Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-450IA2. A specific marker of dihydralazine-induced hepatitis.

Sera from patients with dihydralazine-induced hepatitis were shown to contain anti-liver microsomal autoantibodies (anti-LM) by indirect immunofluorescence. These anti-LM antibodies were different from anti-liver/kidney microsomes (anti-LKM) 1 or 2 autoantibodies which have been previously described. Sera recognized a single 53,000 = Mr polypeptide in human liver microsomes as judged by immunoblotting, and the target antigen was identified as cytochrome P-450IA2 (P-450IA2) by (a) comparison of immunoblotting patterns with anti-human P-450IA2 and anti-rat P-450IA2 and with five anti-LM sera, and (b) specific immunoinhibition of microsomal ethoxyresorufin and phenacetin O-deethylation activities (both P-450IA2 supported reactions) by anti-LM antibodies. Finally, purified human P-450IA2 was recognized by these anti-LM sera. The anti-LM antibodies are specific for the disease because none of the other antisera tested behaved in the same manner as anti-LM, even those from patients treated with dihydralazine and without hepatic disease. A possible role of P-450IA2 in the metabolism of dihydralazine was suggested by competitive inhibition of ethoxyresorufin-O-deethylase observed in microsomal incubations. Thus, a new example is presented in which a cytochrome P-450 may be a target for autoantibodies in drug-induced hepatitis.

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates.

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99 degrees C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or TAQ: plus PWO: DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.COLI: exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.

Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.

Severe and prolonged impairment of mitochondrial beta-oxidation leads to microvesicular steatosis, and, in severe forms, to liver failure, coma and death. Impairment of mitochondrial beta-oxidation may be either genetic or acquired, and different causes may add their effects to inhibit beta-oxidation severely and trigger the syndrome. Drugs and some endogenous compounds can sequester coenzyme A and/or inhibit mitochondrial beta-oxidation enzymes (aspirin, valproic acid, tetracyclines, several 2-arylpropionate anti-inflammatory drugs, amineptine and tianeptine); they may inhibit both mitochondrial beta-oxidation and oxidative phosphorylation (endogenous bile acids, amiodarone, perhexiline and diethylaminoethoxyhexestrol), or they may impair mitochondrial DNA transcription (interferon-alpha), or decrease mitochondrial DNA replication (dideoxynucleoside analogues), while other compounds (ethanol, female sex hormones) act through a combination of different mechanisms. Any investigational molecule should be screened for such effects.

Differences in hepatic drug accumulation and enzyme induction after chronic amiodarone feeding of two rat strains: role of the hydroxylator phenotype?

1. It has previously been shown that the extent of hepatic phospholipidosis induced by chronic amiodarone treatment correlates with the degree of drug accumulation in liver tissue. 2. To investigate a possible influence of pharmacogenetic factors, biochemical and morphological investigations were carried out in two rat strains differing in debrisoquine hydroxylation. 3. Plasma and liver tissue concentrations of amiodarone and its main metabolite, desethyl-amiodarone, were significantly higher in rats with deficient hydroxylation. Microsomal enzyme induction, drug cytochrome P-450 complex formation and typical ultrastructural features of phospholipidosis were only seen in rats with deficient hydroxylation and in a more sensitive species, the guinea-pig. 4. It remains to be seen whether deficient debrisoquine hydroxylation in man is associated with an increased susceptibility to amiodarone side effects.

Formation of inactive cytochrome P-450 Fe(II)-metabolite complexes with several erythromycin derivatives but not with josamycin and midecamycin in rats.

The effects of some macrolides (4 mmoles . kg-1 p.o. daily for 4 days in vivo; 0.3 mM in vitro) on hepatic drug-metabolizing enzymes in rats were compared. One group of macrolides including previously studied compounds (oleandomycin, erythromycin and troleandomycin), as well as several other erythromycin derivatives, showed induction of microsomal enzymes and formation of inactive cytochrome P-450-metabolite complexes in vivo; this formation increased in the order: oleandomycin, erythromycin ethylsuccinate, erythromycin stearate, erythromycin itself, erythromycin propionate, erythromycin estolate and troleandomycin. Troleandomycin and, to a lesser extent, erythromycin and oleandomycin formed cytochrome P-450-metabolite complexes when incubated in vitro with 1 mM NADPH and microsomes from rats pretreated with troleandomycin or phenobarbital, but not with microsomes from control rats or rats treated with 3-methylcholanthrene. In contrast, two other macrolides, josamycin and midecamycin, showed no induction of microsomal enzymes and no detectable formation of cytochrome P-450-metabolite complexes in vivo. In vitro, these macrolides failed to form detectable complexes even with microsomes from rats pretreated with troleandomycin or phenobarbital. Hexobarbital sleeping time was unaffected by preadministration of josamycin or midecamycin (4 mmoles . kg-1 p.o.) 2 hr earlier; the in vitro activity of hexobarbital hydroxylase was not inhibited by 0.3 mM josamycin or midecamycin. We conclude that, unlike several erythromycin derivatives, josamycin and midecamycin do not form inactive cytochrome P-450-metabolite complexes in rats.

