The metabolism of steroids in the fatty liver induced by orotic acid feeding.

1. The metabolism of 4-[4-14C]androstene-3,17-dione, 4-[4-14C]pregnene-3,20-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, 5beta-[7beta-3H]cholestane-3alpha,7alpha-diol and [3H]lithocholic acid was studied in the microsomal fraction of livers from control and orotic acid-treated male rats. 2. As a result of the treatment the orotic acid-fed rats had fatty livers and subnormal concentrations of cholesterol and triglycerides in serum. 3. The 6beta- and 7alpha-hydroxylation of 4-androstene3,17-dione, and the 2alpha-, 2beta- and 18-hydroxylation of 5alpha-androstane-3alpha,17beta-diol, and the 5alpha-reduction of 4-androstene-3,17-dione and 4-pregnene-3,20-dione were decreased by 40--50% in orotic acid-fed rats. Other oxidative and reductive reactions of the steroid hormones were not significantly affected. 4. The 12alpha-hydroxylation of 7alpha-hydroxy-4-cholesten-3-one was decreased by about 50%, whereas the 7alpha-hydroxylation of cholesterol and the 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol were not significantly decreased. The 6beta-hydroxylation of lithocholic acid was stimulated by 40%. 5. The results are discussed in relation to present knowledge of the heapatic drug-metabolizing enzymes and to the recent findings of an abnormal bile acid metabolism in liver disease.     

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.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

Increased expression of cytochrome P450 IIIA2 in male rat liver after dietary vitamin A supplementation

In this study dietary vitamin A supplementation (25 IU/g diet) was assessed for its effect on hepatic microsomal P450 content and on P450 enzyme-specific drug oxidase activities in rats. Intake of the supplemented diet by male rats over a 15-week period resulted in a fivefold increase in hepatic vitamin A stores over those measured in control liver from rats that received a balanced diet without vitamin A supplementation. Serum retinol was unchanged and there was no evidence of hepatocellular injury in any of the animals. There was a 26% increase in P450 content in vitamin A-supplemented rat liver and regioselective androst-4-ene-3,17-dione (androstenedione) and progesterone hydroxylation revealed changes in several P450 pathways. Thus, androstenedione 16 alpha-hydroxylation (P450 IIC11-mediated) and progesterone 21-hydroxylation (P450 IIC6-mediated) were decreased slightly to 80 and 74% of respective control activities while P450 IIA1/2-dependent androstenedione 7 alpha-hydroxylation was slightly increased. In contrast, the 6 beta-hydroxylations of androstenedione and progesterone were increased to 169 and 152% of control following dietary supplementation. Kinetic analysis of androstenedione 6 beta-hydroxylation revealed an increase in maximal reaction velocity (Vmax 4.00 +/- 0.47 vs 2.20 +/- 0.10 nmol/min/mg protein) but the Km was unchanged, suggesting an increase in enzyme concentration. Consistent with this assertion, immunoquantitation of the steroid 6 beta-hydroxylase, P450 IIIA2, revealed a 158% increase in the microsomal expression of this enzyme (9.8 +/- 2.7 vs 6.2 +/- 1.3 ng/micrograms microsomal protein). From these studies it now seems clear that vitamin A, as a dietary additive in nontoxic doses, has the capacity to alter the activity of hepatic microsomal drug oxidases by modulating the expression of P450 enzymes.     

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.     

Studies on rat liver microsomal steroid metabolism using 18O-labelled testosterone and progesterone

