Effects of phenobarbital on metabolism and toxicity of diclofenac sodium in rat hepatocytes in vitro.
Diclofenac sodium (DF-Na) was a nonsteroidal anti-inflammatory drug used in various aspects of inflammatory disease. The purpose of this study was to examine the effects of phenobarbital (PB) on metabolism and toxicity of DF-Na in vitro and explore the potential mechanism of DF-Na induced hepatotoxicity. Rat hepatocytes were isolated by a modification of the two-step in situ collagenase perfusion technique and the harvested rat hepatocytes were cultured with sandwich method. Control or PB (2 mM) pre-treated hepatocytes were incubated with DF-Na (0.1, 0.05 or 0.01 mM) in vitro and cytosolic enzyme leakage levels, cytochrome P450 (CYP) 3A activity, and metabolite content of DF-Na in cell culture medium were measured. The results showed that without any treatment hepatocyte CYP 3A activity gradually decreased with culture time. On day four, CYP 3A activity was 53% of the initial value. The decline of CYP 3A was partially reversed by CYP inducer PB, and the maximum induction of CYP 3A was 2.2-fold over control after continuous exposure of hepatocytes to 2 mM PB for 48 h. Lactic dehydrogenase (LDH), aspartate transaminase (AST), and alanine transamine (ALT) activity and the contents of the DF-Na metabolites 4'-hydroxydiclofenac (4'-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) in media appeared to increase with increasing DF-Na concentrations, though there were no significant differences between DF-Na exposed and control hepatocytes. However, if the hepatocytes first were pre-treated with 2 mM PB for 2 days and then exposed to DF-Na, the concentrations of DF-Na metabolites and the activity of LDH in the media were significantly higher than that of control group. These findings suggest that the hepatotoxicity and metabolism of DF-Na in rat hepatocytes are increased when hepatic CYP 3A activity is increased.
Department of Environmental Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People's Republic of China. wangaiguo@mails.tjmu.edu.cn
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Wang
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Xia
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Yuan
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Yang
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Chen
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Waalkes
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000493:pharmacokinetics* / Q000633:toxicity*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology; D051544:Cytochrome P-450 CYP3A; D004008:Diclofenac / Q000493:pharmacokinetics* / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006900:Hydroxylation; D008297:Male; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004008:Diclofenac; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D010634:Phenobarbital