Sulphotransferases (STs) are a family of closely related enzymes playing a key role in regulation of the bioavailability and activity of important endogenous molecules such as steroid hormones. A relationship between the expression of steroid STs and the diabetic state has been demonstrated in various laboratory animal models, and steroid sulphates such as dehydroepiandrosterone sulphate are known to have anti-diabetic properties. In order to further our understanding of the molecular basis for the association of steroid hormone sulphation and diabetes, we have examined the expression of oestrogen, phenol and dehydroepiandrosterone (DHEA) STs in mice carrying the obesity mutation (ob), which in the homozygous state (ob/ob) produces mice which are obese and diabetic. Our data show that, in male mice, ST activities towards oestrone (E1), oestriol (E3), DHEA and the xenobiotic 1-naphthol are elevated in ob/ob mice, whereas in female mice, only the oestrogen ST activities were elevated, with the DHEA and 1-naphthol ST activities reduced. Using antibodies directed against oestrogen ST, it was demonstrated that the induction of E1 and E3 ST activity in ob/ob mice correlated with the expression of an ST isoenzyme not constitutively expressed in control mouse liver.

OBJECTIVE

Sex hormone-binding globulin (SHBG) is a robust predictor of insulin resistance. Whether this is independent of circulating sex steroid levels remains uncertain. The aim of this study was to investigate the determinants of SHBG in postmenopausal women and whether the relationship between SHBG and insulin resistance is independent of oestrogen and androgen levels.

METHODS

A cross-sectional study of naturally and surgically menopausal women.

METHODS

Seven hundred and sixty three postmenopausal women not using any systemic hormone therapy, mean age 54·4 ± 5·8 years, recruited in the US, Canada, Australia, UK and Sweden between July 2004 and February 2005.

METHODS

Relationships between log-transformed (ln) SHBG and ln homoeostasis model assessment for insulin resistance (HOMA-IR) were explored, taking into account age, body mass index (BMI), blood pressure (BP) and circulating oestradiol, oestrone, testosterone and dihydrotestosterone.

RESULTS

Taking into account age, race, years since menopause, menopause type, BMI, BP, prior postmenopausal hormone use and the sex steroids measured, 34·4% of the variation in SHBG could be explained by the model that included negative contributions by HOMA-IR, BMI and diastolic BP, and a positive contribution by total testosterone (P < 0·001). None of the sex steroids made independent contributions to HOMA-IR, which was best explained by the model that included BMI, SHBG, systolic BP and surgical menopause, with each variable being positively related to HOMA-IR (r(2) = 0·3152, P = 0·03).

CONCLUSIONS

The relationship between SHBG and HOMA-IR, as an estimate of insulin resistance, is not explained by endogenous oestrogen and androgen levels and is, at least in part, independent of BMI in postmenopausal women.

Climacteric symptoms in 120 women were treated with a total of 469 hormone implants (oestradiol 50 mg and testosterone 100 mg) over a period of four years. All patients with a uterus were given an oral progestogen to prevent endometrial hyperplasia. There was a marked response to treatment, hot flushes being improved in all patients, depression in 99% and loss of libido in 92%. Patient acceptability of this type of treatment was good and there were few side effects or complications. After therapy, the serum oestradiol exceeded the serum oestrone but remained within normal limits. When climacteric symptoms returned and re-implantation occurred the serum levels of oestrone, oestradiol, luteinising hormone (LH), follicle stimulating hormone (FSH) and testosterone were within the normal range for the reproductive age. This indicates that the return of symptoms is due to a change in the hormone levels rather than absolute hypo- oestrogenism .

