Mechanism of azathioprine-induced injury to hepatocytes: roles of glutathione depletion and mitochondrial injury.
OBJECTIVE
We sought evidence that azathioprine causes cell death through reduced glutathione (GSH) depletion and mitochondrial injury.


METHODS
Studies were conducted in primary cultures of rat hepatocytes and cultured Hep G2 cells.


RESULTS
Azathioprine toxicity to rat hepatocytes was preceded by depletion of GSH. Prior GSH depletion (by treatment with buthionine sulfoximine) enhanced toxicity whilst supplemental GSH or N-acetylcysteine was protective. In hepatocytes, GSH is consumed during metabolism of azathioprine to 6-mercaptopurine. 6-Mercaptopurine was not toxic to hepatocytes, suggesting that the later steps in azathioprine metabolism were not related to the pathogenic mechanism. In Hep G2 cells, azathioprine did not alter levels of GSH and was not toxic. Ultrastructural studies showed hepatocyte mitochondrial lesions after exposure to azathioprine, but no features of apoptosis. Azathioprine produced rapid and profound depletion of adenosine 5'-triphosphate (ATP). Cyclosporin A and glycine afforded protection against azathioprine toxicity, and Trolox and high-dose allopurinol also attenuated injury.


CONCLUSIONS
The mechanism of azathioprine toxicity to hepatocytes involves depletion of GSH leading to mitochondrial injury with profound depletion of ATP and cell death by necrosis. Cell death was prevented by potent antioxidants, glycine and blocking the mitochondrial permeability transition pore.
Lee
A U
AU
Storr Liver Unit, Westmead Millennium Institute, University of Sydney at Westmead Hospital, 2145, Westmead, NSW, Australia.
Farrell
G C
GC
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors; D000818:Animals; D001379:Azathioprine / Q000378:metabolism / Q000506:poisoning*; D002470:Cell Survival / Q000187:drug effects; D005978:Glutathione / Q000172:deficiency* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D007166:Immunosuppressive Agents / Q000378:metabolism / Q000506:poisoning*; D008297:Male; D008930:Mitochondria, Liver; D028361:Mitochondrial Diseases / Q000473:pathology / Q000503:physiopathology*; D051381:Rats; D017208:Rats, Wistar
D007166:Immunosuppressive Agents; D000255:Adenosine Triphosphate; D005978:Glutathione; D001379:Azathioprine; D019803:Glutathione Disulfide