Metabolic activation of the tricyclic antidepressant amineptine--I. Cytochrome P-450-mediated in vitro covalent binding.

Incubation of [14C]amineptine (1 mM) with hamster liver microsomes resulted in the irreversible binding of an amineptine metabolite to microsomal proteins. Covalent binding measured in the presence of various concentrations of amineptine (0.0625-1 mM) followed Michaelis-Menten kinetics. Pretreatment with phenobarbital increased not only the Vmax, but also the Km, for this binding. Covalent binding required NADPH and molecular oxygen and was decreased when the incubation was made in the presence of inhibitors of cytochrome P-450 such as piperonyl butoxide (4 mM), SKF 525-A (4 mM) or carbon monoxide (80:20 CO-O2 atmosphere). In contrast, binding was increased when microsomes from untreated hamsters were incubated in the presence of 0.5 mM 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrolase. Metabolic activation also occurred in kidney microsomes. In vitro covalent binding to kidney microsomal proteins required NADPH and was decreased by piperonyl butoxide (4 mM) but was not increased by pretreatment with phenobarbital. We conclude that amineptine is activated by hamster liver and kidney microsomes into a chemically reactive metabolite that covalently binds to microsomal proteins.

Metabolic activation of the tricyclic antidepressant amineptine--II. Protective role of glutathione against in vitro and in vivo covalent binding.

Incubation of [11-14C]amineptine (1 mM) with an NADPH-generating system and hamster liver microsomes resulted in the in vitro covalent binding of an amineptine metabolite to microsomal proteins; this binding was decreased by 41-71% in the presence of cysteine, lysine, glycine or glutathione (0.5 mM). An inverse relationship was found between the concentration of glutathione in the incubation mixture (0.25-4 mM) and the extent of covalent binding in vitro, which became undetectable at concentrations of glutathione of 2 mM and higher. Administration of [11-14C]amineptine (300 mg/kg-1 i.p.) to hamsters pretreated with phorone (500 mg/kg i.p.) resulted in the in vivo covalent binding of an amineptine metabolite to hepatic proteins. This binding was increased by phenobarbital-pretreatment and decreased by piperonyl butoxide-pretreatment. After various doses of phorone (150-500 mg/kg), an inverse relationship was found between hepatic glutathione content and in vivo covalent binding. Administration of amineptine alone (300 mg/kg i.p.) depleted hepatic glutathione by 16% only; in these animals, in vivo covalent binding was undetectable from background. Amineptine (300 mg/kg i.p.) did not produce hepatic necrosis, even in hamsters pretreated with phorone and/or phenobarbital. We conclude that physiologic concentrations of glutathione essentially prevent the in vivo covalent binding of an amineptine metabolite to hepatic proteins, and that this binding does not produce liver cell necrosis in hamsters.

Inactivation of human liver cytochrome P-450 by the drug methoxsalen and other psoralen derivatives.

The effects of psoralen derivatives on cytochrome P-450 have been studied in human liver microsomes. CO-binding cytochrome P-450 was decreased by 33% after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM methoxsalen (8-methoxypsoralen). No destruction of cytochrome P-450 was observed when either NADPH or methoxsalen was omitted. A similar (27%) decrease in CO-binding required a 100-times higher concentration of allylisopropylacetamide (2 mM). The activities of 7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase were decreased by about 50% in the presence of 12.5 microM methoxsalen. At this low concentration, neither cimetidine nor SKF 525-A or piperonyl butoxide had any significant inhibitory effect. Monooxygenase activities were also decreased in the presence of 12.5 microM bergapten (5-methoxypsoralen) or 12.5 microM psoralen, but not with 12.5 microM trioxsalen (trimethylpsoralen). CO-binding cytochrome P-450 was not decreased after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM trioxsalen. We conclude that methoxsalen is an extremely potent suicide inhibitor of cytochrome P-450 in human liver microsomes. Bergapten and psoralen are also inhibitory whereas trioxsalen has little effects. In the latter derivative, a methyl group is attached on the furan ring and may hinder its metabolic activation and the inactivation of cytochrome P-450.

