Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.

Deletion of cyclooxygenase 2 in mouse mammary epithelial cells delays breast cancer onset through augmentation of type 1 immune responses in tumors.

Inhibition of cyclooxygenase (COX) 2, which is associated with >40% of breast cancers, decreases the risk of tumorigenesis and breast cancer recurrence. To study the role of COX-2 in breast cancer, we engineered mice that lack selectively mammary epithelial cell (MEC) COX-2 (COX-2 KO(MEC)). Compared with wild type (WT), MEC from COX-2 KO(MEC) mice expressed >90% less COX-2 messenger RNA (mRNA) and protein and produced 90% less of the dominant pro-oncogenic COX-2 product, prostaglandin (PG) E(2). We confirmed COX-2 as the principle source of PGE(2) in MEC treated with selective COX-2 and COX-1 inhibitors. Tumors were induced in mice using medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene. Breast cancer onset was significantly delayed in COX-2 KO(MEC) compared with WT (P = 0.03), equivalent to the delay following systemic COX-2 inhibition with rofecoxib. Compared with WT, COX-2 KO(MEC) tumors showed increased mRNA for Caspase-3, Ki-67 and common markers for leukocytes (CD45) and macrophages (F4/80). Analysis of multiple markers/cytokines, namely CD86, inducible nitric oxide synthase (iNOS), interleukin-6, tumor necrosis factor α (TNFα) and Tim-3 indicated a shift toward antitumorigenic type 1 immune responses in COX-2 KO(MEC) tumors. Immunohistochemical analysis confirmed elevated expression of CD45, F4/80 and CD86 in COX-2 KO(MEC) tumors. Concordant with a role for COX-2 in restraining M1 macrophage polarization, CD86 and TNFα expression were offset by exogenous PGE(2) in bone marrow-derived macrophages polarized in vitro to the M1 phenotype. Our data reveal the importance of epithelial COX-2 in tumor promotion and indicate that deletion of epithelial COX-2 may skew tumor immunity toward type 1 responses, coincident with delayed tumor development.

Elevated concentrations of ethinylestradiol, 17beta-estradiol, and medroxyprogesterone have little effect on reproduction and survival of Ceriodaphnia dubia.

Wastewater effluent contains synthetic and natural hormones, often in complex mixtures, that may be associated with reproductive abnormalities in fish and other aquatic biota. We exposed the sentinel invertebrate Ceriodaphnia dubia to the natural estrogen 17beta-estradiol (E(2)), a synthetic estrogen, ethinylestradiol (EE(2)), and a synthetic progestin, medroxyprogesterone in a 7-day test. These compounds had no significant effect on reproduction or survival even at 10(6) times the concentrations at which reproductive effects have been documented in several fish species. C. dubia is routinely used for screening the toxicity of wastewater effluent. However, in the standard chronic 7-day exposure the endpoints of survival and reproduction were insensitive to several synthetic and natural vertebrate hormones. The C. dubia 7-day chronic toxicity test is probably not a useful monitoring tool for vertebrate hormones and their pharmaceutical analogs unless other sensitive endpoints such as maturation rates, molt frequency, and offspring sex ratios are incorporated in a practical manner.

Regresión de cáncer de mama después del tratamiento hormonal en un modelo murino / Mammary cancer regression after hormonal treatment in a murine model

Carcinomas mamarios murinos, originalmente inducidos por acetato de medroxiprogesterona, con crecimiento autónomo y receptores de estrógenos y progesterona regresionan con estradiol (E2) o con antiprogestágenos. Con el objeto de analizar los mecanismos de la regresión tumoral, estudiamos las características morfológicas y la participación de los reguladores del ciclo celular tales como p21, p27, p53 y MDM2 mediante inmunohistoquímica utilizando dos tumores sensibles al E2 o a los antiprogestágenos y uno sensible al E2 y antiprogestágenos resistente. La regresión se asocia a disminución del parénquima tumoral con aumento del estroma, a un efecto antiproliferativo y proapoptótico y a la inducción de proteínas inhibitorias del ciclo celular tales como p21 y p27. Sin embargo el patrón de expresión de los reguladores del ciclo celular varió. En los tumores sensibles a ambos tratamientos aumentó p21 y p27, los valores basales de p53 fueron altos y los de MDM2 bajos. En el tumor sensible sólo a E2 aumentó únicamente p27 y p21 permaneció en valores bajos acompañados por altos niveles basales de p53 y MDM2. Estos hallazgos sugieren que p21 podría ser esencial para la acción de los antiprogestágenos y que alteraciones en la vía p21/p53 podrían participar en la resistencia al tratamiento hormonal.

