Prevention of hexachlorocyclohexaneinduced neuronal oxidative stress by natural antioxidants Anup Srivastava, T. Shivanandappa Department of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore, Karnataka, India Objective: Decalepis hamiltonii roots are traditionally consumed as general vitalizer and used in ayurvedic medicine preparations. We have isolated/characterized potent antioxidants from the aqueous extract of the root of this plant. In this study, we examined the antioxidant potential of the aqueous extract of the roots of D. hamiltonii (DHAE) against hexachlorocyclohexane (HCH)-induced oxidative stress in four major regions of the rat brain. Methods: The antioxidant activity of the standardized DHAE with known antioxidant constituents was tested against HCH-induced oxidative stress in the major brain regions of 60-day-old adult male Wistar rats. Results: Pretreatment of rats with multiple doses of DHAE, 50 and 100 mg/kg body weight (b.w.), for 7 consecutive days significantly prevented the HCH-induced (single dose −500 mg/kg b.w.) increase in lipid peroxidation, reduction in glutathione, and altered antioxidant enzyme activities viz. superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase in major rat brain regions viz. cortex, cerebellum, midbrain, and brain stem. DHAE, per se, elevated the antioxidant status of the rat brain. Discussion: DHAE shows protective action against HCH-induced oxidative stress in rat brain regions. The protective effect of DHAE could be ascribed to the isolated/characterized antioxidant compounds which could be prospective novel nutraceuticals. Keywords: Antioxidant enzymes, Decalepis hamiltonii, Glutathione, HCH, Lipid peroxidation
Introduction All aerobic organisms are susceptible to oxidative stress simply because the toxic molecular species of oxygen such as superoxide and hydrogen peroxide are produced in mitochondria during respiration.1 Brain is considered highly sensitive to oxidative damage as it is rich in easily peroxidizable fatty acids (like polyunsaturated fatty acid (PUFA)), consumes an inordinate fraction (20%) of the total oxygen for its relatively small weight (2%), and is relatively deficient in antioxidant defenses.2 There is substantial evidence that oxidative stress is a causative or at least ancillary factor in the pathogenesis of major neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis as well as in cases of stroke, trauma, and seizures.3 Decreased level of antioxidant activity and increased Correspondence to: Anup Srivastava, Department of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore, 570020, Karnataka, India. E-mail: [email protected] Current Address: Anup Srivastava, 217, Willow Street, New Haven, CT 06511, USA; T. Shivanandappa, Department of Zoology, University of Mysore, Manasagangotri, Mysore 570006
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lipid peroxidation and oxidative modifications of DNA and proteins especially in substantia nigra of the brain have been reported in patients with Parkinson’s disease. A number of in vitro studies have shown that antioxidants – both endogenous and dietary – can protect nervous tissue from damage by oxidative stress.4 Hexachlorocyclohexane (HCH), an organochlorine insecticide, is widely used in agriculture and public health. Organochlorine pesticides including HCH induce oxidative stress in neural tissues of rat.5 We have earlier shown that HCH causes increased lipid peroxidation and depletion of glutathione (GSH) content apart from altered antioxidant enzyme activities viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) in major regions of the rat brain.6 Involvement of reactive oxygen species (ROS) has been postulated as a possible mechanism for HCH toxicity.7 The brain shows distinct variation in the regional distribution of the antioxidant defenses and metabolic rates that could be
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responsible for the HCH-induced differential oxidative damage in the brain regions.6 Tuberous roots of Decalepis hamiltonii (Wight and Arn.)(Family: Asclepiadaceae) are consumed in southern India as pickles and juice for its alleged health promoting properties. The roots are also used in folk medicine and ayurvedic preparations as general vitalizer and blood purifier.8 We have earlier reported that the roots of D. hamiltonii possess potent antioxidant properties and isolated/characterized the antioxidant constituents which could be associated with their alleged health benefits.9–11 The aqueous extract of the roots of D. hamiltonii contains potent antioxidants (activity-guided purification) namely, 4-hydroxy isophthalic acid, ellagic acid, 14-aminotetradecanoic acid, 4-(1-hydroxy-1-methylethyl)-1-methyl-1, 2-cyclohexane diol, 2-hydroxymethyl-3-methoxybenzaldehyde, 2,4,8 trihydroxybicyclo [3.2.1]octan-3-one (Fig. 1). We have also shown the hepatoprotective potential of the roots of D. hamiltonii in ethanol and CCl4 model systems of liver toxicity.12,13 In this study, we examined the protective potential of the aqueous extract of the roots of D. hamiltonii (DHAE) against HCH-induced oxidative stress in four major regions of the rat brain.
