Search

International audience; Ethanol metabolism causes oxidative stress and lipid peroxidation not only in liver but also in extra-hepatic tissues. Ethanol administration has been shown to cause oxidative degradation and depletion of hepatic mitochondrial DNA (mtDNA) in rodents, but its in vivo effects on the mtDNA of extra-hepatic tissues have not been assessed. We studied the effects of an acute intragastric ethanol administration (5 g/kg) on brain, heart, skeletal muscle, and liver mtDNA in mice. Ethanol administration caused mtDNA depletion and replacement of its supercoiled form by linearized forms in all tissues examined. Maximal mtDNA depletion was about similar (ca. 50%) in all organs studied. It occurred 2 h after ethanol administration in heart, skeletal muscle, and liver but after 10 h in brain. This mtDNA depletion was followed by increased mtDNA synthesis. A secondary, transient increase in mtDNA levels occurred 24 h after ethanol administration in all organs. In hepatic or extra-hepatic tissues, mtDNA degradation and depletion were prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by vitamin E, melatonin, or coenzyme Q, three antioxidants. In conclusion, our study shows for the first time that ethanol metabolism also causes oxidative degradation of the mitochondrial genome in brain, heart, and skeletal muscles. These effects could contribute to the development of (cardio)myopathy and brain injury in some alcoholic patients. Antioxidants prevent these effects in mice and could be useful in persevering drinkers.

International audience; Like other antihuman immunodeficiency virus dideoxynucleosides, stavudine may occasionally induce lactic acidosis and perhaps lipodystrophy in metabolically or genetically susceptible patients. We studied the effects of stavudine on mitochondrial DNA (mtDNA), fatty acid oxidation, and blood metabolites in lean and genetically obese (ob/ob) mice. In lean mice, mtDNA was depleted in liver and skeletal muscle, but not heart and brain, after 6 weeks of stavudine treatment (500 mg/kg/day). With 100 mg/kg/day, mtDNA transiently decreased in liver, but was unchanged at 6 weeks in all organs, including white adipose tissue (WAT). Despite unchanged mtDNA levels, lack of significant oxidative mtDNA lesions (as assessed by long polymerase chain reaction experiments), and normal blood lactate/pyruvate ratios, lean mice treated with stavudine for 6 weeks had increased fasting blood ketone bodies, due to both increased hepatic fatty acid beta-oxidation and decreased peripheral ketolysis. In obese mice, basal WAT mtDNA was low and was further decreased by stavudine. In conclusion, stavudine can decrease hepatic and muscle mtDNA in lean mice and can also cause ketoacidosis during fasting without altering mtDNA. Stavudine depletes WAT mtDNA only in obese mice. Fasting and ketoacidosis could trigger decompensation in patients with incipient lactic acidosis, whereas WAT mtDNA depletion could cause lipodystrophy in genetically susceptible patients.

The effects of nilutamide on the mitochondrial respiratory chain were investigated in rats. In isolated mitochondria, nilutamide (100 microM) inhibited respiration that was supported by substrates feeding electrons into complex I of the respiratory chain but did not inhibit respiration that was supported by substrates donating electrons to complexes II, III or IV. Inhibition of complex I occurred without any lag time. In submitochondrial particles, nilutamide (100 microM) decreased both oxygen consumption mediated by NADH and the oxidation of NADH; addition of superoxide dismutase and catalase did not alleviate inhibition. There was no electron spin resonance evidence for detectable mitochondrial formation of the nilutamide nitro anion free radical by submitochondrial particles or for the formation of iron-nitrosyl complexes with mitochondrial Fe-S clusters in isolated hepatocytes. Severe inhibition of complex I by nilutamide (500 microM) led to upstream inhibition of fatty acid beta-oxidation. Nilutamide (100 microM) decreased the mitochondrial membrane potential and ATP formation in mitochondria energized by malate plus glutamate. In hepatocytes incubated without glucose, nilutamide (500 microM) led to an early (2 hr) drop in cellular ATP and early (4 hr) toxicity. With 5 mM glucose, however, ATP was not decreased and toxicity was mild at these early times. It was concluded that nilutamide itself inhibited the mitochondrial respiratory chain at the level of complex I and decreased ATP in hepatocytes incubated without glucose, which resulted in early toxicity. In the presence of glucose, ATP was not depleted at early times and delayed toxicity was probably the result of an oxidative stress (as previously reported).

