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.

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.

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.

Metabolic activation of the antidepressant tianeptine. II. In vivo covalent binding and toxicological studies at sublethal doses.

Administration of [14C]tianeptine (0.5 mmol/kg i.p.) to non-pretreated hamsters resulted in the in vivo covalent binding of [14C]tianeptine metabolites to liver, lung and kidney proteins; this very high dose (360-fold the human therapeutic dose) depleted hepatic glutathione by 60%, and increased SGPT activity 5-fold. Lower doses (0.25 and 0.125 mmol/kg) depleted hepatic glutathione to a lesser extent and did not increase SGPT activity. Pretreatment of hamsters with piperonyl butoxide decreased in vivo covalent binding to liver proteins, and prevented the increase in SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). In contrast, pretreatment of hamsters with dexamethasone increased in vivo covalent binding to liver proteins, and increased SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). Nevertheless, liver cell necrosis was histologically absent 24 hr after the administration of tianeptine (0.5 mmol/kg i.p.) to non-pretreated or dexamethasone-pretreated hamsters. In vivo covalent binding to liver proteins also occurred in mice and rats, being increased by 100% in dexamethasone-pretreated animals. In vivo covalent binding to liver proteins was similar in untreated female Dark Agouti rats and in female Sprague-Dawley rats. These results show that tianeptine is transformed in vivo by cytochrome P-450, including glucocorticoid-inducible isoenzymes, into chemically reactive metabolites that covalently bind to tissue proteins. The metabolites, however, exhibit no direct hepatotoxic potential in hamsters below the sublethal dose of 0.5 mmol/kg i.p. The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

The drug methoxsalen, a suicide substrate for cytochrome P-450, decreases the metabolic activation, and prevents the hepatotoxicity, of carbon tetrachloride in mice.

Methoxsalen, a potent suicide inhibitor of cytochrome P-450 that can be used in humans, might be of value for the prevention of hepatitis in subjects with carbon tetrachloride poisoning. As a preliminary step, we have determined its effects on the hepatotoxicity of carbon tetrachloride in mice. Several monooxygenase activities, the in vitro covalent binding of carbon tetrachloride metabolites to microsomal proteins, and in vitro microsomal lipid peroxidation initiated by carbon tetrachloride metabolites were decreased by 60-90% in microsomes from mice killed 2 hr after the administration of methoxsalen (250 mumol X kg-1); microsomal lipid peroxidation mediated by endogenous iron and NADPH was not modified. Administration of methoxsalen (250 mumol X kg-1) 30 min before carbon tetrachloride (0.1 ml X kg-1) decreased both the in vivo formation of conjugated dienes in microsomal lipids and the in vivo covalent binding of carbon tetrachloride metabolites to lipids and proteins. This pretreatment completely prevented the hepatotoxicity of carbon tetrachloride. Other cytochrome P-450 inhibitors (cimetidine, SKF 525-A or piperonyl butoxide) given at this low molar dose (250 mumol X kg-1) exerted no protective effect. Methoxsalen (500 mumol X kg-1) was also effective, but only partially, when given 30 min after carbon tetrachloride (0.025 ml X kg-1). We conclude that pretreatment with methoxsalen decreases the metabolic activation of carbon tetrachloride, and completely prevents its hepatotoxicity in mice. Post-treatment with methoxsalen must be given early and is only partially effective in mice.

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

Incubation under air of [14C]tianeptine (0.5 mM) with a NADPH-generating system and hamster, mouse or rat liver microsomes resulted in the in vitro covalent binding of [14C]tianeptine metabolites to microsomal proteins. Covalent binding to hamster liver microsomes required NADPH and oxygen; it was decreased in the presence of the cytochrome P-450 inhibitors, carbon monoxide, piperonyl butoxide (4 mM), and SKF 525-A (4 mM) or in the presence of the nucleophile, glutathione (1 or 4 mM). In vitro covalent binding to hamster liver microsomes was not decreased in the presence of quinidine (1 microM), and was similar with microsomes from either female Dark Agouti, or female Sprague-Dawley rats. In contrast, in vitro covalent binding to hamster liver microsomes was decreased in the presence of troleandomycin (0.25 mM), while covalent binding was increased with microsomes from either hamsters, mice or rats pretreated with dexamethasone. Preincubation with IgG antibodies directed against rabbit liver glucocorticoid-inducible cytochrome P-450 3c(P-450 IIIA4) decreased in vitro covalent binding by 53 and 89%, respectively, with microsomes from control hamsters and dexamethasone-pretreated hamsters, and by 60 and 81%, respectively, with microsomes from control and dexamethasone-pretreated rats. We conclude that tianeptine is activated by hamster, mouse and rat liver cytochrome P-450 into a reactive metabolite. Metabolic activation is mediated in part by glucocorticoid-inducible isoenzymes but not by the isoenzyme metabolizing debrisoquine. In vivo studies are reported in the accompanying paper.

