Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6 beta-, 16 beta-, and 16 alpha-hydroxylase activities at concentrations between 10(-7) and 10(-5) M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a "type II" spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5 alpha-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

Metabolism of dichlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of ten individual dichlorobiphenyls (DCBs) has been investigated as part of a major study of the role of metabolism in the toxicity of polychlorinated biphenyl (PCB) pollutant mixtures. The DCBs were metabolized to monohydroxy and dihydrodiol metabolites and unstable metabolites of intermediate polarity. DCBs with both chloro substituents on the same ring, one or both of which were ortho substituents, were susceptible to the same regioselectivities for hydroxylation by control, phénobarbital (PB)- or β-naphthoflavone (BNF)-induced cytochromes P-450 (principally in the 4-position), with the greatest rates of hydroxylation arising with PB-induced cytochrome P-450. In contrast, DCBs with no ortho chlorosubstituents had regioselectivities for hydroxylation by control and PB-induced cytochrome P-450 which differed from that of BNF-induced cytochromes P-450; the greatest rates of hydroxylation were with BNF-induced systems. DCBs with one chloro substituent on each ring were metabolized, with the site of hydroxylation being under the electronic influence of the chloro substituent. With 4,4'-DCB, 60 per cent of the hydroxylated DCB metabolite underwent an NIH shift [G. Guroff, J. W. Daly, D. M. Jerina, J. Renson, B. Witkop and S. Udenfriend, Science157, 1524 (1967)]. The BNF-induced system produced the highest rates of dihydrodiol fomation that were eliminated by an epoxide hydratase inhibitor. The results indirectly prove that arene oxides are intermediates in DCB metabolism and are possibly the source of DCB mutagenicity. The PCBs 2,4,2'4'- and 3,4,3',4'-tetrachlorobiphenyl induced the same effects as PB and BNF respectively. Thus, PCBs differentially affect the metabolism of their individual components and are, possibly, responsible for enhancing their own toxicity by inducing enhanced rates of formation of arene oxide intermediates.     

Metabolism of monochlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of the monochlorobiphenyls (MCBs) 2-, 3- and 4-chlorobiphenyl, has been investigated as a model for the metabolism of polychlorinated biphenyl pollutants. MCB metabolism was catalyzed by cytochrome P-450, as indicated by a dependence on NADPH and O₂, inhibition by 2-diethylaminoethyl-2,2-diphenylpropylacetate (SKF 525-A), metyrapone and CO, and the formation of type I difference spectra, on the addition of MCBs to microsomes. All MCBs yielded a 4'-monohydroxy MCB as the major metabolite, as determined by mass and nuclear magnetic resonance spectroscopy, dechlorination to 4-hydroxybiphenyl, and high-pressure liquid chromatography retention times. Minor monohydroxy and dihydroxy metabolites were also produced from the MCBs. The regioselectivity of control cytochrome P-450 for metabolism of MCBs at the 4' position was not altered by preinduction of cytochrome P-450 with 2,4,2',4'-tetrachlorobiphenyl (TCB) or cytochrome P-448 with 3,4,3', 4'-TCB. 2-Chlorobiphenyl was metabolized only by control and induced cytochrome P-450; 3- and 4-chlorobiphenyl were metabolized by control and by induced cytochrome P-450 and P-448. Thus, the regioselectivity of metabolism of MCBs is independent of the chlorine position or the form of the induced cytochrome involved, but the extent of metabolism of polychlorinated biphenyls (PCBs) is determined by induction of the hepatic cytochromes P-450.     

Inhibition of rat testicular heme synthesis and depression of microsomal cytochrome P-450 by estradiol benzoate

Twenty-four hours after a single dose (50 μg, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of δ-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [¹³C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 μg, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.     

Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions. Studies with manganese-substituted cytochrome b5

The effects of cytochrome b5 with manganese-protoporphyrin IX substituted for heme were compared with those of native cytochrome b5 and the apoenzyme on the oxygenation of substrates in the reconstituted system containing liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and phosphatidylcholine. Mn-b5, unlike b5, remains essentially fully oxidized in the presence of NADPH and NADPH-cytochrome P-450 reductase under aerobic conditions. The effects of various concentrations of b5 and its derivatives were determined at constant P-450 and reductase concentrations. Cytochrome b5 inhibits benzphetamine demethylation by isozyme 2, the effect increasing up to the highest concentrations tested, and stimulates 7-ethoxycoumarin deethylation by isozyme 2 and acetanilide p-hydroxylation by isozyme 4, the optimal b5:P-450 molar ratio being about 2. In contrast, Mn-b5 inhibits all three reactions and apo-b5 is either inactive or slightly inhibitory. The activities of the three substrates as well as testosterone were determined with P-450 isozymes 2, 3b, 3c, and 4 in the reconstituted system with no additions or with b5 or Mn-b5 present. Cytochrome b5 is stimulatory, inhibitory, or without any effect, the result depending on both the substrate and P-450 isozyme present, whereas Mn-b5 is inhibitory in most instances. Both b5 and its manganese derivative alter the rates of testosterone 6 beta- or 16 alpha-hydroxylation by most of the P-450 cytochromes. The activities are influenced by the molar ratio of reductase to P-450. The Km values of benzphetamine, ethoxycoumarin, and acetanilide are, with one exception, significantly decreased in the presence of b5 or Mn-b5. We conclude that some of the effects of b5 on the oxygenase system are not accounted for by its role as an electron donor to cytochrome P-450.     

