Methotrexate induced mitochondrial injury and cytochrome c release in rat liver hepatocytes

Abstract

Methotrexate (MTX) is a folic acid antagonist that is widely used to treat a variety of diseases. One of the most serious side effects of MTX therapy is hepatotoxicity. The potential molecular cytotoxic mechanisms of MTX toward isolated rat hepatocytes were investigated using Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques. A concentration and time dependent increase in cytotoxicity and reactive oxygen species (ROS) formation and a decrease in mitochondrial membrane potential (MMP) were observed with MTX. Furthermore, a significant increase in MTX (300 μM)-induced cytotoxicity and ROS formation were observed when glutathione (GSH)-depleted hepatocytes were used whereas addition of N-acetylcysteine (a GSH precursor) decreased cytotoxicity. Catalase inactivation also increased MTX-induced cytotoxicity, while the direct addition of catalase to the hepatocytes decreased cytotoxicity. MTX treatment in isolated rat mitochondria caused swelling and significantly decreased adenosine triphosphate (ATP) and GSH content, and cytochrome c release. Potent antioxidants such as mesna, resveratrol and Trolox decreased MTX-induced cytotoxicity and ROS formation and increased MMP. This study suggests that MTX-induced cytotoxicity caused by ROS formation and GSH oxidation leads to oxidative stress and mitochondrial injury in rat hepatocytes.

Keywords:

• Hepatotoxicity
• reactive oxygen species
• glutathione
• oxidative stress
• mitochondrial injury

Acknowledgements

This study was extracted from the PhD thesis of Dr. Abdullah Al Maruf (University of Toronto: 2014). This study was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant.

Disclosure statement

The authors declare that there is no conflict of interest.

Additional information

Funding

This study was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant.