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 Ca2+ 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 Ca2+ 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 Ca2+ 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, Ca2+ 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 Ca2+ entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.

Highlights

  • Acetaminophen (N-acetyl-para-aminophenol, APAP) is a widely used and safe over-the-counter analgesic antipyretic drug (Rumack, 2004)

  • We found that serum deprivation enhanced the Ca2+ responses and cell death induced by 20 mM APAP or 1 mM hydrogen peroxide (H2O2) (Supplementary Figures 5A,B) in human hepatoma cell line (HepG2), as compared with that and tiron (1 mM), suppressed APAP- or H2O2-induced losses of cell viability (A) and increases of cell death (B) in HepG2 cells. (C,D) Selective transient receptor potential (TRP) channels blockers, 1 μM of either CPZ, 2-aminoethyl diphenylborinate (2-APB), CTZ, or AA861 suppressed APAP- or H2O2-induced losses of cell viability (C) and increases of cell death (D) in HepG2 cells. (E,F) Small interfering RNA (siRNA)-mediated knockdown of TRPV1, TRPC1, TRPM2, and TRPM7 suppressed APAP- or H2O2-induced losses of cell viability (E) and increases of cell death (F) in HepG2 cells

  • We found that APAP overdose elicited reactive oxygen species (ROS) production, [intracellular Ca2+ concentration (Ca2+]i) increases, and GSH depletion triggering HepG2 cell death

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Summary

Introduction

Acetaminophen (N-acetyl-para-aminophenol, APAP) is a widely used and safe over-the-counter analgesic antipyretic drug (Rumack, 2004). An accidental APAP overdose can result in potentially lethal hepatotoxicity in both humans and experimental animals (Thomas, 1993; Hinson et al, 2010). It has been established that APAP overdose causes a constellation of co-related cellular events (Hinson et al, 2010). APAP causes mitochondrial dysfunction, deregulation of Ca2+ homeostasis, and DNA fragmentation (Muriel, 2009; Hinson et al, 2010). Though, increased [Ca2+]i in hepatocytes is a consequence of APAP overdose, how GSH depletion/ROS accumulation, [Ca2+]i increases and mitochondrial dysfunction interact to induce APAP overdose-induced hepatocellular death remains elusive

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