Neuroprotective effects of zinc on antioxidant defense system in lithium treated rat brain

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1 Indian Journal of Experimental Biology Vol. 45, November 2007, pp Neuroprotective effects of zinc on antioxidant defense system in lithium treated rat brain Punita Bhalla 1, Vijayta Dani Chadha 1, Rakesh Dhar 2 & D K Dhawan 1* 1 Department of Biophysics, Panjab University, Chandigarh, , India 2 Regional Sophisticate Instrumentation Center, Panjab University, Chandigarh, , India Received 6 June 2006; revised 20 February 2007 With a view to find out whether zinc affords protection against lithium toxicity the activities of antioxidant enzymes and lipid peroxidation profile were determined in the cerebrum and cerebellum of lithium treated female Sprague Dawley rats. Lipid peroxidation was significantly increased in both the cerebrum and the cerebellum of animals administered with lithium for a total duration of 4 months as compared to the normal control group. On the contrary, the activities of catalase and glutathione-s-transferase (GST) were significantly reduced after 4 months of lithium treatment. The activity of superoxide dismutase (SOD) was significantly increased in the cerebrum after 4 months lithium administration, whereas in the cerebellum the enzyme activity was unaffected. No significant change in the levels of reduced glutathione (GSH) was found in either cerebrum or cerebellum after 2 months of lithium treatment. However, 4 months lithium treatment did produce significant changes in GSH levels in the cerebrum and in the cerebellum. Zinc supplementation for 4 months in lithium-treated rats significantly increased the activities of catalase and GST in the cerebellum, showing that the treatment with zinc reversed the lithium induced depression in these enzyme activities. Though, zinc treatment tended to normalize the SOD activity in the cerebrum yet it was still significantly higher in comparison to normal levels. From the present study, it can be concluded that the antiperoxidative property of zinc is effective in reversing the oxidative stress induced by lithium toxicity in the rat brain. Keywords: Cerebellum, Cerebrum, Lithium, Oxidative stress, Zinc Lithium carbonate is one of the mood stabilizing drugs. It is used in the therapeutic treatment of manic depressive psychosis 1,2 but has narrow therapeutic index 3 and an overdose results in toxic side-effects. Lithium effects have been investigated in detail in the brain, intestine, liver, and thyroid functions 4,5. There have been reports of its neurotoxicity occurring even at therapeutic doses. Lithium alters the activities of enzymes superoxide dismutase (SOD) and glutathione peroxidase in the brain 6. Further, malondialdehyde (MDA) levels (a marker for lipid peroxidation) were found to be significantly increased in the kidney following lithium treatment 7. Alterations in the levels of essential and non-essential elements in the rat liver and brain following lithium administration to diabetic, and lead treated rats have been reported 8,9. Significant reduction in the levels of zinc in the serum of lithium-treated rats has also been *Correspondent author Telephone: dhawan@pu.ac.in observed 10. This reduction in zinc levels could be an adverse effect of the long-term lithium therapy. Zinc is an essential trace element required for a broad range of biological activities. It is nontoxic in physiological doses 11. It is known to be associated with metal binding proteins that regulate the functions of zinc as well as of copper 12. Zinc is present in large amounts in the brain and about 10% of the total brain zinc is localized in the glutamate containing synaptic vesicles, and is liberated during synaptic neurotransmitter release 13. Zinc stabilizes the cell membrane structure through its antioxidant actions by regulating the levels of metallothioneins 14, and has also been reported to inhibit spontaneous lipid peroxidation in the rat brain 15. Since chronic treatment with lithium produces increased oxidative stress together with a fall in the levels of zinc, and zinc is known to have antioxidative effects, it is of interest to investigate whether zinc supplementation during lithium therapy can counter oxidative stress by augmenting the antioxidative

