Atrazine-induced alterations in rat erythrocyte membranes: Ameliorating effect of vitamin E

J BIOCHEM MOLECULAR TOXICOLOGYVolume 22, Number 5, 2008

Atrazine-Induced Alterations in Rat ErythrocyteMembranes: Ameliorating Effect of Vitamin EMohan Singh, Rajat Sandhir, and Ravi KiranDepartment of Biochemistry, Panjab University, Chandigarh 160 014, India; E-mail: [email protected]

Received 18 January 2008; revised 29 May 2008; accepted 6 June 2008

ABSTRACT: Erythrocytes are a convenient model tounderstand oxidative damage to the membranes in-duced by various xenobiotics. The objective of thepresent study was to investigate the propensity ofatrazine to induce oxidative stress and its possi-ble attenuation by vitamin E. Experimental animalswere orally administered atrazine (300 mg kg−1 bodyweight, daily) and vitamin E (100 mg kg−1 bodyweight, daily) for a period of 7, 14, and 21 days.Erythrocyte membranes were prepared and analyzedfor acetylcholinesterase (AChE) activity, lipid perox-idation (LPO), and lipid composition. Susceptibilityof erythrocytes to atrazine exposure was further in-vestigated in terms of morphological alterations byscanning electron microscopy (SEM). Results indi-cate that atrazine exposure caused a significant in-hibition of AChE activity and induction of oxida-tive stress in terms of increased malondialdehyde(MDA) levels. Atrazine treatment significantly de-creased total lipid, cholesterol, and phospholipid con-tent of erythrocyte membranes. SEM revealed vary-ing degrees of distortion depending on duration ofatrazine exposure. However, administration of vita-min E ameliorated the oxidative stress and changesin the erythrocyte membranes induced by atrazine.C© 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol22:363–369, 2008; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10:1002/jbt.20249

KEYWORDS: Atrazine; Erythrocyte Membranes; LipidPeroxidation; Vitamin E; Scanning ElectronMicroscopy

INTRODUCTION

Herbicides are the most rapidly growing class ofthe pesticides used because of the increase in labor

Correspondence to: M. Singh.c© 2008 Wiley Periodicals, Inc.

cost and low rotation of crops [1]. Atrazine is a tri-azine herbicide that is used as a selective pre and post-emergence herbicide for the control of weeds in aspara-gus, maize, sorghum, sugarcane, and pineapple fields.It is also used in forestry and for nonselective weed con-trol on noncrop areas. It has been employed extensivelyin agriculture in the United States and worldwide formore than 40 years [2,3]. As a result of its indiscrim-inate use, residues of atrazine are found not only inplants, soil, water, and cultivated ground but also inagricultural products such as fruits, milk, butter, andsugar beet [4]. Earlier studies from our laboratory haveestablished that atrazine exposure led to DNA damagein the rat liver and erythrocytes [5]. Various studies in-dicate that toxic manifestations induced by pesticidesmay be associated with enhanced production of reac-tive oxygen species (ROS) [6,7]. Among ROS, superox-ide anions, hydroxyl radicals, and hydrogen peroxideenhance the oxidative process and induce peroxida-tive damage to membrane lipids. Hydroxyl radicalshave been proposed as initiators of lipid peroxidation(LPO) through an iron-catalyzed Fenton reaction inmembranes [8].

Erythrocytes are highly susceptible to oxidativedamage due to the presence of heme iron, polyunsat-urated fatty acids (PUFA) and oxygen, which may ini-tiate the reactions that induce oxidative changes in thered blood cells. Vitamin E is a lipid soluble antioxidantthat plays an important role in animal health by inac-tivating harmful free radicals produced through nor-mal cellular activities and from various stressors [9].In addition, vitamin E has important role in scaveng-ing free radicals and in stabilizing the cell membranes,thus maintaining their permeability and integrity [10].Because of the health problems induced by many en-vironmental pollutants, many efforts have been madeto evaluate the potency of vitamin E as an antioxidant.The present study was undertaken to evaluate the pos-sible beneficial effects of vitamin E against the atrazine-induced oxidative stress.

