Assessment of xenoestrogenic exposure by a biomarker approach: application of the E-Screen bioassay to determine estrogenic response of serum extracts

Background

Epidemiological documentation of endocrine disruption is complicated by imprecise exposure assessment, especially when exposures are mixed. Even if the estrogenic activity of all compounds were known, the combined effect of possible additive and/or inhibiting interaction of xenoestrogens in a biological sample may be difficult to predict from chemical analysis of single compounds alone. Thus, analysis of mixtures allows evaluation of combined effects of chemicals each present at low concentrations.

Methods

We have developed an optimized in vitro E-Screen test to assess the combined functional estrogenic response of human serum. The xenoestrogens in serum were separated from endogenous steroids and pharmaceuticals by solid-phase extraction followed by fractionation by high-performance liquid chromatography. After dissolution of the isolated fraction in ethanol-DMSO, the reconstituted extract was added with estrogen-depleted fetal calf serum to MCF-7 cells, the growth of which is stimulated by estrogen. After a 6-day incubation on a microwell plate, cell proliferation was assessed and compared with the effect of a 17-beta-estradiol standard.

Results and conclusions

To determine the applicability of this approach, we assessed the estrogenicity of serum samples from 30 pregnant and 60 non-pregnant Danish women thought to be exposed only to low levels of endocrine disruptors. We also studied 211 serum samples from pregnant Faroese women, whose marine diet included whale blubber that contain a high concentration of persistent halogenated pollutants. The estrogenicity of the serum from Danish controls exceeded the background in 22.7 % of the cases, while the same was true for 68.1 % of the Faroese samples. The increased estrogenicity response did not correlate with the lipid-based concentrations of individual suspected endocrine disruptors in the Faroese samples. When added along with the estradiol standard, an indication of an enhanced estrogenic response was found in most cases. Thus, the in vitro estrogenicity response offers a promising and feasible approach for an aggregated exposure assessment for xenoestrogens in serum.

     

Biomarker genes for detecting estrogenic activity of endocrine disruptors via estrogen receptors

Endocrine disruptors (EDs) are compounds used in various industrial products, drugs, and cosmetics. They can be found in the environment and disturb the endocrine and reproductive systems, resulting in adverse effects to humans and wildlife such as birth defects and developmental disorders. Since several EDs have a structure similar to that of endogenous steroid hormones such as estrogens, they intend to have an affinity for steroid hormone receptors and alter hormone-mediated metabolism by binding to these receptors. EDs are therefore a global concern and assays should be developed to efficiently determine whether these compounds are detrimental to biological systems. Diverse experimental methods may help determine the endocrine disrupting potential of EDs and evaluate the adverse effects of a single and/or combination of these reagents. Currently, biomarkers have been employed to objectively measure EDs potency and understand the underlying mechanisms. Further studies are required to develop ideal screening methods and biomarkers to determine EDs potency at environmentally relevant concentrations. In this review, we describe the biomarkers for estrogenicity of EDs identified both in vitro and in vivo, and introduce a biomarker, cabindin-D(9k) (CaBP-9k), that may be used to assess estrogenic activity of EDs.     

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses,prolactin release,and membrane estrogen receptor trafficking in rat pituitary cells

Background  

Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens.     

Combinations of Physiologic Estrogens with Xenoestrogens Alter ERK Phosphorylation Profiles in Rat Pituitary Cells

Background

Estrogens are potent nongenomic phospho-activators of extracellular-signal–regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously.

Objectives

We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs (alkylphenols and bisphenol A) to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol (E₂), estrone (E₁), and estriol (E₃).

Methods

We quantified hormone/mimetic-induced ERK phosphorylations in the GH₃/B6/F10 rat pituitary cell line using a plate immunoassay, comparing effects with those on cell proliferation and by estrogen receptor subtype-selective ligands.

Results

Alone, these structurally related XEs activate ERKs in an oscillating temporal pattern similar (but not identical) to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated (at the lowest, subpicomolar concentrations) and attenuated (at the highest, picomolar to 100 nM concentrations) the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated.

Conclusions

XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important (e.g., development, reproductive cycling, pregnancy, menopause).     

