MATERIALS AND METHODS

Materials
Animals: Fifty adult male albino rats weighting 150-200 g were included in this study. They were divided into 5 equal groups, one control and the other 4 for the drugs. These drugs were given by oral ingestion through an orogastric tube. After four weeks, blood samples were taken from inner canthus of the eye by capillary tubes for measurements of serum alanine transaminase (ALT), serum aspartate transaminase (AST) and total bilirubin. Then all rats of the groups were scarified and dissected for light microscopic examination of their livers.

Drugs:

A Valporic acid and silymarin drug was suspended in 5.0 mL of deionized water, while vitamin E was dissolved in corn oil in accordance to Onyema et al. (2006). All preparations and appropriate concentrations were prepared with the help of a biochemist.

Grouping of animals: Fifty male albino rats were divided in five equal groups as follows:

Method
Anesthesia: After four week of treatment, the animals were deeply anesthetized by ether inhalation in a ball jar. Then, blood samples were collected for different laboratory analysis from inner canthus of the eye by capillary tubes.

Biochemical parameters studied: Blood samples of about 5 cm were obtained for biochemical analysis after four weeks of ingestion of the drugs from all rats.

The following biochemical parameters were used for each sample:

Histopathological study: After four weeks of daily ingestion of the drugs, all rats of each group were anaesthetized then sacrificed by cervical dislocation. After scarification livers were carefully dissected from each rat and were prepared for histological examination according to Carleton et al. (1980).

Statistical analysis: Data analyzed by software statistical computer package (SPSS) version 13. Paired student’s (t) test was employed for comparison between groups. Difference was considered significant at p<0.05 level. All values were expressed as Mean±SD.

RESULTS

In the present study, there was no statistically significant difference between (IA) negative control and (IB) positive control subgroups. Thus, in the subsequent results comparison referred as a control group. Results of biochemical study are shown in Table 1.

Results of histopathological study by light microscopically examination of the liver section of the different groups:

Table 1:	Statistical comparison between studied groups as regard serum AST, ALT and serum bilirubin
M: Mean, SD: Standard deviation, *: Significant, ***: Means very highly significant

Fig. 1:	Photomicrograph of liver tissue from control group (H and E) x 200

Fig. 2(a-c):	Photomicrograph of the liver of group IV: (a) Portal triaditis, (b) Hepatic cells infiltrated with inflammatory cells and (c) Hepatic cells with nuclear degeneration with prominent nucleoli and inflammation of the portal tract (H and E) x 200

Fig. 3(a-c):	Photomicrographs of livers of: (a) Group V: Mild portal triaditis and hepatic cells infiltrated mildly with inflammatory cells, (b) Group VI: Hepatic cells infiltrated mildly with inflammatory cells and (c) Group VII: Hepatic cells infiltrated mildly with inflammatory cells (H and E) x 200

DISCUSSION

Hepatitis more frequently observed in patients taking valporic acid than other antiepileptic drugs (Gopaul et al., 2003).

In the present study, there was no statistically significant difference between (IA) negative control and (IB) positive control subgroups, the histopathological studies of the liver of these groups show normal hepatic lobules and sinusoid and normal bile tract. AST, ALT and total bilirubin of valporic acid group were very highly significant increased in comparison with the control group. These results were in accordance with Tong et al. (2003), who said that these valporic acid drug induce hepatotoxicity by a multiple step mechanism. The exact mechanism responsible for liver injury caused by these drugs remains unknown. It may hypothesize to involve the generation of toxic metabolites and/or reactive oxygen species. The reactions of toxic metabolites with glutathione in mitochondria produce a localized depletion of glutathione that would result in oxidation stress. Oxidative stress precedes the onset of steatosis and necrosis in liver.

Jurima-Romet et al. (1996) and Graf et al. (1998) said that toxic effect of valporic acid by which free radicals have role in cytotoxicity, hepatotoxicity. It is known that free radical reduction lead to decrease in body antioxidant defense and increase in production of free radicals. In addition, at also induces oxidative stress Tang et al. (1997) found that rats show a similar genetically determined valporic acid mediated oxidative damage are generally attributed to the formation of free which act as stimulator of lipid peroxidation and source for destruction and damage to the cell membrane.

