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.
Wang
A G
AG
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
Xia
T
T
Yuan
J
J
Yu
R A
RA
Yang
K D
KD
Chen
X M
XM
Qu
W
W
Waalkes
M P
MP
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