Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6 beta-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver, 6 beta-Hydroxylation was the most important pathway of biotransformation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2 beta-, 15 beta- and 15 alpha-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6 beta-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6 beta-alcohol was the only metabolite formed from AD at a rate greater than 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti-P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2 beta-, 6 beta-, 15 alpha- and 15 beta-positions (by 31-56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6 beta-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6 beta-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6 beta-hydroxylation.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

Effects of clofibrate on the metabolism of progesterone and oestradiol in rat liver microsomal fraction

1. The substrate conversion of [4-(14)C]progesterone and [4-(14)C]oestradiol during incubation with the liver microsomal fraction from both control and clofibrate-treated rats amounted to about 10-15 and 20-25% respectively. 2. The metabolites of progesterone formed by preparations from control rats were hydroxylated in the 16alpha-position (14%), the 6beta-position (12%) and the 2alpha-position (7%). Of the products formed from oestradiol 12% were recovered as a 16alpha-hydroxylated derivative whereas 5% had a 6beta- and 2% a 6alpha-hydroxyl group. 3. Clofibrate affected the microsomal metabolism of both progesterone and oestradiol. It induced 7alpha-hydroxylation of both compounds, metabolic conversions not found in control rats. The 6beta-hydroxylation of progesterone and the 6alpha-hydroxylation of oestradiol were enhanced by a factor of 2 and 3.5 respectively. The 2alpha-hydroxylation, and the 20alpha- and 20beta-hydroxy steroid reduction of progesterone were significantly decreased as were the 16alpha- and the 6beta-hydroxylation of oestradiol.     

Rapid quantitation of testosterone hydroxyl metabolites by ultra-performance liquid chromatography and mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography and mass spectrometry (UPLC/MS) method was developed to simultaneously quantify seven monohydroxyl testosterone metabolites (16alpha-, 2alpha-, 7alpha-, 6alpha-, 2beta-, 6beta-, and 16beta-hydroxyl testosterones) in rat liver microsomes. The UPLC system used a short 1.7-microm particle size column coupled to a Sciex 4000 Q trap in multiple reaction monitor (MRM) mode. All hydroxyl testosterones were resolved within 2.5 min. A 4-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method in rat liver microsomes. This method is applicable to the measurement of the testosterone hydroxylase activity in biological matrices such as the liver microsome incubates.     

Microbial transformation of steroids--II. Transformations of progesterone, testosterone and androstenedione by Phycomyces blakesleeanus

Phycomyces blakesleeanus transformed progesterone, testosterone and androstenedione into mixtures of products. Five monohydroxylated metabolites were obtained in reasonable yields from the progesterone transformation. Only 7 alpha- and 15 beta-hydroxyprogesterone have been reported previously from this organism. We find that it gives these two metabolites and also 6 beta-, 14 alpha- and 15 alpha-hydroxyprogesterone as major products. Five compounds were also purified from testosterone transformation mixtures. Two of these were monohydroxylated, two were ring A dehydrogenation products, and two were oxidised at C-17. The products were identified as 6 beta-hydroxytestosterone, 7 alpha-hydroxytestosterone, androsta-1,4-diene-3,17-dione (1-dehydroandrostenedione), 17 beta-hydroxyandrosta-1,4-diene-3-one (1-dehydrotestosterone) and androstenedione. All five metabolites were produced in reasonable yields, although hydroxylation was the minor transformation in this case. Only two significant products were formed from androstenedione. Both were reduced at C-17; one was also monohydroxylated. They were testosterone and 14 alpha-hydroxytestosterone. The testosterone and androstenedione transformation products have not been reported previously for this organism. We also report for the first time the preparation of P. blakesleeanus cell-free extracts which transformed progesterone reasonably efficiently and faithfully in vitro, although the proportions of each product varied from one extract to another.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.     

Investigation of rat liver microsomal 6 beta-hydroxylation of 4-androstene-3,17-dione and 4-pregnene-3,20-dione using methodology which excludes steroid-3-imine induced introduction of the 6 beta-hydroxyl group

To avoid artefactual 6 beta-hydroxylation of 3-oxo-4-ene steroids due to steroid-3-imine formation and rearrangement a combined extraction and liquid chromatography purification procedure for incubated rat liver microsomes has been worked out. With this procedure no nonenzymatic 6 beta-hydroxylation could be observed. Conventional termination of incubations with male rat liver microsomes (105,000 g sediments) and 4-14 C-labelled 4-androstene-3,17-dione (or progesterone) by solvent extraction and evaporation might lead to a 30% overestimation of the formation of 6 beta-hydroxy-derivatives at substrate saturation. Furthermore this work-up procedure produces 6-oxo-derivatives which were not observed when the new procedure was used. By elimination of the artefactual 6-oxygenation some properties of the male rat liver microsomal 4-androstene-3,17-dione 6 beta-hydroxylase have been studied, and the activity has been compared to the artefact produced by solvent extraction and evaporation. Using the combined extraction-liquid chromatography purification it was demonstrated that the microsomal 6 beta-hydroxylase active on 4-androstene-3,17-dione and progesterone was inhibited to 95% by carbon monoxide. This makes previous suggestions regarding participation of a non cytochrome P450-dependent 4-androstene-3,17-dione 6 beta-hydroxylase less likely.     

