| Summary: | Valproic acid (VPA) therapy is associated with a rare but severe hepatotoxicity. The relationships between the various pathophysiological findings of VPA-induced hepatotoxicity and the role of VPA biotransformation in the induction of hepatotoxicity have not been systematically investigated. The present thesis compared the effects of VPA, synthesized VPA metabolites, and alpha-F-VPA on markers of mitochondrial dysfunction (WST-1), cytotoxicity (LDH), oxidative stress (DCF), and glutathione (GSH) depletion in a novel model of sandwich-cultured rat hepatocytes (SCRH). The contribution of the CYP- and UGT-mediated biotransformation of VPA in VPA-induced toxicity was also examined. Time-dependent effects of VPA on GSH depletion were characterized in relation to the effects of VPA on the WST-1, LDH, and DCF markers. The effects of glutathione supplementation on the attenuation of the markers for VPA-induced toxicities were investigated. Urine samples from children on VPA therapy were assayed to correlate levels of VPA metabolites with the lipid peroxidation marker, 15-F2t-isoprostane. Lastly, the contributions of hepatic CYP-enzymes in the oxidative metabolism of VPA were characterized in human liver microsomes.
Our findings in SCRH indicated that (E)-2,4-diene-VPA was the only exogenously administered metabolite tested that was consistently more toxic than VPA. Consistent with this finding, alpha-F-VPA, which is resistant to bioactivation by several biotransformation pathways, was nontoxic. Chemical inhibition experiments indicated that the CYP- and UGT-mediated metabolism of VPA or the in situ generated VPA metabolites were unlikely involved in the observed VPA-induced toxicities in SCRH. Furthermore, VPA-associated GSH depletion appeared not to be a factor in the mitochondrial dysfunction, but may play a partial role in VPA-induced cytotoxicity. GSH may serve a protective role against VPA-induced oxidative stress in SCRH. In human subjects, the VPA-glucuronide or N-acetylcysteine metabolites were extremely weak but statistically significant predictors of lipid peroxidation in the urine of children receiving VPA. From the reaction phenotyping experiments, CYP2C9 was the major catalyst for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in human liver microsomes, whereas CYP2A6 contributed partially to 3-OH-VPA formation. Overall, these findings add significant knowledge to the role of VPA and its metabolites in the induction of hepatotoxicity and how VPA is metabolized in humans.
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