PGRMC1 can trigger estrogen-dependent proliferation of breast cancer cells: estradiol vs. equilin vs. ethinylestradiol.

Objective: Previous studies have shown that progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer tissue can predict a worse prognosis for breast cancer patients. Moreover, we demonstrated that PGRMC1 can increase the proliferation of progestogens. However, the role of PGRMC1 in terms of estrogen-induced proliferation and comparing different estrogens is still unclear. Methods: Non-transfected and PGRMC1-transfected T-47D cells were stimulated with estradiol (E2), with equilin (EQ), or with ethinylestradiol (EE) at 1, 10, and 100 nmol/l. Increase of proliferation was compared with a control (without estrogens) and with the estrogen-induced stimulation in empty vector cells vs. PGRMC1-transfected cells. Results: The empty vector cells showed significant proliferation (12-15%) with all three estrogens only at the highest concentration, with no relevant differences between the estrogens. PGRMC1-transfected cells showed about three-fold higher proliferation (29-66%), whereby E2 elicited the strongest and EE the lowest proliferating effects, significantly lower compared to E2 and also compared to EQ. No significant differences were seen between E2 and EQ. Conclusions: PGRMC1 increases strongly the estrogen-dependent breast cell proliferation. The proliferating effects of EE may be lower compared to E2 and EQ. This could have importance in comparing hormone therapy and contraception. Thus, PGRMC1 not only could predict the risk using progestogens but also of different estrogens.

Equilin in conjugated equine estrogen increases monocyte-endothelial adhesion via NF-κB signaling.

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17ß-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)ß expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERß or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis.

Equilin displays similar endothelium-independent vasodilator potential to 17ß-estradiol regardless of lower potential to inhibit calcium entry.

Conjugated equine estrogens (CEE) have been widely used by women who seek to relieve symptoms of menopause. Despite evidence describing protective effects against risk factors for cardiovascular diseases by naturally occurring estrogens, little is known about the vascular effects of equilin, one of the main components of CEE and not physiologically present in women. In this regard, the present study aims to compare the vascular effects of equilin in an experimental model of hypertension with those induced by 17ß-estradiol. Resistance mesenteric arteries from female spontaneously hypertensive rats (SHR) were used for recording isometric tension in a small vessel myograph. As effectively as 17ß-estradiol, equilin evoked a concentration-dependent relaxation in mesenteric arteries from female SHRs contracted with KCl, U46619, PDBu or ET-1. Equilin-induced vasodilation does not involve classical estrogen receptor activation, since the estrogen receptor antagonist (ICI 182,780) failed to inhibit relaxation in U46619-precontracted mesenteric arteries. Vasorelaxation was not affected by either endothelium removal or by inhibiting the release or action of endothelium-derived factors. Incubation with L-NAME (NOS inhibitor), ODQ (guanylyl cyclase inhibitor) or KT5823 (inhibitor of protein kinase G) did not affect equilin-induced relaxation. Similarly, indomethacin (COX inhibitor) or blockage of potassium channels with tetraethylammonium, glibenclamide, 4-aminopyridine, or ouabain did not affect equilin-induced relaxation. Inhibitors of adenylyl cyclase SQ22536 or protein kinase A (KT5720) also had no effects on equilin-induced relaxation. While 17ß-estradiol inhibited calcium (Ca2+) -induced contractions in high-K+ depolarization medium in a concentration-dependent manner, equilin induced a slight rightward-shift in the contractile responses to Ca2+. Comparable pattern of responses were observed in the concentration-response curves to (S)-(-)-Bay K 8644, a L-type Ca2+ channel activator. Equilin was unable to block the transitory contraction produced by caffeine-induced Ca2+ release from intracellular stores. In conclusion, equilin blocks L-type Ca2+ channels less effectively than 17ß-estradiol. Despite its lower effectiveness, equilin equally relaxes resistance mesenteric arteries by blocking Ca2+ entry on smooth muscle.

Cellular and molecular actions displayed by estrone on vascular endothelium.

