Autoantibodies against cytochromes P450: role in human diseases.
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Autoantibodies↗
Biomedical subjects
D Pessayre
Publications and source records attributed to D Pessayre.
Toxicity of the antiandrogen flutamide in isolated rat hepatocytes.
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.
Adenosine Triphosphate↗
Interactions of dihydralazine with cytochromes P4501A: a possible explanation for the appearance of anti-cytochrome P4501A2 autoantibodies.
The antihypertensive drug dihydralazine may, on rare occasions, cause immunoallergic hepatitis characterized by anti-cytochrome P450 (P450)1A2 autoantibodies. To understand the first steps leading to this immune reaction, we studied the covalent binding fo dihydralazine metabolites to microsomes from rat and human livers. Upon incubation with NADPH and microsomes, dihydralazine formed metabolites that reacted with heme (as evidenced by destruction of heme, formation of 445-nm light-absorbing complexes, and covalent binding of heme to P450 apoprotein) and covalently bound to microsomal proteins. Formation of these metabolites was shown (by NADPH dependence, induction by beta-naphthoflavone, and immunoinhibition by anti-P4501A antibodies) to be mediated by P4501A. Finally, these metabolites appeared to bind to P4501A2, which produced them. These results support the following scheme for the first steps of this autoimmune reaction: P4501A2 metabolizes dihydralazine into reactive metabolites that then bind to it, forming a neoantigen that triggers an immune response characterized by autoantibodies against P4501A2.
Animals↗
Inhibition by nilutamide of the mitochondrial respiratory chain and ATP formation. Possible contribution to the adverse effects of this antiandrogen.
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).
Adenosine Triphosphate↗
Inhibition by perhexiline of oxidative phosphorylation and the beta-oxidation of fatty acids: possible role in pseudoalcoholic liver lesions.
In an attempt to better understand the mechanisms for pseudoalcoholic liver lesions in human beings, we determined the effects of perhexiline on mitochondrial functions in mice and rats. A first series of studies suggested that protonated perhexiline entered mouse mitochondria along the mitochondrial membrane potential. Release of a proton in the mitochondrial matrix led to uncoupling of oxidative phosphorylation, and accumulation of perhexiline inhibited complexes I and II of the respiratory chain, decreased ATP formation in vitro and decreased the mitochondrial beta-oxidation of long-, medium- and short-chain fatty acids in vitro and in vivo in mice. In cultured rat hepatocytes, exposure for 24 hr to 25 mumol/L perhexiline markedly decreased hepatocellular ATP and cell viability. Exposure to 5 mumol/L perhexiline did not modify ATP and viability but decreased the beta-oxidation of palmitic acid uniformly labeled with carbon 14 by 38%, increased hepatocyte triglyceride levels by 98% and produced microvesicular steatosis after 72 hr of culture. We conclude that perhexiline is concentrated inside mitochondria, where it inhibits both oxidative phosphorylation and the mitochondrial beta-oxidation of fatty acids. These effects may contribute to the development of necrosis, steatosis and possibly certain other pseudoalcoholic liver lesions in human beings.
Adenosine Triphosphate↗
Administration of high doses of human recombinant interleukin-2 decreases the expression of several cytochromes P-450 in the rat.
Human recombinant interleukin-2 (IL-2) administration is being tested in patients with advanced cancer. Its effects on the expression of cytochromes P-450 were determined in rats. IL-2 administration (1-25 x 10(6) U/kg i.v. twice daily for 1 to 4 days) resulted in a time- and dose-dependent decrease in cytochrome P-450 measured by the absorbance of its Fe(++)-CO complex. After 25 x 10(6) U/kg twice daily for 4 days, cytochrome P-450 decreased 44%; immunoreactive cytochrome P-450 1A1 decreased nonsignificantly (22%); but cytochrome P-450 1A2 decreased 68%; 2B1/2, 50%; 2C11, 75%; 2D1, 36%; and 3A, 70%. Aminopyrine N-demethylase activity decreased 53%, ethoxycoumarin O-deethylase 64%, benzo(a)pyrene hydroxylase 71%, ethoxyresorufin O-deethylase 42%, pentoxyresorufin O-dealkylase 81% and erythromycin N-demethylase 56%. In rats treated with 3-methylcholanthrene for 4 days, IL-2 coadministration (25 x 10(6) U/kg i.v. twice daily for 4 days) did not decrease significantly immunoreactive cytochrome P-450 1A1 and 1A2, whereas cytochromes P-450 2B1/2, 2C11 and 3A decreased 39, 54 and 67%, respectively. In rats treated with phenobarbital for 4 days, IL-2 coadministration decreased immunoreactive cytochromes P-450 2B1/2 29%, whereas cytochromes P-450 1A2, 2C11 and 3A decreased 38, 63 and 67%, respectively. We conclude that administration of high doses of IL-2 decreases the expression of several cytochromes P-450 in rats. Microsomal enzyme inducers appear to limit the effects of IL-2 on the induced forms of cytochromes P-450. Because much lower doses are used in humans, their potential effects on drug metabolism cannot be assessed from present results.
