Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) Hull Extract and Active Compounds Inhibit Proliferation of Primary Human Leiomyoma Cells and Protect against Sexual Hormone-Induced Mice Smooth Muscle Hyperproliferation.

Uterine leiomyomas, also known as fibroids, are benign neoplasms of the uterus and have a high incidence rate in women of reproductive age. Hysterectomy or myomectomy is the initial treatment, but fibroids will recur if the patient is still exposed to similar risk factors. Therefore, developing new therapeutic strategies are urgently necessary. In this study, the anti-proliferation effects of each fraction of adlay seeds were evaluated in uterine leiomyomas, and we identified the potential phytochemical compounds. We found that the ethyl acetate fraction of adlay hull (AHE-ea) appeared to be highly efficient in the anti-proliferation of rat uterine leiomyoma ELT3 cells and primary human uterine leiomyoma (hUL) cells. The proliferation of primary human normal uterine smooth muscle (UtSMC) and normal uterine myometrial (hUM) cells were also suppressed by AHE-ea. Two phytosterols, stigmasterol and ß-sitosterol, were identified from AHE-ea fraction. Mice treated with AHE-ea and stigmasterol alone demonstrated reduced diethylstilbestrol/medroxyprogesterone 17-acetate (DES/MPA)-induced uterine myometrial hyperplasia, which is the critical step for the development of leiomyoma. Taken together, our results suggest that the AHE-ea fraction could be considered as a natural plant-based medicine in the prevention or treatment of uterine leiomyoma growth.

The angiotensin receptor blocker, Losartan, inhibits mammary tumor development and progression to invasive carcinoma.

Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2-ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.

Luteolin suppresses development of medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumors in Sprague-Dawley rats.

Postmenopausal women undergoing hormone-replacement therapy containing both progestins and estrogens are at an increased risk of developing breast cancer compared with women taking estrogen alone. We recently demonstrated that medroxyprogesterone acetate, a progestin commonly used for hormone-replacement therapy, accelerates development of mammary carcinogenesis in 7,12-dimethylbenz(a)anthracene­treated Sprague-Dawley rats. Synthetic antiprogestins used to block the deleterious effects of progestins, are themselves associated with toxic side-effects. In order to circumvent this, we used the aforementioned model to identify less toxic natural compounds that may prevent the development of progestin-accelerated tumors. Luteolin, a naturally-occurring flavonoid commonly found in fruits and vegetables, has previously been shown to possess anticancer properties. In our studies, both low (1 mg/kg) and high (25 mg/kg) doses of luteolin significantly suppressed progestin-dependent increases in tumor incidence, while increasing tumor latency and reducing the occurrence of large (>300 mm3) mammary tumors. However, an intermediate dose of luteolin (10 mg/kg), while suppressing the development of large tumors, did not affect either tumor incidence or latency. Immunohistochemical analysis of tumor tissues revealed that all concentrations of luteolin (1, 10, and 25 mg/kg) significantly reduced levels of VEGF within tumors. The suppressive effects of luteolin on tumor incidence and volume, together with its ability to reduce VEGF and blood vessels, persisted even after treatment was terminated. This suggests that luteolin possesses anti­angiogenic properties which could mechanistically explain its capacity to control tumor progression. Thus luteolin may be a valuable, non-toxic, naturally-occurring anticancer compound which may potentially be used to combat progestin-accelerated mammary tumors.

Medroxyprogesterone acetate aggravates oxidative stress and left ventricular dysfunction in rats with chronic myocardial infarction.

The role of estrogens during myocardial ischemia has been extensively studied. However, effects of a standard hormone replacement therapy including 17ß-estradiol (E2) combined with medroxyprogesterone acetate (MPA) have not been assessed, and this combination could have contributed to the negative outcomes of the clinical studies on hormone replacement. We hypothesized that adding MPA to an E2 treatment would aggravate chronic heart failure after experimental myocardial infarction (MI). To address this issue, we evaluated clinical signs of heart failure as well as left ventricular (LV) dysfunction and remodeling in ovariectomized rats subjected to chronic MI receiving E2 or E2 plus MPA. After eight weeks MI E2 showed no effects. Adding MPA to E2 aggravated LV remodeling and dysfunction as judged by increased heart weight, elevated myocyte cross-sectional areas, increased elevated left ventricle end diastolic pressure, and decreased LV fractional shortening. Impaired LV function in rats receiving MPA plus E2 was associated with increased cardiac reactive oxygen species generation and myocardial expression levels of NADPH oxidase subunits. These results support the interpretation that adding MPA to an E2 treatment complicates cardiovascular injury damage post-MI and therefore contributes to explain the adverse outcome of prospective clinical studies.

