BACKGROUND

There is now abundant evidence that inhibition of steroid sulphatase alone or in conjunction with inhibition of aromatase may enhance the response of postmenopausal patients with hormone-dependent breast cancer to this type of endocrine therapy. Additionally, sulphatase inhibition has been proposed to be of potential therapeutic benefit in the immune system and for neuro-degenerative diseases. After the finding that our first highly potent active site-directed steroid sulphatase inhibitor, oestrone-3-O-sulphamate (EMATE), was highly oestrogenic, we proposed non-steroidal coumarin sulphamates such as 4-methylcoumarin-7-O-sulphamate (COUMATE) as alternative non-steroidal steroid sulphatase inhibitors. In this work, we describe how tricyclic coumarin-based sulphamates have been developed which are even more potent than COUMATE, are non-oestrogenic and orally active. We also discuss potential mechanisms of action.

RESULTS

4-Ethyl- (4), 4-(n-propyl)- (6), 3-ethyl-4-methyl- (8), 4-methyl-3-(n-propyl)coumarin-7-O-sulphamate (11); the tricyclic derivatives 665COUMATE (13), 666COUMATE (15), 667COUMATE (17), 668COUMATE (20) and the tricyclic oxepin sulphamate (22) were synthesised. In a placental microsome preparation, all of these analogues were found to be more active than COUMATE in the inhibition of oestrone sulphatase, with the most potent inhibitor being 667COUMATE which has an IC(50) of 8 nM, some 3-fold lower than that for EMATE (25 nM). In addition, 667COUMATE was also found to inhibit DHEA-sulphatase some 25-fold more potently than EMATE in a placental microsome preparation. Like EMATE, 667COUMATE acts in a time- and concentration-dependent manner, suggesting that it is an active site-directed inhibitor. However, in contrast to EMATE, 667COUMATE has the important advantage of not being oestrogenic. In addition, we propose several diverse mechanisms of action for this active site-directed steroid sulphatase inhibitor in the light of recent publications on the crystal structures of human arylsulphatases A and B and the catalytic site topology for the hydrolysis of a sulphate ester.

CONCLUSIONS

A highly potent non-steroidal, non-oestrogenic and irreversible steroid sulphatase inhibitor has been developed. Several mechanisms of action for an active site-directed steroid sulphatase inhibitor are proposed. With 667COUMATE now in pre-clinical development for clinical trial, this should allow the biological and/or clinical significance of steroid sulphatase inhibitors in the treatment of postmenopausal women with hormone-dependent breast cancer and other therapeutic indications to be fully evaluated.

1. Equilibrium dialysis studies have been made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulphophthalein, oestrone sulphate and dehydroepiandrosterone sulphate each bind at the same single primary binding site with association constants of 1.1 X 10(7), 6.6 X 10(5) and 2.6 X 10(5) 1/mol respectively at pH 7.0,IO.16M,4 degrees C. As well as bromosulphophthalein and dehydroepiandrosterone sulphate, a number of strucurally similar organic anions including 2-hydroxyoestradiol-glutathione oestrone glycyronide, N-methyl-4-aminoazobenzene-glutathione and several bile acids, were able to displace oestrone sulphate from ligandin in a manner consistent with competition at a single binding site. From these experiments association constants for the competing ligands were derived; these were inthe range 1 X 10(4)-1 X 10(6) 1/mol. 2. Ligandin was found to bind a number of compounds for which, because of their low aqueous solubilities relative to their binding affinities complete binding isotherms could bot be obtained. These included several steroids (but not cortisol), 20-methylcholanthrene, diethylstilboestrol, oleate and palmitate. Oestrone sulphate was able to compete with these ligands for binding and the results of the competition experiments were interpretable in terms of 1:1 competition at a single binding site. 3. In general the conjugation of non-polar ligands with sulphate or glutathione resulted in increased affinities, but such increases were relatively small (approximately 15% in therms of free energy) implying that the main driving force for the binding of both the conjugated and unconjugated species was the hydrophobic effect. This conclusion is borne out by the observations that both oestrone and its sulphate showed slight increases in affinity with increase in ionic strength, as would be expected for hydrophobic interactions. 4. As well as non-polar compounds and organic anions, ligandin was also found to bind sulphate and glucuronate to a measurable degree, and to interact quite strongly with glutathione. For the latter compound a single binding site was found with an association constant of 1 X 10(5) 1/mol. Glutathione was able to cause the dissociation of the ligandin-oestrone sulphate complex, but this effect was not explicable in terms of simple 1:1 competition. 5. Both oestrone and oestrone sulphare were bound most strongly at pH 6-7, the affinity of the protein for these ligands falling off quite sharply on either side of this maximum. 6. The affinities of ligandin for bromosulphophthalein, steroids and their conjugates, diethylstilboestrol and N,N-dimethyl-4-aminoazobenzene are similar in magnitude to those of serum albumin and aminoazodye-binding protein A (B. Ketterer, E. Tipping, J.F. Hackney and D. Beale, 1976).

