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BioMed Research International
Volume 2014 (2014), Article ID 379748, 7 pages
http://dx.doi.org/10.1155/2014/379748
Research Article

Evaluation of Azathioprine-Induced Cytotoxicity in an In Vitro Rat Hepatocyte System

1Department of Pharmacology & Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8
2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada M5S 3M2

Received 20 February 2014; Accepted 18 June 2014; Published 1 July 2014

Academic Editor: Thomas Minor

Copyright © 2014 Abdullah Al Maruf et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.