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Bhagu R. Bhavnani - One of the best experts on this subject based on the ideXlab platform.
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Equine Estrogens Differentially Prevent Neuronal Cell Death Induced by Glutamate
The Journal of the Society for Gynecologic Investigation: JSGI, 2003Co-Authors: Bhagu R. Bhavnani, Mauricio Berco, Julie BinkleyAbstract:Objective In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17 β -estradiol, 17 α -estradiol, equlin, 17 β -dihydroequilin, 17 α -dihydroequilin, Equilenin, 17 β -dihydroEquilenin, 17 α -dihydroEquilenin, Δ^8-estrone and Δ^8, 17 β -estradiol against glutamate toxicity. Methods The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum, and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1–40 μ M) and glutamate (1–10 mM) were added in a total volume of 100 μL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Reslts were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay. Results The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutanate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Δ ^8-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17 β -estradiol, estrone, and 17 α -estradiol were the least potent. The order of potency was Δ ^8, 17 β -estradiol > Δ ^8-estrone > 17 β -dihydroEquilenin > 17 α -dihydroEquilenin > Equilenin > 17 β -dihydroequilin = equilin > 17 α -dihydroequilin > 17 β -estradiol > estrone > 17 α -estradiol in PC12 cells and Δ ^7, 17 β -estradiol > Δ ^8-estrone > Equilenin = 17 β -dihydroEquilenin > 17 β -dihydroequilin > equilin > 17 α -dihydroEquilenin > 17 α -dihydroequilin > 17 α -estradiol = 17 β -estradiol > estrone in HT22 cells. Conclusion Our data indicate that the neutrotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Δ ^8 estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.
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Potential role of the interaction between equine estrogens, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in the prevention of coronary heart and neurodegenerative diseases in postmenopausal women
Lipids in Health and Disease, 2003Co-Authors: Joel Perrella, Mauricio Berco, Anthony Cecutti, Alan Gerulath, Bhagu R. BhavnaniAbstract:Background An inverse relationship between the level of high-density lipoprotein (HDL) and coronary heart disease (CHD) has been reported. In contrast, oxidized HDL (oHDL) has been shown to induce neuronal death and may play an important role in the pathogenesis of CHD. In the present study we have investigated a: the effect of various equine estrogens on HDL oxidation, b: the inhibition of LDL oxidation by HDL and c: the effect of these estrogens on LDL oxidation in the presence of HDL. Results All 11 equine estrogens tested protected the HDL from oxidation in a concentration dependant manner. Equilenin, 17β-dihydroEquilenin, and 17α-dihydroEquilenin (Δ^6–8-estrogens) were found to be the most potent inhibitors of HDL oxidation. Some of the novel ring B unsaturated estrogens were 2.5 to 4 times more potent inhibitors of HDL oxidation than 17β-estradiol. HDL was found to delay LDL oxidation. The protection of LDL oxidation by HDL is enhanced by the addition of estrogen, with Equilenin being again more potent than 17β-estradiol. Conclusions Equine estrogens can differentially inhibit the oxidation of HDL with the Δ^6–8-estrogens being the most potent antioxidants. The ability of estrogens to enhance HDL's antioxidant activity is to our knowledge the first report of an interaction of estrogen with HDL that results in the delay or inhibition of LDL oxidation. This may be another mechanism by which estrogens may reduce the risk of CHD and neurodegenerative diseases in healthy and younger postmenopausal women.
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Equine estrogens differentially prevent neuronal cell death induced by glutamate.
Journal of the Society for Gynecologic Investigation, 2003Co-Authors: Bhagu R. Bhavnani, Mauricio Berco, Julie BinkleyAbstract:In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17beta-estradiol, 17alpha-estradiol, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, Equilenin, 17beta-dihydroEquilenin, 17alpha-dihydroEquilenin, Delta(8)-estrone(,) and Delta(8),17beta-estradiol against glutamate toxicity. The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum, and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1-40 microM) and glutamate (1-10 mM) were added in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Results were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay. The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutamate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Delta(8)-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17beta-estradiol, estrone, and 17alpha-estradiol were the least potent. The order of potency was Delta(8),17beta-estradiol > Delta(8)-estrone > 17beta-dihydroEquilenin > 17alpha-dihydroEquilenin > Equilenin > 17beta-dihydroequilin = equilin > 17alpha-dihydroequilin > 17beta-estradiol > estrone > 17alpha-estradiol in PC12 cells and Delta(8),17beta-estradiol > Delta(8)-estrone > Equilenin = 17beta-dihydroEquilenin > 17beta-dihydroequilin > equilin > 17alpha-dihydroEquilenin > 17alpha-dihydroequilin > 17alpha-estradiol = 17beta-estradiol > estrone in HT22 cells. Our data indicate that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Delta(8) estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.