Endokrine Disruptoren in Lebensmitteln

It has been hypothesized that chemicals with estrogenic or antiandrogenic activity (xenoestrogens/xenoantiandrogens) may cause developmental, reproductive, and tumorigenic effects in humans and animals. The endocrine disruptor hypothesis is biologically plausible, but evidence for a causal link between environmental exposures to such agents and adverse health effects in humans is limited to prenatal exposures to high doses of the potent estrogen diethylstilbestrol. In principle, there is agreement that risks from endocrine disruptors are determined by time of exposure, dose, and potency. The biological relevance of the known, and usually low concentrations of hormonally active agents in foods, and the question as to which extent xenoestrogens/xenoantiandrogens can indeed exert adverse effects on humans is a controversial issue. This is in part due to uncertainties regarding the role of combination effects and the shape of the dose-response curve at very low concentrations that will result from dietary intake of both synthetic chemicals and phytochemicals. Moreover, information on exposures to certain agents is incomplete and complicates a toxicological risk assessment. Thus, there is a need for further research addressing the question of whether and if so, which compounds and classes of compounds may have an impact on human reproductive health.     

Vitellogenin Induction in Mucus from Brook Trout (Salvelinus fontinalis)

Induction of vitellogenin (VTG) is widely used as a biomarker of exposure of male or immature fish to chemicals that are agonists of the estrogen receptor (i.e., xenoestrogens). Analysis of VTG in samples of epidermal mucosa collected from fish is a non-invasive method for evaluating whether wild fish are exposed to xenoestrogens. In this study, the mean levels of VTG in the mucus of immature brook trout (Salvelinus fontinalis) collected from the Credit River in Ontario, Canada downstream of aging residential septic systems and in an agricultural watershed were 0.67 ng per mg protein, which was significantly elevated relative to the mean VTG levels of 0.22 ng per mg protein in the mucus of immature brook trout collected from a less impacted site. To validate the mucus assay, immature brook trout were exposed in the laboratory to 17α-ethinylestradiol (EE2) at nominal concentrations of 10, 50 and 100 ng/L and VTG levels in mucus from these fish showed a concentration-dependent increase relative to fish from the control treatment. This study illustrates the utility of this non-lethal method for assessing whether wild fish have been exposed in situ to xenoestrogens. Exposures to xenoestrogens from non-point sources may be impacting brook trout populations in urban watersheds in southern Ontario.

     

Xenoestrogen modulation of the immune system: effects of dichlorodiphenyltrichloroethane (DDT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Dichlorodiphenyltrichloroethane (DDT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are complex organic compounds that are frequently found in the environment as a result of agricultural and industrial activities. Both compounds have substantial immune system and endocrine system disrupting activity, acting as estrogen agonists or antagonists (xenoestrogens). Research has demonstrated that exposure to xenoestrogens can result in body weight loss, developmental abnormalities, thymic atrophy, carcinogenesis, and tissue-specific hypoplastic and hyperplastic responses. Although several studies have reported significant adverse effects of these compounds on the endocrine system, very few investigations have focused on the specific mechanisms of action on the immune system. This paper reviews the cellular and molecular mechanisms of DDT- and TCDD-induced toxicity on the endocrine and immune systems, and explores their potential impact on the pathogenesis of immune disease.     

The Short-Chain Fatty Acid Methoxyacetic Acid Disrupts Endogenous Estrogen Receptor-α–Mediated Signaling

Background

Ethylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is methoxyacetic acid (MAA), a short-chain fatty acid that inhibits histone deacetylase activity and alters gene expression.

Objective

Because estrogen signaling is necessary for normal reproductive function and modulates gene expression, the estrogen-signaling pathway is a likely target for MAA; however, little is known about the effects of MAA in this regard.

Methods

We evaluated the mechanistic effects of MAA on estrogen receptor (ER) expression and estrogen signaling using in vitro and in vivo model systems.

Results

MAA potentiates 17β-estradiol (E₂) stimulation of an estrogen-responsive reporter plasmid in HeLa cells transiently transfected with either a human ERα or ERβ expression vector containing a cytomegalovirus (CMV) promoter. This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter. In contrast to its effects on exogenous ER, MAA decreases endogenous ERα expression and attenuates E₂-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.

