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Pesticides are synthetic chemicals that use worldwide for controlling the agricultural and domestic pest. The active component of pesticides is broad and includes Organochlorine, Organophosphate, Carbamate, Pyrethroid and Neonicotinoid compounds. Of all these compounds, Organophosphates are the most widely used insecticides today. They are used in agriculture, the home, gardens, and veterinary practice. More than 100 different OP compounds are formulated as a pesticide which mainly used as insecticide and herbicide (1) Chronic exposure to pesticides especially organophosphates cause many severe problems for human health (2). Studies indicated that the prevalence of diseases such as diabetes, cardiovascular diseases, congenital disabilities, and reproductive disorders had been related to long term exposure to pesticides. Also, chronic neurologic effects such as neurodegenerative disorders include Parkinson, Alzheimer, and Amyotrophic lateral sclerosis (ALS) are suspected of being linked to chronic OP exposure, creating a real concern in the health community (3-6) Occupational exposure to the pesticide is a major concern. Agricultural and pesticide manufacturing factory workers are at risk of exposure to high level of pesticides and its consequence health effect. (7-9).
Paraoxonase-1 (PON1) is a serum esterase which is synthesized by the liver. It is a high-density lipoprotein-associated enzyme that hydrolyzes oxidized phospholipids and capable of detoxifying OP compounds in human serum (10,11). Also, PON1 has a protective effect against free radicals that is considered as a powerful antioxidant (12). Human serum PON1 enzyme has two distinct enzyme activities: arylesterase (AREase) and paraoxonase (PONase) activities toward specific substrates phenylacetate and paraoxone respectively. There is a genetic polymorphism in PON1 gene which severely affects paraoxonase activity of the enzyme, and wide individual variation is observed on paraoxonase activity, Humans can be categorized in three different serum PON1 phenotypes by using enzymatic analysis include AA: low activity, AB: moderate activity, BB: high intensity. The AREase activity has not been affected by genetic polymorphism and usually shows the content of the enzyme in the serum. (13).
 In addition to genetic factors that affect PON1 activity, other factors such as disease conditions and environmental pollutants affect PON1 activity. Inorganic arsenic reduced serum PONase and AREase activity in rats after chronic low dose exposure (14). Studies have been indicated that in several chronic diseases such as diabetes, cancer, and Alzheimer disease, associated with increased systemic oxidative stress, reduced serum PON1 activity has been observed(15,16). Then it is postulated that oxidative stress is associated with reduced PON1 activity (17-19).
 One of the proposed mechanisms of OP toxicity is oxidative stress. It is reported that the OP compounds interact with cellular antioxidant defense and also produce oxygen free radicals which lead to oxidative tissue damage. The oxidative stress markers have been found in the serum of OP pesticide manufacturing factory workers (20,21).
 The metabolism of organophosphates may be affected by the genetic polymorphic variation (Polymorphism) in serum paraoxonase activity in individuals at risk of OP exposure. Then several types of research have been studied the relation between the PON1 polymorphism and the risk of OP poisoning (19,22,23) but little is known about the effect of OP occupational exposure on the plasma PON1 activity and also the relation between OP-induced oxidative stress and PON1 activity.
Aims of the study:
In the present study PChE activity and RBC AChE activity as a biochemical marker of exposure, lipid peroxidation as a biomarker of oxidative stress and serum paraoxonase /arylesterase activity were measured in OP pesticide manufacturing factory workers and also a correlation between these biomarkers were investigated.
 
 
 
 

Materials & Methods

Chemicals
Acetylthiocholine iodide, 5, 5-dithiobis-2-nitrobenzoic acid (DTNB), Malondialdehyde bis dimethyl acetal, phenylacetate, 2-thiobarbituric acid (TBA), paraoxon were purchased from Sigma-Aldrich (USA)
Human subjects
In this cross-sectional study, 21 male workers of a pesticide manufacturing factory at the age range of 23-55 years, with a minimum work history of 1 year and a maximum of 5 years were included. Age- and sex-matched control subjects (n=40) were volunteers from Pathology Laboratory clients that they visited for a routine checkup who were not directly exposed to the pesticides. Every subject was asked to fill out a questionnaire about his health status, the use of medications, occupational history, exposure to pesticides, and so forth, and signed a consent form to participate in this study. Based on the questionnaire, subjects with a history of chronic diseases such as diabetes mellitus and hypertension, cigarette smoking and use of drugs were excluded from the study. A blood sample was obtained from the brachial vein of each subject by an expert person. The project was approved by the Ethics Committee of Tarbiat Modares University (Ethical code: IR.TMU.REC.1394.14), and informed consent was received from all workers and healthy subjects.
Sample preparation
Venous blood was collected into dried heparinized tubes. After blood centrifugation, plasma was separated from the erythrocytes and stored in -70 ̊C until assay. The erythrocytes were washed with normal saline then were suspended in de-ionized water in a volume corresponding to the initial volume of the whole blood and then were frozen for hemolysis.
PChE and RBC AChE activity assay
The activities of AChE and PChE were measured by the Ellman spectrophotometric method (24,25). In this method AChE hydrolyzed acetylthiocholine to produce thiocholine and acetate. The thiocholine reduces the Dithiobis-Nitrobenzoic Acid (DTNB) to nitrobenzoate (TNB) anions, a yellow color compound, which has an absorbance at 405 nm. The increase in absorbance at 405 is proportional to the enzyme activity.  The RBC lysate was diluted 60-fold with a buffer (0.1 M phosphate buffer, pH=7.4). The activity of the enzyme was calculated by the use of the extinction coefficient of TNB anions. The PChE activity in plasma was also measured in the same way except butyrylthiocholine was used as a substrate.
Lipid peroxidation assay
Plasma Lipid peroxidation was estimated by the measurement of Thiobarbituric acid reactive substances (TBARS) concentration which was performed by thiobarbituric acid reaction (TBAR) colorimetric assay. One volume of plasma was mixed with two volumes of TBA reagent (containing 3.75% TCA and 0.0925% TBA) and the mixture incubated at 90°C for 60 min. After rapidly cooling on ice, centrifuged at 1000 g for 10 min and the optical density of supernatant was measured in 540 nm in a plate reader. Because the principal member of TBARS is malondialdehyde (MDA); MDA standard curve was established with using the stable MDA precursor, (Malondialdehyde bis dimethyl acetal) and then the concentration of TBARS is expressed in nmol MDA/mL in plasma (26)
Determination of PON1 enzymatic activities
Paraoxonase activity assay
 Ponase activity was measured with paraoxon as a substrate as previously described (27). Briefly, 40 µl paraoxon (6mM) was added to 150 µl assay buffer (containing 0.132 M Tris-HCl pH 8.5 and 1.32 mM CaCl₂) then immediately after addition of 10µl sample, absorbance was monitored for 5minute at 405 nM by a plate reader. In this assay paraoxonase hydrolysis, the paraoxone to produce p-nitrophenol and the paraoxonase activity was calculated by using the extinction coefficient of p-nitrophenol (18050M⁻¹ cm⁻¹) and units were expressed as micromoles of paraoxon hydrolyzed per minute. The salt stimulated activity was measured by the addition of 1M NaCl in the assay buffer. The percent stimulation of enzyme activity was calculated as follows:
 