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Metabolism of [3H] 17β-dihydroequilin and [3H] 17β-dihydroequilin sulfate in normal postmenopausal women ☆
Steroids, 1994Co-Authors: Bhagu R. Bhavnani, Anthony Cecutti, Duncan WallaceAbstract: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.
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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 Journal of Clinical Endocrinology and Metabolism, 1993Co-Authors: Bhagu R. Bhavnani, A CecuttiAbstract: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 be...
A Cecutti - One of the best experts on this subject based on the ideXlab platform.
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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 Journal of Clinical Endocrinology and Metabolism, 1993Co-Authors: Bhagu R. Bhavnani, A CecuttiAbstract: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 be...
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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 Journal of clinical endocrinology and metabolism, 1993Co-Authors: Bhagu R. Bhavnani, A CecuttiAbstract: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.
Masataka Ihara - One of the best experts on this subject based on the ideXlab platform.
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Asymmetric total synthesis of (+)-Equilenin utilizing two types of cascade ring expansion reactions of small ring systems
Journal of the Chemical Society Perkin Transactions 1, 2000Co-Authors: Masahiro Yoshida, Mohamed Ismail, Hideo Nemoto, Masataka IharaAbstract:Enantioselective synthesis of (+)-Equilenin 1 utilizing two types of cascade ring expansion reactions of small ring systems is described. The first key step is an asymmetric epoxidation–ring expansion reaction of cyclopropylidene derivatives to afford chiral cyclobutanones. We found that both the fructose-derived chiral ketone and the chiral (salen)Mn(III) complex were effective catalysts for the asymmetric induction. The second key step is the palladium-promoted cascade ring expansion–intramolecular insertion reaction of the isopropenylcyclobutanol. Solvents were an important factor for the diastereoselective formation of hydrindanes. By utilizing these methodologies, the asymmetric total synthesis of (+)-Equilenin 1 has been accomplished.
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asymmetric total synthesis of Equilenin utilizing two types of cascade ring expansion reactions of small ring systems
Journal of The Chemical Society-perkin Transactions 1, 2000Co-Authors: Masahiro Yoshida, Mohamed Ismail, Hideo Nemoto, Masataka IharaAbstract:Enantioselective synthesis of (+)-Equilenin 1 utilizing two types of cascade ring expansion reactions of small ring systems is described. The first key step is an asymmetric epoxidation–ring expansion reaction of cyclopropylidene derivatives to afford chiral cyclobutanones. We found that both the fructose-derived chiral ketone and the chiral (salen)Mn(III) complex were effective catalysts for the asymmetric induction. The second key step is the palladium-promoted cascade ring expansion–intramolecular insertion reaction of the isopropenylcyclobutanol. Solvents were an important factor for the diastereoselective formation of hydrindanes. By utilizing these methodologies, the asymmetric total synthesis of (+)-Equilenin 1 has been accomplished.
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a novel strategy for the enantioselective synthesis of the steroidal framework using cascade ring expansion reactions of small ring systems asymmetric total synthesis of Equilenin
Tetrahedron Letters, 1999Co-Authors: Hideo Nemoto, Masahiro Yoshida, Keiichiro Fukumoto, Masataka IharaAbstract:Abstract Enantioselective synthesis of (+)-Equilenin ( 1 ) utilizing a novel strategy is described. The key steps are two cascade ring expansion reactions of small ring systems; 1) chiral (salen)Mn III complex-catalyzed cascade reaction of cyclopropylidene; 2) Pd II -mediated cascade reaction of the cyclobutanol.
Judy L. Bolton - One of the best experts on this subject based on the ideXlab platform.
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The naphthol selective estrogen receptor modulator (SERM), LY2066948, is oxidized to an o-quinone analogous to the naphthol equine estrogen, Equilenin.
