Serum copper alteration after ingestion of an oral contraceptive.

Changes in the concentration of copper in the serum after administration of an oral contraceptive were determined with atomic absorption spectrophotometry. Statistically significant (P = .001) increases were observed in all volunteers.

Acute phase protein in serum of women using hormonal contraceptives.

Acute phase protein precipitating sonatic C-polysaccharide of pneumococci appears in serum of women under treatment with hormonal contraceptives in a significantly higher number of cases when compared with control groups. The summarized results of three preliminary studies show that in 80 control serums there were four positive specimens (5 percent) and in 80 serums from women using hormonal contraceptives there were 58 positive specimens (72.5 percent).

Pituitary tumors in mice after prolonged feeding of synthetic progestins.

An enhanced development of pituitary tumors was observed in virgin female mice of the C57 Leaden strain following repeated oral administration of synthetic progestins. This finding appears to parallel the co-carcinogenic enhancement of mammary tumor development elicited in C3H mice treated repeatedly with progesterone.

Effects of the antifertility drug enovid in five strains of mice, with particular regard to carcinogenesis.

PIP: The antifertility drug, Enovid, was tested for possible carcinogenicity in female mice of 5 specially selected strains: BALB/c, C3H, C3HfB, A, and C57BI. Enovid was chosen for testing since it is one of the most widely used oral contraceptives. The 5 strains of mice provided maximum genetic variation in the test animals. The drug was fed at 3 dose levels: 5 mcg/gm, 10 mcg/gm and 20 mcg/gm of food. The lowest dose did not prevent reproduction. The 10 mcg dose prevented some females from reproducing. The 20 mcg dose prevented all females from reproducing. The strains of mice differed in their response to Enovid. Weight gain was reduced in all strains. Effect on life-span varied, partly because of the tumors. Cervical and vaginal lesions showed invasion of the epithelium into the stroma but was limited, with few exceptions to the BALB/c females. In the BALB/c strain these lesions occurred in controls as well, but showed more progression and a higher incidence with the highest dose of Enovid. None of these lesions appeared grossly as tumors and none had extended beyond the vaginal wall or metastasized. They were observed only on histologic sections. Neither ovarian nor mammary gland tumors were increased in any strain. In the C3H strain such tumors seemed to be inhibited. In the C3HfB strain there was some inhibition of hepatomas and in the BALB/c strain some inhibition of adrenocortical adenoma. Chromophobe adenomas of the hypophysis were significantly increased in old C57BI females treated with the highest dose of Enovid. Offspring of Enovid-treated females showed no abnormalities. Enovid increased the occurrence and may have advanced the progression of epithelial lesions of the cervix and vagina of old BALB/c females. A study of the lesions in untreated females of this strain might help the understanding of carcinoma in situ in women and possibly the appearance of adeno-carcinoma of the vagina of young women whose mothers had been treated with stilbestrol during the first trimester to maintain pregnancy. Other neoplasms in this strain were not increased by the Enovid therapy. In the C3H strain mammary tumors were reduced by the Enovid and those that did occur were found later than in controls. Results from experimental animals should be applied to humans with care. Such results are of greatest value in directing attention to certain areas for investigation.^ieng

Prolactin and the development and progression of early neoplastic mammary gland lesions.

Chronic suppression of prolactin by 2-bromo-alpha-ergocryptine (CB-154) in young nulliparous or mature multiparous C3H mice sharply suppressed the development of preneoplastic mammary gland lesions (hyperplastic alveolar nodules) and markedly inhibited the progression of these preneoplasias to carcinomas. This effect was also observed in C3H mice treated with either 17beta-estradiol or the oral contraceptive Enovid. Chronic CB-154 induced prolactin suppression was more effective than ovariectomy in the suppression of hyperplastic alveolar nodule development and comparable to ovariectomy in the suppression of mammary carcinoma development. Evidence is also provided indicating that human placental lactogen, a peptide chemically and physiologically similar to prolactin, promotes growth both in vitro (organ culture) and in vivo (athymic "nude" mouse) of the epithelium contained in benign human breast tumors. Whether or not human pituitary prolactin is capable of mimicking the mammotrophic action of human placental lactogen and whether a dysplastic, prolactin-sensitive lesion comparable to hyperplastic alveolar nodules exists in the human breast remains to be determined.

