Abdellah Mansouri

Alterations in mitochondrial function, hydrogen peroxide release and oxidative damage in mouse hind-limb skeletal muscle during aging.

Mitochondrial function, hydrogen peroxide generation and oxidative damage were measured in hind-limb skeletal muscle from young (6-8 month) and old (27-29 month) wildtype and heterozygous Mn-superoxide dismutase (MnSOD) knockout mice (Sod2(+/-)). The reduction in MnSOD activity in the Sod2(+/-) mice makes these mice a good model to examine the implications of life-long elevated endogenous mitochondrial oxidative stress on mitochondrial function. ATP production was reduced approximately 30% with age in skeletal muscle mitochondria isolated from wildtype mice, and reduced 40-45% in mitochondria from both young and old Sod2(+/-) mice compared to the young wildtype mice. Release of hydrogen peroxide from skeletal muscle mitochondria increased 40-50% with age in both wildtype and Sod2(+/-) but was not higher in mitochondria from Sod2(+/-) mice. Activities of electron transport Complexes I and V were decreased 25-30% in both young and old Sod2(+/-) mice compared to wildtype mice, and were 25-30% lower in mitochondria from old wildtype and old Sod2(+/-) mice. DNA oxidative damage (oxo8dG levels) increased more than 45% with age and over 130% in the young Sod2(+/-) mice compared to the wildtype mice. These data show that mitochondrial oxidative stress in mouse skeletal muscle is increased with age, leading to alterations in mitochondrial function. In addition, increased oxidative stress generated by reduced activity of MnSOD does not exacerbate these alterations during aging.

An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice

BACKGROUND & AIMSnEthanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks.nnnMETHODSnEffects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [3H]thymidine incorporation, respectively.nnnRESULTSnTwo hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant.nnnCONCLUSIONSnAfter an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics.

Mitochondrial injury in steatohepatitis.

Rich diet and lack of exercise are causing a surge in obesity, insulin resistance and steatosis, which can evolve into steatohepatitis. Patients with non-alcoholic steatohepatitis have increased lipid peroxidation, increased tumour necrosis factor-α (TNF-α) and increased mitochondrial β-oxidation rates. Their in-vivo ability to re-synthesize ATP after a fructose challenge is decreased, and their hepatic mitochondria exhibit ultrastructural lesions, depletion of mitochondrial DNA and decreased activity of respiratory chain complexes. Although the mechanisms for these effects is unknown, the basal cellular formation of reactive oxygen species (ROS) may oxidize fat deposits to cause lipid peroxidation, which damages mitochondrial DNA, proteins and cardiolipin to partially hamper the flow of electrons within the respiratory chain. This flow may be further decreased by TNF-α, which can release cytochrome c from mitochondria. Concomitantly, the increased mitochondrial fatty acid β-oxidation rate augments the delivery of electrons to the respiratory chain. Due to the imbalance between a high electron input and a restricted outflow, electrons may accumulate within complexes I and III, and react with oxygen to form the superoxide anion radical. Increased mitochondrial ROS formation could in turn directly oxidize mitochondrial DNA, proteins and lipids, enhance lipid peroxidation-related mitochondrial damage, trigger hepatic TNF-α formation and deplete antioxidants, thus further blocking electron flow and further increasing mitochondrial ROS formation. Mitochondrial dysfunction plays an important role in liver lesions, through the ROS-induced release of both biologically active lipid peroxidation products and cytokines. In particular, the up-regulation of both TNF-α and Fas triggers mitochondrial membrane permeability and apoptosis. The ingestion of apoptotic bodies by stellate cells stimulates fibrogenesis, which is further activated by lipid peroxidation products and high leptin levels. Chronic apoptosis is compensated by increased cell proliferation, which, together with oxidative DNA damage, may cause gene mutations and cancer.

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 β-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 β-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.

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.

Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.

Although tamoxifen can trigger steatohepatitis, the mechanism of steatosis is unclear. We hypothesized that this DNA-intercalating, cationic amphiphilic drug could accumulate within mitochondria to impair fatty acid oxidation, respiration, and mitochondrial DNA relaxation and synthesis. We studied the in vitro effects of tamoxifen on topoisomerases and mouse liver mitochondria and its in vivo hepatic effects in mice treated for 1 to 28 days with a daily dose of tamoxifen reproducing the plasma concentrations observed in humans. In vitro, tamoxifen inhibited topoisomerase-mediated plasmid DNA relaxation. It accumulated 40-fold inside mitochondria and inhibited both respiration and fatty acid oxidation. In vivo, a single dose of tamoxifen inhibited palmitic acid oxidation and hepatic lipoprotein secretion. Tamoxifen administration also decreased mitochondrial DNA synthesis and progressively depleted hepatic mitochondrial DNA, down to 40% of control values at 28 days. The decrease in mitochondrial DNA-encoded respiratory complexes sensitized mitochondria to the inhibitory effects of tamoxifen on mitochondrial respiration. Hepatic steatosis was absent at 5 days, mild at 12 days, and moderate at 28 days. The fatty acid synthase protein was normally expressed at 12 days but was decreased by 52% at 28 days. In conclusion, tamoxifen decreases hepatic triglyceride secretion, and it accumulates electrophoretically in mitochondria, where it impairs β-oxidation and respiration. Tamoxifen also inhibits topoisomerases and mitochondrial DNA synthesis and progressively depletes hepatic mitochondrial DNA in vivo. These combined effects could decrease fat removal from the liver, thus causing hepatic steatosis despite a secondary down-regulation of hepatic fatty acid synthase expression.