Effects of methoxsalen on the metabolism of acetaminophen in humans.

We reported recently that the drug methoxsalen, a potent suicide inhibitor of hepatic cytochrome P-450, decreases the metabolic activation of acetaminophen and prevents its hepatotoxicity in mice. We have now studied the effects of methoxsalen on the metabolism of acetaminophen in humans. In vitro, 100 microM methoxsalen decreased by 40% the covalent binding of a [3H]acetaminophen metabolite to microsomal proteins after incubation of [3H]acetaminophen with human liver microsomes and an NADPH-generating system. In vivo, a single oral dose of methoxsalen (30 mg), given 3 hr before acetaminophen (1 g), decreased by 38% the partial apparent oral salivary clearance of acetaminophen into glutathione-derived conjugates (the end products of its oxidative metabolism) in nine human volunteers. These observations demonstrate that methoxsalen decreases the metabolic activation of acetaminophen in humans.

Tianeptine, a new tricyclic antidepressant metabolized by beta-oxidation of its heptanoic side chain, inhibits the mitochondrial oxidation of medium and short chain fatty acids in mice.

Tianeptine is a new tricyclic antidepressant which is metabolized mainly by beta-oxidation of its heptanoic side chain. We determined the effects of tianeptine on the mitochondrial oxidation of natural fatty acids in mice. In vitro, tianeptine (0.5 mM) inhibited by only 32% the formation of beta-oxidation products from [1-14C]palmitic acid by hepatic mitochondria, but inhibited by 71% that from [1-14C]octanoic acid and by 51% that from [1-14C]butyric acid. The activity of the tricarboxylic acid cycle, assessed as the in vitro formation of [14C]CO2 from [1-14C]acetylcoenzyme A was decreased by 51% in the presence of tianeptine (0.5 mM). The inhibition of both beta-oxidation and the tricarboxylic acid cycle appeared reversible in mitochondria from mice exposed to tianeptine in vivo but incubated in vitro without tianeptine. In vivo, administration of tianeptine (0.0625 mmol/kg i.p.), decreased by 53 and 58%, respectively, the formation of [14C]CO2 from [1-14C]octanoic acid and [1-14C]butyric acid, but did not significantly decrease that from [1-14C]palmitic acid. After administration of high doses of tianeptine, however, formation of [14C]CO2 from [1-14C]palmitic acid became inhibited as well, transiently after 0.25 mmol/kg and durably (greater than 24 hr) after 0.75 mmol/kg i.p. Hepatic triglycerides were increased 24 hr after administration of 0.75 mmol/kg i.p. of tianeptine, but not after 0.25 mmol/kg i.p. Microvesicular steatosis of the liver was observed in some mice after 0.75 mmol/kg i.p., but not after 0.5 mmol/kg i.p. We conclude that tianeptine inhibits the oxidation of medium- and short-chain fatty acids in mice. Microvesicular steatosis, however, requires very large doses in mice (0.75 mmol/kg i.p., i.e. 600-times the oral dose in humans), and is therefore unlikely to occur in humans.

Inhibitory effects of nilutamide, a new androgen receptor antagonist, on mouse and human liver cytochrome P-450.

The effects of nilutamide were studied first with human liver microsomes. At concentrations expected in the human liver (110 microM), nilutamide inhibited hexobarbital hydroxylase, benzphetamine N-demethylase, benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities by 85, 40, 35 and 25%, respectively. There was no in vitro inhibition of NADPH-cytochrome c reductase activity, no in vitro loss of CO-binding cytochrome P-450, and no spectral evidence for the in vitro formation of a possible cytochrome P-450Fe(II)-nitroso metabolite complex. Other studies were performed with mouse liver microsomes. Nilutamide (550 microM) did not significantly increase the consumption of NADPH by aerobic microsomes, and did not modify the kinetics for the reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase in an anaerobic system. Nilutamide (22 microM) produced either a type I or a type II binding spectrum. Kinetics for the inhibition of hexobarbital hydroxylase were consistent with competitive inhibition. A last series of experiments was performed after administration of nilutamide in mice. Thirty minutes after administration of doses (15 or 30 mumol.kg-1 i.p.) similar to those used in humans, the hexobarbital sleeping time was increased by 40 and 60%, respectively. There was no evidence, however, for the irreversible inactivation of microsomal enzymes since CO-binding cytochrome P-450 and monooxygenase activities remained unchanged in liver microsomes from mice killed 1 or 6 hr after administration of nilutamide (30 mumol.kg-1 i.p.). These results show that nilutamide inhibits hepatic cytochrome P-450 activity, and suggest that inhibition may actually occur after therapeutic doses of nilutamide in humans.