Medroxyprogesterone acetate alone or synergistic with chemotherapy suppresses colony formation and DNA synthesis in C6 glioma in vitro.

We have studied the effects of medroxyprogesterone acetate (MPA) on C6 glioma growth in vitro in order to prove the hypothesis that it could arrest growth and induce drug sensitisation in a glial tumour as it does in breast cancer cells. Plating, thymidine-labelling index, ultra-structure, and soft agar colony growth were determined after incubation with MPA, and/or cisplatin, procarbazine and methotrexate (MTX). MPA (microg/ml) reduced the thymidine-labelling index by 41 and 73% at 48 and 96 h, respectively, and decreased colony growth by 61%. Soft agar colony inhibition by MPA was almost as potent as MTX (0.3 microg/ml), but the latter drug showed very high cytotoxicity. Electron microscopy revealed that in medroxyprogesterone treated cells myeloid bodies developed, but MTX treatment caused mainly necrosis. Medroxyprogesterone increased procarbazine and cisplatin-induced colony growth and S-phase inhibition, but reduced MTX-induced thymidine-labelling inhibition. In conclusion, progesterone may inhibit growth and sensitize to drugs.

Gallstones induced by sex steroids in the female Syrian hamster: duration effects.

Scanning (SEM), and transmission (TEM) electron microscopy were used and correlated to morphologically characterize changes induced in the gallbladder epithelium of female Syrian hamster in response to treatments with estradiol (E) alone, and estradiol with medroxyprogesterone (E + MP). Compared with control (C), the E- and E + MP-treated groups demonstrated alterations in the serum lipid profile as well as significantly decreased body weights. The liver with gallbladder weights, as well as the uterus weights, were significantly increased. Two-month E and E + MP treatment groups exhibited increased number of anionically charged apical granules, and luminal mucoid elements. Contrastingly, the three-month treatment groups demonstrated larger and more gallstone-like deposits as compared to the C and two-month E and E + MP groups. This report presents a comprehensive overview of our previous and current data, including that of other investigators in relation to the various factors and parameters involved in the cholelithiatic process.

Morphological aspects of female Syrian hamster gallbladder induced by one-month sex steroid treatment.

Light (LM), transmission (TEM), and scanning (SEM) electron microscopy were used to characterize morphological changes induced in the gallbladder epithelium of female Syrian hamsters in response to one-month estradiol alone (E) and estradiol with medroxyprogesterone (E + MP) treatments. TEM data were correlated with the SEM observations. Compared with control (C), E- and E + MP-treated hamsters showed significant decreases in body weight, while the liver and gallbladder, and uterus weights increased. Moreover, E treatment induced some subcellular changes (microvilli, nucleus, mitochondria, RER, glycogen, abundant apical granules). The E + MP treatment appeared to exacerbate these similar changes and, in addition, induced apical excrescences and cell shedding. Both E and E + MP gallbladders showed luminal micelles, cellular debris and crystalliths associated with mucus. Simultaneously, an increased acidification of the mucoid content of the apical granules was noticed.

Epithelial surface changes and induction of gallstones in the male Syrian hamster gallbladder as a result of a two-month sex steroid treatment.