Effects of various doses of aminoglutethimide (AG) alone upon adrenal steroidogenesis were studied in normal postmenopausal women, whereas the effects of combined treatment with aminoglutethimide in variable doses together with 40 mg of hydrocortisone were studied in postmenopausal women with advanced mammary cancer and compared to effects of treatment with cortisol alone. Despite the well known inhibitory effect of AG on cortisol biosynthesis, plasma cortisol levels were unaffected by AG in doses of 150-1000 mg/d, probably due to a compensatory increase in ACTH in subjects with an intact pituitary-adrenal axis. The aromatase system appeared to be very sensitive to inhibition by AG, a clearcut inhibition being shown at doses as low as 150 mg/d. Evaluated from the ratio of plasma oestrone (E1) to plasma androstenedione (AN), treatment with AG at a dose of 150 mg/d appeared to reduce the aromatase activity to 33% of the basal value; 250 mg/d resulted in a reduction to 20% and 1 g/d to 5% of basal values. Whereas AG at 150 mg/d did not appear to affect 11 beta-hydroxylase, the latter was clearly inhibited by 250 mg/d and even more so by 1000 mg/d, as indicated by the increase in plasma 11-desoxycortisol and 17-OH progesterone (17-OHP) levels. Due to the increase of the latter, their biosynthetic precursor, AN and to a lesser degree testosterone (TS) levels increased significantly during AG treatment at a dose of 250 or 1000 mg/d. delta 5 steroid levels remained practically unchanged, probably because 11-(as well as the 21-) hydroxylation concerns essentially the delta 4 pathway. During combined treatment with 500-1000 mg/d of AG and cortisol 40 mg/d, AN and TS were significantly higher than during treatment with cortisol alone, suggesting that cortisol had not completely blocked ACTH secretion. E1 and E2 levels were however lower than during treatment with cortisol alone, a consequence of the inhibition of the aromatase activity. Although at a dose of 500-1000 mg/d AG is a highly effective aromatase inhibitor, oestrogen levels during treatment with AG with or without concomitant administration of cortisol are still significantly different from zero. Therefore if one aims at complete elimination of any oestrogen effect, addition of an antioestrogen to AG treatment may be required.

Plasma androstenedione and oestrone concentrations were measured in 72 postmenopasual women. The women included some who had undergone oophorectomy, some with osteoporosis, and normal controls; they were matched for years since menopause. Both hormone concentrations were significantly reduced in the women with osteoporosis. The women who had undergone oophorectomy had hormone concentrations intermediate between the normal and osteoporotic values. Oestrogen deficiency secondary to low androstenedione levels is a major risk factor in postmenopausal osteoporosis, and may be caused by failure of ovarian stromal androgen secretion or some abnormality in the pituitary-adrenal axis.

