Experimental and Toxicologic Pathology 63 (2011) 269–276

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology

0940-29

doi:10.1

n Corr

E-m

journal homepage: www.elsevier.de/etp

Effects on antioxidant status of liver following atrazine exposure and itsattenuation by vitamin E

Mohan Singh n, Rajat Sandhir, Ravi Kiran

Department of Biochemistry, Basic Medical Science Block, Panjab University, Chandigarh 160014, India

a r t i c l e i n f o

Article history:

Received 4 September 2009

Accepted 19 January 2010

Keywords:

Atrazine

Catalase

Glutathione-s-transferase

Glutathione peroxidase

Lipid peroxidation

Superoxide dismutase

Vitamin E

93/$ - see front matter & 2010 Elsevier Gmb

016/j.etp.2010.01.005

esponding author. Tel.: +91 172 2534133; fa

ail address: mohansran2k@yahoo.com (M. Sin

a b s t r a c t

In the present investigation, the effect of atrazine on antioxidant enzymes and body weight was studied

in male Wistar rats. Atrazine (300 mg/kg bw) was administered by gavage for 7, 14 and 21 days.

A significant increase in hepatic lipid peroxidation (LPO) was observed following atrazine administra-

tion. Vitamin E treatment (100 mg/kg bw), on the otherhand, attenuated atrazine-induced LPO in liver.

In addition, vitamin E treatment restored the GSH content and glucose-6-phosophate dehydrogenase

activity that was found to be lowered after atrazine administration. The activities of antioxidant

enzymes: superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase were

significantly increased following atrazine administration and vitamin E treatment could restore these

activities. In conclusion, the results of the study demonstrate that atrazine induces oxidative stress in

terms of enhanced lipid peroxidation. However, vitamin E treatment ameliorated the effects of atrazine

suggesting it as potential antioxidant against atrazine-induced oxidative stress.

& 2010 Elsevier GmbH. All rights reserved.

1. Introduction

Herbicides control or kill plants through a variety of mechan-isms, including the inhibition of biological processes, such asphotosynthesis, mitosis, cell division, enzyme function, rootgrowth, or leaf formation, interference with the synthesis ofpigments, protein or DNA, destruction of cell membranes; or thepromotion of uncontrolled growth (William et al., 1995). Atrazineis a triazine herbicide that is used as a selective pre-emergenceand post-emergence herbicide for the control of weeds inasparagus, maize, sorghum, sugarcane and pineapple. It is alsoused in forestry and for non-selective weed control on non-cropareas. It has been employed extensively in agriculture in the USand worldwide for over 40 years (Worthing 1991; US EPA, 1994).Atrazine is readily absorbed through the gastrointestinal tract. Onadministration of a single dose of atrazine (0.53 mg) to rats bygavage, 20% of it was excreted in feces within 72 h and 80% wasabsorbed across the gastrointestinal tract into the bloodstream(Hayes and Laws, 1990). Human exposure pathways for thischemical include occupational exposure through both inhalationand dermal absorption during its manufacture, its formulationand its application by spraying. Although, atrazine generally haslow level of bioaccumulation in fish, it does accumulate in brain,gall bladder, liver and gut of some fishes (Eisler, 1989). Therefore,consumption of contaminated fish can also contribute to human

H. All rights reserved.

x: +91 172 2541022.

gh).

exposure. Earlier studies from our laboratory have suggested thatatrazine induces genotoxicity in liver and alters erythrocytemembrane structure in rats (Singh et al., 2008a; 2008b).

Previously, Gojmerac et al. (1995) had reported hepaticdegeneration in pigs following atrazine exposure. Pesticides andherbicides induce hepatotoxicity, as liver is major site fordetoxification of these compounds. The detoxification reactionsof atrazine can be divided into phase I and phase II reactions. Themajor phase I metabolic reaction in plants and mammals iscytochrome P450-mediated N-dealkylation, while the phase IIreaction is the glutathione-s-transferases (GST) catalyzed con-jugation with glutathione (GSH) (Elia et al., 2002). Herbicides suchas paraquat are known to exert their effects by inducing oxidativestress in tissues of mammals and fish (Winston and DiGiulio,1991). To prevent oxidation-induced damage, there areeffective antioxidant systems in organisms. Some components ofthese systems are GSH and certain antioxidant enzymes includingfree radical scavenging enzymes, such as glutathione peroxidase(GPx), superoxide dismutase (SOD) and catalase. Other associatedantioxidant enzymes are glutathione reductase (GR) and GST.

Non-enzymatic antioxidants such as a-tocopherol (vitamin E),ascorbate (vitamin C), b-carotene (vitamin A), flavonoids (quer-cetin, rutin, etc.), selenium and thiol containing compounds suchas glutathione (GSH) can also act to overcome the oxidative stress,being a part of total antioxidant system (Sies et al., 1992). VitaminE is an important biological free radical scavenger in the cellmembranes (Horwitt, 1976). In the present investigation westudied whether vitamin E has the potential to attenuate atrazine-induced oxidative stress.

