John A. Holt

Estrogen and progesterone withdrawal increases cerebral vasoreactivity to serotonin in rabbit basilar artery

An in vitro animal model which examines the effects of sex hormone variations during the menstrual cycle on basilar artery reactivity is presented. Three groups of rabbits were utilized: a chronically depleted control group which received no further hormonal treatment after bilateral surgical oophorectomy (O), simulating menopause, and two groups of intact females, one of which was treated to mimic the estrogen and progesterone surge during the luteal phase (H) and the third group which was acutely estrogen and progesterone depleted after the luteal surges to simulate the immediate premenstrual state (W). We show that both acute and chronic estrogen and progesterone withdrawal significantly increase serotonin sensitivity (ED50) in basilar artery rings. There was no difference between groups for maximum contraction (Tmax) to serotonin, nor optimal resting tension. Furthermore, there was no difference in vasoreactivity and contractility to norepinephrine between groups. In order to distinguish between the effects of chronic and acute treatment we examined acute estrogen and progesterone superfusion in basilar artery rings from intact non-treated female rabbits. Acute superfusion of pre-contracted and non-pre-contracted artery segments resulted in significant dilatation only when supraphysiologic concentrations of estrogen and progesterone were used. We conclude that both acute and chronic female sex hormone withdrawal selectively increases cerebral vasoreactivity to serotonin.

Estrogen and progestin binding and changes in secretions by human cervical tissue during the ovarian cycle.

Functional events in the cervix were correlated with sex steroid receptor activity and serum concentrations of sex steroids by measuring cytoplasmic estrogen and progestin binding capacities in cervices collected at hysterectomies performed during the proliferative, midcycle, and secretory phases. Ovarian endocrine status was determined from morphologic criteria for the endometrium as well as from serum concentrations of estrogens and progesterone. Also measured as indices of ovarian endocrine status and cervical function were concentrations of lysozyme, albumin and immunoglobulin G (IgG) in cervical mucus. Early to midfollicur phase specimens had progestin binding in cervical cytosol of 2.3+ or -.6 fmol, and in luteal phase specimens this binding was less than .3 fmol. There were no differences in cytoplasmic estrogen-binding capacities among the 3 phases. Serum estradiol levels were higher (P .05) for luteal phase specimens than for early to midfollicular phase specimens, and the IG, albumin, and lysozyme concentrations of cervical mucus were similar for luteal and early to midfollicular phase specimens, surprisingly. These finding support an hypothesis that progestins reduce cellular responsiveness to the estrogen-receptor complex.

Specific antibodies and immunoglobulins in the oviductal fluid of the rhesus monkey.

Levels of specific antibodies against model antigens, immunoglobulins G and A and also albumin, in oviductal fluid were studied in the rhesus monkey during the periovulatory period. Animals were systemically or intravaginally immunized against T4 coliphages. Attempts to induce ovulation were made with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) monitored by radioimmunoassay of serum estrogen and progesterone. Collection of tubal fluid over 6 to 13 days was accomplished by surgical cannulation using a refrigerated extracorporeal collection device for each side. The results indicated the following: (1) The levels of specific antibodies against T4 coliphage and immunoglobulins (IgG, IgA) in the oviduct fluid averaged approximately one tenth of the serum values and showed a characteristic decrease and subsequent increase by a factor of 4 to 5 during and following treatment with hMG/hCG. The nadir was observed on the first or second day after hCG injection. (2) This pattern was similar in both ovulatory or nonovulatory cycles; therefore, these changes seem to be associated with the changes in serum estrogen levels. (3) There was a striking difference in serum and tubal fluid antibody levels after systemic versus after vaginal immunization by a factor of 10(3) and 10(4); however, the patterns in tubal fluid under treatment with hMG/hCG were very similar. (4) Specific antibodies in oviductal fluid and serum were mainly of the IgG class. (5) A concomitant change of total protein and albumin in oviduct fluid was also observed. The presence of sperm agglutination antibody in oviductal fluid was demonstrated in two monkeys after systemic immunization with homologous spermatozoa. The sperm antibody titers showed a similar pattern of change after hMG/hCG treatment.

