Inhibition of oestrone sulphatase followed by oestrogen removal from tumour cells may be a new form of endocrine therapy of breast cancer in women. We investigated the inhibitory effect of the subchronic administration of oestrone-3-O-sulphamate (EMATE), a steroid sulphatase inhibitor, to ovariectomized rats, to evaluate this method for testing new nonsteroidal inhibitors. EMATE in DMSO was administered both orally and subcutaneously (s.c.) for 7 days at doses of 0.5 and 2.5 mg/kg. In addition the rats were injected s.c. with 0.5 mg oestrone sulphate/kg 26 and 2 h before decapitation under ether anaesthesia. Oestrone sulphatase activity (ESA) was measured radiometrically using [3H]oestrone sulphate as substrate for desulphuration in white blood cells, liver homogenate, microsomes and spleen homogenate. ESA in liver microsomes was found to be nearly 40 times higher than in white blood cells while in spleen ESA was nearly half of that found in liver homogenates and white blood cells. ESA can be inhibited by EMATE down to 50-1.5% of control activity depending on the dose and administration route. The inhibition was in the order, liver homogenate < spleen < liver microsomes < white blood cells, and was more pronounced after s.c. administration of the inhibitor than after oral administration. Ovariectomy was found to be not necessary for oestrone sulphatase-inhibiting studies. Two sequential s.c. injections of oestrone sulphate enhanced the enzyme activities significantly in liver and white blood cells, but not in spleen. In conclusion, white blood cells and liver microsomes of intact female rats can be used for ESA-inhibiting studies. Sulphate-conjugated oestrone can induce oestrone sulphatase in vivo in liver and white blood cells thereby enhancing oestrogen supply in the peripheral organs.

Conversion of oestrone sulphate to oestrone has been suggested to make a major contribution to the level of oestrone found in breast tissues. In order to examine the ability of breast tissues to take up oestrone sulphate (E1S), 3H E1S or E1-35S was infused into postmenopausal women with advanced breast cancer. For 3 subjects infusion of 3H E1S was repeated after treatment with Danazol, a potential inhibitor of oestrone sulphatase activity. After infusion of 3H E1S significant levels of 3H E1S were detected in normal and malignant breast tissues (tissue: plasma ratios 0.14 +/- 0.13 and 0.24 +/- 0.12 respectively, mean +/- S.D., n = 5). Similar 3H E1S tissue: plasma ratios were detected after infusions of 3H E1 indicating that the 3H E1S detected in breast tissues after infusion of 3H E1S may have originated from the hydrolysis of 3H E1S in tissues other than the breast, with subsequent uptake and sulphation in breast tissues. After infusion of E1-35S no significant levels of radioactivity were detectable in normal or malignant breast tissues. Treatment with Danazol had no significant effect on tissue levels of 3H E1S or on the CRE1S E1 or MCR-E1S. It is concluded that oestrone sulphate, as such, is not taken up by breast tissues and that any contribution that oestrone sulphate makes to the oestrogen content of breast tissues will depend upon prior hydrolysis.

The uptake and metabolism of 3H-oestradiol by breast tumour tissue was studied in three postmenopausal women before, after 1 week of treatment with ethynyl-oestradiol (EE, 10 micrograms/day) and again two weeks later after treatment with medroxyprogesterone acetate (MPA, 500mg/day, i.m.). Treatment with EE reduced the tissue: plasma gradients for both 3H-oestradiol and 3H-oestrone and little further change in these ratios occurred after MPA treatment. The proportion of oestrogen present as 3H-oestrone increased in tumours from 2/3 women after treatment with MPA but not EE, although no increase in the activity of oestradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) was detected. Plasma and tissue concentrations of 5-androstenediol, dehydroepiandrosterone and dehydroepiandrosterone sulphate were all reduced after MPA treatment. The results from this study show that treatment with EE and MPA can reduce the uptake of oestradiol by breast tumours. No increase in E2DH activity was detected but it is possible that MPA influences other enzymes involved in oestradiol metabolism.

