Inability to confirm estrogenicity of the heterocyclic amine PhIP in two in vitro assays.

2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is a heterocyclic amine which is found in food after cooking and which is a known mutagen. Reports from several laboratories have proposed that PhIP has estrogenic activity, which would classify PhIP as a xenoestrogen with human exposure via food. We tested PhIP in two cell-based assays for estrogenicity, both based on human cell lines but utilising different outcome measures: ERLUX (reporter-gene activation) and ESCREEN (cell proliferation). PhIP was inactive in both assays at concentrations spanning the picomolar to micromolar range. To eliminate supplier differences as an explanation for the disparity between these results and positive findings in the literature, we purchased PhIP from three suppliers and found no detectable estrogenic activity in any batch. (1)H NMR spectroscopy confirmed the chemical identity of the tested stock solutions. Correct assay performance was confirmed by including positive and vehicle controls on every assay plate, and by demonstrating the expected responses to a panel of known estrogens (estradiol, bisphenol A, and genistein). Our results differ from those in the literature and, whilst the exact reason for this is unknown, we discuss possible explanations of the disparity. Our results provide no in vitro evidence for the classification of PhIP as an estrogen.

Expression profiling of estrogen-responsive genes in breast cancer cells treated with alkylphenols, chlorinated phenols, parabens, or bis- and benzoylphenols for evaluation of estrogenic activity.

We examined expression profiles of estrogen-responsive genes after treatment with alkylphenols (p-cresol (pC), 4-n-ethylphenol (4EP), 4-n-heptylphenol (4HP), 4-t-octylphenol (4OP) and nonylphenol (NP)), chlorinated phenols (4-chlorophenol (4CP), 4-chloro-3,5-dimethylphenol (CDP), 2,4-dichlorophenol (DCP) and pentachlorophenol (PCP)), parabens (methylparaben (MPB), ethylparaben (EPB) propylparaben (PPB) and butylparaben (BuPB)), or bis- and benzoylphenols (bisphenols A and B and p-hydroxybenzophenone (pHBP)) by means of a DNA microarray assay first to evaluate the estrogenic activity of these chemicals and then to understand the structural basis for the activity. By selecting a set of 120 genes showing greater statistical reliability for estrogen, a more reliable assay for each of the chemicals was achieved and, for the chemicals for which data were available, the results were consistent with those of previously reported estrogen receptor-binding and yeast two-hybrid assays except for chlorinated and few other phenols. Evaluation of the chemicals based on gene function indicated that the genes related to proliferation, transcription and transport were mostly up-regulated while significant numbers of genes related to enzymes and signaling were down-regulated. The genes related to transcription showed the highest degree of variation among the six functional categories (enzymes, signaling, proliferation, transcription, transport and others) for the chemicals with relatively high levels of estrogenic activity. These results indicate that the variations in chemicals and their biological effects can be monitored by the appropriate grouping of estrogen-responsive genes.

Effect of natural and synthetic estrogens on a549 lung cancer cells: correlation of chemical structures with cytotoxic effects.

A series of synthetic (nonylphenol, diethylstilbestrol, and bisphenol A) and natural (quercetin, resveratrol, and genistein) phenolic estrogens were investigated for their ability to affect the viability and proliferation of A549 lung cancer cells. To assess and distinguish the cytotoxic effect of individual estrogens, we used both the MTT tetrazolium spectrophotometric method and the fluorescence assay, while the induction of the cell specific apoptotic process was examined by fluorescence microscopy after treatment of cells with SYTO 24 green fluorescent dye. A systematic study of interferences for both fluorescence and MTT methods is presented. The results showed that both natural and synthetic estrogens decreased the viability and proliferation of A549 lung cancer cells in a dose-dependent manner but at different sensitivities. Nonylphenol appeared very different as compared to the other estrogens, acting by inducing the higher inactivation rate of the cells within a very short time. The cytotoxic effect of the estrogens was directly related to their structural and conformational characteristics including chain length, number, and position of hydroxyl groups and degree of saturation.

Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds.

The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17beta-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5alpha-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3'-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities.

