Effects of Tibolone Metabolites on Human Endometrial Cell Lines in Co-culture

In human endometrium, cell proliferation is regulated by ovarian steroids through heterotypic interactions between stromal and epithelial cells populating this tissue. We tested the proliferative effects of tibolone and its metabolites using endometrial co-cultures that mimic the normal proliferative response to hormones. We found that both the Δ4-tibolone metabolite and the pure progestin ORG2058 counteract estradiol-driven epithelial cell proliferation. Surprisingly, the estrogen receptor binding 3-hydroxyl-metabolites of tibolone also counteracted estradiol-driven proliferation. Inhibition of proliferation by 3β-OH-tibolone was abrogated by low doses of the progesterone receptor antagonist mifepristone, This suggests that 3β-OH-tibolone is converted to a progestagenic metabolite. We found that the stromal cells used in the co-cultures express high levels of the ketosteroid dehydrogenase, AKR1C2, which is able to oxidize 3β-OH-tibolone back to tibolone. Thus the unexpected progestagenic effect of 3β-OH-tibolone in these co-cultures may be due to metabolic activity present in the stromal cells of the co-cultures

METABOLISM OF ESTRADIOL, ETHYNYLESTRADIOL, AND MOXESTROL IN RAT UTERUS, VAGINA, AND AORTA: INFLUENCE OF SEX STEROID TREATMENT

This paper is available online at http://dmd.aspetjournals.org ABSTRACT: Estrogen replacement therapy for postmenopausal women consists of an estrogenic and a progestagenic compound. The treatment has a positive estrogenic effect on bone, the cardiovascular system, and vagina but is dependent of the estrogen-progestagen balance in uterus to prevent unwanted proliferation. We were interested in the influence of estrogens and progestagens on estrogen metabolism in target tissues of estrogen replacement therapy. Therefore, we studied the metabolism of estradiol, 17␣-ethynylestradiol, and moxestrol (11␤-methoxy-17␣-ethynylestradiol) in rat uterus, vagina, and aorta. In uterus and vagina, estradiol was converted to estrone, estradiol-3-glucuronide, and estrone-3-glucuronide. These metabolites demonstrate the presence of 17␤-hydroxysteroid dehydrogenase (17␤-HSD) and UDP-glucuronosyl transferase (UDP-GT) in uterus and vagina. We found that the conversion of estradiol by 17␤-HSD in uterus was increased in animals treated with estradiol or with a combination of estradiol and progesterone. The conversion of estradiol in uterus by UDP-GT was estradiol-induced and in contrast, progesterone-suppressed. In the vagina, steroid hormone treatment had no effect on estradiol conversion by 17␤-HSD or UDP-GT. Ethynylestradiol was glucuronidated only, and this was not affected by steroid treatment. Moxestrol was not converted in any of the three organs that were studied, indicating that the 11␤-methoxy substituent renders it a poor substrate for glucuronidation. Overall, the estrogen metabolism, and its regulation by sex steroids, in rat uterus is different compared with human uterus. Therefore, the rat may not be the best-suited model to investigate uterine effects of estradiol-progestagen combined treatment

Human Cytosolic Hydroxysteroid Dehydrogenases of the Aldo-ketoreductase Superfamily Catalyze Reduction of Conjugated Steroids: IMPLICATIONS FOR PHASE I AND PHASE II STEROID HORMONE METABOLISM*S⃞

Aldo-ketoreductase 1C (AKR1C) enzymes catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids. They are Phase I metabolizing enzymes for natural and synthetic steroid hormones. They convert 5α-dihydrotestosterone (Dht, potent androgen) to 3α/β-androstanediols (inactive androgens) and the prodrug tibolone (Tib) to estrogenic 3α/β-hydroxytibolones. Herein we demonstrate for the first time that human AKR1C enzymes (AKR1C1-4) are able to reduce conjugated steroids such as Dht-17β-glucuronide (DhtG), Dht-17β-sulfate (DhtS), and Tib-17β-sulfate (TibS). Product identities were characterized by liquid chromatography-mass spectrometry, and kinetic parameters of the reactions were determined. The product profile of the reduction of each steroid conjugate by the individual AKR1C isoform was similar to that of the corresponding free steroid except for the reduction of DhtG catalyzed by AKR1C2, where a complete inversion in stereochemical preference to 3β-reduction (with DhtG) from 3α-reduction (with Dht and DhtS) was observed. The catalytic efficiency of 3-keto reduction was modestly affected by the presence of a 17-sulfate group but severely impaired by the presence of a 17-glucuronide group for AKR1C1-3 isoforms. AKR1C4, however, showed superior catalytic efficiencies versus the other isoforms, and those were unaffected by steroid conjugation. Our findings provide evidence for alternative pathways of steroid metabolism where the phase I reaction (reduction) occurs after the phase II reaction (conjugation). Specifically, it is indicated that Dht is metabolized to its metabolite 3α-androstanediol-17-glucuronide via the previously unrecognized “conjugation pathway” involving the sequential reactions of UGT2B17 and AKR1C4 in liver but via the conventional “reduction pathway” involving the sequential reactions of AKR1C2 and UGT2B15/17 in prostate

Hormone replacement therapy dependent changes in breast cancer-related gene expression in breast tissue of healthy postmenopausal women

Risk assessment of future breast cancer risk through exposure to sex steroids currently relies on clinical scorings such as mammographic density. Knowledge about the gene expression patterns in existing breast cancer tumors may be used to identify risk factors in the breast tissue of women still free of cancer. The differential effects of estradiol, estradiol together with gestagens, or tibolone on breast cancer-related gene expression in normal breast tissue samples taken from postmenopausal women may be used to identify gene expression profiles associated with a higher breast cancer risk. Breast tissue samples were taken from 33 healthy postmenopausal women both before and after a six month treatment with either 2mg micronized estradiol [E2], 2mg micronized estradiol and 1mg norethisterone acetate [E2+NETA], 2.5mg tibolone [T] or [no HRT]. Except for [E2], which was only given to women after hysterectomy, the allocation to each of the three groups was randomized. The expression of 102 mRNAs and 46 microRNAs putatively involved in breast cancer was prospectively determined in the biopsies of 6 women receiving [no HRT], 5 women receiving [E2], 5 women receiving [E2+NETA], and 6 receiving [T]. Using epithelial and endothelial markers genes, non-representative biopsies from 11 women were eliminated. Treatment of postmenopausal women with [E2+NETA] resulted in the highest number of differentially (p>0.05) regulated genes (16.2%) compared to baseline, followed by [E2] (10.1%) and [T] (4.7%). Among genes that were significantly down-regulated by [E2+NETA] ranked estrogen-receptor-1 (ESR1, p=0.019) and androgen receptor (AR, p=0.019), whereas CYP1B1, a gene encoding an estrogen-metabolizing enzyme, was significantly up-regulated (p=0.016). Mammary cells triggered by [E2+NETA] and [E2] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. In this prospective study, prolonged administration of [E2+NETA] and to a lesser extent of [E2] but not [T] were associated in otherwise healthy breast tissue with a change in the expression of genes putatively involved in breast cancer. Our data suggest that normal mammary cells triggered by [E2+NETA] adjust for steroidogenic up-regulation through down-regulation of the estrogen-receptor pathway. This feasibility study provides the basis for whole genome analyses to identify novel markers involved in increased breast cancer risk