Chemico-Biological Interactions, 2012Co-Authors: Teshome Gherezghiher, Bradley T. Michalsen, R. Esala P. Chandrasena, Johann Sohn, Gregory R J Thatcher, Judy L. BoltonAbstract:Abstract o -Quinone forming estrogens and selective estrogen receptor modulators (SERMs) have been associated with carcinogenesis. LY2066948, a novel SERM in development by Eli Lilly for the treatment of uterine fibroids and myomas, has structural similarity to the equine estrogen Equilenin present in hormone replacement formulations; both contain a naphthol group susceptible to oxidative metabolism to o -quinones. LY2066948 was synthesized and assayed for antiestrogenic activity, and in cell culture was confirmed to be a more potent antiestrogen than the prototypical SERM, 4-hydroxytamoxifen. Oxidation of LY2066948 with 2-iodoxybenzoic acid gave an o -quinone ( t 1/2 = 3.9 ± 0.1 h) which like 4-hydroxyEquilenin- o -quinone ( t 1/2 = 2.5 ± 0.2 h) was observed to be exceptionally long-lived with the potential to cause cytotoxicity and/or genotoxicity. In model reactions with tyrosinase, the catechol metabolites of LY2066948 and Equilenin were products; interestingly, in the presence of ascorbate to inhibit autoxidation, these catechols were formed quantitatively. Tyrosinase incubations in the presence of GSH gave the expected GSH conjugates resulting from trapping of the o- quinones, which were characterized by LC–MS/MS. Incubations of LY2066948 or Equilenin with rat liver microsomes also gave detectable o -quinone trapped GSH conjugates; however, as observed with other SERMs, oxidative metabolism of LY2066948 mainly occurred on the amino side chain to yield the N -dealkylated metabolite. CYP1B1 is believed to be responsible for extra-hepatic generation of genotoxic estrogen quinones and o -quinone GSH conjugates were detected in Equilenin incubations. However, in corresponding incubations with CYP1B1 supersomes, no o -quinone GSH conjugates of LY2066948 were detected. These studies suggest that although the naphthol group is susceptible to oxidative metabolism to long-lived o -quinones, the formation of these quinones by cytochrome P450 can be attenuated by the chemistry of the remainder of the molecule as in the case of LY2066948.
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metabolism of Equilenin in mcf 7 and mda mb 231 human breast cancer cells
Chemical Research in Toxicology, 2001Co-Authors: David C. Spink, Fagan Zhang, Mirza M. Hussain, Barbara H. Katz, Xuemei Liu, D. Hilker, Judy L. BoltonAbstract:Sulfate conjugates of the B-ring unsaturated estrogens, equilin, Equilenin, and 8-dehydroestrone, and their 17α- and 17β-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most...
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Metabolism of Equilenin in MCF-7 and MDA-MB-231 Human Breast Cancer Cells
Chemical research in toxicology, 2001Co-Authors: David C. Spink, Fagan Zhang, Mirza M. Hussain, Barbara H. Katz, Xuemei Liu, D. Hilker, Judy L. BoltonAbstract:Sulfate conjugates of the B-ring unsaturated estrogens, equilin, Equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of Equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of Equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of Equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both Equilenin and 17beta-dihydroEquilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyEquilenin and 4-methoxy-17beta-dihydroEquilenin. Rates of Equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of Equilenin and 17beta-dihydroEquilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroEquilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of Equilenin and 17beta-dihydroEquilenin in extrahepatic, estrogen-responsive tissues.
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The major metabolite of equilin, 4-hydroxyequilin, autoxidizes to an o-quinone which isomerizes to the potent cytotoxin 4-hydroxyEquilenin-o-quinone.
Chemical research in toxicology, 1999Co-Authors: Fagen Zhang, Emily Pisha, Yumei Chen, Li Shen, Yansan Xiong, Rb Van Breemen, Judy L. BoltonAbstract: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.
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Synthesis of the Equine Estrogen Metabolites 2-Hydroxyequilin and 2-HydroxyEquilenin
Chemical research in toxicology, 1999Co-Authors: Fagen Zhang, Judy L. BoltonAbstract:Equilin and Equilenin make up approximately 20% of Premarin which is currently the most popular estrogen replacement therapy. Although there are numerous health benefits of estrogen replacement therapy, there are concerns over the link between estrogen replacement therapy and breast and endometrial cancer risk. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols which are further oxidized to electrophilic/redox active o-quinones which have the potential to both initiate and promote the carcinogenic process. In this investigation, we have synthesized potential metabolites of equilin and Equilenin, 2-hydroxyequilin and 2-hydroxyEquilenin, respectively, as well as their methyl ether metabolites. These compounds were synthesized from commercially available optically pure equilin via a practical and efficient approach; five steps gave 2-methoxyequilin from which 2-hydroxyequilin was prepared by BBr3-catalyzed demethylation in one step. Similarly, treating 2-methoxyequilin with SeO2 followed by demethylation with BBr3 produced 2-hydroxyEquilenin. The structures of the catechols as well as those of their methoxy ethers were unambiguously characterized by one-dimensional and two-dimensional NMR experiments, including 1H, 13C, APT, COSY, HMBC, and HMQC as well as mass spectrometry.