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276270

2. Materials and methods

2.1. Chemicals

Atrazine (technical grade 97.83%) was a gift from MeghmaniIndustries Ltd. (India). Vitamin E (a-tocopheryl acetate, tradename – Evion) was obtained from Merck Pharmaceuticals, India.All other chemicals used were of analytical grade procured fromlocal commercial sources.

2.2. Animals

Male rats (wistar strain), weighing about 100–120 g, wereused throughout the studies. The animals were procured fromthe Central Animal House of the Panjab University. The animalswere housed in polypropylene cages, provided with water andstandard pellet diet ad libitum.

2.3. Experimental design

Animals were randomly segregated into the four groups witheach group having 18 animals. Animals in each group werefurther subdivided into three sub-groups and were treated withatrazine and/or vitamin E daily for a period of 7, 14 and 21 days asdescribed below. Atrazine was given at a dose of 300 mg/kg bodyweight as it was the maximum tolerated dose based on the pilotstudy conducted with various doses of atrazine and also based onthe reported doses (Narotsky et al., 2001). The dose of vitamin Eused was based on the dose that has been shown to be mosteffective in lowering toxicity of various xenobiotics (Yousef et al.,2006). All the experiments performed were according to guide-lines for use and care of laboratory animals and were approved bythe ethical committee of the Panjab University.

Control (vehicle): Animals were given 1 ml of corn oil, orally.Atrazine treated: Animals were given atrazine (300 mg/kg body

weight) dissolved in corn oil, orally.Vitamin E treated: Animals were given vitamin E (100 mg/kg

body weight) dissolved in corn oil, orally.Atrazine+Vitamin E treated: Animals were given atrazine

(300 mg/kg body weight) along with vitamin E (100 mg/kg bodyweight) dissolved in corn oil, orally.

2.4. Body weight and liver weight

The body weight and liver weight of the animals weremeasured at the end of treatment periods.

2.5. Sample collection

At the end of 7 days, 14 days and 21 days, overnight fasted ratswere sacrificed. Livers of the animals were removed, rinsed in icecold isotonic saline (0.9% w/v NaCl), blotted dry, and weighedseparately.

2.6. Preparation of samples

A 10% (w/v) tissue homogenate was prepared in 50 mM TrisHCL (pH 7.4) using Potter–Elvehjem glass homogenizer. Postmitochondrial supernatant (PMS) was prepared by centrifugingthe homogenate at 10,000 rpm for 10 min at 4 1C. The pellet wasdiscarded and supernatant thus obtained was referred to as PMS.Various biochemical parameters were assayed in the homogenateand post mitochondrial supernatant of rat liver.

2.7. Lipid peroxidation (LPO)

LPO in liver was estimated by the TBA reaction with themalonyldialdehyde, a product formed due to peroxidation oflipids by the method of Wills (1966). LPO was expressed as innmol of MDA formed/mg protein, using a molar extinctioncoefficient of MDA as 1.56�105.

2.8. Reduced glutathione (GSH)

Glutathione content was quantified in the PMS obtained fromliver according to the method described by Ellman et al. (1961).Samples were deproteinized by sulfosalicyclic acid followed bythe reaction of sulfhydryl groups of glutathione with DTNB (5, 50-dithiobis-2-nitrobenzoic acid) to produce a yellow coloredproduct 5-thio-2-nitrobenzoic acid, which is measured at412 nm. The results were expressed as nmol GSH/mg protein.

2.9. Antioxidant enzymes

Superoxide dismutase (SOD): SOD activity was assayed in thePMS according to the method of Kono (1978). The method isbased on the formation of blue formazan from the reaction ofnitro-blue tetrazolium (NBT) and superoxide radical (produced inthe incubation medium from hydroxylamine hydrochloride),which is monitored spectrophotometrically at 560 nm. The resultswere expressed as units/mg protein. One unit of superoxidedismutase represents the quantity of enzyme which produces 50%inhibition in the rate of reduction of NBT mediated by hydro-xylamine hydrochloride.

Catalase: Catalase activity was assayed in by the method ofLuck (1971). The results were expressed as mmol of H2O2

decomposed/min/mg protein.Glutathione peroxidase (GSH-Px): GSH-Px activity was mea-

sured in the PMS by the method of Paglia and Valentine (1967) asmodified by Lawrence and Burk (1976). The reaction measures therate of GSH oxidation by H2O2 catalyzed by the GSH-Px present inthe PMS. The rate of GSSG formation is measured by following thedecrease in absorbance at 340 nm as NADPH is converted toNADP+ by glutathione reductase. The results are expressed asnmol of NADPH oxidized/min/mg protein.

Glutathione-s-transferase: The GST activity was assayed accord-ing to the method of Habig et al. (1974). The GST activityis determined by measuring the rate of formation of conjugate ofGSH and 1-chloro-2,4-dinitrobenzene (CDNB), which in turn isdetermined spectrophotometrically at 340 nm. The GST activitywas measured as nmol of CDNB conjugated/min/mg protein.