Estrogenic potential of progestins in oral contraceptives to stimulate human breast cancer cell proliferation.

Most oral contraceptives (OC) contain a progestin in combination with an estrogen, and the progestin component in OC includes one of the following 19-nortestosterone derivatives: norethynodrel; norethindrone; or norgestrel (levonorgestrel). It is well known that estrogens promote the growth of breast cancer. However, progestins have recently also been implicated in the development of breast cancer. We have compared and contrasted the ability of synthetic progestins to stimulate the proliferation of cultured human breast cancer cells and examined their possible mechanism of action. We found that some progestins used in OC were able to stimulate the growth of estrogen receptor-positive (ER+) MCF-7 and T47DA18 human breast cancer cells but not ER- MDA-MB-231, BT-20, and T47DC4 human breast cancer cells. However, two other progestins, MPA and R5020, which are not used in OC, were either not able to stimulate or only slightly stimulated growth. The potency of norethynodrel [median effective dose (EC50) = 4 x 10(-8) M] and norethindrone (EC50 = 3 x 10(-8) M) was greater than norgestrel (EC50 = 2 x 10(-7) M) in MCF-7 cells. E2 (EC50 = 8 x 10(-13) M) was an even more potent stimulator of growth. More importantly, the progestin-induced growth stimulation was blocked by the antiestrogens 4-hydroxytamoxifen and ICI 164,384 but not the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynyl)-estra-4, 9-dien-3-one (RU486). To determine whether the proliferative action of progestins was mediated through the ER, cells were transfected with a chloramphenicol acetyltransferase reporter gene containing an estrogen response element derived from vitellogenin 2A gene. The progestins which stimulated the growth of breast cancer cells also increased chloramphenicol acetyltransferase activity. The induction of chloramphenicol acetyltransferase activity was blocked by the addition of the antiestrogens 4-hydroxytamoxifen and ICI 164,384 but not the antiprogestin RU486. This study provides direct evidence that the 19-nortestosterone derivatives in OC have estrogenic properties and suggests that activation of ER, but not progesterone receptor, is the growth-stimulatory mechanism for these synthetic progestins. Our results may help to explain the conflicting evidence linking OC and breast cancer risk. A rigorous evaluation of the "total" estrogenic potential of OC might produce a better correlation with breast cancer risk.

Progestin regulation of vascular endothelial growth factor in human breast cancer cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the degree of vascularity, progression, and metastasis of breast cancer, and cases of this disease with increased vascular density have a poor prognosis. We show that in T47-D human breast cancer cells, progesterone induces a dose-dependent increase of 3-4-fold in media VEGF levels, with a maximum response occurring at a concentration of 10 nM. This effect is blocked by the antiprogestin RU 486. In addition to progesterone, a number of synthetic progestins used in oral contraceptives (e.g., norethindrone, norgestrel, and norethynodrel), hormone replacement therapy (medroxyprogesterone acetate), and high-dose progestin treatment of breast cancer (megestrol acetate) also increase VEGF in the media of cultured T47-D cells. This effect is hormone specific and is not produced by estrogens, androgens, or glucocorticoids. Collectively, these observations suggest that the increase in VEGF caused by progestins is mediated by progesterone receptors present in T47-D cells. The induction of VEGF by progestins is also cell type specific and does not occur in human breast cancer cell lines MCF-7, ZR-75, or MDA-MB-231, nor in Ishikawa cells derived from a human endometrial carcinoma. This is the first report that progestins regulate VEGF expression in human breast cancer cells and raises the possibility that increased angiogenesis in response to endogenous progesterone or its therapeutically used analogues may play a role in cell growth or metastasis in a subset of human breast tumors.