Elevated latent prenatal steroidogenic activity has been found in the amniotic fluid of autistic boys, based on measuring prenatal androgens and other steroid hormones. To date, it is unclear if other prenatal steroids also contribute to autism likelihood. Prenatal oestrogens need to be investigated, as they play a key role in synaptogenesis and corticogenesis during prenatal development, in both males and females. Here we test whether levels of prenatal oestriol, oestradiol, oestrone and oestrone sulphate in amniotic fluid are associated with autism, in the same Danish Historic Birth Cohort, in which prenatal androgens were measured, using univariate logistic regression (n = 98 cases, n = 177 controls). We also make a like-to-like comparison between the prenatal oestrogens and androgens. Oestradiol, oestrone, oestriol and progesterone each related to autism in univariate analyses after correction with false discovery rate. A comparison of standardised odds ratios showed that oestradiol, oestrone and progesterone had the largest effects on autism likelihood. These results for the first time show that prenatal oestrogens contribute to autism likelihood, extending the finding of elevated prenatal steroidogenic activity in autism. This likely affects sexual differentiation, brain development and function.

Gender-based differences in cardiovascular mortality may be due to a cardio-protective effect of oestrogens on the myocardium. However, mRNA expression of oestrogen receptors in myocardial tissue of the adult heart has yet to be demonstrated. Furthermore, a calcium antagonistic action of 17beta-oestradiol on myocardial tissue has been discussed. Therefore, two subjects were investigated in atrial myocytes of the human, and ventricular myocytes of guinea-pig and rat in this study. (1) Are oestrogen receptors expressed in adult myocardial cells? (2) Is there an influence of oestrogens on the L-type calcium current of cardiac myocytes? Expression of oestrogen receptors was investigated by reverse polymerase chain reaction. L-type calcium current was usually measured by the patch-clamp technique in whole-cell recording mode under selective recording conditions, i.e. overlapping currents were blocked. One series of experiments was performed in perforated patch configuration to avoid internal perfusion. 17beta-oestradiol inhibited L-type calcium current reversibly in all three species. At 10(-5) M, the inhibition was 15-20%. This inhibition was independent of the sex and the species. A full concentration response curve of 17beta-oestradiol on basal L-type current was recorded from female guinea-pig myocytes. The inhibition increased from 2% at 10(-7) M to about 30% at 10(-4) M 17beta-oestradiol. The values could be fitted by a sum of two sigmoidal functions with log EC50 values of -6.5 and -4.9 M and Hill slopes of 2.5 for both. The specificity of the 17beta-oestradiol action was tested by recording the L-type current in the presence of 17alpha-oestradiol and oestrone. 17alpha-oestradiol also inhibited the current, but with a maximal inhibition of only 17%. The concentration-response curve could be fitted by a single sigmoidal function (log EC50 -6-3 M; Hill slope 0.55). Oestrone did not influence the current at all. The decrease in L-type current after the application of 17beta-oestradiol via a rapid perfusion system developed with a time constant of 3-4 s, which was in the same range as that for the influence of isoprenaline. The isoprenaline-stimulated L-type current was much more susceptible to the inhibition by 17beta-oestradiol, i.e. in pre-stimulated cells (1) the inhibitory effect is significantly higher (e.g. at 10(-5) M, inhibition was 36.3% compared with 11.2% in untreated cells) and (2) an inhibitory effect can be seen with oestradiol concentrations as low as 10(-9) M. Although the concentrations needed to gain a calcium antagonistic influence on the basal current were much too high to explain a cardio-protective influence of oestrogens, the presence of oestrogen receptors in cardiac myocytes of all three species, together with the shift in concentration dependence following pre-stimulation by isoprenaline, suggest that myocytes are a potential target for oestrogen.