Paraoxonase activity with 1M NaCl – Basal paraoxonase activity      ×100%
 

Basal paraoxonase activity
 
The percent stimulation is a qualitative property of paraoxonase that separates the AA phenotype from the combined AB and BB phenotypes. The percent stimulation above 60% refers to AB and BB phenotypes, and less than 60% relates to AA phenotype (28). Individuals with AA phenotypes which had low enzyme activity were excluded from the study to reduce the within-group variation and partly to reduce the effect of polymorphism; then mean paraoxonase activity was compared between groups.
Arylesterase activity assay
Arylesterase activity was measured with the phenyl-acetate substrate as described earlier (29). Briefly,5µl of the sample was added to a 195µl substrate solution containing 3.26 mM phenyl-acetate, 100mM Tris–HCl pH=8 and 2 mM CaCl₂ and then the rate of generation of phenol was determined at 270 nm at 37°C, using a continuously recording spectrophotometer. The molar extinction coefficient of 1310 M⁻¹ cm⁻¹ was used for calculations of enzyme activity and units were expressed as micromoles of phenyl-acetate hydrolyzed per minute.
Statistical analysis
Student’s t-test was used to determine the significance of differences observed between groups. F-test was done to determine the normal distribution of variances between groups. The correlation between groups was measured by the Pearson test which measures the statistical relationship, or association, between two continuous variables. p-values of 0.05 or less were considered as significant differences.
 

Results

Paraoxonase Phenotype in the Studied individuals
According to the percent stimulation of paraoxonase activity by salt, the studied individuals were categorized in AA phenotype or combined AB and BB phenotypes. Table 1 shows the phenotype distribution of studied individuals. Only 11 persons from a total 61 persons have AA phenotype with low paraoxonase activity. The data of persons with AA phenotype were excluded from the study to reduce the within-group variation and eliminate the effects of polymorphisms.
Table 1) The phenotype distribution of studied individuals

	Phenotype
	AA	AB and BB
Control	6	34
Workers	5	16
Total	11	50

AA: low activity, BB: high activity, AB: Moderate activity
 
PChE and RBC AChE activity
PChE activity and RBC AChE activity were measured by the Ellman method and results indicated in Figure 1. The significant reduction in both enzyme activity was observed in factory workers (p<0.001). The PChE activity inhibition was about 31.7±5.9%while RBC AChE activity inhibition was 14.7±2.2% (Table 2).

Table 2) Comparison of biochemical parameters between organophosphate pesticide manufacturing factory workers and control unexposed groups

Biochemical parameters	Control(Mean±SD)	Workers(Mean±SD)
PChE activity(U/L)	7482±1466	5105±1599***
AChE activity(U/grHb)	128.5±15.31	109.6±12.22***
Ponase activity(U/L)	86.38±46.28	53.72±37.36*
AREase activity(U/L)	7490±1545	4592±1337***
MDA (nM/ml)	10.42±2.04	16.05±2.5*

PChE(Plasma cholinesterase), AChE(RBC Acetylcholinesterase), Ponase(Paraoxonase), AREase(Arylesterase),MDA(malondealdehyde).
(*) represents significant difference versus control. (t-test,*p<0. 05, ***p<0.001)
  Figure1) correlation between plasma cholinesterase (PChE) activity and plasma Arylesterase activity(A), RBC acetylcholinesterase (AChE) activity and plasma Arylesterase activity(B), plasma lipid peroxidation and plasma Arylesterase activity(C) and plasma lipid peroxidation and plasma Paraoxonase activity(D) (Pearson test, p<0.01)