title: Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes.
abstract: Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by β-oxidation. Another biotransformation pathway involves β-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2',7'-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C₁₂), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it contributes to the toxicity of (E)-2-ene-VPA in PB-pretreated rat hepatocytes.
authors.affiliation: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jayakumar
authors.initials: J
authors.lastname: Surendradoss
authors.forename: Thomas K H
authors.initials: TK
authors.lastname: Chang
authors.forename: Frank S
authors.initials: FS
authors.lastname: Abbott
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D015394:Molecular Structure; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014230:Triazoles / Q000494:pharmacology; D014635:Valproic Acid / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D014230:Triazoles; C033020:1-aminobenzotriazole; D014635:Valproic Acid; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital