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 DOI: 10.1191/0960327102ht238oa

2002 21: 179Hum Exp ToxicolA Ranjbar, P Pasalar and M Abdollahi

manufacturing workersInduction of oxidative stress and acetylcholinesterase inhibition in organophosphorous pesticide

  

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Induction of oxidative stress andacetylcholinesterase inhibition inorganophosphorous pesticidemanufacturing workersA Ranjbar 1, P Pasalar1 and M Abdollahi*,2

1Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran;2Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences,Tehran, Iran

Oxidative stress status and acetylcholinesterase (AChE)activity were studied in blood samples obtained from 45organophosphorous (OP)-formulating pesticide workerswith a minimum work history of 1 year in the agerange of 23±55. Controls were age-matched workers of afood-making factory. They were evaluated for oxidativestress markers, including thiobarbituric acid-reactivesubstances (TBARS) indicator of lipid peroxidation(LPO), ferric-reducing ability of plasma (FRAP) indicatorof total anti-oxidant capacity, total thiol (SH) groupsand gamma glutamyl transpeptidase (GGT) levels inblood and AChE activity in erythrocytes. The resultsshow marked inhibition of AChE activity, increased

TBARS, decreased FRAP and decreased thiol grouplevels in workers. The reduction in activity of AChEcorrelated well with increased TBARS and decreasedFRAP in OP formulators. It is concluded that OP-formulating workers are exposed to more oxidativestress. The measurement of erythrocyte AChE activity inpesticide workers who formulate OPs can be a goodmonitoring factor and is recommended to be performedin a regular manner. Human & Experimental Toxicology(2002) 21, 179±182.

Key words: human; organophosphorous; oxidative stress; pesticides

Introduction

Pesticide poisoning is an important cause of morbid-ity and mortality in many countries. It has beenestimated that there are 3 million cases of severepoisoning and 220 000 deaths every year in theworld.1 It has been well known that pesticide manu-facturing workers, even though using many securitysupplies, may have exposure to pesticides in theworkplace chronically. Occupational exposure occursin the mixing and loading of equipment in pesticidefactories usually. Among pesticides, organophospho-rous (OP) compounds are the most commonly usedinsecticides and some of them have been misused aswar weapons in the world.2 Many dangerous effectsof OPs in acute poisoning cases result from inhibi-tion of blood cholinesterase (ChE) activity includingplasma and erythrocyte.3 It is well known that ace-

tylcholinesterase (AChE) that is located in erythro-cyte membranes can be an indicator of chronicexposure to OPs.4,5 Also there is evidence that AChEinhibition correlates with OP-induced symptoms oftoxicity.4,6 On the other hand, it has been reportedthat OPs may induce oxidative stress on acuteexposure in humans7,8 and animals.9,10 The termoxidative stress refers to the situation of imbalancebetween production of free radicals and anti-oxidantdefense.11 Principally, oxidative stress in human canresult from diminished body anti-oxidants likegamma glutamyl transpeptidase (GGT) or increasedproduction of reactive oxygen species (ROS), whichleads to lipid peroxidation (LPO).11 Measurement ofLPO products, e.g., malondialdehyde (MDA), andtotal anti-oxidant capacity of the blood or totalthiol groups is an effective marker to study oxygenfree radicals (OFRs) effects in the body. Althougha few reports are available on LPO products andanti-oxidant status in experimental animals with OPpesticides, no detailed report is available on workersof OP-formulating factories. Therefore, we were inter-ested to examine whether workers of a pesticide

© Arnold 2002 10.1191/0960327102ht238oa

*Correspondence: Prof Mohammad Abdollahi, Department ofToxicology and Pharmacology, Faculty of Pharmacy, Tehran Uni-versity of Medical Sciences, Tehran 14155-6451, Iran.E -mail: mabdol@hbi.dmr.or.ir

Received 15 September 2001; revised 1 February 2002; accepted 24February 2002

Human & Experimental Toxicology (2002) 21, 179 ± 182

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factory who are chronically exposed to OPs may haveincreased LPO or decreased anti-oxidant capacity inrelation to AChE inhibition, which is known as abiomarker of OPs exposure.

Materials and methods

Chemicals

Acetylthiocholineiodide, quinidine, 5,50-dithiobis-2-nitrobenzoic acid (DTNB), tris base and 1,1,3,30- tetraethoxypropane (Sigma, Dorset, UK), hyamine1622 (benzethonium chloride), 2-thiobarbituric acid(TBA), n-butanol (Merck, Darmstadt, Germany),2,4,6-tripyridyl-s-triazine (TPTZ; Fluka, Milan, Italy),glycylglycine buffer and glupa-c-substrate (Zistche-mie, Tehran, Iran) were used in this study.

