Developmental toxicity and transcriptome analysis of equine estrogens in developing medaka (Oryzias latipes) using nanosecond pulsed electric field incorporation.

Equine estrogens (EQs) are steroidal hormones isolated from the urine of pregnant mares and are used in the formulation of human medications. This study initially investigated the embryonic developmental toxicity of equilin (Eq) and equilenin (Eqn) in medaka (Oryzias latipes). Malformations were observed in embryos exposed to nominal concentrations of 1 and 10 mg/L of Eq and Eqn. Delayed hatching was observed at 1 mg/L of Eq. To further investigate the molecular mechanism of developmental toxicity caused by Eq and Eqn, transcriptome and bioinformatics analyses were performed. Among 2016 and 3855 total differentially expressed genes (DEGs), 1117 DEGs overlapped between Eq. (55.4 % of total DEGs) and Eq. (29.0 % of total DEGs). Gene ontology indicated effects in terms related to blood circulation and cell junctions. Pathway analyses using DEGs revealed that both Eq and Eqn treatments at 10 mg/L affected various KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, such as neuroactive ligand-receptor interaction, mitogen-activated protein kinase signaling, retinol metabolism, and cytokine-cytokine receptor interaction. These results suggest that the disruption of these KEGG pathways is involved in the developmental toxicity of EQs in medaka embryos.

Occurrence and seasonal variation of equine estrogens, equilin and equilenin, in the river water of Japan: Implication with endocrine-disrupting potentials to Japanese medaka (Oryzias latipes).

In this study, we determined the concentration of equine estrogens, such as equilin (Eq) and equilenin (Eqn), in the river water collected from nine research stations in Hokkaido, Japan. The LC-MS/MS analysis revealed that Eq concentrations were 2.7 ±â€¯6.7, 0.22 ±â€¯0.12, and 1.2 ±â€¯0.64 ng/L in Sep 2015, Feb 2016, and Jul 2016, respectively. Eqn had concentration levels similar to those of Eq. Comparison of the concentrations at nine research stations showed that seasonal variation was observed in the detected Eq and Eqn concentration levels. This study was the first to show the occurrences and seasonal variation of Eq and Eqn in the river water of Japan. We further investigated the reproductive and transgenerational effects of Eq in Japanese medaka (Oryzias latipes) exposed to 10, 100, and 1000 ng/L for 21 days and assessed the transcriptional profiles of the estrogen-responsive genes in the livers of both sexes. The reproduction assay demonstrated that 1000 ng/L of Eq adversely affected the reproduction (i.e. fecundity) in the F0 generation and that the hatching of F1 generation fertilized eggs was reduced in the 100 and 1000 ng/L treatment groups. Our qRT-PCR assay revealed that the mRNA expression levels of hepatic vitellogenin 1 and 2, choriogenin L and H, and estrogen receptor α were significantly up-regulated in males exposed to 100 and/or 1000 ng/L of Eq. In contrast, the transcriptional levels of several genes, such as pregnane X receptor and cytochrome P450 3A, were down-regulated in the livers of males after the 21-d exposure. These results suggest that Eq has endocrine-disrupting potential such as reproductive and transgenerational effects by the modulation of hepatic estrogen-responsive genes expression on medaka.

Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1.

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17ß-estradiol (17ß-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3ß, 17ß-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low µM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17ß-dihydroequilenin (17ß-Eqn), 17ß-dihydroequilin (17ß-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17ß-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17ß-Eq was stimulated at celecoxib concentrations below 40 µM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.

Endocrine-disrupting potentials of equine estrogens equilin, equilenin, and their metabolites, in the medaka Oryzias latipes: in silico and DNA microarray studies.

Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine-disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α-dihydroequilin, 17ß-dihydroequilin, 17α-dihydroequilenin and 17ß-dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three-dimensional model of the ligand-binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17ß-dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α-dihydroequilin and 17ß-dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24-h exposure to Eq and Eqn (100 ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer-related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine-disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka.

Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

NMR and computational studies of stereoisomeric equine estrogen-derived DNA cytidine adducts in oligonucleotide duplexes: opposite orientations of diastereomeric forms.

