Beta-hydroxysteroid Oxidoreductase Activity Research Articles

17 beta-hydroxysteroid oxidoreductase activity in intact cells significantly differs from classical enzymology analysis.

This paper summarizes our most recent results of steroid enzyme studies on cultured breast and endometrial cancer cells. It deals mainly with estrogen 17 beta-hydroxysteroid oxidoreductase (17 beta HSOR) activity, which presides over estradiol (E2) and estrone (E1) interconversion, a major metabolic pathway of estrogens. Assessment of either the oxidative or reductive component of 17 beta HSOR was carried out on intact cells by means of an original approach based on reverse phase-high performance liquid chromatography and radioactive detection on line. This system allows the continuous monitoring of both precursor degradation and formation of several radiometabolites to assess rates and direction of steroid metabolism. Overall, hormone-responsive, estrogen receptor (ER)-positive cells, regardless of whether they were derived from breast (MCF7) or endometrial (Ishikawa) tumor tissues, showed a prevalence for reductive metabolism (E1-->E2), whilst oxidative pathways (E2-->E1) were largely dominant in non-responsive, ER-poor mammary (MDA-MB231) and endometrial (HEC-1A) cells. The above estimates of 17 beta HSOR activity were at variance with those obtained using the classical enzymology approach, not only in quantitative terms (being markedly lower using intact cell analysis), but also because the prevalent direction of estrogen metabolism was often reversed. Although striking methodological differences may well account for this discrepancy, intact cell analysis is undoubtedly more similar to the in vivo state than the artificial requirements of classical enzymology procedures.

Properties and regulation of 17 beta-hydroxysteroid oxidoreductase of OVCAR-3, CAOV-3, and A431 cells: effects of epidermal growth factor, estradiol, and progesterone.

Although there is a growing body of evidence that 17 beta-hydroxysteroid oxidoreductase plays a role in the regulation of steroid levels in epithelial tumors of the endometrium and breast, our knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17 beta-hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR-3, CAOV-3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17 beta-hydroxysteroid oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basis of estradiol/testosterone activity ratios, all three cell lines appear to have type 2-like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGF, alone or in combination, were without effect on the enzymatic activity of OVCAR-3 cells. EGF decreased the activity of CAOV-3 cells slightly. In contrast, EGF stimulated A431 17 beta-hydroxysteroid oxidoreductase activity 7-8-fold over a 5-day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGF. With EGF, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGF alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGF to low-affinity receptors on A431 cells.

A possible mechanism for increased breast cell proliferation by progestins through increased reductive 17β‐hydroxysteroid dehydrogenase activity

We have investigated whether progestins may be able to regulate breast cell proliferation by altering the fraction of oestradiol relative to oestrone, using the human breast cancer cell line MCF-7. The ability of the two oestrogens, oestradiol and oestrone, to stimulate breast tumour cell proliferation was investigated. Oestradiol in concentration was of 10-fold greater proliferative potency than oestrone. The progestin MPA increased both reductive and oxidative 17 beta-hydroxysteroid oxidoreductase activity when the tissue culture media pH indicator phenol red was included in the media. When phenol red was excluded from the tissue culture media, MPA increased predominantly the reductive 17 beta-hydroxysteroid oxidoreductase activity, and to a far greater extent than in the presence of phenol red. Other progestins such as levonorgestrel, norethisterone and norethisterone acetate also increased predominantly reductive 17 beta-hydroxysteroid oxidoreductase activity in the absence of phenol red. The action of MPA on reductive 17 beta-hydroxysteroid oxidoreductase activity was increased by treatment with oestradiol to a small but significant extent. We propose that the progestational increase of reductive 17 beta-hydroxysteroid oxidoreductase activity is a possible mechanism by which progestins may increase breast cell proliferation in vivo.

Induction of ovarian cysts in progesterone-synchronized immature rats: evidence that suppression of follicular aromatase activity is not a prerequisite for the induction of cystic follicles.

