Toxicity of low dose azathioprine and 6-mercaptopurine in rat hepatocytes. Roles of xanthine oxidase and mitochondrial injury.
OBJECTIVE
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).
Tapner
Michael J
MJ
Storr Liver Unit, Westmead Millennium Institute, Westmead Hospital, Westmead, NSW 2145, Australia.
Jones
Brett E
BE
Wu
Wan M
WM
Farrell
Geoffrey C
GC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001379:Azathioprine / Q000008:administration & dosage / Q000506:poisoning*; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D002839:Chromans / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D008297:Male; D015122:Mercaptopurine / Q000008:administration & dosage / Q000506:poisoning*; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000378:metabolism / Q000473:pathology*; D009336:Necrosis; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014969:Xanthine Oxidase / Q000378:metabolism*
D000975:Antioxidants; D002839:Chromans; D004791:Enzyme Inhibitors; D000493:Allopurinol; D000255:Adenosine Triphosphate; D015122:Mercaptopurine; D014969:Xanthine Oxidase; D005978:Glutathione; D001379:Azathioprine; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D019803:Glutathione Disulfide