The authors hypothesized that IL-6 production by breast tumour tissues would correlate with OR-positivity, as only those tumours that contain oestrogen receptors (OR) and use oestrogen as a mitogen would benefit from locally increased oestrogen. IL-6 increases the activity of the 17beta-oxidoreductase, which converts oestrone to oestradiol, a process that may contribute to the increased concentration of oestrogen around breast tumours. IL-1alpha upregulates IL-6 production; therefore, the correlation between IL-1alpha and IL-6 immunoreactivity and OR-positivity in paraffin-embedded human breast tumours was further investigated.The results indicate IL-6 immunoreactivity in 40 of 66 paraffin embedded breast tumour specimens, a finding which did not correlate with the clinical evaluation of oestrogen receptor positivity (P=0.32 by Fisher's exact test). However, there was a correlation between IL-6 and IL-1alpha immunoreactivity (P<0.05). To study an in vitro model for this phenomenon, the IL-6 immunoreactivity in available cell lines was tested. Surprisingly, no production of IL-6 protein or mRNA could be detected in any of the cell lines, and this did not change with IL-1alpha stimulation. Therefore, none of the cell lines apparently reflected the immunological potential observed in the majority of surgical specimens.

BACKGROUND

Aminoglutethimide was the first aromatase inhibitor to be used successfully in breast cancer patients. However, this drug also inhibits mineralcorticoid and glucocorticoid synthesis, making co-medication with corticosteroids necessary, and it is often poorly tolerated. The primary objective of this trial was to evaluate the clinical efficacy and tolerability of vorozole, a new non-steroidal oral aromatase inhibitor, in postmenopausal breast cancer patients. The secondary objective was to evaluate the pharmacodynamic activity of the drug.

METHODS

Thirty-four postmenopausal patients previously treated with tamoxifen in the adjuvant setting and/ or for advanced disease were treated with vorozole, 2.5 mg once daily. Patients were monitored with respect to treatment efficacy and safety. Hormonal evaluations were performed at baseline and during the course of treatment in order to evaluate the pharmacodynamic efficacy and safety of vorozole.

RESULTS

According to UICC criteria, there were seven responders, one complete and six partial, for an overall response rate of 21% (95% confidence interval (CI) 9%-38%). The median duration of response was 9.6 months (95% CI 4.6-0), the median time to progression for the entire group was 4.7 months (95% CI 2.9-6.6) and the median survival time was 29.7 months (95% CI 19.1-0). Tolerability was excellent to good in 97% of the patients. Oestradiol and oestrone levels were suppressed to the limit of detection of the assays used. No effect was observed on the other endocrine parameters.

CONCLUSIONS

Our results suggest that vorozole is an effective and safe drug for the treatment of advanced postmenopausal breast cancer following tamoxifen failure.

Human peripheral blood adherent cells (PBAC) incubated with oestrone in high concentration (10(-5) M) release, on exposure to bacterial endotoxin, an amount of tumour necrosis factor (TNF) greater than do cells incubated without the steroid. The finding may imply a non-endocrine hormonal process leading to heightened local TNF responses. This may be the basis of endotoxin hypersensitivity in pregnancy.

The effect of oestrone acetate (in total doses of 5 and 10 mg) on systemic and renal haemodynamics and the renin-angiotensin system has been studied in adult female rats. The administration of 10 mg of oestrogen resulted in a significant fall in renal blood flow associated with significant rises in both renal vascular resistance and mean arterial pressure. No changes were noted in cardiac output or total peripheral resistance at either dose. Whilst the higher dose of oestrogen induced a significant increase in plasma renin activity, no change was noted in animals receiving 5 mg of oestrogen. Both regimens caused significant reductions in plasma and intrarenal renin concentrations. Although renal blood flow correlated with plasma renin activity in animals with a normal renal blood flow, no such correlation was noted in animals with oestrogen-induced reductions in renal blood flow. The present study demonstrates that oestrogen-induced reductions in renal blood flow result from a rise in intrarenal vascular resistance which cannot be accounted for by simultaneous changes in either plasma renin activity or renal renin concentration.