Decrease in hepatic cytochrome P450 after interleukin-2 immunotherapy.

Interleukin-2 (IL-2) has been shown to decrease cytochrome P450 (CYP) mRNAs and proteins in cultured rat hepatocytes, and IL-2 administration decreases CYPs in rats. Although high doses of IL-2 are administered to cancer patients, the effect on human CYPs has not yet been determined. Patients with hepatic metastases from colon or rectum carcinomas were randomly allocated to various daily doses of human recombinant IL-2 (from 0 to 12.10(6) units/m(2)). IL-2 was infused from day 7 to day 3 before hepatectomy and the conservation of a non-tumorous liver fragment in liquid nitrogen. Hepatic CYPs and monooxygenase activities were not significantly decreased in 5 patients receiving daily doses of 3 or 6 10(6) IL-2 units/m2, compared to 7 patients who did not receive IL-2. In contrast, in 6 patients receiving daily doses of 9 or 12 x 10(6) IL-2 units/m2, the mean values for immunoreactive CYP1A2, CYP2C, CYP2E1, and CYP3A4 were 37, 45, 60 and 39%, respectively, of those in controls; total CYP was significantly decreased by 34%, methoxyresorufin O-demethylation by 62%, and erythromycin N-demethylation by 50%. These observations suggest that high doses of IL-2 may decrease total CYP and monooxygenase activities in man.

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.

Polymorphism of dextromethorphan oxidation in a French population.

Genetically-controlled drug oxidation capacity was studied using dextromethorphan, an anti-tussive drug, as the test compound in 103 healthy white French subjects (61 males and 42 females). Phenotyping was performed using the metabolic ratio (MR) calculated as MR = 0-10 h urinary output of dextromethorphan/0-10 h urinary output of dextrorphan, after oral administration of 40 mg (113.6 mumol) of dextromethorphan hydrobromide. The log MR was bimodally distributed: 99 subjects (96.1%) were phenotyped as extensive metabolizers; they had a log MR between -3.1 and -1.1, a urinary output of dextromethorphan below 5 mumol 10 h-1 and a urinary output of dextrorphan above 20 mumol 10 h-1. Four subjects (3.9%) were phenotyped as poor metabolizers; they had a log MR between -0.5 and +0.7, a urinary output of dextromethorphan above 5 mumol 10 h-1 and a urinary out of dextrorphan below 20 mumol 10 h-1.

Effects of clarithromycin on cytochrome P-450. Comparison with other macrolides.

Repeated administration of clarithromycin (0.5 mmol.kg-1 p.o. daily for 5 days) to rats increased markedly the same cytochrome P-450 isoenzyme (P-450p) as that induced by troleandomycin. Clarithromycin, however, did not form cytochrome P-450 Fe(II)-metabolite complexes in vitro with microsomes from clarithromycin-treated rats or in vivo after repeated doses of clarithromycin. Nevertheless, clarithromycin formed cytochrome P-450 Fe(II)-metabolite complexes with microsomes from dexamethasone-treated rats in vitro, or after administration to dexamethasone-treated rats in vivo. Similar effects were observed with roxithromycin. In contrast, erythromycin and troleandomycin formed metabolic complexes when given alone, whereas josamycin, midecamycin and spiramycin did not form complexes, even in dexamethasone-treated rats. We conclude that clarithromycin and roxithromycin induce cytochrome P-450p, but do not form complexes with this isoenzyme, although they do form complexes with other glucocorticoid-inducible isoenzymes. We propose that macrolides may be classified into three groups, those forming complexes when given alone (e.g., erythromycin and troleandomycin), those forming complexes only in glucocorticoid-pretreated rats (clarithromycin and roxithromycin) and those not forming complexes (josamycin, midecamycin and spiramycin).

Mechanism for isaxonine hepatitis. I. Metabolic activation by mouse and human cytochrome P-450.