Kwan Yong Choi - One of the best experts on this subject based on the ideXlab platform.
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Contribution of a low-barrier hydrogen bond to catalysis is not significant in ketosteroid isomerase.
Molecules and cells, 2015Co-Authors: Soo Jang, Hyung Jin Cha, Hee Cheon Lee, Hyeong Ju Lee, Gildon Choi, Sejeong Shin, Bee Hak Hong, Kwan Yong ChoiAbstract:4 -isomers at a rate that ap- proaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 O and C3-O of equi- lenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with Equilenin, suggest- ing that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-Equilenin complex revealed that the distance between Tyr14 O and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activ- ity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI. 1
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Rapid Mapping of Potential Active Site of Protein KSI by Multi-Dimensional NMR: Use of HyTEMPO as an Indirect Probe
2012Co-Authors: Vandana Mehrotra, Yong Nam Joe, Hyung Jin Cha, Hyungju Lee, Kwan Yong Choi, Hee Cheon LeeAbstract:A novel NMR method to rapidly map the enzyme -substrate active sites has been described. Potential active site mapping has been done by competition binding experiments using paramagnetic reagents (HyTEMPO) along with a substrate (or analog) (Equilenin), by 1H-15N HSQC NMR experiments. HyTEMPO induces the line broadening of signals in NMR spectrum. We analyzed the peak height value of cross peaks in 1H-15N HSQC spectrum of KSI with HyTEMPO and Equilenin. Our result revealed that residues in hydrophobic cavity of KSI, particularly active site region, were mainly perturbed by HyTEMPO. Upon the addition of Equilenin as an intermediate analog, few resonances were experienced as signal enhancements. These observations proved that HyTEMPO could map the potential active site of a protein KSI in the present case. This mapping method provides both high accuracy and speed, besides also a possibility of functional studies for unknown proteins in order to solve their difficult three-dimensional crystal structure.
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Rapid Mapping of Active Site of KSI by Paramagnetic NMR
2012Co-Authors: Yong Nam Joe, Hyung Jin Cha, Kwan Yong Choi, Hyeong Ju Lee, Hee Cheon LeeAbstract:Active site mapping has been done for Δ5-3-ketosteroid isomerase (KSI) by analyses of paramagnetic effect on 1H-15N HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediate analog (Equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active site region, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while they experienced full recovery of a lineshape upon the addition of Equilenin. The mapped region was very similar to the active site of KSI as described by the crystal structure. These observations indicate that a combined use of paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site of KSI, and can be applied to the analysis of both active site and function in unknown protein
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Crystal structure of delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with Equilenin settles the correct hydrogen bonding scheme for transition state stabilization.
The Journal of biological chemistry, 1999Co-Authors: Hyun Soo Cho, Gildon Choi, Hyunju Kim, Donghan Lee, Kwang S. Kim, Weon Tae Lee, Kwan Yong ChoiAbstract:Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue Equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of Equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.
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crystal structure of delta 5 3 ketosteroid isomerase from pseudomonas testosteroni in complex with Equilenin settles the correct hydrogen bonding scheme for transition state stabilization
Journal of Biological Chemistry, 1999Co-Authors: Hyun Soo Cho, Gildon Choi, Hyunju Kim, Donghan Lee, Kwang S. Kim, Weon Tae Lee, Kwan Yong ChoiAbstract:Abstract Δ5-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp38 (pK a ∼4.7) was identified as the general base abstracting the steroid C4β proton (pK a ∼12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue Equilenin reveals clearly that both the Tyr14 OH and Asp99 COOH provide direct hydrogen bonds to the oxyanion of Equilenin. The result negates the catalytic dyad mechanism in which Asp99 donates the hydrogen bond to Tyr14, which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr14 OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.