ToxSci Advance Access originally published online on November 1, 2007
Toxicological Sciences 2008 101(2):341-349; doi:10.1093/toxsci/kfm273
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The Mitochondrial Superoxide/Thioredoxin-2/Ask1 Signaling Pathway is Critically Involved in Troglitazone-Induced Cell Injury to Human Hepatocytes
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* Department of Pharmacology, Yong Loo Lin School of Medicine National University of Singapore, Singapore 117595
Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117543
Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut Storrs, Connecticut 06269-3092
1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269-3092. Fax: +1 (860) 486-5792. E-mail: urs.boelsterli{at}uconn.edu.
Received October 3, 2007; accepted October 29, 2007
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Abstract |
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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)/apoptosis signal–regulating kinase 1 (Ask1) signaling pathway, leading to cell death, and that, hence, the mitochondrially targeted radical scavenger, mito-carboxy proxyl (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 (0–100µM), 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 toward the oxidized state and subsequent activation of Ask1. Cell injury, but not the decrease in m, was prevented by cyclosporin A (3µM), 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.
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