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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Azathioprine Acts upon Rat Hepatocyte Mitochondria and Stress-Activated Protein Kinases Leading to Necrosis: Protective Role of N-Acetyl-L-cysteine

Unidad de Toxicología Molecular Hepática, Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, Spain (C.M., M.D.F.-M., J.A.F., O.E., I.D.R., L.G.G.); Farmasierra S.A., Madrid, Spain (T.O., E.A.); Celltech Pharma S.A., Madrid, Spain (C.C.); and Institute of Medical Biochemistry and Chemistry, University of Bari, Bari, Italy (M.L., M.D.P.)

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release. The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

Address correspondence to: Dr. Luis G. Guijarro, Unidad de Toxicología Molecular, Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain. E-mail: luis.gonzalez{at}uah.es

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