A variant in myeloperoxidase promoter hastens the emergence of hepatocellular carcinoma in patients with HCV-related cirrhosis.

BACKGROUND & AIMS: Genetic dimorphisms modulate the activities of several pro- or antioxidant enzymes, including myeloperoxidase (MPO), catalase (CAT), manganese superoxide dismutase (SOD2), and glutathione peroxidase 1 (GPx1). We assessed the role of the G(-463)A-MPO, T(-262)C-CAT, Ala16Val-SOD2, and Pro198Leu-GPx1 variants in modulating HCC development in patients with HCV-induced cirrhosis. METHODS: Two hundred and five patients with HCV-induced, biopsy-proven cirrhosis but without detectable HCC at inclusion were prospectively followed-up for HCC development. The influence of various genotypes on HCC occurrence was assessed with the Kaplan-Meier method. RESULTS: During follow-up (103.2±3.4 months), 84 patients (41%) developed HCC, and 66 died. Whereas the Ala16Val-SOD2 or Pro198Leu-GPx1 dimorphisms did not modulate the risk, HCC occurrence was increased in patients with either the homozygous GG-MPO genotype (HR=2.8 [1.7-4.4]; first quartile time to HCC occurrence: 45 vs. 96 months; LogRank <0.0001) or the homozygous CC-CAT genotype (HR=1.74 [1.06-2.82]; first quartile time to HCC occurrence: 55 vs. 96 months; LogRank=0.02). Compared to patients with neither of these two at risk factors, patients with only the CC-CAT genotype had a HR of 2.05 [0.9-4.6] (p=0.08) and patients with only the GG-MPO genotype had a HR of 3.8 [1.5-9.1] (p=0.002), while patients with both risk factors had an HR of 4.8 [2.2-10.4] (p<0.0001). However, only the GG-MPO genotype was independently associated with the HCC risk in multivariate Cox analysis. CONCLUSIONS: The high activity-associated GG-MPO genotype increases the rate of HCC occurrence in patients with HCV-induced cirrhosis.

Central role of mitochondria in drug-induced liver injury.

A frequent mechanism for drug-induced liver injury (DILI) is the formation of reactive metabolites that trigger hepatitis through direct toxicity or immune reactions. Both events cause mitochondrial membrane disruption. Genetic or acquired factors predispose to metabolite-mediated hepatitis by increasing the formation of the reactive metabolite, decreasing its detoxification, or by the presence of critical human leukocyte antigen molecule(s). In other instances, the parent drug itself triggers mitochondrial membrane disruption or inhibits mitochondrial function through different mechanisms. Drugs can sequester coenzyme A or can inhibit mitochondrial ß-oxidation enzymes, the transfer of electrons along the respiratory chain, or adenosine triphosphate (ATP) synthase. Drugs can also destroy mitochondrial DNA, inhibit its replication, decrease mitochondrial transcripts, or hamper mitochondrial protein synthesis. Quite often, a single drug has many different effects on mitochondrial function. A severe impairment of oxidative phosphorylation decreases hepatic ATP, leading to cell dysfunction or necrosis; it can also secondarily inhibit ß-oxidation, thus causing steatosis, and can also inhibit pyruvate catabolism, leading to lactic acidosis. A severe impairment of ß-oxidation can cause a fatty liver; further, decreased gluconeogenesis and increased utilization of glucose to compensate for the inability to oxidize fatty acids, together with the mitochondrial toxicity of accumulated free fatty acids and lipid peroxidation products, may impair energy production, possibly leading to coma and death. Susceptibility to parent drug-mediated mitochondrial dysfunction can be increased by factors impairing the removal of the toxic parent compound or by the presence of other medical condition(s) impairing mitochondrial function. New drug molecules should be screened for possible mitochondrial effects.

Hepatic mitochondrial DNA depletion after an alcohol binge in mice: probable role of peroxynitrite and modulation by manganese superoxide dismutase.

