Inducers of cytochrome P450 2E1 enhance methotrexate-induced hepatocytotoxicity

Objectives: To study the effect of cytochrome P450 2E1-inducers on methotrexate (MTX)-induced cytotoxicity in human hepatocytes, and investigate the role of silymarin in preventing this toxicity. Design and methods: Cells were exposed to MTX in the presence of either ethanol (EtOH) or acetaminophen (APAP), or either combined with silymarin (S). Apoptosis and necrosis were measured by analyzing 6000 cells/sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. Cytokine expression was measured by RT-PCR. Gluthatione (GSH) content was determined in cytosolic (c) and mitochondrial (m) fractions. Results: MTX + EtOH and MTX + APAP increased MTX cytotoxicity 2.9-fold and 1.9-fold, respectively. S abolished this toxicity. MTX + EtOH increased the release of IL 6, IL 8 and TNF alpha by 1.0, 1.2, and 1.1 times, respectively. Cytokine expression was upregulated versus control for IL 6 (22%), IL 8 (38%), and TNF alpha (29%). Addition of 0.5 mmol/L S downregulated TNF a expression and reduced cytokine release. TNF a increased cytotoxicity by 22%, while anti-TNF alpha antibody eradicated it. MTX + EtOH depleted 45% mGSH (p < 0.001) while S replenished it to 87% (p < 0.001), when both were compared to control levels. Conclusions: Cytochrome P450 2E1-inducers contribute to increase oxidative stress in MTX-exposed cells by increasing TNF alpha and depleting both cGSH and mGSH. This enhances MTX-cytotoxicity and promotes apoptosis. Copyright (C) 1999 The Canadian Society of Clinical Chemists.