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albumin (BSA), tetraethoxypropane were purchased from Sigma Chemical Co. (St Louis, MO, USA). Trichloroacetic acid (TCA), hydrogen peroxide (H2O2), 5,5′ dithiobis(2-nitrobenzoic acid) (DTNB) and other chemicals were purchased from Sisco Research Laboratories, Mumbai, India. All the chemicals used were of highest purity grade available.
Preparation of the root powder and extraction Roots of D. hamiltonii were washed with water, followed by crushing with a roller to separate the inner woody core from the outer fleshy layer. The fleshy portions were pooled, dried at 40°C in a hot air oven and fine powdered. The powder was used for extraction. The aqueous extract was prepared by homogenizing the root powder in warm water (50°C) and allowed to stand for 24 hours, filtered with Whatman paper No. 1, and the filtrate was lyophilized and weighed (17% of root powder). To quantify/characterize the extract composition total polyphenolic content was measured (13.8 mg/g extract) as per the method of Singleton and Rossi.14 Aqueous extract of D. hamiltonii was chosen for this study as it shows highest antioxidant activity among the different solvent extracts.
Animals and treatment Materials and methods Chemicals Technical HCH was obtained from Tata chemicals (Mithapur, India) which had the following composition of isomers: alpha 72%, beta 5%, gamma 13.6%, and delta 8%. Nicotinamide adenine dinucleotide phosphate reduced (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA), GSH, oxidized glutathione (GSSG), GR, cumene hydroperoxide (CHP), pryogallol, bovine serum
Figure 1
Sixty-day-old adult male Wistar rats (180–200 g) were divided into different groups, of eight each. The Institute Animal Ethics Committee guidelines were followed for the animal experiments. In a 90-day dietary study on rats it was established that the root extract of D. hamiltonii is safe to the mammalian system at the highest dose used in this study. Based on the preliminary experiments the neuroprotective dose of DHAE was decided. Neuroprotective activity of DHAE was tested with multiple dose (7 consecutive
Antioxidant compounds present in the aqueous extract of the roots of Decalepis hamiltonii.
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days) oral pretreatment at the doses of 50 and 100 mg/ kg b.w. HCH (1/3 of LD50) was given orally on the 7th day 1 hour after the DHAE administration. HCH dosage was based on preliminary experiments that showed induction of oxidative stress at acute dosage in rat brain.
Experimental design and groupings Group I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV– DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/ kg b.w.); Group V – HCH (500 mg/kg b.w.) in sunflower oil. Animals were sacrificed by anesthesia after 16 hours of HCH administration; the brain perfused with saline was dissected on ice to get different regions viz., cortex, cerebellum, brainstem, and midbrain, which were processed immediately for biochemical assays.
Lipid peroxidation Lipid peroxidation (LPO) in the tissue homogenate was measured by estimating the formation of TBAreactive substances (TBARS).15 Tissue homogenate (10% w/v in 50 mM phosphate buffer, pH 7.4) was mixed with TCA (10%) and TBA (0.34%) and boiled in a water bath for 15 minutes, cooled, and centrifuged. Absorbance of the supernatant was read at
535 nm. TBARS was calculated using tetraethoxypropane as the standard.
Glutathione A 10% (w/v) tissue homogenate prepared in 5% (w/v) trichloroacetic acid, centrifuged at 2000 g for 5 minutes and GSH in the deproteinized supernatant was estimated by Ellman’s reagent with a standard curve.16
Antioxidant enzymes assays Brain tissue was homogenized (10% w/v) in ice-cold 50 mM phosphate buffer ( pH 7.4), centrifuged at 10 000 g for 20 minutes. at 4°C and the supernatant was used to assay the enzyme activities. SOD activity was measured using pyrogallol (2 mM) autoxidation in tris buffer.17 CAT activity was measured using H2O2 (3%) as the substrate in phosphate buffer.18 GPx activity was measured by the indirect assay method using GR. Cumene hydroperoxide (1 mM) and GSH (0.25 mM) were used as substrates and coupled oxidation of NADPH by GR (0.25 U) in tris buffer (50 mM, pH 7.6) was monitored at 340 nm.19 GR activity was assayed in a reaction mixture containing oxidized glutathione (20 mM) and NADPH (2 mM) in potassium phosphate buffer.20 GST activity was assayed by using glutathione (20 mM) and CDNB (30 mM) as the substrates in phosphate buffer, change in absorbance
Figure 2 Protective effect of D. hamiltonii aqueous extract on HCH-induced lipid peroxidation in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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at 344 nm was monitored in a UV–Visible Spectrophotometer.21 Protein content was estimated by the method of Lowry, with bovine serum albumin as the standard.
