrolysis of the substrate. Same procedure was repeated for BChE assay using butyrylthiocholine iodide as substrate. ChE activities were expressed in μmoles/min/ml of plasma.

Statistical analysis

Student′s t-test was used to compare the significance of the mean differences in ChE activity between exposed and control subjects. The values of P < 0.05 were considered significant.

RESULTS

The demographic data of lifestyle habits, type of crop cultivation, pesticides used, and frequency of application collected on both exposed and control subjects are summarized in Table 1. The average age of exposed subjects and controls were 34.6 ± 8.22 and 28.1 ± 9.33 years, respectively. About 53.6% of study subjects were using pesticides weekly once and 21.4% were using weekly thrice for their crop protection. The majority of the workers did not use any protective equipment and a normal cloth was used to cover their face as a mask while spraying. About 68–71% had complained having the symptoms of headache and eye irritation [Table 1].

Table 1.

Characteristics of agricultural workers (n=28)

Table 2 shows the list of commonly used pesticides in the study locations and World Health Organization (WHO) classification. These data were collected in the local market and from the farmer. Majority of the pesticides used were in the categories of moderately hazardous to highly hazardous. Pesticides used were in the chemical group of OP, organochlorine, carbamates, and synthetic pyrethroids.

Table 2.

List of commonly used pesticides in the study area

AChE and BChE activities measured in the blood plasma of exposed and control subjects are given in Table 3. AChE activity among exposed subjects ranged between 1.65 and 3.54 μmoles/min/ml, with a mean concentration of 2.51 μmoles/min/ml, whereas in the control group it ranged between 2.22 and 3.51 μmoles/min/ml. The BChE activity in agricultural workers ranged between 0.16 and 5.2 μmoles/min/ml, with a mean concentration of 1.66 μmoles/min/ml, whereas in the control group it was 2.19–5.06 μmoles/min/ml, with a mean concentration of 3.87 μmoles/min/ml (P < 0.05). The measured levels of AChE and BChE activities in exposed subjects were comparatively less than in the control subjects [Figure 1].

Table 3.

Cholinesterase activity (μmoles/min/ml) among exposed and control group subjects

Figure 1.

Variation in cholinesterase activity between exposed and control groups (P<0.05, t-test)

DISCUSSION

The finding suggests that the AChE activity in agriculture workers was decreased (14%) when compared to that of controls due to the inhibition of AChE activity by pesticides. The inhibition of AChE might have resulted in the accumulation of ACh at the synaptic junctions, which may lead to cytotoxicity. Our result supports the earlier findings of Vidyasagar et al.[12] as they also found inhibited AChE activity in the OP-exposed workers. Occupational exposures to ChE inhibiting pesticides used in India for agricultural pest control can impair the respiratory health of agricultural workers who work in the field.[13]

In agreement with the present study, California agricultural pesticide applicators showed considerable changes in AChE inhibition and low AChE activities due to exposure to pesticides during the high exposure period.[14] Their study also demonstrated that relations exist between change in ChE inhibition and symptoms, especially respiratory symptoms, symptoms of the CNS (analysis including controls), and eye symptoms (internal analysis). Studies by Hillman[15] and Clarke et al.[16] indicated that there was a significant decrease in activity of AChE and BChE found among the OP pesticide sprayers as compared to the controls. Reduction in plasma BChE activity (56%) in the present study supports similar findings observed by various researchers.[17,18]

The use of ChE inhibiting pesticides in the agricultural activity caused the depletion of AChE and BChE activities among workers. From earlier reports and the present results, it can be speculated that decreased plasma ChE activity is due to prolonged exposures to OP pesticides among the study subjects as compared to controls [Figure 1].

Limitations of the study

This study was carried out as part of the academic dissertation and a large number of people could not be included. However, the data generated highlights the effects of pesticide exposures and it would help conduct further studies.

CONCLUSION

The AChE and BChE activities measured were significantly lesser due to multiple exposures to different groups of pesticides used for agricultural activity. Unscientific way of pesticide mixing, improper way of handling pesticides, and entering agriculture field immediately after pesticide application play significant roles in reducing plasma ChE activity. Preventive measures coupled with biomonitoring of pesticide exposure using ChE inhibition as a marker are very much important. This will create awareness among the agriculturists, pesticide manufacturers, agriculture department, etc.