Regulation of human apolipoprotein A-I gene expression by equine estrogens.

Estrogen replacement therapies, such as conjugated equine estrogen (CEE, Premarin), reduce the risk of coronary heart disease among postmenopausal women. In the present study, a HepG2 stable cell line (HepG2/S) that harbors a luciferase reporter gene cassette with the human apolipoprotein A-I (apoA-I) promoter region was used to examine the activity of CEE components in modulating human apoA-I promoter activity. A number of estrogens modulated apoA-I promoter activity, with equilenin (Eqn) being the most potent. Eqn produced a 3-fold increase in apoA-I promoter activity and a similar increase in apoA-I mRNA without affecting its degradation rate. Nuclear runoff assays indicated that the transcription rate of the apoA-I gene was increased 2.5-fold in Eqn-treated cells. When HepG2/S cells were exposed to Eqn, apoA-I protein secretion increased by 80%, whereas the level of secreted apoA-II remained unchanged. Transient transfection studies with human apoA-I promoter constructs derived from pGL3-luciferase reporter plasmid were used to identify the cis-acting element involved in Eqn-mediated induction. The results demonstrated that the apoA-I electrophile/antioxidant response element (EpRE/ARE) might be responsible for the increase in apoA-I promoter activity by Eqn. Cotransfection experiments using estrogen receptor (ERalpha and/or ERbeta) expression vectors have indicated that neither receptor can potentiate the Eqn-mediated induction of apoA-I promoter activity. In addition, mobility shift analysis using antibody against either ERalpha or ERbeta cannot detect the presence of these receptors in the DNA-protein complex. The data indicate that Eqn can induce the promoter activity of the human apoA-I gene, leading to an increase in apoA-I mRNA levels and apoA-I protein secretion through an ER-independent pathway involving apoA-I EpRE/ARE.

Comparison of the antioxidant effects of equine estrogens, red wine components, vitamin E, and probucol on low-density lipoprotein oxidation in postmenopausal women.

OBJECTIVE: Oxidized low-density lipoprotein (LDL) seems to play an important role in the etiology of atherosclerosis. To further study this, we performed two studies: (1) we determined the ability of 10 estrogen components of the drug, conjugated equine estrogen (CEE), trans-resveratrol (t-resveratrol) and quercetin (red wine components), trolox (vitamin E analog), and probucol (a serum cholesterol-lowering drug) to delay or prevent the oxidation of plasma LDL isolated from untreated postmenopausal women, and (2) we assessed the effect of long-term (>1 year) estrogen replacement therapy and hormone replacement therapy on LDL oxidation by ex vivo methods. DESIGN: For the in vivo study, three groups of postmenopausal women were selected based on whether they were on long-term CEE therapy (group A: 0.625 mg CEE; n = 21), on combination CEE plus progestogen therapy (group B: 0.625 mg CEE + 5.0 mg medroxyprogesterone acetate, 10 days; n = 20), or not on any hormone therapy (group C; n = 37). For the in vitro study, only LDL samples obtained from group C were used. The kinetics of LDL oxidation were measured by continuously monitoring the formation of conjugated dienes followed by determination of the lag time. RESULTS: All compounds tested protected the LDL from oxidative damage. The relative antioxidant potency of estrogen components was generally greater than that of the other compounds. The minimum dose (nmoles) required to double the lag time from the control lag time of 57 +/- 2 min was 0.47 for 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta 8 -estrone; 0.6 to 0.7 for Delta 8 -17beta-estradiol, equilenin, and quercetin; 0.9 for 17beta-dihydroequilin and 17alpha-dihydroequilin; 1.3 for equilin, estrone, 17beta-estradiol, 17alpha-estradiol; 1.4 for trolox; 1.9 for probucol; and 3.0 for t-resveratrol. The data from the in vivo study indicate that after long-term estrogen replacement therapy (group A) and hormone replacement therapy (group B), the LDL was significantly ( p < 0.01) protected (higher lag time) against oxidation compared with the control (group C). There was no difference between groups A and B. CONCLUSIONS: The oxidation of LDL isolated from postmenopausal women is inhibited differentially by various estrogens and other antioxidants. The unique ring B unsaturated estrogen components of CEE were the most potent, and t-resveratrol, the red wine component, was the least potent. Long-term CEE or CEE + medroxyprogesterone acetate administration to postmenopausal women protects the LDL against oxidation to the same extent. These combined data support the hypothesis that some of the cardioprotective benefits associated with CEE therapy and perhaps red wine consumption may be due to the ability of their components to protect LDL against oxidative modifications.