2 BHALLA et al.: NEUROPROTECTIVE EFFECT OF ZINC IN RAT BRAIN 955 mechanisms in cerebrum and cerebellum regions of the rat brain. Materials and Methods Rats (40) of Sprague Dawley (SD) strain, weighing g in the age group of 3 to 4 months were obtained from the Central Animal House, Panjab University, Chandigarh. Animals were housed in polypropylene cages under hygienic conditions and were acclimatized to the laboratory environment for at least one week before putting them on different treatments. All procedures were done in accordance with ethical guidelines for care and use of laboratory animals, and protocols were followed as approved by the Experimental Animals Committee. Animals were divided into following four groups of 10 animals each. Group I consisted of untreated rats and served as normal controls; Group II consisted of rats administered with lithium in diet; Group III consisted of rats administered with zinc in drinking water; and group IV consisted of rats co-administrated with lithium and zinc. Animals of group I which served as normal controls were fed standard laboratory feed and water ad libitum. Animals of group II and IV were given lithium in the form of lithium carbonate in diet at a dose of 1.1 g/kg diet 16. Animals of group III and IV were given zinc in the form of zinc sulfate mixed in drinking water at a dose level of 227 mg/l. The animals were weighed before starting different treatments and then after every three days till the end of the study. All the treatments were given for three different durations of one, two, and four months. Lithium levels in the plasma were also estimated and were found to be in the range of meq/lt. Tissue preparation At the end of various treatments, the animals of each group were anesthetized with ether and sacrificed by decapitation; their brains were removed, rinsed in ice-cold isotonic saline, and dissected into two regions (viz. cerebrum-whole cerebral hemisphere and cerebellum). Tissue homogenates (10%; w/v) were prepared in ice-cold 10 mm PBS (phosphate-buffered saline, 0.15 M NaCl), ph 7.4. The homogenates were centrifuged at 1000 g for 10 min at 4 C and the supernatant was used for biochemical assays. For the superoxide dismutase assay, the supernatant was further centrifuged at 12,000 g for 20 min to remove the mitochondrial pellet. Lipid peroxidation The quantitative measurement of lipid peroxidation was performed according to the method of Wills 17. The results were expressed as nmoles malondialdehyde/mg protein. Catalase Catalase was estimated by the U.V. spectrophotometer method described by Luck 18. Superoxide dismutase The assay was performed according to the method of Kono 19. The method is based on the principle of an inhibitory effect of superoxide dismutase on the reduction of nitroblue tetrazolium (NBT) dye by superoxide anions which are generated by photooxidation of hydroxylamine hydrochloride. Reduced glutathione Glutathione content was estimated according to the method of Ellman 20. Glutathione-S-transferase The enzyme activity was assayed by the method of Habig et al 21. Protein estimation Protein contents were estimated by using the method of Lowry et al 22. Statistical analysis Tabulated values represent means ± SD. One way analysis of variance (ANOVA) followed by post hoc student-newman-keuls multiple comparison tests were used to analyze the data from experimental and control groups. Values of P<0.05 were considered as significant. Table 1 Effect of zinc on lipid peroxidation (LPO) status in different regions of lithium treated rats [Valus expressed as nmoles of MDA formed/mg protein are mean ± SD] Control 0.55 ± ± ± ± ± ± 0.02 Lithium treated 0.42 ± ± ± ± ± 0.04 a ± 0.04 a1 Zinc treated 0.49 ± ± ± ± ± ± 0.06 Lithium + Zinc treated 0.42 ± ± ± ± ± 0.02 b ± 0.08 b1 F 3,36 Value a Comparison of all the treatment groups with normal control group. P values: a2,b2 <0.001; a1,b1 <0.05