Effect of tumor promoting contraceptive steroids on growth and drug metabolizing enzymes in rat liver.

Liver tumor formation in rats treated with oral contraceptive steroids for long periods has been associated with the tumor promoting potential of these agents. As other known liver tumor promoters, e.g., phenobarbital and hexachlorocyclohexane, induce liver growth and hepatic monooxygenases, we investigated whether or not estrogens have similar effects. Female rats were treated with a wide range of doses of ethinylestradiol, including human contraceptive doses, which are approximately 1 microgram/kg. The physiological estrogen estradiol was studied for comparison. Also included were norethynodrel and norethisteron and its acetate and enanthate because these human progestins act predominantly estrogenic in rats. Daily s.c. doses of ethinylestradiol (0.5 mg/kg) produced a rapid increase of liver mass, DNA, RNA, and protein which was almost maximal after 7 days. The percentages of parenchymal cells involved in DNA synthesis and mitosis were enhanced up to 20-fold, suggesting parenchymal hyperplasia as the main cause of liver growth. Sinus wall cells showed a proportionate increase of number and DNA synthesis. Likewise, all other steroids tested produced significant increases of liver mass and DNA. For ethinylestradiol and estradiol extrapolated threshold doses were in the range of 1 microgram/kg. These doses are below those used in previous tumor promotion studies in rats. Using 5 different substrates to check monooxygenase activities of isolated liver microsomes, no induction or only very weak induction by estrogens was found. These studies suggest that induction of liver growth may be a property relevant for the tumor promoting activity of estrogens; in contrast, induction of hepatic monooxygenases does not appear to be necessary for liver tumor promotion in the rat.

The estrogenic activity of synthetic progestins used in oral contraceptives enhances fatty acid synthase-dependent breast cancer cell proliferation and survival.

Overexpression of the lipogenic enzyme fatty acid synthase (FAS) is a common molecular feature in subsets of sex-steroid-related tumors including breast carcinomas that is associated with poor prognosis. In this study, we explored whether breast-cancer associated FAS (oncogenic antigen-519) is regulated by the progestin component in oral contraceptives. In addition, we examined the role of FAS hyperactivity on progestin-regulated breast cancer cell proliferation, survival and metastatic properties. We found that in estrogen receptor (ER)- and progesterone receptor (PR)-positive MCF-7 human breast cancer cells, synthetic progestins used in oral contraceptives including norethynodrel (NOR) and norethindrone, induced a dose-dependent increase of FAS enzymatic activity, with a maximum response (> or = 4-fold) occurring at a concentration of 10(-8) M. FAS activity was only slightly stimulated after exposure to two other progestins, medroxy-progesterone acetate (MPA) and megestrol acetate (MGA), which are used in postmenopausal hormone replacement therapy and high-dose progestin treatment therapy. Western blot analyses showed that NOR-induced stimulation of FAS activity correlated closely with NOR-induced up-regulation of FAS protein expression. To determine the role of FAS accumulation following NOR exposure, we pharmacologically examined the requirement for FAS activity in NOR-stimulated breast cancer cell proliferation and survival. The novel small compound C75 (a slow-binding FAS inhibitor) blocked NOR-induced breast cancer cell proliferation in anchorage-dependent assays. More importantly, pharmacological inhibition of FAS activity completely abolished NOR-stimulated soft-agar colony formation of MCF-7 cells. To evaluate the involvement of PR and ER signalings in NOR-induced up-regulation of FAS expression and activity, NOR was used in combination with either the anti-progestin RU486 (mifepristone) or the pure antiestrogen ICI 182,780 (Faslodex). RU486 and ICI 182,780 similarly abolished NOR-induced FAS activation, supporting the notion that PR- and ER-mediated FAS up-regulation might play different roles in NOR-stimulated breast cancer cells. Interestingly, when we evaluated the involvement of PR and ER signalings on NOR-induced breast cancer cell proliferation, the anti-estrogen ICI 182,780, but not the anti-progestin RU486, was found to inhibit NOR-stimulated proliferation and survival of MCF-7 cells in anchorage-dependent and -independent assays. To further determine whether NOR produced their effects via the ER, we evaluated its effects on endogenous ER transcriptional activity by using transient transfection assays with an estrogen-response element reporter construct (ERE-Luciferase). In the absence of E2 stimulation, treatment with NOR dramatically increased the levels of ERE-dependent transcriptional activity. This estrogenic like-effect of NOR was blocked by the addition of ICI 182,780, whereas RU486 failed to inhibit NOR-induced ERE activity. In summary, this study provides direct evidence that: a) a number of synthetic progestins used in oral contraceptives significantly activates breast cancer-associated FAS (OA-519) activity and expression in hormone-dependent breast cancer cells; b) FAS activity is necessary for progestin-induced anchorage-independent growth and survival of human breast cancer cells, and c) activation of ER, but not PR signaling, is the stimulatory mechanism through which synthetic progestins enhance a FAS-dependent proliferative and pro-survival signaling. These findings should be helpful to explain the conflicting evidence linking oral contraceptives and breast cancer risk through the estrogenic activation of tumor-associated FAS (OA-519), a molecular marker associated with poor clinical outcome of breast cancer disease.