Differential neuroprotective effects of equine estrogens against oxidized low density lipoprotein-induced neuronal cell death.

OBJECTIVE: In the present study, the neurotoxic effect of oxidized low density lipoprotein on PC-12 neuronal cells maintained in culture was used to test the neuroprotective effect of several equine estrogens, such as estrone (E(1)), 17beta-estradiol (17beta-E(2)), 17alpha-estradiol (17alpha-E(2)), equilin (Eq), 17beta-dihydroequilin (17beta-Eq), 17alpha-dihydroequilin (17alpha-Eq), equilenin (Eqn), 17beta-dihydroequilenin (17beta-Eqn), 17alpha-dihydroequilenin (17alpha-Eqn), Delta(8)-estrone (Delta(8)-E(1)), and Delta(8),17beta-estradiol (Delta(8),17beta-E(2)). METHODS: The PC-12 cells (10,000 cells/well) were grown on collagen-coated 96-well plates in Dulbecco's Modified Eagle Medium supplemented with 10% horse serum, 5% fetal bovine serum, and 10 mM HEPES. In culture, the cells displayed normal PC-12 morphology and behavior, exhibiting increased dendritic growth and cessation of cell division upon exposure to nerve growth factor. Twenty-four hours after plating, various concentrations (0.1-50 microM) of estrogens were added followed by addition of oxidized low density lipoprotein (5-12.5 microg/well) in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2H-tetrazolium, inner salt) cell proliferation assay. RESULTS: The results indicate that the extent of low density lipoprotein oxidation and concentration of oxidized low density lipoprotein is directly proportional to cell toxicity. The mean +/- standard deviation cell death obtained using 10.0 microg/well of oxidized low density lipoprotein was 53.6% +/- 8.7%. With the exception of 17alpha-estradiol, all estrogens tested were found to be neuroprotective against oxidized low density lipoprotein toxicity in a typical dose-dependent manner. The order of neuroprotective potency was Delta(8)-E(1) (1.2 microM), Delta(8),17beta-E(2) (1.3 microM), Eqn (5.3 microM), 17beta-Eqn (5.3 microM), Eq (6 microM), 17beta-Eq (8.5 microM), E(1) (11 microM), 17beta-E(2) (11 microM), 17alpha-Eq (12 microM), and 17alpha-Eqn (16 microM) followed by 17alpha-E(2) which provided less than 50% protection. CONCLUSION: Our data indicate that the neurotoxic effects of oxidized low density lipoprotein can be inhibited differentially by various estrogens, with the Delta(8) estrogens being the most potent neuroprotectors. These novel findings further suggest that some of the neuroprotective benefits associated with estrogen therapy might occur by the suppression of oxidized low density lipoprotein neurotoxicity. Because estrogens such as Delta(8)-E(1) are relatively less uterotropic and potentially less feminizing than the classic estrogen 17beta-E(2), they may be useful in the prevention of Alzheimer disease and other neurodegenerative diseases in both women and men.

Effect of tamoxifen treatment on the endometrial expression of human insulin-like growth factors and their receptor mRNAs.

