MATERIALS AND METHODS

Growing albino rats of the Wistar strain (135-150 g) were obtained from the Animal House, Department of Biochemistry in the Faculty of Basic Medical Sciences, University of Uyo, Uyo-Akwa Ibom State. The animals were kept in the experimental section of the animal house, in a well ventilated standard laboratory condition of temperature and relative humidity. The animals were fed with normal rat formular (Pfizer Livestock Co. Ltd., Aba, Nigeria). Both the experimental and control animals had free access to both rat chow and water during the experimental period. The animals were randomly divided into 8 groups of 7 rats each. Group 1 animals served as the control and were gavaged normal saline (1.0 mL). Groups 2, 3, 4, 5, 6, 7 and 8 received phenytoin, phenytoin + folic acid, phenytoin + vitamin E, phenytoin + vitamin E + vitamin C, phenytoin + vitamin C, phenytoin + folic acid +vitamin E and phenytoin + vitamin E + vitamin C + folic acid, respectively.

Administration of phenytoin and antioxidant vitamins: Commercially available phenytoin capsules were obtained from Parke-Davis Hoofreg, Vitamin C and folic acid tablets were obtained from Emzor Pharmaceuticals (Nig) Ltd, Lagos, Nigeria while Vitamin E (soft gel) was of Viboost Healthcare Limited, India. The choice of commercial products was to reflect the condition under which these medications are administered. The drugs were administered daily by oral intubation as follows, Phenytoin: 5 mg kg^-1 body weight of rat, folic acid: 70 μg kg^-1 body weight of rat, vitamin C: 1.4 mg kg^-1 body weight of rat and Vitamin E: 10 IU kg ^-1 body weight of rat. The treatments lasted for 4 weeks.

Collection and preparation of samples: The animals were anaesthetized with chloroform and sacrificed following guidelines of our Institutional Ethics Committee on animal studies. Blood samples were collected into plain sample tubes for sera preparation. The blood samples were allowed to clot at room temperature for 2 h. Sera were separated by centrifugation at 3000 g for 5 min using bench top centrifuge (MSE Minor, England). Sera were separated into plain sample tubes and stored in the refrigerator for analysis. All analysis on serum samples were completed within 24 h of sample collection.

Biochemical analysis: Serum proteins, uric acid and hepatic enzymes of clinical significance were measured in serum samples spectrophotometrically, using Randox kits (Randox Laboratory Ltd., Diamond Road, Crumlin, Co. Antrim, United Kingdom, BT29 4QY). Serum catalase activity was determined by the spectrophotometric assay using Bioxytech catalase kit (OxisResearch, Foster City, U. S. A.). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured according to the method of Reitman and Frankel (1957). Serum total protein was analyzed using biuret kit method (Tietz et al., 1995). The absorbances of all the tests were determined using spectrophotometer (HAICH, DR 3000, Germany).

Statistical analysis: Statistical analysis was carried out by employing student‘s t-test and ANOVA post hoc, a probability of p<0.05 was considered significant.

RESULTS

Uric acid and serum catalase levels of rats on phenytoin therapy were estimated to investigate the effects of antioxidant vitamins on phenytoin related hepatotoxicity. Table 1 and 2 shows the influence of phenytoin and antioxidant vitamins supplement on some biochemical indicators of liver injury. The administration of phenytoin alone caused significant (about 100%) increases in AST (p = 0.00) and ALT (p = 0.000) when compared to control animals. Significant increase in serum globulin and decrease in serum total protein and albumin were also observed in the phenytoin treated rats. Serum concentration of uric acid was highly elevated (p = 0.000) while serum catalase activity was significantly reduced following phenytoin treatment. Supplementation of phenytoin treatment with antioxidant vitamins resulted in various degrees of protective changes against phenytoin induced toxicity. The serum activities of AST and ALT were significantly reduced by supplementation with folic acid, vitamin E and/or vitamin C when compared with groups that received no supplement. The phenytoin associated increase in globulin and decrease in albumin level and serum catalase enzyme activity, were reversed by antioxidant vitamin supplementations. However, the elevated level of uric acid in serum was not affected by any of the vitamins. Analysis of data (Fig. 1, 2 and 3) showed significant position correlation between serum aminotransferases and serum globulin (r = 0.481, p = 0.000) and significant negative correlation between the aminotransferases and albumin (r = -0.773, p = 0.000) and catalase activities (r = -0.663, p = 0.000). No correlation between the transaminases and uric acid was observed in the treated groups of animals.

Table 1:	Effects of antioxidant vitamins on phenytoin induced changes in serum proteins
Values are expressed as Mean±SD. *Significantly different values of test group compared to control at p<0.05

Table 2:	Effects of antioxidant vitamins on phenytoin induced changes in serum aminotransferases, catalase and uric acid
Values are expressed as Mean±SD. *’**Significantly different values of test group compared to control at p<0.05 and p<0.01, respectively

