Abstract
Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose- and Ca2+-dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca2+ may protect the hepatocyte against bile acid-induced necrosis.
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Abbreviations
- Cbz-Leu-Leu-Tyr:
-
N-carbonylbenzyloxy-l-leucyl-l-leucyl-l-tyrosine, diazomethyl ketone
- CHAPS:
-
3-[(3-cholamidopropyl)dimethyl, ammonio]-1- propanesulfonate
- CMC:
-
critical micellar concentration
- GCDC:
-
glycochenodeoxycholic acid
- FACS:
-
fluorescence-activated cell sorter
- FCCP:
-
carbonyl cyanide p-trifluoromethoxyphenylhydrazone
- MMPT:
-
mitochondrial membrane permeability transition
- ROS:
-
reactive oxygen species
- TBARS:
-
thiobarbituric acid-reactive substances
- TFP:
-
trifluoperazine
- TMRM:
-
tetramethylrhodamine methylester
- Triton X-100:
-
t-octylphenoxypolyethoxyethanol
- Δψ:
-
mitochondrial electrochemical gradient
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Supported in part by grants from the National Institutes of Health (RO1DK38446 and IP30 DK34914) and the Abbey Bennett Liver Research Fund.
Presented in part at the American Association for the Study of Liver Diseases Annual Meeting, Chicago, IL, U.S.A., November 1996, and published in abstract form (Hepatology 1996: 24:237A).
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Sokol, R., Straka, M., Dahl, R. et al. Role of Oxidant Stress in the Permeability Transition Induced in Rat Hepatic Mitochondria by Hydrophobic Bile Acids. Pediatr Res 49, 519–531 (2001). https://doi.org/10.1203/00006450-200104000-00014
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DOI: https://doi.org/10.1203/00006450-200104000-00014
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