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PubMedDate not supplied

Equine ovarian aromatase: evidence for a species specificity.

Mare granulosa cells and cyclic corpus luteum microsomes are reported to aromatize 19-norandrogens more efficiently than androgens. However, 16 alpha-hydroxytestosterone and epitestosterone were not aromatized by the equine corpus luteum microsomal estrogen synthetase. These results indicate that the equine aromatase system would be different from the human placental microsomal estrogen synthetase, which aromatizes 16 alpha-hydroxyandrogens and epitestosterone but not 19-norandrogens. Furthermore, our data show that the rates of aromatization of androgens and 19-norandrogens were not additive and that 19-norandrogens competitively inhibited the aromatization of androgens, suggesting that a single enzymic system would be involved in the aromatization of androstenedione, 19-norandrostenedione, testosterone, and 19-nortestosterone. Our findings, which are identical to those previously reported for stallion testis and mare placental estrogen synthetases, provide evidence for a strong species specificity of the equine aromatase system.

Androstenedione↗

PubMed2006

Roles of 11beta-hydroxysteroid dehydrogenase in fish spermatogenesis.

In fish spermatogenesis, the main action of progestins is generally regarded as the induction of sperm maturation. Our previous in vitro study demonstrated that a progestin, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), induced the initiation of meiosis in spermatogenesis in the Japanese eel (Anguilla japonica). In the present study, to elucidate the molecular mechanisms underlying the action of DHP, we attempted to clone cDNAs encoding genes whose expression was induced by DHP in eel testis, using cDNA subtraction. One of the cDNAs we isolated encodes eel 11beta-hydroxysteroid dehydrogenase short form (e11beta-HSDsf), and Northern blot and RT-PCR analysis showed that transcripts of e11beta-HSDsf in testis were induced by DHP. The recombinant e11beta-HSDsf had 11beta-dehydrogenase activity, metabolizing cortisol to cortisone, and 11beta-hydroxytestosterone to 11-ketotestosterone (11-KT). In vitro experiments revealed that eel immature testis had 11beta-dehydrogenase activity, and DHP treatment enhanced the activity. To understand the role of 11beta-HSD in spermatogenesis, we examined the direct effects of cortisol on eel spermatogenesis using an organ culture system. Cortisol induced DNA replication in spermatogonia and enhanced the spermatogonial proliferation induced by 11-KT. However, excess cortisol inhibited proliferation. In addition, 11-KT production was induced in testicular fragments incubated with cortisol. These results suggest that optimal levels of cortisol induced spermatogonial mitosis by increasing 11-KT production. Furthermore, two possible roles of DHP on spermatogenesis, via the up-regulation of 11beta-HSD expression, are suggested: positive feedback control of 11-KT production and the modulation of cortisol levels to protect testes from excess circulating cortisol.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1982

Steroid production in testicular tissue of the European eel.

Testicular tissue of normal and hCG-stimulated European eels was incubated in vitro with tritiated progesterone or androstenedione as substrates. The following compounds were isolated and identified: 5 beta-androstane-3,17-dione; 17 beta-hydroxy-5 beta-androstan-3-one; androst-4-ene-3,11,17-trione (adrenosterone); 11 beta-hydroxyandrost-4-ene-3,17-dione; 11 beta-hydroxytestosterone; 3 alpha,11 beta-dihydroxy-5 beta-androstan-17-one, and an additional steroid for which the oxidation product was identified as 5 beta-androstene-3,11,17-trione. Four of these steroids have not been hitherto identified in gonadal tissue of any vertebrate. The pattern of steroid production in this tissue is unique for its 5 beta-reduction, for the appearance of adrenosterone as a major metabolite, and for the lack of production of 11-ketotestosterone, which is a regular metabolite of gonadal tissue of teleosts. Thus, it appears that steroid metabolism in the eel testis deviates considerably from the known pattern of steroid production in gonads of other vertebrates.

Androgens↗

PubMed1991

Androgen metabolism by porcine granulosa cells during the process of luteinization in vitro: identification of 19-oic-androstenedione as a major metabolite and possible precursor for the formation of C18 neutral steroids.

