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. Copyright 2004 Elsevier Ltd.