Identification of 6α- and 7α-hydroxyestrone as major metabolites of estrone and estradiol in porcine uterus.

Maschler, Itzhak; Ball, Peter; Bayerköhler, G.; Gaues, J.; Knüppen, R.

doi:10.1016/0039-128x(83)90025-9

Cited by 10 publications

(6 citation statements)

References 9 publications

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“…Estradiol is metabolized after nuclear passage in porcine endometrium cells by dehydrogenation to estrone (1,2), and subsequent hydroxylation at either 6a-or 7a- (3). Our previous results sug¬ gested a localization of the 17ß-ol dehydrogenase in organdíes resembling lysosomes in size and density, although the pH optimum of the enzyme was in the neutral range.…”

mentioning

confidence: 91%

Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

Adamski¹,

Sierralta²,

Jungblut³

1989

Acta Endocrinologica

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Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17\g=b\ but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 \m=x\ 10\m=-\7 mol/l for estradiol and Km 4.4 \m=x\10\m=-\5mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 \m=x\10\m=-\6mol/1 for estrone and Km 2.1 \m=x\10\m=-\5mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17 000 \m=x\ g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200 000 \m=x\ g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrondense vesicles of 0.15\p=n-\0.20\g=m\min diameter as a common structural element which might harbour the dehydrogenase.Estradiol is metabolized after nuclear passage in porcine endometrium cells by dehydrogenation to estrone (1,2), and subsequent hydroxylation at either 6a-or 7a-(3). Our previous results sug¬ gested a localization of the 17ß-ol dehydrogenase in organdíes resembling lysosomes in size and density, although the pH optimum of the enzyme was in the neutral range. We re-investigated this discrepancy by employing novel techniques for homogenization and organeile separation (4). The ex¬ periments were also thought to clarify whether the prevailing oxidation of estradiol to estrone and the less prominent reversal are catalyzed by one and the same enzyme or by individual entities at differ¬ ent localizations. This might help in exploring the mechanism by which the cell terminates the hor¬ monal stimulus. Materials and MethodsThiamine pyrophosphate was bought from Sigma (Mün¬ chen, FRG); NAD+ and NADPH from Boehringer Mann¬ heim (Mannheim, FRG); Percoll from Pharmacia (Frei¬ burg, FRG); Triton X-100 from Serva (Heidelberg, FRG); xylene-based fluor, estrone, estradiol, estriol and all other grade A chemicals were from E.Merck (Darmstadt, FRG). Tritium-labelled steroids [2,4,6,7-3H]estrone,specific activity 95 Ci/mmol and [6,7-3H] estradiol, specific activity 59 Ci/mmol were from New England Nuclear (Dreieich/FRG) and purified before use. Bond Elut RP-18 (100 mg) solid-phase extraction columns were from Analytichem International (Harbor City, CA); reversed-phase columns LiChrosorb RP-18, RT 250-4, 5 um, and guard columns, RT 4-4, 10 um, were from E.Merck; acetonitrile HPLC-grade was bought from Zinsser (Frankfurt, FRG). Milli-Q H20, Millipore (Eschborn, FRG) was used for HPLC.Endometrium was ...

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mentioning

confidence: 91%

Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

Adamski¹,

Sierralta²,

Jungblut³

1989

Acta Endocrinologica

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“…The catalytical property of 17 -HSD 4, revealing the virtually unidirectional oxidative activity, clearly defines it as a steroid inactivating enzyme (Gurpide & Marks 1981), since it produces estrone which shows little affinity to the estradiol receptor. The conversion of 17 -estradiol to estrone might be complemented by hydroxylations in positions 6 or 7 (Maschler et al 1983) producing steroids devoid of estradiol receptor affinity and permitting fast release from cells after formation. The V max and K m values for EDH are similar to those for estrone hydroxylases (Adamski et al 1994) and allows the metabolic conversion 17 -estradiol<estrone<6 / 7 -hydroxy-estrone without rate-limiting steps.…”