Transmission (TEM) and scanning (SEM) electron microscopic observations were correlated to characterize morphologic changes induced in the gallbladder of male Syrian hamsters following a two-month estradiol (E) and estradiol + medroxyprogesterone (E + MP) treatment. Compared to control (C), E-treated surface epithelial cells show pleomorphism, cytoplasmic vacuolizations, apical granules, excrescences and decapitations, and small gallstone-like deposits. Following both E + MP treatment, a large accumulation of apical granules containing acidic mucoid products, abundant intraluminal deposits and numerous fields of observation suggest that cell debris and mucous condensation could participate in the formation of the large intraluminal gallstone-like deposits detected as a result of this treatment. In control gallbladders these events were never observed. MP added to E also increases liver and gallbladder weight as well as blood lipid levels. These findings complement and confirm other previous data obtained following short steroid treatment in male, ovariectomized and intact female hamsters. In addition, these results support our hypothesis that gallstone nucleation and growth originate from multiple factors, hormonal disturbance, modulation of liver lipid metabolism, production of cell debris and mucus, can be responsible for the initial gallstone nucleation.

Sequentially administered ethinyl estradiol and medroxyprogesterone acetate in the treatment of refractory epithelial ovarian carcinoma in patients with positive estrogen receptors.

The activity of sequentially administered hormonal therapy was investigated over 25 days in 25 patients with epithelial ovarian carcinoma who had estrogen receptor (ERc)-positive tumors. Patients received ethinyl estradiol (EE) (50 micrograms/d) on days 1 to 7 and medroxyprogesterone acetate (MPA) (400 mg/d) on days 8 to 25. Twenty-three patients completed one or more courses of treatment. There were no complete responses (CR). Four partial responses (PR) with durations of 9, 4, 3, and 1 months were seen. Two incomplete responses with durations of 6 and 4 months were also seen. Six patients had stable disease (SD), and 11 patients had progression. The overall response rate was 17% and may represent a modest improvement in response over those in previously published studies conducted with MPA alone. No significant toxic effects were noticed, and some patients reported an improved sense of well-being. However, two patients experienced depression with this treatment. The mean ERc values in responders, patients with SD, and nonresponders were 70.0, 36.7, and 47.9 fmol/mg cytosolic protein, respectively. Future studies of hormonal therapy in patients with ovarian carcinoma should attempt to identify more reliable indices for determining sensitivity to these agents.

Hormone dependence of a mouse mammary tumor line induced in vivo by medroxyprogesterone acetate.

The administration of MPA to virgin female BALB/c mice led to the development of mammary adenocarcinomas, which in further in vivo transplants gave rise to both MPA-dependent and MPA-independent lines. In this paper we chose one of the MPA-dependent lines with high contents of estrogen (ER) and progesterone (PR) receptors, and were able to demonstrate that a) the growth of these tumors could be manipulated by the administration or the withdrawal of the hormonal supply; b) PR were down-regulated in MPA-treated mice; c) progesterone had the same stimulatory effect as MPA on tumor growth; d) tumors did not grow in estrogen-treated mice; e) tumor growth was much lower in males than in females; f) the presence of the ovaries had a positive influence on tumor growth, even in the presence of MPA; g) the withdrawal of progestin pellets in ovariectomized mice usually led to complete remissions followed by regrowth of the tumors after several weeks; and h) the regrowing tumors maintained their steroid receptor pattern and (in 3 out of 4 cases) their hormone-dependent behavior in further passages.

Transforming growth factor-beta activities in 'in vivo' lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice.

We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody.

Mouse mammary tumors induced by medroxyprogesterone acetate: immunohistochemistry and hormonal receptors.

Mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in female BALB/c mice were investigated as to their morphology and immunohistochemistry and their content of steroid, prolactin (PRL), and epidermal growth factor (EGF) receptors. Histologically, these tumors were mainly of ductal origin, since hyperplastic alveolar nodules were observed only in 3 cases. No viral particles were encountered in electron microscopic studies. Estrogen and/or progesterone, PRL, and EGF receptors were detected in MPA-induced tumors, as well as in the occasional spontaneous mammary tumors of multiparous females. EGF was detected, by a radioimmunoassay, in the cystic fluid of 12 mammary adenocarcinomas. MPA treatment was found to induce uterine secretory changes, glandular cystic hyperplasia, and eventually deciduomas that stained strongly for desmin and to a lesser degree for vimentin, suggesting a muscular differentiation. Consequently, MPA-induced adenocarcinomas can be considered as ductal tumors that possess estrogen and/or progesterone, PRL, and EGF receptors. Whether MPA induces tumor growth directly via progesterone receptors remains to be investigated.