Alcohol consumption increases reactive oxygen species (ROS) formation, which can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. To test whether manganese superoxide dismutase (MnSOD) modulates acute alcohol-induced mitochondrial alterations, transgenic MnSOD-overexpressing (MnSOD(+++)) mice, heterozygous knockout (MnSOD(+/-)) mice, and wild-type (WT) littermates were sacrificed 2 or 24 h after intragastric ethanol administration (5 g/kg). Alcohol administration further increased MnSOD activity in MnSOD(+++) mice, but further decreased it in MnSOD(+/-) mice. In WT mice, alcohol administration transiently increased mitochondrial ROS formation, decreased mitochondrial glutathione, depleted and damaged mtDNA, and decreased complex I and V activities; alcohol durably increased inducible nitric-oxide synthase (NOS) expression, plasma nitrites/nitrates, and the nitration of tyrosine residues in complex V proteins. These effects were prevented in MnSOD(+++) mice and prolonged in MnSOD(+/-) mice. In alcoholized WT or MnSOD(+/-) mice, mtDNA depletion and the nitration of tyrosine residues in complex I and V proteins were prevented or attenuated by cotreatment with tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a superoxide scavenger; N(omega)-nitro-l-arginine methyl ester and N-[3-(aminomethyl)benzyl]acetamidine (1,400W), two NOS inhibitors; or uric acid, a peroxynitrite scavenger. In conclusion, MnSOD overexpression prevents, and MnSOD deficiency prolongs, mtDNA depletion after an acute alcohol binge in mice. The protective effects of MnSOD, tempol, NOS inhibitors, and uric acid point out a role of the superoxide anion reacting with NO to form mtDNA-damaging peroxynitrite.

Myeloperoxidase and superoxide dismutase 2 polymorphisms comodulate the risk of hepatocellular carcinoma and death in alcoholic cirrhosis.

UNLABELLED: Alcohol increases reactive oxygen species (ROS) formation in hepatocyte mitochondria and by reduced nicotinamide adenine dinucleotide phosphate oxidases and myeloperoxidase (MPO) in Kupffer cells and liver-infiltrating neutrophils. Manganese superoxide dismutase (MnSOD) converts superoxide anion into hydrogen peroxide, which, unless detoxified by glutathione peroxidase or catalase (CAT), can form the hydroxyl radical with iron. Our aim was to determine whether Ala16Val-superoxide dismutase 2 (SOD2), G-463A-MPO, or T-262C-CAT dimorphisms modulate the risks of hepatocellular carcinoma (HCC) and death in alcoholic cirrhosis. Genotypes and the hepatic iron score were assessed in 190 prospectively followed patients with alcoholic cirrhosis. During follow-up (61.1 +/- 2.7 months), 51 patients developed HCC, and 71 died. The T-262C-CAT dimorphism did not modify hepatic iron, HCC, or death. The GG-MPO genotype did not modify iron but increased the risks of HCC and death. The hazard ratio (HR) was 4.7 (2.1-10.1) for HCC and 3.6 (1.9-6.7) for death. Carriage of one or two Ala-SOD2 allele(s) was associated with higher liver iron scores and higher risks of HCC and death. The 5-year incidence of HCC was 34.4% in patients with both the GG-MPO genotype and one or two Ala-SOD2 alleles, 5.1% in patients with only one of these two traits, and 0% in patients with none of these traits. Corresponding 5-year death rates were 37.6%, 11.6%, and 5%. CONCLUSION: The combination of the GG-MPO genotype (leading to high MPO expression) and at least one Ala-SOD2 allele (associated with high liver iron score) markedly increased the risks of HCC occurrence and death in patients with alcoholic cirrhosis.

MnSOD overexpression prevents liver mitochondrial DNA depletion after an alcohol binge but worsens this effect after prolonged alcohol consumption in mice.

Both acute and chronic alcohol consumption increase reactive oxygen species (ROS) formation and lipid peroxidation, whose products damage hepatic mitochondrial DNA (mtDNA). To test whether manganese superoxide dismutase (MnSOD) overexpression modulates acute and chronic alcohol-induced mtDNA lesions, transgenic MnSOD-overexpressing (TgMnSOD(+++)) mice and wild-type (WT) mice were treated by alcohol, either chronically (7 weeks in drinking water) or acutely (single intragastric dose of 5 g/kg). Acute alcohol administration increased mitochondrial ROS formation, decreased mitochondrial glutathione, depleted and damaged mtDNA, durably increased inducible nitric oxide synthase (NOS) expression, plasma nitrites/nitrates and the nitration of tyrosine residues in complex V proteins and decreased complex V activity in WT mice. These effects were prevented in TgMnSOD(+++) mice. In acutely alcoholized WT mice, mtDNA depletion was prevented by tempol, a superoxide scavenger, L-NAME and 1400W, two NOS inhibitors, or uric acid, a peroxynitrite scavenger. In contrast, chronic alcohol consumption decreased cytosolic glutathione and increased hepatic iron, lipid peroxidation products and respiratory complex I protein carbonyls only in ethanol-treated TgMnSOD(+++) mice but not in WT mice. In chronic ethanol-fed TgMnSOD(+++) mice, but not WT mice, mtDNA was damaged and depleted, and the iron chelator, deferoxamine (DFO), prevented this effect. In conclusion, MnSOD overexpression prevents mtDNA depletion after an acute alcohol binge but aggravates this effect after prolonged alcohol consumption, which selectively triggers iron accumulation in TgMnSOD(+++) mice but not in WT mice. In the model of acute alcohol binge, the protective effects of MnSOD, tempol, NOS inhibitors and uric acid suggested a role of the superoxide anion reacting with NO to form mtDNA-damaging peroxynitrite. In the model of prolonged ethanol consumption, the protective effects of DFO suggested the role of iron reacting with hydrogen peroxide to form mtDNA-damaging hydroxyl radical.

Mitochondrial involvement in drug-induced liver injury.

Mitochondrial dysfunction is a major mechanism of liver injury. A parent drug or its reactive metabolite can trigger outer mitochondrial membrane permeabilization or rupture due to mitochondrial permeability transition. The latter can severely deplete ATP and cause liver cell necrosis, or it can instead lead to apoptosis by releasing cytochrome c, which activates caspases in the cytosol. Necrosis and apoptosis can trigger cytolytic hepatitis resulting in lethal fulminant hepatitis in some patients. Other drugs severely inhibit mitochondrial function and trigger extensive microvesicular steatosis, hypoglycaemia, coma, and death. Milder and more prolonged forms of drug-induced mitochondrial dysfunction can also cause macrovacuolar steatosis. Although this is a benign liver lesion in the short-term, it can progress to steatohepatitis and then to cirrhosis. Patient susceptibility to drug-induced mitochondrial dysfunction and liver injury can sometimes be explained by genetic or acquired variations in drug metabolism and/or elimination that increase the concentration of the toxic species (parent drug or metabolite). Susceptibility may also be increased by the presence of another condition, which also impairs mitochondrial function, such as an inborn mitochondrial cytopathy, beta-oxidation defect, certain viral infections, pregnancy, or the obesity-associated metabolic syndrome. Liver injury due to mitochondrial dysfunction can have important consequences for pharmaceutical companies. It has led to the interruption of clinical trials, the recall of several drugs after marketing, or the introduction of severe black box warnings by drug agencies. Pharmaceutical companies should systematically investigate mitochondrial effects during lead selection or preclinical safety studies.

Prolonged ethanol administration depletes mitochondrial DNA in MnSOD-overexpressing transgenic mice, but not in their wild type littermates.