In order to investigate the possible involvement of oxygen functions in the rat liver microsomal metabolism of progesterone and testosterone these steroids were specifically labelled with 18O in their oxo-functions and incubated with NADPH supplemented 105,000 g sediments. Gas chromatography-mass spectrometry was used to identify the metabolites formed as well as to quantitate the losses of 18O-label. With 18O-labelled testosterone as substrate two of the major monohydroxylated metabolites, i.e. 2 beta- and 6 beta-hydroxytestosterone were shown to have lost about 25 and 50% of their 18O respectively. A complete retention of label was found in 7 alpha- and 16 alpha-hydroxytestosterone. None of the monohydroxylated progesterone metabolites, i.e. the 2 alpha-, 6 beta- and 16 alpha-hydroxyprogesterone had lost any 18O following incubation with 3,20-18O-labelled progesterone. Control incubation (30', 37 degrees C) with buffer and 18O-labelled progesterone and testosterone revealed no exchange of 18O. Thus the partial loss of 3-18O-label during 2 beta- and 6 beta-hydroxylation of testosterone may indicate a covalent interaction between the steroid 3-oxo-group and one or more cytochrome P-450 species in the rat liver microsomes. In view of the potentiating effect of a 3-imine group in spontaneous 6 beta-hydroxylation the present in vitro data suggest that a steroid protein-interaction may occur via a 3-imine group during 6 beta-hydroxylation of testosterone in rat liver microsomes. Analysis of 5 alpha-reduced metabolites of both progesterone and testosterone showed significant losses of 3-18O, but due to the ease with which 3-oxo-5 alpha-steroids exchange their 3-18O with aqueous media an enzymatically induced loss of 3-18O could not be safely established. The 20-oxido-reductase which converted progesterone did not induce a loss of 20- or 3-18O thus indicating that the oxofunctions were not covalently engaged in the enzymatic binding of the steroid.     

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)     

A conspicuous down-regulating effect of morphine on essential steroid hydroxylation reactions and certain drug N-demethylations.

This study was conducted to explore the potency of morphine to induce reductions of specific cytochrome P450 isoenzyme functions. Male Sprague-Dawley rats were treated with escalating doses (20-125 mg/kg per day) of morphine for 2 weeks in order to study the effects on the following cytochrome P450 catalyzed reactions: 16 alpha-hydroxylation of dehydroepienderosterone (DHA) and progesterone; 17 alpha- and 21-hydroxylation of progesterone; N-demethylation of ethymorphine, codeine and morphine as well as O-dealkylation of ethylmorphine and codeine. 16 alpha-Hydroxylation of DHA and progesterone and 17 alpha-hydroxylation of progesterone decreased to 18, 12 and 10% of control activities, respectively. The N-demethylation of ethylmorphine and codeine decreased to 34 and 43% of control activities, respectively. Morphine treatment had no effect on the 21-hydroxylation reactions or the O-dealkylation of ethylmorphine or codeine. A monoclonal antibody (Mab) against rat liver cytochrome P450 2 c/RLM 5 exerted a 66-73% inhibition of the N-demethylation of ethylmorphine and codeine, respectively, whereas the O-dealkylation reactions were not affected. This Mab inhibited the 16 alpha- and 17 alpha-hydroxylation of DHA and progesterone, whereas the 21-hydroxylation reactions were unaffected. The steroid hydroxylation reactions in rat adrenals were not altered upon morphine treatment. Our data suggest that a major part of the 16 alpha- and 17 alpha-steroid hydroxylations are catalyzed by the same (or closely related) cytochrome(s) P450 as the opioid N-demethylation reactions.     

The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The liver microsomal metabolism of [4-14C]cholesterol, endogenous cholesterol, 7 alpha-hydroxy-4-[6 beta-3H]cholesten-3-one, 5-beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol and [3H]lithocholic acid was studdied in control and clofibrate (ethyl p-chlorophenoxyisobutyrate)-treated rats. 2. The extent of 7 alpha-hydroxylation of exogenous [414C]cholesterol and endogenous cholesterol, the latter determined with a mass fragmentographic technique, was the same in the two groups of rats. The extent of 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol was increased by about 60 and 120% respectively by clofibrate treatment. The 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was not significantly affected by clofibrate. The 6 beta-hydroxylation of lithocholic acid was about 80% higher in the clofibrate-treated animals than in the controls. 3. The results are discussed in the context of present knowledge about the liver microsomal hydroxylating system and bile acid formation in patients with hypercholesterolaemia, treated with clofibrate.     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.     

Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome P-450

In order to elucidate the isozyme specificity of complex formation between cytochrome P-450 and the isosafrole metabolite the effect of complex dissociation on different steroid hydroxylation pathways was studied in hepatic microsomal fractions. Isosafrole induction was found to increase the 16 beta- and 7 alpha-hydroxylation of androst-4-ene-3,17-dione approximately 2.8- and 1.7-fold, respectively, whereas the 16 alpha-hydroxylation pathway was decreased to about one-quarter of control activity; 6 beta-hydroxylation was unchanged from control activity. More striking changes were apparent following dissociation of the isosafrole metabolite from its complex with ferricytochrome P-450 by the steroid substrate. Thus an approximate fourfold elevation of 16 beta-hydroxylase activity was observed after displacement and 6 beta-hydroxylation increased about twofold; 7 alpha-hydroxylase activity was decreased to 0.75-fold of undisplaced activity and 16 alpha-hydroxylase activity was unchanged. These data provide convincing evidence that at least two forms of phenobarbital-inducible cytochrome P-450 (cytochromes P-450PB-B and P-450PB/PCN-E) are present to some extent in a catalytically inactive complexed state in isosafrole-induced rat hepatic microsomes. Furthermore, there is now evidence to suggest that the constitutive isozymes cytochrome P-450UT-A and cytochrome P-450UT-F are not complexed to any degree in hepatic microsomes from isosafrole-induced rats.     