Breaks in DNA accompany estrogen-receptor-mediated cytotoxicity from 16α[125I]iodo-17β-estradiol

Strategies for diagnosis and therapy in which sex steroid receptor ligands serve as carriers for radionuclides are attractive because a high incidence of carcinomas of the female genital tract and the breast that are seen clinically have an abundant expression of one or more of the receptor proteins. A radiohalogenated estrogen receptor (ER) ligand, 16α-[123I]iodo-17β-estradiol [123I]E, has met clinical criteria for receptor-mediated diagnostic imaging. Its [125]I-labeled sister nuclide derivative [125I]E decays by orbital electron capture with emission of very-low-energy (Auger) electrons, which gives this latter radiohalogen the potential to serve in pharmaceuticals for radiotherapy; as examples, [125I]deoxyuridine, when incorporated into the DNA molecule, or [125I]E, when bound to the receptor within ER-rich tumor cells, are both cytotoxic in vitro. Whereas the mechanisms and subcellular changes that accompany the cytotoxicity from [125I]deoxyuridine are well documented in the form of aberrations and breaks in the cellular DNA, the effects at the subcellular level causing the cytotoxicity of the sex steroid receptor ligand [125I]E have not been characterized and are the focus of our study. We found that in a standard colonyforming assay the addition of [125I]E to the cultures decreased the survival rate of ER-positive MCF-7 cells in a dose-dependent manner. The decreased survival rate was prevented by the addition of competing excess radioinert ER ligand (diethylstilbestrol); [125I]E did not reduce survival in ER-negative MCF-7 cells. The [125I]E-induced and ER-mediated cytotoxicity was accompanied by aberrations in the DNA components of the nuclei of the cells. These included chromatid and chromosome breaks, gaps, and tri-radial chromosome formation. Our findings add plausibility and credence to the notion that the cytotoxicity imparted by Auger-electron-emitting radioligands for sex steroid receptors is in part attributable to radiodecay that causes double-stranded breakage of DNA.

Estradiol-induced changes in rabbit luteal cell progestin production and cholesterol and cholesterol ester content

Estradiol deprivation in vivo causes accumulation of cholesterol and cholesterol ester in the corpora lutea of pseudo-pregnant rabbits within 24 hours. These accumulations occur concomitantly with an abrupt cessation of progestin production. Retreatment with estradiol restores progestin production and can block further increases in cholesterol accumulation (per mg total tissue protein). Ample quantities of precursor cholesterol ester and cholesterol substrate are present in corpora lutea when progestin falls because of estradiol deprivation. We conclude therefore that the gonadotropic action of estradiol is, in part, because of its stimulation of cholesterol conversion to progestins.

Serum CA 125 and survival of mice inoculated with ovarian carcinoma and treated with antiestrogen, estrogen, or progestin

The human ovarian carcinoma cell line NIH-OVCAR-3 grown in immunodeficient mice has been reported to be sensitive to estrogen medications and to express progestin receptor. To assess the effects of sex steroids on CA 125 production and survival times in these mice, we administered Tamoxifen, estrogen, and progestin. During the first 28 days after inoculation of mice with 2.3 million tumor cells ip, serum CA 125 rose exponentially, reaching 4308 +/- 776 and 3905 +/- 1013 units/ml (mean +/- SEM, P greater than 0.1) in placebo- and Tamoxifen-treated mice, respectively; median survival times were 41 and 39 days, respectively (P greater than 0.1). Uninoculated mice had nondetectable CA 125, and all outlived the inoculated mice. In tumor-inoculated mice, serum CA 125 levels and survival were similar when estrogen or progestin was injected alone and when both were given in combination. We detected no significant differences in production of CA 125 in vitro by tumor cells harvested from ascites fluid when the mice were treated with placebo, estrogen, or progestin. We conclude that, for our model, serial measurements of serum CA 125 provide excellent estimates of the relationship between tumor burden and survival, and that CA 125 production appears unaffected by estrogen, progestin, or Tamoxifen.

Sulfhydryl sensitivity and [125I]-16α-iodo-17β-estradiol binding of estrogen receptor in ovarian epithelial carcinomas

Abstract We find that estrophilin from either the cytosolic or nuclear fractions of ovarian epithelial carcinomas (OVCA) binds irreversibly to controlled-pore glass beads (CPG). The CPG-adsorbed estrophilin releases [3H]-estradiol at 4°C in the presence of 1.25 mM AgNO3; estradiol binding capacity from the nuclear fraction is restored by 10 mM dithiothreitol. The number of available estradiol binding sites in cytosol that are sensitive to AgNO3 correlate with (a) the number of estradiol-inhibitable binding sites found in the 8S region in low-salt sucrose gradients (r = 0.9) but not with (b) estradiol-inhibitable binding in the 4S region (r = 0.3) and thus, as expected, only poorly with (c) estimates of available cytoplasmic estrogen binding sites using a standard dextran-coated charcoal analysis (r = 0.7). Total estrogen binding extracted from the nuclear fraction with 0.5 M KCl and adsorbed to hydroxylapatite agreed with the sulfhydryl blocking reagent sensitive moiety (r = 0.9). Estrophilin from OVCA cytosol or nuclear fractions bound [125I]-16α-iodo-estradiol indistinguishably from [3H]-estradiol. The two forms of radiolabelled-estradiol yielded equivalent data that demonstrated a shift in the estrogen binding moiety from the 8S region to the 4–5S region when 0.5 M KCl was added to gradient analyses of OVCA cytosols. From these observations we conclude that the estrophilin found in OVCA is similar to that found in normal and cancerous tissues of other female reproductive organs and that this estrophilin can bind a biologically active radiohalogenated estrogen potentially useful for imaging or treating these tumors.