The equine estrogens equilin (EQ) and equilenin (EN) are the active components in the widely prescribed hormone replacement therapy formulation Premarin. Metabolic activation of EQ and EN generates the catechol 4-hydroxyequilenin (4-OHEN) that autoxidizes to the reactive o-quinone form in aerated aqueous solutions. The o-quinones react predominantly with C, and to a lesser extent with A and G, to form premutagenic cyclic covalent DNA adducts in vitro and in vivo. To obtain insights into the structural properties of these biologically important DNA lesions, we have synthesized site-specifically modified oligonucleotides containing the stereoisomeric 1'S,2'R,3'R-4-OHEN-C3 and 1'R,2'S,3'S-4-OHEN-C4 adducts derived from the reaction of 4-OHEN with the C in the oligonucleotide 5'-GGTAGCGATGG in aqueous solution. A combined NMR and computational approach was utilized to determine the conformational characteristics of the two major 4-OHEN-C3 and 4-OHEN-C4 stereoisomeric adducts formed in this oligonucleotide hybridized with its complementary strand. In both cases, the modified C adopts an anti glycosidic bond conformation; the equilenin distal ring protrudes into the minor groove while its two proximal hydroxyl groups are exposed on the major groove side of the DNA duplex. The bulky 4-OHEN-C adduct distorts the duplex within the central GC*G portion, but Watson-Crick pairing is maintained adjacent to C* in both stereoisomeric adducts. For the 4-OHEN-C3 adduct, the equilenin rings are oriented toward the 5'-end of the modified strand, while in 4-OHEN-C4 the equilenin is 3'-directed. Correspondingly, the distortions of the double-helical structures are more pronounced on the 5'- or the 3'-side of the lesion, respectively. These differences in stereoisomeric adduct conformations may play a role in the processing of these lesions in cellular environments.

Mixtures of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents.

Most effluents from wastewater treatment works (WwTWs) contain estrogenic chemicals that include steroidal estrogens and xenoestrogens. We investigated the nature of mixtures of estrogenic contaminants taken up by two species of fish exposed to two WwTWs effluents. Sexually immature rainbow trout, Oncorhynchus mykiss, and sexually mature roach, Rutilus rutilus, were exposed to tap water, river water, or one of two estrogenic WwTWs effluents for up to 10 days, when the fish were sacrificed and tissues removed for chemical analysis. Estrogenic contaminants in the bile and gonads were hydrolyzed, concentrated by solid-phase extraction, and fractionated by RP-HPLC. Active fractions were detected and quantified using a yeast estrogen receptor transcription screen (YES assay) and the identities of estrogenic components in the fractions determined by GC-MS. Bile from rainbow trout exposed to either tap water or river water contained low amounts of 17beta-estradiol (E2) and estrone (E1) with a total estrogenic activity (mean+/-standard error) of 10+/-5 and 31+/-9 ng of E2 equivalents/mL (ng of E2eq/mL) for male and female fish, respectively. In effluent-exposed trout the total estrogen content of bile was considerably higher with the following composition and concentrations (ng of E2eq/mL) of individual estrogens: E2 (male, 591+/-125; female, 710+/-207), E1 (male, 338+/-75; female, 469+/-164), ethinylestradiol, EE2 (male, 32+/-2; female, 40+/-6), nonylphenol (NP) and short-chain NP polyethoxylates (male, 21+/-4; female, 22+/-3). An additional estrogenic compound, 17beta-dihydroequilenin (DHQ), was identified for the first time in effluent-exposed fish, and was present in trout bile at concentrations of (male) 40+/-9 and (female) 30+/-5 ng of E2 eq/mL. DHQ, E2, E1, and EE2, but not NP or NP polyethoxylates, were also detected in bile of effluent-exposed roach, and the concentrations of all these steroidal estrogens in ng of E2eq/mL were lower in male (E2, 62+/-2; E1, 35+/-11; EE2, 10+/-2; DHQ, 1+/-1) compared with female (E2, 740+/-197; E1, 197+/-37; EE2, 40+/-6; DHQ, 8+/-2) roach. The synthetic estrogen EE2 was also detected in the testes and ovaries of effluent-exposed roach. This study shows that a mixture of estrogenic contaminants present in WwTWs effluents bioconcentrate in fish tissues, resulting in the induction of vitellogenin, and are likely to contribute to feminizing effects in wild fish living in U.K. rivers. The composition of the mixture of estrogenic contaminants in the bile is species dependent and may determine the susceptibility of fish to the effects of exposure to estrogenic effluents.

