Soy isoflavone exposure through all life stages accelerates 17ß-estradiol-induced mammary tumor onset and growth, yet reduces tumor burden, in ACI rats.

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17ß-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56 %, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20 weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

Progesterone, as well as 17ß-estradiol, is important for regulating AHR battery homoeostasis in the rat uterus.

Several studies indicate that the aryl hydrocarbon receptor (AHR), which plays an important role in mediating the toxicity of many industrial chemicals, plays an important role in the physiology of female reproductive tract organs. This makes it likely that the AHR and additional components of the AHR signalling pathway are under the control of female sex steroids. In a previous study, we could already demonstrate the regulation of many members of the AHR battery by 17ß-estradiol (E2) in the uterus of rats. In this study, we addressed the potential role of progesterone (P4) in this context. In a comparative approach using ovariectomized rats which were treated for 3 days with either vehicle control, E2, progesterone (P4) or the combination of both hormones in addition to sham-operated animals, we could demonstrate that in addition to E2, P4 is also an important factor in regulating AHR signalling in the rat uterus. P4 has effects similar to E2 on uterine Ahr, Arnt and Arnt2 mRNA levels, resulting in a downregulation of these genes, while the E2-mediated downregulation of key AHR response genes Cyp1a1, Gsta2 and Ugt1 is completely antagonized by P4. As with E2, P4 leads to an increase in uterine AHR levels, especially in the endometrial epithelium despite the decrease in corresponding mRNA levels. This indicates a complex gene-specific regulatory network involving E2, P4 and possibly AHR itself to maintain all components of the AHR signalling cascade at the required levels during all stages of the oestrous cycle and pregnancy.

Regulation of uterine AHR battery gene expression by 17ß-Estradiol is predominantly mediated by estrogen receptor α.

The aryl hydrocarbon receptor (AHR) is known to mediate the cellular response to numerous xenobiotics including dioxin. Surprisingly AHR knockout mice provide evidence for the involvement of the AHR signalling cascade in estrogen regulated physiological functions of the female reproductive system. Several studies already aimed to investigate the impact of the AHR mediated xenobiotic response pathway on estrogen receptor (ER) signalling, whereas on contrary availability of data describing the effect of 17ß-Estradiol (E2) on the AHR signalling cascade is rather limited. In this study we observed an inhibitory effect of E2 treatment on uterine Ahr, Arnt, Arnt2, Ahrr, Cyp1a1, Ugt1 and Nfe2l2 gene expression in ovariectomized Wistar rats, whereas Cyp1b1, Nqo1 and Gsta2 displayed an increased transcription. The usage of the ER selective agonists, 16α-LE(2) (ERα selective) and 8ß-VE(2) (ERß selective), enabled us to distinguish between ER subtype specific responses. On mRNA level the observed changes in gene expression were mainly mediated by ERα except for the expression of Nqo1. In most cases the activation of ERß caused effects opposite to the ones observed following activation of ERα. Despite the significant changes in AHR mRNA levels immunohistochemical staining uterine tissue section did not reveal changes of the AHR protein level. Taken together our results validate, support and extend the hypothesis of uterine crosstalk between AHR and ER signalling pathways. Furthermore they give an insight into how the AHR and its related genes may participate in E2 dependent uterine physiological processes and provide another potential mechanism of action for xenoestrogens.

The rat prepubertal uterine myometrium and not the luminal epithelium is predominantly affected by a chronic dietary genistein exposure.

Current knowledge about dietary soy isoflavone-induced hormonal effects and potential priming effects for the responsiveness of the organism to other estrogens is insufficient. The present study examined the effects of pre- and postnatal soy isoflavone exposure on estrogen responsiveness by estrogen receptor agonists in the uteri of prepubertal Wistar rats. To this end, offspring were generated from dams already maintained on three dietary groups, (1) a phytoestrogen-free diet, (2) a soy isoflavone-rich diet with 232 ppm daidzein and 240 ppm genistein or (3) a custom-made diet supplemented with 700 ppm genistein (GEN). Then, F1 females continuously exposed to isoflavones from GD1 to PND21 and non-exposed controls were subjected to an immature uterotrophic assay to compare physiological parameters and the response to subcutaneous treatment with 17ß-estradiol, GEN or an estrogen receptor subtype (ERα and ERß)-specific agonist. Uterine wet weight (UWW), luminal epithelial height (LEH) and myometrial thickness (MMT) were determined. In addition, isoflavone plasma levels and mRNA expression profiles of relevant steroid receptors and of molecular markers for proliferation and estrogenicity were assessed for all groups. The influence of dietary isoflavones on the sensitivity to various estrogenic stimuli in these prepubertal animals was minor. Yet, the uterus of immature rats with high chronic GEN exposure alone showed already an increase in UWW, LEH and MMT. The myometrial response to GEN was more pronounced than that of the luminal epithelium, which may be due to a non-uniform distribution of steroid receptors, in particular the progesterone receptor. In conclusion, although the impact of a continuous, prenatally initiated exposure to dietary isoflavones on the uterine physiology of juvenile rats is modest, the possible priming effects of this exposure for beneficial or adverse late-onset consequences in adults should not be neglected.

Dose-dependent effects of isoflavone exposure during early lifetime on the rat mammary gland: Studies on estrogen sensitivity, isoflavone metabolism, and DNA methylation.

