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Context: Previous studies in postmenopausal women have demonstrated that, after oral administration of norethisterone, a small proportion of the compound is rapidly converted into ethinylestradiol. The shape of the concentration - time curve suggested that this occurred in the liver. The results were confirmed by in vitro investigations with adult human liver tissue. In 2002, it was shown that, after oral treatment of women with tibolone, aromatization of the compound occurred, resulting in the formation of a potent estrogen, 7 alpha-methyl-ethinylestradiol. The result has been called into question, because the adult human liver does not express cytochrome P450 aromatase, which is encoded by the CYP 19 gene. Moreover, it has been claimed that the serum level of 7 alpha-methyl-ethinylestradiol measured by gas chromatography/mass spectrometry was an artifact., Reply: Aromatization of steroids is a complex process of consecutive oxidation reactions which are catalyzed by cytochrome P450 enzymes. The conversion of the natural C19 steroids, testosterone and androstenedione, into estradiol-17beta and estrone is dependent on the oxidative elimination of the angular C19-methyl group. This complex key reaction is catalyzed by the cytochrome P450 aromatase, which is expressed in many tissues of the adult human (e.g. ovary, fat tissue), but not in the liver. However, 19-nortestosterone derivatives are characterized by the lack of the C19-methyl group. Therefore, for the aromatization of these synthetic steroids, the action of the cytochrome P450 aromatase is not necessary and the oxidative introduction of double bonds into the A-ring can be catalyzed by other hepatic cytochrome P450 enzymes. The final key process in the formation of a phenolic A-ring, both in natural androgens and 19-nortestosterone derivatives, is the enolization of a 3-keto group to the C2-C3-enol or the C3-C4-enol moiety, which occurs without the action of enzymes., Conclusion: 19-nortestosterone derivatives (norethisterone, norethynodrel, tibolone) can readily be aromatized in the adult human liver. This leads to the formation of the potent estrogens ethinylestradiol from norethisterone or norethynodrel and 7 alpha-methyl-ethinylestradiol from tibolone. This may have clinical consequences, e.g. the elevated risk of venous thromboembolic disease in premenopausal women treated with high doses of norethisterone for bleeding disorders, or the elevated risk of stroke or endometrial disease in postmenopausal women treated with tibolone.

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.

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.

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.

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.

This study investigated the oxidation of the oral contraceptive 17alpha-ethinylestradiol (EE2) during ozonation. First, the effect of ozone (O3) on the estrogenic activity of aqueous solutions of EE2 was studied using a yeast estrogen screen (YES). It could be shown that O3 doses typically applied for the disinfection of drinking waters were sufficient to reduce estrogenicity by a factor of more than 200. However, it proved impossible to completely remove estrogenic activity due to the slow reappearance of 0.1-0.2% of the initial EE2 concentration after ozonation. Second, oxidation products formed during ozonation of EE2 were identified with LC-MS/MS and GC/MS and the help of the model compounds 5,6,7,8-tetrahydro-2-naphthol (THN) and 1-ethinyl-1-cyclohexanol (ECH), which represent the reactive phenolic moiety and the ethinyl group of EE2. Additionally, oxidation products of the natural steroid hormones 17beta-estradiol (E2) and estrone (E1) were identified. The chemical structures of the oxidation products were significantly altered as compared to the parent compounds, explaining the diminished estrogenic activity after ozonation. Overall,the results demonstrate that ozonation is a promising tool for the control of EE2, E2, and E1 in drinking water and wastewater.

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.

The cellular action of steroid hormones is mediated by specific receptors. Recently, two different estrogen receptors (ER), alpha and beta, have been cloned with a specific tissue distribution. Active estrogen as well as active progestin are compounds of oral hormonal contraceptives and hormone replacement therapy. To examine the regulation of ER-alpha and -beta activities after treatment with synthetic progestins and synthetic and natural estrogens, COS 7 cells were transfected with the vector expressing ER-alpha and -beta in combination with a luciferase reporter vector. ER-alpha activity was upregulated in the presence of synthetic progestins in a dose-dependent manner. Norethisterone, norethynodrel and desogestrel proved to be the most potent stimulatory agents of ER-alpha expression. On the other hand, not all progestins exhibited a stimulatory action on ER-beta activity. Only norgestrel, levonorgestrel, norethynodrel and norethisterone induced ER-beta-activating functions in a dose-dependent manner. Luciferase activity due to estrogen stimulation served as a positive control. Our results indicate that progestins have different effects on the activities of ER-alpha and -beta.

A total of 99 premenopausal and 27 postmenopausal women were evaluated to determine the quantity of glandular proliferation resulting from progestin inhibition of estrogen-primed subjects and of subjects without hormonal stimulation. Endometrial glandular proliferation rates were determined by using mitosis counts, proliferating-cell nuclear antigen (PCNA), and nuclear cyclin (MIB1) immunocytological staining. The endometria of normally cycling premenopausal women, of women who received a synthetic progestin, and of untreated postmenopausal women were studied. In untreated normally cycling premenopausal women, the proliferation of the glandular epithelium was increased during the follicular phase and decreased during the luteal phase. Premenopausal women receiving a synthetic progestin and untreated postmenopausal women who were not estrogen-primed showed minimal epithelial proliferation. Endometrial glandular proliferation is inhibited by endogenous progesterone in premenopausal women. Endometrial proliferation is markedly reduced in premenopausal women receiving a synthetic progestin and in untreated postmenopausal women.

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.

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.

Platelet-activating factor-acetylhydrolase (PAF-AH), the enzyme that inactivates PAF, is decreased in the plasma of both rabbits and humans during the latter stages of pregnancy. The activity of the enzyme was decreased in rats by the administration of 1,3,5(10)-estratrien-17 alpha-ethynyl-3,17 beta-diol (17 alpha-ethynylestradiol) and increased by dexamethasone treatment. In the present study, we have further defined the hormonal regulation of PAF-AH levels in plasma of adult and juvenile rats. Estrone (E1), 17 beta-estradiol (E2), estriol (E3), and various progestins [4-pregnen-6 alpha-methyl-17 alpha-ol-3,20-dione (medroxyprogesterone), 4-estren-17 alpha-ethynyl-17 beta-ol-3-one (norethindrone), and 5(10)-estren-17 alpha-ethynyl-17 beta-ol-3-one (norethynodrel)] were administered to adult rats, and the plasma PAF-AH activities were assayed. E1, E2, and E3 administration to adult female rats lowered the plasma PAF-AH activity, E3 being the most effective. After administration of these estrogens the activity returned to the preinjection level within 4 days. The administration of medroxyprogesterone resulted in a 2-fold increase in plasma PAF-AH activity in adult female rats, and the activity remained elevated for up to 30 days. When adult male rats were treated with similar doses only a 20% increase was observed and the PAF-AH activity returned to control values by day 10. In contrast, norethindrone and norethynodrel administration resulted in a reduction of the enzyme activity in adult female rats. The plasma PAF-AH activity in juvenile male and female rats (3 weeks of age, 45-60 g body wt) was two times higher than that in adult rats of both sexes and spontaneously decreased up to the time of puberty. When juvenile male or female rats were injected with either 17 alpha-ethynylestradiol or medroxyprogesterone, a minimal change in PAF-AH activity was observed. In contrast, when dexamethasone was administered to juvenile male and female rats the plasma PAF-AH activity increased in a manner similar to adult animals. It is suggested that estrogens cause a decrease and medroxyprogesterone an increase in plasma PAF-AH activity. It is suggested that the different responsiveness to medroxyprogesterone between adult female and male rats and juvenile animals may depend on the concentration of the hormone receptor in the tissue responsible for the synthesis of PAF-AH. The decrease in PAF-AH activity after the administration of norethindrone and norethynodrel may be due to the known estrogenic activity of these steroids.

The role of hormones, especially progesterone and synthetic progestins, in the pathogenesis of breast cancer is highly debated and far from being clarified. The observation that differentiation of the mammary gland prior to exposure to a carcinogenic agent inhibits the initiation of mammary carcinomas, and the fact that this protection is mediated by cell kinetic changes induced in the gland parenchyma by hormonal stimuli such as that of pregnancy, led us to determine how ovariectomy and hormonal supplementation affect the cell kinetic characteristics in various terminal ductal structures of an intact animal mammary gland, i.e., terminal end buds, terminal ducts and alveolar buds. Fifty-day-old virgin Sprague-Dawley rats were either ovariectomized or treated for 21 days with norethynodrel and mestranol or medroxyprogesterone acetate in doses of 0.5 mg (low dose - LD), or 5.0 mg (high dose - HD); chorionic gonadotropin (hCG) in doses of 1 or 100 IU/day, or placental lactogen (PL) in a dose of 0.5 mg. Cell kinetics were studied by counting the number of cells incorporating [3H]-thymidine and expressed as the DNA labeling index. In the intact animal, the rate of cell proliferation was highest in terminal end buds, decreased progressively towards the ductal portions and was even lower in alveolar buds. Ovariectomy significantly reduced the proliferative activity of the mammary epithelium, ten-fold in terminal ducts and alveolar buds, and five-fold in terminal end buds. DNA labeling index was significantly reduced in terminal ducts and alveolar buds by all hormones at all doses, except PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbits were fed an atherogenic or normal control diet for 3 months; half of each group were treated with mestranol--norethynodrel (M--N) and half with cottonseed oil vehicle. At the end of the 3-month period rabbits were killed and aortic collagen and elastin synthesis determine. Collagen and elastin synthesis was increased in aortas of rabbits on an atherogenic diet, but those treated with M--N had a lower rate of collagen synthesis and less deposition of cholesterol in the aorta than those administered cottonseed oil. It is suggested that long-term administration of the contraceptive steroid combination may retard the progression of atherosclerosis through the hormone's effect on vascular connective tissue.

Contrasting with the hypertension resulting consistently after DOCA implantation, mature female rats given Enovid daily for 20 weeks had only slight and occasional increases in blood pressure. The pressure elevations remained infrequent and transient even when drinking water was replaced with isotonic saline solution but they became more pronounced when Enovid treatment was initiated at an earlier age. Despite the failure to cause sustained hypertension, the data indicate that Enovid affects blood pressure and that both mestranol and norethynodrel are essential for this effect. Pressor responses to norepinephrine or angiotensin were unaffected even after 20 weeks of pretreatment but those to electrical stimulation of the posterior hypothalamus were increased in rats pretreated with Enovid. Enhanced responsiveness to hypothalamic stimulation occurred only in the awake state and was not demonstratable after the central nervous system (CNS) had been depressed with urethane. These results can be explained by an increased sensitivity of hypothalamic pressor areas produced by Enovid prior to the development of hypertension and the mechanisms involved may be similar to those occurring in spontaneously-hypertensive rats. However, the possibility of hypersensitivity at other sites in the sympathetic vasomotor outflow cannot be ruled out.

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.

The effects of steroidal oral contraceptive norethynodrel plus ethinylestradiol-3-methyl ether (SOC) at a daily dose of 5 mg: 0.06 mg per kg body weight for 28 days on intestinal absorptive functions have been investigated in protein-deficient female albino rats. The administration of this contraceptive caused significant increase in glucose and amino acids uptake but had no effect on calcium and zinc uptake in pair-fed as well as in protein-deficient rat. Further studies carried out on glucose transport system showed that the transport of sodium-dependent glucose was significantly enhanced while that of sodium-independent glucose remained unaltered in drug-treated animals. Kinetic studies of glucose transport in the presence of sodium ions revealed that SOC treatment affected the rate of uptake of glucose by elevating Vmax, but the apparent Kt value remained the same in treated and untreated animals.

Short-term oral contraceptive therapy has been reported to decrease postheparin lipolytic activity (PHLA). Resistance to heparin has been held responsible for this effect. To test several alternative explanations, we studied both PHLA and heparin concentrations in nine control women and nine women receiving long-term estrogen-progestin therapy after they were given heparin intravenously (10 units/kg). There were no significant differences in the concentration of heparin, its rate of disappearance, or calculated space of distribution between control and treated groups. PHLA was depressed (p less than 0.05) by approximately 50% during long-term estrongen-progestin therapy. PHLA disappearance was similar in both groups. Thus, reduced PHLA in women receiving long-term oral contraceptive therapy cannot be related to altered heparin metabolism or to accelerated enzyme disappearance from plasma. Long-term estrogen-progestin administration may decrease the heparin-facilitated release of individual triglyceride hydrolase activities or enhance the affinity of enzyme binding to tissues.

