Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--I. Importance of hydrolytic enzymes on conjugate absorption

The biliary excretion of steroid after administration of [3H]oestrone ([3H]E1), [3H]oestrone glucuronide ([3H]E1G) and [3H]oestrone sulphate ([3H]E1S) into the hepatic portal vein of anaesthetized rats was very rapid with more than 70% of E1S and greater than 80% of E1 and E1G excreted in the first 30 min. There was a lag period in the biliary excretion of E1S, this was less apparent with E1 and absent with E1G. Biliary excretion accurately reflects the amount of steroid in the portal circulation and was therefore used as an assessment of absorption from the gastrointestinal (GI) tract. Absorption (as judged by excretion in bile) was least after administration of each steroid into the stomach. The extent of absorption correlated well with the lipophilicity of the steroids as shown by their relative partition coefficients between n-octanol and pH 6.5 phosphate-buffered saline (E1 greater than or equal to E1S greater than or equal to E1G). There was no significant difference in excretion profile when the steroids were given into the caecum (at 5 h, E1, 46.3 +/- 9.1%; E1G, 42.2 +/- 14.5%; E1S, 39.9 +/- 7.1%). The similarity, despite marked differences in physicochemical properties, suggested conjugate hydrolysis to the parent steroid. In contrast, after administration into the small intestine, excretion of E1 was very rapid and was maximal at 1 h (72.5 +/- 8.0%); E1G showed a near-linear excretion rate (1 h, 14.4 +/- 3.0%; 5 h, 80.0 +/- 11.7%), whereas in comparison E1S excretion was low (1 h, 12.1 +/- 2.4%; 5 h, 36.9 +/- 2.7%). The involvement of hydrolytic enzymes in conjugate absorption was assessed. Ampicillin pretreatment (200 mg/kg/day for 2 days) reduced the absorption of E1G from both the proximal and distal small intestine (by approximately 50%) but had no effect on the absorption of E1S. There was, therefore, evidence that quantitative absorption of E1G requires prior hydrolysis (by mammalian and/or microbial enzymes) but intact absorption of E1S from this region of the tract was implicated. Ampicillin pretreatment reduced the absorption of both conjugates (greater with E1S) from the caecum; hydrolysis clearly precedes absorption from the caecum. The above findings were supported by an in vitro study which showed that ampicillin pretreatment abolished the hydrolysis of E1S by caecal contents but only partially reduced the hydrolysis of E1G. The presence of mammalian glucuronidase enzyme may account for this difference.     

Development of some male characteristics supported by oestrone but not dehydroepiandrosterone in the boar

Prepubertally castrated boars received subcutaneous injections twice weekly, from 13 to 35 weeks of age, of dehydroepiandrosterone (2 mg/5 kg) or oestrone (1 mg/5 kg). Dehydroepiandrosterone did not support the growth and secretory activity of the accessory organs, or induce copulatory behaviour. However, oestrone caused hypertrophy of the prostate, seminal vesicles and bulbourethral glands which was due to an increase in fibrous stromal tissue and not to the secretory epithelium. Oestrone also induced some male mating behaviour patterns in the presence of an oestrous gilt, although penile extrusion and ejaculation did not occur. The morphological and behavioural effects of the steroid treatments were supported by steroid profiles in blood plasma as seen in comparison with androgen and oestrogen values of intact and untreated castrated boars. It is concluded that oestrogen in the intact boar might enhance the secretion of the accessory organs by affecting the neural control of the secretory processes rather than by increasing the amount of secretory epithelium in the glands.     

A direct assay for oestrone sulphate and its use to investigate the effect of ampicillin on plasma levels of oestrone sulphate

A direct radioimmunoassay for measuring plasma levels of oestrone sulphate has been developed using 8-anilino-2-naphthalene sulphonic acid to displace oestrone sulphate from plasma binding proteins. Oestrone sulphate was assayed by using an antiserum raised against glucuronide which cross-reacted 100% with oestrone sulphate. The direct assay gave a good analytical recovery of oestrone sulphate and there was a good correlation (r = 0.82, P less than 0.001) for plasma levels of oestrone sulphate measured by the direct assay and a method involving steroid conjugate extraction and enzyme hydrolysis. The mean (+/- S.D.) plasma level of oestrone sulphate in men was 1100 +/- 280 pg/ml. The effect of taking the antibiotic, Ampicillin, on plasma levels of oestrone sulphate was investigated in four men. Plasma levels of oestrone sulphate were significantly reduced after taking Ampicillin for 5 days. Ampicillin may act to lower plasma levels of oestrone sulphate by reducing the growth of bacteria in the gut or by inhibiting oestrogen sulphotransferase activity.     