Xenoestrogenicity in in vitro assays is not caused by displacement of endogenous estradiol from serum proteins.

The possibility that compounds tested for estrogenicity can compete for binding places on serum proteins and cause an increase of available and very potent endogenous estrogens is of great interest for both the in vitro assay results and the prediction of risk for humans. In in vitro assays, small amounts of estradiol remaining after the charcoal stripping of serum applied in the culture medium could be displaced by the tested compounds, leading to an estrogenic response that might be falsely attributed to the test compound. We have studied the stripping efficiency of charcoal and measured whether reported xenoestrogens can displace estradiol from serum in an in vitro assay using negligible depletion-solid phase microextraction (nd-SPME). Possible competition was also studied with a mathematical exposure model, from which the predictions were compared to the measurements. We found that the common charcoal stripping procedure removed 99% of initially present estradiol. Additionally, our results with charcoal adsorption indicate that charcoal is not useful for serum protein binding assays, as it adsorbs more than the free fraction of ligand. Although the competition model predicted a displacement of estradiol from the serum proteins at the higher applied doses of xenoestrogen, the measurements showed no displacement. Therefore, we conclude that estrogenic responses in the in vitro assay applied here are not caused by displacement of remaining estradiol in the stripped serum. The possibility remains, however, that our displacement hypothesis does apply for estrogen sulfates, as these are present in much higher concentrations than estradiol in stripped serum.

Possible health impact of phytoestrogens and xenoestrogens in food.

Plants produce estrogen-like substances, denominated phytoestrogens, which are present in many human foodstuffs. The consumption of phytoestrogens has been associated with a variety of protective effects. Their relative estrogenic potency combined with their concentrations in food and human plasma indicate biological relevance. However, their biological properties differ from those of estradiol or other endogenous estrogens in humans. For instance, their possible effects on SHBG, inhibition of steroid metabolizing enzymes, anti-proliferative and anti-angiogenetic and other side effects have been described. Furthermore, phytoestrogens can exert estrogenic and antiestrogenic activities at the same time and their potency and metabolism have not been yet elucidated in all cases. In recent decades growing evidence has accumulated on the hormone-like effects of synthetic chemicals that appeared in the environment. The possible impact of xenoestrogens, to which humans are also exposed through the food chain, needs to be further clarified as well. The molecular effects and control mechanisms of these substances, their pharmacokinetics, threshold levels and dose-response differences are issues that require further research before a full assessment of their effect on humans can be drawn. Evaluating the total exposure and impact of this estrogenic effect is very challenging because of the lack of specific knowledge in some areas and the differences in the biological activity among these substances, as pinpointed in this review.

Estrogenic and antiestrogenic activities of flavonoid phytochemicals through estrogen receptor binding-dependent and -independent mechanisms.

Members of the flavonoid class of phytochemicals have previously been demonstrated to possess estrogenic activity in a number of hormonally responsive systems. We have performed the present study to characterize the estrogenic and antiestrogenic activity of flavonoids in the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Using an ER-dependent reporter gene assay and an ER competition binding assay, we have identified phytochemicals possessing estrogenic and antiestrogenic activities, which appeared to correlate directly with their capacity to displace [3H]estradiol from ER. Several flavonoids, including kaempferide, apigenin, and flavone, were distinct, in that their antiestrogenic activity did not appear to correlate with binding to ER, and therefore their suppression of estrogen-mediated gene transactivation and proliferation may occur independent of direct antagonism of the receptor. Further examination in HEK-293 cells transfected with ERalpha or ERbeta demonstrated potent antagonism with kaempferide and apigenin, while flavone was weakly antagonistic only toward ERP. These results suggest that the receptor binding-independent antiestrogenic chemicals may function through alternate signaling pathways as indirect ER modulators in a receptor- and cell type-specific manner. We conclude that antiestrogenic activities of flavonoid phytochemicals may occur through ER binding-dependent and -independent mechanisms and that the binding-independent antiestrogen activity of certain flavonoids is biologically significant in regulation of breast cancer cell proliferation.

