Aromatization of testosterone and 19-nortestosterone by a single enzyme from equine testicular microsomes. Differences from human placental aromatase

Silberzahn, Pierre; Gaillard, Jean‐Luc; Quincey, D.; Dintinger, Thierry; Al-Timimi, Ihsan

doi:10.1016/0022-4731(88)90385-8

Cited by 26 publications

(14 citation statements)

References 24 publications

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“…Thus, published findings have claimed that aromatization of 19-norandrogens is both possible [11][12][13][14] or not possible [7][8][9][10] using various in vitro systems including placental microsomes and granulosa or Leydig cells. Previous publications describing a variety of aromatase preparations and detection methods suggested that both MENT and 19-NT could be aromatized.…”

Section: Discussionmentioning

confidence: 99%

“…Aromatization of C19 androgens such as T and androstenedione (AD) to their C18 aromatic A-ring products, estradiol and estrone (E 1 ), respectively, takes place in three sequential steps [4][5][6]: (1) hydroxylation of the C19-methyl group; (2) a second hydroxylation of the C19-methyl group to form a 19-oxo-compound; (3) cleavage of the bond between C10 and C19 to yield formic acid and the aromatized A-ring product with loss of the hydrogen atoms at the 1β and 2β positions. Although the mechanism of action of human aromatase involves the C19-methyl group, published findings concerning the potential aromatization of 19-norandrogens in vitro and in vivo have been contradictory [7][8][9][10][11][12][13][14]. Most of these studies have employed human placental microsomes or Leydig or granulosa cells which contain other enzymatic activities, and detection methods involving thin layer or gas chromatography, HPLC, or radioimmunoassays.…”

Section: Introductionmentioning

confidence: 99%

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Dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

Attardi

Pham

Radler

et al. 2008

The Journal of Steroid Biochemistry and Molecular Biology

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Dimethandrolone undecanoate (DMAU: 7α,11β-dimethyl-19-nortestosterone 17β-undecanoate) is a potent orally active androgen in development for hormonal therapy in men. Cleavage of the 17β-ester bond by esterases in vivo leads to liberation of the biologically active androgen, dimethandrolone (DMA), a 19-norandrogen. For hormone replacement in men, administration of C19 androgens such as testosterone (T) may lead to elevations in circulating levels of estrogens due to aromatization. As several reports have suggested that certain 19-norandrogens may serve as substrates for the aromatase enzyme and are converted to the corresponding aromatic A-ring products, it was important to investigate whether DMA, the related compound, 11β-methyl-19-nortestosterone (11β-MNT), also being tested for hormonal therapy in men, and other 19-norandrogens can be converted to aromatic A-ring products by human aromatase. The hypothetical aromatic A-ring product corresponding to each substrate was obtained by chemical synthesis. These estrogens bound with high affinity to purified recombinant human estrogen receptors (ER) α and β in competitive binding assays (IC 50 's: 5−12 × 10 −9 M) and stimulated transcription of 3XERE-luciferase in T47Dco human breast cancer cells with a potency equal to or greater than that of estradiol (E 2 ) (EC 50 's: 10 −12 to 10 −11 M). C19 androgens (T, 17α-methyltestosterone (17α-MT), androstenedione (AD), and 16α-hydroxyandrostenedione (16α-OHAD)), 19-norandrogens (DMA,, and 7α-methyl-19-nortestosterone (MENT)) or the structurally similar 19-norprogestin, norethindrone (NET) were incubated at 50 μM with recombinant human aromatase for 10−180 min at 37 °C. The reactions were terminated by extraction with acetonitrile and centrifugation, and substrate and potential product were separated by HPLC. Retention times were monitored by UV absorption, and UV peaks were quantified using standard curves. Aromatization of the positive controls, T, AD, and 16α-OHAD was linear for 40−60 min, and conversion of T or AD was complete by 120 min. The nonsteroidal aromatase inhibitor, letrozole, demonstrated concentration-dependent suppression of T aromatization. Under the same conditions, there was no detectable conversion of DMA, 11β-MNT, or NET to their respective hypothetical aromatic A-ring products during incubation times up to 180 min. Aromatization of MENT and 19-NT proceeded slowly and was limited. Collectively, these data support the notion that in the absence NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of the C19-methyl group, which is the site of attack by oxygen, aromatization of androgenic substrates proceeds slowly or not at all and that this reaction is impeded by the presence of a methyl group at the 11β position.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

Attardi

Pham

Radler

et al. 2008

The Journal of Steroid Biochemistry and Molecular Biology

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“…Estrogen biosynthesis occurs in several tissues and cells of mammals, including adipose tissue, skin fibroblasts, brain, pituitary, prostate, testis, ovary, placenta, and retina (3)(4)(5)(6)(7)(8)(9)(10). P450arom has been immunohistochemically localized in the syncytiotrophoblast of the human placenta (11)(12)(13)(14)(15), in ovaries of human, rat, mice, golden hamster, guinea pig, and cow (11,14,(16)(17)(18)(19)(20)(21)(22), in testes of rat, mouse, Supported in part by a grant from the Association pour la Recherche sur le Cancer (grant no.…”

Section: Introductionmentioning

confidence: 99%

Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.