There are large inter- and intra-individual variations in the serum concentrations of natural and synthetic sex steroids irrespective of the route of administration. Oral ingestion of steroids has a stronger effect on hepatic metabolism than parenteral administration, as the local concentration in liver sinusoids are 4-5 times higher during the first liver passage. Oestradiol and oestrone are interconvertible, dependent on the local concentrations in liver and target organs, and oestrone sulphate serves as a large reservoir. The oestrone/oestradiol ratio has no physiological significance, as oestrone is only a weak oestrogen. Oestrone is both a precursor and a metabolite of oestradiol. Oestriol is extensively conjugated after oral administration. Therefore, the oestriol serum levels are similar after oral intake of 10 mg and after vaginal application of 0.5 mg oestriol resulting in similar systemic effectiveness. Conjugated oestrogens can easily enter the hepatocytes but are hormonally active only after hydrolyzation into the parent steroids. Ethinylestradiol which exerts strong effects on hepatic metabolism and inhibits metabolizing enzymes, should not be used for hormone replacement therapy. Among the progestogens, the progesterone derivatives have less effects on liver metabolism than the norethisterone derivatives (13-methyl-gonanes and 13-ethyl-gonanes). The highly potent 13-ethyl-gonanes are effective at very low doses, because of a slow inactivation and elimination rate due to the ethinyl group.

Both mammary adipose tissue and breast cancers have the ability to aromatize androgens into oestrogens. Such potential may maintain the growth of hormone-dependent tumours. It has therefore been important to determine the effects of new aromatase inhibitors such as formestane, exemestane, anastrozole and letrozole on oestrogen biosynthesis and concentrations of endogenous hormones within the breast. Studies based on in vitro incubations of breast cancer and cultures of mammary adipose tissue fibroblasts demonstrate that these drugs are highly effective inhibitors, with IC50 values ranging between 1 and 50 nM (although the relative efficacy varies between tissues and test systems). Despite this potential, in vitro incubations of breast tissues from patients treated with type II inhibitors such as aminoglutethimide and letrozole can display paradoxically high aromatase activity; this appears to be caused by the reversible nature of the inhibition, coupled with induction/stabilization of the aromatase enzyme. To assess in situ effects within the breast, postmenopausal women with large primary breast cancers have been treated neoadjuvantly with aromatase inhibitors using a protocol that included (i) breast biopsy before treatment, (ii) definitive surgery after 3 months of treatment and (iii) infusion of [3H]androstenedione and [14C]oestrone in the 18 h immediately before biopsy and surgery. With this study design, it has been shown that drugs such as letrozole profoundly inhibit in situ aromatase activity and reduce endogenous oestrogens within the breast.

An isotopic infusion technique has been used in an attempt to determine the contribution that local, in situ, oestrone synthesis makes to the oestrogen content of breast tumours. 3H-Androstenedione and 14C-oestrone were infused into women with advanced breast cancer for 12 hr before operation. At surgery, normal breast and breast tumour biopsy samples were obtained and 3H-androstenedione, 3H-oestrone derived from 3H-androstenedione and 14C-oestrone were isolated and measured. DNA polymerase alpha activity, a marker of cellular proliferation, was also measured to examine whether local synthesis of oestrone exerted a biological effect. The study was repeated after patients had been treated with the aromatase inhibitor, 4-hydroxyandrostenedione, before undergoing further surgery for removal of their tumours. In 4/6 tumours examined, in situ synthesis of 3H-oestrone from 3H-androstenedione accounted for the major part (84.3 +/- 9.0%) of the 3H-oestrone detected, while no significant in situ synthesis occurred in 2 other tumours. Although treatment with 4-hydroxyandrostenedione did not significantly alter the uptake of 3H-androstenedione or 14C-oestrone into breast tissues, in situ formation of 3H-oestrone was only detected in one tumour sample after treatment. DNA polymerase alpha activity decreased in 4/6 tumours after treatment with 4-hydroxyandrostenedione. Overall, however, there was no significant correlation between the level of 3H-oestrone formed in situ and DNA polymerase alpha activity (r = 0.38, NS). It is concluded that in some, but not all, breast tumours in situ formation of oestrone can make an important contribution to the oestrogen content of breast tumours.

OBJECTIVE

Despite overwhelming biological plausibility, evidence for a protective effect of oestrogen on cognitive function in postmenopausal women is inconsistent. This study examines the association between endogenous oestrogen levels and subsequent 4-year decline in cognitive function test performance in community-dwelling older women.