Differential effects of estrogens and progestins on the anticoagulant tissue factor pathway inhibitor in the rat.

Oral contraceptives (OC) and postmenopausal hormone therapy (HT) modulate plasma levels of proteins that regulate blood coagulation. It remains unclear whether the progestin component contributes to these changes. The present study was designed to determine whether progestins modulate two essential plasma anticoagulants, antithrombin (AT) and tissue factor pathway inhibitor (TFPI), in an animal model. Ovariectomized rats were treated orally with three progestins, norethindrone acetate (NETA), trimegestone (TMG), or drospirenone (DSP), either alone or combined with 17alpha-ethyinylestradiol (EE). Plasma AT levels were unchanged. However, TFPI activity was reduced by EE alone (10-100 microg/kg/day) in a dose-dependent manner; NETA (3 or 10 mg/kg/day) reduced TFPI by approximately 40 or approximately 80%, respectively, while TMG and DSP had no effect. NETA and EE effects were blocked by co-administration of ICI-182,780, an estrogen receptor antagonist, suggesting that both responses were likely estrogen receptor-mediated. Reduced TFPI after NETA or EE treatment was not accompanied by changes in TFPI mRNA levels in tissues that express TFPI, but there was a positive correlation between plasma TFPI and total cholesterol. Sex hormone effects on TFPI in this model and as reported in women may help to shift the coagulation balance to a more prothrombotic state. Progestins such as TMG and DSP that lack estrogenic activity could potentially have an improved clinical profile.

p-Toluenesulfonyl isocyanate as a novel derivatization reagent to enhance the electrospray ionization and its application in the determination of two stereo isomers of 3-hydroxyl-7-methyl-norethynodrel in plasma.

In this paper, p-toluenesulfonyl isocyanate has been reported as a novel derivatization reagent with strong nuclephilic reactivity for the hydroxyl compounds. The derivatization for the two pharmacologically active 3-hydroxyl metabolites, 3alpha-hydroxyl-7-methyl-norethynodrel and 3beta-hydroxyl-7-methyl-norethynodrel by p-toluenesulfonyl isocyanate can be accomplished in 2 min under room temperature. The offline derivatization procedure introduced an easily ionizable sulfonylcarbamic ester moiety to the metabolites. This greatly improved the analyte's sensitivity in negative electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 100 pg/ml in plasma. Therefore, a sensitive high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of the two stereo isomers was developed. The method had been validated to be accurate, precise, and sensitive, and can be used for the metabolism pharmacokinetic study of tibolone in human subjects.