Biodistribution, with high uptake by the reproductive tract, of an intraperitoneally infused radiohalogenated steroidal estrogen-receptor ligand☆

We infused [123I]16 alpha-(123I)-iodo-estradiol ([123I]E2) intraperitoneally (i.p.) into swine to study its biodistribution and to explore the i.p. use of radiohalogenated steroid estrogen-receptor (ER) ligands as a potential option for diagnosing and treating intra-abdominal, retroperitoneal, and distant sites of advanced ER-rich malignancies. Fifty to 80% of the radiolabel was absorbed from the peritoneal cavity within 30 minutes, and 30 to 50% of the infused radiolabel was excreted in the urine within 2 hr. The rate of biliary clearance was maximal within 25 minutes. At 3 hr, the ER-rich reproductive tract had greater than 63 times the concentration of radiolabel in blood; the former was blocked by non-labeled competitors for ER. Uptake by non-ER-rich tissues, compared to blood, ranged from 0.7:1 (heart and lungs) to 16:1 (spleen); the omentum, however, exhibited a concentration as high as 64:1, which was not blocked by non-labeled ER ligands. Uptake by ER-rich target tissue remained high when charcoal was used to prevent reabsorption of radiolabel from the digestive tract after its biliary excretion, and when the products of biliary excretion were removed by catheterization of the common bile duct. Neither charcoal nor exteriorization of bile appeared to affect urinary clearance of the radiolabel over the time course of the experiments. Taken together with the recent development of syntheses that yield radiohalogenated sex steroid receptor ligands of high specific activity, our findings are encouraging for the potential application of radiolabeled ligands as i.p. administered pharmaceuticals. The advantage of the i.p. route is that it provides direct uptake of the pharmaceutical by free-floating clusters and individual cancer cells in ascitic fluid, as well as delivery via the circulation to vascularized intra- and/or extraperitoneal metastases.

RAPID LIVER METABOLISM, URINARY AND BILIARY EXCRETION, AND ENTEROHEPATIC CIRCULATION OF 16ALPHA-RADIOIODO-17BETA-ESTRADIOL

The radiohalogenated estrogen 16 alpha-[123I]iodo-17 beta-estradiol ([123I]E2) is emerging as a diagnostic tool for imaging of ER-rich malignant tumors, with potential application for site-directed radiotherapy. Clinical use requires an accurate accounting for the biodistribution of the radioactivity, including an assessment of its enterohepatic circulation. We investigated the metabolism and circulation of [125I]E2 in the enterohepatic system in swine, a pharmacokinetic model that resembles humans. With indicator dilution methods, we found that, after its injection into the portal vein, more than 99% of [125I]E2 was cleared from the blood by the liver during the first pass. Water-soluble metabolites were then partly released into the blood and partly excreted into bile. After injection of [125I]E2 into the external jugular vein, one-third of the radioactivity was excreted in bile and two-thirds in the urine. More than 90% of the radioactivity in urine and bile was that of [125I]E2-glucuronide or [125I]E2-sulfate; only a very small fraction of the excreted radioactivity was from free 125I. Radioactivity in bile collected from one swine after i.v. injection of [125I]E2, and then infused into the proximal duodenum of a second swine, was almost totally absorbed during passage through the intestine at 5-7 hr after infusion. The reabsorbed radioactivity was cleared in the urine.

Prolonged clearance of intraperitoneal 16α-[125I]iodo-17β-estradiol in presence of ascites

Abstract Radioestrogens have potential as adjunct therapeutic agents against ovarian carcinomas, because selected radionuclides can deposit lethal doses of radiation to tumor cells and many ovarian carcinomas and their metastases express estrogen receptors. Because intraperitoneal administration is a possible approach, we investigated absorption from the peritoneal cavity of a radioiodoestradiol after intraperitoneal application in rats with and without ovarian tumors and ascites and compared the distribution of the radioactivity with that obtained after intravenous injection. In the absence of ascites, 70% of the intraperitoneal dose was cleared into the intestine within 2 hours after injection, indicating fast absorption from the peritoneal cavity. In the presence of ascites, clearance of intraperitoneal radioiodoestradiol was considerably slower; at 2 hours after injection, 50% of the injected dose remained in the ascites, mostly as radioiodoestradiol. Uptake of radioactivity in estrogen receptor-rich tissues, e.g., uterus, after intraperitoneal injection was high (about 20: 1 over blood), regardless of the presence of ascites, but moderately lower than that observed after intravenous injection of radioiodoestradiol. (AM J OBSTET GYNECOL 1991;165:1847-53.)