Toxicology 214 (2005) 123–130

Hexachlorocyclohexane differentially alters the antioxidantstatus of the brain regions in rat

Anup Srivastava, T. Shivanandappa∗

Department of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore 570020, India

Received 29 April 2005; received in revised form 6 June 2005; accepted 15 June 2005Available online 19 July 2005

Abstract

Hexachlorocyclohexane (HCH), a highly persistent organochlorine insecticide, is neurotoxic at acute doses and causes degen-erative effects on chronic exposure. HCH has been reported to induce oxidative stress in cells and tissues. Mammalian brainis sensitive to oxidative stress which is implicated in neurodegenerative diseases. Effect of HCH on the brain regions, cortex,cerebellum, midbrain and brainstem, has been investigated by studying the response of antioxidant enzymes in rats treated orallywith HCH at 25 and 100 mg/kg b.w. for 2 weeks. Lipid peroxidation and glutathione depletion was seen in all the brain regionsof HCH treated rats. The brain regions showed distinct variation in the antioxidant enzyme activities. Activities of glutathioneperoxidase, glutathione reductase, glutathione-S-transferase and catalase were markedly induced whereas superoxide dismutasewas inhibited at higher dose in all the brain regions. Marked induction and inhibition of the antioxidant enzymes, especiallyin the cortex and to varying degrees in other brain regions, was seen in HCH treated rats. These biochemical changes suggestv e capacityo remain tob©

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ulnerability to oxidative stress in the brain is region-specific. Whether these changes are adaptive or compromise thf the brain to deal with the HCH-induced oxidative stress that could lead to degenerative neurotoxic manifestationse understood.2005 Elsevier Ireland Ltd. All rights reserved.

eywords:Hexachlorocyclohexane; Brain regions; Antioxidant enzymes; Lipid peroxidation

. Introduction

Hexachlorocyclohexane (HCH), an organochlorinensecticide, widely used in agriculture and public

∗ Corresponding author. Tel.: +91 821 2513210/9342186724;ax: +91 821 2517233.E-mail address:tshivanandappa@yahoo.com

T. Shivanandappa).

health, is considered to be number one environmcontaminant in many parts of the world particulaIndia and China (Li, 1999). HCH enters animal tissuvia food chain, respiration or dermal contact andaccumulated in cells (Zhu et al., 1986; Lopez-Aparicet al., 1988). Technical HCH is a mixture of at leafive isomers of which�-HCH (lindane) is the maiinsecticidal component. Acute toxicity of�-HCH torat is in the range of 100–200 mg/kg b.w. whereas

300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2005.06.005

124 A. Srivastava, T. Shivanandappa / Toxicology 214 (2005) 123–130

of technical HCH is 2.6 g/kg b.w. (Ulman, 1972). Atacute doses, HCH induces neurotoxic effects such asconvulsive seizures and increased neuronal activity(Woolley and Zimmer, 1986), enhanced transmitterrelease (Baker et al., 1985), alterations in the activitiesof the membrane bound enzymes, acetylcholinesterase(Raizada et al., 1994), Na+, K+-ATPase (Parries andHokin-Neaverson, 1985) and Mg2+-ATPase (Sahooet al., 1999). At low subacute exposure, HCH isreported to induce changes in neurotransmitter levels(Sunol et al., 1988; Martinez and Martinez-Conde,1995; Anand et al., 1998). Subchronic effects oftechnical HCH on the brain, however, are not clearlyknown.

Technical HCH is known to produce various degen-erative effects in different organs such as liver, kidney,adrenal and testes (Shivanandappa and Krishnakumari,1981; Shivanandappa et al., 1982; Joseph et al., 1992).Therefore, chronic effects of HCH on tissues mayinvolve non-specific effects, particularly the induc-tion of oxidative stress. Organochlorine pesticidesincluding HCH induce oxidative stress in hepatic(Junqueira et al., 1986; Verma et al., 1992; Bagchi andStohs, 1993), testicular (Samanta and Chainy, 1997;Samanta et al., 1999) and neural (Sahoo and Chainy,1998; Sahoo et al., 2000) tissues of rat. Involvementof reactive oxygen species (ROS) has been postulatedas a possible mechanism for HCH toxicity (Holian etal., 1984; Junqueira et al., 1994; Samanta and Chainy,1997; Srivastava and Shivanandappa, 2005).

ids( cedo tion( -t xi-d s-S t al.,1 iblef ns( -d raini -p asedl s int i eta ea ont ;

Sahoo and Chainy, 1998; Sahoo et al., 2000). In thisstudy, we have investigated the response of the antiox-idant enzymes of the brain regions to oxidative stressinduced by HCH in the laboratory rat.

