title: Evaluation of azathioprine-induced cytotoxicity in an in vitro rat hepatocyte system.
abstract: Azathioprine (AZA) is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using "Accelerated Cytotoxicity Mechanism Screening" techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 μM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS) formation was observed when glutathione- (GSH-) depleted hepatocytes were used. The addition of N-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2) formation and increased % mitochondrial membrane potential (MMP). Addition of N-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N'-diphenyl-p-phenylenediamine), Trolox (a water soluble vitamin E analogue), and mesna (2-mercaptoethanesulfonate), also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury.
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Al Maruf
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Luke
authors.initials: L
authors.lastname: Wan
authors.affiliation: Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8 ; Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada M5S 3M2.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000493:Allopurinol / Q000008:administration & dosage; D000818:Animals; D001379:Azathioprine / Q000008:administration & dosage* / Q000009:adverse effects; D005978:Glutathione / Q000378:metabolism; D006084:Graft Rejection / Q000188:drug therapy; D022781:Hepatocytes / Q000187:drug effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016377:Organ Transplantation; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D000493:Allopurinol; D006861:Hydrogen Peroxide; D005978:Glutathione; D001379:Azathioprine
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