Progesterone receptor induces ErbB-2 nuclear translocation to promote breast cancer growth via a novel transcriptional effect: ErbB-2 function as a coactivator of Stat3.

Progesterone receptor (PR) and ErbB-2 bidirectional cross talk participates in breast cancer development. Here, we identified a new mechanism of the PR and ErbB-2 interaction involving the PR induction of ErbB-2 nuclear translocation and the assembly of a transcriptional complex in which ErbB-2 acts as a coactivator of Stat3. We also highlighted that the function of ErbB-2 as a Stat3 coactivator drives progestin-induced cyclin D1 promoter activation. Notably, PR is also recruited together with Stat3 and ErbB-2 to the cyclin D1 promoter, unraveling a new and unexpected nonclassical PR genomic mechanism. The assembly of the nuclear Stat3/ErbB-2 transcriptional complex plays a key role in the proliferation of breast tumors with functional PR and ErbB-2. Our findings reveal a novel therapeutic intervention for PR- and ErbB-2-positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.

Differential effects of 17beta-estradiol and of synthetic progestins on aldosterone-salt-induced kidney disease.

UNLABELLED: Elevated mineralocorticoid levels and female sex hormones have been shown to confer opposing effects on renal injury, but their combined effects are still unknown. OBJECTIVE: Identify the function of estrogens and of different synthetic progestins on aldosterone salt-mediated renal disease. METHODS: The role of 17beta-estradiol, medroxyprogesterone acetate (MPA), and drospirenone during renal injury was studied in Wistar rats subjected to uni-nephrectomy plus aldosterone salt treatment. RESULTS: Aldo-salt treatment of intact, ovariectomized, and estradiol-treated female rats resulted in remnant kidney hypertrophy without structural damage. Co-treatment with MPA, but not with drospirenone, increased kidney hypertrophy, fluid turnover, sodium retention, and potassium excretion. Medroxyprogesterone acetate also caused glomerular, vascular, tubular, and interstitial lesions that were accompanied by increased blood pressure and enhanced NADPH oxidase (p67phox) and sodium channel (alpha-ENaC) expression. Drospirenone, a progestin with anti-mineralocorticoid function, and spironolactone prevented kidney hypertrophy, hypertension, and sodium retention. Drospirenone and spironolactone also increased renal angiotensin II type 2 receptor expression and relieved aldosterone-induced suppression of serum angiotensin II levels. CONCLUSION: The choice of specific synthetic progestins has profound implications on the development of kidney injury and renal gene expression under conditions of elevated aldosterone serum levels and salt intake.

The MPA mouse breast cancer model: evidence for a role of progesterone receptors in breast cancer.

More than 60% of all breast neoplasias are ductal carcinomas expressing estrogen (ER) and progesterone receptors (PR). By contrast, most of the spontaneous, chemically or mouse mammary tumor virus induced tumors, as well as tumors arising in genetically modified mice do not express hormone receptors. We developed a model of breast cancer in which the administration of medroxyprogesterone acetate to BALB/c female mice induces mammary ductal carcinomas with a mean latency of 52 weeks and an incidence of about 80%. These tumors are hormone-dependent (HD), metastatic, express both ER and PR, and are maintained by syngeneic transplants. The model has been further refined to include mammary carcinomas that evolve through different stages of hormone dependence, as well as several hormone-responsive cell lines. In this review, we describe the main features of this tumor model, highlighting the role of PR as a trigger of key signaling pathways mediating tumor growth. In addition, we discuss the relevance of this model in comparison with other presently used breast cancer models pointing out its advantages and limitations and how, this model may be suitable to unravel key questions in breast cancer.

Differential effects of medroxyprogesterone acetate on thrombosis and atherosclerosis in mice.

