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Investigation of valproic acid-associated oxidative stress and hepatotoxicity Tong, Vincent

Abstract

A serious adverse reaction of valproic acid (VPA), a widely used anti-epileptic, is a rare but potentially fatal hepatotoxicity affecting mostly children under 2 years of age with developmental delay and who are on VPA polytherapy. The mechanism of the hepatotoxicity remains unknown. This thesis describes in vivo and in vitro studies to examine the hypothesis that VPA increases oxidative stress in rats that in turn is associated with liver toxicity. 15-F[sub 2t]- Isoprostaglandin (15-F[sub 2t]-lsoP), a free-radical catalyzed, lipid peroxidation breakdown product of arachidonic acid, was measured as an indicator of oxidative stress. Our findings showed a dose-dependent elevation in plasma and liver 15- F[sub 2t]-lsoP with plasma concentration-time profiles similar to that of the drug in rats given a single high dose of VPA. This increase in 15-F[sub 2t]-lsoP did not involve cytochrome P450-mediated biotransformation even though rats treated with both phenobarbital (PB) and VPA resulted in greater levels of 15-F[sub 2t]-lsoP compared to rats treated with only VPA. These results prompted further mechanistic studies in rats to establish an association between VPA glucuronidation and 15-F[sub 2t]-lsoP levels. VPA glucuronidation, the major VPA biotransformation pathway, correlated with 15-F[sub 2t]-lsoP formation. Levels of both 15-F[sub 2t]-lsoP and VPA-1-O-acyl glucuronide (VPA-G) were elevated by PB and reduced by inhibitors of VPA-glucuronidation in rats after a single dose of VPA. The fluorinated analogue of VPA (afluoro- VPA), which was a poor substrate for glucuronidation, did not elevate 15-F[sub 2t]-lsoP levels. To determine whether there was a temporal relationship between VPA-associated oxidative stress and hepatotoxicity, rats were given high daily doses of VPA for 14 days. VPA elevated levels of 15-F[sub 2t]-lsoP prior to the onset of hepatic necrosis and steatosis. An in vitro model using primary cultured rat hepatocytes also demonstrated that VPA induces oxidative stress as measured by elevated levels of 15-F[sub 2t]-lsoP and 2',7'-dichlorofluorescein (DCF). In hepatocytes with reduced levels of glutathione, these oxidative stress biomarkers were further elevated, and were accompanied by mitochondrial dysfunction and hepatocyte toxicity. The work presented is significant in that it supports the hypothesis that VPA-associated oxidative stress occurs prior to hepatotoxicity and the link of acyl glucuronidation of VPA to the production of reactive oxygen species is unique.

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