In order to investigate the regulation of the hypothalamo-pituitary-gonadal axis during fetal development, sheep fetuses at day 70 of gestation were implanted subcutaneously with a biodegradable implant containing the long-acting gonadotrophin-releasing hormone (GnRH) agonist, buserelin. The treatment of fetuses with a GnRH agonist throughout the last half of gestation (term = 145 days) abolished the increase in plasma LH concentrations that was seen in 2-day-old control lambs in response to an injection of GnRH. This attenuated response was associated with corresponding reductions in the pituitary content of LH and FSH. Immunolocalization studies revealed that pituitary glands from newborn lambs implanted with a GnRH agonist during fetal development were devoid of immunopositive LH- and FSH-containing cells. At birth the testicular weights of GnRH agonist-treated ram lambs were significantly decreased by 40% when compared with controls. This was associated with a 45% reduction in the total number of Sertoli cells per testis. In newborn ewe lambs GnRH agonist treatment had no effect on ovarian weight or on the morphological appearance of the ovaries. GnRH agonist treatment had no effect on the plasma concentrations of progesterone and oestrone in the maternal circulation or on the length of gestation. These results show (1) that GnRH positively regulates the synthesis and secretion of gonadotrophins in the fetus, (2) that reduced fetal gonadotrophic support during the last half of gestation results in a reduction in testicular growth, and (3) that fetal gonadotrophins do not affect maternal steroid secretion.

17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyses the intracellular conversion of oestrone (E1) to oestradiol (E2). E2 is known to be involved in the development and progression of breast cancer and endometriosis. Since 17beta-HSD1 is overexpressed in these oestrogen-dependent diseases, inhibition of this enzyme may be a more target-directed therapeutical approach compared to established medical treatments. For the identification of highly active and selective 17beta-HSD1-inhibitors that are suitable for application as potential therapeutics, there is a need for an appropriate, efficient and reliable screening system. Here, we report the development and application of our screening system using our in house library of potential 17beta-HSD1-inhibitors. Four potent and selective compounds with a good first pharmacokinetic profile were identified.

Both parity and oral contraceptive use are associated with elevated circulating levels of sex hormones, at least transiently, and with increased risk of cervical cancer in human papillomavirus (HPV)-infected women. We directly evaluated whether elevations in the physiologic levels of these hormones predispose to the development of cervical neoplasia. We identified 67 premenopausal and 43 postmenopausal women with cervical intraepithelial neoplasia 2, 3, or cervical cancer >>/=CIN2) diagnosed during enrollment of a population-based cohort of 10 077 women. Four controls, two chosen randomly and two chosen from women testing positive for cancer-associated HPV, were matched to each case on menopausal status, age, days since last menses (pre), or years since menopause (post). Sex hormone-binding globulin, oestradiol, oestrone, oestrone-sulphate, dehydroepiandrosterone sulphate, and progesterone were measured in enrollment plasma. There was no consistent association between the sex hormones and risk of>>/=CIN2. Excluding cases with invasive disease had a minimal impact on results. Though this case-control study was based on a well-defined population, it was limited by reliance on a single measure of hormone levels taken at the time of diagnosis. Nonetheless, our results do not support the hypothesis that plasma levels of sex hormones have an important bearing on the risk of cervical neoplasia in HPV-infected women.

Two-thirds of breast tumours are oestrogen-receptor positive and 60-70% of these tumours respond to interventions that reduce the effects of oestrogen. Until recently, tamoxifen was the drug of choice for the treatment of hormone-responsive early and advanced breast cancer. However, tamoxifen is associated with increased incidences of endometrial cancer and thromboembolic disease, and many tumours eventually become resistant to treatment with tamoxifen. Thus, there is a need for alternative therapies with different mechanisms of action. In postmenopausal women, aromatase inhibitors (AIs) suppress oestrogen levels by inhibiting oestrogen synthesis via the aromatase enzyme pathway. The third-generation AIs (anastrozole, letrozole and exemestane) are more potent than the earlier AIs (aminoglutethimide, formestane and fadrozole) with respect to both aromatase inhibition and oestrogen suppression. While the earlier AIs were unable to show any benefit over megestrol acetate or tamoxifen as second- and first-line therapy, respectively, in postmenopausal women with advanced breast cancer, third-generation AIs have shown significant benefits in both settings. Comparison of aromatase inhibition and oestrogen suppression between the third-generation AIs anastrozole and letrozole showed a small but significantly greater difference in the degree of suppression of oestrone and oestrone sulphate (but not oestradiol), with letrozole. In an open-label trial, there were no significant differences between letrozole and anastrozole for the clinical end points of time to progression (primary end point), time to treatment failure, overall survival, clinical benefit, duration of clinical benefit, time to response, duration of response or objective response rate in patients with confirmed hormone receptor-positive tumours. Together these data suggest that once a certain threshold of aromatase inhibition is reached, small differences in oestrogen suppression between the third-generation AIs do not lead to clinically significant differences in overall efficacy.