Alterations of various cellular defense mechanisms consisting of enzymatic and non-enzymatic components (reduced glutathione) have reported in valporic acid-induced hepatotoxicity (Tong et al., 2005). These results were in accordance with Tong et al. (2003), said that oxidative stress is one of the mechanisms for valporic acid-induced hepatic injury. Majority of normally formed free radicals removed by the action of reduced glutathione. In circumstances where there is a reduction in glutathione results in the initiation of lipid peroxidation resulting in tissue injury. Histopathological examination of the liver shows necro-inflammatory foci in the hepatic lobules and inflammation in the portal tract (portal triaditis).

Khan et al. (2011), who reported that histopathological changes in animals receiving valporic acid was portal inflammation, fatty changes and liver cell necrosis.

Studying valporic acid+silymarin group, AST, ALT and total bilirubin of this group were very highly significant increased in comparison with the control group and were significantly decreased in comparison with the valporic acid group. Histopathological examination of the group liver show mild degree of necrosis and mild inflammation in the portal tract. These results were in accordance with Kosina et al. (2002), who said that; the preclinical studies using different hepatotoxic substances showed that silymarin has multiple actions as a hepatoprotective agent. The antioxidant property and cell-regenerating functions because of increased protein synthesis considered as most important. It provides an important additional protection against oxidative damage. There are varieties of antioxidants in silymarin which protect against disease-causing oxidative damage.

Non-protein thiol is an important defense mechanism in living cells. As a substrate for antioxidant enzymes, i.e., glutathione peroxidase and glutathione reductase, it protects cellular constituents from the damaging effects of peroxidase formed in metabolism and other reactive oxygen species reactions. Aged garlic extract increases cellular glutathione in a variety of cells including those in normal liver and mammary tissue (Tanaka et al., 2004).

AST, ALT and total bilirubin of valporic acid+vitamin E group were highly significant increased in comparison with control group and were significantly decreased in comparison with valporic acid group. Histopathological examination of group livers shows mild degree of necrosis and mild inflammation in the portal tract. These results were agree with Aluclu et al. (2009), who reported that chronic treatment with valporic acid decrease antioxidant level of vitamin E. The related studies have shown that vitamin E has a protection effect against hepatotoxicity resulting from valporic acid application. Vitamin E is a natural antioxidant and extremely effective in detoxifying harmful free radicals in various tissues. It has inhibited hepatocytes lipid peroxidation caused by free radical forming agents and this reflects the ability of tocopherol to scavenge reactive species within the lipid region of the membrane and thereby, prevent cell injury (Tayal et al., 2007). Buchi et al. (1984) has revealed that antioxidant containing α-tocopherol and N, N diphenyl-p-phenylenediamine have been effective in reversing the hepatotoxicity induced by valporic acid in rats. Vitamin E, the major lipid-soluble antioxidant in liver, can scavenge free radicals and preserve membrane integrity. It protects cells and sub-cellular structures from the oxidative damage by decreasing LPO products (Bansal et al., 2005).

Sodergren et al. (2001) said that; antioxidants recognized to scavenge free radicals, might prevent propagation of the drug-induced lipid peroxidation process. Vitamin E is a well-characterized chain-breaking antioxidant with the particular function of preventing lipid peroxidation in membrane systems.

Continuous valporic acid treatment decrease level of antioxidants such as vitamin E and glutathione peroxidase, resulting in oxidative stress (Tamai et al., 1988; Kotch et al., 1995; Amel et al., 2012).

In short, the liver is highly affected by ingestion of valporic acid. However, ingestion of silymarin and/or vitamin E that is naturally occurring antioxidants can decrease this harmful effect of these drugs on the liver. We recommended that the patient on chronic use of valporic acid must do periodic investigation of liver function and they must use silymarin and/or vitamin E to protect their livers.