Toxicity of low dose azathioprine and 6-mercaptopurine in rat hepatocytes. Roles of xanthine oxidase and mitochondrial injury.

BACKGROUND/AIMS To study effects of pharmacologic concentrations of azathioprine and 6-mercaptopurine (6-MP) on rat hepatocytes. METHODS Hepatocytes cultured on matrigel were incubated with azathioprine or 6-MP; effects of putative protective agents were studied. Viability (LDH leakage), reduced (GSH) and oxidized glutathione (GSSG), mitochondrial (mt) GSH, ATP and ultrastructural changes were determined. RESULTS Azathioprine and 6-MP (0.5-5 micromol/l) reduced viability 5-34% at day 1 and 42-92% by day 4. Allopurinol (20 microM) (xanthine oxidase inhibitor) and 2 mM Trolox (vitamin E analog) together provided near complete protection. During culture with azathioprine, GSSG increased before cell death and there was a disproportionate reduction of mtGSH and ATP, together with ultrastructural abnormalities in mitochondria. All changes were prevented by allopurinol and trolox. Discontinuation of 1 micromol/l azathioprine restored ATP levels and arrested cell injury, while culture in glucose-enriched media augmented ATP levels and ameliorated cell death. CONCLUSIONS Clinically relevant concentrations of azathioprine and 6-MP are toxic to rat hepatocyte cultures by a mechanism that involves oxidative stress, mitochondrial injury and ATP depletion. This can lead to irreversible de-energization and cell death by oncosis (necrosis).

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