Alcohol consumption increases reactive oxygen species formation and lipid peroxidation, whose products can damage mitochondrial DNA (mtDNA) and alter mitochondrial function. A possible role of manganese superoxide dismutase (MnSOD) on these effects has not been investigated. To test whether MnSOD overexpression modulates alcohol-induced mitochondrial alterations, we added ethanol to the drinking water of transgenic MnSOD-overexpressing (TgMnSOD) mice and their wild type (WT) littermates for 7 weeks. In TgMnSOD mice, alcohol administration further increased the activity of MnSOD, but decreased cytosolic glutathione as well as cytosolic glutathione peroxidase activity and peroxisomal catalase activity. Whereas ethanol increased cytochrome P-450 2E1 and mitochondrial ROS generation in both WT and TgMnSOD mice, hepatic iron, lipid peroxidation products and respiratory complex I protein carbonyls were only increased in ethanol-treated TgMnSOD mice but not in WT mice. In ethanol-fed TgMnSOD mice, but not ethanol-fed WT mice, mtDNA was depleted, and mtDNA lesions blocked the progress of polymerases. The iron chelator, DFO prevented hepatic iron accumulation, lipid peroxidation, protein carbonyl formation and mtDNA depletion in alcohol-treated TgMnSOD mice. Alcohol markedly decreased the activities of complexes I, IV and V of the respiratory chain in TgMnSOD, with absent or lesser effects in WT mice. There was no inflammation, apoptosis or necrosis, and steatosis was similar in ethanol-treated WT and TgMnSOD mice. In conclusion, prolonged alcohol administration selectively triggers iron accumulation, lipid peroxidation, respiratory complex I protein carbonylation, mtDNA lesions blocking the progress of polymerases, mtDNA depletion and respiratory complex dysfunction in TgMnSOD mice but not in WT mice.

The manganese superoxide dismutase Ala16Val dimorphism modulates iron accumulation in human hepatoma cells.

The Ala/16Val dimorphism incorporates alanine (Ala) or valine (Val) in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD), modifying MnSOD mitochondrial import and activity. In alcoholic cirrhotic patients, the Ala-MnSOD allele is associated with hepatic iron accumulation and an increased risk of hepatocellular carcinoma. The Ala-MnSOD variant could modulate the expression of proteins involved in iron storage (cytosolic ferritin), uptake (transferrin receptors, TfR-1 and-2), extrusion (hepcidin), and intracellular distribution (frataxin) to trigger hepatic iron accumulation. We therefore assessed the Ala/Val-MnSOD genotype and the hepatic iron score in 162 alcoholic cirrhotic patients. In our cohort, this hepatic iron score increased with the number of Ala-MnSOD alleles. We also transfected Huh7 cells with Ala-MnSOD-or Val-MnSOD-encoding plasmids and assessed cellular iron, MnSOD activity, and diverse mRNAs and proteins. In Huh7 cells, MnSOD activity was higher after Ala-MnSOD transfection than after Val-MnSOD transfection. Additionally, iron supplementation decreased transfected MnSOD proteins and activities. Ala-MnSOD transfection increased the mRNAs and proteins of ferritin, hepcidin, and TfR2, decreased the expression of frataxin, and caused cellular iron accumulation. In contrast, Val-MnSOD transfection had limited effects. In conclusion, the Ala-MnSOD variant favors hepatic iron accumulation by modulating the expression of proteins involved in iron homeostasis.

Ibuprofen administration attenuates serum TNF-alpha levels, hepatic glutathione depletion, hepatic apoptosis and mouse mortality after Fas stimulation.

Fas stimulation recruits neutrophils and activates macrophages that secrete tumor necrosis factor-alpha (TNF-alpha), which aggravates Fas-mediated liver injury. To determine whether nonsteroidal anti-inflammatory drugs modify these processes, we challenged 24-hour-fasted mice with the agonistic Jo2 anti-Fas antibody (4 microg/mouse), and treated the animals 1 h later with saline or ibuprofen (250 mg/kg), a dual cyclooxygenase (COX)-1 and COX-2 inhibitor. Ibuprofen attenuated the Jo2-mediated recruitment/activation of myeloperoxidase-secreting neutrophils/macrophages in the liver, and attenuated the surge in serum TNF-alpha. Ibuprofen also minimized hepatic glutathione depletion, Bid truncation, caspase activation, outer mitochondrial membrane rupture, hepatocyte apoptosis and the increase in serum alanine aminotransferase (ALT) activity 5 h after Jo2 administration, to finally decrease mouse mortality at later times. The concomitant administration of pentoxifylline (decreasing TNF-alpha secretion) and infliximab (trapping TNF-alpha) likewise attenuated the Jo2-mediated increase in TNF-alpha, the decrease in hepatic glutathione, and the increase in serum ALT activity 5 h after Jo2 administration. The concomitant administration of the COX-1 inhibitor, SC-560 (10 mg/kg) and the COX-2 inhibitor, celecoxib (40 mg/kg) 1 h after Jo2 administration, also decreased liver injury 5 h after Jo2 administration. In contrast, SC-560 (10 mg/kg) or celecoxib (40 or 160 mg/kg) given alone had no significant protective effects. In conclusion, secondary TNF-alpha secretion plays an important role in Jo2-mediated glutathione depletion and liver injury. The combined inhibition of COX-1 and COX-2 by ibuprofen attenuates TNF-alpha secretion, glutathione depletion, mitochondrial alterations, hepatic apoptosis and mortality in Jo2-treated fasted mice.