Protective effect of 16,16-dimethyl prostaglandin E2 on the hepatotoxicity of bromobenzene in mice.

It has been suggested that 16,16-dimethyl prostaglandin E2 may have a cytoprotective effect in the liver. To assess this hypothesis, we determined the effects of this prostaglandin on the metabolism and toxicity of bromobenzene in mice. Administration of 16,16-dimethyl prostaglandin E2 (50 micrograms/kg s.c., 30 min before, and every 6 hr after, the administration of bromobenzene) did not modify the disappearance curves of unchanged bromobenzene from plasma and liver, and did not modify the amount of bromobenzene metabolites covalently bound to hepatic proteins 1-24 hr after the administration of a toxic dose of bromobenzene (0.36 ml/kg i.p.). The prostaglandin, however, markedly reduced serum alanine aminotransferase activity, the extent of liver cell necrosis, the depletion of glutathione, and the disappearance of cytochrome P-450 after administration of this toxic dose of bromobenzene (0.36 ml/kg i.p.). It also markedly reduced mortality after administration of a lethal dose of bromobenzene (0.43 ml/kg i.p.). We conclude that 16,16-dimethyl prostaglandin E2 can prevent hepatic necrosis without decreasing the covalent binding of bromobenzene metabolites to hepatic proteins. The mechanism for this dissociation between covalent binding and toxicity remains unknown.

Metabolic activation of the new tricyclic antidepressant tianeptine by human liver cytochrome P450.

Incubation of [14C]tianeptine (0.5 mM) with human liver microsomes and a NADPH-generating system resulted in the in vitro covalent binding of a tianeptine metabolite to microsomal proteins. This covalent binding required oxygen and NADPH. It was decreased by piperonyl butoxide (4 mM) by 81%, and SKF 525-A (4 mM) by 87%, two relatively non-specific inhibitors of cytochrome P450, and by glutathione (4 mM) by 70%, a nucleophile. Covalent binding was decreased by 54% in the presence of troleandomycin (0.1 mM), a specific inhibitor of the glucocorticoid-inducible cytochrome P450 IIIA3, but remained unchanged in the presence of quinidine (0.1 mM) or dextromethorphan (0.1 mM), two inhibitors of cytochrome P450 IID6. Preincubation with IgG antibodies directed against cytochrome P450 IIIA3 decreased covalent binding by 65% whereas either preimmune IgG or IgG antibodies directed against P450 IA1, an isoenzyme inducible by polycyclic aromatic compounds, exhibited no significant inhibitory effect. We conclude that tianeptine is activated by human liver cytochrome P450 into a reactive metabolite. This activation is mediated in part by glucocorticoid-inducible isoenzymes but not by P450 IID6 (the isoenzyme which oxidizes debrisoquine) nor by P450 IA1 (an isoenzyme inducible by polycyclic aromatic compounds). The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitor of metabolic activation and as a chain-breaking antioxidant.

Administration of silymarin (800 mg/kg i.p.) 30 min before carbon tetrachloride (18 microL/kg i.p.) did not modify total hepatic levels of CCl4 and metabolites in mice, but decreased by 40% the in vivo covalent binding of CCl4 metabolites to hepatic lipids at 2 hr. This pretreatment decreased by 60% the exhalation of ethane during the first hour after CCl4, and decreased by 50% the incidence of liver cell necrosis. In vitro, silymarin (800 micrograms/mL) decreased by 50 to 70% various monooxygenase activities, and decreased by 20% the covalent binding of CCl4 metabolites to microsomal proteins. Silymarin (800 micrograms/mL) decreased by 70% in vitro lipid peroxidation mediated by CCl4 metabolites, and decreased by 90% peroxidation mediated by NADPH alone. Silibinin, one of the three isomers composing silymarin, also decreased carbon tetrachloride-induced lipid peroxidation; this effect, however, was less than that of silymarin in vitro, and was more transient in vivo. Pretreatment with silibinin (800 mg/kg i.p.) 30 min before CCl4 (18 microL/kg i.p.) did not improve SGPT activity or liver histology at 24 hr. We conclude that silymarin prevents carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice, firstly, by decreasing the metabolic activation of CCl4, and, secondly, by acting as a chain-breaking antioxidant.