Glucose-6-phosphate dehydrogenase (G-6-P D): G-6-P D wasassayed by method of Lohr and Waller (1963). The enzymeactivity is determined by measuring increase in absorbance at340 nm. The results are expressed as nmol NADPH formed/min/mg protein.

2.10. Protein estimation

Protein content was determined according to Lowry et al.(1951) using bovine serum albumin as standard.

2.11. Statistical analysis

All values were expressed as mean7standard deviation (SD)of six animals. Data were analyzed using one way analysis ofvariance (ANOVA) followed by Newman–Keul’s test for the pairwise comparisons between the various treated groups. Valueswith po0.05 were considered statistically significant.

Table 2Effect of vitamin E administration on liver weight of atrazine treated rats.

Treatment Liver weight/100 g body weight(g)

7 days 14 days 21 days

Control 4.8671.13 4.6470.60 4.2570.37

Atrazine 4.6270.38 3.8470.73 3.6970.14n

Vitamin E 4.8570.38 4.01471.05 4.3670.33

Atrazine+Vitamin E 4.7070.55 3.8670.27 3.8970.25

Values are expressed as Mean7SD; n=6

n Significantly different from control group (npo0.05).

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276 271

3. Results

3.1. Body weight and liver weight

The changes in the body weight of the animals of variousgroups are shown in Table 1. A progressive increase in the bodyweight of rats was observed in the control and vitamin E treatedgroups. Maximum gain in the body weight was observed in thevitamin E treated rats as compared to all other groups. Theadministration of atrazine resulted in the loss of body weight ofthe rats. The reduction in body weight was pronounced in animalstreated with atrazine for 21 days. After exposure with atrazine for14 and 21 days there was a decrease of about 15% and 24.98%,respectively. Treatment with vitamin E resulted in improvementin the body weight of the atrazine exposed rats in all the groups.The beneficial effect of vitamin E was observed after 14 days oftreatment. An increase in body weight by 15.39% and 20.53% wasobserved after 14 and 21 days of treatment, respectively. Thus, itcan be concluded that administration of vitamin E along withatrazine has beneficial effect on the body weight of the rats.

The liver weight data for animals in different groups ispresented in the Table 2. Atrazine administration resulted in adecrease in the liver weight at all time points, though the decreasewas statistically significant (po0.05) only after 21 days oftreatment. No significant change was observed in the liverweight of animals when vitamin E was administered along withatrazine.

Fig. 1. Effect of vitamin E administration on lipid peroxidation in liver of atrazine

treated rats. Values are expressed as Mean7SD; n=6; nsignificantly different from

control group (npo0.05); #significantly different from atrazine treated group

(#po0.05).

3.2. Effect on lipid peroxidation

The results of lipid peroxidation are shown in Fig. 1.Malondialdehyde (MDA) is the main oxidation product ofperoxidized polyunsaturated fatty acids and represents animportant index of lipid peroxidation (Elia et al., 2002).Administration of atrazine significantly (po0.05) increasedhepatic MDA levels in rats and the effect was more pronouncedwith time. An increase by 137.0% in MDA levels was observed inthe liver of the atrazine administered rats after 21 days. Vitamin Etreatment by itself resulted in a significant decrease inthiobarbituric acid reactive substances (TBARS) as compared tothe control group after day 14 and day 21 of the treatment(po0.05). Administration of vitamin E along with atrazineresulted in statistically significant (po0.05) decrease in lipidperoxidation in the liver of the rats when compared with animalstreated with atrazine alone. About 9–18% decrease in LPO was

Table 1Effect of vitamin E administration on body weight of atrazine treated rats.

Treatment Body weight (g)

7 days 14 days

Initial Final Initial

Control 100.5277.07 118.0273.56 111.677(+17.50)

Atrazine 102.0075.11 86.90710.07n 105.687(�15.10)

Vitamin E 105.0077.07 120.0075.10 106.667(+15.00)

Atrazine+Vitamin E 101.43710.69 93.5776.26 108.337(�7.86)

Values in parenthesis represent changes in body weight.Values are expressed as Mean

n Significantly different from control group (npo0.05).# Significantly different from atrazine treated group (#po0.05).

observed in the liver of atrazine+vitamin E treated group ascompared to the atrazine administered group. Increased LPO inthe liver of atrazine treated rats suggests that oxidative stress isinduced by atrazine administration.

21 days

Final Initial Final

5.77 140.9779.23 103.3375.81 136.6775.77

(+29.30) (+33.34)

4.22 88.9675.63n 104.4174.62 78.3379.24n

(�16.72) (�26.08)

11.54 146.66711.55 106.53711.55 149.86711.46

(+40.00) (+43.33)

9.83 125.0077.61# 105.0075.00 126.5679.32#

(+16.67) (+21.56)

7SD; n=6

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276272

3.3. Effect on reduced glutathione

GSH displays remarkable metabolic and regulatory versatility.GSH/GSSG is the most important redox couple and plays crucialrole in antioxidant defense system. Results of our experiments(Fig. 2) show that GSH content in the liver of the atrazineadministered rats were significantly decreased (po0.05) ascompared to control group. The GSH content decreased by25.26%, 31.22% and 40.13% in the atrazine treated group after 7,14 and 21 days of the administration, respectively. However, theGSH content was found to be significantly increased in ratstreated with atrazine along with vitamin E.