2. Materials and methods

2.1. Chemicals

Technical HCH was obtained from Tata Chem-icals (Mithapur, India) which had the followingcomposition of isomers: alpha, 72%; beta, 5%;gamma, 13.6% and delta, 8%. Nicotinamide ade-nine dinucleotide phosphate reduced (NADPH),1-chloro-2,4-dinitrobenzene (CDNB), thiobarbi-turic acid (TBA), glutathione (GSH), oxidizedglutathione (GSSG), glutathione reductase (GR),cumene hydroperoxide (CHP), pryogallol, bovineserum albumin (BSA), tetraethoxypropane werepurchased from Sigma Chemical Co. (St. Louis,MO, USA). Trichloroacetic acid (TCA), hydrogenperoxide (H2O2), 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB) and other chemicals were purchased fromSisco Research Laboratories, Mumbai, India. All thechemicals used were of highest purity grade available.

2.2. Animals and pesticide treatment

Ninety-day-old adult male Wistar rats (250–300 g)w veda ofg calHb neds alsw rainsp therd erep

2

atew bar-b ta Mp %)

The brain, being rich in polyunsaturated fatty acPUFA), is a vulnerable target organ for HCH-induxidative stress and the consequent lipid peroxidaHalliwell and Gutteridge, 1989). The brain shows disinct variation in the regional distribution of the antioant biochemical defenses (Brannan et al., 1980; Gosampson et al., 1988; Ansari et al., 1989; Verma e992) and metabolic rates that could be respons

or differential oxidative damage in the brain regioShukla et al., 1988; Hussain et al., 1995). There is evience that vulnerability to oxidative stress in the b

s region-specific (Baek et al., 1999). Neurotoxic comounds induce oxidative stress as evident by incre

ipid peroxidation and altered antioxidant defensehe brain regions (Verma and Srivastava, 2001; Latinl., 2003; Polydoro et al., 2004). Our knowledge on thction of organochlorine pesticides including HCH

he brain regions is rather limited (Hincal et al., 1995

ere divided into three groups. Rats of group I sers control and received the vehicle only. Animalsroups II and III were administered daily techniCH [25 (1/100 LD50) and 100 (1/25 LD50) mg/kgody weight, respectively] suspended in 0.1 ml refiunflower oil by oral intubation for 14 days. Animere sacrificed by diethyl ether anesthesia, the berfused with saline were dissected out and furissected on ice to get different regions, which wrocessed immediately for biochemical assays.

.3. Lipid peroxidation

Lipid peroxidation (LPO) in the tissue homogenas measured by estimating the formation of thioituric acid reactive substances (TBARS) (Ohkawa el., 1979). Tissue homogenate (10% (w/v) in 50 mhosphate buffer, pH 7.4) was boiled in TCA (10

A. Srivastava, T. Shivanandappa / Toxicology 214 (2005) 123–130 125

and TBA (0.34%) for 15 min, cooled and centrifuged.Absorbance of the supernatant was read at 535 nm.TBARS was calculated using tetraethoxypropane as thestandard.

2.4. Antioxidant enzymes

Brain tissue was homogenized (10%, w/v) in ice-cold 50 mM phosphate buffer (pH 7.4), centrifuged at10,000×g for 20 min at 4◦C and the supernatant wasused to assay the enzyme activities.

Superoxide dismutase (SOD) activity was measuredusing pyrogallol (2 mM) autooxidation in tris buffer(Marklund and Marklund, 1974).

Catalase (CAT) activity was measured using H2O2(3%) as the substrate in phosphate buffer (Aebi, 1974).

Glutathione peroxidase (GPx) activity was mea-sured by the indirect assay method using glutathionereductase. Cumene hydroperoxide (1 mM) andglutathione (0.25 mM) were used as substrates and oxi-dation of NADPH by glutathione reductase (0.25 U) intris buffer was monitored at 340 nm (Mannervik, 1985).

Glutathione reductase (GR) activity was esti-mated using oxidized glutathione (2 mM) and NADPH(2 mM) in potassium phosphate buffer (Calberg andMannervik, 1985).

Glutathione transferase (GST) activity was assayedby the method ofWarholm et al. (1985)using glu-tathione (2 mM) and CDNB (3 mM) as the substratesin phosphate buffer, change in absorbance at 344 nmw

2

5%(2 zeds witha

d ofL hes

2

e eb yzed

by Duncan’s multiple range test (Statistica Software,1999), represented by asterisk/s for each level ofsignificance. A difference was considered significantatp< 0.05.

3. Results

No symptoms of neurotoxicity were observed. Thefood consumption and body weight of the HCH treatedrats was not affected.

