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Serum estradiol, estrone, estrone sulfate and sex hormone binding globulin were measured in 10 postmenopausal patients with advanced breast cancer receiving sequential treatment with medroxyprogesterone acetate and megestrol acetate. Treatment with megestrol acetate caused a non-significant reduction in serum estradiol (mean reduction of 19%, 0.05 less than P less than 0.1) but significant reductions in serum estrone (mean reduction of 20%, P less than 0.02) and serum estrone sulfate (mean reduction of 54%, P less than 0.005) compared to treatment with medroxyprogesterone acetate. In contrast, treatment with medroxyprogesterone acetate reduced serum sex hormone binding globulin more compared to treatment with megestrol acetate (mean reduction of 69%, P less than 0.01). These findings suggest that the two progestins have differential effects on serum hormone levels. The finding that treatment with megestrol acetate causes a significant reduction in serum estrone sulfate level warrants further investigations of this potentially important mechanism of action of this drug in advanced breast cancer.

Inhibition of postmenopausal estrogen production by aromatase inhibitors is an established drug treatment modality for postmenopausal breast cancer. In this article postmenopausal estrogen disposition and the alterations caused by treatment with aromatase inhibitors are reviewed. Recent investigations have challenged the hypothesis that aromatization of androstenedione into estrone is the sole production pathway for estrogens in postmenopausal women. The finding that estrogens persist in the plasma of patients receiving aminoglutethimide treatment despite a near total inhibition of the aromatase enzyme suggests that alternative pathways for estrogen synthesis exist. While nonspecific actions of aromatase inhibitors may be disadvantageous, certain effects may also be beneficial. Recent findings that aminoglutethimide may induce estrone sulfate metabolism questions whether this "prototype" aromatase inhibitor might have a dual mechanism of action. The importance of investigating the possible influence of different aromatase inhibitors on all components of estrogen disposition is considered.

17 beta-Hydroxysteroid oxidoreductase, as well as estrone sulfate and dehydroepiandrosterone sulfate sulfatases, were found in the plasma membrane of microvilli of the fetal syncytiotrophoblast. Because of their location, these enzymes may influence feto-maternal transfer of steroids circulating as sulfates, the utilization of sulfated estrogen precursors and the proportion of estrone and estradiol delivered towards fetal and maternal circulations. Microvillar vesicles isolated from human term placentas were disrupted in hypotonic medium to obtain a membrane preparation. A fraction of the estradiol 17 beta-oxidoreductase (E2DH) activity in the vesicle remained associated to the membrane after disruption and treatment with 2 M NaCl. The membrane-associated activity was resistant to inhibition with trypsin and did not react with a polyclonal antibody which neutralized cytosolic E2DH activity. The membrane-associated enzyme was solubilized with a cholate-glycerol buffer solution and purified on Sephadex G-100. The estimated molecular weight of the solubilized enzyme (137 kDa) appears to correspond to a tetramer since it was found to be about twice the size of the cytosolic enzyme. Both enzymes focused in polyacrylamide gels at pH 5.2. The Km relative to E2 of the membrane-associated E2DH (1.3 microM) differs from those of mitochondrial (0.43 microM), microsomal (0.69 microM) and cytosolic (11 microM) fractions. The cytosolic and the microvillar membrane associated 17 beta-hydroxysteroid oxidoreductases also differ in their specificity for C18 and C19 steroid substrates and in their pH dependence patterns. Sulfatases acting on estrone sulfate and dehydroepiandrosterone sulfate in microvillar membranes were insensitive to trypsin and as resistant to washes with 2 M NaCl as alkaline phosphatase. This data indicated that steroid sulfatases are also microvillar membrane associated enzymes of potential physiologic importance in the hydrolysis of estrogen precursors.

Estrone sulfate 16 alpha-hydroxylase activity is undetectable in liver microsomes from fetal guinea-pigs of the English Shorthair variety. Within 2 days of birth, considerable activity is present in both sexes of pigmented and albino animals. In the pigmented group, maximum activity occurs during the second week of life, with the mean values for each of the first 4 weeks, in both sexes, significantly higher than for the corresponding mature (greater than 12-week-old) animals. Immature levels in the albino group are also significantly higher than those of mature albinos. Pigmented females of all ages possess significantly higher activities than do their male counterparts. There are no such sex-related differences in albinos. Pigmented animals of all ages exhibit higher activities than do their albino counterparts. Castration of either sex, pigmented or albino, results in increased enzyme activities as compared with intact or sham-operated controls. Gestation leads to maternal enzyme values which are significantly above those of non-pregnant females, whether pigmented or albino. Beyond the first few days of life, total liver microsomal cytochrome P450 shows no significant change with age, gestation or pigmentation. These data support the conclusion that estrone sulfate 16 alpha-hydroxylase activity in the guinea-pig is markedly diminished, following sexual maturity, by presently unknown factors. This holds for both pigmented and albino animals but the decrease is greater in the latter. This decrease can be reversed by castration in either sex, or by pregnancy and could possibly relate to gonadal-pituitary relationships as demonstrated by others for rat liver hydroxylases.

