ToxSci Advance Access published online on November 1, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm273
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The Mitochondrial Superoxide/Thioredoxin-2/Ask1 Signaling Pathway is Critically Involved in Troglitazone-induced Cell Injury to Human Hepatocytes
,
,1
* Department of Pharmacology, Yong Loo Lin School of Medicine
Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore
Department of Pharmaceutical Sciences, University of Connecticut School of Pharmacy, Storrs, Connecticut
1 Address for correspondence: Urs A. Boelsterli, PhD, University of Connecticut, School of Pharmacy, Dept Pharmaceutical Sciences, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269-3092, USA, Phone: +1 (860) 486-8087, Fax: +1 (860) 486-5792, Email: urs.boelsterli{at}uconn.edu
Received October 3, 2007; revision received October 29, 2007; accepted October 29, 2007
 |
Abstract |
|---|
Although the mechanisms and susceptibility factors of troglitazone-associated idiosyncratic liver injury have not been elucidated, experimental evidence has identified oxidant stress and mitochondrial injury as a potential hazard in vitro. In search of upstream mediators of toxicity, we hypothesized that troglitazone-induced increased mitochondrial generation of superoxide might activate the thioredoxin-2 (Trx2)/Ask1 signaling pathway, leading to cell death, and that, hence, the mitochondrially targeted radical scavenger, mito-CP, would prevent the increase in superoxide net levels and inhibit mitochondrial signaling and cell injury. Immortalized human hepatocytes (HC-04) were exposed to troglitazone (0-100 µM), which caused concentration and time-dependent apoptosis after 12-24 h (ketoconazole-insensitive). We found that troglitazone rapidly dissipated the mitochondrial inner transmembrane potential (
m) and independently increased the net levels of mitochondrial superoxide by 5-fold. This was followed by a shift of the redox ratio of mitochondrial Trx2 towards the oxidized state and subsequent activation of Ask1. Cell injury, but not the decrease in m, was prevented by cyclosporin A (3 µM), indicating that mitochondrial permeabilization, but not membrane depolarization, was causally involved in cell death. Mito-CP not only decreased troglitazone-induced superoxide levels, but also prevented Trx2 oxidation and activation of Ask1 and protected cells from toxic injury. These data indicate that troglitazone, but not its oxidative metabolite(s), produce intramitochondrial oxidant stress that activates the Trx2/Ask1 pathway, leading to mitochondrial permeabilization. Furthermore, the data support our concept that targeted delivery of an antioxidant to mitochondria can inhibit upstream signaling and protect from troglitazone-induced lethal cell injury.
Key Words: troglitazone; mitochondria; oxidative stress; idiosyncratic drug toxicity; thioredoxin-2; mito-CP.