Serum hormone profiles for women and animals with cystic ovaries have led to the hypothesis that elevated serum LH and androgens are involved in the induction of ovarian follicular cysts. To test the ability of LH-like activity to induce cysts, immature rats (bearing progesterone implants to suppress endogenous LH secretion) were assigned to treatment groups that received 0 (control), 0.1, 0.5, or 1.5 IU hCG twice daily for 9 days beginning on day 27 of life. Serum progesterone concentrations were maintained at approximately 156 ng/ml throughout the in vivo treatment period. By the morning of day 36 (day 10 of treatment) the largest follicles in ovaries from control animals were at the small antral stage of development. In contrast, the largest follicles in ovaries from rats receiving 0.1, 0.5, and 1.5 IU hCG were primarily at the large antral, precystic, and cystic stages of development, respectively. On all days tested, progesterone, androstenedione, and estradiol accumulation in medium alone was greater for follicles from rats receiving hCG than for follicles from control rats. Progesterone and androstenedione increased in response to cAMP in vitro in follicular incubations from both control and hCG-treated animals. Only estradiol production by follicles from rats treated with 0.1 or 0.5 IU hCG increased in response to cAMP in vitro. Follicles from all treatment groups produced significantly more estradiol in the presence of a saturating amount of aromatizable substrate than in medium alone. Indeed, on day 36, cystic follicles produced more than 8 ng estradiol when incubated in the presence of either androstenedione or testosterone. In addition, more androstenedione was accumulated in vitro when developing cysts were incubated with exogenous testosterone than when noncystic follicles were incubated under similar conditions. The results of these experiments demonstrate that chronic stimulation by LH-like activity is capable of inducing follicular cysts in a time- and dose-related manner in intact noncycling immature rats. The ability of these cysts to produce 1-2 ng estradiol in medium alone and even greater amounts of estradiol in the presence of exogenous androgen indicates that inhibition of the ability of FSH to induce and stimulate follicular aromatase activity is not a prerequisite for the induction of follicular cysts in these animals. Finally, the increasing ability to accumulate androstenedione in the presence of exogenous testosterone suggests that follicular 17 beta-hydroxysteroid oxidoreductase activity increases in response to chronic stimulation by low doses of LH-like activity during the development of follicular cysts.

17 beta-Hydroxysteroid oxidoreductase activity in the endometrium of normal women and patients with pelvic pain and polycystic ovaries.

17 beta-Hydroxysteroid oxidoreductase (17-OHSD) activity in the endometrium of women with pelvic pain syndrome (PPS) and/or polycystic ovaries (PCO) was compared with that of a control group. In both groups there was a 10-fold increase in 17-OHSD activity in secretory phase tissue compared with that of the proliferative phase, measured by both oxidative and reduction pathways, and a highly significant correlation between the two directions (P less than 0.001). In normal subjects, the ratio of activity measured under oxidative conditions: reducing conditions, at all stages of the cycle except late proliferative phase, was 2.1-2.9. In the late proliferative phase the ratio was 5.5 which was significantly different from other stages of the cycle. Similar ratios were found for the PPS/PCO group (proliferative phase 2.5, secretory phase 5.6); these were also significantly different (P less than 0.01). On the basis of this study oestrogen metabolism in the endometrium of women with PPS and/or PCO appears to be no different from that of normal subjects. Measurement of enzyme activity in high speed soluble and particulate fractions of endometrial homogenate indicated the presence of two activities with different cofactor requirements. Gel filtration chromatography of the soluble fraction revealed a single peak of activity coincident with a molecular weight of 30 kDa with a strong preference for NAD + as cofactor. These preliminary findings suggest the presence of both soluble and particulate forms of 17-OHSD activity in the endometrium.

Steroid modulation of 17 beta-hydroxysteroid oxidoreductase activities in human placental villi in vitro.

Recent data are consistent with the presence of two 17 beta-hydroxysteroid oxidoreductase (17 beta-HOR) activities in placental homogenates. One is localized to intracellular membrane fractions. The conversion of 17 beta-estradiol (E2) to estrone (E1) by this enzyme can be completely inhibited in vitro by C19 and C21 steroids. The second activity is detected in microsomes, but is recovered principally in the cytosol. It also has 20 alpha-HOR activity, but has a high affinity only for C18 steroids. We used this difference to estimate the relative contributions of the two enzymes to E2, E1, and testosterone (T) metabolism by villous tissue in vitro. Fragments of tissue from vaginally delivered placentas (38-40 weeks) were incubated with [3H]E2, [3H]E1, or [3H]T as substrates and various unlabeled steroids as potential inhibitors. Approximately 40% of the E2 to E1 reaction was not inhibited by C19 steroids at 100-200 microM, whereas the conversion of T to androstenedione was inhibited by 90% or more under similar conditions. In contrast, the metabolizable C19 and C21 steroids, 5 alpha-dihydrotestosterone and 20 alpha-dihydroprogesterone, which inhibited the conversion of E1 to E2 by microsomes, stimulated E2 formation from E1 by villi. We conclude that nonspecific 17 beta-HOR accounts for approximately 60% of the E2 to E1 conversion and nearly all of the T to androstenedione conversion in villous tissue fragments. The data also suggest that net E2 formation in villi is catalyzed principally by the C18-specific 17 beta-HOR.