Species differences in 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat, monkey, and human prostate microsomes.

The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6 alpha-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol varied independently of both the 7 alpha- and 7 beta-hydroxylation, which varied in unison. The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with beta-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7 alpha- and the 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol without increasing the 6 alpha-hydroxylation. The 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human and monkey prostate microsomes, but not the 6 alpha-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human prostate microsomes, but not the 6 alpha-hydroxylation, was markedly inhibited (greater than 85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6 alpha-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5 alpha-reductase inhibitors.

5 alpha-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5 alpha-reductase. The inactivation of 5 alpha-dihydrotestosterone involves reversible reduction to 5 alpha-androstane-3 beta,17 beta-diol by 3 beta-hydroxysteroid oxidoreductase followed by 6 alpha-, 7 alpha-, or 7 beta-hydroxylation. 5 alpha-Androstane-3 beta,17 beta-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity (Km approximately 0.5 microM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5 alpha-reductase (4-azasteroid analogs; 10 microM) or inhibitors of 3 beta-hydroxysteroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 microM) had no appreciable effect on the 6 alpha-, 7 alpha-, or 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (10 microM) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1-10 microM) all markedly inhibited 5 alpha-androstane-3 beta,17 beta-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1-10 microM) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole greater than clotrimazole greater than ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a Ki of 0.03 microM. The imidazole-type antimycotic drugs inhibited all three pathways of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Rat liver microsomal progesterone metabolism: evidence for differential troleandomycin and pregnenolone 16 alpha-carbonitrile inductive effects in the cytochrome P-450 III family

The effect of Troleandomycin (TAO) and pregnenolone 16 alpha-carbonitrile (PCN) on the hepatic microsomal progesterone metabolism in the rat is evaluated. Over thirteen hydroxylated progesterone derivatives are detected, including the novel 6 beta, 21-, 6 beta, 16 alpha-, 6 beta, 16 beta- and 2,21-dihydroxy derivatives, suggesting the induction of several cytochrome P-450 isozymes. PCN treatment results overall in an augmented production of progesterone metabolites whereas TAO treatment both induces and represses specific hydroxylase activities. Progesterone metabolism with purified isozymes isolated from liver microsomes from TAO and PCN treated rats differs significantly from that observed with intact microsomes, reflecting the complexity of the induction pattern of the cytochrome P-450 III family.     

Evidence for the involvement of a distinct form of cytochrome P450 3A in the oxidation of digitoxin by rat liver microsomes.

The preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1. J. Biochem. Toxicol., 7 (43-52).) described the regulation of two rat liver microsomal proteins (50- and 51-kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51-kDa 3A protein were usually paralleled by changes in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation. The present study demonstrates that age- and sex-dependent changes in the 50-kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50-kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this conclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone-16 alpha-carbonitrile (PCN), which induces both the 50-kDa and the 51-kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16- to 18-fold) in the 6 beta-hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN-induced rats with chloramphenicol caused a approximately 70% decrease in liver microsomal testosterone 6 beta-hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51-kDa protein) was also inhibited by chloramphenicol in a time- and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized troleandomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: effects of antibodies and chemical inhibitors of cytochrome P450 enzymes.

The purpose of the present study was to test the hypothesis that rat prostate microsomes contain a single cytochrome P450 enzyme responsible for the conversion of 5 alpha-androstane-3 beta,17 beta-diol to a series of trihydroxylated products. The three major metabolites formed by in vitro incubation of 5 alpha-[3H]androstane-3 beta,17 beta-diol with rat prostate microsomes were apparently 5 alpha-androstane-3 beta,6 alpha,17 beta-triol, 5 alpha-androstane-3 beta,7 alpha,17 beta-triol, and 5 alpha-androstane-3 beta,7 beta,17 beta-triol, which were resolved and quantified by reverse-phase HPLC with a flow through radioactivity detector. The ratio of the three metabolites remained constant as a function of incubation time, microsomal protein concentration, ionic strength, and substrate concentration. The ratio of the three metabolites was dependent on pH, apparently because the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol shifted from the 6 alpha- to the 7 alpha-position with increasing pH (6.8-8.0). The V(max) values were 380, 160, and 60 pmol/mg microsomal protein/min for the rate of 6 alpha-, 7 alpha-, and 7 beta-hydroxylation, respectively. Similar Km values (0.5-0.7 microM) were measured for enzymatic formation of all three metabolites, which suggests that formation of all three metabolites was catalyzed by a single, high-affinity enzyme. Testosterone, 5 alpha-dihydrotestosterone, and 5 alpha-androstane-3 alpha,17 beta-diol did not appreciably inhibit the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, suggesting that this enzyme exhibits a high degree of substrate specificity. Formation of all three metabolites was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase (85%) and by a 9:1 mixture of carbon monoxide and oxygen (60%). Several chemical inhibitors of cytochrome P450 enzymes, especially the antimycotic drug clotrimazole, also inhibited the formation of all three metabolites. Polyclonal antibodies that recognize liver cytochrome P450 1A, 2A, 2B, 2C, and 3A enzymes did not inhibit 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity. Overall, these results are consistent with the hypothesis that the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes is catalyzed by a single, high-affinity P450 enzyme. This cytochrome P450 enzyme appears to be structurally distinct from those in the 1A, 2A, 2B, 2C, and 3A gene families.     