In this work we provide evidence that estrone "per se" modulates cellular endothelial growth and survival, events that play key roles in the development of vascular disease. Moreover, under oxidative stress conditions the hormone prevented apoptosis triggered by hydrogen peroxide. Although estrone did not affect E-selectin and VCAM-1 mRNAs synthesis, the hormone prevented the expression of these adhesion molecules induced by the proinflammatory agent LPS. The steroid partially attenuated leukocyte adhesion not only under basal conditions but also in the presence of LPS. Using ICI182780 compound as estrogen receptor antagonist, and PD98059 as MAPK inhibitor we obtained evidence that the mitogenic action of estrone involved the participation of ER and MAPK transduction pathway activation. The presence of estradiol impaired the effect of estrone on cell proliferation and vasoactive production. These results suggest that estrone exhibits a remarkable biological action on endothelial cells, modulating vasoactive production, proliferation, apoptosis, and cell adhesion events.

Molecular clustering of endometrial carcinoma based on estrogen-induced gene expression.

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Effects of the components of hormone therapy on matrix metalloproteinases in breast-cancer cells: an in vitro study.

A combination of E and progestogens significantly increased matrix metalloproteinase (MMP)-2 in both T47D cells (E(2)-medroxyprogesteroneacetate [MPA] and E(2)-P) and MCF-7 cells (E(2)-MPA, E(2)-P, and equilin-MPA). All combinations resulted in higher MMP-9 levels in MCF-7 cells, but higher MMP-9 levels resulted only with equilin-norethinderone in T47D cells.

The estrogen metabolite 17beta-dihydroequilenin counteracts interleukin-1alpha induced expression of inflammatory mediators in human endothelial cells in vitro via NF-kappaB pathway.

In most studies showing cardio- and vasculoprotective effects of estrogens, 17beta-estradiol was used and little information on possible effects of different estrogen metabolites is yet available. We investigated whether particular estrogen metabolites are effective in counteracting inflammatory activation of human endothelium. Human endothelial cells were incubated with 17alpha-dihydroequilenin, 17beta-dihydroequilenin, delta-8,9-dehydroestrone, estrone and 17beta-estradiol and stimulated with interleukin (IL)-1alpha. The expression of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) was determined. 17beta-dihydroequilenin and 17beta-estradiol at a concentration of 1 microM reduced IL-1alpha-induced up regulation of IL-6, IL-8 and MCP-1 close to control levels. When both compounds were used in combination an additive effect was observed. 17alpha-dihydroequilenin and delta-8,9-dehydroestrone showed a similar anti-inflammatory effect only when used at 10 microM whereas estrone had no effect. The effect of 17beta-dihydroequilenin on IL-1alpha-induced production of IL-6, IL-8 and MCP-1 was reversed by the estrogen receptor antagonist ICI 182,780. 17beta-dihydroequilenin also inhibited IL-1alpha-induced translocation of p50 and p65 to the nucleus of the cells. We have identified the estrogen metabolite 17beta-dihydroequilenin, as an inhibitor of inflammatory activation of human endothelial cells. Characterization of specific estrogens--as shown in our study--could provide the basis for tailored therapies, which might be able to achieve vasoprotection without adverse side effects.

B-ring unsaturated estrogens: biological evaluation of 17alpha-Dihydroequilein and novel B-Nor-6-thiaequilenins as tissue selective estrogens.

The pharmacology and SAR of representative equine estogens is described. 17alpha-Dihydroequilenin was found to prevent bone loss after 5 weeks of oral administration to ovariectomized rats. The stereochemical significance of the D-ring and the C/D ring juncture was investigated with a series of benzothiophene-based equilenin analogues.

Comparison of the proliferative effects of estradiol and conjugated equine estrogens on human breast cancer cells and impact of continuous combined progestogen addition.