The present studies were conducted to define the pathway(s) by which androstenedione is metabolized in porcine granulosa cells (pGC) and determine whether metabolism of this steroid is affected by in vitro luteinization. pGC isolated from large preovulatory follicles were cultured for up to 2 days in the presence of 5 microM unlabeled or [4-14C]-labeled androstenedione. Metabolism of androstenedione was assessed by HPLC, using in-line liquid scintillation detection. Metabolite identification was confirmed by gas chromatography-mass spectrometry of HPLC fractions isolated from medium conditioned by granulosa cells (pGCCM) cultured for 48 h in the presence of unlabeled androstenedione. The metabolites identified were 19-oic-androstenedione (3,17-dioxo-4-androsten-19-oic acid), 19-hydroxytestosterone, 19-hydroxyandrostenedione, 19-nor-testosterone, an estrenolone of as yet unproven stereoisomerism, 5(10)-estrene-3 beta, 17 beta-diol, 17 beta-estradiol, testosterone, and 19-nor-androstenedione. Results indicate that 19-nor-androstenedione is artifactually derived from 19-oic-androstenedione as a result of degradation in storage and during isolation. After metabolite identification, studies of the time course of androstenedione metabolism by pGC during in vitro luteinization were conducted. 17 beta-Estradiol and 19-oic-androstenedione were the predominant metabolites, and accumulation of these steroids was virtually identical. Production of these metabolites was maximal during the first 12 h of culture. The accumulation of 5(10)-estrene-3 beta,17 beta-diol and 19-nor-testosterone was maximal at 48 h of culture, with 5(10)-estrene-3 beta,17 beta-diol consistently accumulating in greater concentrations than 19-nor-testosterone. Aromatase activity of pGC was negligible from 36-48 h of culture, as demonstrated by minimal accumulation of 17 beta-estradiol during this period of culture. The accumulation of 19-oic-androstenedione, 5(10)-estrene-3 beta,17 beta-diol, and 19-nor-testosterone was also negligible during this latter time period, suggesting that their formation is associated with aromatase. From these results, pGC from preovulatory follicles undergoing luteinization in vitro lose the ability to convert androstenedione to estrogens. The formation of 19-oic-androstenedione, shown here for the first time, parallels the formation of 17 beta-estradiol, and this acidic steroid is proposed to be a product of aromatase. As reported in previous studies, pGC do produce C18 neutral steroids from exogenous androstenedione. The production of these steroids requires an active aromatase to produce their immediate precursor, which is here hypothesized to be 19-oic-androstenedione. However, their maximal production does not commence until aromatase activity has declined, and it is hypothesized that their production depends on modifications in steroid metabolism associated with luteinization.

Androgens↗

PubMed2004

Ethanol inhibits in-vitro metabolism of nifedipine, triazolam and testosterone in human liver microsomes.

Although extended exposure to ethanol induces CYP3A metabolism in-vivo, the acute effects of ethanol on CYP3A metabolism have not been fully evaluated in-vitro. We assessed the effect of ethanol on CYP3A-mediated biotransformation using human liver microsomes in-vitro with three prototypic CYP3A-mediated reactions: nifedipine to oxidized nifedipine, triazolam to its 1-hydroxy (1-OH TRZ) and 4-hydroxy (4-OH TRZ) metabolites, and testosterone to 6beta-hydroxytestosterone (6beta-OH TST). Ethanol inhibited metabolism of nifedipine (oxidized nifedipine IC50 3 mg dL(-1), where the IC50 value is the inhibitor concentration corresponding to a 50% reduction in metabolite formation velocity), triazolam (1-OH TRZ IC50 1.1 mg dL(-1), 4-OH TRZ IC50 2.7 mg dL(-1)) and testosterone (6beta-OH TST IC50 2.4 mg dL(-1)). The inhibitory potency of ethanol was similar for the three substrates representing the three hypothetical CYP3A substrate categories. The IC50 values obtained were lower than clinically relevant blood alcohol concentrations. In conclusion, ethanol is an inhibitor of human CYP3A metabolism and may contribute to clinically important interactions.

Androgens↗

PubMed1985

Developmental changes in the rate of production of an unusual testosterone metabolite, 4-androstene-3 alpha, 17 beta-diol, by chick liver microsomes.