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364 SINGH, SANDHIR, AND KIRAN Volume 22, Number 5, 2008

MATERIALS AND METHODS

Chemicals

Technical-grade atrazine (97.83%) was a giftfrom Meghmani Industries Ltd. (Ahmedabad, India).Vitamin E (α-tocopheryl acetate, trade name Evion)was obtained from Merck Pharmaceuticals (Mumbai,India). All other chemicals used were of analyti-cal grade and were obtained from local commercialsources.

Animals

Male rats (Wistar strain), weighing about 100–120 g, were used throughout the studies. The animalswere procured from the Central Animal House of theuniversity. The animals were housed in polypropylenecages and were fed standard diet (Ashirwad Industries,Kharar, India) and were given water ad libitum.

Experimental Design

Animals were randomly segregated into the fol-lowing groups, each group having 18 animals. Animalsin each group were further subdivided into three sub-groups and the following treatments were given for aperiod of 7, 14, and 21 days:

• Control (vehicle): Animals were given 1 ml of cornoil, 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 and vitamin E treated: Animals were givenatrazine (300 mg/kg body weight) and vitamin E(100 mg/kg body weight) dissolved in corn oil,orally.

All the experiments were performed according toguidelines for use and care of laboratory animalsand were approved by the ethical committee of theuniversity.

Sample Collection

After completion of treatment periods, blood sam-ples were collected under light ether anesthesia fromsupraorbital sinus using glass capillaries into hep-arinized vacutainers (Becton–Dickinson, Oxford, UK).

Preparation of Erythrocyte Membranes

Erythrocyte membranes were prepared by themethod described by Rhoda [11]. Erythrocyte mem-

branes were prepared by centrifuging the blood sam-ples containing an anticoagulant at 1000 g at 4◦C for10 min. The pellet-containing cells were washed thricewith ice-cold saline. The buffy coat and plasma wereremoved at the end of each centrifugation. The os-motic lysis of erythrocytes was carried out by quicklyadding 10 volumes of distilled water to 1 volume ofpacked cells. After centrifugation at 20,000×g, the pos-themolytic residue was washed twice with 1.0 mM TrisEDTA (pH 7.4), then with 10 mM Tris EDTA (pH 7.4),and finally two times with 2 mM Tris HCl (pH 7.4). Thepreparation thus obtained was membrane residue andusually half of the original packed cell volume of thewashed cells. The erythrocyte membranes were keptfrozen at −60◦C for further analysis.

Biochemical Assays

Acetylcholinesterase. The activity of acetyl-cholinesterase (AChE) in erythrocyte membraneswas determined according to the method of Ellmanet al. [12].

Lipid Peroxidation. The lipid peroxidation in the ery-throcyte membranes was determined by the method ofWills [13]. TBA-MDA chromophore has been taken asan index of lipid peroxidation.

Erythrocyte Membrane Lipid Composition. Total lipidcontent of erythrocyte membranes was estimated ac-cording to the method of Frings and Dunn [14]. Phos-pholipid phosphorus was estimated by the method ofBartlett [15] as modified by Marinetti [16]. Cholesterolwas estimated according to the method of Zlatkis et al.[17].

Protein Estimation. Protein content was determinedaccording to Lowry et al. [18] using bovine serum al-bumin as the standard.

Scanning Electron Microscopy

Blood samples were fixed immediately after col-lection in 2.5% glutaraldehyde prepared in 0.2 M phos-phate buffer (pH 7.2). After 2 h of fixation, the cellswere centrifuged at 1000–1500 rpm. The fixative wasdiscarded and the pellet was resuspended in the phos-phate buffer. This process was repeated 2–3 times, andthe supernatant was discarded every time. The pelletwas suspended in distilled water and again centrifugedand reconstituted for 1–2 times in distilled water. Fi-nally, the pellet was suspended in a minimum amountof distilled water and a drop of sample was smeared onthe metallic SEM stubs, which were loaded with a con-ductive silver tape on their top. The stubs were coatedwith gold to a thickness of 100 A using sputter ioncoater using a gold source for 4–5 min. The samples

J Biochem Molecular Toxicology DOI 10:1002/jbt

Volume 22, Number 5, 2008 AMELIORATING EFFECT OF VITAMIN E 365

were observed under scanning electron microscope(JSM-6100, Jeol, Tokyo, Japan) at the Regional Sophisti-cated Instrumentation Center (RSIC) of the university.