Comparative studies of N-hydroxylation and N-demethylation by microsomal cytochrome P-450

The N-hydroxylation of representative aromatic amines by rabbit liver microsomes was mediated by cytochrome P-450 as demonstrated by the sensitivity to carbon monoxide and other cytochrome P-450 inhibitors. The rate of N-hydroxylation was increased by induction with phenobarbital. Involvement of isozyme LM2 (P-50IIB1) was demonstrated in reconstituted systems. Aromatic N-hydroxylation was substantially faster and more efficient than aliphatic N-hydroxylation, while N-demethylation of aromatic and aliphatic dimethylamines was comparable in rate and efficiency. Aliphatic N-hydroxylation showed no rate increase with increasing pH despite the predicted increase in the concentration of the neutral substrate. The relative rates of N-hydroxylation and N-demethylation were compared for a series of para-substituted aromatic amines. The rate of demethylation of para-substituted N,N-dimethylanilines, as measured both by product formation and by NADPH consumption, correlated with the electronic parameter sigma and with the Hansch lipophilicity parameter pi. N-Hydroxylation of a similar series of anilines did not show a dependence on the electronic parameter but was dependent on the lipophilicity parameter. The differing dependence on the electronic parameter suggests that there are different rate-determining processes of N-oxidation for these two reactions.     

Tetrachloroethylene metabolism by the hepatic microsomal cytochrome P-450 system

The interaction of tetrachloroethylene with hepatic microsomal cytochromes P-450 has been investigated using male Long-Evans rats. The spectral binding of tetrachloroethylene to cytochromes P-450 in hepatic microsomes from uninduced rats was characterized by a K_s of 0.4 mM. The K_s was not affected by phenobarbital induction, but was increased following pregnenolone-16α-carbonitrile induction. The K_M of 1.1 mM, calculated for the conversion of tetrachloroethylene to total chlorinated metabolites by the hepatic microsomal cytochrome P-450 system, was decreased by phenobarbital induction and increased by pregnenolone-16α-carbonitrile induction. The maximum extents of binding (ΔA_max) and metabolism (V_max) of tetrachloroethylene were increased by both phenobarbital and pregnenolone-16α-carbonitrile induction. Induction with β-naphthoflavone was without effect on any of the above parameters. The effects of the inducing agents on tetrachloroethylene-stimulated CO-inhibitable hepatic microsomal NADPH oxidation followed the same trend as their effects on V_max for the metabolism of tetrachloroethylene, although in all cases the extent of NADPH oxidation was 5- to 25-fold greater than the extent of metabolite production. The inhibitors of cytochromes P-450, viz. metyrapone, SKF 525-A, and CO, inhibited the hepatic microsomal binding and metabolism of tetrachloroethylene. Free trichloroacetic acid was found to be the major metabolite of tetrachloroethylene from the hepatic microsomal cytochrome P-450 system. Neither 2.2,2-trichloroethanol nor chloral hydrate was produced in measurable amounts from tetrachloroethylene. A minor but significant metabolite of tetrachloroethylene by cytochrome P-450 was the trichloroacetyl moiety covalently bound to components of the hepatic microsomes. Incubation of tetrachloroethylene. an NADPH-generating system. EDTA and hepatic microsomes was without effect on the levels of microsomal cytochromes P-450, cytochrome b₅, beme, and NADPH-cytochrome c reductase. It is concluded that hepatic microsomal cytochromes P-450 bind and metabolize tetrachloroethylene. The major product of this interaction is trichloroacetic acid, which is also the major urinary metabolite of tetrachloroethylene in vivo. The forms of cytochrome P-450 that bind and metabolize tetrachloroethylene include those induced by pregnenolone-16α-carbonitrile and by phenobarbital. Cytochrome P-448. which was induced in rat liver by β-naphthoflavone, does not appear to spectrally bind or metabolize tetrachloroethylene. The metabolism and toxicity of tetrachloroethylene are considered in relation to other chlorinated ethylenes.     

Purification and characterization of microsomal cytochrome P-450s

1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature.