A large proportion of the beneficial effects that oestrogens demonstrate on the vasculature are believed to be mediated via direct effects on the vascular wall. In this study we compared a number of oestrogenic compounds isolated from pregnant mare's urine including 17beta-oestradiol and oestrone, in terms of their abilities to inhibit stimulated endothelin-1 release from normal human coronary artery endothelial cells (CAEC). We also examined their ability to stimulate expression of constitutive endothelial nitric oxide synthase (eNOS) and explored their effects on cellular angiotensin converting enzyme (ACE). All the oestrogens tested were able to inhibit serum-stimulated ET-1 release. Oestrone and 17alpha-dihydroequilenin failed to significantly affect cellular eNOS levels. 17Beta-oestradiol and oestrone significantly increased cellular ACE levels while 17beta,delta(8,9)-dehydroestradiol decreased cellular ACE. We discuss these observations in terms of their potential clinical relevance and use as a means of screening novel oestrogen-like compounds.

To clarify the mechanism of cervical ripening at term, collagenase activity in human cervical tissue was studied. The effects of steroids and prostaglandins on collagenase activity were also examined. Collagenase activities in cervical tissues obtained from non-pregnant (n = 5) and pregnant women (early-pregnant, n = 3; late-pregnant, n = 14) were measured, with or without the addition of steroids and prostaglandins into the incubation medium prior to the measurement of enzyme activity. The enzyme activity was significantly (P < 0.01-0.05) higher in the cervical tissue obtained from late-pregnant women than that from non-pregnant and early-pregnant women. Collagenase activity was significantly (P < 0.05) elevated when steroid sulphates such as dehydroepiandrosterone sulphate and oestrone sulphate were added to the incubation medium, while the addition of free steroids, prostaglandin E2 and prostaglandin F2 alpha did not alter the activity. These data suggest that conjugated steroid produced in the foetoplacental unit during pregnancy may be involved in cervical ripening through the enhancement of collagenase activity.

The functional relationship between the microsomal cytochrome P450 and 17 beta-hydroxysteroid oxidoreductase (HSOR) enzymes involved in steroid metabolism was investigated in rat liver. In male and female rat hepatic microsomes the NADPH-dependent conversion of androstenedione (AD) to testosterone (T) was approx. 4-fold greater at 6 weeks of age than in 1 week old animals. In hepatic microsomes from 15 week old rats the activity of the HSOR pathway was greater in males than in females (1.51 compared to 0.80 nmol T formed/min/mg protein). However, oestradiol administration to intact adult male rats did not decrease HSOR activity. Thus, androgen is not essential for maintenance of HSOR enzymes. Instead, it is likely that irreversible androgen imprinting of the HSOR enzyme occurs during the prepubertal period. The in vitro characteristics of HSOR activity were also assessed. The Km for NADH-dependent reduction of AD to T was 9.2 microM and the Vmax was 3.0 nmol/min/mg protein but the NAD-mediated formation of AD from T did not follow Michaelis-Menton kinetics. pH markedly influenced HSOR-mediated AD/T interconversion with 17-ketosteroid reduction facilitated at low pH, and 17 beta-hydroxysteroid dehydrogenation about 2-fold more efficient at pH 8.0 than at pH 5.5. Product steroid activation of HSOR activity was noted. 17 beta-Hydroxysteroids, including T and oestradiol, activated the rate of conversion of AD to T and 17-ketosteroids such as oestrone and AD activated the NAD-dependent dehydrogenation of T. Activation was not observed at low steroid substrate concentrations so that it was not possible to analyse this phenomenon by a conventional kinetic approach.

