Idiosyncratic valproic acid-induced hepatotoxicity : an in vitro approach to detection and mechanism of toxicity

The first objective of this study was to develop a rapid, high capacity, and objective in vitro cytotoxicity method for the detection of metabolism-dependent cytotoxicity of a test chemical. The in vitro system uses an external biotransformation system (rabbit liver microsomes plus NADPH) in conjunction with human lymphocytes as the target cells. Viability was determined by assessing plasma membrane integrity using a membrane impermeant fluorescent nucleic acid dye (YO-PRO-1™) and a multiwell plate scanner for fluorescence. Using this technique in combination with a series of antioxidant enzymes and iron chelators, the role of reactive oxygen species in the mechanism of VPA-induced in vitro cytotoxicity was evaluated. Obtained results suggest the generation of H₂O₂ as the sole mechanism of VPA-induced in vitro cytotoxicity and disputes involvement of reactive metabolites as the cause of cell death in this system. The VPA-induced cytotoxicity was completely inhibited by antagonists of poly(ADP-ribose) polymerase (PARP) implicating PARP in the mechanism of VPA-induced cytotoxicity under these conditions. The in vitro generation of H₂O₂ during cytochrome P-450 metabolism of VPA was confirmed by a fluorometric modification of the Nash method. Results suggest that co-dehydrogenation of VPA is the primary reaction leading to the formation and release of H2O2. Therefore, VPA was included in the class of compounds known as partial uncouplers. Lymphocytes isolated from a patient with a history of severe VPA-induced hepatotoxicity demonstrated a higher level of cytotoxicity in the presence of VPA as compared to the age matched controls. Coincubation with catalase afforded complete protection against VPA-induced cytotoxicity in lymphocytes isolated from this patient. The VPA-induced cytotoxicity prior to the initiation of oral treatment with antioxidant vitamins was not different from samples collected post antioxidant treatment. An LC/MS assay was developed to quantify an oxidation product of salicylate, namely 2,3-dihydroxy benzoic acid as a sensitive indication of hydroxyl radical formation. VPA induced a significant increase in the formation of 2,3-dihydroxy benzoic acid with a mean maximal plasma level of 2.5-fold of the control group. To our knowledge the present data, provide the first evidence of in vivo VPA-induced generation of OH*. The induction of oxidative stress by VPA as demonstrated in this study may be responsible or partially contribute to the mechanism of the idiosyncratic hepatotoxicity associated with VPA therapy. To construct a better in vitro model for mechanistic studies of VPA-associated hepatotoxicity, biotransformation of 4-ene VPA in two commonly used hepatocyte culture systems (type I collagen and Matrigel) as well as species differences in guinea pigs and rats was evaluated. Although primary hepatocytes cultured on Matrigel matrix were observed to have better phenotypic characteristics, the levels of the GSH conjugated metabolites were higher in the collagen I system. Species differences in the epoxidation of 4-ene VPA and in the formation of the isomeric GSH conjugates derived from 2,4-diene VPA were apparent.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat