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Valproic acid biotransformation and toxicity in sandwich-cultured rat hepatocytes Surendradoss, Jayakumar


Valproic acid (VPA) is a widely prescribed broad-spectrum antiepileptic drug. Clinical use of VPA is associated with a rare, but possibly fatal, idiosyncratic hepatotoxicity. The mechanism of VPA hepatotoxicity is not known, but it may involve reactive metabolites of VPA. Using a sandwich-cultured rat hepatocyte model, the present work investigated the toxicity of two specific VPA metabolites, (E)-2,4-diene-VPA and valproyl-1-O-β acyl glucuronide (VPA-G), and their role in the hepatocyte toxicity of VPA. The overall experimental strategy was to modulate the in situ formation of these two metabolites and determine the consequences on VPA toxicity in sandwich-cultured rat hepatocytes. VPA toxicity was assessed by markers such as 2′,7′-dichlorofluorescein formation (oxidative stress), BODIPY 558/568 C12 accumulation (steatosis), lactate dehydrogenase release (necrosis), and cellular content of total glutathione (antioxidant status). (E)-2-ene-VPA, which is a β-oxidation metabolite of VPA, was also included in the experiments concerning (E)-2,4-diene-VPA, as it formed relatively large amounts of (E)-2,4-diene-VPA. Based on the modulatory experiments with phenobarbital and 1-aminobenzotriazole, in situ generated (E)-2,4-diene-VPA did not appear to contribute to VPA toxicity in sandwich-cultured rat hepatocytes. However, the results from (E)-2-ene-VPA experiments indicated the toxic potential of in situ generated (E)-2,4-diene-VPA in sandwich-cultured rat hepatocytes, when generated at high concentrations. As part of this study, a sensitive and rapid ultra-high performance liquid chromatography – tandem mass spectrometry method for the quantification of VPA-G in hepatocyte culture medium was developed, validated, and applied successfully to quantify in situ concentrations of VPA-G. From a comprehensive screening of several known inducers of uridine 5'-diphospho-glucuronosyltransferase enzymes, this study identified β-naphthoflavone, L-sulforaphane, and phenobarbital to be effective in increasing the in situ formation of VPA-G from VPA in sandwich-cultured rat hepatocytes. According to the findings with β-naphthoflavone and borneol, in situ generated VPA-G did not appear to be toxic to sandwich-cultured rat hepatocytes and was unlikely to contribute to the hepatocyte toxicity of VPA. Overall, the results of the present study add value to the existing knowledge on the role of reactive metabolites of VPA in VPA hepatotoxicity. Future studies should investigate the role of VPA-CoA thioester formation on VPA toxicity in sandwich-cultured rat hepatocytes.

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