Regulation of cytochrome P-450-dependent catechol estrogen formation in rat liver microsomes. Evidence for involvement of estrogen receptors

Experiments were conducted to evaluate whether estrogen 2-hydroxylase activity in liver microsomes, the main pathway for oxidative metabolism of estrogens in the rat, is regulated by administration of synthetic estrogens. Ovariectomized rats were treated with ethinylestradiol (EE), 100 micrograms s.c. for 3 days. Liver microsomes from EE-treated animals showed a 2-fold increase over control in estrogen 2-hydroxylase activity measured over a substrate concentration range of 0.5 to 50 microM. Double-reciprocal plots of enzyme activity as a function of substrate concentration were linear; apparent Vmax values were 2-fold greater in microsomes from EE-treated animals while apparent Km values for control and EE preparations were not different. Administration of the triphenylethylene antiestrogen tamoxifen (TAM), 100 micrograms s.c. for 3 days, did not affect microsomal catechol estrogen formation activity, and apparent Km and Vmax values were comparable with controls. When EE and TAM were co-administered, no increase in microsomal estrogen 2-hydroxylase was observed, and apparent Km and Vmax values were not different from either control of TAM-treated preparations. Thus, acute administration of EE was associated with a specific increase in the apparent Vmax of estrogen 2-OHase activity, and this effect was not observed when TAM was co-administered with the estrogen.     

Involvement of cytochrome P-450c in alpha-naphthoflavone metabolism by rat liver microsomes

Metabolism of alpha-naphthoflavone (ANF) is increased markedly in rat liver microsomes by 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two inducers of cytochromes P-450c and P-450d (P-450c and P-450d). Although several indirect lines of evidence in the literature suggest that ANF is metabolized by P-450c, Vyas et al. [J. Biol. Chem. 258:5649-5659 (1983)] reported that ANF metabolism by 3-MC-induced rat liver microsomes was only partially inhibited by antibodies against P-450c. Our laboratory has previously reported clastogenic effects of metabolites of ANF, and in the present study we reexamined the role of P-450c in ANF metabolism by both uninduced and TCDD-induced rat liver microsomes, using monospecific polyclonal antibodies to P-450c and P-450d. ANF metabolism was inhibited to different extents in TCDD-induced microsomes by different preparations of anti-P-450c. One lot of anti-P-450c produced only 50% inhibition of ANF metabolism in TCDD-induced microsomes, whereas another lot of anti-P-450c inhibited ANF metabolism by 80%. Anti-P-450d had no effect on ANF metabolism. Neither anti-P-450c nor anti-P-450d inhibited ANF metabolism in uninduced rat liver microsomes. In a reconstituted enzyme system, purified P-450c metabolized ANF 47 and 510 times more rapidly than P-450d and P-450b, respectively. Metabolites resulting from oxidation at 7,8- or 5,6-positions (7,8-dihydro-7,8-dihydroxy-ANF, 5,6-dihydro-5,6-dihydroxy-ANF, 5,6-oxide-ANF, and 6-hydroxy-ANF) were formed by all preparations of microsomes. An unknown toxic ANF metabolite was formed only with a reconstituted P-450c system and with 3-MC- or TCDD-induced microsomes. Our results indicate that P-450c is responsible for the majority of the metabolism of ANF in TCDD-induced microsomes, whereas other constitutive isozymes are responsible for the metabolism seen in uninduced liver microsomes. The variable inhibition of ANF metabolism with different lots of anti-P-450c probably reflects the differences in the proportion of antibodies to different epitopes important in the binding or metabolism of this substrate.     