Effect of calcium channel modulators on temperature regulation in ovariectomized rats.

Clinical studies evaluating a calcium channel modulator, gabapentin, for the treatment of vasomotor symptoms have been reported. The present studies evaluated three calcium channel modulators in ovariectomized (OVX) rodent models of temperature regulation. Gabapentin, reported to interact with the alpha(2)delta subunit of voltage-sensitive calcium channels and the L-type voltage-gated calcium channel blockers, verapamil and nifedipine, were examined. These series of experiments demonstrated that orally administered gabapentin, verapamil and nifedipine all acutely and dose-dependently lower tail skin temperature in both models of OVX-induced thermoregulatory dysfunction. These compounds all had a rapid onset of action, however, the efficacy of all three calcium channel modulators is less than that observed following chronic estrogen treatment. Additionally, these compounds were also tested in a telemetric rat model measuring core body temperature to evaluate any temperature effects on internal core temperature. The present data suggests that gabapentin, verapamil and nifedipine all act to globally alter temperature regulation in steroid-dependent models of thermoregulatory function.

Bioaccumulation of 14C-17alpha-ethinylestradiol by the aquatic oligochaete Lumbriculus variegatus in spiked artificial sediment.

A bioaccumulation study was performed with the endobenthic freshwater oligochaete Lumbriculus variegatus MULLER exposed to the radiolabelled synthetic steroid 17alpha-ethinylestradiol (14C-EE2) in a spiked artificial sediment. Concentration of total radioactivity increased constantly and almost linearly during 35 days of exposure. The accumulation factor normalised to worm lipid content and sediment TOC (AFlipid/OC) was 75 at the end of the uptake period, but a steady state was not reached. Uptake kinetics were calculated fitting the measured AFs to a kinetic rate equation for constant uptake from sediment using iterative non-linear regression analysis. After 10 days of elimination in contaminant-free sediment 50% of the accumulated total radioactivity was excreted by the worms. Extracts from L. variegatus sampled at the end of the uptake phase were analysed by thin layer chromatography (TLC). The results showed that 6% of the total radioactivity incorporated by the worms was 14C-EE2. After treatment of extracts with beta-glucuronidase the amount of 14C-EE2 increased to 84%. These results suggest that L. variegatus has the potency to accumulate high amounts of conjugated EE2. Hence, a transfer of EE2 to benthivores and subsequent secondary poisoning of predators might be possible.

An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio.

The aim of the present study was to elucidate how full life-cycle exposure to estrogens impacts zebrafish development and reproduction, compared to partial life-cycle exposure only, and whether the estrogen-induced effects in zebrafish are reversible or irreversible. Zebrafish were exposed in a flow-through system to an environmentally relevant concentration (3 ng/L) of the synthetic estrogen 17alpha-ethinylestradiol (EE2) either from fertilization until the all-ovary stage of gonad development (i.e., 42 d postfertilization [DPF] in our experiment) or from fertilization until the reproductive stage (i.e., 118 DPF). Reversibility of the estrogen-induced effects was assessed after 58 d of depuration in EE2-free water until 176 DPE Early life exposure led to a lasting induction of plasma vitellogenin (VTG) in adult females but altered neither the sex ratio nor the reproductive capabilities. Full life-cycle exposure resulted in elevated VTG concentrations and caused gonadal feminization in 100% of exposed fish and thus inhibited reproduction. Two types of ovaries were observed in continuously exposed adult fish, immature ovaries with primary growth stage oocytes only and mature ovaries containing the full range of all oocyte maturation stages. Fish with immature ovaries had plasma VTG levels like control males, while fish with mature ovaries had female-like VTG levels. The effects of full life cycle exposure were at least partly reversible, and 26% of fish of the previous all-female cohort developed fully differentiated testes. These findings suggest that continuous estrogen exposure had arrested the developmental transition of the gonads of genetic males from the early all-ovary stage to functional testes. After the exposure had ceased, however, these males apparently were able to accomplish testicular differentiation.