BACKGROUND AND PURPOSE: The risk for cardiovascular events including venous and arterial disease and stroke is elevated after treatment with estrogen and medroxyprogesterone acetate (MPA) in postmenopausal women. Here, we have investigated the effect of MPA on arterial thrombosis and atherosclerosis in a murine model of atherosclerosis. EXPERIMENTAL APPROACH: Apolipoprotein E (ApoE)-/- mice were bilaterally ovariectomized and treated with placebo, MPA (27.7 microg day(-1)) and MPA + 17-beta-oestradiol (E2; 1.1 microg day(-1)) for 90 days, on a Western-type diet. Thrombotic response was measured in a photothrombosis model, platelet activation by fluorescence activated cell sorting (FACS) analysis (CD62P) and thrombin generation by the endogenous thrombin potential (ETP). Furthermore, aortic plaque burden and aortic root plaque composition were determined. KEY RESULTS: MPA and MPA + E2-treated animals showed an aggravated thrombotic response shown by significantly reduced time to stable occlusion. The pro-thrombotic effect of MPA was paralleled by increased ETP whereas platelet activation was not affected. Furthermore, MPA + E2 reduced the number of cells positive for alpha-smooth muscle actin and increased hyaluronan in the plaque matrix. Interestingly, total plaque burden was reduced by MPA but unchanged by MPA + E2. CONCLUSION AND IMPLICATIONS: Long-term treatment with MPA and MPA + E2 increased arterial thrombosis despite inhibitory effects of MPA on atherosclerosis in ApoE-deficient mice. Increased thrombin formation, reduced smooth muscle content and remodelling of non-collagenous plaque matrix may be involved in the pro-thrombotic effects. Thus, MPA exhibits differential effects on arterial thrombosis and on atherosclerosis.

Progesterone increases airway eosinophilia and hyper-responsiveness in a murine model of allergic asthma.

BACKGROUND: Sex hormones might affect the severity and evolution of bronchial asthma. From existing literature, there exists, however, no convincing evidence for either exacerbation or improvement of allergic symptoms by progesterone. OBJECTIVE: This study was aimed to explore the effect of exogenously administered progesterone in a mouse model of allergic asthma. METHODS: BALB/c mice were sensitized to ovalbumin (OVA) by intraperitoneal injections with OVA followed by chronic inhalation of nebulized OVA or physiologic saline (Sal). Medroxyprogesterone acetate or placebo was instilled daily into the oesophagus before and during the inhalatory OVA challenge phase. RESULTS: Progesterone worsened allergic airway inflammation in OVA-challenged mice, as evidenced by enhanced bronchial responsiveness to inhaled metacholine and increased bronchial eosinophilia. Elevated airway eosinophilia corresponded with higher bronchial and systemic IL-5 levels in the progesterone group. The ratio of IL-4/IFN-gamma levels in bronchoalveolar lavage fluid and numbers of eosinophil colony-forming units in the bone marrow were also elevated in the latter group. Progesterone, however, did not influence allergen-specific IgE production, nor did it affect bronchial responses in Sal-challenged mice. CONCLUSION: Our data show that exogenously administered progesterone aggravates the phenotype of eosinophilic airway inflammation in mice by enhancing systemic IL-5 production. Progesterone also increases bronchial hyper-reactivity.

Novel murine mammary epithelial cell lines that form osteolytic bone metastases: effect of strain background on tumor homing.

We have developed a series of novel mammary epithelial cell lines from tumors arising in strain 129 mice, with the ultimate goal of evaluating the role of host factors in the development of bone metastases. Mammary tumors were induced in mice with subcutaneously implanted medroxyprogesterone acetate (MPA) pellets followed by administration of DMBA by oral gavage. Mammary tumor development was efficient in the 129 strain and was independent of osteopontin (OPN) expression. Epithelial cell lines were isolated from these tumors; surprisingly, these cells did not form tumors upon inoculation into the mammary fat pad of syngeneic mice, even when MPA was present. One OPN-deficient cell line was selected for further study; full transformation of these cells required expression of both polyoma middle T and activated ras. These doubly transfected cells, 1029 GP+Er3, grew in soft agar, and formed hormone-independent tumors efficiently in the mammary fat pad that spontaneously metastasized to several soft tissue sites but not to the bone. Derivatives of these cells were isolated from tumors arising in the fat pad and from a lung metastasis (r3T and r3L, respectively): these cells formed tumors more rapidly in the fat pad than the parental GP+Er3 cells. Upon left ventricle injection, the r3T and r3L cells formed osteolytic bone metastases in 129 mice, with few metastases seen in other organs. These tumors filled the marrow cavity, and caused extensive destruction of both cortical and trabecular bone. Intriguingly, in an alternative syngeneic host, (129xC57B1/6) F1, osteolytic bone metastases were not seen on x-ray; instead extensive liver metastasis was present in these mice, indicating that genetic factors in these two strains regulate tumor cell homing and distribution during metastasis. These cell lines provide an important new tool in the study of bone metastasis, particularly in elucidating the role of host factors in the development of these lesions, as the 129 mouse strain is frequently used for genetic manipulations in the mouse.