Fig. 3. Effect of vitamin E administration on the activity of superoxide dismutase

in liver of atrazine treated rats. Values are expressed as Mean7SD; n=6;nsignificantly different from control group (npo0.05); #significantly different from

atrazine treated group (#po0.05).

3.4. Effect on superoxide dismutase

Changes in the activities of SOD of the liver of rats afteradministration of atrazine and/or vitamin E are depicted in theFig. 3. Administration with atrazine resulted in increased activityof the enzyme in the liver at all time points. However, increase inactivity was not significant after 7 days of exposure. A significant(po0.05) increase in the activity of SOD was observed after14 and 21 days of treatment (Fig. 3). The activity increasedby 28.71% and 45.3% after 14 and 21 days of atrazineadministration, respectively. However, administration of vitaminE along with atrazine restored the activity of the enzyme tocontrol values. A significant improvement was observed in theliver SOD activity after 14 days (22.99%) and after 21 days(19.88%) as compared to atrazine treated rats.

3.5. Effect on catalase

The alterations in the catalase activity after different treat-ments in rats is shown in Fig. 4. The catalase activity wassignificantly (po0.05) increased in the liver of rats treated withatrazine. The increase was 31.17% and 47.49% after 14 and 21 daysof atrazine administration, respectively. However, administrationof vitamin E along with atrazine resulted in significant decrease

Fig. 2. Effect of vitamin E administration on glutathione content (reduced) in the

liver of atrazine treated rats. Values are expressed as Mean7SD; n=6;nsignificantly different from control group (npo0.05); #significantly different

from atrazine treated group (#po0.05).

Fig. 4. Effect of vitamin E administration on the activity of catalase in liver of

atrazine treated rats. Values are expressed as Mean7SD; n=6; nsignificantly

different from control group (npo0.05); #significantly different from atrazine

treated group (#po0.05).

(po0.05) in the catalase activity in the liver after 14 and 21 daysof atrazine exposure.

3.6. Effect on glutathione peroxidase

It is clearly evident from the data in Fig. 5 that atrazineadministration led to an increase in the activity of glutathioneperoxidase (GPx) in the liver as compared to the control group.The increase in the activity of the enzyme was statisticallysignificant (po0.05) after 7 days of treatment and was found tobe dependent on duration of exposure. It was observed that therewas an increase by 44.09% (14 days) and 142.54% (21 days) in the

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276 273

liver of the atrazine exposed group as compared to the controlgroup. A significant (po0.05) improvement in the glutathioneperoxidase activity was observed after 14 and 21 days by vitaminE treatment to atrazine exposed animals.

3.7. Effect on glutathione-s-transferase

In the present investigation, glutathione-s-transferase (GST)activity increased significantly in the liver of the rats exposed toatrazine (Fig. 6). Time dependent increase was observed followingatrazine exposure. Maximum increase was observed after 21 daysof the administration (95.49%). Administration of vitamin E alongwith atrazine resulted in decrease in the activity of enzyme by

Fig. 5. Effect of vitamin E administration on the activity of glutathione peroxidase

in liver of atrazine treated rats. Values are expressed as Mean7SD; n=6;nsignificantly different from control group (npo0.05); #significantly different from

atrazine treated group (#po0.05).

Fig. 6. Effect of vitamin E administration on the activity of glutathione-s-

transferase in liver of atrazine treated rats. Values are expressed as Mean7SD;

n=6; nsignificantly different from control group (npo0.05); #significantly different

from atrazine treated group (#po0.05).

Fig. 7. Effect of vitamin E administration on the activity of glucose-6-dehydro-

genase in liver of atrazine treated rats. Values are expressed as Mean7SD; n=6;nsignificantly different from control group (npo0.05); #significantly different from

atrazine treated group (#po0.05).

35.52% in the liver of the rats as compared to atrazine exposedgroup.

3.8. Effect on glucose-6-phosphate dehydrogenase

Fig. 7 shows the changes in the activity of the hepatic glucose-6-phosphate dehydrogenase (G6PD) in the rats after varioustreatments. From the data it is clearly evident that atrazineadministration resulted in significant decrease in the activity ofthe enzyme in rat liver after all the treatment periods. Thedecrease was most pronounced after 21 days of treatment. Adecrease by 15.25% (14 days) and 31.62% (21 days) was observedfollowing atrazine administration as compared to respectivecontrol groups. Vitamin E showed its anti-oxidative effect byimproving the levels of enzyme in liver nearly to the control levelswhen administered along with atrazine. A significant (po0.05)increase by 29.93% was found after 21 days of treatment ascompared to atrazine administered group.

