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

J Hepatol. 2004 Mar;40(3):454-63. doi: 10.1016/j.jhep.2003.11.024.


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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Allopurinol / pharmacology
  • Animals
  • Antioxidants / pharmacology
  • Azathioprine / administration & dosage
  • Azathioprine / poisoning*
  • Cell Differentiation
  • Cell Survival
  • Cells, Cultured
  • Chromans / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Glutathione / metabolism
  • Glutathione Disulfide / metabolism
  • Hepatocytes / drug effects*
  • Hepatocytes / enzymology*
  • Hepatocytes / metabolism
  • Hepatocytes / pathology
  • Male
  • Mercaptopurine / administration & dosage
  • Mercaptopurine / poisoning*
  • Microscopy, Electron
  • Mitochondria, Liver / metabolism
  • Mitochondria, Liver / pathology*
  • Necrosis
  • Oxidative Stress
  • Rats
  • Rats, Wistar
  • Time Factors
  • Xanthine Oxidase / metabolism*


  • Antioxidants
  • Chromans
  • Enzyme Inhibitors
  • Allopurinol
  • Adenosine Triphosphate
  • Mercaptopurine
  • Xanthine Oxidase
  • Glutathione
  • Azathioprine
  • 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
  • Glutathione Disulfide