Human subjectsIn this retrospective cohort study, 45 male workers ofa pesticide factory at the age range of 23±55 years,with a minimum work history of 1 year and amaximum of 5 years, were included in this study.These workers were active in five work shifts weeklystarting at 7:30 a.m. and ending at 4 p.m. The mainwork of the workers was formulating of OPs duringlast 1 year. Age- and sex-matched control subjects(n=45) were volunteers from a food packaging plantbased on the socio-economic and nutrition status,who were not directly exposed to pesticides. Everysubject was asked to fill-out a questionnaire concern-ing his/her health status, the use of medications,occupational history, exposure to pesticides, and soforth, and sign a consent form to participate in thisstudy. Based on the questionnaire, subjects withhistory of diabetes mellitus, cigarette smoking, hyper-tension, other chronic illnesses or use of drugs wereexcluded from the study. Five milliliters of bloodfrom study subjects was obtained from the brachialvein.

AChE assay

Heparinized blood collected from subjects was cen-trifuged to separate red cells. Hemolysates wereprepared and frozen until assayed. The rate of hydro-lysis of acetylthiocholine by red cell suspension was

measured at 440 nm by the reaction of thiocholineiodide with DTNB, which gives the yellow 5-thio-2-bisnitrobenzoate anion as described previously.12

Thiobarbituric acid-reactive substances (TBARS)measurement

Plasma samples were mixed with trichloroacetic acid(20%) and the precipitate was dispersed in H2SO4

(0.05 M). TBA (0.2% in 2 M sodium sulfate) wasadded and heated for 30 min in boiling water bath.TBARS adducts were extracted by n-butanol andabsorbance was measured at 532 nm.13

Ferric-reducing ability of plasma (FRAP) assay

Anti-oxidant capacity of blood was determined bymeasuring the ability of plasma to reduce Fe3+ toFe2+. The complex between Fe2+ and TPTZ gives ablue color with absorbance at 593 nm.14

Total SH groups assay

Total SH groups of plasma were measured spectro-photometrically at 412 nm using DTNB as thereagent.15

GGT activity measurement

The absorbance of 5-amino-2-nitrobenzoate, whichresulted from reaction of L-®-glutamyl-3-carboxyl-4-nitranilide with glycylglycine in the presence of GGT,was measured at 405 nm.16

Statistical analysis

Student’s t-test was used to determine the significanceof differences observed between study groups. F-testwas done to determine the normal distribution ofvariances between groups. The correlation betweengroups was measured by Pearson test. P values greaterthan 0.01 were considered insignificant.

Results

The levels (mean‹SD) of TBARS, thiol groups, AChE,GGTand FRAPare demonstrated in detail in Table 1. Asignificant increase (P<0.001) in LPO was observedin pesticide workers, as recognized by determinationof TBARS. The values for pesticide workers andcontrols were 11.98‹0.75 and 7.60‹0.74 nmol/mL,

Table 1 Oxidative stress parameters and AChE activity in control and OP formulators

Control workers (n=45) OP workers (n=45) Student’s t-test F-test

Mean‹SD 95% CI Mean‹SD 95% CI t df P F P

AChE (kU/L) 14.93‹1.56 10.91 ±11.69 11.30‹1.3 14.46 ±15.40 11.99 88 <0.001 1.446 0.113TBARS (nmol/mL) 7.62‹0.74 7.40±7.84 11.98‹0.75 11.76 ±12.20 28.09 88 <0.001 1.006 0.493FRAP (·mol/mL) 1.60‹0.05 1.59±1.62 1.38‹0.05 1.36±1.39 19.83 88 <0.001 1.027 0.464GGT (U/L) 8561‹136 8521±8602 8765‹335 8564±8765 1.92 88 n.s. 6.06 44Total SH groups (mM) 0.25‹0.01 0.246 ±0.254 0.14‹0.01 0.133 ±0.140 42.33 88 <0.001 1.525 0.083

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respectively. The FRAP level was significantly lower(P<0.001) among pesticide workers in comparison tocontrols. The values for workers and controls were1.38‹0.05 and 1.60‹0.05 ·mol/mL, respectively.TotalSH groups of pesticide workers were significantly(P<0.01) lower than that of controls, 0.14‹0.01 versus0.25‹0.01 mM. AChE activity of pesticide workers(11.30‹1.3 kU/L) was significantly (P<0.001) lowerthan that of controls (14.93‹1.56 kU/L). No significantchange in GGT activity of pesticide workers and con-trols was observed. In pesticide workers, inhibitionof AChE activity correlated well with TBARS in-crease (r2=0.45, P<0.001, df=43) and FRAP decrease(r2=0.38, P<0.001, df=43). There was also significantcorrelation between TBARS increase and FRAP de-crease (r2=0.5, df=43, P<0.01).

