Indian Journal of Experimental Biology Vol. 45, November 2007, pp. 954-958
Neuroprotective effects of zinc on antioxidant defense system in lithium treated rat brain Punita Bhalla1, Vijayta Dani Chadha1, Rakesh Dhar2 & D K Dhawan1* 1
Department of Biophysics, Panjab University, Chandigarh, 160 014, India Regional Sophisticate Instrumentation Center, Panjab University, Chandigarh, 160014, India
2
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 psychosis1,2 but has narrow therapeutic index3 and an overdose results in toxic side-effects. Lithium effects have been investigated in detail in the brain, intestine, liver, and thyroid functions4,5. 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 brain6. Further, malondialdehyde (MDA) levels (a marker for lipid peroxidation) were found to be significantly increased in the kidney following lithium treatment7. 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 reported8,9. Significant reduction in the levels of zinc in the serum of lithium-treated rats has also been ______________ *Correspondent author Telephone: +91-172-2534119 E-mail:
[email protected]
observed10. 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 doses11. It is known to be associated with metal binding proteins that regulate the functions of zinc as well as of copper12. 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 release13. Zinc stabilizes the cell membrane structure through its antioxidant actions by regulating the levels of metallothioneins14, and has also been reported to inhibit spontaneous lipid peroxidation in the rat brain15. 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
BHALLA et al.: NEUROPROTECTIVE EFFECT OF ZINC IN RAT BRAIN
mechanisms in cerebrum and cerebellum regions of the rat brain. Materials and Methods Rats (40) of Sprague Dawley (SD) strain, weighing 110-130 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 diet16. 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 0.6-0.8 mEq/lt. Tissue preparation ⎯ At the end of various treatments, the animals of each group were anesthetized
Group
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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 Wills17. The results were expressed as nmoles malondialdehyde/mg protein. Catalase ⎯ Catalase was estimated by the U.V. spectrophotometer method described by Luck18. Superoxide dismutase ⎯ The assay was performed according to the method of Kono19. 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 Ellman20. Glutathione-S-transferase ⎯ The enzyme activity was assayed by the method of Habig et al21. Protein estimation ⎯ Protein contents were estimated by using the method of Lowry et al22. 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