The effect of tamoxifen (Tam) treatment on the endometrial expression of IGF-I and II/IGF-IR and IIR mRNAs was assessed by measuring the levels of expression of these mRNAs in the endometrial samples of Tam-treated postmenopausal breast cancer patients by RT-PCR assays. The levels of these mRNAs were compared with those in normal endometrium and in Type I and Type II endometrial carcinomas (EC). The promoter-specific transcript profiles of IGF-I and II were also elucidated. The levels of IGF/IGF-R mRNAs in the endometrium from Tam-treated patients were found to be comparable with the high levels of these mRNAs observed in the proliferative and early secretory phase endometrium and in the samples of Type I EC. These results indicate that Tam acts as an estrogen agonist in inducing the endometrial expression of these genes. Tam stimulated transcription from the multiple promoters of IGF-I and II, with the exception of IGF-II P2 promoter, in which case the transcription across exon 4 appeared to be inhibited. The profiles of IGF-I and II transcripts of endometrium from Tam-treated patients were similar to Type I EC but differed considerably from those of Type II EC. These results suggest that the Tam-associated ECs are likely to be similar to type I EC and will therefore have a more favorable prognosis.

Comparison of pharmacokinetics of a conjugated equine estrogen preparation (premarin) and a synthetic mixture of estrogens (C.E.S.) in postmenopausal women.

OBJECTIVE: To compare the pharmacokinetics and relative bioavailabilities of key estrogen components of Premarin (Wyeth-Ayerst, Canada) with those of a generic conjugated estrogen preparation, C.E.S. (synthetic mixture of estrogens; ICN, Montreal, Canada) in healthy postmenopausal women. METHODS: We conducted a randomized, single-dose, two-treatment, three-period crossover study in 41 postmenopausal women. After an oral dose (2 x 0.625 mg) of Premarin or C.E.S., plasma concentrations of unconjugated and total estrone (E(1)), equilin (Eq), 17beta-estradiol (17beta-E(2)), 17beta-dihydroequilin (17beta-Eq), Delta(8)-esterone (Delta(8)-E(1)) and Delta(8),17beta-estradiol (Delta(8),17beta-E(2)) were measured over 72 hours using gas chromatography and mass spectroscopy. RESULTS: After administration of C.E.S., E(1), Eq, and 17beta-Eq appeared in blood at a significantly faster rate (lower t(max)) than after Premarin. The rapid appearance of estrogens after C.E.S. was associated with significantly higher (14-61%) C(max) values. In contrast to the high C(max) values, the area under the curve (AUC)(infinity) of unconjugated and total Eq, and 17beta-Eq were significantly lower after C.E.S., whereas those of E(1) were significantly higher. Although, the t(max) values for 17beta-E(2) were lower and the C(max) values higher after C.E.S., only the C(max) of unconjugated 17beta-E(2) was significantly different after Premarin. Unconjugated and total Delta(8)-E(1) and its main metabolite, Delta(8),17beta-E(2), were detectable in plasma only after administration of Premarin. The geometric mean ratio (GMR) (C. E.S./Premarin) of bioavailability parameters indicated that all C(max) and t(max) values for the unconjugated and total E(1), Eq, 17beta-E(2), and 17beta-Eq fell outside the regulatory requirement that the 90% confidence intervals of GMRs of two products be within 80% and 125%. Similarly, with the exception of total E(1) and total Eq, none of the AUC(t) or AUC(alpha) of the remaining estrogens meets the required regulatory standards of bioequivalence. CONCLUSIONS: C.E.S. is not bioequivalent to Premarin. Because C.E.S. also is not pharmaceutically equivalent to Premarin, it cannot be assumed to be therapeutically equivalent. Until long-term clinical trials with C.E.S. demonstrate its efficacy, extrapolation of the long-term benefits described for Premarin to C.E.S. would be risky and questionable.

Loss of IGF-II imprinting in endometrial tumors: overexpression in carcinosarcoma.

