The long-term objective of the research outlined in this application is to better understand the role of drug interactions in the rare, but potentially fatal hepatotoxicity caused by valproic acid (VPA), and to account for the unexplained observation that recent febrile illness represents a predisposing factor in VPA-mediated liver injury. Particular emphasis will be placed on the involvement of tumor necrosis factor (TNF), an endogeneous cytokine released in response to bacterial and viral infections, in depleting hepatic stores of glutathione (GSH) and rendering liver tissue more vulnerable to drug-induced injury. The hypothesis to be tested is that VPA and hepatotoxic VPA metabolites act synergistically with other antiepileptic drugs and TNF to cause oxidative stress and liver damage. This hypothesis will be tested by a number of in vitro and in vivo studies, the specific aims of which are as follows: (A) To develop improved methodology for the analysis of GSH and GSSG in biological specimens. (B)1. To demonstrate that liver injury results from the treatment of animals with a combination of VPA, phenytoin/carbamazepine and TNF at non-hepatotoxic doses of each component. (B)2. To demonstrate that the liver injury in (B)1 is associated with depletion of GSH. (B)3. To determine whether treatment with NAC (i) protects against the liver injury in (B)1 and (ii) attenuates the depletion of GSH in (B)2. (C)1-3. In parallel to Aims (B)1-3, to demonstrate that a similar phenomenon occurs with delta4-VPA (a potent hepatotoxic metabolite of VPA). (D)1. To investigate the mechanism by which VPA inhibits GSSG reductase. (D)2. To investigate the role of a delta2(E),4-VPA-CoA as a reactive metabolite of delta4-VPA in mitochondria. (D)3. To compare the respective abilities of delta4-VPA and its non-hepatotoxic isomer delta2(E)-VAP to deplete GSH and cause oxidative stress. The experimental approach will utilize the rat as an animal model for in vivo studies, and will employ both intact hepatocytes and cell-free preparations from rat liver for in vitro work. New analytical techniques, based on electrospray mass spectrometry, will be developed to assess changes in GSH homeostasis, and to investigate the production of chemically-reactive, potentially toxic, CoA thioester metabolites of VPA in liver mitochondria. The proposed studies on the involvement of TNF as an endogeneous mediator of drug-induced liver damage is novel feature of the proposed research, and should provide important information on the role of this cytokine in exacerbating oxidative stress caused by foreign compounds. The project will also focus on the toxicological consequences of the interplay between fatty acid and xenobiotic metabolism. Collectively, these studies are intended to provide a basic understanding of the role of drug interactions and prodromal illness in VPA liver toxicity, which should lead to the development of safer, improved therapy with VPA and related antiepileptics.
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