Steatohepatitis-inducing drugs trigger cytokeratin cross-links in hepatocytes. Possible contribution to Mallory-Denk body formation.

Mallory-Denk bodies (MDB) are hepatocyte inclusions containing cytokeratin 8 (CK8) which can develop, along with other steatohepatitis lesions, in patients treated with amiodarone, perhexiline maleate or 4,4'-diethylaminoethoxyhexestrol. These drugs accumulate lipids, whose subsequent peroxidation liberates reactive by-products, like malondialdehyde (MDA). The formation of MDB has been previously reproduced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine or griseofulvin administration which cross-link CK8 by tissue transglutaminase, thus forming an entangled network, from which MDB progressively arise. The present study depicts the mechanisms initiating MDB formation by steatohepatitis-inducing drugs. Short incubation of hepatocytes with amiodarone (50 microM), 4,4'-diethylaminoethoxyhexestrol (50 microM) or perhexiline maleate (25 microM) increased the pool of CK8 monomers and increased cell calcium to activate Ca(++)-dependent transglutaminases which cross-linked the CK8 monomers into CK8-containing oligomers. The present study also provides the first evidence that MDA might directly participate in MDB formation, as this reactive agent cross-linked purified CK8 or albumin in vitro, disrupted the cytokeratin network of isolated hepatocytes, and bridged CK8 molecules. In conclusion, steatohepatitis-inducing drugs increase cell calcium and activate tissue transglutaminase, which cross-links CK8 to form a molecular scaffold, from which MDB might secondarily arise. Malondialdehyde also cross-links CK8, albeit through a different mechanism, and might also contribute to MDB formation.

Prediction of drug-induced liver injury in humans by using in vitro methods: the case of ximelagatran.

OBJECTIVE: To investigate the possible mechanisms underlying the liver enzyme elevations seen during clinical studies of long-term treatment (>35 days) with ximelagatran, and investigate the usefulness of pre-clinical in vitro systems to predict drug-induced liver effects. METHODS: Ximelagatran and its metabolites were tested for effects on cell viability, mitochondrial function, formation of reactive metabolites and reactive oxygen species, protein binding, and induction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) gene expression or nuclear orphan receptors. Experimental systems included fresh and cryopreserved hepatocytes, human hepatoma cell lines (HepG2 and HuH-7) and subcellular human liver fractions. RESULTS: Loss of cell viability was only seen in HepG2 cells at ximelagatran concentrations 100 microM and in cryopreserved human hepatocytes at 300 microM, while HuH-7 cells were not affected by 24 h exposure at up to 300 microM ximelagatran. Calcium homeostasis was not affected in HepG2 cells exposed to ximelagatran up to 300 microM for 15 min. There was no evidence for the formation of reactive metabolites when cell systems were exposed to ximelagatran. ALT and AST expression in human hepatoma cell lines were also unchanged by ximelagatran. Mitochondrial functions such as respiration, opening of the transition pore, mitochondrial membrane depolarization and beta-oxidation were not affected by ximelagatran or its metabolites. CONCLUSION: Ximelagatran at concentrations considerably higher than that found in plasma following therapeutic dosing had little or no effect on cellular functions studied in vitro. The in vitro studies therefore did not elucidate the mechanism by which ximelagatran induces liver effects in humans, possibly because of limitations in the experimental systems not reflecting characteristics of the human hepatocyte, restricted exposure time, or because the primary mechanism for the observed clinical liver effects is not on the parenchymal liver cell.