Simultaneous determination of testosterone metabolites in liver microsomes using column-switching semi-microcolumn high-performance liquid chromatography

A sensitive and selective column-switching semi-microcolumn high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of testosterone and eight of its metabolites (6alpha-, 6beta-, 16alpha-, 16beta-, 7alpha-, 2alpha-, and 2beta-hydroxytestosterone, and androstenedione) in liver microsomes. After incubation for 10 min, testosterone and its metabolites were extracted from the microsomes with ethyl acetate, and the extract was evaporated to dryness. The residue was dissolved in the mobile phase and loaded onto the HPLC system. The analytes were first concentrated in a precolumn and subsequently transferred to the analytical column, where they were separated using linear gradient elution. A UV detector set at 254 nm was used to detect the analytes. This newly developed method clearly separated TES and the metabolites with high resolution and was found to be reproducible with intra- and interday variability of <10.7%. This method has been subsequently used to determine the testosterone hydroxylation activities catalyzed by 15 different recombinant CYP isozymes. The results confirmed the formation of stereoselectively hydroxylated metabolites by each CYP isozyme.     

Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1.

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16 alpha-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male greater than female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2 beta-, 6 beta-, and 15 beta-hydroxylation of testosterone.     

Cytochrome P450 3A4-catalyzed testosterone 6beta-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.     

Bioconversion of 16 alpha-hydroxyprogesterone to 16 alpha-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

Using newborn rat adrenal cells in primary culture, 16 alpha-hydroxyprogesterone was bioconverted into numerous 16 alpha-hydroxylated steroids. The method of analysis of these steroids comprised the association of column and thin-layer chromatography to gas chromatography-mass spectrometry in order to obtain the mass spectra of pure compounds. The identified compounds resulted principally from the enzymatic reactions of 21-hydroxylation 11 beta-hydroxylation and reduction of the 20-oxo and 3-oxo-4-ene groups. Minor metabolites resulted from 18-hydroxylation and 6 beta-hydroxylation of the substrate. The metabolism of 16 alpha-hydroxyprogesterone is similar to that of progesterone in the same cell-culture system; however, there are two exceptions. The 21-hydroxylation of 16 alpha-hydroxyprogesterone occurs at a rate similar to that of its 11 beta-hydroxylation, whereas the 21-hydroxylation of progesterone is faster than its 11 beta-hydroxylation. The ratio of 11 beta- to 18-hydroxylation of 16 alpha-hydroxyprogesterone is about 3, whereas the ratio of 11 beta- to 18-hydroxylation of progesterone, 20 alpha-dihydroxyprogesterone and DOC is between 1./ and 2. It is most likely the rate of 18-hydroxylation which is decreased by the hydroxyl group at C-16. The use of adrenal cell cultures is a practical, simple method for the preparation of a variety of 16 alpha-hydroxylated steroids from a single substrate. Its adaptation to the production of important amounts of 16 alpha-hydroxylated corticosteroids will permit the study of their biological activity.     

Alternate hydroxylating activities in newborn rat adrenal cells in primary culture

During the course of a study to produce reference compounds, the metabolism of tetrahydrogenated derivatives (ring A reduced) of progesterone, 6 alpha-hydroxyprogesterone, 11-deoxycorticosterone and corticosterone in newborn rat adrenal cells in primary culture was studied. Analysis of the metabolites was made by gas chromatography-mass spectrometry. Most products resulted from the enzymatic reactions of 11 beta-, 18- and 21-hydroxylation, reduction of the 20-oxo group and oxidoreduction of the 3-hydroxyl group. However, unexpected metabolites were produced from the incubation of 3 beta, 5 alpha-tetrahydroprogesterone and 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. They resulted from the 16 alpha-hydroxylation of the precursors and probably from the 15 alpha-, 16 beta- and 17 alpha-hydroxylation of 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. These hydroxylating activities are weak and were not detected from the endogenous steroidogenesis. They were not detected either from the incubation of exogenous steroids with a 3-oxo-4-ene structure or from steroids with a 21-hydroxyl substituent. They result only from substrates showing diminished or no affinity towards the 11 beta/18- and 21-steroid hydroxylase systems. These unusual hydroxylations could be catalyzed by monooxygenase systems in the endoplasmic reticulum similar to those present in the liver or by the monooxygenase systems specific to steroidogenesis. In particular, the reaction specificity of cytochrome P-450(11) beta could be altered by the presence of a 6 alpha-hydroxyl group in a tetrahydrogenated steroid.