Statistical analysis The data were expressed as means ± SE of eight observations (n = 8) and significant difference between each of the groups was statistically analyzed by Duncan’s multiple range test (Statistica Software, 1999, Statsoft Inc., Tulsa, USA). A difference was considered significant at P < 0.05.
Results Lipid peroxidation Increased LPO was observed in cortex (22%) and cerebellum (26%) but it was not significant in brain stem and midbrain of HCH-treated rats. DHAE pretreatment in dose-dependent manner ameliorated the HCH-induced LPO. DHAE, per se, decreased the basal level of lipid peroxidation in cortex, cerebellum, and midbrain (Fig. 2).
Antioxidant enzymes Effect of HCH on the antioxidant enzymes also showed distinct regional variation. A significant decrease in the activity of SOD in all the brain regions of HCH-treated rats was seen. Brain stem (40%) and cortex (37%) showed marked decrease
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followed by midbrain and cerebellum. DHAE, prevented the HCH induced decrease in SOD activity dose dependently in all the brain regions studied, except for DHAE low dose in cerebellum (Fig. 3). CAT activity was markedly increased in all the brain regions of HCH-treated rats. The increase in activity was highest in the cortex (4 fold) followed by cerebellum (73%), brain stem (53%), and lowest in midbrain (33%). DHAE, both doses, prevented the increase in CAT activity induced by HCH in all brain regions except for brain stem (Fig. 4). An induction of GPx activity by HCH treatment in all the brain regions was observed, which was in the following order: cerebellum (119%) > cortex (110%) > brain stem (48%) > midbrain (34%). DHAE, both doses, prevented the increase in GPx activity induced by HCH in all the brain regions studied (Fig. 5). Marked induction of GR in the brain regions of the HCH-treated rats was seen, which was highest in the cortex (>4 fold) followed by cerebellum (67%), brain stem (39%) and, least in the midbrain (30%). DHAE prevented the HCH-induced increase in GR activity in cortex and cerebellum but not in midbrain and brain stem (Fig. 6). Similarly, GST activity was elevated in the brain regions of HCH-treated rats except cerebellum. Different brain regions responded to a different degree, with cortex (3 fold) showing highest induction followed by brain stem (30%) and least in midbrain. DHAE could only prevent the severe increase in the
Figure 3 Protective effect of D. hamiltonii aqueous extract on HCH-induced decrease in the SOD activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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Figure 4 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in CAT activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
Figure 5 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in GPx activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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Figure 6 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in GR activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
GST activity in the brain cortex of rats treated with HCH (Fig. 7). DHAE significantly prevented the toxic insult induced by HCH as measured by
antioxidant enzyme activities in the rat brain. The multiple dose treatment of DHAE, as such, enhanced the antioxidant status of the rat brain.
Figure 7 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in GST activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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Figure 8 Protective effect of D. hamiltonii aqueous extract on HCH-induced decrease in GSH in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
Glutathione A significant reduction in GSH content was observed in all the brain regions of HCH-treated rats. In HCHtreated rats, cortex showed maximum reduction, and brain stem, the least. DHAE pretreatment not only prevented the decrease in GSH due to HCH treatment but also enhanced GSH levels in the cortex and cerebellum of the rat brain (Fig. 8).