The genomic imprinting of the maternal allele defines the monoallelic expression of the IGF-II gene in most human tissues. The loss of imprinting (LOI) leading to biallelic overexpression of IGF-II has been reported in several human malignancies, including uterine leiomyosarcoma. To ascertain if LOI occurs in endometrial malignancies, the allelic expression of the IGF-II gene was examined in samples of normal human endometrium (n=22) and endometrial tumors (n=12) by assessing the ApaI polymorphism in cDNA segments amplified by RT-PCR. The biallelic overexpression of IGF-II mRNA, involving activation of all four (P1-P4) promoters, was detected in one normal endometrium and in one endometrial carcinosarcoma. Low level biallelic expression of IGF-II was also detected in two samples of hormone-unresponsive/Type II endometrial carcinomas. The level of IGF-I mRNA in these four samples was low. The IGF-IR mRNA was overexpressed in all endometrial cancers including the carcinosarcoma sample, but not in normal endometrium. These data suggest that LOI associated with overexpression of IGF-II and concomitant overexpression of IGF-IR may play a role in the rare carcinosarcoma of the endometrium.

Discordant expression of insulin-like growth factors and their receptor messenger ribonucleic acids in endometrial carcinomas relative to normal endometrium.

The inappropriate expressions of insulin-like growth factors (IGF-I and II) and IGF-I receptor (IGF-IR) are implicated in the malignant growth of many cancers. To determine changes, if any, in the levels of expression of IGFs and IGF receptor genes in neoplastic endometrium, relative to normal endometrium, the mRNA levels of IGF-I and II and of IGF-IR and IIR were measured in samples of endometrial carcinomas (EC) and normal endometrium, through all phases of the menstrual cycle, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. In normal endometrium, the mRNA levels of IGF-I were elevated in the proliferative and early secretory phases. The IGF-II mRNAs were relatively high in the proliferative phase, but unaltered through early and late secretory phases. Significantly elevated levels of IGF-II transcripts were observed during the menstrual phase, suggesting a possible role of IGF-II in endometrial regeneration. A positive correlation between the levels of IGF-I and IGF-IR mRNAs, apparent in the samples of normal endometrium, was not observed in endometrial carcinomas. The IGF-IR and IIR mRNA levels were elevated in endometrial carcinoma samples. On the other hand, the IGF-I and II mRNA levels were conspicuously low in many carcinoma samples, which were not associated with hyperplasia (type II EC), but relatively elevated in two other carcinoma samples, associated with adenomatous hyperplasia (type I EC). These results albeit with few samples suggest the possibility that the overexpressed receptor, IGF-IR, could be activated differently in two types of endometrial carcinomas, namely ligand-dependently in type I ECs and ligand-independently in type II ECs.

Biologic effects of delta-8-estrone sulfate in postmenopausal women.

OBJECTIVE: To determine in postmenopausal women the biological effects of delta-8-estrone sulfate, a novel estrogen component of Premarin (Wyeth-Ayerst, Philadelphia, PA). METHODS: An open-label, nonrandomized study of six healthy postmenopausal women was conducted. Each subject took 0.125 mg of delta-8-estrone sulfate daily for 8 weeks. Blood samples were collected at day 0 (baseline) and once a week for 8 weeks. Urine was collected on day 0 and at weeks 2, 3, 5, 6, 7, and 8. Serum gonadotropins (follicle-stimulating hormone/luteinizing hormone), plasma binding proteins (corticosteroid-binding globulin/sex hormone-binding globulin), a marker for bone turnover (urinary n-telopeptide), and markers for cardiovascular effects (cholesterol, low-density lipoprotein, high-density lipoprotein(c), low-density lipoprotein oxidation, and rate of diene formation) were measured. RESULTS: Follicle-stimulating hormone levels decreased from 84.0 +/- 8.5 to 67.0 +/- 8.5 mlU/mL (P = .02), whereas luteinizing hormone levels did not change. Corticosteroid-binding globulin levels increased from 3.30 +/- 0.16 to 4.10 +/- 0.16 mg/dL (P = .02), and no change in sex hormone-binding globulin was noted. The n-telopeptide levels decreased an average of 31% from 40.7 +/- 4.9 to 28 +/- 7.0 nmol/L bone collagen equivalents/mmol/L creatinine (P = .03). Plasma diene concentration and diene production rate decreased by 34% and 40%, respectively; these changes were not significantly different from baseline values. In contrast, a significant (P = .03) 68% increase in the lag time for low-density lipoprotein(c) oxidation (38.5 +/- 5.5 minutes versus 64.8 +/- 8.5 minutes) was observed. No significant change occurred in total cholesterol, high-density lipoprotein(c), and low-density lipoprotein(c). CONCLUSION: Small doses (0.125 mg) of delta-8-estrone sulfate have profound estrogenic effects in postmenopausal women. The changes observed in n-telopeptide levels and the lag-time delay in oxidation of low-density lipoprotein(c) indicate that this estrogen contributes toward the overall beneficial effects on bone and cardiovascular system associated with Premarin therapy.

