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.

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.

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.

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.

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.

Embryotoxicity and maternal serum concentrations of medroxyprogesterone acetate (MPA) in baboons (Papio cynocephalus).

A single dose of MPA (Depo-Provera; Upjohn Co.; USA) was administered intramuscularly on day 27 (+/- 2) of gestation to three dosage groups of pregnant baboons. The dosage expressed as mg/kg corresponded to 1 (2.5 mg/kg), 10 (25 mg/kg) and 40 (100 mg/kg) times the human contraceptive dose equivalent (HDE) based on body weight. Injectable MPA was not teratogenic in baboons at 1x HDE. The teratogenicity was confined to the higher doses which included malformations of the external genitalia at 10x and 40x HDE and adrenal gland hypoplasia at 40x HDE. The maternal serum MPA concentrations were high during the critical period of adrenal and genital development. The pattern of MPA concentrations in maternal sera was similar between animals in the low dose group (10x HDE). In contrast, animals in the high dose group (40x HDE) showed interanimal variations in maternal serum concentrations of MPA. This study has demonstrated that injectable MPA is not teratogenic in baboons at human contraceptive dose equivalent and, even at higher doses, does not result in any nontarget organ malformations.

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.

High dose medroxyprogesterone acetate (MPA) treatment in metastatic carcinoma of the breast: a dose-response evaluation.

The results of controlled clinical trial that used high doses of medroxyprogesterone acetate (MPA) in the treatment of metastatic breast cancer are reported. Two treatment reigmens were used: regimen A, 500 mg daily with a total dose of 30 g; regimen B, 1,000 mg daily with a total dose of 60 g. The overall response rates were similar, with no statistically significant difference between the two treated groups. Regimen A (lower dosage group) reached a remission rate of 44%, whereas regimen B (higher dosage group) had a remission rate of 41%. The mean duration of response was 8 months with regimen A and 9 months with regimen B. The advantages of the lower dosage regimen as opposed to the higher dosage regimen of MPA in the treatment of advanced breast cancer are discussed.

PIP: A controlled clinical trial that used high doses of medroxyprogesterone acetate (MPA) in the treatment of metastatic breast cancer was conducted. Therapy consisted of 2 treatments: regimen A, 500 mg daily with a total dose of 30 g; regimen B, 1000 mg daily with a total dose of 60 g. From June 1975 to September 1976, 101 patients entered into the study and were randomly allocated into the 2 treatment groups. Both treatment groups were comparable in terms of age, menopausal status, free interval, and dominant site of lesions. Selection of patients was done according to the following criteria: histologically proved progressive metastatic carcinoma of the breast, without any treatment for at least 2 months; no prior hormonal manipulation; performance status 50 or more, and life expectancy longer than 3 months; measurable disease. Overall response rates were similar, with no statistically significant difference between the 2 treated groups. Regimen A reached a remission rate of 44%; regimen B had a remission rate of 41%. The mean duration of response was 8 months with regimen A and 9 months with regimen B. Both regimens were well tolerated. Clinical toxicity was mild with both dosages of MPA. The main side effect was gluteus abscess, with a higher incidence in group B. This was probably due to the greater amount of injected drug suspension in the 1000 mg/day regimen. The incidence of thrombophlebitis and vaginal bleeding was negligible.

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.

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.

Embryotoxicity of a single dose of medroxyprogesterone acetate (MPA) and maternal serum MPA concentrations in cynomolgus monkey (Macaca fascicularis).

A single dose of MPA (Depo-Provera; Upjohn Co., Kalamazoo, Michigan) was administered intramuscularly to 12 time-mated pregnant cynomolgus monkeys on day 27 (+/- 2) of gestation at 25 mg/kg or at 100 mg/kg. Maternal blood samples were collected immediately prior to MPA injection and then at regular intervals until cesarean section at term (day 152 +/- 3). Infants in both dose groups had external genital abnormalities. Female infants in the low-dose groups had partial or complete labial fusion, prominent median raphe, and clitoral hypertrophy; at high doses (100 mg/kg), the female infants had complete labial fusion and a distinct penile urethra. MPA had an opposite effect on external genitalia of male infants. The penis was short and the scrotal swelling was absent or less conspicuous, and two males had hypospadias. The adrenal glands were significantly smaller (P less than 0.05) in infants of both sexes treated with 100 mg/kg. One of the infants treated with 25 mg/kg of MPA had a muscular ventricular septal defect. Serum concentrations of MPA were determined by radioimmunoassay in eight pregnant monkeys. In the 25 mg/kg group the patterns of MPA profiles in the serum were similar in all four animals. An initial peak occurred at 24-48 hr postinjection (2.7-9.6 ng/ml), followed by a slight decrease at 3 days postinjection (gestational day 30), and then a steady increase to maximum levels of 10-14 ng/ml occurring between gestational days 37 and 50. Serum levels gradually declined to concentrations below 5 ng/ml by midgestation in three of four monkeys. By comparison, both the patterns and magnitude of MPA concentration showed great interanimal variation in the 100 mg/kg group. MPA was present in cord blood at measurable concentrations in infants at both dose groups; the levels ranged from 0.6 to 8.3 ng/ml, corresponding to 40-72% of the maternal concentrations. These results demonstrate that a single injection of MPA during early pregnancy causes selective embryotoxicity in both male and female fetuses. Presence of high levels of MPA in maternal sera during the critical period of genital development can cause specific genital defects; however, the exact mechanism by which MPA causes these paradoxical genital abnormalities is unknown.