Discussion HCH has been reported to cause lipid peroxidation/ oxidative stress in the tissues of rat.5 Brain is considered highly vulnerable to oxidative stress than other organs of the body.22 This study adds to the evidence that the vulnerability to oxidative stress of the rat brain is region-specific. Cortex and cerebellum showed increase in LPO but not brain stem and midbrain, which reflects the differential sensitivity of the brain regions to oxidative stress. This observation is in agreement with our earlier report of sub-chronic treatment of HCH to rats.6 The exact reason for such variation is not clear but the relatively higher amount of myelin (low in PUFA) in midbrain and brain stem could explain this difference.23 Induction of LPO by HCH may involve cytochrome P450dependent microsomal metabolism, which generates highly reactive metabolite pentachlorocyclohexene or due to ROS produced by respiratory burst.24 DHAE pretreatment prevented the HCH-induced LPO,
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which could be attributed to its free radical quenching antioxidant constituents. Oxidative stress is efficiently neutralized by synchronized actions of different cellular antioxidants (like SOD, CAT, GPx, GR, GST, and GSH) in mammalian cells. Altered antioxidant profile of the brain regions may indicate an adaptive biochemical response to HCH-induced oxidative stress.5,25 The decrease in GSH content and SOD activity; and increase in CAT, GPx, GR, and GST by HCH treatment in rat brain is in agreement with earlier reports.5–6 DHAE pretreatment could restore the depletion in GSH content and improve the altered antioxidant enzyme activities induced by HCH in rat brain. DHAE pretreatment, per se, boosted the antioxidant status in all the regions of the rat brain but alteration in the antioxidant status by HCH was due to the oxidative stress which was marked by increased LPO and decreased GSH level.5,26 The precise mechanism by which the plant extracts enhance the antioxidant profile is not well understood. Some studies report that the augmentation of antioxidant status by aqueous plant extracts is orchestrated by interaction with the antioxidant response elements (AREs) that transcriptionally regulate the genes related to antioxidant enzymes.27 It has also been shown that the γ-glutamylcystein synthetase (γ-GCS), a key enzyme in the GSH synthesis, is also transcriptionally regulated by AREs.28 The natural
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antioxidants like resveratrol, curcumin, quercetin, epigallocatechin 3-gallate, rosmarinic acid, etc. have been demonstrated to ameliorate neuronal oxidative in different models. Many of these natural antioxidants are not only active scavengers of free radicals but also act as modulators of pro-survival or pro-apoptotic signaling pathways. There is a possibility that the antioxidant compounds present in DHAE are interacting with AREs in vivo, which needs further research. Antioxidant intervention is gaining significance as therapeutic approach for treatment of neurological disorders. As natural plant products have minimal adverse effects in contrast to the side effects of a number of synthetic drugs they are being employed in antioxidant therapy for neurodegenerative disorders.29 Dietary antioxidants and herbal extracts can considerably contribute to the modulation of complex mechanisms of neurodegenerative diseases.30 This study for the first time demonstrates the protective potential of D. hamiltonii roots against HCHinduced neuronal oxidative stress. Because failure to cope with oxidative stress is a common factor in the aetiology of many diseases DHAE’s effects on the improvement of the antioxidant response could provide an explanation for the health promoting properties attributed to it. The antioxidant compounds isolated are prospective novel nutraceuticals and could be good candidates for use as therapeutic agents for treating neurodegenerative diseases involving oxidative stress.
Acknowledgements This work was done at Central Food Technological Research Institute, Mysore, India. The authors wish to thank the Director of the institute for his keen interest in this study. The first author acknowledges Council for Scientific and Industrial Research, New Delhi for awarding the research fellowship.
References 1 Balaban RS, Nemoto S, Finkel T. Mitochondria oxidants and aging. Cell 2005;120:483–95. 2 Chong ZZ, Li F, Maiese K. Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease. Prog Neurobiol 2005;75:207–46. 3 Cui K, Luo X, Xu K, Ven Murthy MR. Role of oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants. Prog Neuropsychopharmacol Biol Psychiat 2004;28:771–99. 4 Lau FC, Shukitt-Hale B, Joseph JA. The beneficial effects of fruit polyphenols on brain aging. Neurobiol Aging 2005;26: 128–32. 5 Sahoo A, Samanta L, Chainy GBN. Mediation of oxidative stress in HCH-induced neurotoxicity in rat. Arch Environ Cont Toxicol 2000;39:7–12. 6 Srivastava A, Shivanandappa T. Hexachlorocyclohexane differentially alters the antioxidant status of the brain regions in rat. Toxicology 2005;214:123–30.