4. Discussion

The decrease in the body weight following atrazine adminis-tration is in accordance with reports in literature of decrease inbody weight following exposure to pesticides (Kennedy, 1986;Sharma et al., 2005). Roloff et al. (1992) observed decrease in bodyweight after the atrazine administration in mice. The decrease inthe body weight after atrazine administration could be due toreduced diet intake or due to necrotic changes in the various bodytissues (Gojmerac et al.,1995). The gain in body weight observedafter vitamin E administration to atrazine exposed animals mightbe due to the protective effect of vitamin E against atrazinetoxicity. Numerous reports are available in the literature showingprotective effect of antioxidants against the pesticide-inducedtoxicity. Improvement in the body weight of the animals after theadministration of antioxidants like vitamin C and zinc inxenobiotics induced stress have been reported by Patel andChinoy (1997). Leibovitz et al. (1990) have reported significant

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276274

increase in body weight of the rats after supplementation withantioxidants like vitamin E, b-carotene and selenium.

Exposure of experimental animals to pesticides is knownto induce lipid peroxidation in various tissues, which is respon-sible for the adverse biological effects (Sharma et al., 2005;El-Demerdash et al., 2004; Kamboj et al., 2006). Mechanism ofpesticide toxicity has been usually associated with the increase oflipid peroxidation in liver (Sharma et al., 2005; Datta et al., 1994).Reactive oxygen species such as superoxide anions, hydroxylradicals and hydrogen peroxide enhance the oxidative processand induce peroxidative damage to membrane lipids. IncreasedLPO may be one of the molecular mechanisms involved in theatrazine-induced toxicity.

Vitamin E treatment attenuated atrazine-induced lipid perox-idation in liver. Previous studies from our laboratory suggestedthe protective role of vitamin E against the atrazine-inducedgenotoxicity in the liver and biochemical changes in theerythrocytes membranes (Singh et al., 2008a; 2008b). Protectiverole of antioxidants has also been suggested in the case of toxicityinduced by other pesticides (Numan et al., 1990; Bagchi et al.,1993). Vitamin E allows free radicals to abstract a hydrogen atomfrom the antioxidant molecule rather than from polyunsaturatedfatty acids (PUFA), thus breaking the chain of free radicalreactions. The resulting antioxidant radical is relatively a non-reactive species (Pascoe et al., 1987). The present results indicatethat vitamin E treatment results in decreased LPO in the liver andprotects it from atrazine-induced oxidative stress.

A Significant decrease in GSH content in the liver of rats afteratrazine exposure indicated pro-oxidant conditions in the liver.Decrease in GSH levels after administration of various pesticidesis well documented in literature (Konstantinova and Russanov,1999; Hazarika et al., 2001; Thapar et al., 2002; Prasamthi et al.,2005; Singh et al., 2006). It has been reported by variousresearchers that GSH plays an important role in protecting cellsfrom xenobiotic-induced tissue injury (Reed and Fariss, 1984; Wuet al., 2004). The reduced levels of GSH in the atrazineadministered rats could be the result of either increasedutilization of GSH for conjugation and/or participation of GSH asan antioxidant in terminating free radicals produced due toatrazine-induced toxicity. Administration of vitamin E along withatrazine resulted in restoration of GSH levels to nearly controlvalues.

Cells have wide array of enzymatic and non-enzymaticantioxidant defense systems. The enzymatic antioxidant systemincludes superoxide dismutase (SOD), catalase, glutathioneperoxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GST), glucose-6-phosphate dehydrogenase, etc.These enzymes play an important role in countering the oxidativestress induced due to the formation of reactive oxygen species.

SOD provides the first line of defense against oxygen derivedfree radicals. SOD activity decreases oxidative stress by dismuta-tion of O2

� (McCord and Fridovich, 1969). The increase insuperoxide dismutase activity after atrazine administrationappears to be a adaptive response to increased generation ofreactive oxygen species. It has been reported in the literature thatexposure of animals to xenobiotics increases SOD activity invarious tissues (Datta et al., 1992; John et al., 2001). The increasein the activity of SOD in our study reflects compensatorymechanism to increased oxidative stress. Vitamin E, being anantioxidant, reduces the oxidative stress and hence normalizesSOD activity to some extent.

Endogenous H2O2 may be converted either by catalase orglutathione peroxidase to H2O (Kehrer, 1993) or otherwise it maygenerate the highly reactive free hydroxyl radical (OH�) by theFenton reaction (Harber and Weiss, 1934), which is widelybelieved to be responsible for oxidative damage (Fridovich,

1978; Chance et al., 1979; Halliwell and Gutteridge, 1984). Theelevated activity of catalase is an adaptive response like SODagainst enhanced generation of free radicals (Koner et al., 1998).Vitamin E showed the ameliorating effect on the atrazine-inducedincrease in catalase activity.

GPx converts H2O2 or other lipid peroxides to water orhydroxyl lipids and in the process GSH is converted to oxidizedglutathione (GSSG). We observed increase in GPx activityfollowing atrazine exposure. Sharma et al., (2005) reported anincreased glutathione peroxidase activity in the liver of rats aftertreatment with dimethoate. However, Elia et al. (2002) havereported significant increase in the activity of hepatic GPx but nochange in the activity of GPx in the gills of fish following atrazineexposure. The results of our study indicate that atrazine-induceddecrease in the GSH content could be due to increase in theactivity of GPx. The observed increase in the activity ofglutathione peroxidase upon atrazine administration might bethe natural mechanism to counteract the pro-oxidant effect ofatrazine toxicity. Administration of vitamin E along with atrazineshowed protective effect against atrazine-induced oxidativestress.