Animals↗
Evaluation of human blood lymphocytes as a model to study the effects of drugs on human mitochondria. Effects of low concentrations of amiodarone on fatty acid oxidation, ATP levels and cell survival.
Human lymphocytes were assessed as a cellular model for determining the effects of drugs on human mitochondria. Formation of total oxidized 14C-products was maximal with 1 mM [U-14C]palmitic acid, was linear for 90 min, linear with the number of lymphocytes, and decreased by 95% and 77% in the presence of 30 microM rotenone and 2 mM KCN. Seven drugs were tested which had previously been shown to inhibit beta-oxidation in animals; all decreased formation of total oxidized 14C-products by human lymphocytes, but with different IC50 values: 35 microM with amiodarone, 2.75 mM with tetracycline and amineptine, 3.75 mM with tianeptine, and more than 10 mM for valproic acid and the ibuprofen enantiomers. Formation of [14C]CO2 either increased or decreased, in relation to the various effects of these drugs on coupling, beta-oxidation, and the tricarboxylic acid cycle. There was a general trend for some relationship between inhibition of fatty acid oxidation and loss of cellular ATP. Those compounds, however, which uncoupled oxidative phosphorylation (2,4-dinitrophenol, amiodarone, ibuprofen) and/or inhibited the mitochondrial respiratory chain (amiodarone, rotenone, KCN) resulted in comparatively higher ATP depletion. Amiodarone, a drug which produces several effects (uncoupling, inhibition of beta-oxidation, of the tricarboxylic acid cycle and of the respiratory chain), caused a dramatic decrease in cellular ATP and cell viability at low concentrations (20-100 microM). Both these effects were prevented by the addition of 5 mM glucose, a substrate for anaerobic glycolysis. We conclude that human lymphocytes may be a useful model for assessing the effects of drugs on human mitochondrial function. IC50 values determined with this model may not necessarily apply, however, to other cells.
Adenosine Triphosphate↗
Decreased mitochondrial oxidation of fatty acids in pregnant mice: possible relevance to development of acute fatty liver of pregnancy.
Severe impairment of the beta-oxidation of fatty acids, as a consequence of a single factor or a combination of different causes, leads to microvesicular steatosis of the liver. In an effort to understand the mechanism(s) leading to the development of acute fatty liver of pregnancy in some women, we determined the effects of pregnancy on the mitochondrial oxidation of fatty acids in mice. In vivo, the rate of oxidation of the whole fatty-acid chain length was determined by measuring the rate of exhalation of [14C]CO2 after intragastric administration of a tracer dose of [U-14C]palmitic acid. [14C]CO2 exhalation was not significantly decreased at 14 days of gestation, but it had declined by 40% at 18 days of gestation (i.e., 24 to 48 hr before delivery). The rate of first beta-oxidation cycle was assessed by measuring the rate of [14C]CO2 exhalation after administration of [1-14C]octanoic acid, [1-14C]butyric acid or [1-14C]palmitic acid. [14C]CO2 exhalation had declined by 60%, 46%, and 24% after administration of [1-14C]octanoic acid, [1-14C]butyric acid and [1-14C]palmitic acid, respectively, in 18-day-pregnant mice. Total hepatic lipids and triglycerides, expressed per gram of liver, remained unchanged in 18-day-pregnant mice. In vitro, the rate of mitochondrial beta-oxidation (expressed per milligram of protein) had decreased by 47% at 18 days' gestation with [U-14C]palmitic acid as substrate and by 33% with [1-14C]octanoic acid but remained unchanged with [1-14C]palmitic acid. The activity of the tricarboxylic acid cycle, assessed by the formation of [14C]CO2 from [1-14C]acetic acid, had decreased by 24%. We conclude that the mitochondrial oxidation of fatty acids decreased during late-term pregnancy in mice as a consequence of both decreased mitochondrial beta-oxidation of medium-chain fatty acids, and decreased activity of the tricarboxylic acid cycle. We suggest that this effect, in combination with other factors, may contribute to the development of fatty liver of pregnancy in some pregnant women.