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures.

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.

Human Ovarian 17β-Hydroxysteroid Oxidoreductase Activity: A Comparison of Normal and Polycystic Ovarian Tissues*

Ovarian tissue and ovarian venous blood were obtained from women undergoing wedge resection, and ovarian tissue was obtained from normally menstruating women who had an oophorectomy for medical reasons. A morphological evaluation was made of the wedged tissue. 17 beta-Hydroxysteroid oxidoreductase levels were determined as the 17-ketoreductase (17-KR) and 17 beta-dehydrogenase activities in both normal and wedged tissue. Plasma androstenedione (A) and testosterone (T) levels were measured in the ovarian venous blood. Low, but measurable, 17 beta-hydroxysteroid oxidoreductase activity was found in the mitochondria, microsomes, and cytosol. With whole, cell-free homogenates, mean 17-KR activity was not significantly different in normal tissues and polycystic tissues; mean 17-KR activity of corpora lutea was significantly greater than that of the other tissues. Mean 17 beta-dehydrogenase activity was not significantly different from 17-KR activity in the tissues studied. Ovarian venous A levels were higher than the reported mean ovarian venous A levels of normal women with only one exception; approximately half of the ovarian venous T levels were higher than reported mean ovarian venous T levels of normal women. The morphology of the wedge sections did not correlate well with the biochemical data.

Human skin androgen metabolism and preliminary evidence for its control by two forms of 17 beta-hydroxysteroid oxidoreductase.

Human forehead skin incubated in vitro is known to metabolize testosterone to 17-oxosteroids faster than the reverse reaction, while axillary skin rapidly metabolizes androstenedione to 17 beta-hydroxysteroids, such as testosterone and 5 alpha-dihydrotestosterone. While this has been confirmed using a larger number of patients, some indication has been found that 17 beta-hydroxysteroid oxidoreductase activity declines with age in the axilla. The relative rates of 17 beta-oxidation and reduction (direction of operation of skin 17 beta-hydroxysteroid oxidoreductase activity) were not altered by variety of incubation conditions. Large amounts of a membrane-bound 17 beta-hydroxysteroid oxidoreductase, showing preference for NAD as coenzyme and testosterone (rather than androstenedione) as steroid substrate, were found in forehead skin from one patient. On the other hand, the main axillary skin enzyme in skin from another patient was soluble and showed preference for NADP and androstenedione. It is postulated that 17 beta-oxidation and reduction in skin is controlled by the relative amount, the coenzyme preferences and the kinetic properties of these two enzymes.

Metabolism of androstenedione by human platelets: a source of potent androgens.

The metabolism of tritium-labeled androstenedione by human platelets was studied in vitro. The following metabolites were identified: testosterone, 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, and 5 alpha-androstane-3 alpha,17 beta-diol. The rates of formation of these metabolites remained linear with time of incubation up to 2 h and with increased platelet protein concentration up to 14.6 mg/ml. Estrogens and 5 beta-reduced metabolites were not formed by platelets. The major enzyme systems involved in the metabolism of androstenedione in human platelets were the 17 beta-hydroxysteroid oxidoreductase and 5 alpha-reductase activities; in addition, 3 alpha-and 3 beta-hydroxysteroid oxidoreductase activities were also present in these megakaryocyte fragments. Thus, it appears that platelets are a potential site of extraglandular conversion of androstenedione to potent androgens in man. It is also possible that androgens formed within the platelet may affect platelet function by regulating their sensitivity to aggregating stimuli.

Estrogen metabolism in neural tissues of rabbits: 17 beta - hydroxysteroid oxidoreductase activity.

Incubation of adult rabbit neural tissue homogenates with (3H)-estrone and (3H)-estradiol revealed that the conversion of estrone to estradiol is higher in both male and female animals than estradiol to estrone. Both 17 oxidation and reduction are higher in male animals than in females. However, it is observed that the quotient of estrone leads to estradiol/estradiol leads to estrone for pituitary tissue and hypothalamus are higher in females than in males. There is no such dimorphism in cerebral cortex. The overall metabolism in pituitary is higher than in hypothalamus and cortex in both sexes. These results suggest that 17 beta hydroxysteroid oxidoreductase activity may play an important role in the regulation of estrogen function in neuroendocrine tissues.