OBJECTIVES: So far, most epidemiological studies investigating breast cancer risk and hormone replacement therapy have been conducted with conjugated equine estrogens (CEE). Recent trials indicate that the addition of progestogens may increase breast cancer risk. In the present study, we compared the effects of the human estrogen 17beta-estradiol (E(2)) with those of the main equine components of CEE, i.e. equilin (Eq) and 17alpha-dihydroequilin (Dheq) on the proliferation of human breast cancer cells. The proliferative effect of progestogen addition was also investigated. MATERIALS AND METHODS: The well-established human breast cancer cell line MCF-7 was used as an in vitro model. The proliferative effect of E(2), Eq and Dheq was tested in the concentration range 0.01-10 nmol/l. The progestogens progesterone, medroxyprogesterone acetate (MPA) and norethisterone (NET) were continuously combined with 0.1 nmol/l estrogen at concentrations of 0.01 nmol/l, 1 nmol/l, 0.1 mumol/l and 10 mumol/l. Proliferation was measured after 7 days by the adenosine triphosphate (ATP) chemosensitivity test. RESULTS: All three estrogens increased the proliferation of MCF-7 cells by between 40 and 180%. The most proliferatively potent estrogen was E(2), followed by Eq and Dheq, which showed a slightly lower proliferative activity than E(2). The addition of progesterone inhibited E(2)-induced proliferation by about 30%, but only at the high non-physiological concentration of 10 mumol/l. All three progestogens inhibited Eq-induced proliferation, although their effect tended to be low, with values between 5 and 40%. No progestogen reduced Dheq-induced proliferation by more than 20%. In contrast, MPA slightly increased the proliferation rate by about 5% at the high physiological concentration of 0.1 mumol/l when combined with Dheq. The same held true when MPA and NET were added at the high pharmacological concentration of 10 mumol/l, causing increases of about 10%. CONCLUSIONS: Our results indicate that equine estrogens have a proliferative action similar to that of 17beta-estradiol. Continuous addition of progestogens does not result in any major reduction of proliferative potency. Some progestogens may even enhance the estrogen-induced proliferation of pre-existing breast cancer cells, particularly when combined with certain equine estrogens. However, in none of the tested circumstances do progestogens increase the proliferative effect of estradiol, and progesterone has no deleterious effect even at pharmacological levels, in contrast to progestogens.

Equine estrogens differentially prevent neuronal cell death induced by glutamate.

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, 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. 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 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. 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 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. CONCLUSIONS: 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.

Protection against gp120-induced neurotoxicity by an array of estrogenic steroids.

gp120, the coat protein of HIV, can be neurotoxic and is thought to contribute to AIDS-related dementia complex. Such toxicity involves activation of glutamate receptors, mobilization of free cytosolic calcium, and generation of oxygen radicals. We have previously shown that the estrogen 17beta-estradiol, in concentrations of 100 nM or higher, lessens the neurotoxicity of gp120 in hippocampal and cortical cultures, blunts gp120-induced calcium mobilization, and lessens the oxidative consequences. In this study, we examined the protective potential of other estrogens. We found gp120 neurotoxicity in hippocampal cultures to be significantly lessened by estrone, equilin and estriol, although with an order of magnitude less potent than 17beta-estradiol. We also found all four estrogens to blunt gp120-induced calcium mobilization, with estriol being more efficacious than the other three estrogens. These findings give insight both into the mechanisms of estrogenic protection (e.g. receptor-dependent versus independent actions) as well as into the potential therapeutic use of estrogens against AIDS-related dementia complex.

Equine estrogens induce apolipoprotein E and glial fibrillary acidic protein in mixed glial cultures.

Premarin, which contains several equine estrogens, as well as estradiol (E2) as a minor component, is widely used for replacement therapy of estrogen deficits, but little is known of its direct actions on brain cells. In mixed glial cultures, apolipoprotein E (apoE) and glial fibrillary acidic protein (GFAP) are induced by estrogens. GFAP induction showed an inverted-U shape E2 dose response, with a maximum induction at 1 pM, whereas apoE mRNA induction was greatest at 100 pM. GFAP and ApoE mRNAs were induced by equine estrogens in the following order: E2=equilin>estrone>17 alpha-dihydroequilenin. However, the induction of apoE secretion by 17 alpha-dihydroequilenin was as effective as by the other estrogens. The greater response of apoE secretion than GFAP mRNA induction to 17 alpha-dihydroequilenin might be therapeutically important because of the glial scarring during brain lesions, in which GFAP induction has a major role in inhibiting neurite outgrowth, whereas apoE secretion supports neurite outgrowth.

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.

A two-year, double-blind comparison of estrogen-androgen and conjugated estrogens in surgically menopausal women. Effects on bone mineral density, symptoms and lipid profiles.