In rodents the dominant phase I metabolic reaction foratrazine is cytochrome P450-mediated N-dealkylation as demon-strated by urine analysis and liver fraction studies (Lang et al.,1996; Hanioka et al., 1999a; 1999b). Phase II biotransformation ofatrazine may also occur: several studies have demonstrated GSHconjugation of the parent compounds by rat and mouse liverfractions (Timchalk et al., 1990; Egaas et al., 1993). Thoughmetabolic pathways of atrazine in humans have not been fullycharacterized, presence of atrazine mercapturates in human urineindicates that GSH conjugation is an important route ofbiotransformation of atrazine in humans (Lucas et al., 1993;Jaeger et al., 1998; Buchholz et al., 1999). In the present study,atrazine induced the activity of GST in the liver of rats. Increasedactivity of the GST suggested increased production of glutathioneconjugates or metabolites that resulted in decreased content ofthe GSH in the liver. Various workers have reported increasedactivity of GST in rats after administration of pesticides (Schoketand Vincze, 1990; Thapar et al., 2002; El-Demerdash et al., 2004;Singh et al., 2006). Sharma et al., (2005) have reported increase inthe activity of hepatic GST after dimethoate administration torats. This can be understood in a view of the fact that pesticidesget detoxified mainly through GST catalyzed reaction, utilizingGSH. Pesticides are expected to induce the activity of GST as apotent protective mechanism of the organism. Present investiga-tions suggest that vitamin E treatment tends to reduce theatrazine-induced toxicity.

Glucose-6-phosophate dehydrogenase functions as the firstand rate-limiting enzyme in the pentose phosphate pathway,responsible for generation of NADPH in a reaction coupled to theoxidation of glucose-6-phosophate. The major role of NADPH(generated by G6PD) is the regeneration of reduced glutathione,which detoxifies hydrogen peroxide and oxygen radicals in thecell (Weksler et al., 1990; Deutsch, 1983). Thus, decrease in theactivity of G6PD enzyme may be one of the causes of increasedoxidative stress, observed after atrazine exposure. The presentresults are in accordance with the previous studies from ourlaboratory for various pesticides in rats (Thapar et al., 2002; Singhet al., 2006). Administration of vitamin E along with atrazinereduces the atrazine-induced oxidative stress by recovering G6PDactivity.

The molecular mechanism involved in the generation of freeradicals by atrazine in liver is not known. The reports availablefrom studies with plants suggest that atrazine-induced toxicitycould be related to inefficient activation of singlet oxygen (1O2)quenching pathways leading to 1O2 accumulation. In addition,

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276 275

atrazine treatment also increased hydrogen peroxide (H2O2)content, while reducing gene expression and enzymatic activitiesrelated to two major H2O2-detoxification pathways (Ramel et al.,2009).

5. Conclusion

From the above observations it can be concluded that exposureof atrazine results in increased oxidative stress and alteredantioxidant status of the liver. Administration of vitamin E alongwith atrazine resulted in partial normalization of the toxic effectsof atrazine thus highlighting the protective action of vitamin E.

References

Bagchi D, Hassoun EA, Bagchi M, Stohs SJ. Protective effects of antioxidants againstendrin-induced hepatic lipid peroxidation, DNA damage and excretion ofurinary lipid metabolites. Free Radical Biol Med 1993;15:217–22.

Buchholz BA, Fultz E, Haack KW, Vogel JS, Gilman SD, Gee SJ, Hammock BD, Hui X,Wester RC, Maibach HI, HPLC-accelerator MS. measurement of atrazinemetabolites in human urine after dermal exposure. Anal Chem1999;71:3519–25.

Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs.Physiol Rev 1979;59:527–605.

Datta C, Gupta J, Sengupta D. Interactions of organophosphorous insecticidesphosphamidon and malathion on lipid profile and acetylcholinesterase activityin human erythorocyte membrane. Indian J Med Res 1994;100:87–9.

Datta J, Gupta (Dasgupta) J, Sarkar A, Sengupta D. Effects of organophosphorusinsecticide phosphomidon on antioxidant defense components of humanerythrocytes and plasma. Indian J Exp Biol 1992;30:65–7.

Deutsch J. Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, Bergmeyer J,editors. Methods of enzymatic analysis. Berlin: Verlagsgerellschaff; 1983. p.190–6.

Egaas E, Skaare JU, Svendsen NO, Sandvik M, Falls JG, Dauterman WC, Collier TK,Netland J. A comparative study of effects of atrazine on xenobioticsmetabolizing enzymes in fish and insects, and of the in vitro phase-II atrazinemetabolism in some fish, insects, mammals, and one plant species. CompBiochem Physiol C 1993;106:141–9.