The metabolism of isaxonine was first investigated in mice. Incubation, under air, of [2-14C] isaxonine (1 mM) with mouse liver microsomes and an NADPH-generating system resulted in the irreversible binding of a [14C] isaxonine metabolite to microsomal proteins; binding required active microsomes, NADPH and oxygen, it was inhibited by 4 mM piperonyl butoxide or by a CO-O2 (80:20) atmosphere. In the presence of various concentrations of isaxonine (0.125-2 mM), binding followed Michaelis-Menten kinetics; the Vmax was increased by both phenobarbital and 3-methylcholanthrene pretreatments. In vivo, 2.5 hr after the administration of [2-14C] isaxonine (4 mmol X kg-1 i.p.), a [14C] isaxonine material was irreversibly bound to mouse liver proteins; this binding was decreased by piperonyl butoxide and increased by phenobarbital or 3-methylcholanthrene pretreatments. Irreversible binding also occurred in the kidney. Unlike their effects in the liver, piperonyl butoxide and phenobarbital did not modify significantly in vitro metabolic activation by kidney microsomes and in vivo covalent binding to kidney proteins; pretreatment with 3-methylcholanthrene increased both in vitro and in vivo binding in the kidney. In a second series of experiments, in vitro metabolic activation was demonstrated with human liver microsomes; as in mice, covalent binding required NADPH and was markedly inhibited by piperonyl butoxide. We conclude that isaxonine is activated by mouse and human cytochromes P-450 into a reactive metabolite. In vivo covalent binding to mouse liver and kidney proteins appears to result mainly from the in situ binding of the metabolite formed in each organ.

Mechanism for isaxonine hepatitis. II. Protective role of glutathione and toxicological studies in mice.

Coincubation of 0.4 mM [3H]glutathione with 4 mM isaxonine , an NADPH-generating system, glutathione S-transferase and mouse liver microsomes, followed by thin-layer chromatography of the incubation mixture, resulted in the appearance of a 3H-labeled peak with characteristics consistent with a glutathione- isaxonine metabolite adduct: this peak was absent if either isaxonine or the NADPH-generating system was omitted and was decreased if the transferase was omitted. In vivo, the concentrations of hepatic glutathione and glutathione disulfide were markedly decreased 2.5 hr after administration of isaxonine (4 mmol X kg-1 i.p.); this depletion of glutathione was prevented essentially by pretreatment with piperonyl butoxide. In vitro, addition of 4 mM glutathione decreased markedly the amount of [14C] isaxonine metabolite that bound to microsomal proteins during incubation of 1 mM [2-14C] isaxonine with hepatic microsomes and an NADPH-generating system. In vivo, pretreatment with diethylmaleate decreased further hepatic glutathione concentration and markedly increased the amount of [14C] isaxonine metabolite covalently bound to hepatic proteins, 2.5 hr after administration of [2-14C] isaxonine (4 mmol X kg-1 i.p.). Administration of isaxonine (4 mmol X kg-1 i.p.) decreased hepatic cytochrome P-450 concentration, but failed to produce liver cell necrosis, even in mice pretreated with phenobarbital, 3-methylcholanthrene or diethylmaleate, despite high levels of in vivo covalent binding in pretreated animals. We conclude that the reactive metabolite of isaxonine may be conjugated with glutathione or may covalently bind to hepatic proteins. The metabolite, however, has limited hepatotoxic potential in mice.

Inhibition of rat liver estrogen 2/4-hydroxylase activity by troleandomycin: comparison with erythromycin and roxithromycin.

Administration of troleandomycin (0.5 mmol.kg-1 p.o. daily for 5 days) decreased by 61% and 36%, respectively, the estradiol and ethinylestradiol 2/4-hydroxylase activities of hepatic microsomes from male Sprague-Dawley rats killed 2 hr after the last dose. This decrease did not appear to be due to the in vivo formation of the inactive cytochrome P-450 p Fe(II)-metabolite complex, since disruption of this complex with potassium ferricyanide did not increase estrogen hydroxylase activities. Troleandomycin administration, however, essentially suppressed cytochrome P-450 UT-A (one of the P-450 forms involved in the hydroxylation of estrogens) and resulted in the appearance of cytochrome P-450 forms whose estradiol hydroxylase activity was inhibitable by troleandomycin in vitro. Similarly, troleandomycin (2 mM) inhibited by 60% estradiol and ethinylestradiol 2/4-hydroxylase activities in microsomes from dexamethasone-treated rats, although it had no inhibitory effect in microsomes from control rats. In contrast, erythromycin and roxithromycin (2 mM) exerted no inhibitory effect, even in microsomes from dexamethasone-treated rats. In vivo, these macrolides (0.5 mmol.kg-1 p.o. daily for 5 days) decreased moderately cytochrome P-450 UT-A levels and estradiol 2/4-hydroxylase activity, and did not modify ethinylestradiol 2/4-hydroxylase activity. We conclude that the administration of troleandomycin, but not that of erythromycin or roxithromycin, decreases ethinylestradiol 2/4-hydroxylase activity in male rat liver microsomes, as a possible consequence of decreased cytochrome P-450 UT-A levels and of the induction of glucocorticoid-responsive P-450 forms whose ethinylestradiol hydroxylase activity is inhibitable by troleandomycin.

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.