Potentiation of the vitellogenic response to 17alpha-ethinylestradiol by cortisol in the fathead minnow Pimephales promelas.

The effects of elevated plasma cortisol levels on vitellogenin (VTG) induction were examined in the fathead minnow (Pimephales promelas) in an attempt to evaluate the potential influence of stress on this commonly used biomarker of estrogenicity. Two separate experiments were conducted in which fish plasma cortisol was elevated to various levels for 14 d by noninvasive additions of cortisol to the aquaria water. Fathead minnows were exposed to either cortisol alone, 17alpha-ethinylestradiol (EE2) alone, or a combination of the two hormones, and plasma levels of VTG as well as liver expression of VTG mRNA were measured. Both experiments gave similar results, with an exposure to 4 ng/L of EE2 resulting in significantly greater levels of plasma VTG in the presence of, compared to that in absence of, cortisol, whereas exposure to cortisol alone at concentrations between 144 and 800 microg/L had no effect on plasma VTG levels. This potentiation of the EE2-induced vitellogenesis by cortisol was dose-dependent, with plasma VTG reaching 125, 167, and 295% of the values obtained with EE2 alone when 144, 360, and 800 microg/L of cortisol, respectively, were added to the water. Liver mRNA results were consistent with plasma VTG, although they generally were more variable. The present study demonstrates that cortisol does not independently induce vitellogenesis but can potentiate estrogen-induced VTG synthesis in fathead minnow. The implications of these findings for the use of VTG as a biomarker of estrogenicity are discussed.

Relationship between ethinylestradiol-mediated changes in endocrine function and reproductive impairment in Japanese medaka (Oryzias latipes).

Many biochemical endpoints currently are used to describe endocrine function in fish; however, the sensitivity of these parameters as biomarkers of impaired reproduction or sexual development is not well understood. In the present study, adult Japanese medaka (Oryzias latipes) were assessed for reproductive output and endocrine function, including circulating steroid concentrations, ex vivo steroidogenesis from the gonads, aromatase activity, hepatic estrogen receptor (ER), and plasma vitellogenin (VTG) after exposure to 0, 0.2, 5, 500, and 2,000 ng/L of 17alpha-ethinylestradiol (EE) for 14 d. The EE altered these biochemical responses at various sites along the hypothalamus-pituitary-gonadal axis at concentrations as low as 0.2 ng/L, but it only depressed reproductive function at concentrations of 500 ng/L or greater. Offspring also had reduced ability to hatch at 500 ng/L of EE, but this concentration did not produce any other observed changes in development or sexual phenotype. The reproductive parameters correlated well with VTG, ER, and gonadosomatic index (GSI) in both sexes of adult medaka, which could be indicative of the ER-mediated mode of action for EE. Vitellogenin and ER were elevated at higher concentrations of EE in both sexes, whereas GSI was decreased. Overall, most biochemical endpoints were more sensitive than reproduction or development to exposure, indicating that reproductive function may be relatively protected.

Evaluation of genotoxic potential of norethynodrel in human lymphocytes in vitro.

The genotoxicity study of a synthetic progestin norethynodrel, was carried out on human lymphocytes chromosomes using sister chromatid exchanges (SCEs), replication index (RI) and chromosomal aberrations (CAs) as parameters. The study was carried out in the presence and absence of metabolic activation (S9 mix). Norethynodrel was studied at three different concentrations (20, 40 and 60 microg/ml of peripheral blood lymphocyte culture) and was found non-genotoxic in the absence of metabolic activation. But in the presence of S9 mix norethynodrel increased SCE (p<0.03) and CA (p<0.005) frequencies and inhibits lymphocyte proliferation (p<0.03) at 60 microg/ml. The results suggest a genotoxic and cytotoxic effect of norethynodrel in human lymphocytes in vitro in the presence of S9 mix.