Testosterone is produced by the chick embryo testis from the 13th day of incubation. We have investigated the ability of microsomes prepared from the fetal and neonatal liver to metabolize testosterone and have found that the principal metabolite generated by microsomes in the presence of NADPH is 4-androstene-3 alpha, 17 beta-diol. The rate of production of this metabolite declined sharply over the time of hatching. Conversely, 16 alpha-hydroxytestosterone production increased transiently just after hatching. Our findings indicate that chick liver microsomes contain a 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid: NAD(P) oxidoreductase, EC 1.1.1.50) whose activity changes during development.

Androstenediols↗

PubMed1987

A quantitative study of steroid bioconversions in the testis of the African catfish, Clarias gariepinus (Burchell), under natural spawning and natural and cultivated non-spawning conditions.

Quantitative aspects of bioconversions in the testes of the African catfish (Clarias gariepinus) were studied in vitro by incubation of tissue with [3H]pregnenolone or [3H]androstenedione. During the breeding period, spawning and non-spawning animals were collected from their natural habitat, the Hula nature reserve, in northern Israel. In the same period, non-spawning animals were collected from a fish pond in the same region. It was shown that spawning was accompanied by significant changes in steroid bioconversions, i.e. a reduction in androgen synthesis, especially of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone and an increase in the production of C21-steroids, especially progesterone, 17 alpha-hydroxyprogesterone and a pregnenolone ester. These changes resulted from a decreased contribution of the cytochrome P-450 enzymes 17 alpha-hydroxylase, C17-20-lyase and 11 beta-hydroxylase. A rise in plasma gonadotrophin concentration was observed only in spawning catfish. In the absence of such an increase in plasma gonadotrophin, steroid synthesis in the testes of non-spawning feral and pond catfish was primarily directed towards the production of 11-oxygenated androgens and 5 beta-pregnane-3 alpha,17 alpha,20 alpha-triol. It is suggested that spawning is induced by gonadotrophin and the ensuing change in steroidogenesis. It is possible that husbandry conditions inhibit the necessary increase in gonadotrophin release.

Animals↗

PubMed2000

[Viability and differentiation of human hepatocytes immunoprotected by macroencapsulation and transplanted in rats].

OBJECTIVES: To determine the viability and differentiation of human hepatocytes immunoprotected by encapsulation and transplanted in rats without immunosuppression. METHODS: Freshly isolated human hepatocytes were encapsulated in hollow fibers and transplanted in the peritoneal cavity of immunocompetent rats. The fibers were explanted for analysis at D3, D7 and D14 following transplantation. Morphological features under light and electron microscopy and gene expression were compared to those of non-transplanted encapsulated hepatocytes (D0). Human cytochrome P450 3A and albumin mRNAs were quantified by Northern blot. Cytochrome P450 3A proteins were detected by Western blot and cytochrome P450 3A enzyme activity was assessed by measuring the formation of 6beta-hydroxytestosterone by high performance liquid chromatography. RESULTS: Transplanted hepatocytes were more than 60 % viable and exhibited morphological criteria of hepatocytic differentiation up to D7. Albumin and cytochrome P450 3A transcripts were also detected up to D14. At D3 and D7, albumin mRNA levels were of 30 %, compared to control D0 hepatocytes, while cytochrome P450 3A5 and cytochrome P450 3A4 mRNA levels were 65 % and 0 %, respectively. Cytochrome P450 3A immunoreactivity was detected by Western blot up to D14 and 6beta-hydroxylase activity was 17 % at D3 compared to D0, supporting with disappearance of cytochrome P450 3A4 mRNA. CONCLUSIONS: Human hepatocytes remain viable for a short period, following encapsulation and intraperitoneal transplantation in rat. Other experimental conditions need to be tested to prevent or delay a decrease in hepatocyte specific gene expression.

Animals↗

PubMed2001

Multisite kinetic models for CYP3A4: simultaneous activation and inhibition of diazepam and testosterone metabolism.