Comparative study of cytochrome P-450 in liver microsomes. A form of monkey cytochrome P-450, P-450-MK1, immunochemically cross-reactive with antibodies to rat P-450-male

Cytochrome P-450, designated as P-450-MK1, which is cross-reactive with antibodies to rat P-450-male, was purified to an electrophoretical homogeneity from liver microsomes of the untreated male crab-eating monkey. The molecular weight of P-450-MK1 was estimated to be 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The oxidized form of P-450-MK1 showed a peak at 418 nm, indicating that this cytochrome is in a low spin state. The carbon monoxide-bound reduced form showed a peak at 451 nm. The first 22 amino acid residues of the NH2-terminal sequence of P-450-MK1 was fairly homologous to those of P-450-male (75% identity, not including unidentified amino acid residues). Unlike the P-450-male, P-450-MK1 did not exhibit catalytic activities for testosterone 2 alpha- and 16 alpha-hydroxylations and catalyzed testosterone 6 beta-hydroxylation. It is, therefore, suggested that although the spectral and immunochemical properties and the N-terminal amino acid sequence of P-450-MK1 were similar to those of P-450-male, the physiological functions of P-450-MK1 may be somewhat different from those of P-450-male. Comparison of the physico-chemical properties of P-450-MK1 with those of P-450-D1 and P-450-HM2, which are cross-reactive with anti-P-450-male antibodies, purified from liver microsomes of dogs and humans, respectively, are also discussed.     

Influence of ethanol and benzene on cytochrome P-450 fractions in rat liver microsomes

The repartition and characteristics of liver microsomal cytochrome P-450 fractions from benzene- or ethanol-treated rats were compared to those observed either in untreated animals, or in rats treated by classic inducers, namely phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone. DEAE-cellulose chromatography allowed the separation of four main cytochrome P-450 fractions called A (nonabsorbed), Ba, Bb, and Bc (successively eluted by a NaCl gradient). In control rats, and in ethanol- and benzene-treated animals, fractions A were predominant; phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, and benzene induced Bb fractions. Enzymatic and immunological methods allowed a characterization of those cytochrome P-450 fractions. Fractions A are similar in all cases, and mainly active towards aniline. This aniline hydroxylase activity is especially increased by ethanol. As a rule, fractions Ba and Bc behave similarly and exhibit rather low monooxygenase activities. On the contrary, fractions Bb differ from each other as a function of the inducer. Phenobarbital-induced Bb fraction is different from all other Bb fractions and especially active towards benzphetamine. 3-Methylcholanthrene- and beta-naphthoflavone-induced Bb fractions are identical, but they are different from all other Bb fractions, and especially active towards 7-ethoxycoumarin. Fraction Bb induced by benzene is different from those induced by classic inducers, but may be identical to the Bb fraction of control animals. As a whole, benzene and ethanol appear to display inducing properties different from those of phenobarbital or polycyclic aromatic hydrocarbon-like inducers.     

Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes

Assay conditions in determining total cytochrome P-450 in four laboratories were compared. Although the determination was derived from the original Omura and Sato method in each laboratory, the four standard protocols differed slightly, resulting in considerable differences in the results. Since the cytochrome P-450 content is usually expressed per mg protein, the protein assay conditions were evaluated as well. Furthermore, we compared the cytochrome P-450 values obtained by the CO- and the dithionite (DT)-difference methods. The effect of a number of variables in the assay was investigated. The influence of the storage temperature of the microsomes was ascertained as well as effects of the gassing time with CO and the time between addition of dithionite, CO-gassing and the recording of the difference spectra. After evaluating these variables a standard operation procedure was established. Using this procedure the interlaboratory coefficient of variation for total cytochrome P-450 was 4.8%, a value which was comparable to the intralaboratory coefficients of variation. The final results also show that the millimolar extinction coefficient for the DT-difference method is higher than for the CO-difference method.     