Discussion

Despite the use of many protective tools in thepesticide industries, it is possible for everybody inthe workplace to be exposed to pesticides fumes,liquids or powder during the job. Measuring plasma(pseudocholinesterase) or erythrocyte (true ChE)activities is a good marker of exposure in OP-for-mulating industries. It is believed that the AChE,which is an erythrocyte membrane-bound enzyme,is more stable than the pseudo one and it is muchless variable due to exposure to anti-ChEs.17,18

Serum ChE may return to normal within a fewweeks because it is rapidly replaced by newenzymes synthesized in the liver.17 Therefore, meas-urement of erythrocyte AChE activity can be amarker of chronic exposure to OP pesticides anti-ChEs. Our previous study indicated that there is acorrelation between erythrocyte AChE inhibition andcomplained symptoms of toxicity in OP-formulatingpesticide workers.4 In the present study, AChE activ-ities of pesticide workers have been inhibited incomparison to that of controls (Table 1). In the meantime, these workers had a significantly increasedLPO and decreased thiol groups and anti-oxidantcapacity; the former and the latter were in goodcorrelation with inhibited activity of AChE. Role ofLPO and resulting oxidative stress has been reportedfor pesticides and OP exposure in animals. It wasreported that Bidrin as an OP induces renal tubularepithelial cell toxicity through elevation of ROS asrevealed by increased MDA formation and LPO.9

Also it has been reported that 15-day treatment ofrats by Quinalphos as an OP causes testicular tissuedamage due to free radical-mediated LPO.10 Inhumans, it has been reported that in acute malathionpoisoning, the level of TBARS, superoxide dismu-tase (SOD), catalase (CAT) and GGT are increased. In

the meantime, they measured AChE activity but theydid not find a correlation between AChE inhibitionand level of TBARS.7 Also there is evidence thatsubchronic exposure to malathion causes anincrease in the MDA and activities of SOD, CAT,glutathione peroxidase (GPx), glutathione reductase(GR) and glutathione S-transferase (GST) in theblood, suggesting that OPs may induce oxidativestress leading to generation of free radicals andalterations in anti-oxidants or OFR-scavengingenzymes.19 The role of oxidative stress and LPO asthe main mechanism of OP-induced myopathy20 andneurotoxicity21 in rats has been also found. In thepresent study, the anti-oxidant capacity of blood wasmeasured by FRAP assay. Since in FRAP assay theglutathione (GSH) level is not measured, thiolgroups were measured, which had a significantreduction, too. Proteins with sulphydryl groups areimportant in the defense against free radicalsbecause of the specific binding of transition metals,thereby reducing free radical generation. GSH is anearly consequence of oxidative stress. Oxidation ofGSH to glutathione disulfide (GSSG) may also resultin formation of mixed disulfides with sulphydrylgroups in proteins. The decreased FRAP level andthiol groups show that there is suppression in totalanti-oxidant capacity of the body. In supporting theabove-mentioned animal studies, the elevated con-centration of TBARS might be due to increasedperoxidation of membranes. Since oxidative stressis a balance between free radical production andanti-oxidant activityÐand regarding our resultsabout significant correlation between increasedTBARS and decreased anti-oxidant capacityÐit isconfirmed that the raised TBARS is due to decreasedanti-oxidant activity of the blood.

Conclusion

Pesticide workers are exposed to more oxidativestress as evidenced by increased concentrations ofplasma TBARS and changes in anti-oxidant status.The measurement of erythrocyte AChE activity inpesticide workers who formulate OPs is a good tox-icity monitoring factor and is recommended to beperformed in a regular manner. To overcome oxida-tive stress in pesticide workers, supplementation ofanti-oxidant vitamins may have beneficial effects.

Acknowledgement

The authors convey their great thanks to Dr AlirezaSedighi for his kind contribution in finding studysubjects.

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