Almadhidi

Seralini²,

Fresnel³

et al. 1995

J Histochem Cytochem.

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Estrogens are the major steroids produced by equine gonads.To identrfy the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immuneblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromtase (P45Oamm) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for IntroductionThe conversion of androgens to estrogens, termed aromatization, is catalyzed by P45Oarom enzyme, the product of the gene called CYP19 in humans (1). Recently, Osawa et al. (2) showed that aromatase also acts as estrogen 2-hydroxylase and postulated that this enzyme could be responsible for the high levels of catechol estrogens and 19-hydroxyandrogens during pregnancy. The enzymatic complex, which is located in the endoplasmic reticulum, includes the heme glycoprotein P450arom and the flavoprotein NADPH-cytochrome P450 reductase.Estrogen biosynthesis occurs in several tissues and cells of mammals, including adipose tissue, skin fibroblasts, brain, pituitary, prostate, testis, ovary, placenta, and retina (3-10). P450arom has been immunohistochemically localized in the syncytiotrophoblast of the human placenta (11)(12)(13)(14)(15), in ovaries of human, rat, mice, golden hamster, guinea pig, and cow (11,14,(16)(17)(18)(19)(20)(21)(22), in testes of rat, mouse, Supported in part by a grant from the Association pour la Recherche sur le Cancer (grant no. 6933) and by a grant from the Ligue Nationale Fransaise contre le Cancer (Comitt de la Manche). and bear (23)(24)(25), in human prostate (6), in the brain of quail, rat, and mouse (26-28), and in the retina of goldfish (10).In mare, the concentration of estradiol-l7fl, which is the main estrogen in the follicular fluid, is greatly increased in the large follicles compared with the small follicles (8,29). The mare cyclic corpus luteum aromatizes androgens very efficiently (30), although only low levels of circulating estradioL17fl are present during the luteal phase (31,32). In comparison to other species (33,34), the stallion testis secretes very high amounts of estrogens, essentially as estrogen-3-sulfates. Plasma estrogens markedly increase with age (35), together with the number and volume of Leydig cells (36). However, the cellular localization of aromatase has not yet been established in equine ovary and testis.We have previously shown that equine aromatase exhibits some distinct properties compared with the human enzyme: the equine testicular, placental, and ovarian estrogen synthetase aromatizes androgens and 19-norandrogens with similar velocity, but not 16a-hydroxytestosterone or epitestosterone, in contrast to the human placental enzyme, which aromatizes androg...

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“…The equine estrogen synthetase enzyme aromatizes 19-norandrogens with a slightly higher velocity than androgens, but has a markedly weaker affinity for 19-norandrogens than for androgens [26,[42][43][44][45]. In contrast, HPMES aromatizes 19-norandrogens poorly [50], but has a much higher affinity for NT than does the equine estrogen synthetase (KJ KmM of 30 versus 158) [26].…”

Section: Discussionmentioning

confidence: 99%

“…MNT acetate, which had been selected for clinical trial, produced a masculinization and a regression of the breast cancer in treated patients [39-411. We have previously reported that, during the process of estrogen formation by equine estrogen synthetase, 19-hydroxyandrogen and 19-oxoandrogen are released and stereospecific loss of lp, 28 hydrogen atoms occurs [42-441. However, in contrast to the human estrogen synthetase, equine estrogen synthetase aromatizes 19-norandrogens very efficiently [26, [42][43][44][45]. Taking the above observations into account, we investigated the ability of the human and equine microsomal or purified estrogen synthetases to aromatize a 7a-methylated 19-norandrogen.…”

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confidence: 99%

Different in vitro metabolism of 7 α‐methyl‐19‐nortestosterone by human and equine aromatases

Moslemi

Dintinger

Dehennin

et al. 1993

European Journal of Biochemistry

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The ability of human and equine placental microsomes to aromatize 7α‐methyl‐19‐nortestoster‐one (MNT) was studied. Kinetic analysis indicates that MNT shares the androgen‐binding site of human and equine placental microsomal aromastases. Human placental microsomal estrogen synthetase had about a 2.5‐fold higher relative affinity for MNT than the equine placental enzyme (KiMNT/Km androstenedione of 32 versus 87). However, MNT was not metabolized by human placental microsomes, whereas it was very actively metabolized by equine placental microsomes. Further studies using purified equine cytochrome P‐450arom indicated that the presence of a 7α‐methyl group and the absence of a C19 methyl group did not impair its conversion by the purified enzyme. The product of this reaction was separated and identified as 7α‐methylestradiol by gas chromatography coupled to mass spectrometry.

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Aromatization of testosterone and 19-nortestosterone by a single enzyme from equine testicular microsomes. Differences from human placental aromatase

Cited by 26 publications

References 24 publications

Dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

Dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.

Different in vitro metabolism of 7 α‐methyl‐19‐nortestosterone by human and equine aromatases

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