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.     

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.     

Comparison of human fetal hepatic and adrenal cytochrome P450 activities with some major gestational steroids and ethylmorphine as substrates.

The immunoidentified human fetal liver and adrenal microsomal contents of cytochromes P450IIIA and P450XVIIA1 were compared to the metabolism of steroids and ethylmorphine. In fetal liver microsomes, 16 alpha-hydroxylation of dehydroepiandrosterone (DHA) was catalyzed at a high rate in almost all investigated specimens and accompanied by a high ethylmorphine N-demethylase activity. Progesterone 16 alpha- and 17 alpha-hydroxylation was found only in the livers with the highest DHA 16 alpha-hydroxylation activities, while 21-hydroxylation of progesterone was catalyzed only occasionally in these samples. In fetal adrenal microsomes, 21-hydroxylation of progesterone to 11-desoxycorticosterone (DOC) and 11-desoxycortisol (DOCOL) was catalyzed. In contrast to fetal liver, the adrenals also catalyzed the 17 alpha-hydroxylation of pregnenolone and the formation of DHA from 17 alpha-OH-pregnenolone. 16 alpha-hydroxylation of DHA and ethylmorphine N-demethylation were modest in the adrenals. P450IIIA/HLp was immunoidentified in all investigated liver specimens except two (18/20) in which no ethylmorphine N-demethylation or 16 alpha-hydroxylation of DHA was found. P450XVIIA1 bands were observed in 8/20 blots of liver specimens, but there was no correlation between the density of these bands and the 17 alpha-hydroxylation of progesterone. All 11 fetal adrenal samples catalyzed DHA 16 alpha-hydroxylation, although only 8 were positive for P450IIIA/HLp. All investigated adrenals were positive in regard of the P450XVIIA1 band, except one (8/9) with a low 17 alpha-hydroxylation of progesterone. All adrenal specimens catalyzed 21-hydroxylation of progesterone and contained P450C21 bands in immunoblots and all samples catalyzed the formation of DOC and DOCOL from progesterone. Our findings in the fetal livers show a correlation between the DHA 16 alpha-hydroxylation and immunoidentified P450IIIA/HLp bands. In adrenals, there was a correlation between the immunoidentified P450XVIIA1 bands and the 17 alpha-hydroxylation of progesterone.     

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.     

Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice

Since tumor cells show abnormal fatty acid composition, it is likely that their desaturase systems were affected to some extent. Although desaturase activities in experimental tumors have been evaluated, to our knowledge, fatty acid desaturases in human neoplasms and particularly in human tumors grown in nude mice have not been assessed yet. We have therefore, chosen a rapidly growing human lung mucoepidermoid carcinoma (HLMC) grown in nude mice to study microsomal fatty acid desaturation and chain elongation activities. Tumor microsomal proteins were incubated with unlabeled malonyl-CoA and one of the following fatty acids: [1-14C]palmitic (16:0), [1-14C]linoleic (18:2), alpha-[1-14C]linolenic (alpha-18:3), and unlabeled gamma-linolenic (gamma-18:3) plus [2-14C]malonyl-CoA. Data show that HLMC microsomes were capable to desaturate 16:0, alpha-18:3, and dihomogammalinolenic acids (20:3) by delta 9, delta 6 and delta 5 desaturase, respectively; however, delta 6 desaturase activity on [14C]18:2 was not detected. The microsomal elongation system was active in all fatty acid series tested except for 18:2. These findings show that the undetectable activity for 18:2 desaturation is not exclusively found in experimental tumors.     