The hepatotoxicity of flutamide, an antiandrogen that produces hepatitis in some human recipients, was studied in isolated rat hepatocytes. Flutamide (1 mM) led to the covalent binding of reactive electrophilic metabolites to male rat hepatocyte proteins. It decreased the reduced glutathione (GSH)/glutathione disulfide ratio and total protein thiols. This was associated with an early increase in phosphorylase a activity (a Ca(++)-dependent enzyme) and a decrease in cytoskeleton-associated protein thiols, the formation of plasma membrane blebs, the release of lactate dehydrogenase (LDH) and a loss of cell viability. Both covalent binding and LDH release were decreased by piperonyl butoxide (an inhibitor of cytochrome P450) and increased by dexamethasone pretreatment (which induces cytochrome P450 3A). The toxicity was increased by beta-naphthoflavone (which induces cytochrome P450 1A). Hepatocytes from female rats (which lack cytochrome P450 3A2) exhibited lower covalent binding and lower LDH release. The addition of cystine (a GSH precursor) increased hepatocellular GSH and decreased LDH release in male hepatocytes. The administration of a diet deficient in sulfur-containing amino acids had the opposite effects; it produced toxicity with 100 microM flutamide. Flutamide (50 microM) markedly inhibited respiration (mainly at the level of complex I) in isolated male rat liver mitochondria and flutamide (1 mM) decreased ATP levels in isolated male rat hepatocytes. It was concluded that flutamide is toxic to rat hepatocytes as a result of the cytochrome P450 (3A and also 1A)-mediated formation of electrophilic metabolites, whose damaging effects are further aggravated by the inhibitory effect of flutamide on mitochondrial respiration and ATP formation.

Incubation of the antiandrogen nilutamide with rat liver microsomes and NADPH, under anaerobic conditions, led to the formation of the nitro anion free radical, as indicated by electron spin resonance spectroscopy. The steady-state concentration of the nitro anion free radical was not decreased by SKF 525-A, carbon monoxide or metyrapone (3 inhibitors of cytochrome P-450) but was decreased by NADP+ or p-chloromercuribenzoate (2 inhibitors of NADPH-cytochrome P-450 reductase). Under aerobic conditions, the nitro anion free radical was reoxidized by oxygen, and the electron spin resonance signal was not detected. This redox cycle was associated with NADPH oxidation, consumption of oxygen, and formation of superoxide anion, hydrogen peroxide and glutathione disulfide. Under anaerobic conditions, nilutamide was further reduced to chemically reactive metabolites. Anaerobic incubation of [3H]nilutamide (0.1 mM) with rat liver microsomes and a NADPH-generating system resulted in the in vitro covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADPH-cytochrome P-450 reductase inhibitors (methylene blue, 2'-adenosine monophosphate) or in the presence of the nucleophile glutathione, but was unaffected by cytochrome P-450 inhibitors (SKF 525-A, CO). Covalent binding was decreased markedly under aerobic conditions. We conclude that nilutamide is reduced by microsomal NADPH-cytochrome P-450 reductase into a nitro anion free radical. In the presence of oxygen, this nitro anion free radical undergoes redox cycling with oxygen, and forms reactive oxygen species. In anaerobiosis, it is reduced further to covalent binding species.