The observation that the susceptibility of the mammary gland to chemical carcinogenesis is inversely related to its level of hormonally induced differentiation led us to test whether treatment of virgin rats with an estrogenic-progestagenic hormone combination protected the gland against this carcinogenesis. Virgin Sprague-Dawley rats aged 45, 55, 65, or 75 days had implanted subcutaneously for 21 days a pellet containing norethynodrel-mestranol (NM) (98.5%-1.5%) at two doses, a physiological or low dose (LD) of 0.5 mg, equivalent to the dose used in Enovid for contraception in humans, and a pharmacological or high dose (HD) of 5.0 mg. Twenty-one days after NM pellet removal, the mammary glands of 5 animals per group were examined for the number of terminal end buds (TEBs), terminal ducts (TDs), alveolar buds (ABs) and lobules, and the DNA labeling index (DNA-LI). The remaining animals received 8 mg 7,12-dimethylbenz(a) anthracene (DMBA)/100 g body weight, and tumorigenesis was evaluated at 24 weeks. The percentage of TEBs decreased with age, and further with NM treatment at both doses. Treatment did not significantly modify the percentage of TDs, but increased that of ABs in most groups. The DNA-LI of TEBs remained constant, even during aging and after treatment, whereas both aging and treatment reduced DNA-LI in TDs and ABs. Tumor incidence declined with increasing age from 75% to 44% in the 45 and 75 day-old control groups respectively. Adenocarcinoma incidence followed the same trend. NM treatment had a dose-related protective effect against development of tumors in general and of adenocarcinomas in particular. LD treatment resulted in a marginally significant reduction in adenocarcinoma incidence, whereas HD-treated animals were 0.24 times as likely as controls to develop carcinomas. There was a statistically significant correlation between the percentage of TEBs present in the gland at the time of carcinogen administration and the incidence of adenocarcinomas. It was concluded that treatment of virgin rats with the hormone combination norethynodrel-mestranol resulted in long lasting structural changes in the mammary gland which protected this organ from a subsequent carcinogenic insult.

The immunodepressive activity of widely employed steroid contraceptive agents (SCA's) was evaluated in mice and rats on various humoral and cell-mediated responses. Three different progestagens (lynestrenol, norethindrone or norethynodrel) were administered in combination with the estrogen mestrahese SCA's could significantly reduce the severity of allergic encephalomyelitis in rats, variations in their capacity to interfere with primary response to sheep erythrocytes in mice and anti-mouse erythrocyte autoantibody formation were found. Treatments employed with these SCA's did not reduce tumor allograft resistance in mice and the anti-sheep erythrocyte response in rats. In addition, otherwise immunosuppressive treatments with these SCA's did not modify the mouse response to a T-independent antigen. The possible clinical significance of these results is discussed.

Norethynodrel proved to have moderately potent effects on hepatic function in female rats. It causes (a) loss of body mass, (b) increase in the relative liver mass, (c) rise in the protein content of liver, (d) increase in the cytochrome P-450 level, (e) reduction in the length of pentobarbital-induced sleep, and (f) increase in the rate of biotransformation of (i) aniline and (ii) aminopyrine. In contrast, ethynyl estradiol was shown to have little or no effect as judged by the same criteria, but, like norethynodrel, progesterone and estradiol, caused loss of body mass.

Oral contraceptives were administered cyclically to 96 female rhesus monkeys for 5 years. Forty-eight animals received Enovid-E and 48 were treated with Ovulen at doses 1, 10, and 50 times the human dose. Ophthalmic lesions did not occur at any of the dose levels employed. Significant fundic lesions, including papilledema, venous dilatation, venous retinal thrombosis, or arterial retinal thrombosis, did not develop in any of the treated animals.

To assess whether structural modifications on the A-ring of norethisterone (NET) could modify its antigonadotropic potency, comparative studies using NET, 5 alpha-dihydro NET (5 alpha-NET) and its 3 beta,5 alpha and 3 alpha,5 alpha tetrahydro derivatives in castrated adult rats were undertaken. The antigonadotropic effect of these compounds was evaluated by measuring the serum and pituitary immunoreactive concentrations of LH and FSH following their chronic sc administration to animals depleted of progesterone receptors. The results demonstrated that 3 beta,5 alpha-NET and 5 alpha-dihydro-NET exhibited a significantly greater gonadotropic inhibiting activity as compared with that of their parent compound. The simultaneous administration of tamoxifen with 3 beta,5 alpha-NET resulted in a significant diminution of its antigonadotropic potency, particularly for LH. These data indicate that the potent antigonadotropic effect of 3 beta,5 alpha-NET metabolite was mediated via oestrogen receptors. The LH inhibitory activity of 5 alpha-dihydro-NET was not suppressed by the non-steroidal antioestrogen administration, thus suggesting that 5 alpha-NET might exert its effect via androgen receptors. The overall data were interpreted as demonstrating that metabolic conversion products of NET exhibit potent antigonadotropic effect. The data are consistent with an A-ring enhancement of the antigonadotropic potency of this synthetic progestin and open an alternate approach to the development of fertility regulating agents.