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7 Srivastava A, Shivanandappa T. Causal relationship between HCH cytotoxicity, ROS induction and Na+,K+-ATPase inhibition in EAT cells. Mol Cell Biochem 2006;86:87–93. 8 Nayar RC, Shetty JKP, Mary Z, Yoganarshihan SN. Pharmacognostical studies on the root of Decalepis hamiltonii Wt. and Arn. and comparison with Hemidesmus indicus (L.) R. Br. Proc Indian Acad Sci 1978;87:37–48. 9 Srivastava A, Harish R, Shivanandappa T. Novel antioxidant compounds from the aqueous extract of the roots of Decalepis hamiltonii (Wight and Arn.) and their inhibitory effect on lowdensity lipoprotein oxidation. J Agric Food Chem 2006;54: 790–5. 10 Srivastava A, Shereen , Harish R, Shivanandappa T. Antioxidant activity of the roots of Decalepis hamiltonii (Wight & Arn.). LWT-Food Sci Technol 2006;39:1059–65. 11 Srivastava A, Rao LJM, Shivanandappa T. Isolation of ellagic acid from the aqueous extract of the roots of Decalepis hamiltonii: antioxidant activity and cytoprotective effect. Food Chem 2007;103:224–33. 12 Srivastava A, Shivanandappa T. Hepatoprotective effect of the root extract of Decalepis hamiltonii against carbon tetrachloride-induced oxidative stress in rats. Food Chem 2010;118:411–7. 13 Srivastava A, Shivanandappa T. Hepatoprotective effect of the aqueous extract of the roots of Decalepis hamiltonii against ethanol-induced oxidative stress in rats. Hepatol Res 2006;35: 267–75. 14 Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Viticult 1965;16:144–58. 15 Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 1979;95:351–8. 16 Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophy 1959;82:70–7. 17 Marklund S, Marklund G. Involvement of the superoxide anion in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974;47:469–74. 18 Aebi H. Catalase. In: Bergmeyer HU, (ed.) Meth enzymol anal. Vol. 2. Weinheim: Verlag Chenie; 1974. p. 674–8. 19 Mannervik B. Glutathione peroxidase. In: Miester A, (ed.) Meth enzymol. Vol. 113. FL: Academic Press; 1985. p. 490–5. 20 Calberg I, Mannervik B. Glutathione reductase. In: Meister A, (ed.) Methods in enzymology. Vol. 113. FL: Academic Press; 1985. p. 484–90. 21 Warholm M, Guthenberg C, Bahr CV, Mannervik B. Glutahtione transferase from human liver. In: Meth enzymol. Vol. 113. Deerfield Beach, FL: Academic Press, 1985. p. 499–504. 22 Reeve AK, Krishnan KJ, Turnbull DM. Age related mitochondrial degenerative disorders in humans. Biotechnol J 2008;3: 750–6. 23 Macevilly CJ, Muller DPR. Lipid peroxidation in neural tissues and fractions from Vitamin E-deficient rats. Free Rad Biol Med 1996;20:639–48. 24 Videla LA, Barros SBM, Junqueira VBC. Lindane-induced liver oxidative stress. Free Rad Biol Med 1990;9:169–79. 25 Sahoo A, Chainy GBN. Acute hexachlorocyclohexaneinduced oxidative stress in rat cerebral hemisphere. Neurochem Res 1998;23:1079–84. 26 Ferguson LR. Role of plant polyphenols in genomic stability. Mutat Res 2001;475:89–111. 27 Moskaug JO, Carlsen H, Myhrstad MC, Blomhoff R. Polyphenols and glutathione synthesis regulation. Am J Clin Nutr 2005;81:277S–83S. 28 Joshi G, Perluigi M, Sultana R, Agrippino R, Calabrese V, Butterfield DA. In vivo protection of synaptosomes by ferulic acid ethyl ester (FAEE) from oxidative stress mediated by 2,2azobis(2-amidino-propane)dihydrochloride (AAPH) or Fe(2+)/ H(2)O(2): Insight into mechanisms of neuroprotection and relevance to oxidative stress-related neurodegenerative disorders. Neurochem Int 2005;48:318–27. 29 Butterfield D, Castegna A, Pocernich C, Drake J, Scapagnini G, Calabtrese V. Nutritional approaches to combat oxidative stress in Alzheimer’s disease. J Nutr Biochem 2002;13:444–9. 30 Wang YJ, He F, Li XL. The neuroprotection of resveratrol in the experimental cerebral ischemia. Chin J Med Gen 2003;83:534–6.
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Copyright of Nutritional Neuroscience is the property of Maney Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Introduction All aerobic organisms are susceptible to oxidative stress simply because the toxic molecular species of oxygen such as superoxide and hydrogen peroxide are produced in mitochondria during respiration.1 Brain is considered highly sensitive to oxidative damage as it is rich in easily peroxidizable fatty acids (like polyunsaturated fatty acid (PUFA)), consumes an inordinate fraction (20%) of the total oxygen for its relatively small weight (2%), and is relatively deficient in antioxidant defenses.2 There is substantial evidence that oxidative stress is a causative or at least ancillary factor in the pathogenesis of major neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis as well as in cases of stroke, trauma, and seizures.3 Decreased level of antioxidant activity and increased Correspondence to: Anup Srivastava, Department of Food Protectants and Infestation Control, Central Food Technological Research Institute, Mysore, 570020, Karnataka, India. E-mail: [email protected] Current Address: Anup Srivastava, 217, Willow Street, New Haven, CT 06511, USA; T. Shivanandappa, Department of Zoology, University of Mysore, Manasagangotri, Mysore 570006