Pharmacokinetics and pharmacodynamics of conjugated equine estrogens: chemistry and metabolism.

Conjugated equine estrogens (Premarin), are used extensively for estrogen replacement therapy and prevention of osteoporosis and cardiovascular disease in postmenopausal women. Premarin contains at least 10 estrogens that are the sulfate esters of the ring B saturated estrogens: estrone, 17beta-estradiol, 17alpha-estradiol, and the ring B unsaturated estrogens: equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, and delta-8-estrone. Bioassays and estrogen receptor binding studies indicate that all 10 estrogens are biologically active. Moreover, individual components, such as equilin sulfate, delta-8-estrone sulfate, 17beta-dihydroequilin sulfate and estrone sulfate, have potent estrogenic effects. Estrogen sulfates can be absorbed directly from the gastrointestinal tract; however, hydrolysis of the sulfates also occurs in the gastrointestinal tract, and the unconjugated estrogens formed are readily absorbed. After absorption, these estrogens are sulfated rapidly and circulate in this form. The pharmacokinetics of these estrogens indicate that the unconjugated estrogens are cleared from the circulation at a faster rate than their sulfate ester forms. In postmenopausal women, the 17-keto derivatives of these estrogens are metabolized to the more potent 17beta-reduced products. The extent of this activation is nearly 10 times higher with some ring B unsaturated estrogens. The 17beta-reduced metabolites are cleared from the blood at a slower rate than their corresponding 17-keto derivatives. In the human endometrium, equilin is metabolized to 2-hydroxy and 4-hydroxy equilin, with 2-hydroxylation being predominant. In contrast, 2-hydroxy and 4-hydroxy estradiol are formed in equal amounts. Similarly, 16alpha-hydroxylation occurs with both types of estrogens; however, with the ring B saturated estrogens, the 17-keto steroid 16alpha-hydroxy estrone was the major urinary metabolite, whereas with the ring B unsaturated estrogens, the 17beta-reduced steroids, such as 16alpha-hydroxy-17beta-dihydroequilin and 16alpha-hydroxy-17beta-dihydroequilenin, were the major metabolites. This difference in metabolism may be important as it has been suggested that 16alpha-hydroxy estrone (alpha-ketol structure) can form covalent adducts with macromolecules and that it may be oncogenic. These types of interactions will not occur with the 16alpha-hydroxylated-17beta-reduced metabolites of ring B unsaturated estrogens. Since all of the estrogens present in Premarin have estrogenic activity, the pharmacological effects of Premarin are a result of the sum of these individual activities. Therefore, preparations lacking some of these important components may not offer the same degree of beneficial effects as Premarin.

Pharmacokinetics and pharmacodynamics of a novel estrogen delta8-estrone in postmenopausal women and men.

