Gastroenterology

Gastroenterology

Volume 128, Issue 1, January 2005, Pages 96-107
Gastroenterology

Basic-liver, pancreas, and biliary tract
Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells

https://doi.org/10.1053/j.gastro.2004.10.045Get rights and content

Background & Aims: Alcohol consumption exacerbates liver injury in chronic hepatitis C, and enhanced mitochondrial oxidative stress is one possible mechanism. The aim of this study was to determine whether hepatitis C virus core protein and alcohol-inducible cytochrome P450 2E1 contribute to reactive oxygen species production and cytotoxicity in human hepatoma cells. Methods: Huh-7 cells expressing core protein, cytochrome P450 2E1, or both were exposed to 0.1 mmol/L tertiary butyl hydroperoxide, tumor necrosis factor α, and/or 25 mmol/L ethanol. Cytotoxicity, reactive oxygen species production, glutathione content, and mitochondrial membrane potential were measured. Results: Expression of core/cytochrome P450 2E1 synergistically enhanced cell death induced by either tertiary butyl hydroperoxide or tumor necrosis factor α. After tertiary butyl hydroperoxide treatment, total reactive oxygen species production was increased more than 3-fold compared with cells that did not express core and cytochrome P450 2E1. Mitochondrial depolarization and reduced glutathione depletion occurred as well, and cell death was prevented by inhibition of mitochondrial permeability transition or caspase activity. Confocal microscopy showed that the mitochondria themselves were the origin of the reactive oxygen species. In the absence of core/cytochrome P450 2E1 expression, mitochondrial changes and cell death did not occur. Ethanol treatment further decreased mitochondrial reduced glutathione content and exacerbated mitochondrial reactive oxygen species production, depolarization, and cell death. All these effects were prevented by the antioxidant N-acetylcysteine. Conclusions: Mitochondrial reactive oxygen species production is induced by hepatitis C virus core and cytochrome P450 2E1, resulting in a reduction of mitochondrial antioxidant capacity and sensitivity to oxidants and tumor necrosis factor α. Alcohol further depletes mitochondrial reduced glutathione, which exacerbates depolarization and cell death. Sensitization of mitochondria to oxidative insults is thus a potential mechanism for alcohol-related exacerbation of liver injury in chronic hepatitis C.

Section snippets

Establishment of cell lines

Huh-7/191-20 cells with stable, conditional expression of HCV core protein have been previously described.19 Core protein expression in these cells is induced by withdrawal of tetracycline. Human CYP2E1 complementary DNA excised from plasmid pCI-2E1 (provided by Dr. A. I. Cederbaum) was inserted into the EcoRI restriction site of the pcDNA6/V5-HisB vector (Invitrogen, Carlsbad, CA) to form the plasmid pcDNA-2E1. Huh-7/191-20 cells were transfected by using the LipofectAMINE PLUS reagent

Establishment of core/cytochrome P450 2E1 cell lines

We transfected the pcDNA-2E1 plasmid into human hepatoma cells that express the full-length HCV core protein under the control of a tetracycline suppressible promoter.18, 19 Clone L14 preserved tightly regulated expression of core protein 48–96 hours after the withdrawal of tetracycline (Figure 1A and B) and showed expression of CYP2E1. Induction of core protein did not affect the expression of CYP2E1 (Figure 1C). Immunofluorescence microscopy showed homogenous expression of core in both L14

Discussion

The objective of this study was to generate a cellular model system and use it to determine factors that account for the synergistic effects of alcohol and hepatitis C. Because alcohol consumption increases CYP2E1 and the viral core protein increases cellular ROS, we specifically examined how core, CYP2E1, and alcohol interact with mitochondrial function and cell survival. The results show that there are primarily 2 different synergistic effects operating at the level of the mitochondria.

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  • Cited by (0)

    Supported by Grant AA12863 from the National Institute on Alcohol Abuse and Alcoholism. K.L. is a recipient of the John Mitchell Hemophilia of Georgia Liver Scholar Award of the American Liver Foundation.

    1

    K.O. and M.K. contributed equally to this work.

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