Some substrates of cytochrome P450 (CYP) 3A4, the most abundant CYP in the human liver responsible for the metabolism of many structurally diverse therapeutic agents, do not obey classical Michaelis-Menten kinetics and demonstrate homotropic and/or heterotropic cooperativity. The unusual kinetics and differential effects observed between substrates of this enzyme confound the prediction of drug clearance and drug-drug interactions from in vitro data. We have investigated the hypothesis that CYP3A4 may bind multiple molecules simultaneously using diazepam (DZ) and testosterone (TS). Both substrates showed sigmoidal kinetics in B-lymphoblastoid microsomes containing a recombinant human CYP3A4 and reductase. When analyzed in combination, TS activated the formation of 3-hydroxydiazepam (3HDZ) and N-desmethyldiazepam (NDZ) (maximal activation 374 and 205%, respectively). For 3HDZ, V(max) values remained constant with increasing TS, whereas the S(50) and Hill values decreased, tending to make the data less sigmoidal. Similar trends were observed for the NDZ pathway. DZ inhibited the formation 6beta-hydroxytestosterone (maximal inhibition, 45% of control), causing a decrease in V(max) but no significant change to the S(50) and Hill values, suggesting that DZ may inhibit via a separate effector site. Multisite rate equation models have been derived to explore the analysis of such complex kinetic data and to allow accurate determination of the kinetic parameters for activation and inhibition. The data and models presented are consistent with proposals that CYP3A4 can bind and metabolize multiple substrate molecules simultaneously; they also provide a generic solution for the interpretation of the complex kinetic data derived from CYP3A4 substrates.

Algorithms↗

PubMedDate not supplied

Investigation of conjugated metabolites of 4-hydroxyandrost-4-ene-3,17-dione in patient urine by liquid chromatography-atmospheric pressure ionization mass spectrometry.

Metabolism of the anticancer drug 4-hydroxyandrost-4-ene,3,17-dione (4OHA) was studied in cancer patients by HPLC-MS-MS. 40HA was administered orally to a breast cancer patient. The drug was extensively metabolized and was excreted in the urine as the 4OHA-glucuronide, 3 alpha-hydroxy-5 beta-androstan-4,17-dione (3 alpha OHA)-sulfate (or 4-hydroxytestosterone-sulfate) and 3 alpha,17-dihydroxy-5 beta-androstan-4-one (3,17-OHA)-sulfate conjugates in the 4 hr posttreatment sample. Other metabolites include 4OHA-sulfate, 3 alpha OHA-glucuronide, and 3,17-OHA-monoglucuronide. When 4OHA was given to the prostatic cancer patients intramuscularly, different metabolites were observed as compared with the female studies. The most noticeable difference is the absence of 4OHA-sulfate in both 24 and 48 hr posttreatment urine samples. The drug was eliminated mainly as 4OHA-glucuronide, 3 alpha OHA-sulfate, and 3,17-OHA-monosulfate. Other metabolites that have been detected include 3 alpha OHA-glucuronide, 3,17-OHA-glucuronide, 3,17-OHA-disulfate, and an unknown metabolite. The variation observed in metabolism could be attributed to a different route of drug administration (oral and intramuscular) and sex difference among the patients. This study describes the utilization of HPLC-MS-MS for monitoring the 4OHA conjugates and provides the first evidence of the presence of 4OHA-sulfate and its analogs in patient urinary extracts.

Androstenedione↗

PubMed2005

[Network for the collection and distribution of Japanese liver tissues resected surgically for drug development and research use].

We focused on the establishment of a trial procedure for the collection and distribution of Japanese liver tissues obtained from waste surgical resections for drug development and research use. The following procedures were prepared for this project: the pretreatment of liver tissues before storage, their storage at 4 degrees C, the transport of liver samples, the setting up of a communication network among the participating hospitals and laboratories and the approval of each ethics committee. Thirteen liver samples (1.6-7.6 g) obtained from patients whose livers were excised due to cirrhosis, hepatocellular carcinoma, cholangiocarcinoma, or metastasis from colorectal carcinoma and were donated for research. Informed consent was obtained from every patient. Freshly isolated human hepatocytes were prepared from nine liver samples (viability 34.3-86.1%). Four samples were unsuitable to prepare hepatocytes. The profile of testosterone metabolism as 6beta-, 2beta-, 16beta-, 16alpha- and 2alpha-hydroxytestosterone and androstenedione in freshly isolated hepatocytes was shown to be specific for human liver. The 6beta-hydroxylation activity catalyzed by CYP3A4/5 indicated a high level of metabolism (139-996 pmol/min/million cells). Levels of 7-ethoxycoumarin O-deethylation and glucronidation activities were sufficient for analysis in freshly isolated human hepatocytes. We conclude that liver tissues from waste surgical resections supplied from a participating hospital can constitute a valuable source of freshly isolated human hepatocytes for drug development and safety evaluation.

