Alan H. Gerulath

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

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

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

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

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

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

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

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

Effect of progesterone and estradiol-17β on nucleic acid synthesis in vitro in carcinoma of the endometrium

Nucleic acid synthesis was studied in 26 tumor specimens, during a one-hour in vitro incubation with the addition of progesterone and estradiol-17beta. On the response scale used, deoxyribonucleic acid and ribonucleic acid synthesis decreased linearly as the logarithmic concentration of either hormone increased. Inhibition of nucleic acid synthesis was highly significant (P less than 0.001) at 80 microgram per milliliter of progesterone and at 40 microgram per milliliter of estradiol. No evidence of synergism was found when the two hormones were combined at these concentrations. In Grade I and II tumors, the effect when both hormones were combined approximated the sum of effects of the individual hormones. In Grade III tumors, little difference was seen between treatment groups.

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

BackgroundAn 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.ResultsAll 11 equine estrogens tested protected the HDL from oxidation in a concentration dependant manner. Equilenin, 17β-dihydroequilenin, and 17α-dihydroequilenin (Δ6–8-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.ConclusionsEquine estrogens can differentially inhibit the oxidation of HDL with the Δ6–8-estrogens being the most potent antioxidants. The ability of estrogens to enhance HDLs 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.

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.

Pharmacokinetics of 17β-Dihydroequilin Sulfate in Normal Postmenopausal Women Under Steady State Conditions

Objective: In the present study, the constant infusion of [3H]17β-dihydroequilin sulfate ([3H]17β-EqS) was used to estimate the metabolic clearance rate (MCR) of 17β-dihydroequilin sulfate (17β-EqS) and to measure the conversion of this estrogen to equilin sulfate (EqS), equilenin sulfate (EqnS), 17β-dihydroequilenin sulfate (17β-Eqn) in normal postmenopausal women. Methods: In seven healthy postmenopausal women, infusion of [3H]17β-EqS was started 30 minutes after a priming dose and continued at a constant rate of 10-20 μCi/hour, for 3-6 hours. Three blood samples were taken during and at the end of infusion. From the plasma, unconjugated and sulfate-conjugated estrogens, 17β-EqS, EqS, EqnS, 17β-EqnS, Eq, Eqn, 17β-Eq, and 17β-Eqn were isolated and purified by high performance liquid chromatography. The MCR of 17β-EqS and the conversion ratios and transfer constants (p) for precursor (17β-EqS) to products were calcualted. Results: The mean MCR of 17β-EqS was calculated to be 797 ± 90 L/day or 506 ± 60 L/m2 per day. The mean conversion ratio (CRPRE-PRO BB) was 2.4 ± 0.4 for EqS, 0.3 ± 0.04 for EqnS, 0.25 ± 0.03 for 17β-EqnS, 0.09 ± 0.02 for Eq, 0.03 ± 0.01 for Eqn, 0.08 ± 0.02 for 17β-Eq, and 0.03 ± 0.01 for 17β-Eqn. In both the sulfate-conjugated and unconjugated forms, the most abundant metabolite formed was Eq. Based on the previously reported MCR of EqS (170 L/M2 per day) and 17β-Eq (1252 L/M2 per day), the transfer constants [ρ]BB were calculated to be 0.8 ± 0.10 and 0.20 ± 0.03, respectively. The results indicate that a large portion of 17β-EqS is converted to EqS and the more potent estrogen 17β-Eq. The ratio of ρEqS-17β-EqS to ρ17β-EqS-EqS was calculated to be 0.8 ± 0.1 and represents the extent of C-17-oxidation and reduction and indicates that substantial amounts of 17β-reduced metabolites will still be presnet in the blood although the oxidation reaction was somewhat greater. Conclusion: The data indicate that, compared with the classic estrogens, the in vivo metabolism of ring B unsaturated estrogens is complex. Thus, although the amount of 17β-EqS originally present in the conjugated equine estrogens is small, the pharmacokinetics and pharmacodynamics of EqS, 17β-EqS, and the extensive interconversions between these estrogens support the hypothesis that the major in vivo activity of the EqS present in conjugated equine estrogens is expresed through its metabolism to 17β-EqS and 17β-Eq. Furthermore, the increased estrogenic activity associated with this drug may in part be due to the formation of these 17β-reduced metabolites.

Vulvodynia and Vestibulitis

Chronic vulvar pain was first described in the late 1800s as hyperesthesia by Skene [1] with further reports by Thomas and Munde [2] and Kelly in the 1920s [3]. In the 1970s, the terms burning vulvar syndrome, psychosomatic vulvovaginitis and vestibular adenitis became popular.

EFFECT OF PROGESTERONE AND ESTRADIOL-17β ON NUCLEIC ACID SYNTHESIS IN VITRO IN CARCINOMA OF THE ENDOMETRIUM

Nucleic acid synthesis was studied in 26 tumor specimens, during a one-hour in vitro incubation with the addition of progesterone and estradiol-17beta. On the response scale used, deoxyribonucleic acid and ribonucleic acid synthesis decreased linearly as the logarithmic concentration of either hormone increased. Inhibition of nucleic acid synthesis was highly significant (P less than 0.001) at 80 microgram per milliliter of progesterone and at 40 microgram per milliliter of estradiol. No evidence of synergism was found when the two hormones were combined at these concentrations. In Grade I and II tumors, the effect when both hormones were combined approximated the sum of effects of the individual hormones. In Grade III tumors, little difference was seen between treatment groups.