Developmental effects of prenatal exposure to bisphenol a on the uterus of rat offspring.

Exposure to estrogenic compounds during critical periods of fetal development could result in adverse effects on the development of reproductive organs that are not apparent until later in life. Bisphenol A (BPA), which is employed in the manufacture of a wide range of consumer products, is a prime candidate for endocrine disruption. We examined BPA to address the question of whether in utero exposure affects the uterus of the offspring and studied the expression and distribution of the estrogen receptors alpha (ERalpha) and beta (ERbeta), because estrogens influence the development, growth, and function of the uterus through both receptors. Gravid Sprague-Dawley dams were administered by gavage either 0.1 or 50 mg/kg per day BPA or 0.2 mg/kg per day 17alpha-ethinyl estradiol (EE2) as reference dose on gestation days 6 through 21. Female offspring were killed in estrus. Uterine morphologic changes as well as ERalpha and ERbeta distribution and expression were measured by immunohistochemistry and Western blot analysis. Striking morphologic changes were observed in the uterine epithelium of postpubertal offspring during estrus of the in utero BPA-treated animals (the thickness of the total epithelium was significantly reduced). ERalpha expression was increased in the 50-mg BPA and EE2-treated group. In contrast, we observed significantly decreased ERbeta expression in all BPA- and EE2-treated animals when compared with the control. In summary, these results clearly indicate that in utero exposure of rats to BPA promotes uterine disruption in offspring. We hypothesize that the uterine disruption could possibly be provoked by a dysregulation of ERalpha and ERbeta.

Exaggeration of experimental hypertension in rats by contraceptive steroids (Enovid).

The possibly additive pressor effects of contraceptive steroids were studied by treating hypertensive rats chronically with Enovid. Renal hypertensive rats were unaffected by drug treatment during the first 5 weeks, but, from the 6th to the 8th week, Enovid-treated rats had much higher systolic pressures than those given corn oil alone. However, these differences dwindled and became insignificant from the 9th to the 16th week despite continued treatment. Subsequently, other rats were pretreated with Enovid or corn oil for 5 weeks before hypertension was induced by implanting deoxycorticosterone acetate (Doca). In contrast to the equivocal results obtained previously, Doca hypertension was consistently more pronounced in rats treated with Enovid than in those given corn oil. This pressure difference was later verified by direct measurement of phasic aortic pressures from indwelling catheters and by the postmortem finding that Enovid-treated rats had larger hearts than corn oil-treated ones. The exact mechanism by which Enovid enhances Doca hypertension is still undetermined, but sympathetic hyperactivity was considered an unlikely explanation, since responses to posterior hypothalamic stimulation, norepinephrine, or ganglion blockade with pentolinium were unaltered.

Response of heart rate to acute administration of isoproterenol in rats treated chronically with norethynodrel, ethinyl estradiol, and both combined.

Chronic (17 to 20 weeks) administration of ethinyl estradiol alone (36 mug/kg/day), and in combination (27 or 50 mug/kg/day) with several doses of norethynodrel (135, 165 or 233 mug/kg/day), attenuated the increase in heart rate accompanying acute SC administration of the beta-adrenergic agonist, l-isoproterenol (50 or 100 mug/kg), to female rats. Dietary administration of an oral contraceptive containing mestranol and norethynodrel (7.5 mg/kg/food) was also accompanied by an attenuated response to isoproterenol. A significant inverse linear relationship was observed between the logarithm of the dose of estrogen received by each group and either heart rate or change in heart rate measured at 10, 20 and 30 min after administration of isoproterenol. Thus, the antagonistic relationship between the dose of estrogen administered chronically and responsiveness of heart rate to a test dose of isoproterenol suggests a reduced beta-adrenergic responsiveness in estrogen-treated rats.