Aged↗

PubMed2006

Thin-layer chromatography analysis of human CYP3A-catalyzed testosterone 6beta-hydroxylation.

Testosterone and other steroid hormones have been studied as prototypic examples of endogenous substrates for hepatic cytochrome P450 (P450) enzymes. CYP3A enzymes from various species, including human, metabolize testosterone by a 6beta-hydroxylation reaction, which is unique to this P450 subfamily. A thin-layer chromatographic method is described for the determination of 6beta-hydroxytestosterone formed enzymatically by incubation of [14C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes. 14C-labeled enzymatic products are applied to silica gel thin-layer plates, which are developed sequentially with methylene chloride:acetone (80:20) followed by chloroform, ethyl acetate, and absolute ethanol (80:20:14). Metabolite quantification is performed by autoradiography and liquid scintillation counting. This method is applicable to enzymatic studies for the determination of CYP3A-dependent testosterone 6beta-hydroxylation activity in both human and animal liver microsomes.

Autoradiography↗

PubMedDate not supplied

cDNA-directed expression of rat P450s IIA1 and IIA2. Catalytic activities toward steroids and xenobiotics and comparison with the enzymes purified from liver.

Cytochromes P-450IIA1 and IIA2 are steroid hydroxylases that are expressed in rat liver. The cDNAs for these enzymes were recently sequenced and compared. To study and compare the catalytic activities of IIA1 and IIA2, their cDNAs were inserted into a vaccinia virus expression vector and expressed in human hepatoma Hep G2 cells. IIA2 was able to efficiently catalyze ethoxycoumarin O-deethylation and propoxycoumarin O-depropylation, while IIA1 was inactive toward these substrates. Neither enzyme could catalyze ethoxy- and pentoxyresorufin dealkylation reactions. Both cDNA-expressed IIA1 and IIA2 metabolize testosterone and these activities were quantitatively and qualitatively similar to those obtained with the purified enzymes. IIA1 produced 7 alpha-hydroxy, 6 alpha-hydroxy, and delta 6-testosterone at ratios of 9:0.5:0.5 while IIA2 formed 15 alpha-hydroxytestosterone, an unknown metabolite and four minor metabolites. Progesterone metabolism was also studied. IIA1 yielded a 9.5:0.5 ratio of 7 alpha-hydroxy and 6 alpha-hydroxyprogesterone, while IIA2 produced at least six metabolites. These studies establish the conditions and verify the reliability and accuracy of the vaccinia virus expression system for studies on the enzymology of IIA1 and IIA2.

Animals↗

PubMed1978

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.

Adenoma↗

PubMed1989

[Prolactin and specific binding of testosterone in cultured cells of the seminal vesicle of Gobius niger L].

Cultured seminal vesicle cells of Gobius niger L., precultured for about 15 days, are tested for their Testosterone-binding capacity. This whole cell system shows a specific binding of the androgen, reaching saturation in the presence of increasing amounts of ligand and the Scatchard Plot indicates a good affinity (KD = 4.4 x 10(-9) M), involving a number of sites of 5.06 x 10(-15) mole/culture. The possible existence of a second binding-site population with lower affinity and greater number of sites remains to be demonstrated. At 18 degrees C, the time course shows a maximal binding after about 60 min. of incubation, followed by a rapid decline at 90 min. The competition experiments involving estradiol, 11-keto- and 11-hydroxytestosterone indicate an effective if not total specificity to these steroids. The cross-reaction percentages at the 50% binding level are respectively 10.9, 8.5, 4.02%. Ovine Prolactin treatment of the cultures for 6 days before the binding experiment significantly improves the specific binding level of testosterone if compared to the controls (p .019, p .005). This result indicates that the Prolactin-Testosterone synergistic data already obtained on the seminal vesicles of Siluridae and Gobiidae is explained by a direct effect of the Prolactin on the androgen receptor level in the seminal vesicle cell.

Analysis of Variance↗

PubMed1989

Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormones and cyclosporine.

Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr approximately 52,000) was expressed in each of 40 individual specimens examined. In about 10-20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr approximately 52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a lambda gt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with lambda max at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6 beta-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15 beta-hydroxytestosterone), comprising up to approximately 20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (M1 and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.

Amino Acid Sequence↗

PubMed1988

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.

Androstenedione↗

PubMed1987

Purification and characterization of human placental aromatase cytochrome P-450.

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.

Androgens↗