Recently a tenth equine estrogen, identified as the sulfate ester of delta8-estrone has been reported to be present in Premarin (a conjugated equine estrogen preparation), and because of its unique ring B unsaturated structure (conjugated double bond in the B ring), we have, in the present study, determined its pharmacokinetics in postmenopausal women and men, its interaction with uterine estrogen receptors and its uterotropic activity. After the administration of [14C]delta8-estrone, blood was drawn at various time intervals, and the plasma fractionated into the unconjugated sulfate and glucuronide fractions. The disappearance of radioactivity as delta8-estrone from plasma can be described as a function of two exponentials. The half-lives of the first and second components were 5+/-0.2 and 40.4 min, respectively. The mean metabolic clearance rate calculated (MCR), was 1711+/-252 l/d m2. From the unconjugated fraction, delta8-17beta-estradiol was also isolated and identified. From the sulfate conjugated fraction, delta8-estrone sulfate and delta8-17beta-estradiol sulfate were isolated in almost equal amounts. No other metabolites of delta8-estrone was detectable in the plasma. Both delta8-estrone and delta8-17beta-estradiol bind with human endometrial and rat uterine estrogen receptors with high affinity. The binding affinities of delta8-17beta-estradiol for human endometrial and rat uterine cytoplasmic receptors were 4 and 25 times higher than those of the parent estrogen delta8-estrone, respectively. Administration of delta8-estrone and delta8-17beta-estradiol (2 microg/100 g body weight) to immature rats significantly (P< 0.05) increased the uterine weight compared to the controls. These data demonstrate that delta8-estrone has estrogenic activity, and that it is further metabolized in man to a single more potent estrogen, delta8-17beta-estradiol. The extent of this activation by 17beta-reduction appears to be greater than that observed with other estrogens. Both estrogens circulate as sulfate conjugates and are very slowly eliminated from the circulation. These data further suggest that delta8-estrone and its major metabolite delta8-17beta-estradiol can contribute to the overall in vivo biological effects of Premarin.

Plasma concentrations of beta-endorphin in trained eumenorrheic and amenorrheic women.

OBJECTIVE: To characterize the pattern of plasma beta-endorphin throughout the normal menstrual cycle and test the hypothesis that beta-endorphin concentrations are elevated in trained women with amenorrhea compared with trained and sedentary eumenorrheic women. DESIGN: Cohort analytic study. SETTING: Academic research environment. PARTICIPANTS: Healthy female volunteers: 10 eumenorrheic sedentary, 11 eumenorrheic trained, and 11 amenorrheic trained women. INTERVENTIONS: Blood samples were collected three times per week for either one complete menstrual cycle (eumenorrheic sedentary and trained subjects) or for a 4-week period (amenorrheic trained subjects). MAIN OUTCOME MEASURE: Plasma beta-endorphin concentrations. RESULTS: beta-Endorphin levels varied considerably across the sampling period and were not associated with menstrual status, gonadotropin, or gonadal steroid concentrations. Average beta-endorphin levels were not different between the follicular and luteal phases for menstruating subjects, but were greater in the eumenorrheic athletes. Compared with eumenorrheic sedentary subjects, plasma beta-endorphin levels were higher in the athletic groups, regardless of menstrual status. CONCLUSION: There were no cycle-related beta-endorphin changes. Eumenorrheic and amenorrheic athletes have higher beta-endorphin concentrations that may reflect adaptations to intense training and not exercise-associated amenorrhea.

Endometrial transcripts of human insulin-like growth factors arise by differential promoter usage.

By the application of RT-PCR, we have demonstrated that in the human endometrium mRNAs for insulin-like growth factors, IGF-I and II, and their receptors are expressed not only in the intact endometrium, but also in the freshly isolated stromal and epithelial cells. The expression of multiple transcript forms of the IGF-I and II at various phases of the menstrual cycle, occurs by differential use of all four IGF-I transcriptional start sites, and two of the four known promoter sites of the IGF-II gene. The complete spectrum of transcripts is displayed by the proliferative phase and the menstrual phase endometrium. During the secretory phase, the exon 1 upstream start site of the IGF-I gene and the P2 promoter of the IGF-II gene are not used. Irrespective of the phase of the menstrual cycle, the stromal cells always display the same transcriptional patterns of both growth factor genes as those of the intact endometrium. In contrast, the epithelial cells do not express IGF-I transcript originating from the exon 2 upstream initiation site. These results indicate that the expressions of the IGF-I and II genes in the intact endometrium and stromal and epithelial cells are modulated at the transcriptional level during the menstrual cycle by differential usage of promoters and start sites.