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).

Steroid sulphatase (STS) inhibitors have been developed primarily for the treatment of hormone-dependent breast cancer, but may also have utility for the treatment of a number of androgen-dependent skin conditions. STS regulates the hydrolysis of steroid sulphates, such as oestrone sulphate (E1S) and dehydroepiandrosterone sulphate, (DHEAS). Liberated oestrone (E1) can be converted to biologically active oestradiol (E2) while dehydroepiandrosterone (DHEA) can undergo reduction to testosterone or aromatisation to E1. In this study the ability of the STS inhibitor STX64 (BN83495) and its N,N-dimethyl analogue (STX289) to inhibit liver and skin STS when applied orally or topically to nude mice was examined. Oral administration at 1 and 10 mg/kg resulted in almost complete inhibition of skin and liver STS. When applied topically to the dorsal neck region at 1.0 and 10 mg/kg not only skin but, unexpectedly, also liver STS was effectively inhibited. An investigation into the metabolism of these two compounds by HepG2 liver carcinoma cells, with high-performance liquid chromatography (HPLC) analysis, was also undertaken. In the presence of HepG2 cells a similar degree of desulphamoylation of STX64 (68%) or de-N, N-dimethylsulphamoylation of STX289 (66%) occurred over a 3h period. In the absence of cells, however, STX289 was resistant to de-N, N-dimethylsulphamoylation whereas STX64 was completely desulphamoylated, demonstrating the more favourable pharmaceutical profile of STX289 for development for topical application. It is concluded that both STX64 and STX289 are not only effective inhibitors of skin STS, but also liver STS when applied topically. These findings suggest that it may be possible to develop a formulation for the percutaneous administration of STS inhibitors, but also that this class of compound may have therapeutic potential for the treatment of a number of skin disorders.

Whether the same or distinct steroid sulphatases (STS) are involved in the hydrolysis of alkyl and aryl steroid sulphates remains controversial. We have examined the ability of a placental steroid sulphatase to hydrolyse oestrone sulphate and/or dehydroepiandrosterone sulphate (DHA-S) by expressing the enzyme in COS-1 cells. Using either intact cells or broken cell preparations, the expressed sulphatase was found to hydrolyse both oestrone sulphate and DHA-S. The catalysis of oestrone sulphate and DHA-S by the expressed sulphatase was almost completely abolished by the steroid sulphatase inhibitor, oestrone-3-O-sulphamate. It is concluded that both alkyl and aryl steroid sulphates can be hydrolysed by the same steroid sulphatase.

The introduction of transdermal and other parenteral delivery systems has broadened the range of options for hormone replacement therapy (HRT). Oral oestrogen is the most common initial therapy; however, direct absorption of oestradiol via the skin results in an oestradiol-oestrone ratio similar to that found in the pre-menopausal state. Both oral and transdermal oestrogen therapy have been shown to be equally effective in relieving climacteric symptoms, and in preventing osteoporosis or modifying some cardiovascular disease risk factors, although transdermal therapy tends to have fewer unwanted effects than oral. Satisfactory circulating oestradiol levels are achieved with skin patches, transdermal gel, or crystalloid oestradiol implants, and adding progestogen to protect the endometrium is well established. Sequential therapy with HRT usually produces a regular bleed, which is a major cause of patient dissatisfaction. The ideal HRT regimen is probably unobtainable, but the development of SERMs and other regimens that avoid bleeding will encourage long-term use.