Puberty is a key stage in mammalian ontogeny, involving endocrinological, physiological and behavioural changes, moderated by intrinsic and extrinsic factors. Thus, not all individuals within one population achieve sexual maturity simultaneously. Here, using the European badger (Meles meles) as a model, we describe male testosterone and female oestrone profiles (using Enzyme-immunoassays) from first capture (3 months, post-weaning) until 28 months (attaining sexual maturity and final body size), along with metrics of somatic growth, scent gland development and maturation of external reproductive organs as well as intra-specific competition. In both sexes, endocrinological puberty commenced at ca. 11 months. Thereafter, cub hormone levels followed adult seasonal hormone patterns but at lower levels, with the majority of cubs reaching sexual maturity during their second mating season (22-28 months). Interestingly, there was evidence for two endocrinological phenotypes among male cubs (less evident in females), with early developers reaching sexual maturity at 11 months (first mating season) and late developers reaching sexual maturity at 22-26 months (second mating season). Early developers also attained a greater proportion of their ultimate adult size by 11 months, exhibiting faster growth rates than late developers (despite having similar adult size). Male cubs born into larger social groups tended to follow the late developer phenotype. Our results support the hypothesis that a minimum body size is required to reach sexual maturity, which may be achieved at different ages, even within a single population, where early maturity can confer individual fitness advantages and enhance population growth rate.

Our objective was to evaluate pigs injected with human chorionic gonadotropin (hCG) as a potential model to study the effect of high testicular steroid levels on the variation in indolic compounds. Pre-selection of the sires based on their plasma skatole levels was used, to evaluate whether the response to hCG would be affected. Out of 34 entire male pigs, 17 pigs were injected with hCG and the remaining pigs were injected with sterile saline four days prior to slaughter. HCG injection increased the levels of testicular steroids (androstenone, testosterone, dehydroepiandrosterone sulphate, oestrone sulphate and oestrone; p<0.001), and indole levels in plasma and fat (p<0.05). Skatole levels in fat increased slightly but not significantly (p=0.107), whereas skatole levels in plasma were not affected. The correlation coefficients between androstenone and skatole levels in fat were 0.54 in the hCG-injected group and non-significant in the control group. Skatole levels in fat were not correlated with testicular steroids in either hCG-injected pigs or controls. Skatole and indole levels in fat were positively correlated in both groups. We concluded that hCG-injected pigs might be useful for studying factors responsible for the role of testicular steroids in the occurrence of high indole levels. Pre-selection of sires based on plasma skatole levels affected both the levels of indolic compounds and testicular steroids, especially in fat, which might have suppressed the response to hCG. Whether hCG affects skatole or not is therefore not fully elucidated.

In order to provide information on the endocrine effects of vasectomy, unconjugated pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol were analysed in the blood plasma of twenty Mexican men on two occasions before and 1, 3, 6 and 12 months after vasectomy. Vasectomy appeared to be associated with a significant decrease in the plasma levels of pregnenolone, dehydroepiandrosterone and androstenedione and a significant increase in the levels of dihydrotestosterone and oestrone. A probably significant increase in oestradiol levels took place 12 months after vasectomy but not before. No consistent changes were found in testosterone (up to 12 months) or in FSH and LH levels (up to 6 months) after vasectomy. The unconjugated steroids indicated above, except oestrone, were also estimated, whenever possible, in seminal plasma specimens obtained from thirty-nine subjects (including the twenty indicated above) on the same occasions. Vasectomy was associated with a highly significant decrease of seminal plasma dihydrotestosterone levels on all occasions and a significant decrease in androstenedione levels after 6 and 12 months. After 12 months there was a decrease in dehydroepiandrosterone and an increase in oestradiol; these changes were both probably significant. In another preliminary study, the levels of pregnenolone sulphate, dehydroepiandrosterone sulphate, testosterone glucuronide, testosterone sulphate and dihydrotestosterone sulphate were estimated before and 1 month after vasectomy in the seminal plasma of fourteen to seventeen subjects. Testosterone glucuronide fell, probably significantly, but other conjugates were unchanged. The data indicate that vasectomy may be associated with significant changes in the circulating and in seminal plasma levels of several steroids. The gradual nature of some of the changes observed suggests the necessity of conducting in several centres large-scale, long-term studies on vasectomized subjects and on a carefully matched control group. During the last decade vasectomy has been widely practised in several parts of the world as a method of fertility control. However, information on the endocrine effects of this intervention appears to be scanty. In most of the human studies reported, a small number of individuals were investigated and the studies have been confined to the assessment of the short-term effects of the operation. Moreover, the hormonal indices assessed by the various investigators have been limited, in most cases, to gonadotrophins and testosterone in blood. The present study was designed to assess in the same subjects the levels of a number of unconjugated steroids, FSH and LH on two occasions before and 1, 3, 6 and 12 months after vasectomy. The studies were extended to include steroid analyses in seminal plasma in the hope that such assays might yield information as to the effects of vasectomy on the distribution of steroids in the fluids of the male reproductive tract.

