Patrick Diel

Tissue-specific estrogenic response and molecular mechanisms

Estrogens exert profound effects on growth, differentiation, and function of many reproductive tissues. They also affect other tissues, including bone, liver, cardiovascular system, and brain. In the last few years it has been demonstrated that several synthetic estrogens can act in a tissue-specific manner. The first example of such a selective estrogen receptor modulator (SERM) was tamoxifen, for which an estrogen agonist-like activity in the endometrium and bone was seen to occur simultaneously with an estrogen antagonist activity in the breast. The mechanisms by which the same compound can exert tissue-specific agonist and antagonist actions are still being investigated. Important aspects include the interaction of the ligand with the two estrogen receptor subtypes and the interaction of these ligand-receptor complexes with effectors, which include different DNA response elements and important coregulator proteins. In addition to well-documented effects on gene transcription, there is evidence that estrogen receptors and other estrogen binding proteins are involved in some rapid, non-genomic effects of estrogens in target cells. For these reasons it is important to point out that a toxicological evaluation of endocrine modulators should include an analysis of potential SERM-like properties.

Estrogenic isoflavones in rodent diets

Many rodent diets contain components such as soy isoflavones (daidzein and genistein) known to have estrogenic properties. The dietary background of phytoestrogens may modulate some responses to environmental estrogens when these compounds are tested in rodent bioassays. Thus, and since only few data were available on the phytoestrogen content of rodent diets commonly used in European laboratories, it was of interest to analyze the daidzein and genistein contents of our standard animal feeds. Isoflavone contents were determined in seven batches of rodent chow (from two suppliers in Germany, Altromin and Ssniff) by high-performance liquid chromatography, and also analyzed in six rodent diets from the United States. The soy-based rodent diets from Germany contained isoflavone (daidzein plus genistein) concentrations in the range of 0.3-0.55 mg/g feed. These isoflavone contents are similar to those analyzed in the US rodent diets, and similar to values reported by others, including one particular lot of feed (with 0.35 mg isoflavones per g) which produced a large uterotrophic response in immature ovariectomized rats [Environ. Health Perspect., 106 (1998) 369]. Coumestrol was found in a sample of commercial rabbit food at rather high levels (0.27 mg/g), but, this phytoestrogen was not detected (<1 microg/g feed) in any of the other samples we analyzed. The soy components in our rodent diet produce a measurable background of daidzein and genistein in blood of female DA/Han rats, total isoflavones (aglycone plus conjugates) ranging between 90 and 290 ng/ml plasma. The ovariectomized animals kept on this chow, showed no signs of estrogenization of the reproductive tract (uterus, vagina), and responded normally to (xeno-)estrogen administration in a uterotrophic assay [J. Steroid Biochem. Mol. Biol., 73 (2000) 1]. Moreover, ovariectomized Wistar rats on our standard rodent diet (Ssniff R/M H) had lower uterine weights than animals kept on the isoflavone-free (solvent extracted) chow; both groups of rats responded to genistein administration with an increase in uterine weights. These results suggest that--albeit the sensitivity of the rodent uterotrophic assay is not reduced by the use of a diet containing soy isoflavones at commonly encountered levels--attention should be given to a variable dietary phytoestrogen background.

The differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and proliferation in the rat is associated with a substance specific modulation of uterine gene expression

In this study the ability of the phytoestrogen genistein (GEN) to regulate proliferation in the rat uterine tissue and the associated molecular mechanisms were investigated in a dose and time dependent manner. A single administration of GEN induced a rapid increase of the uterine weight during the first 24 h. In contrast to E2, treatment with GEN for 3 days did not result in a further increase of the uterine weight. GEN only marginally effected the thickness of the uterine epithelium and the expression of epithelial proliferating cell nuclear antigen (PCNA). Whereas, estrogen sensitive genes were modulated significantly, the expression of key genes involved in the regulation of proliferation (PCNA, ERalpha /ERbeta ratio) remained unaffected by GEN. Our results indicate that GEN has only a limited ability to activate molecular mechanisms involved in the induction of proliferation whereas estrogen sensitive genes are induced in a estrogen like manner.