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

Prenatal toxicity of medroxyprogesterone acetate in rabbits, rats, and mice.

Medroxyprogesterone acetate (MPA) was given once daily sc at 0.1-3,000 mg/kg/day for 3, 6, or 9 consecutive days during gestation days 7 to 15 to CD1 and A/J mice, and New Zealand (NZ) and Dutch Belted (DB) rabbits, and during days 8 to 16 to CD rats. Malformations attributable to MPA did not occur in fetuses of mice or rats exposed to the largest dosage tested. However, 1, 3, or 10 mg/kg on days 13 to 15 to NZ rabbits resulted in 6, 28, and 42% cleft palate, respectively. Comparable cleft palate frequencies were seen in DB offspring.

Depo-Provera--ethical issues in its testing and distribution.

Ethical issues relating to the use of the injectable contraceptive in developed and developing countries alike involve public policy decisions concerning both criteria for testing a new drug and individual choices about using a specific form of contraception approved for national distribution. Drug testing consists of an important but still evolving set of procedures. Depo-Provera is not qualitatively different from any other drug and some unpredictable risks are inevitable, even after extensive animal experiments and clinical trials. In assessing the risks and benefits of Depo-Provera use, epidemiological data from large-scale human use is now beginning to become more important than data from animal experiments and clinical trials. The consumer's best interest is central to any ethically responsible system of drug distribution. Systems of informed choice are needed, even in societies where illiteracy remains common and medical services are weak. In the case of a contraceptive, the risks of non-use leading to unintended pregnancy, which can result in high mortality, are relevant as well as the side-effects of the method. An attempt, therefore, is made here to categorise those issues which are universal and those which are country-specific.

PIP: Ethical issues relating to the use of the injectable contraceptive in developed and developing countries alike involve public policy decisions concerning both criteria for testing a new drug and individual choices about using a specific form of contraception approved for national distribution. Drug testing consists of an important but still evolving set of procedures. Depo-Provera is not qualitatively different from any other drug and some unpredictable risks are inevitable, even after extensive animal experiments and clinical trials. In assessing the risks and benefits of Depo-Provera use, epidemiological data from large scale human use is now beginning to become more important than data from animal experiments and clinical trials. The consumer's best interest is central to any ethically responsible system of drug distribution. Systems of informed choice are needed, even in societies where illiteracy remains common and medical services are weak. In the case of a contraceptive, risks of nonuse leading to unintended pregnancy, which can result in high mortality, are relevant as well as are the side effects of the method. An attempt, therefore, is made here to categorize those issues which are universal and those which are country specific.^ieng

A potential mechanism in medroxyprogesterone acetate teratogenesis.

PIP: MPA (medroxyprogeste)rone acetate) has been shown to be te)ratogenic in rabbits but not in rats or mice (Andrew and Staples 1977). Since normal steroid action appears to be mediated, in large part, through interaction with specific steroid receptors, it was postulated that the species difference in teratogenicity might be due to a difference in the interaction of MPA with target cells. A primary event in steroid-cell interaction is the binding of a steroid to intracellular receptors. Studies were initiated to measure the specific nature of MPA binding to glucocorticoid and progestin receptors in appropriate rat and rabbit target tissues. The competition of MPA with 3H-dexamethasone binding in liver cytosol (glucocorticoid receptor) and with 3H-progesterone binding in uterine cytosol (progesterone receptor) was determined. In rabbit liver cytosol, MPA was as effective at competing for specific dexamethasone binding as the natural glucocorticoids and considerably more effective than the nonspecific steroids. In rat liver cytosol MPA was only 10% as effective as the natural glucocorticoids and the competition could not be distinguished from that of nonspecific steroids. A similar species difference was not seen in uterine cytosol; MPA competed with progesterone in a similar fashion in both rat and rabbit. These data demonstrate a distinct species difference in the competitive nature of MPA for the glucocorticoid receptor but not for the progestin receptor. The results suggest that MPA, or possibly a metabolite, may be teratogenic in rabbits by binding with specific glucocorticoid receptors to inhibit or alter normal steroidal function in embryo-fetal development.^ieng