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lipid peroxidation and oxidative modifications of DNA and proteins especially in substantia nigra of the brain have been reported in patients with Parkinson’s disease. A number of in vitro studies have shown that antioxidants – both endogenous and dietary – can protect nervous tissue from damage by oxidative stress.4 Hexachlorocyclohexane (HCH), an organochlorine insecticide, is widely used in agriculture and public health. Organochlorine pesticides including HCH induce oxidative stress in neural tissues of rat.5 We have earlier shown that HCH causes increased lipid peroxidation and depletion of glutathione (GSH) content apart from altered antioxidant enzyme activities viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) in major regions of the rat brain.6 Involvement of reactive oxygen species (ROS) has been postulated as a possible mechanism for HCH toxicity.7 The brain shows distinct variation in the regional distribution of the antioxidant defenses and metabolic rates that could be
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responsible for the HCH-induced differential oxidative damage in the brain regions.6 Tuberous roots of Decalepis hamiltonii (Wight and Arn.)(Family: Asclepiadaceae) are consumed in southern India as pickles and juice for its alleged health promoting properties. The roots are also used in folk medicine and ayurvedic preparations as general vitalizer and blood purifier.8 We have earlier reported that the roots of D. hamiltonii possess potent antioxidant properties and isolated/characterized the antioxidant constituents which could be associated with their alleged health benefits.9–11 The aqueous extract of the roots of D. hamiltonii contains potent antioxidants (activity-guided purification) namely, 4-hydroxy isophthalic acid, ellagic acid, 14-aminotetradecanoic acid, 4-(1-hydroxy-1-methylethyl)-1-methyl-1, 2-cyclohexane diol, 2-hydroxymethyl-3-methoxybenzaldehyde, 2,4,8 trihydroxybicyclo [3.2.1]octan-3-one (Fig. 1). We have also shown the hepatoprotective potential of the roots of D. hamiltonii in ethanol and CCl4 model systems of liver toxicity.12,13 In this study, we examined the protective potential of the aqueous extract of the roots of D. hamiltonii (DHAE) against HCH-induced oxidative stress in four major regions of the rat brain.
Prevention of HCH-induced neuronal oxidative stress by natural antioxidants
albumin (BSA), tetraethoxypropane were purchased from Sigma Chemical Co. (St Louis, MO, USA). Trichloroacetic acid (TCA), hydrogen peroxide (H2O2), 5,5′ dithiobis(2-nitrobenzoic acid) (DTNB) and other chemicals were purchased from Sisco Research Laboratories, Mumbai, India. All the chemicals used were of highest purity grade available.
Preparation of the root powder and extraction Roots of D. hamiltonii were washed with water, followed by crushing with a roller to separate the inner woody core from the outer fleshy layer. The fleshy portions were pooled, dried at 40°C in a hot air oven and fine powdered. The powder was used for extraction. The aqueous extract was prepared by homogenizing the root powder in warm water (50°C) and allowed to stand for 24 hours, filtered with Whatman paper No. 1, and the filtrate was lyophilized and weighed (17% of root powder). To quantify/characterize the extract composition total polyphenolic content was measured (13.8 mg/g extract) as per the method of Singleton and Rossi.14 Aqueous extract of D. hamiltonii was chosen for this study as it shows highest antioxidant activity among the different solvent extracts.
Animals and treatment Materials and methods Chemicals Technical HCH was obtained from Tata chemicals (Mithapur, India) which had the following composition of isomers: alpha 72%, beta 5%, gamma 13.6%, and delta 8%. Nicotinamide adenine dinucleotide phosphate reduced (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA), GSH, oxidized glutathione (GSSG), GR, cumene hydroperoxide (CHP), pryogallol, bovine serum
Figure 1
Sixty-day-old adult male Wistar rats (180–200 g) were divided into different groups, of eight each. The Institute Animal Ethics Committee guidelines were followed for the animal experiments. In a 90-day dietary study on rats it was established that the root extract of D. hamiltonii is safe to the mammalian system at the highest dose used in this study. Based on the preliminary experiments the neuroprotective dose of DHAE was decided. Neuroprotective activity of DHAE was tested with multiple dose (7 consecutive
Antioxidant compounds present in the aqueous extract of the roots of Decalepis hamiltonii.
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days) oral pretreatment at the doses of 50 and 100 mg/ kg b.w. HCH (1/3 of LD50) was given orally on the 7th day 1 hour after the DHAE administration. HCH dosage was based on preliminary experiments that showed induction of oxidative stress at acute dosage in rat brain.
Experimental design and groupings Group I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV– DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/ kg b.w.); Group V – HCH (500 mg/kg b.w.) in sunflower oil. Animals were sacrificed by anesthesia after 16 hours of HCH administration; the brain perfused with saline was dissected on ice to get different regions viz., cortex, cerebellum, brainstem, and midbrain, which were processed immediately for biochemical assays.