The purpose of this study was to evaluate, without using radioisotopes, the peripheral contribution of dehydroepiandrosterone (D) to estrogens and to androstenedione (A) in patients with hypogonadotropic hypogonadism associated with weight loss (HH) and in normal menstruating women (N). Unlabelled D was infused for 48 h in 12 normal women and in 12 women affected by HH. Plasma levels of D, dehydroepiandrosterone sulfate (DS), A, estrone (E1), estrone sulfate (E1s) and estradiol (E2) were measured before and after 48 h of infusion. Metabolic clearance rates of D (MCRD), production rates of D (PRD), and increases in plasma concentration of DS, A, E1, E1s and E2, relative to the corresponding increase in plasma concentration of D, were determined. The baseline plasma levels of all steroids studied were found to be significantly lower in the patient group than in the control. The MCRD in the normal and the HH groups were similar (1420 +/- 340 l/day versus 1670 +/- 569 l/day, P greater than 0.05). No significant difference was found in PRD between the 2 groups (mean +/- SD 10.3 +/- 5 versus 13.3 +/- 5.5 mg/day, P greater than 0.05). Administration of D increased the levels of estrogen in the normal group but not in the HH group. The relative increase in plasma levels of DS resulting from infusion of D (delta cDS/delta cD) was found to be larger in the HH group than in the normal group (40.4 +/- 17 versus 26.3 +/- 11.8, P less than 0.05). Furthermore, relative increases in plasma levels of A derived from infusion of D were larger in the HH group than in the normal group (0.0495 +/- 0.0021 versus 0.192 +/- 0.0071, P less than 0.001). We conclude from these results that in the HH patients there is a blockage of the peripheral conversion of D to E1 and E1s and an enhancement of the peripheral conversions of D to DS and to A. These metabolic changes may account for the androgenization of the patients under study.

R-27 cells, a tamoxifen-resistant clone of MCF-7 mammary cancer cells, were used to study the effect of tamoxifen and its derivatives (4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen) on the conversion of estrone sulfate to estradiol. The present data indicate that (1) tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen inhibit the uptake of the radioactivity after incubation of these triphenylethylene derivatives with [3H]-estrone sulfate; (2) there is a significant decrease of the conversion of estrone sulfate to estradiol by these antiestrogens; (3) the concentrations of estradiol (cytosol + 0.6 M KCl nuclear extract) which are 293 +/- 50 pg/mg DNA in the control studies (estrone sulfate alone), diminish to 26 +/- 5 pg/mg DNA after addition of tamoxifen, to 9 +/- 2 with 4-hydroxytamoxifen, to 24 +/- 7 with N-desmethyltamoxifen and to 32 +/- 6 with cis-tamoxifen. It is concluded that estrone sulfate can play an important role in the biological responses to estrogens in this breast cancer cell line and tamoxifen and its derivatives block the conversion of estrone sulfate to estradiol. The decrease in concentration of estradiol could be explained by the presence of the estrogen receptor system but other ways of the action of antiestrogens remain to be explored.

The turnover of estrone, estrone sulfate and ethinylestradiol∗ was studied in the whole rat and in isolated rat liver cells with regard to the sequential steps involved in hepatic uptake, metabolism and secretion of estrogens. After i.v. injection of estrone or estrone sulfate (5 nmol/250 g rat), the maximal biliary excretion of both compounds is observed within 20 min, and the fractions of biliary glucuronides (about 79%) and sulfates (about 19%) derived from the two estrogens, are almost identical. Isolated liver cells have preserved their ability to hydroxylate and conjugate estrogens and to release the newly formed conjugates. The major difference between the hepatocellular turnover of estrone and estrone sulfate is uptake, which proceeds much slower with the conjugated form. Uptake of estrone sulfate is followed by deconjugation. This can explain the similar conjugate patterns of metabolites derived from estrone and estrone sulfate in vivo. The free estrogens are conjugated within the isolated hepatocytes. The conjugates are released from the cells mainly as glucuronides (about 35%) and sulfates (about 13% in the case of estrone). The conjugation of ethinylestradiol is faster than conjugation of estrone and can be depressed by inhibitors of microsomal hydroxylation.

Plasma levels of estrone and estrone sulfate were measured in 16 postmenopausal women with advanced breast cancer before and during chronic aminoglutethimide therapy. Aminoglutethimide caused a significant reduction in plasma estrone (mean 36%, P less than 0.025) and estrone sulfate (mean 65%, P less than 0.001). The estrone/estrone sulfate ratio was increased by a mean of 84% (P less than 0.0025). These results suggest that aminoglutethimide influences plasma estrone sulfate by mechanisms unrelated to aromatase inhibition. The findings in this study are consistent with previous results suggesting that aminoglutethimide treatment enhances the rate of estrone sulfate metabolism. This biochemical effect of aminoglutethimide treatment could be a contributory factor to the drugs mechanism of action.

The metabolic fate of estradiol benzoate (estradiol 3-benzoate) was studied in intact and biliary fistula dogs. The steroid used was labeled with 3H at position 6 and 7 of the steroid nucleus and with 14C at the carbonyl carbon of the benzoyl group, thus affording the opportunity to ascertain the loss of the benzoyl group and the fate of both labels. The averages of the radioactivity excreted, given as a percentage of the amounts injected, and the standard deviations were as follows: 11.9 ± 1.1% of the 3H and 40.5 ± 3.5% of the 14C were recovered in the urine of intact animals after 7 h. In dogs with biliary fistulas, the total radioactivity excreted was 6.3 ± 0.3% of the 3H and 27.5 ± 2.5% of the 14C in the urine, and 16.0 ± 0.5% of the 3H and 1.05 ± 0.05% of the 14C in the bile after 7h. The concentration of estradiol benzoate in the blood was also investigated. The dominant conjugates of estrogens in the urine were the sulfates. Hippuric acid, benzoic acid, 17-epiestradiol sulfate and estrone sulfate were detected in the urine. The dominant conjugates in the bile were the glucosiduronates and the sulfates. Estradiol 3-glucosiduronate, estrone glucosiduronate, 17-epiestradiol 3-sulfate and estrone 3-sulfate were detected. These results indicate the immediate elimination of 3-benzoyl group of estradiol benzoate after injection. They also indicate that the resulting hippuric acids and benzoic acids are excreted mostly in the urine, and the enterohepatic circulation of the hydrolyzed steroid is observed to some extent.

