Abstract

The hepatotoxic metabolite of the anticonvulsant drug valproic acid (VPA), namely (E)-2-propyl-2,4-pentadienoic acid (E)-2,4-diene VPA), is known to react with glutathione (GSH) in vivo. Although glutathione-S-transferase (GST) was suspected of being the catalyst for this conjugation reaction, this was yet to be confirmed. In this study, GST activities were detected in the hepatic cytosolic and sonic-disrupted mitoplast fractions isolated from male Sprague-Dawley rats by using 1-chloro-2,4-dinitrobenzene as a substrate. An elevation of GST activities by 45 to 100% was observed after pretreatment of rats with phenobarbital (PB). Subsequently, these apparent GST activities were examined for their effects on the in vivo conjugation of GSH with N-acetyl-S-((E)-2-propyl-2,4-pentadienoyl)cysteamine (2,4-diene VPA-NACA), a structural mimic of (E)-2,4-diene VPA coenzyme A thioester. Reaction products were identified and quantitated by combined liquid chromatography-tandem mass spectrometry. The GST-mediated conjugation of GSH with 2,4-diene VPA-NACA produced two structural isomers via either 5,6- or 1,6-addition of GSH. Only the 1,6- addition product was found for the spontaneous conjugation reaction (control). Quantitatively, GSH conjugates formed in the cytosolic fraction were 23-fold that of control. An additional 1.5-fold enhancement was observed in the cytosolic fraction from PB-treated rats. The production of the GSH conjugates was increased by 2-fold for reactions involving the sonic-disrupted mitoplasts, either from untreated or PB-treated rats. Partially purified GST was found to catalyze the conjugation reactions in a fashion similar to that of the isolated subcellular fractions. No reaction with GSH could be detected for the free acid form of (E)-2,4-diene VPA. As was the case with the in vitro data, two structural isomers of GSH conjugates were detected in the bile of rats that received (E)-2,4-diene VPA. These results indicate that in vivo production of the GSH conjugates of (E)-2,4-diene VPA is most likely catalyzed by GST enzymes, with the esterified diene being essential for the conjugation reaction. In a separate experiment, 2,4-diene VPA-NACA was observed to alkylate reduced oxytocin through one or both cysteine residues. Thus, the toxicity of (E)-2,4-diene VPA might be produced via either GST-promoted depletion of cellular GSH, or a direct modification of key proteins, or both.