Recent insights into the effect of natural and environmental estrogens on mammary development and carcinogenesis.

The present work reviews recent findings related to the action of steroidal (physiological) estrogens on normal mammary gland development and carcinogenesis, as well as effects of related environmental mediators (phyto- and xeno-estrogens), the role of which remains controversial. Orchestration by estrogen receptors (i.e. ERα and ERß) and coregulators of growth, apoptosis and differentiation of epithelial cells, directed our analysis. The bidirectional coordination between epithelium and stroma in parallel with maintenance of stemness are also investigated. The relevance of nuclear and extranuclear localization of ERs and other eventual estrogen binding sites, mediating differential actions in regard to these various topics, is critically addressed to delineate the importance of direct and indirect activation procedures and delicate feedback loops (ligand-induced or/and cross-talk activation, respectively). The inclusion of the outlined regulatory concepts in drug design programs for the prevention and treatment of breast cancer may have potent effects.

The effect of suspended particles coated by humic acid on the toxicity of pharmaceuticals, estrogens, and phenolic compounds.

The sorption characteristics of 10 organic chemicals, categorized as pharmaceuticals, estrogens and phenols, onto synthetic suspended particle (i.e., alumina) coated with humic acid were investigated according to their octanol-water partition coefficient (K(ow)). Chemical analyses were performed with gas chromatography and mass spectrometry (GC/MS) and high performance liquid chromatography (HPLC). The effects of particles on the toxicity reduction were evaluated using bioassay tests, using Daphnia magna and Vibrio fisheri for phenols and pharmaceuticals, and the human breast cancer cell MCF-7 for estrogens. Sorption studies revealed that 22 and 38% of octylphenol and pentachlorophenol, respectively, were removed by suspended particle, whereas 2,4-dichlorophenol was not removed, which was directly proportional to the logK(ow) value. Similar to the sorption tests, suspended particles significantly reduced the acute toxicities of octylphenol and pentachlorophenol to D. magna and V. fisheri (p<0.01), but there was no significant difference in the toxicity of 2,4-dichlorophenol to D. magna (p=0.8374). Pharmaceuticals, such as ibuprofen, gemfibrozil and tolfenamic acid, showed no discernible sorption to the suspended particle, with the exception of diclofenac, which revealed 11% sorption. For estrogens, such as estrone, 17beta-estradiol and 17alpha-ethynylestradiol, the results indicated no reduction in the sorption test. This may be attributed to the polar interaction by functional groups in sorption between pharmaceuticals and estrogens and suspended particles. In the bioassays, presence of suspended particles did not significantly modify the toxicity of pharmaceuticals (regardless of their K(ow) values) to D. magna, V. fisheri or E-screen.

Estrogens and antiestrogens activate hPXR.

The pregnane X receptor (PXR, NR1I2) and the estrogen receptors (ERalpha, NR3A1 and ERbeta, NR3A2) bind a large number of compounds, including environmental pollutants and drugs, which exhibit remarkably diverse structural features. This prompted us to investigate if ER ligands could be PXR activators. We focused our attention on known estrogens from various chemical classes: physiological and synthetic estrogens and antiestrogens, plant and fungus estrogens, and other man-made chemicals belonging to phthalate plasticizers, surfactant-derived alkylphenols and cosmetics. Altogether, nearly 50 compounds were thus analyzed for their ability to activate human PXR in stably transfected cells, HGPXR cells, derived from HeLa cells and expressing luciferase under the control of a chimeric hPXR. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 and 2B6 expressions in a primary culture of human hepatocytes. A significant proportion (54%) of compounds with estrogenic activity or able to bind ER were found to be hPXR activators: in particular, antiestrogens, mycoestrogens and phthalates. An even greater proportion is observed if estrogenic pesticides are included. Altogether, these results raise the question of the meaning and consequences of compounds with double PXR/ER activation ability.

Gene expression profile in the livers of rats orally administered ethinylestradiol for 28 days using a microarray technique.

To identify genes showing responses to estrogen exposure in the livers of animals in a repeated oral dose toxicity study, dose-dependent gene expression profiles were analyzed using high-density oligonucleotide microarrays in Sprague-Dawley rats of both sexes administered ethinylestradiol (EE) for 28 days at concentrations of 0, 0.01, 0.1, and 1.0 ppm in the diet. Among 3776 genes examined, examples showing increased expression on EE-treatment were detected predominantly in females. Genes showing dose-dependent up-regulation with greater than five-fold change at 1.0 ppm from the control levels were found to, respectively, number 4 in males, and 24 in females. Most of the latter exhibited relatively high basal expression as well as low variability, and many exhibited clear dose-dependence. Genes showing dose-dependent down-regulation were rather few, and many of those affected exhibited relatively low expression levels with large variation between animals, like genes showing dose-unrelated expression patterns in both sexes or dose-dependent up-regulation in males. Considering that detection of changes in endocrine-linked organs and estrous cyclicity is only possible at the high dose of 1.0 ppm, up-regulation of genes dose-dependently in females provides a sensitive tool to detect estrogenic effects in the rat liver in the framework of the 28-day toxicity study.