The effect of medroxyprogesterone acetate on bone marrow and testis during cytotoxic chemotherapy.

The effect of medroxyprogesterone acetate (MPA) on the mitotic activity of bone marrow and testis during chemotherapy was investigated experimentally in an animal study. A total of 120 male Swiss albino mice were included in this study. Six groups were formed, each consisting of 20 mice. Low-dose MPA (LD-MPA) (15 mg/kg), high-dose MPA (HD-MPA) (100 mg/kg), LD-MPA plus cyclophosphamide (CP) (65 mg/kg), HD-MPA plus CP (65 mg/kg), and CP (65 mg/kg) were administered to the test groups and no drug was administered to the control group. Bone marrow samples and testis were examined for mitotic activity rate (MAR) on days 0, 18, 22, 26, and 30. In groups with regimens containing CP, MAR of hematopoietic cells in bone marrow was suppressed significantly (p<0.05). There was no difference in MAR of hematopoietic cells in bone marrow between the groups given MPA or not (p>0.05). Mitotic activity rate of the testis cells was significantly suppressed in groups with regimens containing MPA (p<0.05). In conclusion, MPA inhibited mitotic activity of testis, but there was no effect on the mitotic activity of bone marrow. These data do not seem to confirm the hypothesis of a myeloprotective effect of MPA.

Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas.

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.

Progesterone receptor involvement in independent tumor growth in MPA-induced murine mammary adenocarcinomas.

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.

Estrogen-induced hepatic toxicity and hepatic cancer: differences between two closely related hamster species.

AIMS/BACKGROUND: Estrogen is known to affect hepatobiliary function; however, it is unusual for high serum levels of estrogen to actually result in clinically detectable hyperbilirubinemia. Women affected by cholestatic jaundice during pregnancy share this genetic susceptibility with two Cricetulus hamsters, the Armenian hamster (Cricetulus migratorius) and the Chinese hamster (Cricetulus griseus). Nevertheless, the pathophysiologic process responsible for this estrogen induced icterus may be different in women and hamsters. The present study compares various facets of estrogen-induced icterus in these two closely related hamsters. METHODS: Hamsters were injected with various estrogens and the acute and chronic effects on liver were monitored by measuring changes in serum constituents and by observing changes in hepatic structure as seen grossly and by light and electron microscopy. RESULTS: In previous studies, hepatic tumors developed in most Armenian hamsters after chronic estrogen treatment, but in the present study, the livers of Chinese hamsters were remarkably free of neoplastic change under similar conditions. Also, when compared with the responses in the Armenian hamsters, signs of hepatic destruction and regeneration were less prevalent in estrogen-treated Chinese hamsters, and they were less susceptible to the effects of estrogen (because larger doses of estrogen were required to produce icterus and the bilirubin levels were lower and of shorter duration). In contrast to the findings in Armenian hamsters, bile canaliculi were severely affected in livers of estrogen-treated Chinese hamsters, and hepatic microvesicular steatosis, indicative of an unusual lipodystrophy caused by estrogen, was prominent. An additional lesion peculiar to the Chinese hamster was striking sinusoidal dilatation, which may be analogous to the oral contraceptive-induced sinusoidal dilatation in humans. CONCLUSIONS: Although these two hamster species are genetically similar, the genes activated by the estrogen receptor show remarkable heterogeneity when their respective livers are examined. Comparisons within these species may provide information about the specific gene activation responsible for particular pathologic events.

Promoter effect of medroxyprogesterone acetate (MPA) in N-methyl-N-nitrosourea (MNU) induced mammary tumors in BALB/c mice.

The promoter effect of medroxyprogesterone acetate (MPA) on mammary carcinogenesis in female BALB/c mice was investigated using methylnitrosourea (MNU) as initiator. Nine out of 43 animals developed mammary carcinomas in the group treated with MNU (50 mg/kg) and MPA (administration of 40 mg every 3 months) starting 1 week after MNU administration. No tumors appeared in controls receiving only MNU or MPA during the time course of the experiment (9 months). The tumors were lobular adenocarcinomas showing different degrees of squamous differentiation with low or undetectable estrogen and progesterone receptors, and expressing epidermal growth factor receptors. These results support the hypothesis that MPA promotes the growth of MNU induced lesions.

Effects of chronic hormone replacement on the renin-angiotensin system in cynomolgus monkeys.

