AMELIORATING EFFECT OF VITAMIN-E AGAINST CADMIUM (CD) INDUCED BRAIN OXIDATIVE DAMAGE IN ALBINO RATS

Bhuvaneswari Devi C 1 and Kiran Kumari, K 2 . 1. Department of Cell Biology and Neuroscience, University of California Riverside, CA, USA. 2. Department of Zoology, S.P.W. Degree and PG College, Affiliated to Sri Venkateswara University, Tirupati517502, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (7), 1214-1223 1215 2003). Acute inhalation of sufficient exposure of cadmium can cause both a chemical pneumonitis and pulmonary edema from the toxic effect to the alveolar epithelium and endothelium (Newman et al., 1996). Within 24h of exposure, workers develop shortness of breath, fever, and fatigue, which can progress to pulmonary edema and death (Fernandez, 1996). Chronic exposure to cadmium dusts and fumes has been suspected as a cause of emphysema, obstructive lung disease, pulmonary fibrosis, and lung cancer. Exposure of adult rats to low or moderate doses of Cd induced lipid peroxidation (LP) in all tissues, mainly lung and brain (Manca, 1991). Monroe and Halvorsen, 2006 provided evidence that CdCl2 increases oxidative stress in nervous cells. In experimental studies with animals, cadmium can induce neurotoxicity with a wide spectrum of clinical entities including neurological disturbance (Viaene et al., 2000). Changes in the normal neurochemistry of the brain (Gutierrez- Reyes et al., 1998), several authors have shown that antioxidants should be one of the important components of an effective treatment of cadmium poisoning (Valko et al., 2006). Accordingly, to prevent undesired oxidative damage induced by reactive species, organisms are equipped with several lines of antioxidant defence. Vitamin C is an aqueous phase antioxidant has been established for many decades and has several important roles. It modulates intracellular redox status through maintaining sulfhydryl compounds, including glutathione, in their reduced state. It has been suggested to repair protein hydroperoxides, through regeneration of parent amino acids by reduction. Also, it serves to maintain membrane alpha tocopherol and enzyme activities including hepatic mixed function oxidase activity (Suberlich et al., 1994). Gupta and Kar, (1998) reported that vitamin C can prevent increased lipid peroxidation levels resulting from cadmium toxicity. Vitamin C could serve as an effective antioxidant against restraint stress induced pro-oxidant status and increase the antioxidant enzyme activity in rat brain (Zaidi et al., 2004). In view of the above studies mentioned the present study has implicated the importance of antioxidants such as vitamin E in protecting living organism against the toxic effect of cadmium exposure in the brain regions of young Albino rats.

Materials And Methods:-
Chemicals:-Cadmium (Cd) and Vitamin E were selected as test chemicals. The chemicals used in this study namely Thiobarbutric acid, Glutathione oxidized, NADPH, DTNB, Reduced glutathione, Epinephrine were obtained from Sigma, USA. The remaining chemicals obtained from Qualigens and Loba Chemie, India.

Procurement and maintenance of experimental animals:-
The young and adult albino rats (1 month and 3 months old) (Wistar) were purchased from Sri Venkateswara traders, Bangalore and maintained in the animal house of Watson Life Sciences, Tirupati. The animals were housed in transparent plastic cages with hardwood bedding in a room maintained at 28 o ±2 o C and relative humidity 60±10% with a 12 hour light/day cycle. The animals were fed in the laboratory with standard pellet diet supplied by Sri Venkateswara traders, Bangalore and water ad libitum. The protocol and animal use were approved by Y.V. University, India.