OBJECTIVE: To compare the effects of two doses of conjugated equine estrogen (CEE) and two of esterified estrogen plus methyltestosterone (E + A) in surgically menopausal women. STUDY DESIGN: A two-year, parallel-group, double-blind study of 311 women who were randomly assigned to one of four regimens: (1) CEE, 0.625 mg/d; (2) CEE, 1.25 mg/d; (3) esterified estrogens, 0.625 mg, + methyltestosterone, 1.25 mg/d; or (4) esterified estrogens, 1.25, + methyltestosterone, 2.5 mg/d. Study parameters were symptoms, lipids, bone mineral density, side effects and safety. RESULTS: All treatments prevented loss of bone in the spine and hip. The higher E + A dose increased spine and hip BMD more than other treatments (P < .002). All treatments improved menopausal symptoms, with non-significantly greater improvements in well-being and sexual interest in the E + A groups. Similar and significant decreases in low-density lipoprotein were observed in all groups, but high-density lipoprotein and triglycerides were increased only in the unopposed estrogen groups (P < .05). Hirsutism was uncommon and similar in all groups at two years. Discontinuation rates and reasons for withdrawal from the study were similar in both groups. No clinically significant side effects or laboratory test abnormalities were seen. CONCLUSION: As compared to estrogen alone, E + A significantly improved BMD and was well tolerated in surgically menopausal women.

Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens.

The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequilin (4-OHEQ)] and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit glutathione S-transferase (GST). Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without the need of oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diadduct. The behavior of 4-OHEQ was found to be more complex than 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene. In contrast, the endogenous catechol estrogens, 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE), were without effect unless tyrosinase was present to convert the catechols to o-quinones. Scavengers of reactive oxygen species and metal chelators had no effect on GST inhibition by catechol estrogens with the exception of the catalase which protected GST activity. Kinetic studies showed that 4-OHEN was a potent irreversible inactivator of GST. Preincubation of the enzyme with 4-OHEN showed a time-dependent increase in inhibitory effect, and gel filtration did not restore GST activity confirming the irreversible nature of the enzyme inactivation. Analysis of the Kitz-Wilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki = 620 microM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 = 7.3 x 10(-)3 s-1). These data suggest that 4-OHEN is an irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. The inhibition mechanism likely involves oxidation of the catechol estrogens to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.

Effects of estrogen on susceptibility to oxidation of low-density and high-density lipoprotein in postmenopausal women.

OBJECTIVE: To investigate the effects of estrogen on the susceptibility to oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in postmenopausal women. METHODS: A total of 23 postmenopausal women were treated with 0.625 mg of conjugated equine estrogen daily for 3 months. Blood samples were obtained before and after therapy. Plasma levels of total cholesterol and triglyceride and the concentrations of cholesterol, triglyceride, phospholipid in LDL and HDL were determined enzymatically and the levels of apolipoprotein A-I, A-II in HDL and apolipoprotein B in LDL were measured by turbidimetric immunoassay. The isolated LDL and HDL were incubated at 37 degrees C for 24 h with CuSO4 5 mumol/l and the lipid peroxide concentration of LDL and HDL was measured. RESULTS: Estrogen significantly reduced the plasma level of total cholesterol and significantly increased the plasma level of triglyceride. The LDL concentrations of cholesterol, phospholipid and apolipoprotein B were significantly decreased following estrogen therapy. The triglyceride level of LDL did not change significantly. The HDL concentrations of cholesterol, triglyceride, phospholipid and apolipoprotein A-I and A-II were all significantly elevated after estrogen therapy. Estrogen significantly inhibited the peroxidation of LDL at 50-2000 micrograms of LDL protein (14.17 +/- 4.17-11.49 +/- 1.42 nmol/200 micrograms of LDL protein, P < 0.001) and of HDL (4.49 +/- 1.74-3.37 +/- 1.24 nmol/200 micrograms of HDL protein, P < 0.03) induced by their incubation in the presence of CuSO4. CONCLUSIONS: Estrogen inhibited the susceptibility of LDL and HDL to oxidative modification and favorably affected lipid metabolism by reducing the number of LDL particles and increasing the number of HDL particles in plasma that were resistant to oxidation.

Equilin, a principal component of the estrogen replacement therapy premarin, increases the growth of cortical neurons via an NMDA receptor-dependent mechanism.