The regulation of oestrone sulphate formation in breast cancer cells.

The formation of oestrone sulphate has been examined in MCF-7 (oestrogen receptor positive, ER+) and MDA-MB-231 (ER negative, ER-) breast cancer cells. Using intact cell monolayers and a physiological substrate concentration, progesterone (1 microM) and dexamethasone (1 microM) both increased oestrone sulphate formation in MCF-7 cells. In MDA-MB-231 cells, dexamethasone, but not progesterone, increased conjugate formation. A number of growth factors, cytokines and human serum albumin (HSA), which have previously been found to regulate oestrogen synthesis, were also examined for their ability to regulate oestrone sulphate formation. In MCF-7 cells epidermal growth factor, acidic and basic fibroblast growth factors, insulin-like growth factor-type I and insulin all stimulated oestrone sulphate formation. The cytokines, tumour necrosis factor alpha (TNFalpha) and interleukin-1beta also increased conjugate formation in the ER+ cells, as did HSA. In contrast, in MDA-MB-231 cells TNFalpha was without effect and HSA inhibited oestrone sulphate formation. The ability to modulate oestrone sulphate formation in ER+ cells may be an important mechanism to limit the availability of oestrogen to interact with the ER.     

The origin of oestrone sulphate in normal and malignant breast tissues in postmenopausal women.

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.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

Inhibition of oestrone sulphatase activity by tibolone and its metabolites.

Tibolone is a 19-nortestosterone derivative commonly used in hormone replacement therapy. Although tibolone and its 3alpha/beta-hydroxy metabolites exert oestrogenic effects on bone and the vasomotor system, they do not appear to stimulate breast tissue proliferation. It has been proposed that the lack of an oestrogenic effect on breast tissues may result from the inhibition of oestrone sulphatase (E1-STS) in this tissue by tibolone and its metabolites. In this study we have examined the ability of tibolone and its metabolites to inhibit E1-STS activity in intact breast cancer cells, its effect on E1-STS activity in placental microsomes and also the expression of E1-STS mRNA in more detail. As the major proportion of hydroxytibolone metabolites circulate in a sulphated form, the ability of the 3alpha-sulphate and 3alpha,17beta-disulphate metabolites to inhibit E1-STS activity was also examined. In MCF-7 cells, tibolone and its 3beta-hydroxylated metabolite were relatively potent inhibitors; they inhibited activity by 48 % and 46 %, respectively. In these cells, the 3alpha-sulphate and 3alpha,17beta-disulphate metabolites of tibolone inhibited E1-STS activity by 95% and 79% at 10 microM, respectively. No effects of tibolone or its metabolites on the expression of E1-STS mRNA in MCF-7 cells were detected. Using T-47D breast cancer cells, evidence was obtained that the sulphated metabolites of tibolone could continue to inhibit E1-STS activity after removal of the drugs and extensive washing of cells. In placental microsomes, however, the 3beta-hydroxy metabolite was the most potent inhibitor with an IC50 of 20.5 microM; the sulphated metabolites were less potent. Neither tibolone nor its metabolites had any inhibitory effect on the conversion of oestrone to oestradiol in breast cancer cells. Results from this study have confirmed that tibolone and its metabolites can inhibit E1-STS activity. This may explain the absence of breast stimulation as observed in clinical studies.     

The use of nonradiolabelled steroid infusions to investigate the origin of oestrone sulphate in postmenopausal women

Infusion of nonradiolabelled dehydroepiandrosterone sulphate (DHA-S) has been used to investigate the possible formation of oestrone sulphate via a sulphated conjugate of androstenedione. The metabolic clearance rate (MCR) of DHA-S also was measured and the mean value (25 1/24h) was similar to values reported using isotopic techniques. Although conversion of DHA-S to 5-androstenediol, a steroid with oestrogenic properties, was detected during infusion of DHA-S, there were no significant increases in plasma levels of conjugated androstenedione or oestrone sulphate. The MCR's oestrone sulphate measured using infusion of nonradiolabelled steroid in two menopausal women were 99 1/24h and 121 1/24h. For one woman, the production rate of oestrone sulphate, calculated from the conversion of oestrone and oestradiol to oestrone sulphate (151 nmol/day) was similar to the measured production rate of oestrone sulphate (144 nmol/day). It is concluded that in menopausal women, oestrone sulphate is derived from conversion of oestrone and oestradiol with no formation occurring via conjugated androstenedione.     