Induction of mammary adenocarcinomas by medroxyprogesterone acetate in BALB/c female mice.

In a previous paper we reported that medroxyprogesterone acetate (MPA) decreased the incidence of foreign body tumorigenesis in BALB/c mice but that mammary adenocarcinomas appeared in some of the females. The experiment was repeated in 245 virgin females as follows: (1) 40 mice treated with 40 mg of MPA depot s.c. every 2 months during a whole year; (2) 117 mice bearing a foreign body (FB) and treated with MPA; (3) 46 mice bearing a FB; (4) 42 non-treated mice. Mammary adenocarcinomas developed in 16/40 in group 1 and 30/117 in group 2; no mammary tumors appeared in either control groups. The tumors were infiltrating adenocarcinomas often affecting more than one mammary gland; metastases were occasionally observed. Animals killed after 1 year of MPA treatment presented deciduomas. MPA also decreased the incidence of FB-induced sarcomas, confirming previous results.

Inhibitory effect of medroxyprogesterone acetate on foreign body tumorigenesis in mice.

This paper reports on the investigation of the effect of medroxyprogesterone acetate (MPA) on foreign body tumorigenesis that resulted from sc implantation of a glass cylinder. Adult BALB/c mice of both sexes bearing the foreign body were separated into groups. Group 1 received 40 mg MPA sc every 2 months during 1 year, in the vicinity of the glass cylinder; group 2 received the same MPA treatment in the contralateral flank; and group 3 received no hormonal treatment. Sarcomas developed in 4 of 39, 9 of 41, and 17 of 39 mice, respectively. With the use of an evaluation based on the number of high-risk mice per time interval, the MPA inhibitory effect was found to be statistically significant in both groups: 26, 53, and 79% tumor incidence, respectively. A decrease in the rate of tumor development also was observed but only in mice treated with MPA in situ. An unexpected side effect of continuous MPA administration in females was the appearance of adenocarcinomas.

The Depo-Provera debate. Commentary on the article "Depo-Provera, a critical analysis".

A widely publicized article has in recent months caused a great deal of concern among individuals interested in responsible promotion of family planning. The article contains a long series of factual errors, distortions and biased quotations. This commentary presents evidence, based on current knowledge, that Depo-Provera is a satisfactory contraceptive with several advantages and some disadvantages, and poses no more unresolved problems than oral contraceptives. There is no evidence that, at contraceptive doses, it increases the risk of cancer, impairs bone mineralization, "shocks" the hypothalamus, damages the liver or the immune system, or causes premature aging. Studies to date have not shown damaging effects on infants exposed to the drug in utero or via breast milk. To most women, disruption of the menstrual cycle, the major side effect, is not a health hazard. Finally, women in various parts of the world have shown to be quite capable of choosing for themselves whether or not the advantages of the drug can overcome the disadvantage of almost certain menstrual disturbance.

Medroxyprogesterone acetate.

PIP: This monograph review on medroxyprogesterone acetate (MPA) includes chemical and physical data (synonyms and trade names), structural and molecular formulae and molecular weight, chemical and physical properties of MPA, and the production, use, occurrence, and analysis of MPA. Production of MPA, which is not known to occur naturally, can be accomplished by 4 main chemical reactions, and several patents for synthesis routes have been issued; these reactions are summarized. MPA is used orally for treatment of secondary amenorrhea and dysfunctional uterine bleeding in the U.S. It is also administered for treatment of endometriosis. Other countries use MPA as an injectable contraceptive, and Japan uses it in treatment of threatened abortion. MPA also has various veterinary uses. Typical analytical methods for determining MPA purity and chemical clarity are outlined tabularly. Biological data relevant to the evaluation of carcinogenic risk to humans of MPA are also presented. In experimental animals (mice and dogs), MPA produced mammary tumors in dogs when injected intramuscularly. MPA is also reported to have teratogenic effects in some animal species. Human data consist of 1 epidemiological study on breast nodule development and 2 studies of the development of dysplasias and carcinoma in situ of the cervix. These studies have conflicting results and methodological problems, but it is concluded that there is limited evidence for the carcinogenicity of MPA in dogs. No conclusions can be made about its carcinogenicity for humans based on the extant studies.^ieng