Phospholipase A2 (PLA2) activity was measured in endometrium and amnion by a double isotope ratio technique using 1-palmitoyl-2-oleoyl phosphatidylcholine as substrate in the presence and absence of a range of unconjugated steroids and steroid sulphates (0.2-6.4 X 10(-4) M). In the presence of 0.1% Triton, PLA2 activity was inhibited by the majority of steroids tested, pregnenolone sulphate being the most effective (12.9 +/- 3.0% control activity) while oestradiol sulphate, oestrone and testosterone had only a minimal or no effect (99.1 +/- 19.0, 85.4 +/- 4.4 and 104.2 +/- 16.3% control respectively). In the absence of Triton, the inhibitory effect of the free steroids was reduced or absent but oestradiol sulphate and testosterone sulphate stimulated activity by 2-13 and 1.5-3 times respectively. The effect was dose related, linear with time and independent of the stage of the menstrual cycle. Inhibition by pregnenolone sulphate, dehydroepiandrosterone (DHA sulphate and oestrone sulphate was maintained in the absence of Triton (24.9 +/- 3.8, 67.1 +/- 10.1 and 87.2 +/- 13.8% control respectively). In amnion all 5 steroid sulphates caused a marked stimulation of PLA2 activity in both the presence and absence of Triton. The effect was greatest without Triton and at 6.4 X 10(-4) M, testosterone, pregnenolone, oestrone, DHA and oestradiol sulphates increased PLA2 activity 20, 15, 12, 10 and 6-fold respectively. These findings indicate a direct action of steroid sulphates on PLA2 activity in endometrium and amnion.

Minces of the prostate and seminal vesicles of the mature boar were incubated with the following major testicular steroids of the pig: [3H]dehydroepiandrosterone, [3H]5-androstene-3 beta, 17 beta-diol, [3H]oestrone, [3H]oestradiol-17 beta and their respective sulphate conjugates (excluding oestradiol-17 beta). Incubations were also carried out with [3H]testosterone, [3H]5 alpha-dihydrotestosterone and [3H]5 alpha-androstanediols. Minces of the epididymides were incubated with [3H]dehydroepiandrosterone and [3H]oestrone sulphates. The prostate and seminal vesicles converted dehydroepiandrosterone predominantly to weak androgens, whereas 5-androstene-3 beta, 17 beta-diol was primarily converted to testosterone; testosterone and its 5 alpha-reduced metabolites were metabolized in a manner typical of androgen end target organs. Unconjugated oestrone and oestradiol-17 beta were interconverted by the prostate and seminal vesicles. The metabolism of C19 steroid sulphates was less than 1% in all incubations; some oestrone sulphate, however, was converted to unconjugated oestrone and oestradiol, particularly by the caput epididymidis. The significance of these results is discussed in relation to recent studies in vivo.