Regulation of both the outgrowth and the survival of neurons involved in cognitive function can have a significant impact on the function of neural networks involved in memory and other cognitive processes. Results of this investigation demonstrated that 17beta-estradiol and the estrogenic steroids estrone, estriol, mestranol, and equilin induced significant increases in cortical nerve cell growth. Of the neurotrophic estrogenic steroids, equilin was most efficacious. We therefore conducted an extensive analysis of equilin-induced neurotrophism. Equilin induced highly significant increases in the growth of both the macro and micro features of cortical nerve cell morphology. The growth-promoting effects of equilin were present in both serum-containing and serum-free media, indicating that the growth-promoting effect of equilin is direct and not dependent upon factors present in serum. Analysis of the regional selectivity of equilin-induced neurotrophism in the cerebral cortex demonstrated that equilin significantly increased the growth of neurons from the frontal, temporal, and occipital regions, with neurons from the parietal region also influenced, though more modestly. We pursued the mechanism of equilin-induced neurotrophism and found that the growth-promoting effects of equilin were completely abolished in the presence of the glutamatergic NMDAreceptor antagonist AP5. Equilin is a major component of Premarin, the leading prescribed pharmaceutical for estrogen replacement therapy for postmenopausal women in the United States. Results of this investigation have the potential of influencing the application and design of therapeutic agents for the prevention of cognitive decline in estrogen deficient women and for the prevention of Alzheimer's disease in postmenopausal women, a group that comprises a large sector of the population, the size of which will continue to grow in the coming decades.

17alpha-Dihydroequilenin increases hippocampal dendritic spine density of ovariectomized rats.

The effects of estradiol and 17alpha-dihydroequilenin on the apical dendrite spine density of pyramidal cells of the CA1 region of rat hippocampus were compared. 17alpha-Dihydroequilenin was as effective as estradiol in increasing spine densities relative to controls. 17alpha-Dihydroequilenin is not uterotrophic like estradiol but does have beneficial effects on the cardiovascular system, suggesting that it may be an effective single-agent hormone replacement therapy to treat menopausal symptoms and reduce chronic disease risk in menopausal women.

Biologic effects of 17 alpha-dihydroequilin sulfate.

OBJECTIVE: To determine the independent biologic effects of 17 alpha-dihydroequilin sulfate. DESIGN: Prospective randomized study. SETTING: University of Southern California Medical Center. PATIENTS(S): Twenty-one postmenopausal women, mean age 50 +/- 2 (+/-SEM) years, and mean body mass index 27 +/- 2. INTERVENTION(S): Women were randomized to receive daily oral doses of either 1.25 mg of estrone sulfate (E1S), 0.2 mg of 17 alpha-dihydroequilin sulfate, or a combination. Three blood and urine samples were obtained before and after 30 and 90 days of treatment. RESULT(S): After 30 and 90 days of treatment, E1S alone increased sex hormone-binding globulin (SHBG) levels significantly, 19.7% +/- 6.0% and 61.3% +/- 13.0%, whereas 17 alpha-dihydroequilin sulfate reduced SHBG levels, 20.8% +/- 68% and 12.4% +/- 7.5%, respectively. Nevertheless, the combination of E1S and 17 alpha-dihydroequilin sulfate significantly increased SHBG levels, 103% +/- 27.9% and 98.2% +/- 19.1%, compared with baseline at 30 and 90 days. Fewer changes were evident with corticosteroid-binding globulin (CBG). After 90 days of treatment, CBG levels significantly increased 30.9% +/- 5.5% with E1S, decreased by 7.2% +/- 5.0% with 17 alpha-dihydroequilin sulfate, and, with the combination, significantly increased by 10.5% +/- 2.4% compared with baseline. Changes in lipids and lipoproteins were more variable. However, high-density-lipoprotein cholesterol increased significantly with E1S at 30 and 90 days compared with baseline, 96.5% +/- 39% and 91.5% +/- 22.6%, and with the combination increased 66.4% +/- 13.3% and 79.2% +/- 24.4%, respectively. Fewer changes were evident with 17 alpha-dihydroequilin sulfate alone, decreasing 4.4% +/- 22% and 2.6% +/- 21.3%. Urinary ratios of bone collagen equivalents-creatinine and calcium-creatinine decreased in all three groups. However, the combination group resulted in a significantly greater percentage decrease in bone collagen equivalents-creatinine than with E1S alone. CONCLUSIONS(S): 17 alpha-Dihydroequilin sulfate could modify some of the first-pass effects of conjugated equine estrogens and act synergistically with other conjugated equine estrogens to reduce bone resorption.

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.