METHODS

Longitudinal cohort study.

METHODS

Three hundred and forty-three postmenopausal women (median age 70 years).

METHODS

Between 1984 and 1987, serum for measurement of sex hormones was obtained along with relevant covariates. Cognitive function was assessed in 1988-1991 and again in 1992-1996 using the Category Fluency test, the Mini-Mental Status Exam (MMSE) and Trail Making Test B (Trails B).

RESULTS

Women in the highest tertile of oestrone and bioavailable oestradiol had respectively 1.75 (95% CI 1.02, 3.07) and 1.79 (95% CI 1.04, 3.10) higher odds of 4 year decline in Category Fluency, a test of frontal lobe function, compared to those in the lowest tertile, independent of age and education. The 20% of women with highest tertile levels of both oestrone and bioavailable oestradiol had a twofold higher odds of verbal fluency loss (OR = 2.17; 95% CI 1.21, 3.89). Adjustment for testosterone levels or for obesity-related factors associated with high endogenous oestrogens (higher body mass index, waist girth, and triglycerides and lower high-density lipoprotein cholesterol) did not alter results. Neither oestrogen was associated with change in MMSE or Trails B scores.

CONCLUSIONS

Higher endogenous oestrogen levels were associated with a greater decline in verbal fluency in postmenopausal women. This association was not explained by elevated androgens or by obesity or obesity-related factors.

Serum levels of sex hormone binding globulin (SHBG), testosterone, free testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone sulphate, oestradiol, oestrone, oestrone sulphate, FSH, and LH were measured in 20 steroid sulphatase-deficient men with recessive X-linked ichthyosis and in normal men. The serum oestrone sulphate level was significantly higher than normal in the patients (P less than 0.0001). In affected men, there was a tendency towards higher dehydroepiandrosterone sulphate levels and no decline with age was seen in the patients as opposed to normal men (interaction: P less than 0.025). Serum androstenedione, and oestradiol levels were lower than normal in the patients (P less than 0.0005 and P = 0.055, respectively), while their LH level was higher than normal (P less than 0.0005). The serum levels of SHBG, total and free testosterone, dihydrotestosterone, oestrone, and FSH were not significantly different from normal in the icthyotic patients. We suggest that the observed abnormalities in these patients are a consequence of the enzyme deficiency which severely impairs the ability of tissues to hydrolyse steroid sulphates.

Letrozole is an orally competitive aromatase inhibitor. This double-blind, randomised, multicentre trial was carried out to evaluate the endocrine effects of two doses of letrozole, 0.5 mg versus 2.5 mg orally daily, in postmenopausal advanced breast cancer patients progressing after tamoxifen. The pharmacokinetics of letrozole was also assessed. 46 patients entered the trial, 22 on letrozole 0.5 mg and 24 on 2.5 mg. A significant suppression of oestrone and oestradiol levels was achieved by both letrozole doses. Neither letrozole dose induced any changes in cortisol and aldosterone production at rest or after Synacthen stimulation. Androstenedione, testosterone, 17 alpha-OH progesterone, triiodothyronine (T3) thyroxine, (T4) and thyroid-stimulating hormone (TSH) plasma levels did not show any significant changes. Sex hormone binding globulin (SHBG), follicle-stimulating hormone (FSH) and luteinising hormone (LH) levels increased significantly over time. Plasma letrozole concentrations increased until reaching steady-state values after 1 month at the dose of 0.5 mg and after 2 months at 2.5 mg. In conclusion, both letrozole doses suppressed oestrogen levels without affecting adrenal activity.

