Basic-liver, pancreas, and biliary tractHepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells
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.
References (56)
- et al.
Natural history of liver fibrosis progression in patients with chronic hepatitis C
Lancet
(1997) - et al.
Alcohol potentiates hepatitis C virus replicon expression
Hepatology
(2003) - et al.
Hepatic lipid peroxidation in hereditary hemochromatosis and alcoholic liver injury
J Lab Clin Med
(1999) - et al.
Superoxide dismutase in patients with chronic hepatitis C virus infection
Free Radic Biol Med
(1998) - et al.
Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis
J Hepatol
(1995) - et al.
Serum ferritin and hepatic glutathione concentrations in chronic hepatitis C patients related to the hepatitis C virus genotype
J Hepatol
(1999) - et al.
DNA oxidative damage in leukocytes correlates with the severity of HCV-related liver diseasevalidation in an open population study
J Hepatol
(2001) - et al.
Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor
Gastroenterology
(1998) - et al.
Role of Kupffer cells, endotoxin and free radicals in hepatotoxicity due to prolonged alcohol consumptionstudies in female and male rats
J Nutr
(1997) - et al.
Contribution of mitochondria to oxidative stress associated with alcoholic liver disease
Free Radic Biol Med
(2002)
Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol
Hepatology
Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus
Gastroenterology
Mitochondrial injury, oxidative stress and antioxidant gene expression are induced by hepatitis C virus core protein
Gastroenterology
Cellular response to conditional expression of hepatitis C virus core protein in Huh7 cultured human hepatoma cells
Hepatology
Moderate alcohol consumption increases oxidative stress in patients with chronic hepatitis C
Hepatology
Establishment of a recombinant hepatic cell line stably expressing alcohol dehydrogenase
Arch Biochem Biophys
Mitochondrial damage induced by conditions of oxidative stress
Free Radic Biol Med
A novel cytotoxicity screening assay using a multiwell fluorescence scanner
Toxicol Appl Pharmacol
Mitochondria and cells produce reactive oxygen species in virtual anaerobiosisrelevance to ceramide-induced apoptosis
FEBS Lett
A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1)
Biochem Biophys Res Commun
Acetaldehyde impairs mitochondrial glutathione transport in HepG2 cells through endoplasmic reticulum stress
Gastroenterology
Bimodal targeting of microsomal CYP2E1 to mitochondria through activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation
J Biol Chem
Mitochondrial permeability transitiona common pathway to necrosis and apoptosis
Biochem Biophys Res Commun
Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)
Biochem Pharmacol
Ethanol cytotoxicity to a transfected HepG2 cell line expressing human cytochrome P4502E1
J Biol Chem
Cytotoxicity and apoptosis produced by arachidonic acid in Hep G2 cells overexpressing human cytochrome P4502E1
J Biol Chem
Oxidative stress on mitochondria and cell membrane of cultured rat hepatocytes and perfused liver exposed to ethanol
Gastroenterology
Ethanol stimulates the production of reactive oxygen species at mitochondrial complexes I and III
Free Radic Biol Med
Cited by (124)
Mitochondrial Dysfunction and Signaling in Chronic Liver Diseases
2018, GastroenterologyOxidative Stress in Hepatitis C Infection
2018, The Liver: Oxidative Stress and Dietary AntioxidantsThe Effects of Alcohol on Other Chronic Liver Diseases
2016, Clinics in Liver DiseaseOxidative stress attenuates lipid synthesis and increases mitochondrial fatty acid oxidation in hepatoma cells infected with hepatitis C virus
2016, Journal of Biological ChemistryMitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein
2015, Toxicology and Applied PharmacologyCitation Excerpt :These proteins participate in viral replication and also influence cellular functions of the host. Oxidative stress is commonly observed following HCV infection and is caused by increased levels of cellular reactive oxygen species (ROS) or by changes in cellular antioxidant capacities (Choi and Ou, 2006; Oberley, 2002; Otani et al., 2005). In particular, HCV core protein is known to be closely associated with the mitochondria and causes the increase in host ROS production, lipid peroxidation (Lau et al., 1998; Moriya et al., 2001; Okuda et al., 2002) and mitochondrial Ca2 + uptake.
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.