SCOPE: Isoflavone (ISO) exposure during adolescence modulates 17ß-estradiol (E2) sensitivity of the adult mammary gland. The present study investigated the dose dependency of these effects focusing on proliferation, estrogen receptor dependent and independent gene expression, as well as DNA methylation and ISO metabolism. METHODS AND RESULTS: Female Wistar rats were lifelong exposed to an ISO-depleted diet or to diets enriched with a soy ISO extract (ISO-rich diet (IRD)) causing plasma concentrations as observed minimally (IRDlow) and maximally (IRDhigh) in Asian women. The extract was characterized by both phytochemical analysis and E-Screen. Rats were ovariectomized at postnatal day (PND) 80 and treated with E2 from PND94 to 97. In contrast to uterine response, body weight and visceral fat mass were affected by ISO. In the mammary gland, both E2-induced proliferation (proliferating cell nuclear antigen staining) and estrogen receptor activation (progesterone receptor staining) were significantly reduced by IRDhigh but not by IRDlow, which however attenuated Gdf15 mRNA expression. DNA methylation analysis revealed significant differences in the promoter regions of Aldhl1, Extl1, and WAP between IRDhigh and ISO-depleted diet. CONCLUSION: Lifelong exposure to ISO results in dose-dependent differential effects on proliferation, gene expression, and DNA methylation in rat mammary glands. Yet, a decrease in estrogen responsiveness was only achieved by IRDhigh.

In utero and postnatal exposure to isoflavones results in a reduced responsivity of the mammary gland towards estradiol.

SCOPE: Exposure scenarios during different stages of development of an organism are discussed to trigger adverse and beneficial effects of isoflavones (ISO). The aim of this study was to investigate how in utero and postnatal ISO exposure modulates the estrogen sensitivity of the mammary gland and to identify the underlying molecular mechanisms. METHODS AND RESULTS: Therefore, rats were exposed to either ISO-free (IDD), ISO-rich (IRD) or genistein-rich diet (GRD), up to young adulthood. Proliferative activity (PCNA expression) in the mammary gland at different ages and the estrogen sensitivity of the mammary gland to estradiol (E2) or genistein (GEN) in adult ovariectomized animals was determined and compared with different treatments. Treatment with E2 resulted in a significant lower proliferative and estrogenic response of the mammary gland in IRD and GRD compared with IDD. This correlates to a change in the gene expression pattern and a decrease in the ratio of estrogen receptor alpha (ERα) beta (ERß CONCLUSIONS: Our results provide evidence that in utero and postnatal exposure to a diet rich in ISO but also to GEN reduces the sensitivity of the mammary gland toward estrogens and support the hypothesis that in utero and postnatal ISO exposure reduces the risk to develop breast cancer.

Long-term dietary isoflavone exposure enhances estrogen sensitivity of rat uterine responsiveness mediated through estrogen receptor alpha.

The outcome of long-term exposure to dietary isoflavones on estrogen sensitive tissues is discussed controversially. We performed a study on tissue specific effects of lifelong isoflavone exposure on the rat uterus with exposure being initiated prenatally. We compare the effects of the dietary isoflavones, genistein (GEN) and daidzein, or GEN alone to those of isoflavone free diet. Therefore, one group received a phytoestrogen-free diet (PE-free), one an isoflavone-high diet (ISO-high) and one the PE-free diet supplemented with GEN (GEN-rich) throughout their whole lifetime. In ovariectomized adult females a uterotrophic assay was performed, comparing 17beta-estradiol, GEN and two estrogen receptor subtype-specific agonists. The uterus wet weight, the uterine epithelial heights, and uterine markers for proliferation, estrogenicity and estrogen-dependent water channels were determined on mRNA and protein level. The dietary ISO pre-exposure results in a much stronger uterine weight increase following external ERalpha-mediated estrogenic stimuli than seen in the PE-free group. These strongly increased effects were not exclusively due to proliferation hence proliferation associated parameters were almost identical in all groups. Additionally, gene expression analysis showed that estrogen-dependent water channels are highly affected by ISO-containing diets. In conclusion, the lifelong dietary ISO ingestion enhances severely the uterine responsiveness to ERalpha-mediated estrogenic stimuli in female rats. While the uterine proliferation rate was not affected, the water homeostasis was highly affected. Our data clearly demonstrate that estrogen responsiveness is highly modulated by dietary isoflavones. Whether this estrogen sensitivity shift is beneficial or adverse to health remains to be elucidated. However, this is highly relevant for interpreting data from regional differences in endocrine cancer.

Estradiol regulates aryl hydrocarbon receptor expression in the rat uterus.

Several authors have investigated the role of aryl hydrocarbon receptor (AHR) in the reproductive tract, but there are no data available whether 17beta-estradiol (E2) regulates expression of members of the AHR pathway in the uterus. We therefore examined the mRNA expression of Ahr as well as the genes of the AHR dimerization partners ARNT1 and ARNT2 and the AHR regulated genes Cyp1a1 and Gsta2 in the uterus of ovariectomized rats after administration of E2 at two different doses. The data show that Ahr mRNA expression is downregulated while AHR protein amounts increased in all uterine tissue compartments. In addition we observed a downregulation of Arnt1, Arnt2 and Cyp1a1 while Gsta2 mRNA expression is upregulated by E2 in a dose-dependent manner. These results show that members of the AHR pathway are regulated by E2 in the uterus. AHR may therefore play an important role in the mediation of uterine estrogenic effects.

Long-term effects of dietary isoflavones on uterine gene expression profiles.

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects. Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 microg/g diet) and an ISO-high diet (232 microg daidzein and 240 microg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring. We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018+/-350 mg/kg BW) than with ISO-high diet (497+/-133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects. In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.