3.1. Lipid peroxidation

Dose-dependent increase in lipid peroxidation(LPO) was observed in all the brain regions being highin the cortex and cerebellum (23–25%) compared tothat of midbrain and brain stem (17–18%) (Fig. 1).

3.2. Antioxidant enzymes

The distribution of the antioxidant enzymes in thebrain regions of normal (control) rats shows distinctvariation (Table 1). SOD activity was highest in thebrain stem (stem) and lowest in the midbrain whereasCAT activity was high in the midbrain but low in thecortex. Activities of GPx, GR and GST on the otherhand were highest in midbrain but low in cerebellumand cortex respectively (Table 1).

lsos ntd ons

F ida-t .23,c in.

as monitored in a UV–vis spectrophotometer.

.5. Glutathione

A 10% (w/v) tissue homogenate prepared inw/v) trichloroacetic acid, centrifuged at 2000×g for0 min and glutathione (GSH) in the deproteiniupernatant was estimated by Ellman’s reagentstandard curve (Ellman, 1959).Protein content was estimated by the metho

owry et al. (1951)with bovine serum albumin as ttandard.

.6. Statistics

All the data are expressed as means± S.E. ofight observations (n= 8) and significant differencetween each of the groups was statistically anal

Effect of HCH on the antioxidant enzymes ahowed distinct regional variation. A significaecrease in the activity of SOD in the brain regi

ig. 1. Effect of repeated HCH administration on lipid peroxion in different regions of the rat brain. Control values: cortex 1erebellum 1.07, stem 1.42, midbrain 1.31 nmol MDA/mg prote

126 A. Srivastava, T. Shivanandappa / Toxicology 214 (2005) 123–130

Table 1Regional distribution of glutathione and antioxidant enzymes in the rat braina

Cortex Cerebellum Brain Stem Midbrain

SOD (units/mg protein) 0.33 + 0.01 b 0.56 + 0.01 c 1.25 + 0.05 d 0.24 + 0.01 aCAT (nmol H2O2/mg protein) 0.9 + 0.02 a 1.19 + 0.04 b 1.86 + 0.05 c 3.16 + 0.03 dGPx (nmol NADPH/mg protein) 11.16 + 0.58 b 3.36 + 0.2 a 14.13 + 0.19 c 30.94 + 0.87 dGR (nmol NADPH/mg protein) 51.59 + 0.31 a 104.49 + 1.25 c 93.75 + 0.96 b 196.11 + 1.69 dGST (nmol CDNB conjugate/mg protein) 56.43 + 0.85 a 83.08 + 1.31 b 131.53 + 1.55 c 318.08 + 1.87 dGSH (mg/g protein) 14.15 + 0.14 a 15.69 + 0.12 b 16.54 + 0.18 c 14.53 + 0.2 a

Values with non-identical letters are significantly different (p< 0.05) by DMRT.a The values are from the control group.

of HCH treated rats was seen. Stem and cortex showedmarked decrease (36–38%) followed by midbrain andcerebellum (27–29%) at higher dose (100 mg/kg b.w.)of HCH (Fig. 2).

CAT activity was markedly increased in the brain ofHCH treated rats. The increase in activity was highestin the cortex (390%) followed by cerebellum (52%),stem (45%) and lowest in midbrain (23%) at higherdose of HCH (Fig. 3).

A dose-dependent induction of GPx activity in allthe studied brain regions was observed which wasin the following order: cortex (105%) > cerebellum(94%) > stem (40%) > midbrain (20%) at higher doseof HCH (Fig. 4).

Marked induction of GR in the brain regions of theHCH treated rats was seen which was highest in thecortex (337%) followed by cerebellum (158%), mid-brain (31%) and least in the stem (19%) at higher doseof HCH (Fig. 5).

Similarly, GST activity was elevated in the brainregions of HCH treated rats. Different regions

F theb en inT

Fig. 3. Effect of HCH on the activity of catalase(nmol H2O2/mg protein) in the brain regions of rat (seeTable 1forcontrol values).

responded to different degree, with cortex showinghighest induction (175%) followed by stem (24%) leastin midbrain and cerebellum (13–15%) at higher doseof HCH (Fig. 6).

Overall, induction of CAT, GPx, GR, and GST inthe cortex of HCH treated rats was a distinct feature.

Fig. 4. Effect of HCH administration on the activity of glutathioneperoxidase (nmol NADPH/mg protein) in the brain regions of rat (seeTable 1for control values).

ig. 2. Activity (units/mg protein) of superoxide dismutase inrain regions of rats administered HCH (control values are givable 1).

A. Srivastava, T. Shivanandappa / Toxicology 214 (2005) 123–130 127

Fig. 5. Glutathione reductase activity (nmol NADPH/mg protein) inthe brain regions of rats treated with HCH (seeTable 1for controlvalues).