Estrogen sulfates are quantitatively the most important form of circulating estrogens during the menstrual cycle and in the post-menopausal period. Huge quantities of estrone sulfate and estradiol sulfate are found in the breast tissues of patients with mammary carcinoma. It has been demonstrated that different estrogen-3-sulfates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate) can provoke important biological responses in different mammary cancer cell lines: there is a significant increase in progesterone receptor. On the other hand, no significant effect was observed with estrogen-17-sulfates. The reason for the biological response of estrogen-3-sulfates is that these sulfates are hydrolyzed, and no sulfatase activity for C17-sulfates is present in these cell lines. [3H]Estrone sulfate is converted in a very high percentage to estradiol (E2) in different hormone-dependent mammary cancer cell lines (MCF-7, R-27, T-47D), but very little or no conversion was found in the hormone-independent mammary cancer cell lines (MDA-MB-231, MDA-MB-436). Different anti-estrogens (tamoxifen and derivatives) and another potent anti-estrogen: ICI 164,384, decrease the concentration of estradiol very significantly after incubation of estrone sulfate with the different hormone-dependent mammary cancer cell lines. No significant effect was observed for the uptake and conversion of estrone sulfate in the hormone-independent mammary cancer cell lines. Progesterone provokes an important decrease in the uptake and in estradiol levels after incubation of [3H]estrone sulfate with the MCF-7 cells. It is concluded that in breast cancer: (1) Estrogen sulfates can play an important role in the biological response of estrogens; (2) Anti-estrogens and progesterone significantly decrease the uptake and estradiol levels in hormone-dependent mammary cancer cell lines; (3) The control of the sulfatase and 17 beta-hydroxysteroid dehydrogenase activities, which are key steps in the formation of estradiol in the breast, can open new possibilities in the treatment of hormone-dependent mammary cancer.

To elucidate the main metabolic pathways which lead to the foeto-placental biosynthesis of estetrol (I), we investigated the 15 alpha- and 16 alpha-hydroxylations of potential precursors of this estrogen in the human fetal liver. We determined the 15 alpha- and 16 alpha-hydroxylation capacity of the fetal liver for each precursor by GC-MS. The results suggest that estetrol is derived only from estradiol sulfate (II) and DHEA sulfate (III). 15 alpha-Hydroxy-androstenedione (IV) can no longer be regarded as a good precursor of estetrol. The phenolic pathway appears to be a more likely route than the neutral pathway, even when derived from DHEA sulfate.

When the plasma concentrations of estrone sulfate (E1S) were measured in five menstrual cycles, the highest concentrations were found on the day of LH peak (14.25 nmol/l +/- 2.94 [SE]). Peak levels of E1S were 20 times higher than the highest E2 levels measured (0.769 +/- 0.276 nmol/l). To determine whether E1S can be metabolized by adult and fetal tissues we examined estrone (E1) sulfatase activity in brain and other tissues. E1 Sulfatase activity was present in all tissues studied including adult endometrium, fat and skin. When the rate of sulfatase activity was measured in homogenates of fetal hypothalamus, frontal cortex and pituitary (n = 4), the hypothalamic activity (306.0 +/- 39.1 [SE] pmol/min/mg protein) was significantly higher than that of the frontal cortex (127.4 +/- 19.4, P less than 0.002) or pituitary (193.7 +/- 43.3, P less than 0.03). This was not apparent in the adult (n = 2) where the enzyme activity was similar in the hypothalamus (413.9 +/- 27.3) and frontal cortex (446.3 +/- 82.2) and lower in the pituitary (98.2 +/- 19.2). The Km for E1 sulfatase in the fetal frontal cortex was 28.9 microM. The high E1 sulfatase activity in estrogen responsive target tissues, particularly fetal hypothalamus, accompanied by a large circulating reservoir of E1S, suggest that this enzyme could possibly have a regulatory role in controlling the level of intracellular estrogens and in modulating their intracellular function.

The acylation of steroid sulfates by heating at 70°C for 0.5 h with heptafluorobutyric anhydride occurs in quantitative yield. The ester formed is the same as that obtained when the free steroid was so treated with heptafluorobutyric anhydride. Proof was provided by classical chemical analyses. The steroid sulfates of cord blood plasma were extracted and separated on Sephadex LH-20. Acylation with heptafluorobutyric anhydride was followed by gas chromatography with detection by electron capture. Results of analyses of steroid sulfates in human cord blood show the presence of estriol and 16α-hydroxydehydroepiandrosterone.