Lipid peroxidation Lipid peroxidation (LPO) in the tissue homogenate was measured by estimating the formation of TBAreactive substances (TBARS).15 Tissue homogenate (10% w/v in 50 mM phosphate buffer, pH 7.4) was mixed with TCA (10%) and TBA (0.34%) and boiled in a water bath for 15 minutes, cooled, and centrifuged. Absorbance of the supernatant was read at
535 nm. TBARS was calculated using tetraethoxypropane as the standard.
Glutathione A 10% (w/v) tissue homogenate prepared in 5% (w/v) trichloroacetic acid, centrifuged at 2000 g for 5 minutes and GSH in the deproteinized supernatant was estimated by Ellman’s reagent with a standard curve.16
Antioxidant enzymes assays Brain tissue was homogenized (10% w/v) in ice-cold 50 mM phosphate buffer ( pH 7.4), centrifuged at 10 000 g for 20 minutes. at 4°C and the supernatant was used to assay the enzyme activities. SOD activity was measured using pyrogallol (2 mM) autoxidation in tris buffer.17 CAT activity was measured using H2O2 (3%) as the substrate in phosphate buffer.18 GPx activity was measured by the indirect assay method using GR. Cumene hydroperoxide (1 mM) and GSH (0.25 mM) were used as substrates and coupled oxidation of NADPH by GR (0.25 U) in tris buffer (50 mM, pH 7.6) was monitored at 340 nm.19 GR activity was assayed in a reaction mixture containing oxidized glutathione (20 mM) and NADPH (2 mM) in potassium phosphate buffer.20 GST activity was assayed by using glutathione (20 mM) and CDNB (30 mM) as the substrates in phosphate buffer, change in absorbance
Figure 2 Protective effect of D. hamiltonii aqueous extract on HCH-induced lipid peroxidation in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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at 344 nm was monitored in a UV–Visible Spectrophotometer.21 Protein content was estimated by the method of Lowry, with bovine serum albumin as the standard.
Statistical analysis The data were expressed as means ± SE of eight observations (n = 8) and significant difference between each of the groups was statistically analyzed by Duncan’s multiple range test (Statistica Software, 1999, Statsoft Inc., Tulsa, USA). A difference was considered significant at P < 0.05.
Results Lipid peroxidation Increased LPO was observed in cortex (22%) and cerebellum (26%) but it was not significant in brain stem and midbrain of HCH-treated rats. DHAE pretreatment in dose-dependent manner ameliorated the HCH-induced LPO. DHAE, per se, decreased the basal level of lipid peroxidation in cortex, cerebellum, and midbrain (Fig. 2).
Antioxidant enzymes Effect of HCH on the antioxidant enzymes also showed distinct regional variation. A significant decrease in the activity of SOD in all the brain regions of HCH-treated rats was seen. Brain stem (40%) and cortex (37%) showed marked decrease
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followed by midbrain and cerebellum. DHAE, prevented the HCH induced decrease in SOD activity dose dependently in all the brain regions studied, except for DHAE low dose in cerebellum (Fig. 3). CAT activity was markedly increased in all the brain regions of HCH-treated rats. The increase in activity was highest in the cortex (4 fold) followed by cerebellum (73%), brain stem (53%), and lowest in midbrain (33%). DHAE, both doses, prevented the increase in CAT activity induced by HCH in all brain regions except for brain stem (Fig. 4). An induction of GPx activity by HCH treatment in all the brain regions was observed, which was in the following order: cerebellum (119%) > cortex (110%) > brain stem (48%) > midbrain (34%). DHAE, both doses, prevented the increase in GPx activity induced by HCH in all the brain regions studied (Fig. 5). Marked induction of GR in the brain regions of the HCH-treated rats was seen, which was highest in the cortex (>4 fold) followed by cerebellum (67%), brain stem (39%) and, least in the midbrain (30%). DHAE prevented the HCH-induced increase in GR activity in cortex and cerebellum but not in midbrain and brain stem (Fig. 6). Similarly, GST activity was elevated in the brain regions of HCH-treated rats except cerebellum. Different brain regions responded to a different degree, with cortex (3 fold) showing highest induction followed by brain stem (30%) and least in midbrain. DHAE could only prevent the severe increase in the
Figure 3 Protective effect of D. hamiltonii aqueous extract on HCH-induced decrease in the SOD activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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Figure 4 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in CAT activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
Figure 5 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in GPx activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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Figure 6 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in GR activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
GST activity in the brain cortex of rats treated with HCH (Fig. 7). DHAE significantly prevented the toxic insult induced by HCH as measured by
antioxidant enzyme activities in the rat brain. The multiple dose treatment of DHAE, as such, enhanced the antioxidant status of the rat brain.