Fig. 6. Glutathione transferase activity (nmol GS-CDNB/mg pro-tein) in the brain regions of rats treated with HCH (control values aregiven inTable 1).

3.3. Glutathione

A significant reduction in GSH content wasobserved in the brain regions of rats of the higher dose

Fig. 7. Effect of HCH on glutathione content (�g/mg protein) indifferent regions of the rat brain (control values are given inTable 1).

(100 mg/kg b.w.) group. The cortex showed maximumreduction (27%) and the stem, the least (17%) (Fig. 7).

4. Discussion

Potential deleterious effects of xenobiotics aremanifested in tissues due to peroxidation of mem-brane lipids, particularly the PUFA (Halliwell andGutteridge, 1985; Numan et al., 1990; Yang andDiSilvestro, 1992; Bagchi and Stohs, 1993). HCH hasbeen reported to cause lipid peroxidation in the tissuesof rat (Yang and DiSilvestro, 1992; Junqueira et al.,1994; Samanta and Chainy, 1997). Brain is consideredhighly vulnerable to oxidative stress than other organsof the body as it consumes high amounts of oxygen,contains high amounts of PUFA and low levels ofantioxidant enzymes (Somani et al., 1996). Further,the highly lipophilic nature of HCH makes brain themost prone target. Our results show that HCH causedoxidative stress in the brain regions of rats as evidentfrom the induction of LPO. Among the brain regions,cortex and cerebellum showed high LPO as comparedto the midbrain and stem. A somewhat similar trend inLPO induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) in the brain regions of rat has been reported(Hassoun et al., 2003). The biochemical basis forregional variation in LPO in the brain regions is notclear. It is believed that production of reactive oxygenspecies (ROS) is associated with the synthesis ofn yH O.D ainr pidp wsl ,1 vilym ed tobM

atebv teo nyi ofo 1;K int vel

eurotransmitters (Weber, 1994). ROS induction bCH in the brain regions could contribute to LPifferences in the fatty acid composition of the br

egions could also account for this pattern of lieroxidation since white matter rich in myelin sho

ower PUFA than the grey matter (Svennerholm968). The midbrain and stem are relatively heayelinated regions and therefore could be expecte more resilient to peroxidative stress (Macevilly anduller, 1996).Under normal physiological conditions, a delic

alance exists between the rate of formation of H2O2ia dismutation of O2

•− by SOD activity and the raf removal of H2O2 by CAT and GPx. Therefore, a

mpairment in this pathway will affect the activitiesther enzymes in the cascade (Sinet and Garber, 198ono and Fridovich, 1992). For example, reduction

he activity of SOD will result in an increased le

128 A. Srivastava, T. Shivanandappa / Toxicology 214 (2005) 123–130

of O2•−, while decrease in the activities of CAT and

GPx will lead to accumulation of H2O2 in the cell,which leads to peroxidation of membrane lipids viaFenton-type reaction. Our data support earlier reportsof decreased SOD activity by HCH in the cerebral cor-tex of rats (Sahoo and Chainy, 1998; Sahoo et al., 2000).Further, this study shows that SOD activity was reducedin all the brain regions, the cortex and stem being mostaffected in terms of scavenging of O2

•−. Interestingly,TCDD has also been reported to suppress SOD activityin the brain regions of rat (Hassoun et al., 2003) whichimplies that common mechanisms of oxidative stresscould be involved. High induction of CAT and GPxin the brain regions, in our study, indicates enhancedbiochemical defenses to scavenge the over productionof H2O2. This is consistent with our results showingincreased production of TBARS (LPO) by HCH whichis indicative of corresponding overproduction of freeradicals.

Induction of GR could be viewed as a mechanismfor replenishment of GSH, an efficient antioxidantmolecule important for cells to withstand the toxiceffects of xenobiotics or their metabolites. Depletionof GSH content in the brain regions by HCH is con-sistent with the earlier reports on the cerebral cortex(Barros et al., 1988; Sahoo et al., 2000). Depletionof GSH affects the metabolic detoxication of lipidhydroperoxides formed due to HCH action in the brain(Kosower and Kosower, 1979). GSH-conjugation ofHCH metabolites is known (Portig et al., 1979). Induc-t theb ndi

ainc onald uldi nsa za nf essc itterp eb ante oneta tives vityo ay

also indicate an adaptive biochemical response toHCH-induced oxidative stress (Carvalho et al., 2001).

Acknowledgements

The authors wish to thank the Director of the insti-tute for his keen interest in this study and Mr. R. Ravi forhis help in statistical analysis. The first author acknowl-edges Council for Scientific and Industrial Research,New Delhi for awarding a research fellowship.

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