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes).

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.

Comparative assessment of endocrine modulators with oestrogenic activity. II. Persistent organochlorine pollutants.

Risk assessments of synthetic chemicals with oestrogen-like activity must take into account the high dietary levels of natural endocrine modulators in food. In view of current regulations of the European Union, a hygiene-based margin of safety (HBMOS) for xeno-oestrogens was defined as a quotient of estimated human daily intakes weighted by relative rodent in vivo potencies of the compounds. Such comparisons of intakes and potencies of natural isoflavones, with short half-lives, with those of polychlorinated organic pollutants (POP) displaying significant toxicokinetic accumulation, deserves the special consideration of toxicokinetics. For slowly accumulating compounds such comparison is much more favourable when based on comparative blood and tissue levels, not on scenarios of daily exposures. Observing these principles, the present communication extends the HBMOS concept to POP, using o,p'-DDT, the oestrogenic component of DDT mixtures, as a prototype. An HBMOS of 137 is derived for o,p'-DDT indicative of a sufficient margin of safety to ensure the absence of risk to human health due to its hormonal action, under exposure conditions now prevailing in Western countries.

Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens.

Many chemicals in surface waters and sediments have recently been discovered to have estrogenic/antiestrogenic activity. Among these compounds, known as 'endocrine disrupters', are natural and synthetic hormones, phytoestrogenes and a variety of industrial chemicals, such as certain detergents and pesticides. These substances are supposed to affect the development and reproduction in wildlife and humans and may also be involved in the induction of cancer. In order to assess the estrogenic/antiestrogenic potential of pure compounds and complex environmental samples we compared an array of in vitro test systems, (i) two luciferase reporter gene assays using transgenic human MVLN cells (derived from MCF-7 cells) and HGELN cells (derived from HeLa cells); (ii) a competitive binding assay with recombinant human estrogen receptors (ER) alpha and beta; and (iii) a proliferation assay with MCF7-cells (E-Screen). The sensitivity of the assays for 17-beta-estradiol decreased in the order: MVLN-cells=E-Screen>HGELN-cells>binding to ER-alpha>binding to ER-beta. A good correlation was obtained between the estrogenic potencies of 11 compounds (17-beta-estradiol (E(2)), estrone (E(1)), estriol (E(3)), ethinylestradiol (EE(2)), diethylstilbestrol (DES), coumestrol, beta-sitosterol, genistein, 4-nonylphenol, 4-octylphenol, bisphenol A) in the three tissue culture assays. The relative potencies of the compounds obtained by the cell free binding assays were one to two orders of magnitude higher compared with the cell culture assays. The phytoestrogens showed a preference to bind to ER-beta, but only genistein showed a much lower activity in the E-Screen (growth induction in breast cancer cells) compared with the luciferase induction in MVLN and HGELN-cells.

Monitoring of estrogen mimics by a recombinant yeast assay: synergy between natural and synthetic compounds?

Properties of mixtures of compounds exhibiting estrogenic potential have been questioned in the past. Synergistic effects of endocrine disrupters have been proposed, but could never be confirmed. In this study, the transactivational potential of xenoestrogens and phytoestrogens has been evaluated in a yeast test system. Pesticides such as endosulfan, dieldrin, atrazine, and the main metabolites, desethylatrazine and desisopropylatrazine, have been tested and their behavior as mixtures is compared to the behavior of the single compounds. Our results are in contrast to a report (Tran et al., 1996) on the inhibitive effects of xenoestrogens on 17 beta-estradiol-dependent transactivation. Phytoestrogens have been investigated in a similar manner. A synergistic effect could not be confirmed for both, xenoestrogens and phytoestrogens. These compounds are either weak estrogens or completely lack estrogenic potential. Their endocrine disrupting potential in more complex systems must be therefore attributed to other molecular mechanisms such as to metabolic modification or interference with steroidogenesis. This study shows that yeast systems are useful tools for monitoring pure estrogenic properties.

Evaluation of quantitative structure-activity relationship methods for large-scale prediction of chemicals binding to the estrogen receptor.

