Evaluation of acetylcholine esterase activity in the blood of workers exposed to organophosphate and carbamate insecticides by an electrometric method

Introduction:Organophosphate and carbamate insecticides pose major environmental pollution problems and health hazards to people and animals. These insecticides inhibit cholinesterase (ChE) activity in the nervous tissues and neuromuscular junctions. The measurement of blood ChE is a useful tool for monitoring exposure to organophosphate and carbamate insecticides. The purpose of the present study was to use a modified electrometric technique for measuring blood ChE in workers exposed to the organophosphate and carbamate insecticides in Kirkuk, Iraq. Method: A modified electrometric method was used to measure ChE activity in the whole blood of male workers (n = 40) exposed to organophosphate and carbamate insecticides, for a duration of not less than six years. Healthy male volunteers (n =12) not exposed to insecticides served as controls. Following in vitro inhibition of pseudo cholinesterase by quinidine sulfate, true cholinesterase activity was estimated in the blood of the subjects. After in vitro addition of the organophosphate (chlorpyrifos and methidathion, 0.5 and 1 μM) and carbamate (carbaryl, 5 and 10 μM) insecticides to the reaction mixtures, inhibitions of blood ChE were also determined. Results: Mean values of ChE activities (pH/20 min) in the whole blood of healthy non-exposed subjects and insecticide-exposed workers were 1.41and 1.2, respectively. Whole blood ChE activities of the exposed workers was significantly lower than those of healthy individuals. Conclusions: These findings indicate the usefulness of the modified electrometric method for monitoring blood ChE activity in insecticide-exposed workers and there was a significant effect of these Organophosphate and carbamate insecticides on the activity of Ach esterase in workers blood. .


Introduction
Acetylcholinesterase (AChE) is an important enzyme present in the synaptic clefts of the central nervous system of living organisms [1]. It hydrolyses the neurotransmitter acetylcholine and facilitates the proper functioning of muscular system and it is used as a marker for cholinergic neural function [2]. AChE has been subject of keen interest for several decades and the detailed studies carried out in the past have revealed that the AChE activity could be significantly inhibited by organophosphorus (OP) pesticides used in veterinary practice, agriculture, medicine, industry and chemical warfare agents [4,3].Organophosphate and carbamate insecticides are widely used in public health, veterinary practice, and in agriculture [4,5]. They pose major environmental pollution problems and health hazards to people and animals [ 3,[6][7][8]. These insecticides inhibit cholinesterase (ChE) activity in the nervous tissues and neuromuscular junctions, causing an accumulation of acetylcholine at the nerve endings which subsequently produces signs of toxicosis characterized by nicotinic, muscarinic, and central nervous system effects [8,. Various colorimetric and electrometric (potentiometric) methods are available to determine blood cholinesterase activity [01,12]. One of the main methods for measuring blood cholinesterase activity is the electrometric method which is based on the hydrolysis of acetylcholine and the production of cholinesterase activity of workers exposed to inhibitors of this enzyme [07]. The purpose of our study was to further evaluate and apply the modified electrometric method for measuring blood ChE activities in workers exposed to organophosphate and carbamate insecticides in Kirkuk, Iraq .

Subjects and Methods
Forty male workers, in contact with and exposed to carbamate and organophosphate insecticides daily and for a period of working duration not less than 6 years, were included in the study. Their ages ranged between 20-50 years .Apparently healthy volunteers (n=12), who had no history of exposure to anti ChE insecticides for at least six months before blood sampling served as controls. The volunteers were from Kirkuk province, Iraq. Their consents were obtained for the blood examination. The modified eletrometric method of Mohammad (2007) was used to determine whole blood ChE activity. For a typical assay condition, the reaction mixture in a 10-ml beaker contained 3 ml distilled water, 0.2 ml whole blood, and 3 ml pH 8.1 barbital-phosphate buffer.The pH of the mixture (pH1) was measured with a glass electrode using a pH meter (Consort, Belgium) before 0.1 ml of aqueous solution of the substrate acetylcholine iodide (7.1%) was added to the reaction mixture that was incubated at 37°C for 20 minutes. At the end of the incubation period, we measured the pH of the reaction mixture (pH2). The enzyme activity was calculated as follows: • ChE activity (pH/20 minutes) = (pH1 -pH2) -pH of blank.
The blank was without the blood aliquot. The barbital-phosphate buffer solution consisted of The significance of ChE inhibition in the plasma or erythrocytes of each subject was statistically evaluated using unpaired Student's t-test. The level of significance was at P < 0.05.

Results
Tables 1 shows the mean, SD, and related statistics for whole blood ChE activities in apparently healthy subjects (non-exposure group) and workers exposed to insecticides. Whole blood ChE of the workers was significantly below that of the control group (Table 1) .

Table (1): Cholinesterase Activities (ΔpH/20 minutes) in the Blood of healthy volunteers and workers exposed to insecticides
The insecticides (chlorpyrifos, methidathion, and carbaryl) in a concentration-dependent manner variably inhibited bloodChE activities in vitro ( Table 2).

Discussion
This study introduces (for the first time) normal ChE activities of the blood of apparently healthy male and workers who exposed to organophosphate and carbamate insecticides in Kirkuk province (Iraq) as determined by a simple modified electrometric method. It is in agreement with the findings of Ahmed and Mohammad, 2007 [18] in a different Iraqi region (Mosul).
The widespread use of pesticides in agriculture results in continuous exposure of human populations. Agriculture workers are prone to long-term exposure to relatively low levels of organophosphate agents. These workers are daily exposed, use little protection due to cultural and economic reasons, and underestimate the toxicity of organophosphate. Poisoning has frequently resulted from the use of organophosphorus pesticides, the compounds usually having been absorbed dermally or by inhalation during application or during subsequent work in the fields.
Measurement of blood and tissue cholinesterase activities is a useful tool for monitoring exposure to organophosphate and carbamate insecticides and diagnosing their poisoning [10,19]. Usually a 20-30% decrease in serum cholinesterase activity suggests exposure to anticholinesterases [10]. More than 50% inhibition of cholinesterase activity supports the diagnosis of poisoning and indicates a hazardous condition [11].
Based on the findings of the present study and others [16,18,20], it can be assumed that the present electrometric method could have practical applications in human to detect ChE inhibition following exposure to anti-ChE insecticides. This method has a short one stepincubation time and it is sensitive enough, cheap and simple. The normal reference range values of plasma, erythrocyte and whole blood ChE activities of the healthy human volunteers have been reported using the presently described electrometric method [17,20].
In vitro inhibition of whole blood ChE activities by chlorpyrifos, methidation (organophosphates), and by carbaryl (a carbamate) is in agreement with the reported anti ChE actions of these insecticides [16,17,21,22]. These results and previous in vitro and in vivo ChE inhibition studies suggest the sensitivity of the modified method in detecting ChE inhibition caused by organophosphates and possibly carbamates [14,20,[23][24][25] Measurement of blood ChE activity in people is a non-invasive biomarker method for monitoring poisoning or exposure to organophosphate and carbamate insecticides [21,26,27].
These results further support and expand previous findings, and the modified method was validated for determining ChE activities in the blood of people [17,20]. Furthermore, the organophosphate and carbamate insecticides decreased Che activities in several animal species [23][24][25]28].
In conclusion, the present study extends the usefulness of the described electrometric method by detecting ChE inhibition in workers exposed to insecticides