OBJECTIVE: To characterize the effects of estrogen, estrogen combined with progestin, and no treatment in ovariectomized cynomolgus monkeys during long-term reproductive hormone replacement. METHODS: Forty-five surgically postmenopausal cynomolgus monkeys fed a lipid-lowering diet were administered a conjugated equine estrogen (Premarin, 7.2 micrograms/day for the first 8 months, then 166 micrograms/day for the remaining 22 months), alone or in combination with 650 micrograms/day medroxyprogesterone acetate (Cycrin) for 30 months, or left with no hormone replacement therapy. Animals were anesthetized with ketamine-pentobarbital, and samples were taken for measurements of plasma renin activity, angiotensin converting enzyme activity, and angiotensin peptides, angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)]. RESULTS: Chronic replacement therapy with estrogen resulted in a significant elevation of the plasma renin activity [11.7 +/- 2.0 ng/ml per h control versus 22.8 +/- 4.6 ng/ml per h with estrogen (P < 0.05) versus 32.8 +/- 4.9 ng/ml per h with combination therapy (P < 0.01)], whereas estrogen or combination therapy caused a significant reduction in angiotensin converting enzyme activity [229 +/- 8 nmol/ml per min control versus 189 +/- 10 nmol/ml per min with estrogen (P < 0.05) versus 196 +/- 11 nmol/ml per min with combination therapy (P < 0.05)]. Both of these changes in angiotensin processing enzymes observed during replacement therapy resulted in significant increases in plasma Ang I levels [46.7 +/- 12.5 pg/ml control versus 175.5 +/- 65.9 pg/ml with estrogen (P < 0.05) and 561.7 +/- 373.6 pg/ml with combination therapy (P < 0.05)]. Plasma Ang II and Ang-(1-7) levels were not significantly changed. The mean blood pressure did not change with either treatment. CONCLUSION: These studies reveal that, although chronic estrogen replacement activates renin activity and Ang I, it causes a shift in the processing of angiotensin peptides such that the concurrent reduction in angiotensin converting enzyme activity leads to unchanged plasma Ang II levels. Thus, the potentially harmful effects of estrogen-induced hyperreninemia are balanced by its actions interfering with the formation of the vasoactive product Ang II.

Formation of DNA adducts by cyproterone acetate and some structural analogues in primary cultures of human hepatocytes.

Recently, we have shown that the therapeutically-used progestin and antiandrogen cyproterone acetate (CPA) causes the formation of high levels of DNA adducts in rat hepatocytes and rat liver [J. Topinka, U. Andrae, L.R. Schwarz, T. Wolff, Cyproterone acetate generates DNA adducts in rat liver and in primary rat hepatocyte cultures, Carcinogenesis 14 (1993) 423-427: J. Topinka, B. Binkova, L.R. Schwarz, T. Wolff, Cyproterone acetate is an integral part of hepatic DNA adducts induced by this steroidal drug, Carcinogenesis 17 (1996) 167-169; S. Werner, J. Topinka, T. Wolff, L.R. Schwarz, Accumulation and persistence of DNA adducts of the synthetic steroid cyproterone acetate in rat liver, Carcinogenesis 16 (1995) 2369-2372; J. Topinka, B. Binkova, H.K. Zhu, U. Andrae, I. Neumann, L.R. Schwarz, S. Werner, T. Wolff, DNA damaging activity of the cyproterone acetate analogues chlormadinone acetate and megestrol acetate in rat liver, Carcinogenesis 16 (1995) 1483-1487]. Its structural analogues, chlormadinone acetate (CMA) and megestrol acetate (MGA) were much less active in this respect [J. Topinka, B. Binkova, H.K. Zhu, U. Andrae, I. Neumann, L.R. Schwarz, S. Werner, T. Wolff, DNA damaging activity of the cyproterone acetate analogues chlormadinone acetate and megestrol acetate in rat liver, Carcinogenesis 16 (1995) 1483-1487]. In the present study we addressed the question whether these compounds and two further analogues, medroxyprogesterone acetate (MPA) and progesterone, induce the formation of DNA adducts in primary cultures of human hepatocytes. Incubation of CPA with human hepatocytes from two male and four female donors induced the formation of significant levels of CPA-DNA adducts detectable by 32P-postlabeling. The by far most prevalent DNA adduct is most likely identical with adduct A formed in CPA-treated rats. DNA binding was found even at 0.03 microM CPA, the lowest concentration used. The maximum effect occurred at about 10 microM in 5 of the 6 cell preparations. At this concentration 480 and 2690 adducts x 10(-9) nucleotides were detected in hepatocytes of the two male donors and 1072, 816, 613 and 659 adducts x 10(-9) nucleotides in the hepatocytes of the four female donors after an exposure of 6 h with CPA. Extending the incubation time to 20 h resulted in a roughly three-fold higher binding. CMA and MGA were assayed in two of the liver cell preparations from the female donors. At a concentration of 20 microM and an incubation time of 6 h, DNA adduct levels for CMA were 21 and 43% and for MGA 31 and 65% of the levels observed with 20 microM CPA. In contrast, DNA binding of MPA amounted to less than 1% of that observed with CPA and DNA binding of the natural occurring progestin progesterone was below the level of detection. The results point to a genotoxic risk associated with the therapeutic use of CPA and possibly of CMA and MGA. Furthermore, the findings suggest that the significant genotoxicity observed with CPA, MGA and CMA is associated with the presence of the double bond in position 6-7 of the steroid, which is absent in MPA and progesterone.

