Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line.
Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca2+ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca2+ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 microM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2',7'-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N'-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca2+ concentration, which was completely prevented by the antioxidants. An intracellular Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca2+ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca2+ increase and induction of apoptosis were significantly inhibited by an extracellular Ca2+ chelator or Na+/Ca2+ exchanger blockers (bepridil and benzamil), whereas neither Ca2+ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca2+ through the activation of reverse mode of Na+/Ca2+ exchanger. However, tBOOH decreased intracellular Na+ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca2+ concentration by direct activation of the reverse mode of Na+/Ca2+ exchanger, rather than indirect elevation of intracellular Na+ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca2+ may mediate this action of tBOOH. These results further suggest that Na+/Ca2+ exchanger may be a target for the management of oxidative hepatotoxicity.
Kim
J A
JA
College of Pharmacy, Yeungnam University, Kyongsan, Korea.
Kang
Y S
YS
Lee
S H
SH
Lee
Y S
YS
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D010655:Phenylenediamines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors* / Q000502:physiology; D014407:Tumor Cells, Cultured; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D000975:Antioxidants; D002614:Chelating Agents; D016877:Oxidants; D010655:Phenylenediamines; D017382:Reactive Oxygen Species; D019831:Sodium-Calcium Exchanger; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D020122:tert-Butylhydroperoxide; D012964:Sodium; C004845:N,N'-diphenyl-4-phenylenediamine; D002118:Calcium; D000111:Acetylcysteine