The pure antiestrogen ICI 182780 is more effective in the induction of apoptosis and down regulation of BCL‐2 than tamoxifen in MCF‐7 cells

There is increasing evidence that induction of apoptosis by antihormones is an important mechanism in regard to their growth inhibitory action on hormone dependent tumors. In this report we have compared the efficiency of tamoxifen (Tam) and the pure antiestrogen ICI 182780 (ZM) to induce apoptosis in the estrogen dependent breast cancer cell line MCF‐7. Clear evidence for induction of apoptosis could be demonstrated after treatment with both antiestrogens. Application of the pure antiestrogen ZM led to a significantly higher induction of apoptosis compared to the partial agonistic compound Tam. The ability of the two compounds to induce apoptosis correlated with their growth inhibitory action. On the molecular level administration of ZM led to a time dependent steady decrease of BCL‐2 mRNA and protein. Administration of Tam also initially decreased the expression of BCL‐2. In contrast to ZM treatment, BCL‐2 expression increased again after 8 h of incubation with Tam. After 96 h Tam treated cells expressed BCL‐2 levels nearly as high as untreated cells. In general, ZM decreased BCL‐2 levels more effectively than Tam. Our results demonstrate that ZM and Tam possess quantitative and qualitative differences in their ability to down regulate BCL‐2 expression. The higher ability of the pure antiestrogen to down regulate BCL‐2 expression may explain the superiority of the pure antiestrogen to induce apoptosis and to inhibit the growth of MCF‐7 cells.

In vivo test systems for the quantitative and qualitative analysis of the biological activity of phytoestrogens.

Many compounds of plant origin with the ability to bind to the estrogen receptor have been identified in the last decades. One of the most extensively used in vivo assays to characterise the estrogenic potency of these phytoestrogens and mechanisms of their action is the rodent uterotrophic assay. Various protocols exist for this test system, using immature, hypophysectomized, or ovariectomized rats and mice and oral or subcutaneous administration of the test compound. However, just monitoring the ability of a compound to stimulate uterine growth is not sufficient to characterize its estrogenicity. Over the last decades, an increasing number of estrogen sensitive tissues has been identified. Moreover, a variety of different molecular mechanisms have been discovered for the action of estrogens, including non-genomic actions. Therefore, an in vivo test design for estrogenicity should include an analysis of several estrogen sensitive parameters in different estrogen sensitive tissues. To distinguish between agonistic and antagonistic properties of a substance, combinations of the test compound with estrogens and antiestrogens should be analyzed. A reasonable supplement to this enhanced uterotrophic assay are selected estrogen sensitive tumor models, which can be used to test for potential chemopreventive properties of phytoestrogens.

Selective estrogen receptor-β activation stimulates skeletal muscle growth and regeneration

There is increasing evidence suggesting that estrogens augment skeletal muscle regeneration processes after injury. To study the contribution of estrogen receptors α and β (ERα and ERβ) during muscle regeneration, skeletal muscles of ovariectomized (OVX) rats, as well as ERa‐ and ERβ‐knockout (αErko and βErko) mice, were injured with a myotoxin (notexin). OVX rats were simultaneously treated with the ER‐selective ligands genistein, ERα agonist 16α‐LE2 (alpha), ERβ agonist 8β‐VE2 (beta), or 17β‐estradiol (E2). OVX rats showed significantly elevated serum creatine kinase (CK) activity after muscle injury compared to intact sham‐treated animals. Treatment with ER ligands significantly reduced CK activity. TNF‐α, IL‐10, and MCP‐1 expression served to characterize immune responses. Treatment with all ER ligands, but particularly E2 and beta, reduced TNF‐α, but elevated MCP‐1 and IL‐10 expression. PCNA and MyoD expression served to define satellite cell activation and proliferation and were found to be up‐regulated by beta and E2. To further study muscle regeneration responses, expression of the embryonic myosin heavy chain (MHC) was analyzed. Beta and E2 but not alpha increased embryonic MHC expression compared to OVX. The absence of ERβ in βErko mice negatively affected CK activity levels and expression of satellite cell and muscle regeneration markers (MHC embryonic, MyoD, Pax7) compared with aErko and wild‐type mice. In a classic Hershberger assay using male rats, beta stimulated muscle growth, accompanied by a strong induction of IGF‐1 expression. Our data provide evidence that ERβ signaling is involved in the regulation of skeletal muscle growth and regeneration by stimulating anabolic pathways, activating satellite cells and modulating immune responses.—Velders, M., Schleipen, B., Fritzemeier, K. H., Zierau, O., Diel, P. Selective estrogen receptor‐β activation stimulates skeletal muscle growth and regeneration. FASEB J. 26, 1909‐1920 (2012). www.fasebj.org

Nitrate and nitrite in the diet: How to assess their benefit and risk for human health