2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents.

3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.     

Purification and characterization of microsomal cytochrome P-450s

1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature. 2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents. 3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.     

Inhibition of in vitro microsomal lipid peroxidation by isoflavonoids

In a comparative study the inhibition of microsomal lipid peroxidation induced by an Fe2+-ADP-complex and NADPH by naturally occurring isoflavones and their reduced derivatives (isoflavanones and isoflavans) has been examined. It is found that the isoflavanones are more active than the parent isoflavones and the isoflavans are by far the most potent inhibitors. In our in vitro test system 6,7,4'-trihydroxy- and 6,7-dihydroxy-4'-methoxyisoflavans (IC50 values 1.3 X 10(-6) and 1.1 X 10(-6) mol/l respectively) surpass the inhibitory effect of alpha-tocopherol, (+)-cyanidanol-3 and BHA (butylated hydroxyanisole). In order to establish a structure-activity relationship, a few more isoflavans have been included in the investigation.     

Source of the oxygen atom in the product of cytochrome P-450-catalyzed N-demethylation reactions

The source of the oxygen atom in the product of the cytochrome P-450-catalyzed N-demethylation of N-methylcarbazole was determined by mass spectral analysis of the carbinolamine precursor of formaldehyde formed during incubation in oxygen 18-enriched medium. Initial experiments demonstrated that N-(hydroxymethyl)carbazole, the carbinolamine product of the metabolism of N-methylcarbazole, did not exchange oxygen with solvent water. When N-methylcarbazole was incubated in oxygen 18-enriched medium with purified cytochrome P-450 in the presence of either purified NADPH-cytochrome P-450 reductase and NADPH, cumene hydroperoxide, t-butyl hydroperoxide, or peracetic acid, there was no incorporation of oxygen 18 from the medium into N-(hydroxymethyl)carbazole. These results clearly demonstrate that the oxygen atom inserted into N-methylcarbazole by cytochrome P-450 to yield N-(hydroxymethyl)carbazole does not come from the medium and show that the N-demethylation reactions catalyzed by cytochrome P-450 proceed in a manner similar to hydroxylation reactions, with the oxygen atom in the product being derived from the oxidant.     

Induction of hepatic microsomal cytochrome P-450 by mirex and kepone.

The chlorinated insecticides, mirex and Kepone, pose a threat to human health as a consequence of their pollution of the environment. We investigated their potential to affect synergistically the toxicity of other xenobiotics and the pharmacological function of drugs by induction of hepatic microsomal enzymes. Male rats were induced by ip injection of mirex (50 or 5 mg/kg/day for 5 days) or Kepone (10 or 1 mg/kg/day for 5 days). Metabolic activity was tested with warfarin and biphenyl using high-performance liquid chromatographic assays. The high doses of both compounds induced cytochrome P-450 with absorbance bands (reduced, CO complex) at 449 nm. Cytochrome concentrations were enhanced twofold relative to controls. Mirex resembled 3-methylcholanthrene and benzo[a]pyrene by inducing formation of 6-hydroxywarfarin but differed in not inducing 8-hydroxywarfarin. Kepone resembled phenobarbital in inducing 7-hydroxywarfarin but differed in its effects on the other metabolites. The low dose of mirex induced higher amounts of 4′-hydroxywarfarin than did the high dose. The metabolite profiles with high and low doses of Kepone also showed marked variations from one another. Mirex and Kepone are carcinogenic in rats and mice but, in contrast to the polycyclic aromatic carcinogens, do not markedly enhance the activity of microsomal biphenyl 2-hydroxylase relative to biphenyl 4-hydroxylase. We conclude that mirex and Kepone induce hepatic mixed-function oxidase profiles which differ from one another and from the classical inducers, phenobarbital and 3-methylcholanthrene. Mirex apparently only induces one of the enzymes induced by 3-methylcholanthrene. The enzyme profiles arising from the insecticides are dose dependent and will thus potentiate qualitatively differing effects depending on the level of ingestion.     

Inhibition of cytochrome P-450-dependent oxidation reactions by MAO inhibitors in rat liver microsomes

The inhibition of cytochrome P-450 dependent hydroxylations of bufuralol (BH) and antipyrine, and O-deethylation of 7-ethoxycoumarin (7-ECOD) by several monoamine oxidase inhibitors (MAOIs) was investigated in rat liver microsomes. According to their IC50 values, clorgyline was the most potent inhibitor while toloxatone, the only reversible MAOI in this study, was the least potent. A great variability of inhibitory potencies was found, even in the same chemical class of MAOIs. Irreversible inhibition of BH and 7-ECOD has been studied. Rapid irreversible inhibition occurred in some cases, and this could be responsible for in vivo inhibition after repeated dosing of these MAOIs.