The proximate causes of the contraceptive effect of lactation are still a matter of productive debate. This study sought to disentangle the relative impact that intense breast-feeding practices and maternal nutrition have on the regulation of ovarian function in nursing women. A mixed-longitudinal, direct-observational, prospective study was conducted of the return to postpartum fecundity in 113 breast-feeding, well-nourished Toba women. A sub-sample of 70 women provided data on nursing behaviour, daily activities, diet quality and urinary levels of oestrone and progesterone metabolites. Well-nourished, intensively breast-feeding Toba women experienced a relatively short period of lactational amenorrhoea (10.2 +/- 4.3 months) and a high lifetime fertility (TFR=6.7 live births/woman). Duration of lactational amenorrhoea was not correlated with any of the nursing parameters under study or with static measures of maternal nutritional status. The results indicated that the pattern of resumption of postpartum fertility could be explained, at least partly, by differences in individual metabolic budgets. Toba women resumed postpartum ovulation after a period of sustained positive energy balance. As the relative metabolic load hypothesis suggests, the variable effect of lactation on postpartum fertility may not depend on the intensity of nursing per se but rather on the energetic stress that lactation represents for the individual mother.

BACKGROUND

The second to fourth digit ratio (2D:4D) is used as a marker of prenatal sex hormone exposure. The objective of this study was to examine whether circulating concentrations of sex hormones and SHBG measured in adulthood was associated with 2D:4D.

METHODS

This analysis was based on a random sample from the Melbourne Collaborative Cohort Study. The sample consisted of of 1036 men and 620 post-menopausal women aged between 39 and 70 at the time of blood draw. Concentrations of circulating sex hormones were measured from plasma collected at baseline (1990-1994), while digit length was measured from hand photocopies taken during a recent follow-up (2003-2009). The outcome measures were circulating concentrations of testosterone, oestradiol, dehydroepiandrosterone sulphate, androstenedione, Sex Hormone Binding Globulin, androstenediol glucoronide for men only and oestrone sulphate for women only. Free testosterone and oestradiol were estimated using standard formulae derived empirically. Predicted geometric mean hormone concentrations (for tertiles of 2D:4D) and conditional correlation coefficients (for continuous 2D:4D) were obtained using mixed effects linear regression models.

RESULTS

No strong associations were observed between 2D:4D measures and circulating concentrations of hormones for men or women. For males, right 2D:4D was weakly inversely associated with circulating testosterone (predicted geometric mean testosterone was 15.9 and 15.0 nmol/L for the lowest and highest tertiles of male right 2D:4D respectively (P-trend = 0.04). There was a similar weak association between male right 2D:4D and the ratio of testosterone to oestradiol. These associations were not evident in analyses of continuous 2D:4D.

CONCLUSIONS

There were no strong associations between any adult circulating concentration of sex hormone or SHGB and 2D:4D. These results contribute to the growing body of evidence indicating that 2D:4D is unrelated to adult sex hormone concentrations.

In order to examine whether plasma samples may be used for steroid analysis after long periods of storage, cortisol, testosterone, oestrone and oestradiol were remeasured in samples, which had been analysed 1.3-10.8 years earlier. The method for the measurement of these steroids was unchanged over this period. The results demonstrate that at a temperature of -25 degrees C steroids remained stable. Only cortisol and testosterone concentrations showed a small, insignificant decrease (6-9%) after 3 to 4 years of storage. These differences are well within the range of the precision of the method (interassay variation), which over a period of 11 years was 9.4%, 8.0%, 10.0% and 9.5% for cortisol, testosterone, oestrone and oestradiol, respectively. It is concluded that steroid hormones in human plasma are stable in our laboratory, and that they might be analysed even after more than 10 years of storage at -25 degrees C.