Daily urine samples were collected from 5 female golden lion tamarins (Leontopithecus rosalia) over a period of 3 or more months, and urinary oestrogen concentrations were determined by radioimmunoassay. Four females exhibited regular patterns of oestrogen excretion, with a peak-to-peak periodicity of 19.6 +/- 1.4 days. Levels of oestrogen excretion tended to vary between, but not within, individual females. Post-partum oestrogen patterns included periods of clear oestrogen cyclicity before conception, with dramatic elevations in oestrogen excretion following conception. Oestrone was the predominant urinary oestrogen excreted by female lion tamarins. Enzyme hydrolysis with Helix pomatia beta-glucuronidase/sulphatase was an efficient method of liberating conjugated oestrogens in tamarin urine. Urinary oestrogen determinations can provide useful information about reproductive status in female lion tamarins.

Use of the aromatase inhibitor aminoglutethimide is limited by its lack of selectivity for aromatase and its toxicity. Newer agents are more selective, but do not always offer improved inhibition of aromatase. Indirect comparison of their activity in inhibiting aromatase and suppressing plasma oestrogens indicates that aminoglutethimide, rogletimide, formestane, and fadrozole inhibited aromatase activity by 74-91%, with reported falls in oestradiol level of 58-76%. In contrast, the new-generation oral once-daily aromatase inhibitors anastrozole (Arimidex) and letrozole were of a similar activity, inhibiting aromatase activity by over 96%, with a concomitant fall in oestradiol and oestrone levels of at least 80%. Anastrozole at the recommended clinical dose of 1 mg daily also suppressed oestrone sulphate levels by 93.5%; activity with anastrozole 10 mg daily was not statistically significantly different. The new generation of aromatase inhibitors, as typified by anastrozole, thus offers effective and convenient aromatase inhibition which correlates well with decreases in the levels of plasma oestrogens.

Preovulatory bovine follices (n = 73) were collected at different times after the onset of oestrus until shortly before ovulation, which occurred at 24 +/- 1 X 4 h after the peak concentration of LH in the peripheral blood. Non-atretic antral follicles (n = 9) of 15-19 mm were also collected from cows during the luteal phase of the oestrous cycle. Follicular fluid concentrations of dehydroepiandrosterone, androstenedione and oestrone, and of LH, FSH and prolactin were compared in 2-h periods relative to the LH plasma peak. Before the LH surge the concentrations of the steroids were much higher than in non-atretic luteal-phase follicles of similar size. From 0 to 6 h after the LH peak the steroid concentrations decreased sharply to remain low until ovulation; only that of androstenedione increased again after 14 h to remain constant. The ratio between the concentrations of androstenedione and dehydroepiandrosterone remained constant until 14 h after the LH peak; at 14 h it increased about 4-fold and remained high until ovulation. The ratio between the oestrone and androstenedione concentration increased gradually to a 10-fold higher value until at 14 h an abrupt decrease was observed. These changes indicate that after the LH peak androgen production is directly inhibited and, at a slower rate, the aromatizing activity. Androstenedione appeared to be the major aromatase substrate. Before the plasma LH peak the follicular fluid concentration of FSH was higher than in luteal-phase follicles; the concentrations of LH and prolactin were not different from those in luteal-phase follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

The stimulative effect of FSH on aromatase activity was investigated in ovaries of fetal (on days 17 and 18 of gestation) and neonatal mice (on days 0, 3, 6 and 9 after birth). Two to six ovaries, were cultured for 48 h in 2 ml of Medium 199 supplemented with insulin (5 micrograms/ml) and [1 alpha, 2 alpha, 6 alpha, 7 alpha, beta-3H]4-androstene-3,17-dione (0.35 microM) in the presence or absence of porcine FSH (0.5 units/ml) and the amount of [3H]oestradiol-17 beta and [3H]oestrone produced was estimated. In the presence of FSH, aromatase activity per ovary, which was found in all fetal and neonatal ovaries examined, increased with age. In the absence of FSH, however, the production of oestrogens could be demonstrated only in ovaries from 3- to 9-day old mice. FSH increased the aromatase activity by up to 10-fold. In spite of the stimulative effect of FSH on aromatase activity, FSH exerted no significant effect on DNA synthesis of the ovaries. The formation of primordial follicles could not be observed histologically in ovaries of fetal mice on day 17 of gestation, although the ovaries of 6- and 9-day old mice contained multilayered follicles. These results show that FSH stimulates the aromatase activity of the mouse ovary even before the formation of primordial follicles and that the stimulative effect of FSH on ovarian aromatase is not due to the proliferation of ovarian cells.