Statistical Analysis

All values have been expressed as mean ± standarddeviation (SD) of six observations. Data were analyzedusing one way analysis of variance (ANOVA) followedby Newman–Keuls test for the pairwise comparisonsbetween the various treated groups. Values havingp < 0.05 were considered as statistically significant.

RESULTS

Effect on Acetylcholinesterase Activity

Effect of in vivo administration of atrazine and vita-min E on the activity of AChE in erythrocytes is shownin Figure 1. Atrazine treatment resulted in inhibition ofthe AChE activity in membranes isolated from erythro-cytes. A significant (p < 0.05) decrease in AChE activitywas observed after 7 days (22.32%), 14 days (25.58%),and 21 days (26.96%) on atrazine exposed rats as com-pared with control animals. Vitamin E administrationalong with atrazine resulted in a significant (p < 0.05)increase in the activity of AChE ranging between 12%and 14% as compared with the animals treated withatrazine alone.

Effects on Lipid Peroxidation

The results of lipid peroxidation are shown inFigure 2. Treatment of atrazine increased significantly

FIGURE 1. Effect of vitamin E administration on the activity ofacetylcholinesterase in erythrocyte membranes from atrazine-treatedrats. Values are expressed as mean ± SD; n = 6. ∗Significantly differentfrom the control group (∗ p < 0.05). #Significantly different from theatrazine-treated group (# p < 0.05).

(p < 0.05) MDA levels in erythrocyte membranes, andthe increase was more pronounced with the increasein the duration of atrazine treatment. An increase by218.29% in MDA levels was observed in erythrocytes ofatrazine-treated rats after 21 days of treatment. VitaminE treatment alone resulted in a significant decreasein MDA levels as compared with the control group(p < 0.05). Administration of vitamin E along withatrazine resulted in a significant (p < 0.05) decrease inLPO in erythrocyte membranes when compared withanimals treated with atrazine alone. The increase inLPO in erythrocyte membranes suggests that oxidativestress is induced by atrazine.

Effects on Membrane Lipid Composition

Figure 3 shows changes in the total lipid contentof erythrocyte membranes. Atrazine administration re-sulted in a decrease in the total lipid content of erythro-cyte membranes in all the groups. However, the effectwas significant only after 21 days of atrazine treatment.Twenty-four percent decrease in the total lipid contentof erythrocyte membranes was observed after 21 daysof atrazine administration. Vitamin E administrationalong with atrazine restored the total lipid content oferythrocyte membranes to normal level after 21 days ascompared with the atrazine-treated group. The presentfindings show that vitamin E alleviates the effect ofatrazine when given along with atrazine.

Figure 4 shows the effect of atrazine and atrazineand vitamin E administration on the cholesterol contentof erythrocyte membranes. The cholesterol content oferythrocyte membranes was found to decrease after the

FIGURE 2. Effect of vitamin E administration on lipid peroxidationin erythrocyte membranes from atrazine-treated rats. Values are ex-pressed as mean ± SD; n = 6. ∗Significantly different from the controlgroup (∗ p < 0.05). #Significantly different from the atrazine-treatedgroup (# p < 0.05).



FIGURE 3. Effect of vitamin E administration on total lipid contentof erythrocyte membranes from atrazine-treated rats. Values are ex-pressed as mean ± SD; n = 6. *Significantly different from the controlgroup (*p < 0.05). #Significantly different from the atrazine-treatedgroup (# p < 0.05).

atrazine treatment. A significant decrease, of 20.12%and 32.94%, was observed after 14 and 21 days ofatrazine administration, respectively. Administrationof vitamin E along with atrazine resulted in an increasein the cholesterol content in erythrocyte membranesas compared with atrazine-treated rats. The cholesterolcontent increased by 33.85% and 57.02% after 14 and21 days of administration of atrazine, respectively. Theresults show that vitamin E administration along withatrazine has beneficial effect on restoring the lipid com-position of erythrocyte membranes.