Hyaluronidase activity in human semen: correlation with fertilization in vitro.

OBJECTIVES: To characterize the relationship between hyaluronidase activity and the currently used methods of assessing sperm function and to determine whether the measurement of hyaluronidase activity can provide a reliable index of sperm fertilizing capacity in man. DESIGN: Nonrandomized prospective study. SETTING: Tertiary referral IVF and andrology clinics affiliated with the University of Toronto. SUBJECTS: Four hundred eight samples were collected, 248 from men undergoing investigation in andrology and fertility clinics and 160 from men participating in an IVF program. INTERVENTIONS: Semen samples were treated with NP-40 in buffer to extract hyaluronidase and applied to a circular well cut into a petri dish containing a mixture of hyaluronic acid and agar. Enzyme activity was assessed by measuring the area of substrate hydrolysis. RESULTS: Significant positive correlations were found between hyaluronidase activity and sperm concentration, motility, and the percentage of sperm with normal morphology in the studied samples. In the IVF samples, hyaluronidase activity was found to be related significantly to the fertilization rate. Moreover, we were able to establish values of hyaluronidase below which no fertilization occurred and above which fertilization of at least one oocyte was achieved. CONCLUSION: These results suggest that measurement of hyaluronidase activity may provide a useful method for assessing the integrity of the acrosomal enzyme system, providing a simple and reliable predictor of the fertilizing potential of human sperm.

Metabolism of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate in normal postmenopausal women.

The metabolism of 17 beta-dihydroequilin and 17 beta-dihydroequilin sulfate was investigated after intravenous administration of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate to postmenopausal women. Urine was collected for 3 days and 46.2 +/- 10.5% and 54.5 +/- 8.7% of the injected dose of [3H] 17 beta-dihydroequilin and [17 beta-3H]dihydroequilin sulfate was excreted in the urine respectively. The estrogens present in urine were extracted and fractionated into unconjugated, sulfate, and glucuronide conjugated forms. With both precursors, the major amount (63-64%) of metabolites were excreted in the urine conjugated with glucuronic acid. From the unconjugated, sulfate, and glucuronide fraction, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, equilenin, and equilin were isolated. The conversions with both precursors were similar and 17 beta-dihydroequilenin was the major metabolite isolated from all three fractions; however, the highest levels of all four metabolites were present in the glucuronide fraction. Along with these identifiable metabolites, a large amount (51-81%) of radioactivity was present in the form of metabolites which are more polar than any of the known ring-B unsaturated estrogens. These appear to be polyhydroxy 17 beta-reduced ring-B unsaturated estrogens which remain to be identified. The in-vivo formation of equilenin and 17 beta-dihydroequilenin indicates the presence of the enzyme 6.8(9) steroid dehydrogenase in humans.

Pharmacokinetics of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin in normal postmenopausal women.

The MCRs of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin were determined in normal postmenopausal women by single iv injection of either 17 beta-[3H]dihydroequilin sulfate ([3H]17 beta-EqS) or 17 beta-[3H]dihydroequilin ([3H]17 beta-Eq). After the administration of [3H]17 beta-EqS, blood was drawn at various time intervals, and the plasma obtained was fractionated into the unconjugated, sulfate, and glucuronide fractions. The bulk of radioactivity was present in the sulfate fraction, and from this [3H]17 beta-EqS, [3H]equilin sulfate, [3H]equilenin sulfate, and 17 beta-[3H]dihydroequilenin sulfate were isolated and purified, and their concentrations were measured. The disappearance of [3H]17 beta-EqS from plasma can be described as a function of two exponentials. The half-life of the initial fast component was 5 +/- 0.2 min; this component represents the distribution and transfer from a space, with a mean volume (V1) of 6 +/- 0.5 L. The value for the rate constant (k) of total removal from this space was 300 +/- 20 U/day, of which 35 +/- 2% was irreversible. The mean half-life of the slower component of 17 beta-EqS was 147 +/- 15 min, and the mean MCR was 376 +/- 93 L/day.m2. Similarly, after the administration of [3H]17 beta-Eq, the disappearance of radioactivity as 17 beta-Eq from plasma also had two components. The half-lives of the fast and slow component were 5.5 +/- 0.8 and 45 +/- 2.0 min, respectively. The MCR of 17 beta-Eq was 1252 +/- 103 L/day.m2. From both series of experiments, unconjugated and sulfate-conjugated equilin, equilenin, and 17 beta-dihydroequilenin were isolated and purified, and their concentrations were measured. No 17 alpha-reduced metabolites were detected. These results indicate that 17 beta-EqS is cleared twice as fast as equilin sulfate (MCR, 176 L/day.m2), whereas the more potent estrogen 17 beta-Eq is cleared 2 times slower than equilin. The slower elimination and greater estrogenic activity of 17 beta-Eq support the hypothesis that the major in vivo activity of equilin sulfate present in conjugated equine estrogen preparations is expressed via its metabolites 17 beta-EqS and 17 beta-Eq.