Metabolism of sodium oestrone [35S]sulphate in the guinea pig

Intraperitoneal administration of sodium oestrone [(35)S]sulphate to male and female free-ranging guinea pigs is followed by excretion of most of the radioactivity mainly as inorganic [(35)S]sulphate in the urine within 72h. The remainder of the radioactivity in the urine was found in oestrone [(35)S]sulphate, two unidentified metabolites (A and B) and traces of oestradiol-17beta 3-[(35)S]sulphate. When injected intraperitoneally into animals with bile-duct and bladder cannulae, most of the dose was excreted in the bile. Unchanged oestrone [(35)S]sulphate was the main biliary component excreted in males and females, but the latter also excreted appreciable amounts of oestradiol-17beta 3-[(35)S]sulphate and metabolites A and B. The urine from these animals also contained these metabolites, inorganic [(35)S]sulphate and also oestrone [(35)S]sulphate, but in small amounts. Metabolite A was present only in samples from males. Whole body radioautography pinpointed the liver and kidney as the possible sites of metabolism of the ester. The ester underwent little desulphation in the isolated perfused female guinea-pig liver and in animals in which kidney function had been eliminated, and was excreted unchanged in the bile. These results and the observed low oestrogen sulphatase and arylsulphatase C activities found in guinea-pig liver and kidney support the view that the two enzymes are identical.     

Evolution of tissue levels of steroids: oestradiol-17 beta, oestrone and progesterone in the macaque myometrium during gestation

In 15 cynomolgus monkeys between days 30-160 of gestation, tissue levels of oestrone (E1), oestradiol 17 beta (E2) and progesterone (P4) were assayed by RIA in the myometrium and the placenta. Myometrial samples were subdivided as follows: inner and outer layers adjacent to the placental area (IMP and OMP) and inner and outer layers from antiplacental areas (IMAP and OMAP). Steroid levels (ng/g wet wt) were in the range of known plasma values (ng/ml) and there was no asymmetric distribution of steroids between the various locations. When the results obtained in all the layers were pooled the gestational profiles indicated a decrease of E1 between days 80-130, whereas at the same time E2 and P4 increased. The ratio P4/E2 was 8 on day 40, 17 on day 80 and 9 on day 160. In the placenta, levels of E2 and P4 were 4 times higher, levels of E1 10 times higher than in the myometrium. Gestational profiles of the three steroids in the placenta increased from day 30 to day 160. Myometrium steroid content therefore does not appear to be a simple reflection of steroid diffusion from the site of production.     

A comparison between plasma oestrone sulphate concentration and doppler ultrasound as methods for pregnancy diagnosis in sows

Plasma oestrone sulphate (E(1)S) concentration and doppler ultrasound as methods of pregnancy diagnosis in sows were compared. Using either method, pregnancy was accurately detected (test sensitivity > 94% for pregnant sows). E(1)S was a better predictor of nonpregnant animals (test specificity 78 vs 66%, respectively; P < 0.01) and could be used at least 1 wk earlier than doppler ultrasound (24 to 30 d vs 35 d postservice, respectively). E(1)S concentration was not an accurate predictor of litter size.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

Obligatory role of the endocardial endothelium in the increase of myocardial distensibility induced by endothelin-1