Cumulative exposure to oestrogen has been linked to increased risk of breast cancer. Whilst oestrogens induce cancers in rodent bioassays it is unclear whether the mechanisms involved are genotoxic and/or epigenetic. The cytokinesis block micronucleus (CBMN) and the alkaline single cell-gel electrophoresis 'Comet' assays were used to examine MCF-7 cells for chromosomal damage and DNA single-strand breaks (SSBs), respectively. The comet-forming activities of oestrogens were also tested in a 72 h primary culture of cells isolated from freshly expressed breast milk. Micronuclei (MN) were scored in 500 binucleate cells per treatment and SSBs were quantified by comet tail length (CTL) (microm). Effects on mitotic rate (per cent binucleate cells) and cell viability (per cent plating efficiency) were also assessed. beta-Oestradiol, oestrone and oestriol were tested for genotoxicity in the 10(-10)-10(-4) M and 10(-10)-10(-2) M concentration ranges in the CBMN and Comet assays, respectively. Beta-Oestradiol, following 24 h treatment but not 120 h treatment, induced increases (up to 3-fold) in MN at a concentration of 10(-9) M. Oestrone induced dose-related increases in MN (up to 5-fold) following both 24 and 120 h treatment, whereas oestriol appeared not to induce MN. All three oestrogens induced dose-related increases in per cent binucleate cells suggesting that they enhance mitotic rate. In the Comet assay both beta-oestradiol and oestrone induced dose-related increases in SSBs (up to 7-fold over control CTL) and were significantly comet-forming (P < 0.0001) at concentrations as low as 10(-9) and 10(-8) M, respectively, whereas oestriol was less genotoxic. All three oestrogens were significantly comet-forming (P < 0.0001) in a primary culture of breast milk cells, suggesting that they can damage the target cells from which breast cancers may eventually arise.

To test the hypothesis that high levels of endogenous oestrogens increase the risk for developing breast cancer, concentrations of oestrone, oestradiol and oestriol were measured in 24 h urine samples from 1000 women participants in a prospective study of breast cancer on the island of Guernsey. Sixty-nine subjects were diagnosed with breast cancer subsequent to urine collection. Among women who were premenopausal at the time of urine collection, cases excreted less oestrogen than controls; the odds ratios (95% CI) for breast cancer in the middle and upper thirds of the distribution of oestrogen excretion, in comparison with the lower third (reference group, assigned odds ratio = 1.0), were 0.5(0.2-1.2) and 0.4(0.2-1.1) respectively for oestrone, 0.8(0.4-1.8 and 0.4(0.2-1.1) for oestradiol, 0.7(0.3-1.6) and 0.7(0.3-1.6) for oestriol and 0.9(0.4-2.0) and 0.5(0.2-1.3) for total oestrogens. Among women who were post-menopausal at the time of urine collection, the trend was in the opposite direction, with an increase in risk associated with increased oestrogen excretion; the odds ratios were 0.9(0.3-2.2) and 1.1(0.5-2.8) for oestrone, 0.8(0.3-2.3) and 1.9(0.8-4.6) for oestradiol, 1.5(0.6-3.9) and 1.8(0.7-4.6) for oestriol and 0.9(0.4-2.6) and 1.9(0.7-4.7) for total oestrogens. The trends of increasing risk with increasing oestrogen excretion among post-menopausal women were statistically significant for oestradiol (P = 0.022) and for total oestrogens (P = 0.016). We conclude that high levels of endogenous oestrogens in post-menopausal women are associated with increased breast cancer risk, but that the relationship of oestrogens in premenopausal women with risk is unclear.

To assess the relation between urinary endogenous sex steroid levels and the risk of postmenopausal breast cancer, a nested case-cohort study was conducted within a large cohort (the DOM cohort) in the Netherlands (n=9,349). Until the end of follow-up (1 January 1996), 397 postmenopausal breast cancer cases were identified and a subcohort of 424 women was then taken from all eligible women. Women using hormones were excluded, leaving 364 breast cancer cases and 382 women in the subcohort for the analyses. Concentrations of oestrone, oestradiol, testosterone, 5alpha-androstane-3alpha, 17beta-diol and creatinine were measured in first morning urine samples, which had been stored since enrolment at -20 degrees C. A Cox proportional Hazards model was used, with Barlow's adjustment for case-cohort sampling, to estimate breast cancer risk in quartiles of each of the, creatinine corrected, hormone levels, the lowest quartile being the reference group. Women with higher levels of all four of the hormones were at increased risk for postmenopausal breast cancer (highest vs lowest quartile: incidence rate ratio for oestrone (IRR(oestrone)=2.5, 95% CI: 1.6-3.8; IRR(oestradiol)=1.5, 95% CI: 1.0-2.3; IRR(testosterone)=1.6, 95% CI: 1.0-2.4; IRR(5alpha-androstane-3alpha, 17beta-diol)=1.7, 95% CI: 1.1-2.7). In conclusion, women with higher excretion levels of both oestrogens and androgens have an increased risk of breast cancer.