Optical spectral studies of ebselen interaction with cytochrome P-450 of rat liver microsomes

Interaction of ebselen, an anti-inflammatory compound of low toxicity, with rat liver cytochrome P-450 is used as a model system to quantify possible interactions of seleno-organic compounds with sulfhydryl groups of intracellular membrane-bound proteins. Ebselen induces a unique difference spectrum (maximum at 405 nm, minima at 385 and 425 nm) after addition to microsomes under in vitro conditions. This spectrum indicates an interaction with the thiolate anion at cytochrome P-450; it can be blocked by previous addition of dithioerythritol. With uninduced microsomes, addition of ebselen converts maximally 50% of the cytochrome P-450 to P-420 in a time-dependent (nearly complete effect within 10 min) and concentration-dependent manner (halfmaximal effect with 50 microM at 1 nmol/ml cytochrome P-450 concentration) in vitro. In phenobarbital- and 3-methylcholanthrene-induced microsomes, 73% and 64%, respectively, of cytochrome P-450 are converted to P-420 in presence of 200 microM ebselen. It is assumed that only certain isoenzymes of the total hepatic cytochrome P-450 are accessible to ebselen. Bovine serum albumin at physiological concentrations and sulfhydryl compounds such as dithioerythritol are effective in preventing this cytochrome P-450 inactivation by ebselen. Specificity studies reveal that variation of the N-substituent in the benzisoselenazolone system does not influence cytochrome P-450 inactivation, whereas ebselen derivatives with methylated or glucuronidated selenium moiety as well as diselenides do not convert cytochrome P-450 to P-420. It is concluded that benzisoselenazolones are able to interact with sulfhydryl groups of membrane-associated proteins in vitro.     

Inhibition of p-nitroanisole O-demethylation in perfused rat liver by oleate

p-Nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats was rapidly and reversibly inhibited by sodium oleate (0.3 to 0.6 mM). Xylitol partially reversed this inhibitory effect. The inhibition was not mediated by a direct effect of oleate on microsomal components since concentrations of oleate ranging up to 1.0 mM did not affect p-nitroanisole O-demethylation by isolated microsomes. Infusion of 0.6 mM oleate did not alter the measured intracellular NAD+/NADH ratio but did cause a significant increase in the intracellular NADP+/NADPH ratio. A significant decrease in the ATP/ADP ratio was also observed. Oleoyl CoA inhibited p-nitroanisole O-demethylation in microsomes (Ki about 30 microM), and both oleoyl CoA and palmitoyl CoA inhibited the energy-linked nicotinamide nucleotide transhydrogenase in submitochondrial particles (Ki about 1 microM). Thus, inhibition of mixed-function oxidation in the intact liver by oleate is most likely mediated by oleoyl CoA. Oleoyl CoA inhibits mixed-function oxidation in the intact liver by acting directly on cytochrome P-450 and by decreasing generation of NADPH via inhibition of key enzymes of the citric acid cycle and the energy-linked transhydrogenase.     

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.     

Biotransformation of lovastatin. II. In vitro metabolism by rat and mouse liver microsomes and involvement of cytochrome P-450 in dehydrogenation of lovastatin

Metabolism of lovastatin, a new cholesterol-lowering drug, by liver microsomes from rats and mice was investigated. Liver microsomes from rats catalyzed biotransformation of lovastatin at a rate of 3 nmol/mg of protein/min, whereas the rate of metabolism was 37% higher with liver microsomes from mice. The profiles of metabolites were similar, but the relative abundance of individual metabolites was species dependent. Hydroxylation at the 6'-position was the principal pathway of lovastatin biotransformation, whereas hydroxylation at the 3"-position of the side chain was a minor pathway. In both species the 6'-beta-hydroxy-lovastatin accounted for half of the total metabolism. Liver microsomes from rats produced 2- to 4-fold higher amounts of the other three metabolites, namely, 6'-exomethylene-, 3"-hydroxy-, and the hydroxy acid form, than mouse liver microsomes. The conversion of lovastatin to the novel 6'-exomethylene metabolite was catalyzed by cytochrome P-450 since it required microsomes and NADPH and was inhibited by SKF-525A, metyrapone, and 2,4,-dichloro-6-phenylphenoxyethylamine (DPEA). Furthermore, neither 6'-beta-hydroxy-lovastatin nor the 6'-hydroxymethyl analogs could be demonstrated to be intermediates in the formation of the 6'-exomethylene metabolite. The hydroxy acid form of lovastatin was not a substrate for liver microsomes from either species.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Metabolism of the "mixed" cytochrome P-450 inducer hexachlorobenzene by rat liver microsomes