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues.

In on-going studies of 'classical' and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-2H(5)]-testosterone and [16,16,17-2H(3)]-5,7-androstadiene-3 beta,17 beta-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [2H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography-mass spectrometry (GC-MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [2H(5)]-Testosterone was converted into [2H(4)]-oestradiol-17 beta, [2H(4)]-oestrone and [2H(3)]-6-dehydro-oestradiol-17 alpha in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse. On the basis of GC-MS characteristics (M(+) m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 alpha was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M(+)). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [2H(4)]-17 beta-dihydroequilin was also shown, and a biosynthetic pathway is proposed. In static incubations of placental microsomal fractions, the 17 beta-dihydro forms of both equilin and equilenin were shown to be major metabolites of [2H(3)]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17 beta-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17 beta-isomer of dihydroequilenin. Confirmation of the identity of 17 beta-dihydroequilin and 17 beta-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17 beta-dihydroequilin) and 412 (17 beta-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3 beta,17 beta-diol are proposed.

Structure of the ternary complex of human 17beta-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP+.

Excess 17beta-estradiol (E2), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which catalyzes the reduction of inactive estrone (E1) to the active 17beta-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E2 in breast tumor tissues. We present here the structure of the ternary complex of 17beta-HSD1 with the cofactor NADP+ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17beta-HSD1-catalyzed reduction of E1 to E2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-A resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP+ and catalytic residues Tyr-155 and Ser-142 is different from that of E2 in the 17beta-HSD1-E2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17beta-HSD1 is the basis for inhibition of E1-to-E2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.

The major metabolite of equilin, 4-hydroxyequilin, autoxidizes to an o-quinone which isomerizes to the potent cytotoxin 4-hydroxyequilenin-o-quinone.

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with a slight increased cancer risk. Previously, we showed that equilenin, a minor component of Premarin (Wyeth-Ayerst), was metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. In this study, we have compared the chemistry of the major catechol metabolite of equilin (4-hydroxyequilin), which is found in several estrogen replacement formulations, to the equilenin catechol (4-hydroxyequilenin). Unlike endogenous catechol estrogens, both equilin and equilenin were primarily converted by rat liver microsomes to 4-hydroxylated rather than 2-hydroxylated o-quinone GSH conjugates. With equilin, a small amount of 2-hydroxyequilin GSH quinoids were detected (4-hydroxyequilin:2-hydroxyequilin ratio of 6:1); however, no peaks corresponding to 2-hydroxyequilenin were observed in incubations with equilenin. These data suggest that unsaturation in the B ring alters the regiochemistry of P450-catalyzed hydroxylation from primarily 2-hydroxylation for endogenous estrogens to 4-hydroxylation for equine estrogens. 4-Hydroxyequilenin-o-quinone reacts with GSH to give two mono-GSH conjugates and one di-adduct. The behavior of 4-hydroxyequilin was found to be more complex than 4-hydroxyequilenin as conjugates resulting from 4-hydroxyequilenin were detected in addition to the 4-hydroxyequilin-GSH adducts. The mechanism of decomposition of 4-hydroxyequilin likely involves isomerization to a quinone methide which readily aromatizes to 4-hydroxyequilenin followed by autoxidation to 4-hydroxyequilenin-o-quinone. Similar results were obtained with 2-hydroxyequilin, although, in contrast to 4-hydroxyequilenin, 2-hydroxyequilenin does not autoxidize and the reaction stops at the catechol. Since 4-hydroxyequilin is converted to 4-hydroxyequilenin and 4-hydroxyequilenin-o-quinone, similar effects were observed for this equine catechol, including consumption of NAD(P)H likely by the 4-hydroxyequilenin-o-quinone, depletion of molecular oxygen by 4-hydroxyequilenin or its semiquinone radical, and alkylation of deoxynucleosides and DNA by 4-hydroxyequilenin quinoids. Finally, preliminary studies conducted with the human breast tumor cell line MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar, whereas 4-hydroxyequilenin was a much more potent cytotoxin ( approximately 30-fold). These results suggest that the catechol metabolites of equine estrogens have the ability to cause alkylation/redox damage in vivo primarily through formation of 4-hydroxyequilenin quinoids.

Conjugated estrogens--the natural SERMs.

Tissue selective and metabolite replacement therapy may become a new aspect in hormone replacement therapy (HRT). In addition to the naturally secreted hormones, there are also the later formed metabolites that exert a characteristic pharmacological profile. This mechanism is well known in thyroid replacement therapy, when triiodothyronine, the metabolite of thyroxine, is added to substitution therapy. The same is true for testosterone replacement therapy, when dihydrotestosterone is used for replacement. Also in menopausal HRT these aspects will gain tremendous importance. Progesterone metabolites have a strong clinical potency as neurosteroids, and estradiol metabolites are important factors in angiogenesis and angiostasis. Conjugated estrogens consist of different metabolites such as 16-hydroxy-equilin, which has no angiogenetic effect compared with 16-hydroxy-estrone. Estrone sulfate, the main component in conjugated estrogens, can be activated into estrone and 17 beta-estradiol in a tissue specific manner. This aspect will become of interest in clinical practice with HRT.

Superior and distinct antioxidant effects of selected estrogen metabolites on lipid peroxidation.

The effect of the estrogen metabolites, 4-hydroxyestrone and 17alpha-dihydroequilin (metabolites of estradiol-17beta and equilin, respectively), were examined for antioxidant effects on plasma and lipoprotein lipid peroxidation . Lipid peroxidation was evaluated by products of both fatty acid (thiobarbituric acid-reactive substances [TBARS]) and cholesterol (oxysterols) oxidation from lipoproteins or whole plasma. Although all estrogens significantly reduced lipid peroxidation, 4-hydroxyestrone was far more potent than either equilin or 17alpha-dihydroequilin in inhibiting TBARS formation in lipoproteins induced by Cu2+. Similar effects were also noted on TBARS formation in THP-l macrophages in culture. However, 17alpha-dihydroequilin (along with equilin) strongly inhibited oxysterol formation, whereas 4-hydroxyestrone was ineffective. These studies suggest that different estrogens might act preferentially on distinct lipid substrates in exhibiting antioxidant effects.

Metabolism of equilin sulfate in the dog.

The metabolism of equilin sulfate was determined in female dogs receiving 2.5 mg/kg of [3H]equilin sulfate alone or in a preparation that contained all the components that are present in the conjugated equine estrogen product Premarin. The pharmacokinetic parameters of total radioactivity indicated that the drug is rapidly absorbed and it has a moderate half-life in plasma. The total radioactivity in plasma following administration of [3H]equilin sulfate as part of a mixture of conjugated equine estrogens had significantly lower peak concentration (Cmax), a lower area under the curve (AUC), a longer terminal half-life (t1/2) and a longer mean residence time (MRT) than when [3H]equilin sulfate was given alone, indicating that the other components in the conjugated equine estrogen preparation altered the pharmacokinetics of equilin sulfate. An average of 26.7 +/- 4.4% of the administered radioactive dose was excreted in urine of dogs receiving [3H]equilin sulfate. Again, a significantly lower percentage (21.4 +/- 6.3%, P = 0.023) was eliminated in urine of dogs receiving [3H]equilin sulfate in the conjugated equine estrogen preparation, indicating that the absorption of equilin sulfate was perhaps altered by the other components in the conjugated equine estrogen preparation. Metabolite profiles of plasma and urine were similar. Equilin, equilenin, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, 17 alpha-dihydroequilenin and 17 alpha-dihydroequilin were present in both matrices. 17 beta-Dihydroequilin and equilin were the two major chromatographic peaks in plasma samples. 17 beta-Dihydroequilenin and 17 beta-dihydroequilin were the major metabolites in urine. In conclusion, following oral administration of [3H]equilin sulfate to dogs, the radioactivity is rapidly absorbed. The disposition of equilin sulfate is altered by the other components that are present in the conjugated equine estrogen preparation Premarin. The reduction of the 17-keto group and aromatization of ring-B are the major metabolic pathways of equilin in the dog.

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.

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.

Metabolism of [3H]equilin-[35S]sulfate and [3H]equilin sulfate after oral and intravenous administration in normal postmenopausal women and men.

The absorption of equilin sulfate and equilin from the gastrointestinal tract was determined in normal men after the ingestion of [3H]equilin-[35S]sulfate or a mixture of [3H]equilin and equilin-[35S]sulfate, while the metabolism of equilin sulfate was investigated after iv administration of [3H]equilin sulfate to postmenopausal women. After the oral administration of [3H]equilin-[35S]sulfate, equilin sulfate containing both 3H and 35S was isolated from plasma; however, only in the first sample taken at 10 min was the 3H/35S ratio the same as that of the [3H]equilin-[35S]sulfate ingested. The 3H/35S ratio then increased, and by 12 h only traces of equilin-[35S]sulfate were detectable. Similarly, after the ingestion of [3H]equilin and equilin-[35S]sulfate, [3H]equilin-[35S]sulfate was isolated from plasma. The 3H/35S ratio was at all time points greater than the 3H/35S ratio of the ingested mixture. Analysis of urine indicated that over 98% of 35S was not associated with any steroid and was most likely inorganic sulfate. After iv administration of [3H] equilin sulfate to postmenopausal women, equilin, equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin were isolated from the urine. These results indicate that 1) some of the orally administered equilin sulfate was absorbed from the gut without prior hydrolysis, 2) some equilin sulfate was hydrolyzed in the gut before absorption; 3) equilin was absorbed more efficiently than equilin sulfate; 4) equilin absorbed was readily sulfated and circulated in this form; and 5) equilin sulfate was extensively metabolized, and the metabolites were excreted in the urine mainly conjugated with glucuronic acid.

Pharmacokinetics of equilin and equilin sulfate in normal postmenopausal women and men.

The MCRs of equilin sulfate and equilin were determined in normal postmenopausal women and a normal man by single iv injections of either [3H]equilin sulfate or [3H] equilin. After the administration of [3H]equilin sulfate, 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]equilin sulfate, [3H]17 beta-dihydro-equilin sulfate, [3H]equilenin sulfate, and [3H]17 beta-dihydroequilenin sulfate were isolated and purified, and their concentrations were measured. The disappearance of radioactivity from plasma as equilin sulfate can be described as a function that is the sum of two exponentials. The initial fast component (half-life, 5.2 +/- 1.2 min) represents distribution and transfer from a space, with a mean volume of 12.4 +/- 1.6 liters. The mean value for the rate constant of total removal from the initial volume is 163 +/- 19 U/day, of which 15.8 +/- 2% is irreversible. The mean half-life of the slower component of equilin sulfate is 190 +/- 23 min, and the mean MCR is 176 +/- 44 liters/day . m2. Similarly, after the administration of [3H]equilin to a normal postmenopausal woman and a man, the disappearance of radio-activity from plasma as equilin could be fitted by a single straight line, consistent with a one-compartment system. The half-life of equilin was approximately 19-27 min, and the MCR of equilin was calculated to be 1982 liters/day/m2 in the normal man and 3300 liters/day/m2 in the normal postmenopausal woman. The bulk of [3H]equilin was very rapidly metabolized to mainly equilin sulfate. Small amounts of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin were also isolated from the plasma. The in vivo formation of 17 beta-dihydroequilin and its sulfate may be of importance, as this estrogen is approximately 8 times more potent as a uterotropic agent than equilin sulfate.