Robert A. Meigs

Cytochrome P-450 and steroid biosynthesis in the human placenta.

Abstract 1. 1. Cytochrome P-450 was detected and measured in human term placenta and its role in steroid hormone biosynthesis by this endocrine tissue was examined. 2. 2. The cytochrome P-450 concentration in mitochondria was found to be several times that in microsomes but no qualitative difference in properties of cytochrome P-450 located in different particulate fractions was found. 3. 3. Side-chain cleavage of cholesterol, catalyzed by mitochondrial preparations, was inhibited by CO in the dark. Light partially reversed this inhibition. 4. 4. CO did not inhibit the aromatization of androst-4-ene-3,17-dione by microsomes under conditions of limiting oxygen supply. 5. 5. The participation of cytochrome P-450 in these key steps in the biosynthesis of steroid hormones by the human placenta is discussed.

The formation of progesterone by the human placenta

Abstract The net synthesis of progesterone by human placental homogenates and isolated mitochondria has been described for the first time. The availability of possible blood borne precursors has been screened for in maternal and fetal sera extracts. There appears to be enough pregnenolone in fetal blood to act as one potential source of progesterone formation. Although there is adequate cholesterol present in blood supplying the placenta, this cannot be demonstrated to be a significant source without radioactive tracer studies. This appears to be due to an adequate endogenous precursor, already within the placenta, which is tentatively identified as cholesterol itself. Cholesterol declines as progesterone is formed by the mitochondria. The factor which most affects progesterone formation under these conditions is TPNH. It is hoped to use this isolated steroid synthesizing system to explore some of the possible mechanisms for the endocrine control of pregnancy.

Aromatization of steroids by mitochondrial preparations from human term placenta

Abstract Structural and functional aspects of the association of aromatizing activity with placental mitochondrial preparations were examined. Total mitochondrial preparations, previously characterized with respect to their capacity for oxidative phosphorylation and for progesterone synthesis, converted androst-4-ene-3, 17-dione to estrogens with specific activities averaging one-half those of corresponding microsomal fractions. Heavy mitochondria separated by differential centrifugation showed specific activities 16% of microsomal activities. Metabolically active mitochondria, selectively isolated by calcium phosphate loading combined with sucrose density gradient fractionation, also displayed significant aromatizing activity despite considerable inactivation by the experimental procedures. An exogenous NADPH-generating system supported maximum rates of mitochondrial aromatization. No stimulation of aromatization was induced when mitochondrial permeability barriers were disrupted by 11 mM CaCl 2 although cholesterol side-chain cleavage activity was doubled by this treatment. Mitochondrial dehydrogenase systems required exogenous NADP + for effective support of aromatization. Under these conditions, Ca 2+ stimulated aromatization when malate served as metabolic substrate but had no effect on the support of aromatization by citrate or isocitrate. Thus, aromatizing activity of placental mitochondria appears to be firmly associated with or localized in the external mitochondrial compartment and to be less directly integrated with internal metabolic processes for NADPH generation than is the mixed-function oxidase system for cholesterol side-chain cleavage.

The constitutive 7-ethoxycoumarin O-deethylase activity of human placental microsomes: Relationship to aromatase

The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes.

ESTROGEN-INDUCED 16-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN RAT KIDNEY.

The activity of an enzyme system which readily converts estriol to 16-ketoestradiol is present only in the kidneys of mature female rats and is absent in the kidneys of male rats and immature rats of either sex. The full enzyme activity occurs in kidneys of rats of either sex at any stage of maturity after administration of estradiol for 2 weeks. The time course of the appearance and disappearance of the enzyme, the absence of detectable inhibitors and activators, and evaluation of cofactor requirements suggest that this may be an example of enzyme induction by the hormone. Furthermore, the enzyme activity itself represents a metabolic pathway for estriol metabolism whose potential quantitative significance has not heretofore been recognized.

Inhibitor specificity of the placental microsomal oxidase system responsible for the aromatization of epitestosterone (17α-hydroxy-4-androsten-3-one)

Human placental microsomes converted epitestosterone to estradiol-17 alpha at rates of 23-48 pmol/min X mg protein with a Km of 113 microM. Activity was inhibited 70-90% by concentrations of CO, metyrapone, n-octylamine, 7,8-benzoflavone and 7-ethoxycoumarin which had no effect on the aromatization of 4-androstene-3, 17-dione. Conversely, cyanide and azide were more effective inhibitors of the conversion of the latter androgen. A variety of neutral steroids inhibited the aromatization of epitestosterone with 19-norsteroids being particularly effective, but competitive effects could not be demonstrated. Both 17 beta-hydroxy-4-estren-3-one and 16 alpha-hydroxy-4-androstene-3,17-dione caused a mixed inhibition. A number of phenolic steroids were also inhibitory with 16-oxo compounds being particularly effective. Inhibition by estrone was non-competitive (Ki = 16 microM). The aromatization of epitestosterone resembles placental microsomal oxidase activities against estrone and benzo [a]pyrene in its inhibitor specificity and epitestosterone may be the native substrate for an oxidase also active in the metabolism of aromatic xenobiotic chemicals.