Estradiol-17β (estradiol) labeled with either [ 3 H] or [ 14 C], was administered either by the intravenous or by the oral route to normal female beagle dogs. Blood samples from peripheral and portal veins and lymph samples from the lymphatic duct were obtained at increasing time intervals and radioactivity as estrone, estradiol, estrone sulfate, estrone glucuronide and estradiol glucuronides measured. Following intravenous administration the disappearance of estradiol from the blood could be described as a function which was the sum of three exponentials. Radioactivity as estrone appeared rapidly and then disappeared in a manner quantitatively and qualitatively similar to estradiol. The major conjugates circulating were estrone and estradiol glucuronides; the sulfates appeared to be relatively minor components. Following oral administration (labeled estradiol in 20% ethanolic solution) radioactivity appeared rapidly, to reach peak levels in 3–16 min in the peripheral blood, as both estradiol and estrone. Although about 70% of the administered radioactivity appeared in the blood, only about 10% of that was absorbed as estradiol. Estrone and estradiol glucuronides made up the majority of the radioactivity in the peripheral blood. When we measured the radioactivity in the portal blood, it was apparent that conjugation occurred primarily in the intestinal wall and to a lesser extent in the liver. We were able to find measurable quantities of radioactivity as estrone, estradiol, their glucuronides and estrone sulfate in the lymph. Most of this radioactivity arose from the estradiol administered by the intravenous route and little estradiol was absorbed from the intestine via the lymph. The intestines appear to be responsible for most of the first pass metabolism, both conjugation and conversion to estrone, of estradiol administered via the gastrointestinal tract.

The effect of androgens on the conversion of estradiol (E2) and estrone (E1) from estrone-3-sulfate (E1-S) was studied in explants of normal human term placentas. Explants incubated in medium supplemented with 2.0 microM E1-S showed that 50 microM dihydrotestosterone (DHT) stimulated E2 production 15-fold above control values after 0.5h, but neither 50 microM methyltestosterone (MT) nor 50 microM diethylstilbestrol (DES) had any effect. HCG (5.0 i.u./ml), alone or in combination with one of the androgens, did not influence the E2 production. When the explants were incubated in medium with 0.25 microM E1-S (the average concentration reported for late pregnancy plasma), DHT (0.5-50 microM) caused a dose- and time-dependent increase in E2 production, while E1 production and the combined accumulation of E2 and E1 were slightly inhibited by all doses of DHT during the 0.5-4h incubation. When E1 (0.1 microM) was used as substrate, DHT caused a dramatic dose- and time-dependent shift in the E1-E2 equilibrium towards E2. The results indicate that during late pregnancy, a particular class of androgens may increase the production of the more bioactive E2 from the circulating E1-S; the mode of action may be an enhanced conversion of E1 to E2.

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.

Although cystic lesions of the breast are not precancerous per se, statistical studies have indicated that this condition predisposes a 2- to 4-fold greater risk for breast cancer. Seeking a hormonal etiology to this correlation, investigators have analyzed breast cyst fluid ( BCF ) for steroids and have compared the levels to those in the blood. The 17-ketosteroids-androsterone, dehydroisoandrosterone and their sulfates are elevated in BCF . The same is true for estrone sulfate and estradiol-3-sulfate. We have found the most dramatic differences with estriol-3-sulfate (E3-3S), the concentration of which ranged from 187-6134 pg/ml in over 40 specimens analyzed, whereas in 12 serum specimens from normal women, E3-3S was barely detectable. The origin of E3-3S is not known. [3H]E3-3S is not concentrated in BCF following its injection into an arm vein. The blood half-life of [3H]E3-3S is much lower than that of estrone sulfate. Samples of breast nipple aspirates from normal women were also analyzed for E3-3S. None could be detected. The best explanation of the data accumulated thus far is that E3-3S is synthesized at the epithelial lining of the cyst and released into the BCF , from which its efflux is inefficient.

The fecal and urinary excretion of orally administered [4-14C]estrone-3-sulfate was studied in germfree (GF) rats, conventional (CV) rats and gnotobiotic rats selectively associated with estrone-desulfating and/or cecal-volume reducing microorganisms. The time required to excrete 50% of the total label recovered (t 1/2) was 22 h in CV rats vs 32 h in GF rats. Gnotobiotic rats selectively associated with a cecal volume-reducing flora (CRF rats) excreted the label even faster (t 1/2 = 13 h) than CV rats. Association of GF rats as well as CRF rats with estrone-desulfating microorganisms (termed S1 + S2 + R9 rats and CRF + S1 + S2 + R9 rats, respectively) led to a slower excretion of labeled products (t 1/2 = 38 h in S1 + S2 + R9 rats and t 1/2 = 27 h in CFR + S1 + S2 + R9 rats). Intestinal microbial desulfation also increased the relative part of the urinary excretion from 4% in GF rats to 8% in S1 + S2 + R9 rats and from 3% in CRF rats to 9% in CFR + S1 + S2 + R9 rats. We conclude that intestinal microbial desulfation enhances the enterohepatic circulation of orally administered estrone-3-sulfate.

Biosynthesis of estrogen was studied in human endometrial epithelial glands and stromal cells. Intact epithelial glands and stromal cells were isolated from human normal proliferative and secretory endometrial specimens. The isolated glands and stromal cells were separately incubated with 10nM [3H]testosterone in nutrient medium for 20 h in an incubator. Only estrone and estradiol were formed in stromal cells and in epithelial glands in the proliferative phase. Four types of estrogens: estrone, estradiol, estrone sulfate and estradiol-3-sulfate were isolated from the secretory epithelial glands. The capacity of aromatization is higher in stromal cells than that in glands. The highest capacity of aromatization was found in stromal cells in the proliferative phase, 58 ± 30 fmol/mg protein ( x ± SD , n = 10 ) followed in order by stromal cells in the secretory phase, 30 ± 18 (n = 6), and the epithelial glands in the proliferative and secretory phase, 22 ± 12 (n = 10) and 4.3 ± 1.2 (n = 6), respectively. These results indicate that the aromatase in endometrium is distributed in both stromal cells and epithelial glands. The capacity of aromatization varies in the 2 phases of the menstrual cycle with a significant decrease in activity of the epithelial glands in the secretory phase. The capacity of aromatization was also examined when stromal cells were incubated with various concentrations of [3H]T. The apparent Km, estimated from the concentration of Δ4-androgens in cells, is about 27 nM, indicating that the endometrial aromatase has a considerably high affinity to Δ4-androgens.