Acyl-CoA Dehydrogenase↗
Cytochromes P-450 in human hepatocyte plasma membrane: recognition by several autoantibodies.
BACKGROUND: Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of human hepatocytes was examined. METHODS: (1) Plasma membranes with microsomal contamination < 1%, as judged from the activities of glucose-6-phosphatase and NADH-cytochrome c reductase, were prepared. (2) After exposure of uncut, fixed hepatocytes to antibodies, immunofluorescence and immunoperoxidase studies were performed. RESULTS: (1) The specific content of cytochrome P-450 in plasma membrane was 9% of that in microsomes. Plasma membranes showed NADPH-cytochrome c reductase and mono-oxygenase activities; immunoblots showed the presence of cytochromes P-450 1A2, 2C, 2D6, 2E1, and 3A4; cytochromes P-450 1A2, 2D6, and 2C were also recognized by anti-liver microsome and anti-liver/kidney microsome type 1 and type 2 autoantibodies, respectively. (2) Immunofluorescence and immunoperoxidase labeling of the plasma membrane was observed with the three auto-antibodies and with anti-cytochrome P-450 1A2, 2C, 2E1, or 3A4. CONCLUSIONS: It is concluded that cytochromes P-450 are present and functional in the plasma membrane of human hepatocytes and that anti-cytochrome P-450 autoantibodies recognize epitopes expressed on the outer surface.
Autoantibodies↗
Increased ethane exhalation, an in vivo index of lipid peroxidation, in alcohol-abusers.
Ethane exhalation was measured in 42 control subjects, 52 patients with various non-alcoholic liver diseases, and 89 alcohol abusers who had been admitted to hospital for alcohol withdrawal and assessment of liver disease (six with normal liver tests, 10 with steatosis with or without fibrosis, six with alcoholic hepatitis, 29 with cirrhosis, 34 with both cirrhosis and alcoholic hepatitis, and four with both cirrhosis and a hepatocellular carcinoma). Ethane exhalation was similar in control subjects and in patients with non-alcoholic liver diseases, but was five times higher in alcohol abusers. Ethane exhalation in alcohol abusers was significantly, but very weakly, correlated with the daily ethanol intake before hospital admission, and the histological score for steatosis, but not with the inflammation or alcoholic hepatitis scores. Ethane exhalation was inversely correlated with the duration of abstinence before the test. In nine alcoholic patients, the exhalation of ethane was measured repeatedly, and showed slow improvement during abstinence. Ethane exhalation was significantly but weakly correlated with the Pugh's score in patients with alcoholic cirrhosis. It is concluded that the mean ethane exhalation is increased in alcohol abusers. One of the possible mechanisms may be the presence of oxidizable fat in the liver. The weak correlation with the Pugh's score is consistent with the contribution of many other factors in the progression to severe liver disease.
Adult↗
[Physiopathology of drug-induced hepatopathies].
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Acute Disease↗
[Drug-induced steatosis, phospholipidosis and pseudo-alcoholic liver disorders].
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Chemical and Drug Induced Liver Injury↗
Generation of free radicals during the reductive metabolism of nilutamide by lung microsomes: possible role in the development of lung lesions in patients treated with this anti-androgen.
The pulmonary metabolism of nilutamide, a nitroaromatic anti-androgen drug leading to pulmonary lesions in a few recipients, has been investigated in rats. Incubation of nilutamide (1 mM) with rat lung microsomes and NADPH under anaerobic conditions led to the formation of the nitro anion free radical, as indicated by ESR spectroscopy. The steady state concentration of this radical was not decreased by CO or SKF 525-A (two inhibitors of cytochrome P450), but was decreased by NADP+ (10 mM) or p-chloromercuribenzoate (0.47 mM) (two inhibitors of NADPH-cytochrome P450 reductase activity). Anaerobic incubations of [3H]nilutamide (0.1 mM) with rat lung microsomes and a NADPH-generating system resulted in the in vivo covalent binding of [3H]nilutamide metabolites to microsomal proteins; covalent binding required NADPH; it was decreased in the presence of NADP+ (10 mM), or in the presence of the nucleophile glutathione (10 mM), but was unchanged in the presence of carbon monoxide. Under aerobic conditions, in contrast, the nitro anion free radical was reoxidized by oxygen, and its ESR signal was not detected. Covalent binding was essentially suppressed. Instead, there was consumption of NADPH and oxygen, and production of superoxide anion and hydogen peroxide. We conclude that nilutamide is reduced by rat lung microsomes NADPH-cytochrome P450 reductase into a nitro anion free radical. In anaerobiosis, the radical is reduced further to covalent binding species. In the presence of oxygen, in contrast, this nitro anion free radical undergoes redox cycling, with the generation of reactive oxygen species.