Formation of an inactive cytochrome P-450Fe(II)-metabolite complex after administration of amiodarone in rats, mice and hamsters.

Administration of amiodarone hydrochloride (50-150 mg/kg i.p. daily) to rats, mice or hamsters resulted in the in vivo formation of a cytochrome P-450Fe(II)-amiodarone metabolite complex absorbing at 453 nm, unable to bind CO and biologically inactive. In rats, the amount of complex present in hepatic microsomes was small 24 hr after administration of a single dose of amiodarone (100 mg/kg i.p.) but was increased 2.5-times by pretreatment with phenobarbital and 8-times by pretreatment with dexamethasone phosphate. In addition, the complex increased linearly with time as the doses of amiodarone were repeated daily. When both enhancing factors were combined (treatment for 3 days with both dexamethasone and amiodarone), the amount of complex present in liver microsomes reached 0.78 nmol/mg protein or 40% of total cytochrome P-450 in rats. In these rats, in vitro disruption of the complex with potassium ferricyanide suppressed its Soret peak at 453 nm, increased by 70% the CO-binding spectrum of dithionite-reduced microsomes, and restored several monooxygenase activities. The 453 nm-absorbing complex was also formed in vitro upon incubation of amiodarone or N-desethylamiodarone with NADPH, EDTA and microsomes from dexamethasone-treated rats. The formation of the complex was smaller with microsomes from phenobarbital-treated rats and was not detected with microsomes from control rats. We conclude that amiodarone forms an inactive cytochrome P-450Fe(II)-metabolite complex in rats, mice and hamsters.

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.

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.

Formation of an inactive cytochrome P-450 Fe(II)-metabolite complex after administration of troleandomycin in humans.

In rats, it has been shown that troleandomycin induces its own transformation into a metabolite forming an inactive complex with reduced cytochrome P-450. To determine whether similar effects occur in humans, we studied hepatic microsomes from 6 untreated patients and 6 patients treated with troleandomycin, 2 g per os daily for 7 days. In the treated patients, NADPH-cytochrome c reductase activity was increased by 48%; total cytochrome P-450 concentration was also increased, but 33% of total cytochrome P-450 was complexed by a troleandomycin metabolite. The cytochrome P-450 Fe(II)-metabolite complex exhibited properties identical to those of the inactive complex formed in rats: it exhibited a Soret peak at 456 nm, was unable to bind CO, and was destroyed by addition of 50 microM potassium ferricyanide. We also measured the clearance of antipyrine in 6 other subjects. This clearance was decreased by 45% when measured again on te seventh day of the troleandomycin treatment. We conclude that repeated administration of troleandomycin induces microsomal enzymes, produces an inactive cytochrome P-450 Fe(II)-metabolite complex, and decreases the clearance of antipyrine in humans.

Effects of erythromycin on hepatic drug-metabolizing enzymes in humans.

In rats, erythromycin has been shown to induce microsomal enzymes and to promote its own transformation into a metabolite which forms an inactive complex with reduced cytochrome P-450. To determine whether similar effects also occur in humans, we studied hepatic microsomal enzymes from six untreated patients and six patients treated with erythromycin propionate, 2 g per os daily for 7 days. In the treated patients, NADPH-cytochrome c reductase activity was increased; the total cytochrome P-450 concn was also increased but part of the total cytochrome P-450 was complexed by an erythromycin metabolite. The concn of uncomplexed (active) cytochrome P-450 was not significantly modified and the activity of hexobarbital hydroxylase remained unchanged. We also measured the clearance of antipyrine in six other patients; this clearance was not significantly decreased when measured again on the seventh day of the erythromycin propionate treatment. We conclude that the administration of erythromycin propionate induces microsomal enzymes and results in the formation of an inactive cytochrome P-450-metabolite complex in humans. However, the concn of uncomplexed (active) cytochrome P-450 and tests for in vitro and in vivo drug metabolism were not significantly modified.