Hexachlorobenzene (HCB) was metabolised by phenobarbital-induced liver microsomes from male rats to pentachlorobenzene, pentachlorophenol, tetrachloro-1,2-benzenediol and tetrachloro-1,4-benzenediol (1:88:2:9). Metabolites were identified and quantified by electron capture g.l.c. Structures were confirmed by selective ion monitoring g.l.c.-m.s. The formation of pentachlorophenol was dependent on the presence of NADPH and O2 and inhibited by CO, SKF 525A and metyrapone. Conversion of HCB to pentachlorophenol was stimulated by pretreatment of rats with phenobarbital (PB) but not by 3-methylcholanthrene (3-MC), or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the conversion of pentachlorophenol to tetrachloro-1,4-benzenediol was markedly induced by 3-MC but poorly by PB. HCB, Aroclor 1254 and isosafrole stimulated both hydroxylations. The cytochrome P-450c inhibitor 9-hydroxyellipticine inhibited conversion of pentachlorophenol to tetrachlorobenzenediols by HCB and beta-naphthoflavone induced micromes. In addition to hydroxylation reactions, evidence was obtained for the conjugation of HCB with glutathione catalysed by a microsomal glutathione transferase. Radioactivity from [14C]HCB was bound to microsomal protein during aerobic incubations. Binding was inhibited by GSH and N-acetyl-cysteine. Preliminary studies suggested that the reactive species was derived from tetrachloro-1,4-benzoquinone. No correlation was found between levels of metabolites or covalent binding produced by the two sexes and the marked sex dependent hepatic porphyrogenic and carcinogenic effects of HCB.     

Interactions of styrene and styrene oxide with partially purified cytochrome P-450 and P-448 from rat liver microsomes

Styrene and styrene 7,8-oxide were able to bind both to partially purified cytochrome P-450 isolated from phenobarbital (PB)-treated rat liver and to cytochrome P-448 from liver microsomes of 3-methylcholanthrene (3-MC)-treated rats.In the presence of either purified preparation or “fresh” microsomes from PB- or 3-MC-treated animals, styrene produced a characteristic Type I difference spectrum as did styrene 7,8-oxide with “fresh” microsomes from PB rats. In other experiments, the addition of styrene oxide produced spectra which resembled Type I spectra but were somewhat shifted to longer wavelengths.A comparison of the binding parameters for the interaction of styrene or styrene 7,8-oxide with partially purified preparations and “fresh” microsomes indicated that the binding is catalyzed by more than one type of P-450 hemoprotein and that the binding affinity is slightly reduced by the purification procedure. The addition of phosphatidylcholine was unable to restore the binding parameters.     

Effect of diabetes on rat liver cytochrome P-450: Evidence for a unique diabetes-dependent rat liver cytochrome P-450

Detergent-solubilized hepatic microsomal fractions from alloxan diabetic rats exhibited a 52,000 molecular weight hemeprotein band that was not present in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of identically solubilized hepatic microsomal fractions from normal, 3-methylcholanthrene- or phenobarbital-treated rats. This 52,000 mol. wt hemeprotein band disappeared from the protein profile of insulin-treated diabetic rat liver to yield the SDS-PAGE profile of normal rat liver. When P-450 hemeproteins were purified by lauric acid affinity and hydroxylapatite chromatography from solubilized microsomes, only the diabetic rat had a 52,000 mol. wt P-450. This distinct 52,000 mol. wt diabetes-induced P-450 interacted with type II compounds to yield a 2-fold greater absorbance change than was observed with the purified P-450s from either the normal or the chemically induced rats. The properties of this unique 52,000 mol. wt P-450 suggest that it may be the catalytic component responsible for the increased rate of type II substrate (aniline) metabolism observed in the diabetic rat.     