Aromatase activity was measured in homogenates from steroid sulphatase deficient and steroid sulphatase positive placentae. The activity of the aromatase complex was determined from the rate of formation of [14C] oestrone plus [14C] oestradiol when [14C] testosterone was incubated with a rate-limiting quantity of homogenate. A reduced level of aromatase activity was found in vitro in 70% of steroid sulphatase-deficient placentae tested, but the deficiency was much less complete than that of steroid sulphatase. The mean (+/- SD) aromatase activity of steroid sulphatase-deficient placentae was 380 +/- 180 pmol product/h/g tissue (n = 10), significantly lower (P less than 0.001, t-test) than the mean aromatase activity of steroid sulphatase positive placentae (701 +/- 70 pmol product/h/g tissue, n = 10). Seventy per cent of the steroid sulphatase deficient placentae showed lower aromatase activity than that of normal placentae stored for a comparable period of time. Assay imprecision and the sex of the foetus were excluded as reasons for the diminished aromatase activity found. It may arise through an abnormal gene product and consequent alterations in the structure of the microsomal membrane in which both aromatase complex and steroid sulphatase enzymes are retained.

The concentrations of five steroids in samples of spermatic venous blood collected from 17 men undergoing ligation of varicocoeles were compared with those in samples from the antecubital vein. There was evidence of testicular secretion of testosterone, androstenedione, oestradiol-17 beta and oestrone, since the ratios of the mean concentrations in spermatic venous plasma to those in peripheral venous plasma were 77.2, 9.1, 28.7 and 1.6 respectively. The testicular secretion of oestrone sulphate was minimal; the ratio of the mean concentrations in spermatic and peripheral plasma was 1.07. These results support the view derived from isotope dilution studies that almost all oestrone and oestrone sulphate in the circulation is derived from peripheral conversion of other precursor steroids.

Mid-trimester human fetal brain cytosol incubated with [3H]-oestradiol gave a radioactive peak on 5% polyacrylamide gels, which migrated with the bromophenol blue marker (anodic peak) and which could be suppressed by the addition of 100 times molar excess of unlabelled ligand to the incubate. This anodic peak could only be destroyed by proteolytic enzymes if they were added at the beginning of the incubation, but not subsequently, indicating that it did not represent a protein-bound oestradiol. Competition studies show that the anodic peak can be suppressed with natural oestrogens and ethinyloestradiol, but not by the synthetic oestrogens, androgens and progestins tested. Butanol extraction of incubates, followed by t.l.c. in a number of systems, indicates that both oestrone and oestradiol are sulphated. The parent steroids could be liberated by hydrolysis of incubates with either 1N sulphuric acid or aryl sulphatase. This conjugation may effectively curtail the action in fetal tissues of high levels of oestrogens and hence play a role in brain sexual differentiation in the human.

In vitro studies have previously shown that the activity of the aromatase enzyme system, which is responsible for the conversion of androstenedione to oestrone, can be stimulated by natural and synthetic glucocorticoids and also by ACTH. In view of the potential physiological importance of such a regulatory mechanism we have examined the effect of administration of dexamethasone, and of ACTH on the conversion of androstenedione to oestrone in vivo. The transfer constants for the conversion of androstenedione to oestrone [( rho]AEBU) measured in two women before administration of dexamethasone were 1.0% and 1.1% and after were 0.9% and 1.2%. Similarly no increase in conversion of androstenedione to oestrone [( rho]AE1BB) was detected after ACTH stimulation (pre = 0.74%, post = 0.77%). It is concluded from this study that glucocorticoids and ACTH do not have a role in regulating aromatase activity in vivo.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.

Polyclonal antisera raised against two different azobenzoyl-oestrone derivatives were analysed to investigate both the latency/intensity relationship of the immune response and the influence of antigen presentation on the specificity of the antisera elicited. Elongation of the azo-bridge of the hapten ([p(carboxyphenyl)-azo]-1,3,5[10]- oestratrien-3 ol-17 one) with a short aliphatic chain (4-amino-n-butyric acid) resulted in a marginal increase in the antibody yield, without affecting the time required to attain the maximum titre. The increased flexibility and mobility of the extended azo-bridge was shown to result in the appearance of antisera which cross-reacted with oestrogens with D ring structures different to that of oestrone. Antiserum fractionation by affinity chromatography through a stationary phase exposing the carrier protein determinants, as modified by the addition of the coupling bridge and the phenol ring, resulted in a reduction in its specificity. These findings are discussed with regard to the phenomena underlying the specificity of a polyclonal antiserum.

We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both 'ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab.