title: Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line.
abstract: Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca(2+) homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca(2+) entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca(2+) entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype-targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca(2+) influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5'-dithiobis (2-nitrobenzoic acid)-2biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1, TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP-induced Ca(2+) entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan.
authors.forename: Heba
authors.initials: H
authors.lastname: Badr
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kozai
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityKyoto, Japan; World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto UniversityKyoto, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Sakaguchi
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityKyoto, Japan; Laboratory of Environmental Systems Biology, Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto UniversityKyoto, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Numata
authors.affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityKyoto, Japan; World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto UniversityKyoto, Japan; Laboratory of Environmental Systems Biology, Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto UniversityKyoto, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
keywords: Ca2+ entry; HepG2; TRP channels; acetaminophen; cell death; oxidative stress