Medroxyprogesterone acetate accelerates the development and increases the incidence of mouse mammary tumors induced by dimethylbenzanthracene.

Chemical induction of mammary tumors in mice requires usually a long latency period and is often complicated by high non-mammary tumor related mortality. Classically hormone stimulation has been used as the means to increase tumor incidence. The synthetic progestin medroxyprogesterone acetate (MPA) was postulated by some authors to increase mammary tumor incidence in various rodent models. However, controversy exists regarding the role of MPA in experimental and human carcinogenesis. In our study we tested the use of a protocol of combined MPA- and dimethylbenz[a]anthracene (DMBA) treatment for the obtention of mammary tumors with a short latency and with a lower toxicity than the classical multiple dose DMBA protocol. MPA was very effective in accelerating the development and increasing the incidence of mammary tumors induced by DMBA in CD2F1 mice. MPA by itself did not produce any mammary tumors. The mean latency for tumor development from the end of carcinogen treatment was 99 +/- 51 days in the group that received a combination of MPA and four DMBA doses. This group showed significantly earlier mammary tumor incidence (P < 0.0001) and higher tumor numbers than the groups that received only DMBA. Mammary tumors were also analyzed for effects on the mutation rate affecting the Ha-ras and Ki-ras genes. Our data is consistent with MPA probably increasing the number of target cells at risk for mutation by the chemical carcinogen DMBA and possibly promoting the faster development of tumors.

Evaluation of the mutagenicity of medroxyprogesterone acetate in vitro and in vivo.

Medroxyprogesterone acetate (CAS 71-58-9) is used for cyclic hormone substitution during the menopause and in hormone therapy of breast cancer. The test substance was investigated for its mutagenic potential in a series of test systems according to the current EC guidelines. There were no indications for a mutagenic potential of medroxyprogesterone acetate.

Effect of sialoadenectomy on medroxyprogesterone-acetate-induced mammary carcinogenesis in BALB/c mice. Correlation between histology and epidermal-growth-factor receptor content.

To evaluate the possible involvement of the salivary glands in the modulation of medroxyprogesterone (MPA)-induced mammary tumorigenesis, 48 sialoadenectomized virgin BALB/c female mice and 47 controls were treated with 40mg MPA depot s.c. every 3 months for 1 year. Mammary tumors developed in 11 sialoadenectomized and in 34 control mice with similar latencies. In both groups, 75% of the tumors were ductal and progestin-dependent (PD) while the remainder were lobular and progestin-independent (PI). Epidermal growth factor (EGF) levels were measured in salivary glands (SG-EGF) and serum (S-EGF) in both groups. MPA induced a significant increase in SG-EGF and in S-EGF that became evident only after 1 month of MPA treatment. No increase in S-EGF was detected in MPA-treated sialoadenectomized mice, indicating that salivary glands are the major source of S-EGF. The presence of EGF receptors (EGF-R) was investigated in ductal PD and PI tumor lines and compared with 8 PI tumor lines of lobular origin. A significant difference in EGF-R content was found between lobular and ductal tumors. No increase in EGF-R was noted when ductal tumors became autonomous. EGF-R did not correlate with tumor growth rate and there was an inverse correlation between EGF-R and steroid receptors. When the effect of sialoadenectomy on tumor growth was tested in vivo in syngeneic transplants of 2 ductal PD, 1 ductal PI and 2 lobular PI mammary adenocarcinomas, it was not found to be significant when compared with the controls. It may be concluded that SG-EGF plays an important role in the induction of mammary adenocarcinomas by MPA, while it has no significant effect on the growth of established tumors.