Section: Mutations In the Hsd17b4 Genementioning

confidence: 99%

Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome

Launoit¹,

Adamski²

1999

Journal of Molecular Endocrinology

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Six types of human 17 -hydroxysteroid dehydrogenases catalyzing the conversion of estrogens and androgens at position C17 have been identified so far. The peroxisomal 17 -hydroxysteroid dehydrogenase type 4 (17 -HSD 4, gene name HSD17B4) catalyzes the oxidation of estradiol with high preference over the reduction of estrone. The highest levels of 17 -HSD 4 mRNA transcription and specific activity are found in liver and kidney followed by ovary and testes. A 3 kb mRNA codes for an 80 kDa (737 amino acids) protein featuring domains which are not present in the other 17 -HSDs. The N-terminal domain of 17 -HSD 4 reveals only 25% amino acid similarity with the other types of 17 -HSDs. The 80 kDa protein is N-terminally cleaved to a 32 kDa enzymatically active fragment. Both the 80 kDa and the N-terminal 32 kDa (amino acids 1-323) protein are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with -3-hydroxyacyl-coenzyme A (CoA). The enzyme is not active with -stereoisomers. The central part of the 80 kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80 kDa protein (amino acids 597-737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The HSD17B4 gene is stimulated by progesterone, and ligands of PPAR (peroxisomal proliferator activated receptor alpha) such as clofibrate, and is down-regulated by phorbol esters. Mutations in the HSD17B4 lead to a fatal form of Zellweger syndrome.

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“…It has been known for decades that E 2 could be metabolized to 6␣-OH-E 2 and 6␤-OH-E 2 in both animals and humans (Mueller and Rumney, 1957;Breuer et al, 1966). A later study also reported that 6␣-OH-E 1 and 6␤-OH-E 1 were the major metabolites formed after incubation of E 1 with porcine uterine endometrial tissues (Maschler et al, 1983). In our recent study when E 2 was the substrate (Lee et al, 2001), formation of both 6␣-and 6␤-OH-E 2 by human liver microsomes was observed.…”

Section: Discussion Liver Microsomal Hydroxylation Of E 1 At Various mentioning

confidence: 99%

NADPH-Dependent Metabolism of Estrone by Human Liver Microsomes

Lee

Mills²,

Kosh³

et al. 2002

The Journal of Pharmacology and Experimental Therapeutics

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We characterized the NADPH-dependent metabolism of estrone (E 1 ) by liver microsomes of 21 male and 12 female human subjects. The structures of 11 hydroxylated or keto metabolites of E 1 formed by human liver microsomes were identified by chromatographic and mass spectrometric analyses. 2-Hydroxylation of E 1 was the dominant metabolic pathway with all human liver microsomes tested. E 1 is more prone to form catechol estrogens (particularly 4-OH-E 1 ) than 17␤-estradiol (E 2 ) and the average ratio of E 1 4-hydroxylation to 2-hydroxylation (0.24) was slightly higher than the ratio of E 2 4-to 2-hydroxylation (0.20, P Ͻ 0.001). An unidentified monohydroxylated E 1 metabolite (y-OH-E 1 ) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6␤-OH-E 1 , 16␣-OH-E 1 , and 16␤-OH-E 1 were also formed in significant quantities. 16␣-Hydroxylation was not a major pathway for E 1 metabolism. The overall profiles for the E 1 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. Hepatic CYP3A4/5 activity in both male and female liver microsomes correlated strongly with the rates of formation of several hydroxyestrogen metabolites. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of human CYP3A4 or CYP3A5 with [ 3 H]E 1 and NADPH. Notably, human CYP3A5 has very high relative activity for E 1 4-hydroxylation, exceeding its activity for E 1 2-hydroxylation by ϳ100%. It will be of interest to determine the potential biological functions associated with any of the E 1 metabolites identified in our present study.The endogenous estrogens 17␤-estradiol (E 2 ) and estrone (E 1 ) undergo extensive metabolism in the body (their structures and possible sites for oxidative metabolism are illustrated in Fig.

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Identification of 6α- and 7α-hydroxyestrone as major metabolites of estrone and estradiol in porcine uterus.

Cited by 10 publications

References 9 publications

Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome

NADPH-Dependent Metabolism of Estrone by Human Liver Microsomes

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