Genetic polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in patients with alcohol-induced cirrhosis.

Manganese superoxide dismutase (MnSOD) converts the superoxide anion into H(2)O(2), which, unless it is detoxified by glutathione peroxidase 1 (GPx1), can increase hepatic iron and can react with iron to form genotoxic compounds. We investigated the role of Ala/Val-MnSOD and Pro/Leu-GPx1 polymorphisms on hepatic iron accumulation and hepatocellular carcinoma development in patients with alcoholic cirrhosis. Genotypes were determined in 162 alcoholic patients with cirrhosis but without hepatocellular carcinoma initially, who were prospectively followed up for hepatocellular carcinoma development. We found that patients with two Val-MnSOD alleles (slow H(2)O(2) production) and two Pro-GPx1 alleles (presumably quick H(2)O(2) detoxification) had a lower risk of hepatocellular carcinoma development than other patients (chi(2) trend test, P = 0.001; log-rank, P = 0.0009). Indeed, hepatocellular carcinoma percentage was 0% in subjects with this "2Val-MnSOD/2Pro-GPx1" genotype versus 16%, 27%, and 32% in "2Val-MnSOD/1or2Leu-GPx1," "1or2Ala-MnSOD/2Pro-GPx1," and "1or2Ala-MnSOD/1or2Leu-GPx1" patients, respectively. The percentage of patients with stainable hepatic iron increased progressively with these genotypic associations: 22%, 28%, 50%, and 53%, respectively (chi(2) trend test, P = 0.005). Stainable iron was a risk factor for hepatocellular carcinoma (log-rank, P = 0.0002; relative risk, 3.40). In conclusion, polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in French alcoholic patients with cirrhosis.

High doses of stavudine induce fat wasting and mild liver damage without impairing mitochondrial respiration in mice.

OBJECTIVE: Stavudine (d4T), a nucleoside reverse-transcriptase inhibitor (NRTI), can induce lipoatrophy, fatty liver, hyperlactataemia and abnormal liver tests. NRTI toxicity is usually ascribed to mitochondrial DNA (mtDNA) depletion and impaired mitochondrial respiration. However, NRTIs could have effects unrelated to mtDNA. Recently, we reported that 100 mg/kg/day of d4T stimulated fatty acid oxidation (FAO) in mouse liver, and reduced body fatness without depleting white adipose tissue (WAT) mtDNA. We hypothesized that higher d4T doses could further reduce adiposity, while inhibiting hepatic FAO. METHODS: Mice were treated for 2 weeks with d4T (500 mg/kg/day), L-carnitine (200 mg/kg/day) or both drugs concomitantly. Body fatness was assessed by dual energy X-ray absorptiometry, and investigations were performed in plasma, liver, muscle and WAT. RESULTS: D4T reduced the gain of body adiposity, WAT leptin, whole body FAO and plasma ketone bodies, and increased liver triglycerides and plasma aminotransferases with mild ultrastructural abnormalities in hepatocytes. Plasma lactate and respiratory chain activities in tissues were unchanged. Stearoyl-CoA desaturase (SCD-1), an enzyme negatively regulated by leptin, was overexpressed in liver. High doses of beta-aminoisobutyric acid (BAIBA), a d4T catabolite, increased plasma ketone bodies. Although L-carnitine did not correct body adiposity, it prevented d4T-induced impairment of FAO and liver abnormalities. CONCLUSIONS: D4T overdosage triggers fat wasting, leptin insufficiency and mild liver damage, without causing respiratory chain dysfunction. Overexpression of SCD-1 reduces fatty acid oxidation and overcomes the stimulating effect of BAIBA on hepatic FAO. L-carnitine does not correct leptin insufficiency but prevents d4T-induced impairment of FAO and liver damage.

