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

Abstract

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.

Glutathione-S-transferase genotypes and the adverse effects of azathioprine in young patients with inflammatory bowel disease

“…36,41 GST polymorphisms could modulate the cytotoxic action of azathioprine, influencing not only glutathione levels but even modulating the conversion of azathioprine to 6-mercaptopurine, as pointed out previously.…”

Preventive role of chamomile flowers and fennel seeds extracts against liver injury and oxidative stress induced by an immunosuppressant drug in rats

“…The mechanism of AZA toxicity to hepatocytes involves the depletion of GSH leading to mitochondrial injury with profound depletion of ATP and cell death by necrosis. [9] Furthermore, GSH is responsible for ROS scavenging. Therefore, the decrease in GSH induced by AZA administration may be caused by the exhaustion of GSH during ROS scavenging.…”

“…AZA toxicity to rat hepatocytes in vitro and the mechanism of AZA toxicity to hepatocytes and decreasing its viability involves the depletion of glutathione (GSH) leading to mitochondrial injury with profound depletion of adenosine triphosphate (ATP) and cell death by necrosis were reported. [8,9] Plants rich in natural polyphenolic compounds were intensely studied in recent years due to their potent anticarcinogenic, antioxidant, and immunomodulatory properties. Chamomile (Matricaria recutita L.) is one of the most widely used medicinal plants in the world.…”

High-Dose Acetylcysteine in Idiopathic Pulmonary Fibrosis

“…In recent publications, acetylcysteine has been shown to protect hepatocytes from azathioprine-induced toxicity by replenishing intracellular glutathione concentrations. 49,50 Our trial used prednisone and azathioprine as standard therapy for all patients, as proposed by the International Idiopathic Pulmonary Fibrosis Consensus Statement. 21 The mortality up to one month after completion of the study or withdrawal from the study was rather low (9 percent in the acetylcysteine group and 11 percent in the placebo group).…”