Figure 7 Protective effect of D. hamiltonii aqueous extract on HCH-induced increase in GST activity in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
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Figure 8 Protective effect of D. hamiltonii aqueous extract on HCH-induced decrease in GSH in major regions of the rat brain. Treatments: I – Control; Group II – DHAE (100 mg/kg b.w.) (7 days); Group III – DHAE (50 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group IV – DHAE (100 mg/kg b.w.) (7 days) + HCH (500 mg/kg b.w.); Group V – HCH (500 mg/kg b.w.). *Indicates statistical significance (P < 0.05).
Glutathione A significant reduction in GSH content was observed in all the brain regions of HCH-treated rats. In HCHtreated rats, cortex showed maximum reduction, and brain stem, the least. DHAE pretreatment not only prevented the decrease in GSH due to HCH treatment but also enhanced GSH levels in the cortex and cerebellum of the rat brain (Fig. 8).
Discussion HCH has been reported to cause lipid peroxidation/ oxidative stress in the tissues of rat.5 Brain is considered highly vulnerable to oxidative stress than other organs of the body.22 This study adds to the evidence that the vulnerability to oxidative stress of the rat brain is region-specific. Cortex and cerebellum showed increase in LPO but not brain stem and midbrain, which reflects the differential sensitivity of the brain regions to oxidative stress. This observation is in agreement with our earlier report of sub-chronic treatment of HCH to rats.6 The exact reason for such variation is not clear but the relatively higher amount of myelin (low in PUFA) in midbrain and brain stem could explain this difference.23 Induction of LPO by HCH may involve cytochrome P450dependent microsomal metabolism, which generates highly reactive metabolite pentachlorocyclohexene or due to ROS produced by respiratory burst.24 DHAE pretreatment prevented the HCH-induced LPO,
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which could be attributed to its free radical quenching antioxidant constituents. Oxidative stress is efficiently neutralized by synchronized actions of different cellular antioxidants (like SOD, CAT, GPx, GR, GST, and GSH) in mammalian cells. Altered antioxidant profile of the brain regions may indicate an adaptive biochemical response to HCH-induced oxidative stress.5,25 The decrease in GSH content and SOD activity; and increase in CAT, GPx, GR, and GST by HCH treatment in rat brain is in agreement with earlier reports.5–6 DHAE pretreatment could restore the depletion in GSH content and improve the altered antioxidant enzyme activities induced by HCH in rat brain. DHAE pretreatment, per se, boosted the antioxidant status in all the regions of the rat brain but alteration in the antioxidant status by HCH was due to the oxidative stress which was marked by increased LPO and decreased GSH level.5,26 The precise mechanism by which the plant extracts enhance the antioxidant profile is not well understood. Some studies report that the augmentation of antioxidant status by aqueous plant extracts is orchestrated by interaction with the antioxidant response elements (AREs) that transcriptionally regulate the genes related to antioxidant enzymes.27 It has also been shown that the γ-glutamylcystein synthetase (γ-GCS), a key enzyme in the GSH synthesis, is also transcriptionally regulated by AREs.28 The natural
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antioxidants like resveratrol, curcumin, quercetin, epigallocatechin 3-gallate, rosmarinic acid, etc. have been demonstrated to ameliorate neuronal oxidative in different models. Many of these natural antioxidants are not only active scavengers of free radicals but also act as modulators of pro-survival or pro-apoptotic signaling pathways. There is a possibility that the antioxidant compounds present in DHAE are interacting with AREs in vivo, which needs further research. Antioxidant intervention is gaining significance as therapeutic approach for treatment of neurological disorders. As natural plant products have minimal adverse effects in contrast to the side effects of a number of synthetic drugs they are being employed in antioxidant therapy for neurodegenerative disorders.29 Dietary antioxidants and herbal extracts can considerably contribute to the modulation of complex mechanisms of neurodegenerative diseases.30 This study for the first time demonstrates the protective potential of D. hamiltonii roots against HCHinduced neuronal oxidative stress. Because failure to cope with oxidative stress is a common factor in the aetiology of many diseases DHAE’s effects on the improvement of the antioxidant response could provide an explanation for the health promoting properties attributed to it. The antioxidant compounds isolated are prospective novel nutraceuticals and could be good candidates for use as therapeutic agents for treating neurodegenerative diseases involving oxidative stress.
Acknowledgements This work was done at Central Food Technological Research Institute, Mysore, India. The authors wish to thank the Director of the institute for his keen interest in this study. The first author acknowledges Council for Scientific and Industrial Research, New Delhi for awarding the research fellowship.
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