We studied oestrogen binding sites in blood mononuclear cells from healthy blood donors, patients with leukaemia or systemic lupus erythematosus, and in thymocytes, using the dextran-coated charcoal assay and Scatchard analysis of binding data. Using 3H-labelled oestradiol as ligand, inaccurate results were obtained which could be related to the low amounts of binding sites. Using 125I-labelled ligand, saturable oestradiol binding sites could be demonstrated in low amount (mean value 2.1 fmol/mg of cytosolic protein) and high affinity (mean Kd value 0.26 nM; mean Ka value 3.85 X 10(9) M-1). The binding could be inhibited by unlabelled oestradiol but not with oestrone, dihydrotestosterone, cortisol and the progestin-receptor ligand Org 2058. We conclude that blood mononuclear cells and thymocytes contain true oestrogen receptors. This conclusion supports current hypotheses on the involvement of such receptors in oestrogen-mediated modulation of the immune system.

Oestrone (E1), oestradiol (E2), testosterone (T), androstenedione (A) and cortisol (F) as well as LH and the percentage of binding of E1, E2, T and F in plasma were measured and compared in normal young and old male subjects and in male patients with fatty liver, chronic hepatitis and cirrhosis of the liver. The alterations seen were most marked in the cirrhotic patients, but were partially also found in patients with fatty liver and in normal old subjects: a definite increase in E1, a smaller increase in E2, a decrease in T and a rise in LH. F remained unchanged. The ratios of E2/T and E1/T were higher in cirrhotic patients than in healthy young subjects. As the percentage of bound T in plasma rose, the oestrogen/androgen imbalance was greater in patients with liver disease and in old subjects than the ratio of total hormone plasma concentration indicates. The biological relevance of the extremely high E1 plasma concentrations in patients with cirrhosis of the liver is not known. It is suggested that the combination of elevated E1 and E2 and reduced T, which is strongly bound by increased sexual hormone binding globulin (SHBG) may be responsible for gynaecomastia and hypogonadism in chronic liver diseases. As similar alterations of steroid plasma concentrations and their binding to plasma proteins are found both in patients with liver disease and in old men, these changes may be caused by the same mechanism: namely an altered liver function.

BACKGROUND

We assessed adult hypothalamic-pituitary-ovarian function following treatment with chemotherapy and cranial irradiation for childhood acute lymphoblastic leukaemia.

METHODS

The patients (n = 12) had median age at diagnosis of 4.7 years, and at assessment of 20.8 years. They collected a daily urine sample over two to five consecutive menstrual cycles (total of 41 cycles) for analysis of LH and steroid excretion. Blood sampling and ovarian ultrasound examination was performed in the early follicular phase. Sixteen healthy women with regular menstrual cycles were recruited as controls.

RESULTS

Urinary LH excretion was significantly lower in patients throughout the cycle, particularly during the LH surge (P < 0.0001). The length of the luteal phase was significantly shorter in patients than in normal controls (12.2 +/- 0.3 versus 13.6 +/- 0.4 days, P = 0.01) with a high prevalence of short (< or =11 days) luteal phases (15/39 cycles). Luteal phase pregnanediol excretion was slightly but not significantly lower. Follicular and luteal phase excretion of oestrone was lower in patients than in controls (P = 0.01). Early follicular phase plasma oestradiol was also lower in the patient group (P = 0.032) although LH, FSH, inhibin A and B concentrations were similar.

CONCLUSIONS

These data indicate that treatment for childhood leukaemia results in subtle ovulatory disorder in some patients, probably related to cranial irradiation. Follow-up of these women is required to detect any effect on reproductive potential.