Fig. 1:	Correlation graph of catalase versus AST

Fig. 2:	Correlation graph of uric acid versus AST

Fig. 3:	Correlation graph of uric acid versus catalase

DISCUSSION

Immunological responses to the drug and excessive generation of reactive intermediates are thought to be the probable mechanisms of phenytoin toxicity. Although, clinical evidence of immunologic hypersensitivity to phenytoin have been demonstrated by the presence of rashes, fever, lymphademopathy and eosinophilia (Howard et al., 1991; Havill, 1998), molecular interactions of phenytoin or its reactive metabolic products with tissues that could result in generation of inflammatory modulators and markers of tissue destruction such as uric acid, have never been demonstrated. Uric acid is a major product of purine catabolism, a reaction catalyzed by xanthine oxidase. Increased serum concentrations of uric acid have been associated with conditions of increased nucleic acid turnover as in cancers, infections, trauma, chemotherapy and inflammatory diseases (Wu and Wu, 2007, 2008; Conen et al., 2004). In this study we demonstrated that xanthine oxidase, a microsomal enzyme known to generate Reactive Oxygen Species (ROS) and uric acid (Berry and Hare, 2004), might play a role in phenytoin induced liver injury. Recently evidence have shown that ROS and/or their products may modulate cell signaling pathways of inflammatory response such as transcription factor activation (NF-KB and NrF2) and apoptosis (Haddad et al., 2000; Jacobi et al., 2005) and that failure of inflammatory cell to undergo apoptosis has been implicated in persistence of the inflammatory response. Chronic exposure to oxidative stress could therefore be associated with resistance of inflammatory cells to apoptosis (D’Angio and Finkelstein, 2000; Ruggiero et al., 2007).

Enzymes and protein makers of liver injury were measured in the experimental animals and controls. AST, ALT, uric acid and globulin were significantly raised while serum albumin and catalase activity were significantly reduced by the administration of phenytoin. These findings suggest hepatocellular damage by the drug. Aspartate and alanine aminotransferases are known to be elevated in serum during liver damage and/or during increased cellular proliferation. Increased synthesis of immunoglobulin and acute phase reactants following tissue injury may contribute to elevated level of globulin fraction of the serum proteins and impaired synthesis of albumin by the hepatocytes during phenytoin treatment may be responsible for decrease albumin level in the serum. Serum catalase activity in the rats receiving phenytoin becomes depressed probably because of over consumption of catalase by the excessive amount of reactive oxygen and arene oxides species. Decreased catalase and reduced glutathione levels had been reported following phenytoin toxicity (Navarova et al., 2005). Increase in serum uric acid concentrations of rat receiving phenytoin therapy also support the likelihood of phenytoin induced tissue injury. Phenytoin is a known microsomal enzymes inducer and xanthine oxidase is one of them. This enzymes catalyzes the degradation of hypoxanthine to xanthine, then to uric acid and in the process produces superoxide radicals which are converted to hydrogen peroxide (Hille and Nishino, 1995). These reactions also contribute to the oxidation state of individuals receiving phenytoin therapy in addition to the effects of phenytoin arene oxides.

Supplementation of phenytoin treatment with vitamins C and E and folic acid resulted in various degrees of protection against phenytoin-induced liver damage as evident by reduction in the levels of the liver enzymes, namely AST and ALT. However, the serum levels of uric acid in all phenytoin exposed animals were significantly higher than those of unexposed animals, thus suggesting that the protection offered by these antioxidant vitamin supplementations might not have been medicated by inhibition of induction of xanthine oxidase by phenytoin but may be related to vitamins’ ability to quench the effects of the reactive oxygen species and phenytoin arene oxide intermediate on cellular macromolecules. This was further supported by the finding that catalase activities in animal receiving the vitamin supplementations were restored to values above that of control animals. Significant negative correlation was observed between the transaminases and serum catalase activities but was not however observed between transaminases activities and uric acid concentrations. This suggests that, although liver injury following phenytoin treatment increased proportionally to depressed catalase activities (oxidative stress) in the controls and vitamin treated animals, uric acid concentration was not affected by changes in transaminases activities following antioxidants supplement. Induction of xanthine oxidase enzyme by phenytoin which resulted in increased uric acid synthesis did not appear to be affected by antioxidants treatment, however, the reactive oxygen species generated by the enzyme reactions were quenched by the antioxidants thus preventing excessive oxidative cellular damage.

In vivo and in vitro studies indicate that phenytoin mediated liver injuries may involve, at least in part peroxidase catalysed bioactivation of phenytoin to a reactive free radical intermediate which, if not detoxified, may initiated oxidative stress leading to oxidation of liver cell lipid, protein and DNA molecules (Winn and Wells, 1997). Highly reactive oxygen species, such as hydroxyl radicals (^•OH) could be generated by the phenytoin radicals via Fenton reactions. In vivo catalytic iron may be found loosely bound to membrane lipids, DNA and phosphate complexes to enhance fenton reaction (Smith et al., 2004). While OH^* is generally thought to be a primary intermediate of in vivo damage, reactive oxygen species are ultimately responsible for cellular damage if these are not detoxified by cytoprotective enzymes or antioxidants. Lipid peroxidation and protein oxidation and degradation by these radicals generally lead to structural and functional changes (Kehrer and Lund, 1994). This study has shown that supplementation of phenytoin treatment with antioxidant vitamins could ameliorate phenytoin related hepatic damage but however do not affect phenytoin induced uric acid synthesis by xanthine oxidase. The mechanism of phenytoin associated liver damage may therefore involve excessive generation of reactive species and that these oxidant species may be contributed significantly by the reactions of xanthine oxidase.