In order to examine effects of oral contraceptives (OC) on the microsomal drug matabolizing enzymes in a state of protein malnutrition, weanling Sprague-Dwayley female rats were fed diets containing 3.5% (low protein, LP), 26% (normal protein, NP) or 42% (high protein, HP) casein for 224 days and norethynodrel plus mestranol for the last 182 days. LP rats were smaller in body weight than NP and HP rats and the latter two groups showed depressed weight gain and hypertrophy of liver and kidney due to OC. Concentrations of microsomal proteins and cytochrome P-450 were lowered by LP diet and OC did not induce cytochrome P-450. Activity of biphenyl-4-hydroxylase was lowered in LP and HP rats compared to NP group in which activity of this enzyme was significantly decreased due to OC. The diets alone had no effect on the activity of p-nitrobenzoate reductase, however, its activity was enhanced by OC only in NP rats. LP diet caused reduction of 4-methylumbelliferone glucuronly transferase activity which was lowered by OC in NP as well as in LP rats. It is concluded that NP diet more than either LP or HP diet exposed rats to the modification of microsomal drug metabolizing enzymes by OC.

The action of subtoxic doses of armine and nibufin administered alone and combined with steroid drugs on the rat reproductive function and uterine tissue RNA content was studied experimentally. The subtoxic doses of the drugs increased sharply the luteinizing hormone releasing factor content in the hypothalamus, as well as the synthesis and excretion of luteinizing and follicle-stimulating hormones and RNA content in the uterine tissue. Estrogen-progestin drugs, particularly infecundin, exerted a protective action in armine- and nibufin-produced abnormalities of the reproductive function.

This study was instituted to determine the effect of a progestin, norethynodrel, on the induction of experimental carcinogens of the hamster cheek pouch. Thirty-six Syrian hamsters were divided into two groups. One group received 2 mg./kg./day of norethynodrel in subcutaneous injections, and the other group received nothing. The right cheek pouches of both groups were painted three times a week with the chemical carcinogen 9,10-dimethyl-1,2-benzanthracene in a 0.05 percent acetone solution. After 9 weeks of painting, the first tumors developed in the hormone-treated hamsters, while none appeared in the control group until after 10 weeks. After 15 weeks, 100 percent of both groups had tumors. The average latent period was 11.8 weeks for the treated group and 12.7 weeks for the control group (p = less than 0.05 percent, Student's test). Norethynodrel significantly decreased the induction time of the neoplasms.

The serum level of pregnancy-associated alpha2-globulin was studied in 70 healthy women taking three different types of oral contraceptives [40 Bisecurin: containing 1.0 mg of Ethynodiol diaceticum +0.05 mg Ethynilestradiol; 20 Infecundin: containing 2.5 mg of Norethynodrel +0.1 mg of Mestranol; 10 Cervicundin: containing of 0.5 mg Ethynodiol diaceticum]. Elevated amounts of serum level of pregnancy associated alpha2-globulin could be detected in 37 of the 40 women taking Bisecurin and in all 20 taking Infecundin. Cervicundin however, failed to induce any change in the serum protein pattern of the 10 subjects studied. Serum levels of pregnancy-associated alpha2-globulin were higher in the Infecundin-taking (53 mg %), than in the Bisecurin-taking group (29 mg %). This shows the oestrogen dependency of the pregnancy-associated alpha2-globulin and points to a possible implication of this globulin in the immunosuppressive processes.

Male and female, normotensive Wistar:Kyoto rats, spontaneously hypertensive rats (SHR), and Stroke-prone SHR (SHR/SP) with severe hypertension were treated with a contraceptive drug (Enovid) from the time of weaning until they became 180 days old. Although the Envoid-treated SHR and SHR/SP had exacerbations of their high blood pressure, none of the animals developed renal or cerebral damage. Chronic treatment with Enovid caused testicular and ovarian atrophy and a significant increase in circulating corticosterone. It is suggested that the potentially deleterious effects, e.g., stroke, hypertension, hyperglycemia, hyperlipidemia, etc., of chronic treatment with contraceptive drugs is largely determined by pre-existing genetically endowed proneness or resistance.