Nitrate is a natural constituent of the human diet and an approved food additive. It can be partially converted to nitrogen monoxide, which induces vasodilation and thereby decreases blood pressure. This effect is associated with a reduced risk regarding cardiovascular disease, myocardial infarction, and stroke. Moreover, dietary nitrate has been associated with beneficial effects in patients with gastric ulcer, renal failure, or metabolic syndrome. Recent studies indicate that such beneficial health effects due to dietary nitrate may be achievable at intake levels resulting from the daily consumption of nitrate-rich vegetables. N-nitroso compounds are endogenously formed in humans. However, their relevance for human health has not been adequately explored up to now. Nitrate and nitrite are per se not carcinogenic, but under conditions that result in endogenous nitrosation, it cannot be excluded that ingested nitrate and nitrite may lead to an increased cancer risk and may probably be carcinogenic to humans. In this review, the known beneficial and detrimental health effects related to dietary nitrate/nitrite intake are described and the identified gaps in knowledge as well as the research needs required to perform a reliable benefit/risk assessment in terms of long-term human health consequences due to dietary nitrate/nitrite intake are presented.

Dose- and route-dependent hormonal activity of the metalloestrogen cadmium in the rat uterus

The toxic heavy metal cadmium (Cd) is regarded as a potential endocrine disruptor, since Cd exerts estrogen-like activity in vitro and can elicit some typical estrogenic responses in rodents upon intraperitoneal (i.p.) injection. But estrogenic effects have not been documented in vivo with other more relevant routes of exposure, although it is known that Cd absorption and distribution in the body is strongly affected by the application route. Therefore, we investigated its hormonal activity in ovariectomized Wistar rats after oral administration of CdCl(2) (0.05-4 mg/kg b.w. on 3 days by gavage and 0.4-9 mg/kg b.w. for 4 weeks in drinking water) in comparison with i.p. injection of CdCl(2) (0.00005-2 mg/kg b.w.). Uterus wet weight, height of uterine epithelium, and modulation of estrogen-regulated gene expression, i.e. uterine complement component 3 (C3), were determined, and also Cd-levels in uterus and liver were measured by atomic absorption spectrometry. The analysis revealed pronounced differences in Cd tissue levels and hormonal potency for the two routes of administration: a single i.p. injection of Cd increased dose-dependently uterine wet weight and thickness of the uterine epithelium. Interestingly, C3 mRNA expression in the uterus was down regulated at low doses of CdCl(2) (0.00005-0.05 mg/kg b.w.), but strongly stimulated at the highest dose of 2 mg/kg b.w. Other than i.p. injection, oral treatment with Cd, by gavage or in drinking water, did neither increase uterine wet weights nor epithelial thickness. But, both 3-day- and 4-week oral Cd administration resulted in a dose-dependent stimulation of C3 expression in the uterus, significant at and above 0.5 mg/kg b.w. In summary, our data demonstrate an estrogenic effect in the uterus upon i.p. injection of Cd, but considerably lower hormonal potency with oral administration: short and long-term oral treatment with Cd did not affect uterus weight or histology, whilst on the molecular level, an induction of estrogen sensitive uterine gene expression was observed, albeit at dose levels far exceeding those of dietary exposure in humans.

Physical activity and estrogen treatment reduce visceral body fat and serum levels of leptin in an additive manner in a diet induced animal model of obesity

Estrogen replacement and physical activity have been demonstrated to reduce the risk to develop a metabolic syndrome in postmenopausal women. In this study we investigate the combined effects of endurance training and estrogen substitution in a rat animal model of diet induced obesity. Effects on lipid and glucose metabolism were evaluated. Ovariectomized (OVX) or sham-operated (SHAM) female Wistar rats were fed with a high fat diet (HF) for 9 weeks. After 3 weeks of overnutrition the OVX rats either remained sedentary, performed treadmill training, received 17β-Estradiol (E(2)), or combined treatment. The OVX rats had a greater increase in body weight and serum levels of cholesterol, triglyceride and low-density lipoprotein cholesterol (LDL). These parameters could be reduced by E(2) and more effectively E(2) in combination with exercise. Also the increase of visceral body fat and leptin could be improved by E(2) and exercise. This combination showed synergistic effects. Serum levels of insulin could be reduced by exercise training, E(2) substitution revealed no significant changes. Our results indicate that ovariectomy increases the susceptibility to develop obesity. In addition they show that the combination of hormone replacement therapy (HRT) and physical activity may influence parameters related to lipid metabolism positively in an additive manner. The results of this study provide evidence that the combination of HRT with physical activity could be a very effective strategy to prevent the development of a metabolic syndrome induced by overnutrition.

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

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.