Three different QSAR methods, Comparative Molecular Field Analysis (CoMFA), classical QSAR (utilizing the CODESSA program), and Hologram QSAR (HQSAR), are compared in terms of their potential for screening large data sets of chemicals as endocrine disrupting compounds (EDCs). While CoMFA and CODESSA (Comprehensive Descriptors for Structural and Statistical Analysis) have been commercially available for some time, HQSAR is a novel QSAR technique. HQSAR attempts to correlate molecular structure with biological activity for a series of compounds using molecular holograms constructed from counts of sub-structural molecular fragments. In addition to using r2 and q2 (cross-validated r2) in assessing the statistical quality of QSAR models, another statistical parameter was defined to be the ratio of the standard error to the activity range. The statistical quality of the QSAR models constructed using CoMFA and HQSAR techniques were comparable and were generally better than those produced with CODESSA. It is notable that only 2D-connectivity, bond and elemental atom-type information were considered in building HQSAR models. Since HQSAR requires no conformational analysis or structural alignment, it is straightforward to use and lends itself readily to the rapid screening of large numbers of compounds. Among the QSAR methods considered, HQSAR appears to offer many attractive features, such as speed, reproducibility and ease of use, which portend its utility for prioritizing large numbers of potential EDCs for subsequent toxicological testing and risk assessment.

Estrogenic activity of natural and synthetic estrogens in human breast cancer cells in culture.

We investigated the estrogenic activity of various environmental pollutants (xenobiotics), in particular the xenoestrogen o,p-DDT, and compared their effects with those of endogenous estrogens, phytoestrogens, and mycoestrogens on estrogen receptor binding capacity, induction of estrogen end products, and activation of cell proliferation in estrogen-sensitive human breast cancer cells in monolayer culture. We also quantified the levels of phytoestrogens in extracts of some common foods, herbs, and spices and in human saliva following consumption of a high phytoestrogen food source (soy milk) to compare phytoestrogen abundance and bioavailability relative to the reported xenoestrogen burden in humans. Results show that natural endogenous estrogens, phytoestrogens, mycoestrogens, and xenoestrogens bind estrogen receptor (ER) in intact cells, but demonstrate marked differences in their ability to induce end products of estrogen action and to regulate cell proliferation. All of the different classes of estrogens stimulated cell proliferation at concentrations that half-saturated ER, but only some classes were able to induce estrogen-regulated end products. Genistein, a common phytoestrogen found in soy foods, differed from the xenoestrogen DDT in its effects on cell proliferation and ability to induce estrogen-regulated end products. Moreover, we found that many of the foods, herbs, and spices commonly consumed by humans contain significant amounts of phytoestrogens, and consumption of soy milk, a phytoestrogen-rich food, markedly increases the levels of phytoestrogens in saliva. In conclusion, our in vitro results predict that a diet high in phytoestrogens would significantly reduce the binding of weak xenoestrogens to ER in target tissues in vivo.

Relative carcinogenic activity of various synthetic and natural estrogens in the Syrian hamster kidney.

Both synthetic and natural estrogens have been studied for their ability to induce renal carcinomas in castrated male hamsters after 9.0 months of treatment. Tumor foci were detected in frozen serial sections stained histochemically for estrase activity. Both diethylstilbestrol (DES) and 17 beta-estradiol had equal ability (100%) to induce renal tumors [approximately 20.5 +/- 3 (S.E.) tumor foci] in these animals. Hexestrol induced the same incidence and number of renal carcinoma foci as DES or 17 beta-estradiol. However, alpha -dienestrol and DES 3,4-oxide showed an 86 to 88% incidence of renal tumors in hamsters (approximately 10.8 +/- 3). When equilin and d-equilenin, components of therapeutic conjugated estrogens, were tested, only equilin had a 76% incidence of renal tumor foci (5.5 +/- 0.9). The ability of these stilbene and steroidal estrogens to compete for renal tumor estrogen receptor generally correlated well with their ability to cause renal tumorigenesis in the hamster with one notable exception. Although ethinyl estradiol competed as well as did DES or 17 beta-estradiol for estrogen receptor, had similar ability to induce renal progesterone receptor, and led to similar high serum prolactin levels as either DES or 17 beta-estradiol, it had only weak carcinogenic activity (21%) in the hamster kidney (0.6 +/- 0.5 foci). These data represent the first detailed analysis of the relative carcinogenic activity of different estrogens within a given tumor-inducing system, and based on the carcinogenicity data of hexestrol and alpha-dienestrol presented herein, they suggest that epoxidation of the olefinic double bond and the p-quinone metabolite of DES probably are not involved significantly in its carcinogenic activity. Moreover, the poor carcinogenic activity of ethinyl estradiol in this system, despite strong estrogenicity, suggests that estronic activity alone may not be sufficient to effect renal tumorigenesis in the hamster.