Cytotoxicity of unsaturated metabolites of valproic acid and protection by vitamins C and E in glutathione-depleted rat hepatocytes

• Published in: Toxicology (Amsterdam) Vol. 112; no. 1; pp. 69 - 85
• Main Authors: Jurima-romet, Malle, Abbott, Frank S., Tang, Wei, Huang, Hide S., Whitehouse, larry W.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 01.08.1996; Amsterdam Elsevier Science
• Subjects: Analysis of Variance; Animals; Anticonvulsants - metabolism; Anticonvulsants - toxicity; Ascorbic Acid - pharmacology; Biological and medical sciences; Cells, Cultured; Chromatography, Liquid; Cytochrome P-450 Enzyme System - biosynthesis; Cytotoxicity; Dose-Response Relationship, Drug; Drug Synergism; Drug toxicity and drugs side effects treatment; Enzyme Induction - drug effects; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - toxicity; Glutathione; Glutathione - metabolism; Homeostasis; L-Lactate Dehydrogenase - metabolism; Liver - cytology; Liver - drug effects; Liver - enzymology; Male; Mass Spectrometry; Medical sciences; Metabolism; Methionine - analogs & derivatives; Methionine - toxicity; Pharmacology. Drug treatments; Phenobarbital - toxicity; Pregnenolone Carbonitrile - toxicity; Rat hepatocytes; Rats; Rats, Inbred F344; Toxicity: digestive system; Valproic acid; Valproic Acid - metabolism; Valproic Acid - toxicity; Vitamin C; Vitamin E; Vitamin E - pharmacology; 5-GS-3-ene VPA; Cytotoxicity; MRM; 5-GS-4-hydroxy VPA lactone; 4-ene VPA; LDH; Vitamin E; Vitamin C; CYP; GSH; Glutathione; BSO; PBS; LC/MS/MS; Valproic acid; Metabolism; DEM; PB; 2-ene VPA; α-F-4-ene VPA-Gln; 2,4-diene VPA; (E)-2,(Z)-3′-diene VPA; Rat hepatocytes; PCN; α-F-4-ene VPA; VPA (valproic acid); Ascorbic acid; Rat; Metabolite; Toxicity; Liver; Rodentia; Vitamin; Detoxication; α-Tocopherol; Anticonvulsant; Antioxidant; In vitro; Dose activity relation; Unsaturated compound; Prevention; Vertebrata; Mammalia; Hepatocyte; Animal; Mechanism of action
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/0300-483X(96)03352-5

Valproic acid (VPA) and the unsaturated metabolites, 2-ene VPA and (E)-2,(Z)-3′-diene VPA, demonstrated dose-dependent cytotoxicity in primary cultures of rat hepatocytes, as evaluated by lactate dehydrogenase (LDH) leakage. Cellular glutathione (GSH) was depleted by adding buthionine sulfoximine (BSO) to the culture medium. Induction of cytochrome P450 by pretreatment of rats with phenobarbital or pregnenolone-16α-carbonitrile enhanced the cytotoxicity of parent VPA in BSO-treated hepatocytes. The cytotoxicity of 4-ene VPA was apparent in BSO-treated hepatocytes with detectable loss of cell viability at 1 μM of added 4-ene VPA. Depletion of cellular GSH also increased the cytotoxicities of 2-ene VPA and (E)-2,(Z)-3′-diene VPA.The cytotoxicity of 2-ene VPA was comparable to or higher than that of VPA, producing loss of viability at concentrations ≥5 mM. Time-course evaluation of hepatocyte response to 4-ene VPA in the GSH-depleted state revealed a delayed cytotoxicity with no effect during the first 12 h of exposure followed by a pronounced toxicity between 12 and 14 h. Two major GSH conjugates of 4-ene VPA metabolites, namely 5-GS-4-hydroxy VPA lactone and 5-GS-3-ene VPA, were detected in 4-ene VPA treated hepatocytes. Consistent with this finding, a 50% decrease in cellular GSH levels was observed following 4-ene VPA treatment. Under similar conditions, neither toxicity nor the GSH conjugated metabolite were detected in cells treated with the α-fluorinated 4-ene VPA analogue (α-F-4-ene VPA). The antioxidants, vitamin C and vitamin E, demonstrated a cytoprotective effect against 4-ene VPA-induced injury in GSH-depleted hepa-tocytes. These results are in support of hepatocellular bioactivation of VPA via 4-ene VPA to highly reactive species, which are detoxified by GSH. The susceptibility of hepatocytes to VPA metabolite-mediated cytotoxicity depends on cellular GSH homeostasis.