Eisler R. Atrazine hazards to fish, wildlife, and invertebrates: a synoptic review. USfish and wildlife service. Biol Rep 1989;85(1):18.

El-Demerdash FM, Yousef MI, Kedwany FS, Baghdadi HH. Role of alpha-tocopheroland beta-carotene in ameliorating the fenvalerate-induced changes inoxidative stress, hemato-biochemical parameters, and semen quality of malerats. J Environ Sci Health B 2004;39:443–59.

Elia AC, Waller WT, Norton SJ. Biochemical response of bluegill sunfish (Lepomismacrochirus, Rafinesque) to atrazine induced oxidative stress. Bull EnvironContam Toxicol 2002;68:809–16.

Ellman GL, Courtney KD, Andres Jr. V, Feather-Stone RM. A new and rapidcolorimetric determination of acetylcholinesterase activity. Biochem Pharma-col 1961;7:88–95.

Fridovich I. In: Proyer WQ, editor. Free radicals in biology, vol 1. New York:Academic Press; 1978. p. 239.

Gojmerac T, Kartal B, Zuric M, Curic S, Mitak M. Serum biochemical andhistopathological changes related to the hepatic function in pigs followingatrazine treatment. J Appl Toxicol 1995;15:233–6.

Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-transferase. The first enzymaticstep in mercapturic acid formation. J Biol Chem 1974;249:7130–9.

Halliwell B, Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals anddisease. Biochem J 1984;219:1–14.

Hanioka N, Jinno H, Tanaka-Kagawa T, Nishimura T, Ando M. In vitro metabolism ofchlorotiazines: characterization of simazine, atrazine, and propazine metabo-lism using liver microsomes from rats with various cytochrome P450 inducers.Toxicol Appl Pharmacol 1999a;156:195–205.

Hanioka N, Jinno H, Tanaka-Kagawa T, Nishimura T, Ando M. In vitro metabolism ofsimazine, atrazine, and propazine by hepatic cytochrome P450 enzymes of rat,mouse and guinea pig and oestrogenic activity of chlorotriazines and theirmain metabolites. Xenobiotica 1999b;29:1213–26.

Harber F, Weiss J. The catalytic decomposition of dehydrogen peroxide by ironsalts. Proc R Soc London A 1934;147:332.

Hayes WJ, Laws ER. Handbook of pesticide toxicology. Classes of pesticides, vol. 3.New York: Academic Press, Inc.; 1990.

Hazarika A, Sarkar SN, Kataria M. Subacute toxicity of anilofos, a new organopho-sphorus herbicide in male rats: effect on lipid peroxidation and ATPaseactivity. Indian J Exp Biol 2001;39:1113–7.

Horwitt MK. Vitamin E: a reexamination. Am J Clin Nutr 1976;29:569–78.Jaeger LL, Jones AD, Hammock BD. Development of an enzyme-linked immuno-

sorbent assay for atrazine mercapturic acid in human urine. Chem Res Toxicol1998;11:342–52.

John S, Kale M, Rathore N, Bhatnagar D. Protective effect of vitamin E indimethoate and malathion induced oxidative stress in rat erythrocytes. J NutrBiochem 2001;12:500–4.

Kamboj A, Kiran R, Sandhir R. Carbofuran-induced neurochemical and neurobe-havioural alterations in rats: attenuation by N-acteylcysteine. Exp Brain Res2006;170:567–75.

Kehrer JP. Free radicals as mediator of tissue injury and disease. Crit Rev Toxicol1993;23:21–48.

Kennedy Jr GL. Chronic toxicity, reproductive, and teratogenic studies withoxamly. Fundam Appl Toxicol 1986;7:106–18.

Koner BC, Banerjee BD, Ray A. Organochlorine pesticide-induced oxidative stressand immune suppression in rats. Indian J Exp Biol 1998;36:395–8.

Kono Y. Generation of superoxide radical during autoxidation of hydroxylamineand an assay for superoxide dismutase. Arch Biochem Biophys 1978;186:189–95.

Konstantinova SG, Russanov EM. Studies on paraquat-induced oxidative stress inrat liver. Acta Physiol Pharmacol Bulg 1999;24:107–11.

Lang D, Criegee D, Grothsen A, Saalfrank RW, Bocker RH. In vitro metabolism ofatrazine, terbuthylazine, ametryne and terbutryne in rats, pigs, and humans.Drug Metab Dispos 1996;24:859–65.

Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient ratliver. Biochem Biophys Res Commun 1976;71:952–8.

Leibovitz B, Hu ML, Tappel AL. Dietary supplements of vitamin E, beta-carotene,coenzyme Q10 and selenium protect tissues against lipid peroxidation in rattissue slices. J Nutr 1990;120:97–104.

Lohr GW, Waller HD. Glucose 6 phosphate dehydrogenase. In: Bergmeyer HU,editor. Methods of enzymatic analysis. New York and London: AcademicPress; 1963. p. 744.

Lowry OH, Rosenbrough NJ, Farr AL, Randall RL. Protein measurement with thefolin phenol reagent. J Biol Chem 1951;193:265–75.