Manganese superoxide dismutase dimorphism and iron overload, hepatocellular carcinoma, and death in hepatitis C virus-infected patients.

BACKGROUND & AIMS: A genetic dimorphism encodes for either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD), and modulates its mitochondrial import and activity. It has been shown that the presence of at least 1 Ala-encoding allele is more frequent in alcoholic patients with cirrhosis than in controls, and increases the risks of liver iron overload, hepatocellular carcinoma (HCC), and death in these patients. The aim of this study was to assess the influence of the Ala-9Val MnSOD dimorphism on the same parameters and events in hepatitis C virus (HCV)-infected patients. METHODS: We compared the MnSOD genotypic distributions in 94 control subjects and 165 patients with HCV-related cirrhosis. Patients were included at the time of liver biopsy examination showing cirrhosis, and were followed-up prospectively. The mean time of follow-up evaluation was 85.7 +/- 43.8 months. RESULTS: The distribution of MnSOD genotypes in HCV-infected patients (25% Val/Val homozygotes, 44% Ala/Val heterozygotes, and 31% Ala/Ala homozygotes) did not differ from the distribution in controls (P = .3). MnSOD genotypes did not influence survival (log-rank test, P = .6; relative risk 1.0; 95% confidence interval, 0.6-1.6) or the risk of HCC occurrence (log-rank test, P = .3; relative risk, 1.1; 95% confidence interval, 0.8-1.6). CONCLUSIONS: Contrary to previous findings in French alcoholic patients, the Ala-encoding MnSOD allele is represented equally in controls and patients with HCV-related cirrhosis, and it does not significantly influence the risks of liver iron overload, HCC, or death in these patients.

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor.

Doxorubicin, cis-diamminedichloroplatinum (II) and 5-fluorouracil used in chemotherapy induce apoptosis in Hep3B cells in the absence of p53, p73, and functional Fas. Since mediators remain unknown, the requirement of PKC delta (PKCdelta) and c-Abl was investigated. Suppression of c-Abl or PKCdelta expression using SiRNAs impaired PARP cleavage, Gleevec and/or rottlerin inhibited the induction of the subG1 phase and the increase of reactive oxygen species level. Co-precipitations and phosphorylations to mitochondria of c-Abl, PKCdelta and Bcl-X(L/s) were induced. A depolarization of the mitochondrial membrane and activations of caspase-2 and -9 were observed. We propose that, in the absence of p53, p73 and Fas, genotoxic drugs could require both PKCdelta and c-Abl to induce apoptosis through the mitochondrial pathway.

Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.

Calorie-enriched diet and lack of exercise are causing a worldwide surge of obesity, insulin resistance and lipid accretion in liver (i.e. hepatic steatosis), which can lead to steatohepatitis. Steatosis and nonalcoholic steatohepatitis (NASH) can also be induced by drugs such as amiodarone, tamoxifen and some antiretroviral drugs, including stavudine and zidovudine. There is accumulating evidence that mitochondrial dysfunction (more particularly respiratory chain deficiency) plays a key role in the physiopathology of NASH whatever its initial cause. In contrast, the mitochondrial beta-oxidation of fatty acids can be either increased (as in insulin resistance-associated NASH) or decreased (as in drug-induced NASH). However, in both circumstances, generation of reactive oxygen species (ROS) by the damaged respiratory chain can be augmented. ROS generation in an environment enriched in lipids in turn induces lipid peroxidation which releases highly reactive aldehydic derivatives (e.g. malondialdehyde) that have diverse detrimental effects on hepatocytes and other hepatic cells. In hepatocytes, ROS, reactive nitrogen species and lipid peroxidation products further impair the respiratory chain, either directly or indirectly through oxidative damage to the mitochondrial genome. This consequently leads to the generation of more ROS and a vicious cycle occurs. Mitochondrial dysfunction can also lead to apoptosis or necrosis depending on the energy status of the cell. ROS and lipid peroxidation products also increase the generation of several cytokines (TNF-alpha, TGF-beta, Fas ligand) playing a key role in cell death, inflammation and fibrosis. Recent investigations have shown that some genetic polymorphisms can significantly increase the risk of steatohepatitis and that several drugs can prevent or even reverse NASH. Interestingly, most of these drugs could exert their beneficial effects by improving directly or indirectly mitochondrial function in liver. Finding a drug, which could fully prevent oxidative stress and mitochondrial dysfunction in NASH is a major challenge for the next decade.