Animal exposure to Cd and Vitamin E:-
The young albino rats (3 months) were exposed to Cd (Low dose: 1.5 mg/kg/body weight and High dose: 3mg/kg/body weight). A separate batch of low dose and high dose of Cd exposed rats received vitamin-E (5mg/kg/body weight) intraperitoneally for a week. The control animals received only deionized water without Cd. After the period of dosage, the animals were sacrificed through cervical dislocation and the tissues were stored at -80 0 C for the further biochemical analysis.
Biochemical Studies:-Preparation of Brain Mitochondrial Fraction:-Brain mitochondrial fractions were prepared following Lai and Clark, 1979. Briefly, the tissue was homogenized in 5 volumes (w/v) of SET buffer (0.25 M sucrose, 10 mM Tris-HCl, and 1 mM EDTA, pH 7.4). The homogenate was first centrifuge at 800 g for 10 min at 4°C, and then the supernatant was centrifuged at 10,000 g for 20 min at 4°C. Then the pellet of mitochondrial fraction was suspended in SET buffer.
Glutathione peroxidase (GPx) activity:-GPx activity in the mitochondrial fraction of rat kidney was assayed as described by Rotruck et al., (1973). The reaction mixture contained 0.2 ml of EDTA, 0.2ml of 4mM sodium azide, 0.2ml of glutathione reduced, 0.2ml of H 2 O 2 , 0.4ml of 0.32M Sodium pyrophosphate buffer (pH-7.0), 0.1ml of enzyme source. Then the reaction mixture 1216 was incubated at 37º C for 10 min. Then the reaction was arrested by adding of 0.5 ml 10% TCA. Then centrifuged at 2000 rpm for 10 min. To 0.5 ml of supernatant, 3.0 ml of 0.3M disodium hydrogen phosphate and 1.0 ml of DTNB were added and the reaction was read at 412 nm in spectrophotometer. The enzyme activity was expressed as µ mole/min/mg protein.

Glutathione reductase (GR):
GR activity in the mitochondrial fraction of rat brain was assayed as described by Staal et al., (1969). The reaction mixture in a final volume of 3.0ml contained, 1.0ml of 0.3 M Sodium phosphate buffer (pH-6.8), 0.5ml of 250 mM EDTA, 0.5ml of 12.5 mM GSSG, 0.7ml of distilled water, 0.2ml of 30mM NADPH and 0.1ml of enzyme extract. Changes in absorbance were recorded at 340 nm in a spectrofluorometer. The enzyme activity was expressed as µ moles of NADPH oxidised/mg protein/min.

Xanthine oxidase (XO) activity:-
Xanthine oxidase activity was estimated by the method given in Worthington Manual (2004). The assay mixture contained 1.9ml of phosphate buffer (pH 7.5), 1.0 ml of hypoxanthine and 0.1 ml of enzyme source. Increase in absorbance was recorded and absorbance was recorded at 290 nm from the linear curve. The rate is proportional to enzyme concentration within limits of 0.01-0.02 units per test. The activity was expressed as moles of µ urate formed /mg protein/min.
The reaction mixture in a final volume of 3.0 ml contained: 150 mM phosphate buffer (pH 7.5), 1 mM CDNB, 5 mM glutathione (GSH) and an appropriate amount of enzyme protein. The reaction was initiated by the addition of GSH and incubated at 37°C. The formation of a thioether by the conjugation of CDNB to GSH was monitored at 340 nm in a spectrofluorometer. Thioether concentration was determined from the slopes of initial reaction rates. The activity was expressed as µ moles of thioether formed/mg protein/min, where one unit of enzyme activity is defined as one µ moles of thioether formed/mg protein/min.

Lipid peroxidation;-
The level of lipid peroxidation in the tissues was measured in terms of malondialdehyde (MDA; a product of lipid peroxidation) content and determined by using the thiobarbituric acid (TBA) reagent. The reactivity of TBA is determined with minor modifications of the method adopted by Hiroshi et al., (1979).
To 2.5 ml of homogenate, 0.5 ml of saline (0.9% sodium chloride), 1.0 ml of (20% w/v) trichloroacetic acid (TCA) were added. The contents were centrifuged for 20 minutes on a refrigerated centrifuge at 4000 x g. To 1.0 ml of supernatant, 0.25 ml of TBA reagent was added and the contents were incubated at 95°C for 1hr. 1ml of n-butanol was added to it. After thorough mixing, the contents were centrifuged for 15 minutes at 4000 g in a refrigerated centrifuge. The organic layer was transferred into a clear tube and its absorbance was measured at 532 nm. The rate of lipid peroxidation was expressed as µ moles of malondialdehyde formed/g wet wt. of tissue.