Mitochondrial GSH determines the toxic or therapeutic potential of superoxide scavenging in steatohepatitis

BACKGROUND & AIMSnSteatohepatitis (SH) is associated with mitochondrial dysfunction and excessive production of superoxide, which can then be converted into H(2)O(2) by SOD2. Since mitochondrial GSH (mGSH) plays a critical role in H(2)O(2) reduction, we explored the interplay between superoxide, H(2)O(2), and mGSH in nutritional and genetic models of SH, which exhibit mGSH depletion.nnnMETHODSnWe used isolated mitochondria and primary hepatocytes, as well as in vivo SH models showing mGSH depletion to test the consequences of superoxide scavenging.nnnRESULTSnIn isolated mitochondria and primary hepatocytes, superoxide scavenging by SOD mimetics or purified SOD decreased superoxide and peroxynitrite generation but increased H(2)O(2) following mGSH depletion, despite mitochondrial peroxiredoxin/thioredoxin defense. Selective mGSH depletion sensitized hepatocytes to cell death induced by SOD mimetics, and this was prevented by RIP1 kinase inhibition with necrostatin-1 or GSH repletion with GSH ethyl ester (GSHee). Mice fed the methionine-choline deficient (MCD) diet or MAT1A(-/-) mice exhibited reduced SOD2 activity; in vivo treatment with SOD mimetics increased liver damage, inflammation, and fibrosis, despite a decreased superoxide and 3-nitrotyrosine immunoreactivity, effects that were ameliorated by mGSH replenishment with GSHee, but not NAC. As a proof-of-principle of the detrimental role of superoxide scavenging when mGSH was depleted transgenic mice overexpressing SOD2 exhibited enhanced susceptibility to MCD-mediated SH.nnnCONCLUSIONSnThese findings underscore a critical role for mGSH in the therapeutic potential of superoxide scavenging in SH, and suggest that the combined approach of superoxide scavenging with mGSH replenishment may be important in SH.

Lipopolysaccharide-induced mitochondrial DNA depletion.

Hepatic energy depletion has been described in severe sepsis, and lipopolysaccharide (LPS) has been shown to cause mitochondrial DNA (mtDNA) damage. To clarify the mechanisms of LPS-induced mtDNA damage and mitochondrial alterations, we treated wild-type (WT) or transgenic manganese superoxide dismutase-overerexpressing (MnSOD(+++)) mice with a single dose of LPS (5 mg/kg). In WT mice, LPS increased mitochondrial reactive oxygen species formation, hepatic inducible nitric oxide synthase (NOS) mRNA and protein, tumor necrosis factor-alpha, interleukin-1 beta, and high-mobility group protein B1 concentrations. Six to 48 h after LPS administration (5 mg/kg), liver mtDNA levels, respiratory complex I activity, and adenosine triphosphate (ATP) contents were decreased. In addition, LPS increased interferon-β concentration and decreased mitochondrial transcription factor A (Tfam) mRNA, Tfam protein, and mtDNA-encoded mRNAs. Morphological studies showed mild hepatic inflammation. The LPS (5 mg/kg)-induced mtDNA depletion, complex I inactivation, ATP depletion, and alanine aminotransferase increase were prevented in MnSOD(+++) mice or in WT mice cotreated with 1400W (a NOS inhibitor), (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride, monohydrate (a superoxide scavenger) or uric acid (a peroxynitrite scavenger). The MnSOD overexpression delayed death in mice challenged by a higher, lethal dose of LPS (25 mg/kg). In conclusion, LPS administration damages mtDNA and alters mitochondrial function. The protective effects of MnSOD, NOS inhibitors, and superoxide or peroxynitrite scavengers point out a role of the superoxide anion reacting with NO to form mtDNA- and protein-damaging peroxynitrite. In addition to the acute damage caused by reactive species, decreased levels of mitochondrial transcripts contribute to mitochondrial dysfunction.

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

BACKGROUND & AIMSnGenetic 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.nnnMETHODSnTwo 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.nnnRESULTSnDuring 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.nnnCONCLUSIONSnThe high activity-associated GG-MPO genotype increases the rate of HCC occurrence in patients with HCV-induced cirrhosis.

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.