Use of a monoclonal antibody specific for rabbit microsomal cytochrome P-450 3b to characterize the participation of this cytochrome in the microsomal 6 beta- and 16 alpha-hydroxylation of progesterone

A monoclonal antibody was developed that is specific for the 3b electrophoretic class of rabbit liver microsomal cytochrome P-450 as judged by immunoprecipitation and subsequent electrophoretic analysis. The antibody is inhibitory of catalytically distinct, variant forms of P-450 3b prepared from New Zealand White or IIIVO/J rabbits, respectively. Peptide mapping of the immunopurified P-450 3b from NZW and IIIVO/J microsomes indicates that a characteristic difference between the variant forms is exhibited by the antigen. In addition, a competitive assay indicates that the binding properties of the antibody do not differ substantially toward the variant forms of P-450 3b. The inhibitory antibody was used to examine the contribution of P-450 3b to the microsomal 16 alpha- and 6 beta-hydroxylation of progesterone. The antibody inhibits 40-70% of the 16 alpha-hydroxylase activity of microsomes from either New Zealand White or IIIVO/J rabbits. In contrast, it does not inhibit 6 beta-hydroxylation catalyzed by microsomes prepared from strain IIIVO/J but does inhibit this reaction as catalyzed by microsomes from most New Zealand White rabbits. The antibody also inhibits the increased 16 alpha-hydroxylase activity of IIIVO/J microsomes observed in the presence of 5 beta-pregnane-3 beta,20 alpha-diol, an allosteric effector of this variant form of P-450 3b. Use of this monoclonal antibody provides a link between the observed properties of the purified, variant forms of P-450 3b and microsomal metabolism. These results indicate that the antibody can be used to phenotype variant forms of P-450 3b in microsomal fractions.     

Decreased liver cytochrome P-450 in rats caused by norethindrone or ethynyloestradiol.

1. 19-Nor-17alpha-pregna-1,3,5(10)-trien-20-yne-3,17-diol (ethynyloestradiol) or 17beta-hydroxy-19-nor-17alpha-pregn-4-en-20-yn-3-one (norethindrone) but not 17alpha-ethyl-17beta-hydroxy-19-norandrost-4-en-3-one (norethandrolone) caused a time-dependent loss of cytochrome P-450 when incubated in vitro with rat liver microsomal fractions and NADPH-generating systems. 2. The enzyme system catalysing the norethindrone-mediated loss of cytochrome P-450 had many characteristics of the microsomal mixed-function oxidases. It required NADPH and air, and was inhibited by Co. However, it was unaffected by 1 mM-compound SKF 525A. 3. In microsomal fractions from phenobarbitone-pretreated rats the norethindrone-mediated loss of cytochrome P-450 was increased relative to controls. The norethindrone-mediated cytochrome P-450 loss was less pronounced when the animals were pretreated with 3beta-hydroxy-pregn-5-en-2-one 16alpha-carbonitrile (pregnenolone 16alpha-carbonitrile). Pretreatment with 3-methylcholanthrene rendered the animals resistant to the norethindrone effect. 4. Administration in vivo [100mg/kg, intraperitoneally] of norethindrone or ethinyl oestradiol also produced a time-dependent loss of liver cytochrome P-450. Norethandrolone had a similar, though much less-marked, effect. All three steroids lead to an induction of 5-aminolaevulinate synthase and an accumulation of porphyrins in the liver. 5. The loss of cytochrome P-450 and the accumulation of porphyrins in the liver 2 h after the administration of norethindrone to female rats was similar to that seen in males. 6. Rats pretreated with phenobarbitone and given norethindrone or ethynyloestradiol (100mg/kg, intraperitoneally) formed green pigments in their livers. These had characteristics similar to the green pigments produced in the livers of rats after the administration of 2-allyl-2-isopropylacetamide. No green pigments could be extracted from the livers of control rats or those given norethandrolone, oestradiol or progesterone.     

Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini

An improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15 beta,16 beta-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7 alpha- and 15 alpha-hydroxylation of 1 and 7 alpha-hydroxylation of 2, while B. malorum gave 7 beta-hydroxylation of both the substrates. The stability of the enzymatic activity was higher in the presence of co-substrates (i.e., glucose or mannitol) allowing for repeated batches of the biotransformations. The yields of 7 alpha,15 alpha-dihydroxy-1 production were improved obtaining 5.8 gl(-1) (recovered product) from 7.0 gl(-1) of substrate. The structures of the hydroxylated products were assigned by a combination of two-dimensional NMR proton-proton and proton-carbon correlation techniques.