The effect of a long-term (14 days) combined introduction of mestranol and noiethinodrel in contraceptive doses on the activity of pentosophosphate routes enzymes of the carbohydrates metabolism, that of acid and basic ribonucleases and on the level of summary nucleinic acids in the uterus and liver in sexually mature female-rats was studied. It was found that the concentration of summary nucleinic acids in the tissues increases under the effect of the drug, the oxidative reactions enzymes become more active, while the activity of non-oxidative reactions enzymes of the pentosophosphate route and of acid and basic endoribonucleases in inhibited.

Receptivity of female rats has been investigated under chronic experimental conditions. A receptivity index has been worked out for quantitative evaluation. It proved suitable for disclosing stimulatory (2.5 mg noretynodrel, oestradiol propionate) as well as inhibitory (etynodiol diacetate) effects. 2.5 mg noretynodrel has a biphasic effect; causing an inhibition and later an increase in receptivity. Receptivity of untreated female rats is low and can be increased by various steroids; the best treatment was 50 micrograms oestradiol monopropionate administered twice weekly, but this resulted in an inverse mating reaction, with frequent copulations in every phase of the cycle. In untreated females the number of copulations varied widely. Previous deliveries did not influence receptivity and frigidity was also observed.

Chronic treatment with ethynyl estradiol alone (48-50 mug/kg/day) and in combination with several doses of norethynodrel either attenuated or prevented the increase in tail skin temperature accompanying sC. administration of isoproterenol (200 mug/kg b.wt.) to male and female rats. Dietary administration of an oral contraceptive containing norethynodrel and mestranol (7.5 mg/kg of food) was also accompanied by an attenuated response to isoproterenol. Attenuation of the tail skin temperature response was present in all groups after 3 to 5 weeks of treatment but was detectable in the oral contraceptive-treated group after 1 week of treatment. All steroids and combinations, except norethynodrel administered alone (154 mug/kg/day), reduced body weight gain.

Experiments on female rats showed the blocking of the M-cholinoreactive system with amizyl to significantly contribute to the estrogen/norsteroid contraceptive effect during the postcoital periods. This effect was accompanied by decrease in the gonadotropin level and by the change in the LH/FSH ratio, this creating an unfavourable background for implantation of the fertilized ovicell in the endometrium. There was a change in the transport rate in the tube and a delay in the decidual reaction. Changes in the rate of the ovicell transport were not accompanied by distrubances in the process of fertilization or with the cytotoxic action. Mestranol and ethynylestradiol in combination with norethynodrel (1:20) and with the central cholinolytic amizyl were agents with future prospects for short-term postcoital contraception.

Female rats treated chronically with ethynylestradiol (36 micrograms/kg per day) alone, and in combination with the progestational agent, norethynodrel (253 micrograms/kg per day), cooled significantly faster than controls when lightly restrained and exposed to air at 5 degrees C. Rate of cooling of rats given only norethynodrel was similar to that of the control group. In other studies, rate of oxygen consumption was determined for all groups during acute exposure to cold (14 degrees C). All estrogen-treated groups achieved the same maximal rate of oxygen consumption as control and norethynodrel-treated groups during cold exposure, but cooled significantly faster. Two groups of female rats were treated chronically with ethynylestradiol at two separate doses (36 and 61 micrograms/kg per day). An untreated group served as controls. Rate of oxygen consumption of all animals were measured during restraint and exposure to cold (18 degrees C). The estrogen-treated groups again achieved the same maximal rate of oxygen consumption as the control group, but also cooled significantly faster despite the fact that the cold stress was less severe than in the previous experiment. That estrogen-treated rats cooled faster than controls in both studies despite achieving a maximal rate of heat production which did not differ from controls suggests that reduced cold tolerance of estrogen-treated rats may be related to increased heat loss.

The administration of two oral contraceptives to female dogs for 5 years did not produce ocular lesions. Corneal and lenticular opacities occurred with equal frequency in control and treated groups, and fundic lesions, including papilledema, venous dilatation, and venous or arterial retinal thrombosis, were not produced by doses of Enovid-E or Ovulen 1, 10, and 25 times the human dose.