Partial Beclin 1 silencing aggravates doxorubicin- and Fas-induced apoptosis in HepG2 cells.

AIM: To investigate the role of Beclin 1 on the susceptibility of HepG2 cells to undergo apoptosis after anti-Fas antibody or doxorubicin treatment. METHODS: Beclin 1 silencing was achieved using RNA interference. DNA ploidy, the percentage of apoptotic cells and the mitochondrial membrane potential were assessed by flow cytometry. Levels of Beclin 1, Bcl-X(L) and cytochrome c, and the cleavage of poly (ADP-ribose) polymerase (PARP) were assayed by using Western blots. RESULTS: Beclin 1 expression decreased by 75% 72 h after Beclin 1 siRNA transfection. Partial Beclin 1 silencing significantly increased the percentage of subG1 cells 24 and 40 h after treatment with doxorubicin or anti-Fas antibody, respectively, and this potentiation was abrogated by treatment with a pan-caspase inhibitor. Partial Beclin 1 silencing also increased PARP cleavage, mitochondrial membrane depolarization and cytosolic cytochrome c. The pro-apoptotic consequences of partial Beclin 1 silencing were not associated with a decline in Bcl-X(L) expression. CONCLUSION: Partial Beclin 1 silencing aggravates mitochondrial permeabilization and apoptosis in HepG2 cells treated with an anti-Fas antibody or with doxorubicin.

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes.

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.

Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.

Liver steatosis, which involves accumulation of intracytoplasmic lipid droplets, is characteristic of hepatitis C virus (HCV) infection. By use of an in vivo transgenic murine model, we demonstrate that hepatic overexpression of HCV core protein interferes with the hepatic assembly and secretion of triglyceride-rich very low density lipoproteins (VLDL). Core expression led to reduction in microsomal triglyceride transfer protein (MTP) activity and in the particle size of nascent hepatic VLDL without affecting accumulation of MTP and protein disulfide isomerase. Hepatic human apolipoprotein AII (apo AII) expression in double-core/apo AII transgenic mice diminished intrahepatic core protein accumulation and abrogated its effects on VLDL production. Apo AII and HCV core colocalized in human HCV-infected liver biopsies, thus testifying to the relevance of this interaction in productive HCV infection. Our results lead us to propose a new pathophysiological animal model for induction of viral-related steatosis whereby the core protein of HCV targets microsomal triglyceride transfer protein activity and modifies hepatic VLDL assembly and secretion.

The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria.

A genetic dimorphism encodes for either alanine (Ala) or valine (Val) in the mitochondrial targeting sequence (MTS) of human manganese superoxide dismutase (MnSOD) and has been reported to modulate the risk of some cancers, neurodegenerative diseases and severe alcoholic liver disease. Although functional consequences of this dimorphism on MnSOD activity have not been assessed, computer models predict a partial alpha-helix structure for the Ala-MnSOD/MTS, but a beta-sheet structure for the Val-variant, which could hamper mitochondrial import. To investigate this hypothesis, we studied the in-vitro import of chimaeric proteins composed of either one of the MnSOD/MTS fused to the mouse dihydrofolate reductase (DHFR) protein, and the import of the two human MnSOD precursor variants into rat liver mitochondria. Compared to Ala-proteins, the Val-MnSOD/MTS-DHFR precursor and Val-MnSOD precursor were both partly arrested within the inner mitochondrial membrane. The Ala-MnSOD precursor generated 30-40% more of the active, matricial, processed MnSOD homotetramer than the Val-MnSOD precursor. These results show that the Ala-MnSOD/MTS allows efficient MnSOD import into the mitochondrial matrix, while the Val-variant causes partial arrest of the precursor within the inner membrane and decreased formation of the active MnSOD tetramer in the mitochondrial matrix.