Quantitative requirements for NADPH in the support of aromatization by human placental microsomes

Suitable incubation conditions were developed for reduced pyridine nucleotide protection and regeneration to permit quantitative assessment of the NADPH requirement for steroid aromatization by human placental microsomes. 10 mM dithiothreitol was found to protect NADP(H) from microsomal nucleotide pyrophosphatase and 2 mM nicotinamide mononucleotide was utilized to control nucleotide glycohydrolase activity. Under these assay conditions, the initial rates of aromatization obtained with restricted NADPH levels were critically dependent upon both the amount and the source of exogenous NADPH-regenerating dehydrogenase system. With excess Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, an apparent Km for NADPH of 0.20 microM was observed for aromatization which is significantly below all previous estimates of the NADPH requirement and which is at greatest only one-tenth the Km value for NADPH utilization by NADPH-cytochrome c reductase. These findings suggest a potential regulatory role for both NADPH-generating and NADPH-accepting enzymes in the support of estrogen biosynthesis.

The constitutive 7-ethoxycoumarin 0-deethylase of human placental microsomes: relationship to the intermediary steps in steroid aromatization

All oxidative functions of aromatase, i.e., estrogen production, 19-oxygenated androgen production and 7-ethoxycoumarin deethylation, were inhibited in parallel in placental microsomes from non-smokers by the mechanism-based, time-dependent inactivators (suicide substrates) 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione. In contrast, the aromatase suicide substrate androst-4-ene-3,6,17-trione had little or no effect on the conversion of androst-4-ene-3,17-dione to 19-hydroxyandrost-4-ene-3,17-dione or on the conversion of the latter to 3,17-dioxoandrost-4-en-19-al while severely limiting the capacity for estrogen production from androst-4-ene-3,17-dione and 19-hydroxyandrost-4-ene-3,17-dione in such microsomal preparations. Androst-4-ene-3,6,17-trione, therefore, appears to uncouple the 19-hydroxylation of androgens from estrogen synthesis. This agent also produced only a minimal inhibition of 7-ethoxycoumarin deethylation, indicating that this major constitutive transformation of a xenobiotic chemical is associated with the steroid 19-hydroxylating function of the aromatase system.

The support of steroid aromatization by mitochondrial metabolic activities of the human placenta

The role of mitochondrial hexokinase (EC 2.7.1.1.) and mitochondrial ATP synthesis in the utilization of glucose for the support of estrogen biosynthesis was examined in placental mitochondrial preparations supplemented with NADP+, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 4-androstene-3,17-dione. With 14 mitochondrial preparations, rates of steroid aromatization supported by 100 mM glucose and 20 mM ATP had a mean of 65.7 +/- 7.1 (SD) % of rates achieved with saturating levels of glucose 6-phosphate. ADP, but not AMP, could substitute for ATP in this system. Aromatization supported by glucose and high concentrations of ADP was inhibited by AMP but not by 2,4-dinitrophenol or oligomycin. Glucose also supported mitochondrial aromatization when combined with a respiratory chain-linked metabolic substrate (glycerol 3-phosphate) and a limiting concentration of ADP (2 mM). This support was inhibited by 2,4-dinitrophenol, the p-trifluoromethoxyphenylhydrazone of carbonyl cyanide, oligomycin and atractyloside. Thus, glucose metabolism by mitochondrial hexokinase, utilizing ATP generated either by oxidative phosphorylation or mitochondrial adenylate kinase (EC 2.7.4.3), can be coupled with a soluble NADPH-generating system to provide effective support of mitochondrial estrogen synthesis.

Cytochrome P-450 Levels of Immature Rat Ovaries During Early Follicular Development: Quantification, Cellular and Subcellular Distribution, Enzymatic Activity and Response to FSH In Vivo and In Vitro

Absolute values of cytochrome P-450 (cyt. P-450) content were determined by dual beam spectrophotometry in mitochondrial and microsomal fractions of granulosa cells and stroma (theca and interstitial cells) obtained from ovaries of immature rats treated with diethylstilbestrol (DES) or with DES and FSH. Cholesterol side-chain cleavage activity and aromatase activity were also measured in relevant cell fractions. The cyt. P-450 content in the granulosa cell mitochondria was significantly increased in FSH-treated animals (cyt.P-450 in nmols/mg protein, mean +/- SE: DES-treated 0.027 +/- 0.00213, N = 9; DES/FSH-treated 0.0668 +/- 0.0120, N = 13, P = 0.014). The increased cyt. P-450 content was associated with an increase in cholesterol side-chain cleavage activity confirming the functional relevance of the spectrophotometric measurements. The increase in cyt. P-450 content in stroma after FSH treatment was not statistically significant but there was a significant increase in cholesterol side-chain cleavage activity. The cholesterol side-chain cleavage activity of intact granulosa cells from secondary follicles of DES-treated rats was significantly stimulated by in vitro exposure to FSH during 2 hr assay incubations. A significant cyt. P-450 content was detected in the microsomal fraction of granulosa cells from DES-treated rats which was not associated with aromatase activity. Granulosa cell microsomal cyt. P-450 content was not significantly altered by in vivo FSH treatment despite a dramatic increase in aromatase activity of such microsomal preparations.