Phytoestrogens in health and disease.

Phytoestrogens are compounds found in a wide variety of plant foods that historically are said to exhibit estrogen-like activity and, more recently, have been reported to display both estrogenic and anti-estrogenic effects. Population-based studies have been interpreted to suggest that consumption of a phytoestrogen-rich diet is protective against breast, prostate, and bowel cancer and cardiovascular disease and ameliorates estrogen-deficiency symptoms in postmenopausal women. Consequently, there is a global movement towards increased consumption of phytoestrogen-rich foods and tabletized concentrated isoflavone extracts are being heavily promoted. Evaluating the effects and hence the potential benefits and risks of phytoestrogens is a complex task. The interindividual diversity and complexity in dietary phytoestrogen absorption and metabolism make the bioactivity of these compounds unpredictable. Epidemiological studies of relationships between phytoestrogens and cancer and cardiovascular disease that take into account confounding factors are scarce. Results of many of the in vitro and in vivo studies are conflicting and confusing. These compounds do not simply mimic the effects of human steroidal estrogen but rather demonstrate both similar and divergent actions. The ultimate actions of these compounds in specific cells are determined by many factors, including the relative levels of estrogen receptor (ER) alpha and ER beta and the diverse cocktail of co-activators and co-repressors present in any given cell type. Therefore, effects vary according to the phytoestrogen studied, cell line, tissue, species, and response being evaluated. Overall, it is naive to assume that exposure to these compounds is always good; inappropriate or excessive exposure may be detrimental. Extensive documentation of the specific intracellular effects of the various phytoestrogens in different tissues, the relationships between timing and duration of exposure and disease, and results from prospective randomized studies in humans of their clinical effects and potential side effects are essential. Only then can widespread recommendations regarding the dietary and pharmacological intake of these compounds be made.

Hormone-modulating herbs: implications for women's health.

Women in the United States are increasingly turning to botanical medicines to treat conditions throughout their life cycles. Many herbs traditionally used for women's health conditions have been found to contain phytoestrogens. Phytoestrogens and their metabolites can bind estrogen receptors and can have both estrogenic and anti-estrogenic effects. Many women are attracted to the idea of using phytomedicine as an alternative to hormone replacement therapy. It is unclear, however, whether these herbs are safe for women at risk for breast cancer or its recurrence. This paper considers the estrogenicity of herbs such as black cohosh (Cimicifuga racemosa) and the implications for women's health.

Dietary supplement-drug interactions.

Recent surveys show that 18% of adults in the United States use prescription drugs concurrently with herbal or vitamin products, placing an estimated 15 million patients at risk of potential drug-supplement interactions. Despite this widespread concurrent use of conventional and alternative medicines, documented drug-herb interactions are sparse. This review focuses on possible interactions between drugs and herbal medicines used for phytoestrogen-hormone and antiplatelet-oral anticoagulant therapy. Interactions with phytoestrogens are purely speculative, based on competitive estrogen-receptor binding or antiestrogenic effects. In contrast, several case reports document bleeding complications with Ginkgo biloba, with or without concomitant drug therapy. Case reports are also suggestive of interaction between warfarin and dong quai or Panax ginseng. Recommendations for counseling patients at highest risk of adverse interactions are given.

Clinical review 92: Phytoestrogens.

We have reviewed the literature regarding the food sources, potency, population intakes, and known biological effects of phytoestrogens in humans using MEDLINE data base from the years 1975-1996. Over 600 articles pertinent to the metabolism of phytoestrogens, including female reproduction (in particular menstruation and menopause), cardiovascular disease, osteoporosis, and cancer were assessed including relevant case control or cohort studies, as well as randomized trials and review articles. Epidemiological studies regarding human data were included, as well as human cell line and animal studies when there were no relevant human data available. We conclude that phytoestrogens exhibit physiological effects in humans. Mild estrogenic changes occur in postmenopausal women. Benefits are seen regarding hypercholesterolaemia. Epidemiological, animal, and in vitro data encourage further assessment of the role of phytoestrogens in cancer prevention.