The data presented in Figure 5 show that in vivoadministration of atrazine to rats results in a decreasein the content of phospholipids in erythrocyte mem-branes. A significant decrease was observed in phos-pholipid content after 14 and 21 days of atrazine treat-ment. A decrease by 16.76% (14 days) and 37.20%

FIGURE 4. Effect of vitamin E administration on cholesterol contentof erythrocyte membranes from atrazine-treated rats. Values are ex-pressed as mean ± SD; n = 6. ∗Significantly different from the controlgroup (∗ p < 0.05). #Significantly different from the atrazine-treatedgroup (# p < 0.05).

FIGURE 5. Effect of vitamin E administration on phospholipidscontent of erythrocyte membranes from atrazine-treated rats. Valuesare expressed as mean ± SD; n = 6. ∗Significantly different from thecontrol group (∗ p < 0.05). #Significantly different from the atrazine-treated group (# p < 0.05).

(21 days) was observed after atrazine treatment as com-pared with the control groups. Administration of vi-tamin E along with atrazine resulted in 22.86% and57.92% increase in the phospholipid content of the ery-throcyte membranes as compared with groups treatedwith atrazine for 14 days and 21 days, respectively.

Morphological Changes in Erythrocytes

Morphological changes in erythrocytes were stud-ied for various groups using SEM, and the results areshown in electron micrographs. It is evident from theelectron micrograph that the erythrocytes of the controlgroup were perfect discocytes (D), that is, typical bicon-cave disks (Figure 6). Stomatocytes (St) were seen rarelyin the control group. Erythrocytes of rats treated with

FIGURE 6. Scanning electron micrograph of erythrocytes from ratsof the control group at 2500×, 10 kV.



FIGURE 7. A scanning electron micrograph of erythrocytes fromrats after administration of atrazine for 21 days at 2500×, 10 kV.

atrazine showed significant morphological changes ascompared with the control group (Figure 7). Some ofthe major alterations were mild to moderate distor-tion in shape, significant ruptured membranes, andechinocyte formation. Some of the erythrocytes showedcentral or peripheral protuberances. These deforma-tions might have occurred due to the underlying de-formation of cytoskeleton. A scanning electron micro-graph of erythrocytes of atrazine and vitamin E-treatedgroup (Figure 8) shows improved erythrocytes topog-raphy as compared with the atrazine-treated group.Extent of protuberances decreased significantly as com-pared with the control group.

FIGURE 8. A scanning electron micrograph of erythrocytes fromrats after administration of atrazine and vitamin E for 21 days at2500×, 10 kV.

DISCUSSION

Inhibition of AChE by carbamates andorganophosphates is well documented in the literature[19–22]. Kale et al. [23] found that erythrocyte andserum AChE activities were markedly decreased aftercypermethrin and/or fenvelrate treatment. Althoughthere are no reports in the literature on inhibitionof AChE by atrazine administration in experimentalanimals, it has been reported that atrazine has someneuromuscular toxicity at high doses, causing motorincoordination, limb paralysis, and respiratory distressin laboratory animals [24]. Atrazine was given at a doseof 300 mg/kg body weight as it was the maximumtolerated dose based on the pilot study conducted withvarious doses of atrazine and also based on the reporteddoses [25]. In the present investigation, a decrease inthe activity of erythrocyte AChE (membrane-bound)might be due to altered composition and structureof the RBCs membrane. The partial recovery of theactivity of AChE upon co-administration of vitamin Ecan be attributed to its antioxidant property.

MDA is the main oxidation product of peroxidizedpolyunsaturated fatty acids, and the increase in theMDA level is an important index of lipid peroxida-tion [26]. Exposure of animals to pesticides is known toinduce lipid peroxidation in various tissues, which isresponsible for the adverse biological effects in exper-imental animals [7,21,22]. A mechanism of pesticidetoxicity has been usually associated with the increasein lipid peroxidation in the liver as well as erythrocytemembranes [7,27,28]. The increase in LPO observed inthe present study may be one of the molecular mecha-nisms involved in the atrazine-induced toxicity.