Amiodarone has been shown to produce microvesicular steatosis of the liver in some recipients. We have determined the effects of amiodarone on the mitochondrial oxidation of fatty acids in mice. In vitro, the formation of 14C-acid-soluble beta-oxidation products from [U-14C]palmitic acid by mouse liver mitochondria was decreased by 92% in the presence of 125 microM amiodarone and by 94% in the presence of 125 microM N-desethylamiodarone. Inhibition due to 100 or 150 microM amiodarone persisted in the presence of 5 mM acetoacetate, whereas acetoacetate totally relieved inhibition due to 15 microM rotenone. In vivo, exhalation of [14C]CO2 from [U-14C]palmitic acid was decreased by 31, 40, 58 and 78%, respectively, in mice receiving 19, 25, 50 and 100 mg.kg-1 of amiodarone hydrochloride 1 hr before the administration of [U-14C]palmitic acid. One hour after 100 mg.kg-1, the exhalation of [14C]CO2 from [1-14C]palmitic acid, [1-14C]octanoic acid or [1-14C]butyric acid was decreased by 78, 72 and 53%, respectively. Exhalation of [14C]CO2 from [1-14C]palmitic acid was normal between 6 and 9 hr after administration of 100 mg.kg-1 of amiodarone hydrochloride, but was still inhibited by 71 and 37%, 24 and 48 hr after 600 mg.kg-1. Twenty four hours after the latter dose of amiodarone, hepatic triglycerides were increased by 150%, and there was microvesicular steatosis of the liver. We conclude that amiodarone inhibits the mitochondrial beta-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice.

The effects of the R-(-) and S-(+)ibuprofen enantiomers were first studied in vitro with mouse liver mitochondria incubated in the presence of various concentrations of exogenous coenzyme A. In the presence of a low concentration of coenzyme A (2.5 microM), the R-(-)enantiomer (which forms an acylcoenzyme A) inhibited stereoselectively the beta oxidation of [1-14C]palmitic acid but not that of [1-14C]palmitoyl-L-carnitine (which can directly enter the mitochondria). In the presence, however, of a concentration of coenzyme A (50 microM) reproducing that present in liver cell cytosol, both enantiomers (2 mM) slightly inhibited the beta oxidation of [1-14C]palmitic acid and markedly inhibited the beta oxidation of [1-14C]octanoic acid and [1-14C]butyric acid. In vivo, both enantiomers (1 mmol.kg-1) similarly inhibited the formation of [14C]CO2 from [1-14C]fatty acids. Both enantiomers similarly decreased plasma ketone bodies. Both similarly increased hepatic triglycerides, and both produced mild microvesicular steatosis of the liver. We conclude that both ibuprofen enantiomers inhibit beta oxidation of fatty acids in vitro and in vivo. In addition, the R-(-)enantiomer may stereoselectively sequester coenzyme A; at low concentrations of coenzyme A in vitro, this may stereoselectively inhibit the mitochondrial uptake and beta oxidation of long chain fatty acids.

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) decreases cytochrome P-450 (CYP) expression in rat hepatocytes. Because IL-2 increases c-Myc in lymphocytes and because c-myc overexpression represses several genes, we postulated that the IL-2/IL-2R interaction may increase c-Myc and thereby down-regulate CYP in hepatocytes. Cultured rat hepatocytes were exposed for 24 h to IL-2 (350 U/ml) and other agents. IL-2 increased c-myc mRNA and protein but decreased total CYP and the mRNAs and proteins of CYP2C11 and CYP3A. The IL-2-mediated c-myc overexpression and CYP down-regulation were prevented by 1) genistein (a tyrosine kinase inhibitor that blocks the initial transduction of the IL-2R signal), 2) retinoic acid, butyric acid, or dimethyl sulfoxide (three agents that block c-myc transcription), or 3) an antisense c-myc oligonucleotide (which may cause rapid degradation of the c-myc transcript). It is concluded that IL-2 causes the overexpression of c-myc and the down-regulation of CYPs in rat hepatocytes. Block of c-myc overexpression, at three different levels with five different agents, prevents CYP down-regulation, suggesting that c-myc overexpression may directly or indirectly repress CYP in hepatocytes.