Oxidation of glycerol to formaldehyde by rat liver microsomes. Effects of cytochrome P-450 inducing agents

Glycerol was shown recently to be metabolized to formaldehyde by microsomes from chowfed control rats (Winters et al., Biochem Biophys Res Commun 153: 612-617, 1988). In the present study, experiments were carried out to evaluate the oxidation of glycerol by microsomes isolated from rats treated with inducers of different isozymes of cytochrome P-450. The oxidation of glycerol to formaldehyde was increased in microsomes from rats treated with pyrazole, ethanol or acetone relative to their respective controls, but not after treatment with phenobarbital or 3-methylcholanthrene. This reaction was sensitive to inhibition by carbon monoxide and was inhibited by compounds known to be effective substrates for P-450j, e.g. aniline, ethanol, pyrazole and 4-methylpyrazole. Treatment with pyrazole caused an increase in Vmax for glycerol oxidation but did not affect affect the Km (about 15 mM) for glycerol, as compared to saline controls. Evidence that the product of glycerol metabolism is formaldehyde was provided by the observation that this product served as a substrate for the glutathione-dependent formaldehyde dehydrogenase, and the amount of formaldehyde detected was identical to that detected by the Nash reaction. By utilizing [14C]glycerol, and coupling the formaldehyde dehydrogenase reaction to the formate dehydrogenase reaction, 14CO2 could be detected, indicating that the formaldehyde produced was derived from the added glycerol. These results suggest that that glycerol is not metabolically inert when added to microsomes but serves as an effective substrate for the cytochrome P-450j isozyme, extending the alcohol substrate specificity of this enzyme to poly-ols. The production of formaldehyde from glycerol may require caution since glycerol is often present in microsomal or reconstituted systems.     

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl₄-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species.     

Oxidation of 1,1,1,2-tetrafluoroethane in rat liver microsomes is catalyzed primarily by cytochrome P-450IIE1.

1,1,1,2-Tetrafluoroethane (R-134a), a nonozone-depleting alternative air-conditioning refrigerant and propellant for pharmaceutical preparations, is oxidatively defluorinated by rat hepatic microsomes. In this report we show that induction of cytochrome P-450IIE1 in rats, by pyridine administration, resulted in an 8-fold increase in the rate of R-134a metabolism by hepatic microsomes (Vmax 47 vs. 6 nmol F-/mg microsomal protein/15 min). Furthermore, when data were normalized for P-450 content, a 4-fold increase in R-134a metabolism was noted for IIE1-enriched microsome preparations. In contrast, phenobarbital and Aroclor 1254 decreased the specific activity of hepatic microsomes for this function. The microsomal content of P-450IIE1, as evaluated by Western blot, was elevated significantly only in microsomes from pyridine-treated rats. p-Nitrophenol and aniline, which are metabolized at high rates by rat P-450IIE1, decreased the rate of R-134a defluorination by hepatic microsomes; Dixon plot analysis indicated competitive inhibition with a Ki of 36 microM p-nitrophenol or 115 microM aniline. Pyridine also potently induced defluorination of R-134a catalyzed by rabbit liver microsomes. Studies with individual P-450 isozymes purified from rabbit liver showed that the phenobarbital- and polycyclic hydrocarbon-induced isozymes (IIB1 and IA2) defluorinated R-134a at negligible rates (1.9 and 0.4 nmol F-/nmol P-450/60 min, respectively). In contrast, P-450IIE1 catalyzed defluorination of R-134a at a relatively high rate (16.2 nmol F-/nmol P-450/60 min); isozyme IA1, which also is induced by nitrogen-containing heterocycles such as pyridine, was somewhat active (5.3 nmol F-/nmol P-450/60 min).(ABSTRACT TRUNCATED AT 250 WORDS)