Androgen Antagonists↗
Mechanism for the hepatotoxicity of the antiandrogen, nilutamide. Evidence suggesting that redox cycling of this nitroaromatic drug leads to oxidative stress in isolated hepatocytes.
The nitroaromatic drug nilutamide has been shown previously to undergo redox cycling in aerobic rat liver microsomes, being reduced by NADPH-cytochrome P-450 reductase to a nitro anion-free radical which reacts with oxygen, to regenerate the parent drug, and form a superoxide anion dismuted to hydrogen peroxide. In the present study, the effects of nilutamide on isolated rat hepatocytes have been determined. After 6 and 8 hr of incubation with 0.5 mM nilutamide, lactate dehydrogenase was released in the incubation medium, and cell viability was decreased markedly. Consistent with a redox cycle producing reactive oxygen species, nilutamide increased nonmitochondrial (cyanide-resistant) oxygen consumption; the toxicity of nilutamide occurred sooner and was more extensive in the presence of sodium azide (an inhibitor of catalase). Consistent with an oxidative stress, the toxicity of nilutamide was associated with depletion of reduced glutathione, increased levels of glutathione disulfide, increased Ca(++)-dependent phosphorylase a activity, oxidation and accumulation of cytoskeleton-associated proteins and formation of blebs; toxicity was prevented by glutathione precursors, thiol reductants and/or antioxidants, such as L-cystine, L-cysteine, N-acetyl-L-cysteine, dithiothreitol, N,N'-diphenyl-p-phenylene-diamine and alpha-tocopherol. Feeding the animals with a diet supplemented with 2% L-cystine increased the initial glutathione stores of hepatocytes and prevented nilutamide toxicity. It is concluded that nilutamide is toxic to isolated rat hepatocytes, as a probable consequence of an oxidative stress due to the redox cycling of this nitroaromatic compound.
Animals↗
Effects of various tetracycline derivatives on in vitro and in vivo beta-oxidation of fatty acids, egress of triglycerides from the liver, accumulation of hepatic triglycerides, and mortality in mice.
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Animals↗
Nilutamide inhibits mephenytoin 4-hydroxylation in untreated male rats and in human liver microsomes.
1. The effects of nilutamide (an anti-androgen with a hydantoin moiety) on the 4-hydroxylation of mephenytoin were studied in rat liver microsomes. Nilutamide, at a concentration expected in human liver (100 microM) during prolonged administration of nilutamide, inhibited by 40% mephenytoin (0.3 mM) 4-hydroxylase activity in liver microsomes from untreated male rats, but not in microsomes from untreated female rats, or in microsomes from dexamethasone-treated male or female rats. 2. Administration to male rats of nilutamide, in doses (20 mg/kg i.p. twice daily) known to reproduce plasma concentrations observed in human therapeutics, decreased by 60% the 24 h urinary excretion of 4-hydroxymephenytoin after administration of mephenytoin (15 mg/kg oral). 3. Nilutamide (100 microM) markedly inhibited mephenytoin 4-hydroxylase activity in human liver microsomes. Inhibition kinetics were consistent with mixed inhibition. It is concluded that nilutamide inhibits mephenytoin 4-hydroxylase activity in untreated male rats and in human liver microsomes. It is suggested that inhibition is likely to occur in vivo in humans receiving therapeutic doses of nilutamide.
Androgen Antagonists↗
[Calcium in hepatocytes: physiology and physiopathology].
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Calcium↗
[Atrium-related hepatitis. Report of four cases].
Four patients developed acute hepatitis after receiving Atrium, an association of phenobarbital, febarbamate and difebarbomate, for the treatment of tremor or for the prevention of alcohol withdrawal symptoms. Hepatitis occurred 1 to 3 months after treatment. Asthenia was the unique clinical manifestation. Marked increase in serum aminotransferases and gamma-glutamyltranspeptidase levels were the main biological features. Histological examination showed liver cell necrosis in two cases, prominent in the centrolobular area in one case. There was no case of hepatic failure. Atrium withdrawal was followed by complete recovery within 6 to 12 weeks. The mechanism of Atrium hepatotoxicity remains unknown.
Aged↗