Metabolic clearance rate of equilin sulfate and its conversion to plasma equilin, conjugated and unconjugated equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin in normal postmenopausal women and men under steady state conditions.

The constant infusion of [3H]equilin sulfate ([3H]EqS) was used to estimate the MCR of equilin sulfate (EqS) and to measure the conversion of this estrogen to equilin (Eq), equilenin (Eqn), equilenin sulfate (EqnS), 17 beta-dihydroequilin (17 beta-Eq), 17 beta-dihydroequilin sulfate (17 beta-EqS), 17 beta-dihydroequilenin (17 beta-Eqn), and 17 beta-dihydroequilenin sulfate (17 beta-EqnS) in normal postmenopausal women and men. Infusion of [3H]EqS was started in five postmenopausal women and two men 30 min after a priming dose and continued at a constant rate of 12-15 microCi/h for 3 h. Blood samples were taken 15 min before the end of infusion, at the end of the infusion, and 15 min after the end of infusion. Unconjugated and sulfate-conjugated Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated from plasma. The mean MCR of EqS was calculated to be 280 +/- 24 L/day or 170 +/- 18 L/day.m2. The mean conversion ratios for precursor EqS to product 17 beta-EqS, EqnS, 17 beta-EqnS, 17 beta-Eq, Eq, Eqn, and 17 beta-Eqn were 0.300, 0.190, 0.100, 0.020, 0.016, 0.008, and 0.004 respectively. In both the sulfate-conjugated and unconjugated forms, 17 beta-Eq was the most abundant metabolite formed. 17 beta-Eq estrogen is a potent uterotropic agent and has a much higher affinity for estrogen receptors than Eq. Its formation may be of importance in the overall biological activity of EqS present in conjugated equine estrogen preparations.

Metabolism of [3H]equilin in normal and malignant human endometrium and in endometrial adenocarcinoma transplanted into nude mice.

One of the main components of conjugated equine estrogens is equilin sulfate and this estrogen in postmenopausal women is metabolized to 17 beta-dihydroequilin, 17 beta-dihydroequilenin and equilenin. To investigate the possibility that some of these estrogens may be formed directly in the target tissues, we studied the in vitro metabolism of [3H]equilin in various types of normal and malignant human endometrium, including adenocarcinoma grown in athymic nude mice. The results indicate that normal and neoplastic human endometrium can form the above three metabolites. The highest level of 17 beta-reduced products were isolated from the normal secretory endometrium. Equilenin was the most abundant metabolite isolated from both the normal and malignant endometrium. The formation of [3H]equilenin indicates the presence of a 6,8(9) steroid dehydrogenase-isomerase in the human endometrium. The formation of 17 beta-dihydroequilin in the endometrium may be of importance as this estrogen is 8 times more potent as a uterotrophic agent than equilin and estrone.

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus.

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.

Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium.

The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1 beta-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1 beta-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used for detection of very low levels of aromatase activity.