3 956 INDIAN J EXP BIOL, NOVEMBER 2007 Results and Discussion In the present study, four months lithium treatment significantly enhanced the lipid peroxidation both in the cerebrum and in the cerebellum (Table 1). The magnitude of increase was higher in the cerebrum. The results are thus in accordance with the current studies on cellular injury by long-term lithium administration which have implicated peroxidation of polyunsaturated fatty acids, leading to the degradation of phospholipids, which is considered as an index of cellular deterioration 24. The present result showed that zinc countered the lipid peroxidation produced by lithium. This would appear to be due to zinc s antiperoxidative properties. This is of interest to note that Chan et al 25. have demonstrated that zinc is involved in destruction of free radicals through cascading enzyme systems. In general, the mechanism of antioxidation by zinc can be divided into acute and chronic effects. Chronic effects involve exposure of an organism to zinc on a long-term basis, resulting in induction of some other substance that is the ultimate antioxidant, such as the metallothioneins. These zinc-metallothioneins may serve as an efficient antagonist in inhibiting lipid peroxidation in the brain. Studies have shown that zinc causes inhibition of both endogenous as well as induced lipid peroxidation to stabilize biomembranes 26. The present data showed that the activity of catalase was decreased significantly by lithium (Table 2). The decrease was observed both in the cerebrum and in the cerebellum in lithium-treated animals. The specific activity of superoxide dismutase was, however, found to be significantly increased after lithium administration for 4 months in both the brain regions studied (Table 3). The balance between antioxidant enzymes, superoxide dismutase and catalase, is relevant for cell function 27. However, in the present study, the antioxidant balance in the brain has been altered by lithium administration which may perturb the brain cell normal functioning. The decrease in the activity Table 2 Effect of zinc on catalase activity in different regions of lithium treated rats [Values expressed as μmoles of H 2 O 2 decomposed/min./mg protein are mean ± SD] Control ± ± ± ± ± ± 1.85 Lithium treated ± ± ± ± ± 6.19 a ± 2.16 a2 Zinc treated ± ± ± ± ± ± 4.26 Lithium + Zinc treated ± ± ± ± ± ± 4.60 a1,b2 F 3,36 Value a Comparison of all the treatment groups with normal control group. P values: a1 <0.05; a2b2 <0.001 Table 3 Effect of zinc on superoxide dismutase (SOD) activity in different regions of lithium treated rats [Values expressed as I.U. of SOD are means ± SD] Control ± ± ± ± ± ± 2.41 Lithium treated ± ± ± ± ± 2.09 a ± 3.00 Zinc treated ± ± ± ± ± 2.93 a ± 3.15 Lithium+Zinc treated ± ± ± ± ± 1.21 a1,b ± 4.04 F 3,36 Value a Comparison of all the treatment groups with normal control group. P values: a1 <0.05, a2b2 <0.001

4 BHALLA et al.: NEUROPROTECTIVE EFFECT OF ZINC IN RAT BRAIN 957 of catalase, may lead to perturbation in the antioxidant defense. Following zinc administration, the altered levels of enzymes tended to be normalized because of antioxidative property of zinc. Similar reports indicating the antioxidative properties of zinc have been reported earlier Zinc plays an important role in the antioxidant cellular defences being a structural element of nonmitochondrial form of the enzyme superoxide dismutase 31. In the presence of zinc, superoxide dismutase is able to reduce the superoxide radicals to hydrogen peroxide 32. The levels of reduced glutathione were found to be significantly increased in animals administered with lithium for a time period of 4 months (Table 4). The increase was observed in both the brain regions studied. The increased levels of reduced glutathione in lithium-administered animals would suggest an increased detoxification capacity of the brain. Most of the reduced glutathione in the brain is localized in glial cells rather than in neurons 33 suggesting that lithium administration affects the glial cells. Furthermore in the present experiment, a decrease in glutathione-s-transferase following lithium treatment both in the cerebrum and in the cerebellum was observed (Table 5). The observed augmentation of reduced glutathione levels and glutathione-s transferase activity following zinc treatment may be explained by the zinc s property of inducing metallothionein (S-rich protein) as a free radical scavenger, or its indirect action in reducing the levels of oxygen reactive species 34. In conclusion, the data from the present study show that zinc is effective in alleviating the lithium induced toxicity in the rat brain. The effect of zinc may be attributed to its property of inducing metallothionein (S-rich protein) which is a free radical scavenger, or to its indirect action on oxidative stress leading to a reduction in oxygen reactive species. Table 4 Effect of zinc on reduced glutathione (GSH) content in different regions of lithium treated rats. [Values expressed as µmoles GSH/gm tissue are means ± SD] Control 2.85 ± ± ± ± ± ± 0.14 Lithium treated 2.96 ± ± ± ± ± 0.22 a ± 0.20 a2 Zinc treated 3.00 ± ± ± ± ± ± 0.09 Lithium + Zinc treated 3.20 ± ± ± ± ± 0.09 a2, b ± 0.18 a2 F 3,36 Value a Comparison of lithium and zinc treated group with normal control group. P values: b1 <0.05; a2 <0.001 Table 5 Effect of zinc on glutathione-s-transferase (GST) activity in different regions of lithium treated rats. [Values expressed as µmoles of conjugate formed/min./mg protein are means ± SD] Control 0.27 ± ± ± ± ± ± 0.01 Lithium treated 0.18 ± ± ± ± ± 0.01 a ± 0.01 a1 Zinc treated 0.13 ± ± ± ± ± ± 0.02 Lithium + Zinc treated 0.18 ± ± ± ± ± a ± 0.01 b1 F 3,36 Value a Comparison of lithium and zinc treated group with normal control group. P values: a1b1 <0.05

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