Hepatocellular uptake of the endogenous sulfate ester estrone sulfate was characterized by use of isolated liver cells: (1) Uptake was saturable with an apparent K M of 0.8 μM and a V max of 0.3 nmol/mg protein × min. (2) Uptake was inhibited by the sulfhydryl (SH-) poisons mersalyl and dinitrofluorobenzene. (3) Taurocholate and estrone sulfate competed for uptake. (4) Activation energy was 67 KJ/mol; pH optimum was within physiological limits. (5) Inhibition of mitochondrial respiration lowered the uptake rate by 93%. (6) Ouabain and extracellular K + decreased the uptake rate. These data indicate that estrone sulfate uptake into rat hepatocytes is energy dependent and carrier mediated. Mutual competition for uptake of estrone sulfate and taurocholate suggests that both steroids are taken up by a single carrier.

Estrone and dehydroepiandrosterone (DHA) sulfatase activities were studied in the uterus and liver of female guinea-pigs (albino variety). The two activities were found in particulates, with the highest specific activity in microsomes. The effects of pH, buffers, temperature and the non-competitive inhibition of DHA sulfate on estrone sulfatase provided arguments for the existence of two distinct sulfatases. However, acrylamide gel electrophoresis of the solubilized microsome sulfatases gave a single peak for the two activities. In the uterus, the apparent Km of estrone and DHA sulfatases were 26.4 and 15.6 μM. Solubilized microsomal estrone sulfatase was inhibited by unconjugated steroids. The apparent Km of estrone sulfatase in liver was 10.7 μM. Estrone and DHA sulfatase activities were consistently lower in liver than in uterus and no DHA sulfatase activity was detected in fetal liver. In the uterus, the same sulfatase activities were found in female fetuses, castrated or mature females. Estrone sulfatase was significantly increased in the uterus of pregnant females (60–65 days gestation). Estrone sulfate was injected in vivo into mature castrated females. A significant increase in uterine weight and in uterine progesterone receptors was observed. The cytosol progesterone receptors were characterized by their Kd (1.40 nM) and by sucrose density gradient. It is concluded that the variations of estrone sulfatase activity in target tissues like the uterus may control the intracellular levels of biologically active estrogens.

Estrogen sulfatase (ES) and estrogen sulfotransferase (ESFT) activities were measured in a group of primary breast tumors. The mean value of ES activities, measured in 66 breast tumor specimens, was 0.9 nmol of estrone formed from estrone sulfate/mg tissue protein per hr regardless of the hormone receptor status of the specimen. However, the average value of the ESFT activity, expressed in nmol of estradiol-3-sulfate (E2S) formed from estradiol (E2)/mg of cytosol protein per hr, was found to be significantly higher in ER +/PGR + tumors (n = 26, 0.18 +/- 0.15, means +/- SD) than in ER -/PGR - tumors (n = 31, 0.08 +/- 0.06, P less than 0.005). Normal breast tissues also contain ES and ESFT but the activities were lower than those in tumors. When fresh breast tumor tissue fragments were incubated with radioactive E2 (0.4 nM) and E2S (3 nM) separately, E2 was not sulfurylated appreciably while E2S was extensively hydrolyzed to free estrogens indicating that the combined effect of ES and ESFT in breast tumor is favored towards the hydrolysis of estrogen sulfate. These results imply that the circulating estrogen sulfate could be utilized as the precursor of active estrogen to promote the cell growth in hormone sensitive tumors.

The uptake of steroids by human red blood cells was investigated with an incubation technique and with equilibrium dialysis, and the following observations were made: (1) In the sequence pregnenolone, 20α-dihydroprogesterone, testosterone, androstenedione a decrease in binding affinity of erythrocytes suspended in Krebs-Ringer-bicarbonate buffer was observed. The ratio of bound to unbound steroid did not vary significantly with the steroid concentration. Most of the bound steroid was removed by washing the red blood cells several times with isotonic buffer, indicating that steroids might be adsorbed to the cell surface. 1. (2) The percentage of progesterone bound to erythrocytes in a 5% (v/v) plasma solution increased with increasing concentrations (0–75 ng/ml) of progesterone. With progesterone concentrations > 75 ng/ml the distribution of progesterone between the plasma proteins and erythrocytes remained constant. In studies with the whole blood 25–30% of added [ 3 H]-pregnenolone was bound to the red blood cells, but only 0–10% [ 3 H]-progesterone was bound to the erythrocytes. 2. (3) Experiments on the reduction rate of DHA and DHA sulfate, and of estrone and estrone sulfate, by intact and hemolysed human erythrocytes indicate a difference in permeability of the intact cell membrane for steroid sulfates and the corresponding free steroids. 3. (4) In binding studies with a total hemolysate, most of the steroid was associated with proteins of the membrane-free hemolysate and only a small fraction was bound to the membrane. The combining affinity, expressed as the ratio of bound to unbound steroid per unit amount of protein was higher for hemoglobin-free membranes than for a membrane-free hemolysate. The binding of testosterone and progesterone by a soluble protein fraction from erythrocyte membranes is higher than could be expected on the basis of dialysis studies with intact membranes. Testosterone was bound in a specific way by a fraction from a membrane-free hemolysate. containing 17β-hydroxysteroid dehydrogenase activity. An apparent association constant for binding of testosterone by this fraction was found to be in the order of magnitude of 10 8 I (mol protein) −1 . The testosterone-binding fraction isolated from the membrane-free hemolysate was further characterized with gel chromatography. The results of these studies suggest that the uptake of steroids by intact erythrocytes can be explained in terms of (a) adsorption and/or binding to the membrane, (b) diffusion into the cell, and (c) binding inside the cell.

Analysis of urinary metabolites of [6,7- 3 H]-estrone and of [6,7- 3 H]-estrone-3-glucosiduronate was performed in intact guinea-pigs. The preliminary separation of urinary metabolites was carried out by submitting these metabolites to countercurrent distribution. The further separation of each fraction thus obtained was achieved by means of DEAE-Sephadex A-25 column chromatography. The separated peak was then hydrolyzed with the enzyme and the free steroid released was identified by reverse isotope dilution. Thus, both estrone-3-glucosiduronate and 17β-estradiol-3-glucosiduronate were identified as major urinary metabolites of estrone and of estrone-3-glucosiduronate. Concomitant studies in vitro with homogenates of the kidney, small intestine and liver showed that the small intestine and liver readily conjugated estrone as glucosiduronates and the kidney was a rather poor conjugating organ. The kidney and liver were capable of forming estrone-3-glucosiduronate as well as 17β-estradiol-3-glucosiduronate, whereas estrone-3-glucosiduronate was the only glucosiduronate formed by the small intestine. From these results, it was concluded that in this species not only the liver but the small intestine and kidney participated in the metabolic conjugation of estrone and that a part of estrone-3-glucosiduronate was converted to 17β-estradiol-3-glucosiduronate extrahepatically at least in the kidney.

Seven women with senile dementia-Alzheimer's type (SDAT) were treated with conjugated estrogen [main content: estrone sulfate (E1-S)], at a dose of 1.25 mg/day over a 6-week period. A New Screening Test for Dementia developed by Japanese National Institute of Mental Health (NS) and the scores of Hasegawa Scale for dementia (HS) were performed every 3 weeks. Six women showed improvements in NS (P less than 0.05) and 5 women showed improvements in HS. Untreated women with SDAT did not show any improvement. Serum E1-S was measured by a direct radioimmunoassay. Serum E1-S was 911 +/- 156 pg/ml in 7 women with SDAT and lower than that of 7 normal women (1020 +/- 216 pg/ml). Following the treatment, serum E1-S increased to a level of 21.1 +/- 8.1 ng/ml. Estrone and estradiol-17 beta also increased. The results suggest a possibility for the future clinical use of estrogen for senile dementia, after careful clinical research trials including the side effects.

Estrone sulfate (E1-S) in the serum and tissues of patients with breast cancer or endometrial cancer was measured by a direct radioimmunoassay without hydrolysis. The concentration of E1-S in breast cancer tissue was 1.64 +/- 0.28 ng/g wet wt (+/- SE), lower than in surrounding normal breast tissue (4.46 +/- 1.23). Estradiol-17 beta(E2)/E1-S was higher in endometrial cancer tissue than normal endometrial tissue. Estrone sulfatase activity in breast cancer tissue was 0.81 +/- 0.23 nmol/h/mg protein, higher than in surrounding normal breast tissue (0.35 +/- 0.11). These results suggest that E1-S, which is abundant in the peripheral circulation, is hydrolyzed by sulfatase in breast cancer tissue or endometrial cancer tissue and liberates free estrogens, which may stimulate the growth of these malignant tumors.

Characteristics and activities of estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHAS) sulfatases were studied in epithelium and stroma of benign hyperplastic tissues from human prostates. Tissues were obtained by suprapubic prostatectomy, and epithelium and stroma were separated mechanically by standard techniques. The assay procedure comprised homogenization in Tris-buffer, incubation of the homogenate with [3H]E1S or [3H]DHAS, separation of free steroids from nonhydrolyzed steroid sulfates by extraction with ether, and their final quantification by LSC. The main results were: (1) The pH-optimum of the sulfatase was found at pH 7.0. (2) The highest specific sulfatase activity was found in the epithelium and was associated with its nuclear fraction. (3) Michaelis-Menten constants Km (microM) were 8.7 +/- 1.4 (7) and 4.3 +/- 0.8 (5), maximum velocity rates Vmax (nmol/h x mgDNA) were 47.4 +/- 8.8 (7) and 8.4 +/- 1.5 (5) for E1S and DHAS, respectively (means +/- SEM (n]. (4) The enzymatic cleavage of E1-sulfate was competitively inhibited by DHA-sulfate and vice versa with inhibition constants Ki (microM) of 4.0 +/- 0.5 (2) for E1S and 2.7 +/- 0.4 (2) for DHAS. On the basis of these findings, possible roles of steroid sulfate-sulfatases in forming precursors of active androgens and estrogens from the high amounts of E1S and DHAS in blood are discussed.

Plasma estrone, estradiol, estrone sulfate, androstenedione, testosterone and sex hormone binding globulin were measured in 14 patients (3 postmenopausal women, 11 men) with tuberculosis who received rifampicin. During treatment a moderate, but significant increase in the plasma level of estradiol (mean increase 32%, P less than 0.01) and estrone (mean increase 13%, P less than 0.01) were seen. In contrast, plasma estrone sulfate was significantly reduced (mean reduction of 25%, P less than 0.05). No alteration in plasma testosterone was observed, but there was a slight (mean 15%) increase in plasma androstenedione of borderline significance (P = 0.052). In eight patients, from whom all tuberculostatic treatment except rifampicin had been withdrawn, plasma sex hormone binding globulin was found to be increased by 75% by rifampicin treatment. Further, the results obtained in this part of the study confirmed the alteration in plasma estrone sulfate to be caused by rifampicin alone without any contribution from other tuberculostatic drugs. While plasma estradiol could be increased due to elevation of sex hormone binding globulin, plasma estrone was probably increased secondary to the increase in plasma androstenedione. A reduced plasma estrone sulfate level suggests that rifampicin enhances the rate of estrone sulfate metabolism. The possibility that treatment with drugs which reduce plasma estrone sulfate might be beneficial for hormone dependent cancers is discussed.

We administered [6,7-3H]estradiol p.o. and [4-14C]estradiol i.v. simultaneously to 5 women 22-35 years of age. Fourteen blood samples were collected over 480 min, and all urine was collected for 96 h. The blood samples were analyzed for radioactivity as estradiol, estrone, estrone-sulfate and estradiol glucuronide. The urine samples were analyzed for radioactivity as the glucuronide and pH 1 hydrolyzable conjugates of estradiol, estrone, estriol, 16 alpha-hydroxy-estrone, 2-hydroxy-estrone, 2-hydroxy-estradiol, 2-methoxy-estradiol and 2-methoxy-estrone. The major circulating estrogen, after either estradiol, p.o. or i.v. administration, was estrone sulfate; approximately 50% of estradiol administered by either route being converted to and measured as estrone sulfate in the blood. Following oral administration about twice as much estradiol was converted to and measured as estradiol glucuronide in the blood as after i.v. administration. Of the estradiol administered p.o., only 10% was absorbed into the blood as estradiol the rest being metabolized prior to absorption. After estradiol, p.o., the major radioactive compounds in the urine were the glucuronides of estrone and estradiol, but after estradiol, i.v., the conjugates of estrone, estradiol and estriol were present to about the same extent as the conjugates of the 2-oxygenated compounds. Following p.o., considerable metabolism of estradiol administration occurs in the splanchnic tissue, much of it in the intestinal wall.

Metabolites of injected [3H]-estrone-3-sulfate were investigated in urine, gall bladder bile, liver and kidney of mature pigmented and albino male and non-pregnant female guinea pigs (English Shorthair variety). Considerable formation of estriol, 16α-hydroxyestrone and 16β-hydroxyestrone disulfates, as well as estriol and 16α-hydroxyestrone monosulfates occurred in pigmented females. Monoglucuronides and monosulfates of estrone and estradiol-17β (estradiol) were also observed. No 16-hydroxysteroid monosulfates were detected in pigmented males but disulfates were present in urine, to a lesser extent than for females. No hydroxysteroid monosulfates were found in albinos of either sex and, although small amounts of a disulfate-like fraction occurred in urine, the steroid moieties were not identified. Pigmented females formed lesser amounts of hydroxysteroids from injected [3H]-estrone or estradiol than from estrone sulfate. These metabolites were not detected in the other animal groups. Qualitative patterns of estrone and estradiol monoglucuronides and monosulfates were similar in pigmented and albino animals, regardless of the precursor injected.

The aromatization of norandrogens was investigated with highly purified preparations of Leydig cells from mature male pigs. Cell incubations with norandrostenedione and nortestosterone gave rise to large amounts of estrone sulfate in the medium as determined directly by a specific radioimmunoassay (RIA). Estrogen production was at least equal to that seen with androstenedione and testosterone as substrates. Similar findings were made with cells in culture for 5 days before addition of the androgen substrates in a 4 h-test of aromatase activity. Stimulation of estrogen formation was noted when cells were exposed for 48 h to either hCG (0.5 i.u.) or FGF-β (10 ng) daily, as a pretreatment, before adding androstenedione for the test of aromatase activity. Little or no increase was seen with norandrostenedione or nortestosterone as substrate. Further evidence for estrogen production was obtained from HPLC separations of metabolites of cell incubations with norandrostenedione and [ 14 C]nortestosterone monitored by RIA and radioactivity, respectively. It is suggested that norandrogens could serve as important substrates for aromatization in the boar testes.

Normal AXC/SSh rat ventral prostate and clonally derived AXC/SSh rat prostate cancer cells were evaluated for ability to metabolize estrone sulfate (E1S), estrone glucuronide (E1G), or dehydroepiandrosterone sulfate (DHEAS). Both normal and malignant prostate cells converted E1S to estrone. Neither normal nor malignant prostate cells had significant ability to metabolize DHEAS to DHEA, indicating differential specificity of prostate sulfatases(s) for estrogen and androgen sulfates. Both normal and neoplastic prostate cells possess beta-glucuronidase which hydrolyzed E1G to estrone. To assess potential physiologic consequences of these enzymatic activities, we determined the effect of steroid conjugates on in vitro proliferation of selected clonal lines of AXC/SSh rat prostate cancer cells. DHEAS, 10(-6) to 10(-9) M in decade intervals, did not affect in vitro proliferation of AXC/SSh prostate cancer cells; however, 10(-5) M DHEAS decreased in vitro proliferation of these cells. Neither E1S nor E1G, 10(-5) to 10(-9) M in decade intervals, affected in vitro proliferation of AXC/SSh prostate cancer cells. These findings suggest that low residual levels of steroid conjugates, which are not removed by charcoal stripping of serum, do not affect demonstrated in vitro androgen modulation of AXC/SSh rat prostate cancer cell proliferation (Cancer Res. 46, 3775-3781, 1986).

The activity of steroid 21-sulfatase, the enzyme that catalyzes the hydrolysis of deoxycorticosterone sulfate (DOC-SO4) is demonstrable in human placenta. Thus, it is possible that this placental enzyme, by way of the hydrolysis of either DOC-SO4 or 21-hydroxypregnenolone mono- or di-sulfate of fetal origin, may be important in the biosynthesis of DOC, which is present in the plasma of pregnant women in high concentration. To investigate this issue further, we evaluated steroid 21-sulfatase activity in microsomal preparations of a sulfatase-deficient placenta. Immediately after delivery, at term, of a living male fetus with sulfatase deficiency, a microsomeenriched fraction of placental tissue was prepared; sulfatase activity was evaluated by use of three substrates, viz. dehydroisoandrosterone sulfate (DS), estrone sulfate (E1-SO4), and DOC-SO4, in various concentrations. Similar incubations were conducted with aliquots of a microsome-enriched fraction prepared from placental tissue of a normal fetus that was delivered, at term, within minutes of the time of delivery of the infant with sulfatase deficiency. In microsomal fractions from the normal placenta, each of the steroid sulfates was hydrolyzed. In the absence of microsomes, and in the presence of microsomal fractions from the sulfatase-deficient placenta, the hydrolysis of DOC-SO4 and DS was not detected. Moreover, in microsomes prepared from the sulfatase-deficient placenta, E1-SO4 was hydrolyzed at a rate that was only 10% of that in incubations with microsomal preparations of the normal placenta. We conclude that with sulfatase deficiency, the placenta is deficient not only in sulfatase activity for steroid-3-sulfates but for steroid 21-sulfates e.g. DOC-SO4, as well.

The preparation and antigenic properties of estrone sulfate-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through the C-6 position on the steroid nucleus have been described. Antibody raised against antigen in the rabbit possessed high affinity (KA = 1.86 × 109 M−1) and excellent specificity to estrone sulfate, exhibiting no significant cross-reactions with other estrogen sulfates (< 1%) and no cross-reactivities with free estrogens, their glucuronides and other related steroids (< 0.001%).

The methods commonly used to evaluate conjugated steroids require hydrolysis and chromatographic purification. To avoid these steps, a simple method involving selective solid phase extraction and RIA using a highly specific antiserum for estrone sulfate (E1S) has been evolved. A Bond-Elut C2 cartridge was used for solid phase extraction of estrone (E1) and E1S; recoveries were 80 and 90% respectively. The intra- and inter assay precision of the assay at 3 serum levels, were 6.5, 10.4 and 4.4 and 12.7, 13.9 and 7.4% respectively. Accuracy, tested by linearity and recovery tests, was acceptable. A good correlation exists between a conventional enzymatic method and the proposed method. The latter is less time consuming and more reliable, thus providing a rapid assay to evaluate E1 and E1S in the same serum sample.

The intestinal content, the mucosa and the rest of the intestinal wall of germfree (GF) and conventional ( CVL ) rats were tested for in vitro hydrolysis of [3H]estrone sulfate. In homogenates from GF rat intestine some estrone sulfate hydrolysis was detected in those from the proximal small intestine (PSI) (4.2 +/- 0.1% hydrolyzed after 4 h), but not in those from the distal small intestine (DSI) and the caecum. Estrone sulfate was also hydrolyzed by the homogenates of the mucosa and the rest of the intestinal wall from each of the segments tested (PSI: 12.8 +/- 0.4% (mucosa) and 21.5 +/- 2.1 (wall); DSI: 8.2 +/- 0.9% (mucosa) and 17.3 +/- 1.7% (wall); caecum: 8.8 +/- 1.6% (mucosa) and 17.3 +/- 0.5% (wall) ). In the homogenates of CVL rat intestine, the estrone sulfatase activity in the rest of the intestinal wall did not differ considerably from the values for GF rats, when expressed per mg protein of the homogenate. The mucosa of the CVL rats, however, showed higher rates of hydrolysis than the mucosa of the GF rats. The microbial estrone sulfatase activity in the intestinal content of CVL rats, tested by anaerobic incubation, was high in the caecum (91.7 +/- 6.6% after 4 h), but very low in the PSI (2.2 +/- 0.7%) and DSI (1.3 +/- 0.5%). Serial dilutions of the caecal content also showed higher viable numbers of estrone sulfate hydrolyzing bacteria. These results add further weight to the suggestion that estrone sulfate may be absorbed from the small intestine, but has to be hydrolyzed in the caecum by the gut microflora prior to absorption.

The liver of a rhesus monkey was perfused in vivo with [3H]-estrone sulfate (E1SO4). The injection was made via the portal vein and blood was collected from the hepatic vein, using a heart catheter. Analysis for free and conjugated metabolites was done. The sulfate fraction represented 2 3 of the recovered activity, indicating a slow rate of desulfiirylation. Conversion of estrone sulfate to free estrogens was indicated. 2-hydroxy-estrogens were found in substantial amounts in the conjugate fractions. Double conjugates of estriol and 2-hydroxy-estriol in the form of sulfoglucosiduronates were indicated. Evidence for retention of E1SO4 by the monkey liver was presented.