This study investigated how the endocardial endothelium (EE) and particularly endothelial type B (ET(B)) receptors influence the effects of endothelin-1 (ET-1) on diastolic distensibility. ET-1 (0.1, 1, and 10 nM) was tested in rabbit papillary muscles (Krebs-Ringer; 1.8 mM CaCl2, 35 degrees C) (i) with intact EE (n = 10), (ii) with damaged EE (0.5% Triton X-100, n = 11), and (iii) in the presence of RES-701-1 (selective endothelial ET(B1) receptor antagonist, 1 microM, n = 6). Additionally, increasing doses (0.1 nM to 1 microM) of Sarafotoxin S6c (SRTXc, a selective ET(B) receptor agonist) and IRL-1620 (a selective endothelial ET(B1) agonist) were studied (i) in muscles with intact EE (n = 7 and n = 6, respectively) and (ii) after damaging the EE (n = 8 and n = 7, respectively). In papillary muscles with intact EE, ET-1 induced dose-dependent positive inotropic and lusitropic effects. At 10 nM, active tension (AT) increased 78% +/- 17%, maximum velocity of tension rise (dT/dt(max)) increased 82% +/- 10%, and maximum velocity of tension decline (dT/dt(min)) increased 77% +/- 17%. These effects were maintained when ET-1 was given after damaging the EE (AT increased 70% +/- 12%, dT/dt(max) increased 93% +/- 14%, and dT/dt(min) increased 56% +/- 14%), but were significantly reduced in the presence of RES-701-1 (AT increased 30% +/- 6%, dT/dt(max) increased 37% +/- 7%, and dT/ dt(min) increased 29% +/- 9%). ET-1 reduced resting tension (RT) and increased diastolic distensibility by 3% +/- 1%, 5% +/- 1%, and 9% +/- 2% (at 0.1, 1, and 10 nM, respectively) in muscles with intact EE. This effect was abolished after damaging the EE or in the presence of RES-701-1. In muscles with intact EE, SRTXc had no significant effects, whereas, when given after damaging the EE, SRTXc (1 microM) increased inotropy and lusitropy (AT increased 116% +/- 24%, dT/dt(max) 110% +/- 28%, and dT/dt(min) 88% +/- 19%) without affecting RT. IRL-1620 dose-dependently decreased AT, dT/dt(max), and dT/dt(min) in muscles with intact EE-effects that were abolished after EE damage. No significant effects were elicited by IRL-1620 in RT. ET-1-induced increase in myocardial distensibility, previously shown to be mediated by ET(A) receptor stimulation, requires an intact EE and active endothelial ET(B1) receptors.     

17 beta-hydroxysteroid dehydrogenase activity in leiomyoma and myometrium and its relationship to concentrations of oestrone, oestradiol and progesterone throughout the menstrual cycle

The activity of the enzyme 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSD) and concentrations of oestrone (E1), oestradiol (E2) and progesterone have been measured in leiomyoma and myometrium obtained at different stages of the menstrual cycle. Apart from conversion of E2 to E1 in the proliferative phase, no significant difference in enzyme activity was noted between normal and tumour tissue. However, interconversion in both tissues was shown to be higher in the secretory than the proliferative phase of the menstrual cycle. E1 concentrations were significantly higher (P less than 0.01) in leiomyoma than in myometrium, obtained during the proliferative phase. Concentrations of both oestrogens, in some tumour and normal tissues, were higher in the proliferative than the secretory phase. Secretory phase tissues contained higher concentrations of progesterone than those obtained in the proliferative phase of the menstrual cycle. Considerable differences in both enzyme activity and steroid concentrations were noted in different areas of the same tumour.     

Change in the relative importance of oestrone and oestradiol synthesis by the rat ovary between fetal and prepubertal stages

Ovaries of rat fetuses at 20 and 21 days and of neonatal rats at 5 and 14 days were cultured in the presence of [3H]testosterone, and the conversion percentages into oestrone (E1) and oestradiol (E2) were determined by double isotopic dilution combined with recrystallization to constant specific activity. Insignificant in the 20- or 21-day-old fetus, oestradiol synthesis increased relative to oestrone synthesis in the 5-day-old neonate (E1/E2 = 3.4) and still more at the stage of 14 days (E1/E2 = 0.78). FSH had no effect on oestrogen synthesis at the 4 stages investigated.     

Involvement of the Gi/o/cGMP/PKG pathway in the AT2-mediated inhibition of outer cortex proximal tubule Na+-ATPase by Ang-(1-7)

The molecular mechanisms involved in the Ang-(1-7) [angiotensin-(1-7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1-7) has a biphasic effect on the proximal tubule Na+-ATPase activity, with the stimulatory effect mediated by the AT1 receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na+-ATPase by Ang-(1-7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT1 receptor-mediated stimulation. In this condition, Ang-(1-7) at 0.1 nM inhibited the Na+-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT2 receptor, and by 1 muM GDP[beta-S] (guanosine 5'-[beta-thio]diphosphate), an inhibitor of G protein. Ang-(1-7) at 0.1 M induced [35S]GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and 1 mug/ml pertussis toxin, an inhibitor of G(i/o) protein, reversed the Ang-(1-7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1-7) on the Na+-ATPase activity was completely reversed by 0.1 microM LY83583, an inhibitor of guanylate cyclase, and by 2 muM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1-7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 microM LY83583. Taken together, these data indicate that Ang-(1-7) inhibits the proximal tubule Na+-ATPase by interaction with the AT2 receptor that subsequently activates the G(i/o) protein/cGMP/PKG pathway.