Conclusions

These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses. Given the established role of the ER in normal reproductive function, the effects of MAA on the endogenous ER reported here are consistent with the reproductive abnormalities observed after EGME exposure and suggest that these toxicities may be due, at least in part, to attenuation of endogenous ER-mediated signaling.     

Mimicking of Estradiol Binding by Flame Retardants and Their Metabolites: A Crystallographic Analysis

Background: Brominated flame retardants (BFRs), used in many types of consumer goods, are being studied because of concerns about possible health effects related to endocrine disruption, immunotoxicity, reproductive toxicity, and neurotoxicity. Tetrabromobisphenol A (TBBPA), the most widely used BFR, and human metabolites of certain congeners of polybrominated diphenyl ether (e.g., 3-OH-BDE-47) have been suggested to inhibit estrogen sulfotransferase, potentially affecting estrogen metabolism.Objectives: Our primary goal was to understand the structural mechanism for inhibition of the hormone-metabolizing enzyme estrogen sulfotransferase by certain BFRs. We also sought to understand various factors that facilitate the binding of flame retardants in the enzyme binding pocket.Methods: We used X-ray crystallography to obtain atomic detail of the binding modes of TBBPA and 3-OH-BDE-47 to estrogen sulfotransferase for comparison with binding of the endogenous substrate estradiol.Results: The crystal structures reveal how BFRs mimic estradiol binding as well as the various interactions between the compounds and protein residues that facilitate its binding. In addition, the structures provide insights into the ability of the sulfotransferase substrate binding pocket to accommodate a range of halogenated compounds that satisfy minimal structural criteria.Conclusions: Our results show how BFRs or their metabolites can bind to and inhibit a key hormone-metabolizing enzyme, potentially causing endocrine disruption.Citation: Gosavi RA, Knudsen GA, Birnbaum LS, Pedersen LC. 2013. Mimicking of estradiol binding by flame retardants and their metabolites: a crystallographic analysis. Environ Health Perspect 121:1194–1199; http://dx.doi.org/10.1289/ehp.1306902     

Screening for Chemical Contributions to Breast Cancer Risk: A Case Study for Chemical Safety Evaluation

Background

Current approaches to chemical screening, prioritization, and assessment are being reenvisioned, driven by innovations in chemical safety testing, new chemical regulations, and demand for information on human and environmental impacts of chemicals. To conceptualize these changes through the lens of a prevalent disease, the Breast Cancer and Chemicals Policy project convened an interdisciplinary expert panel to investigate methods for identifying chemicals that may increase breast cancer risk.

Methods

Based on a review of current evidence, the panel identified key biological processes whose perturbation may alter breast cancer risk. We identified corresponding assays to develop the Hazard Identification Approach for Breast Carcinogens (HIA-BC), a method for detecting chemicals that may raise breast cancer risk. Finally, we conducted a literature-based pilot test of the HIA-BC.

Results

The HIA-BC identifies assays capable of detecting alterations to biological processes relevant to breast cancer, including cellular and molecular events, tissue changes, and factors that alter susceptibility. In the pilot test of the HIA-BC, chemicals associated with breast cancer all demonstrated genotoxic or endocrine activity, but not necessarily both. Significant data gaps persist.

Conclusions

This approach could inform the development of toxicity testing that targets mechanisms relevant to breast cancer, providing a basis for identifying safer chemicals. The study identified important end points not currently evaluated by federal testing programs, including altered mammary gland development, Her2 activation, progesterone receptor activity, prolactin effects, and aspects of estrogen receptor β activity. This approach could be extended to identify the biological processes and screening methods relevant for other common diseases.

Citation

Schwarzman MR, Ackerman JM, Dairkee SH, Fenton SE, Johnson D, Navarro KM, Osborne G, Rudel RA, Solomon GM, Zeise L, Janssen S. 2015. Screening for chemical contributions to breast cancer risk: a case study for chemical safety evaluation. Environ Health Perspect 123:1255–1264; http://dx.doi.org/10.1289/ehp.1408337     

Structural and Functional Profiling of Environmental Ligands for Estrogen Receptors

Background: Individuals are exposed daily to environmental pollutants that may act as endocrine-disrupting chemicals (EDCs), causing a range of developmental, reproductive, metabolic, or neoplastic diseases. With their mostly hydrophobic pocket that serves as a docking site for endogenous and exogenous ligands, nuclear receptors (NRs) can be primary targets of small molecule environmental contaminants. However, most of these compounds are chemically unrelated to natural hormones, so their binding modes and associated hormonal activities are hardly predictable.Objectives: We conducted a correlative analysis of structural and functional data to gain insight into the mechanisms by which 12 members of representative families of pollutants bind to and activate the estrogen receptors ERα and ERβ.Methods: We used a battery of biochemical, structural, biophysical, and cell-based approaches to characterize the interaction between ERs and their environmental ligands.Results: Our study revealed that the chemically diverse compounds bound to ERs via varied sets of protein–ligand interactions, reflecting their differential activities, binding affinities, and specificities. We observed xenoestrogens binding to both ERs—with affinities ranging from subnanomolar to micromolar values—and acting in a subtype-dependent fashion as full agonists or partial agonists/antagonists by using different combinations of the activation functions 1 and 2 of ERα and ERβ.Conclusions: The precise characterization of the interactions between major environmental pollutants and two of their primary biological targets provides rational guidelines for the design of safer chemicals, and will increase the accuracy and usefulness of structure-based computational methods, allowing for activity prediction of chemicals in risk assessment.Citation: Delfosse V, Grimaldi M, Cavaillès V, Balaguer P, Bourguet W. 2014. Structural and functional profiling of environmental ligands for estrogen receptors. Environ Health Perspect 122:1306–1313; http://dx.doi.org/10.1289/ehp.1408453     

Endocrine disruptors: update on xenoestrogens

Endocrine disruptors and their possible impact on human and animal health have become a topic of discussion and an area of active research in toxicology. A focus has been on xenoestrogens, i.e., environmental chemicals with estrogenic activity. In principle, there is agreement that such compounds, in high doses, may cause developmental, reproductive and tumorigenic effects (“hazard”). A matter of controversy is the question of risks associated with xenoestrogens under realistic (low) exposure scenarios; this is due to uncertainty on how to assess the interactions of exogenous compounds with the endocrine system and its complex regulation. Our overview will address topics including: consequences from previous clinical use of the potent estrogen diethyl- stilbestrol with particular emphasis on dose-response relationships, other observations in humans exposed to estrogenic chemicals in an occupational context, and available information on exposure levels of synthetic and naturally occurring estrogens in the diet. Together with a critical appraisal of methods to detect and quantitate the estrogenic activity of synthetic and naturally occurring chemicals, novel aspects in the risk assessment for endocrine active compounds are discussed. Received: 4 October 1999 / Accepted: 20 February 2000     

Literature review of instruments to assess health-related quality of life during and after menopause

Background and objectives: Menopause is a physiological event occurring in women at about the age of 50. It signals the end of the reproductive years and is associated with signs of estrogen deficiency having a considerable impact on womens health-related quality of life (HRQoL). The most common form of treatment is hormone replacement therapy (HRT). Studies have shown negative events can arise from long-term use of HRT. The aim of this review is to determine if there are any HRQoL instruments that address the impact of menopausal symptoms including positive and negative effects of HRT. Methods: The following eight instruments were identified: Greene Climacteric Scale, Womens Health Questionnaire (WHQ), Qualifemme, Menopause-Specific QOL Questionnaire (MENQOL), Menopausal Symptoms List (MSL), Menopause Rating Scale (MRS), Menopausal Quality of Life Scale (MQOL), and the Utian Quality of Life Scale (UQOL). Results: All instruments reviewed proved to be reasonably structured and have their place in applied research. None were found that addressed all aspects of the impact of HRT on HRQoL. Conclusion: In order to capture the possible short-term side effects of HRT on HRQoL, it is necessary to modify one or more of the existing instruments or develop a new instrument applicable in many different countries and languages.     

Environmental exposure to xenoestrogens and oestrogen related cancers: reproductive system,breast, lung,kidney, pancreas,and brain

The role of steroids in carcinogenesis has become a major concern in environmental protection, biomonitoring, and clinical research. Although historically oestrogen has been related to development of reproductive system, research over the last decade has confirmed its crucial role in the development and homeostasis of other organ systems. As a number of anthropogenic agents are xenoestrogens, environmental health research has focused on oestrogen receptor level disturbances and of aromatase polymorphisms. Oestrogen and xenoestrogens mediate critical points in carcinogenesis by binding to oestrogen receptors, whose distribution is age-, gender-, and tissue-specific. This review brings data about cancer types whose eatiology may be found in environmental exposure to xenoestrogens. Cancer types that have been well documented in literature to be related with environmental exposure include the reproductive system, breast, lung, kidney, pancreas, and brain. The results of our data mining show (a) a significant correlation between exposure to xenoestrogens and increased, gender-related, cancer risk and (b) a need to re-evaluate agents so far defined as endocrine disruptors, as they are also key molecules in carcinogenesis. This revision may be used to further research of cancer aetiology and to improvement of related legislation. Investigation of cancers caused by xenoestrogens may elucidate yet unknown mechanisms also valuable for oncology and the development of new therapies.     

Treatment-limiting decisions, comorbidities, and mortality in the emergency departments: A cross-sectional elderly population-based study

Background

Older adults experience a higher risk of death in the emergency departments (EDs), in part, as a result of their comorbidities. A treatment-limiting decision is often reported for older adults who die in the EDs. The Charlson Comorbidity Index (CCI) is a validated method for the scoring of comorbidities. Whether an association between the CCI and treatment-limiting decisions exists remains unknown.

Objective

To determine whether the CCI was associated with the treatment-limiting decisions made for older patients who die in the EDs.

Methods

A total of 2,095 patients >65 years old who died in the EDs in France and Belgium were prospectively included between 2004 and 2005. The recorded data included: 1) the CCI score; 2) patient age; 3) gender; 4) living in senior housing facilities; 5) hospitalizations occurring in the previous year; 6) presence of functional limitations (according to the Knaus classification); 7) chronic diseases; and 8) presence of organ failure(s). A treatment-limiting decision was defined as a predetermined choice not to implement therapies that would otherwise be required to sustain life.

Results

A treatment-limiting decision was identified in 993 (47%) patients. Fully-adjusted logistic regression model showed that a CCI ?? 5 (OR=25.56 with P=0.037), age ??85 years (OR=20.33 with P<0.001), living in an institution (OR=0.15 with P=0.017), hematologic (OR=6.92 with P=0.020) and respiratory disease (OR=0.17 with P=0.046), and neurologic causes (OR=0.20 with P=0.010) of organ failure were significantly associated with treatment-limiting decisions.

Conclusion

An elevated CCI score (??5) was associated with a treatment-limiting decision in elderly patients evaluated in the EDs. Further research is needed to corroborate this finding.     

Assessment of thyroid,testes, kidney and autonomic nervous system function in lead-exposed workers

Summary The objective of the study was to assess whether moderate occupational exposure to lead may be associated with early changes in potential target organs (thyroid, testes, kidney, autonomic nervous system). Workers exposed to lead in a lead acid battery factory (n = 98; mean blood lead 51 g/dl, range 40–75 g/dl) and 85 control workers were examined. None of the indicators of kidney function (in urine: retinol-binding protein, ₂-microglobulin, albumin,N-acetyl--d-glucosaminidase; in serum: creatinine, ₂-microglobulin), endocrine function (follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, thyroxine, triiodothyronine) and autonomic nervous system (R-R interval variations on the electrocardiogram) were correlated with lead exposure (blood lead or duration of exposure) or showed significantly different mean values between the exposed group and controls. These results and an assessment of the published data suggest that compliance with the Directive of the Council of the European Communities on lead exposure (health surveillance in workers whose lead in blood exceeds 40 g/dl and removal from exposure when blood lead exceeds 70–80 g/dl) would prevent the occurrence of significant biological changes in the majority of lead-exposed workers.