Lucas AD, Jones AD, Goodrow MH, Saiz SG, Blewett C, Seiber JN, Hammock BD.Determination of atrazine metabolites in human urine: development of abiomarker of exposure. Chem Res Toxicol 1993;6:107–16.

Luck H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. NewYork: Academic Press; 1971. p. 885–93.

McCord JM, Fridovich I. Superoxide dismutase: an enzymatic function forerythrocuprein (hemocuprein). J Biol Chem 1969;244:6049–55.

Narotsky MG, Best DS, Guidici DL, Cooper RL. Strain comparisons of atrazine-induced pregnancy loss in the rat. Reprod Toxicol 2001;15:61–9.

Numan IT, Hassan MQ, Stohs SJ. Protective effect of antioxidants against endrin-induced lipid peroxidation, glutathione depletion and lethality in rats. ArchEnviron Contam Toxicol 1990;19:302–6.

Paglia DE, Valentine WN. Studies on quantitative and quantitative characterizationof erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69.

Pascoe G, Olafsdottir K, Read DJ. Vitamin E protection against chemical inducedcell injury. I. Maintenance of cellular protein thiols as a cytoprotectivemechanism. Arch Biochem Biophys 1987;256:150–8.

Patel D, Chinoy NJ. Synergistic action of ascorbic acid and calcium in mitigation offluoride induced toxicity in uterus of mice. Indian J Environ Toxicol 1997;7:16–9.

Prasamthi K, Muralidhara, Rajini PS. Fenvelrate induced oxidative damage in rattissues and its attenuation by dietary sesame oil. Food Chem Toxicol2005;43:299–306.

Ramel F, Sulmon C, Bogard M, Couee I, Gouesbet G. Differential patterns of reactiveoxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biol. 2009;9:28.

Reed DJ, Fariss MW. Glutathione depletion and susceptibility. Pharmacol Rev1984;36:25S–33S.

Roloff BD, Belluck DA, Meisner LF. Cytogenetic studies of herbicide interactionin vitro and in vivo using atrazine and linuron. Arch Environ Contam Toxicol1992;22:267–71.

Sharma Y, Bashir S, Irshad M, Nag TC, Dogra TD. Dimethoate-induced effects onantioxidant status of liver and brain of rats following subchronic exposure.Toxicology 2005;215:173–81.

Sies H, Stahl W, Sundquist AR. Antioxidant functions of vitamins. Vitamins E and C,beta-carotene, and other carotenoids. Ann NY Acad Sci 1992;669:7–20.

Singh M, Kaur P, Sandhir R, Kiran R. Protective effects of vitamin E against atrazine-induced genotoxicity in rats. Mutat Res 2008a;654:145–9.

Singh M, Sandhir R, Kiran R. Atrazine induced alterations in rat erythrocytemembranes: ameliorating effect of vitamin E. J Biochem Mol Toxicol2008b;22:363–9.

Singh M, Sandhir R, Kiran R. Erythrocyte antioxidant enzymes in toxicologicalevaluation of commonly used organophosphate pesticides. Indian J Exp Biol2006;44:580–3.

Schoket B, Vincze I. Dose related induction of rat hepatic drug metabolizingenzymes by diuron and chlorotoluron, two substituted phenyl-urea herbi-cides. Toxicol Lett 1990;50:1–7.

Thapar A, Sandhir R, Kiran R. Acephate induced oxidative damage in erythrocytes.Indian J Exp Biol 2002;40:963–6.

Timchalk C, Dryzga MD, Langvardt PW, Kastl PE, Osborne DW. Determination ofthe effect of tridiphane on the pharmacokinetics of [14C]-atrazine followingoral administration to male Fischer 344 rats. Toxicology 1990;6:27–40.

US Environmental Protection Agency. Atrazine, simazine and cyanosine: notice ofinitiation of special review. Fed Regist 1994;59:60412–43.

Weksler BB, Moore A, Tepler J. Hematology. In: Andreoli TE, Carpenter CCJ, Plum F,Smith LH, editors. Cecil essentials of medicine. Philadelphia: WB SaundersCompany; 1990.

M. Singh et al. / Experimental and Toxicologic Pathology 63 (2011) 269–276276

William RD, Burrill LC, Ball D, Miller TL, Parker R, Al-Khatib K, Callihan RH, EberleinC, Morishita DW. Pacific northwest weed control handbook, OR. Corvallis:Oregon State University Extension Service; 1995. 358.

Wills ED. Mechanism of lipid peroxide formation in animal tissues. Biochem J1966;99:667–76.

Winston GW, Di Giulio RT. Prooxidant and antioxidant mechanisms in aquaticorganisms. Aquat Toxicol 1991;19:116–37.

Worthing CR. The pesticide manual, 9th ed. Farnham: British Crop ProtectionCouncil; 1991.

Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and itsimplications for health. J Nutr 2004;134:489–92.

Yousef MI, Awad TI, Mohamed EH. Deltamethrin-induced oxidative damage andbiochemical alterations in rat and its attenuation by Vitamin E. Toxicology2006;227:240–7.