Estimation of protein content:-
Protein content of the kidney was estimated by the method of Lowry et al., (1951). 1% (W/V) homogenate was prepared in 0.25 M ice cold sucrose solution. To 0.5 ml of crude homogenate, 1ml of 10% TCA was added and the samples were centrifuged at 1000 g for 15 min. The residue was resuspended in 0.5 ml of 1N NaOH. And 4 ml of alkaline copper reagent was added followed by 0.4 ml of folin-phenol reagent (1:1 folin: H 2 O). The color was measured at 600 nm in a UV-Vis spectrophotometer (Hitachi model U-2000) against blank. The protein standard graph was prepared using Bovine serum albumin. The protein content of the tissues was calculated using the standard graph.

Statistical treatment of the data:-
The mean and standard deviation (SD), analysis of variance (ANOVA) was calculated using standard statistical software package.

Discussion:-
Cadmium has been recognized as one of the most toxic environmental and industrial pollutant. Cadmium is ubiquitous toxic metal that may induce oxidative damage by disturbing the prooxidant-antioxidant balance in tissues was observed in rats treated with cadmium (Kostic et al., 1993;Zikic et al., 1998;Pavlovic et al., 2001). The severity of intoxication depends upon the route, dose, and duration of exposure to the metal (Ognjanovic et al., 1995).
Our data showed that significant decrease in the activities of GPX, GR, XO and increased GST activity in both doses of Cd exposure. However, the effect was more in high dose exposed animals compared to the low dose exposed animals. And moreover, among the brain regions studied, cerebral cortex was found to be more vulnerable to the Cd induced neurotoxicity compared to the other brain regions. And we also observed a significant increase in lipid peroxidation (i.e., increased MDA level) in all the three brain regions of cd-exposed rats. Further, inhibition of antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase in cadmium-exposed animals suggests disturbed antioxidant/pro-oxidant ratio, resulting in oxidative stress. A number of antioxidant defense systems, both enzymatic and nonenzymatic, operate to control excessive levels of ROS.
The brain is particularly sensitive to oxidative damage because of its high rate of oxidative metabolism (Pajovic et al., 2003). Cd also enhances the production of free radicals in the brain of adult rats and interferes with the antioxidant defense system that in turn leads to a Cd-induced alteration of the structural integrity of lipids (Shukla et al., 1996). In adult rats exposed to Cd, there was an increase of LP in the corpus striatum and cerebral cortex (Pal et al., 1993). (Nishimura et al., 2006) reported that Cd can enter the brain parenchyma and neurons and causes neurological alterations in humans (Rose et al., 1992) and animal models (Lukawski et al., 2005), leading to lower attention, hyper nociception, and olfactory dysfunction and memory deficits.
The GPx activity with Vitamin-E co-administration along with cadmium, this could be due to the supplements enhanced the antioxidative defense system protected against cadmium toxicity (Shaikh et al., 1999).  (Waisberg et al., 2003). The activity of LP was significantly lowered in rats co-supplemented with Vit-E supplement. This is due to the antioxidant and antioxidant defense systems protects cells from Cd-induced toxicity (Shaikh et al.,1999). The LP activity was much more lowered in rats supplemented with Vitamin E. This could be due to the Vitamin E was liposoluble antioxidant, has key role in scavenging free radicals and stabilizing the cell membranes thus maintaining their permeability. Moreover, it is known that the antioxidant (Vitamin E) may act synergistically preventing lipid peroxidation and cell destruction (El Demerdash et al., 2004). Since Vitamin E (α-tocopherol) is primarily located in cell membranes, it is possible that α-tocopherol modifies the kinetics of distribution of cadmium in cell membranes, such that its delivery to neuron or glia cells may be impeded. However, our observation that cadmium concentration in the nervous system was not reduced by α-tocopherol supplementation suggests that the benefits of α-tocopherol treatment were not a result of changes in cadmium distribution in the nervous system. Rather our findings are consistent with the view that protective effects of αtocopherol against sub chronic high dose cadmium exposure might be due to its antioxidant properties, and this may have a major impact on improving the quality of life of individuals suffering from neurotoxicity caused by cadmium exposure.