There are no reports to show the protective roleof antioxidants in atrazine toxicity. However, the pro-tective role of antioxidants has been suggested in thecase of toxicity induced by other pesticides [29,30].Gultekin et al. [31] have shown that pretreatment ofrats with melatonin or a combination of vitamins Eand C in repeated doses prior to the administrationof chlorpyrifos–ethyl reduced pesticide-induced LPO.Vitamin E allows free radicals to abstract a hydrogenatom from the antioxidant molecule rather than fromPUFA, thus breaking the chain of free radical reactions.The resulting antioxidant radical is relatively an unre-active species and is nontoxic [32]. Vitamin E depletederythrocytes were shown to be more susceptible to LPOand lysis than those on normal diet [33]. The presentresults indicate that vitamin E treatment results in de-creasing LPO in erythrocyte membranes and thus pro-tects them from atrazine-induced oxidative stress.

The decrease in the lipid content of the erythrocytemembranes of atrazine-administered animals maybe attributed to their decreased phospholipid and



cholesterol content. In the literature, there are a fewreports regarding the effect of pesticides on the lipidcomposition of erythrocyte membranes. Datta et al.[27] have reported that the phospholipid content ofthe erythrocyte membranes was reduced after the invitro insecticide treatment. The increase in choles-terol/phospholipid ratios was observed after acephatetreatment in rat erythrocyte membranes [28]. Kambojet al. [34] have demonstrated that the cholesterol tophospholipid in rat brain was found to be increased af-ter carbofuran exposure, which is a major determinantof membrane fluidity. From the present observations,it is suggested that atrazine exposure induces oxida-tive stress that results in the increase in lipid peroxi-dation and a decrease in the phospholipid content ofthe erythrocyte membranes. The changes in the choles-terol and phospholipid contents may be responsible forthe changes in the activities of the membrane-boundAChE.

Morphological changes in erythrocytes observedduring SEM studies correlate well with the observedbiochemical changes. Morphological alterations in-duced by atrazine toxicity may be related to its effect onLPO, the decreased phospholipid and cholesterol con-tent of erythrocytes membranes. Oxidative stress hasbeen shown to alter cell morphology significantly. Ery-throcytes exposed to model prooxidants such as t-butylhydroperoxide have been documented to show vari-ous degrees of structural deformations owing to spe-cific oxidative transformation of the cytoskeleton [35].Different studies have suggested that morphologicalchanges in erythrocytes are possibly due to oxidativestress induced by administration of pesticides [28,36–38]. It has also been reported that changes in the mem-brane lipid composition might lead to morphologicalchanges in blood cells in response to administrationof various pesticides [39,40]. Diplock et al. [41] havesuggested that vitamin E is responsible for structurallystabilizing biomembranes due to the physico-chemicalinteractions between α-tochopherol and unsaturatedfatty acids. Morphology of erythrocytes was almostnormalized when vitamin E was administered alongwith atrazine.

CONCLUSIONS

The present study revealed that administration ofatrazine to rats produced alterations in the activity ofmembrane-bound enzyme acetylcholinesterase alongwith lipid composition of the erythrocyte membranes.This was accompanied with the increase in lipid per-oxidation in the erythrocyte membranes. Atrazine ad-ministration resulted in the decrease in the total lipids,phospholipids, and cholesterol content. The altered

membrane lipid composition of erythrocyte after treat-ment of atrazine is responsible for changes in the ac-tivity of membrane-bound AChE. Scanning electronmicroscopy of erythrocytes treated with atrazine fordifferent time periods showed significant morpholog-ical changes as compared with erythrocytes from thecontrol groups. These changes in the topography oferythrocytes may be attributed to an increase in LPOand changes in lipid composition of the membranes.The administration of vitamin E along with atrazine re-stored the AChE activity, lipid peroxidation, lipid com-position, and morphology of the erythrocytes. Thus,present investigation suggests that the biochemical andmorphological alterations induced by atrazine toxi-city can be prevented by the coadministration withvitamin E.

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Atrazine-induced alterations in rat erythrocyte membranes: Ameliorating effect of vitamin E

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