To evaluate the prevalence of hyperprolactinaemia in PCO patients and its possible correlation with a steroid pattern, we studied prolactin secretion (basal and after TRH stimulation) in 40 women affected by typical PCO. LH, FSH, testosterone, oestradiol, oestrone, DHEA-s and 17-OHP serum levels were also evaluated. Twenty-one patients had prolactin (Prl) values in the normal range both in baseline conditions and after TRH stimulation; 10 patients had normal basal values of Prl but an exaggerated response to TRH stimulation; 9 patients had high Prl basal values and an exaggerated response to TRH. The presence of hyperprolactinaemia was associated with increased serum levels of oestrone (P less than 0.01), DHEA-s (P less than 0.01) and 17-OHP (P less than 0.05). In conclusion, hyperprolactinaemia is as relatively frequent condition which affects almost half the patients suffering from PCO and is probably related to an increase of serum oestrogens, mostly oestrone. Moreover, in patients with PCO and hyperprolactinaemia, the production of some other steroids is also affected.

Tumour and normal breast tissue was obtained from postmenopausal breast cancer patients following [3H]oestradiol infusion (50 mu Ci over a 12 h period). The fraction of radioactivity present as oestradiol or oestrone was measured and the results expressed both as the ratio of oestradiol-oestrone and as the percentage oestrogen present as oestrone, and the findings compared with in vitro measurements of 17 beta-hydroxysteroid dehydrogenase activity. Concentrations of 5-androstene-3 beta, 17 beta-diol, dehydroepiandrosterone and its sulphate and testosterone were measured and related to oestradiol metabolism. The study demonstrated that tumour tissue is less able to metabolise oestradiol to oestrone than is normal breast tissue and indicated that the ability of the tissue to detoxify oestradiol may be dependent on cofactor availability. The results also supported the possibility that increased tissue concentrations of adrenal androgens inhibit oestradiol and thus increase tissue exposure to oestradiol.

Phase I studies have demonstrated that exemestane, an irreversible oral aromatase inhibitor, is able to suppress circulating oestrogen levels. In our previous experience, doses ranging from 2.5 to 25 mg induced a similar suppression of oestrogens. The aim of this study was to identify the minimum effective exemestane dose on the basis of endocrine activity. 20 evaluable postmenopausal advanced breast cancer patients were randomly given exemestane 0.5, 1, 2.5 or 5 mg, in double-blind conditions. Oestrone (E1), oestradiol (E2), oestrone sulphate (E1S), gonadotrophins, sex-hormone binding globulin and dehydroepiandrosterone sulphate serum levels were evaluated from the first day of treatment to the 7th, 14th, 28th and 56th day. Serum E1, E2 and E1S levels were suppressed by all doses starting from day 7; the degree of inhibition versus baseline was 25 up to 72% for E1, 30 up to 62% for E2 and 16 up to 52% for E1S, with higher doses achieving greater suppression; these changes were maintained over time. A significant increase in FSH and LH levels was observed for all doses. Treatment tolerability was satisfactory. The endocrine effects of exemestane appear to be dose related and 0.5 and 1 mg are ineffective for adequately suppressing circulating oestrogens.

We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and androstenedione (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 14 alpha-hydroxy-4-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.

Of 35 male survivors of myocardial infarction, aged 24-48 years, 34 had higher plasma-oestradiol levels than age-matched controls. 29 of the 35 had higher oestrone levels than controls.