Article pubs.acs.org/crt
Combination of Metabolomics with Cellular Assays Reveals New Biomarkers and Mechanistic Insights on Xenoestrogenic Exposures in MCF‑7 Cells Sarah Potratz,*,† Patrick Tarnow,† Harald Jungnickel,† Sven Baumann,‡,§ Martin von Bergen,‡,∥,⊥ Tewes Tralau,† and Andreas Luch† †
Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany ‡ Department of Molecular Systems Biology, UFZ−Helmholtz-Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany § Institute of Pharmacy, Faculty of Biosciences, Pharmacology and Psychology, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany ∥ Institute of Biochemistry, Faculty of Biosciences, Pharmacology and Psychology, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany ⊥ Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Fredrik Bajers Vej 7K, 9220 Aalborg, Denmark S Supporting Information *
ABSTRACT: The disruptive potential of xenoestrogens like bisphenol A (BPA) lies in their 17β-estradiol (E2)-like binding to estrogen receptors (ERs) followed by concomitant modulation of ER target gene expression. Unsurprisingly, most endocrine testing systems focus on the quantification of canonical transcripts or ER-sensitive reporters. However, only little information is available about the corresponding metabolomic changes in vitro. This knowledge gap becomes particularly relevant in the context of potential mixture effects, for example, as a consequence of coexposure to potentially estrogenically active pollutants (e.g., Cd2+). Such effects are often difficult to dissect with molecular tools, especially with regard to potential physiological relevance. Metabolomic biomarkers are well-suited to address this latter aspect as they provide a comprehensive readout of whole-cell physiology. Applying a targeted metabolomics approach (FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic exposure in MCF-7 cells. Cells were treated with E2 and BPA in the presence or absence of Cd2+. Statistical analysis revealed a total of 11 amino acids and phospholipids to be related to the compound’s estrogenic potency. Co-exposure to Cd2+ modulated the estrogenic profile. However, the corresponding changes were found to be moderate with cellular assays such as the E-screen failing to record any Cd2+-specific estrogenic effects. Overall, metabolomics analysis identified proline as the most prominent estrogenic biomarker. Its increase could clearly be related to estrogenic exposure and concomitant ERα-mediated induction of proliferation. Involvement of the latter was confirmed by siRNA-mediated knockdown studies as well as by receptor inhibition. Further, the underlying signaling was also found to involve the oncoprotein MYC. Taken together, this study provides insights into the underlying mechanisms of xenoestrogenic effects and exemplify the strength of the complementary use of metabolomics and cellular and molecular assays.
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INTRODUCTION
The appropriate assessment of chemical compounds exerting endocrine activity has been haunting toxicology ever since the term “endocrine disruptor” was introduced in 1993.1 One of the most intensively investigated substances in this field is © 2016 American Chemical Society
Special Issue: Systems Toxicology II Received: March 31, 2016 Published: August 12, 2016 883
DOI: 10.1021/acs.chemrestox.6b00106 Chem. Res. Toxicol. 2017, 30, 883−892
Article
Chemical Research in Toxicology
address coexposures of ubiquitous environmental contaminants such as BPA20 and Cd2+ as toxicology still tends to assess these as individual substances, not out of ignorance but limited by assays and tools available. With the respective intracellular targets comprising hormone receptors as well as a wide variety of signaling pathways, coexposure is likely to lead to cumulative, synergistic, or antagonistic effects. While a recent study addressed the metabolomics effects of E2 on MCF-7 cells,21 this is the first study that addresses metabolomic changes caused by BPA in human mammalian cells, elucidating the similarities and differences following treatment of MCF-7 cells with BPA or E2 in the presence or absence of Cd2+. In previous studies, the latter caused synergistic effects during coexposure with benzo[a]pyrene.22 The metabolomics analysis yielded several biomarkers indicative of (xeno)estrogenic exposure with the data subsequently being used to obtain some further insights into the underlying pathways of toxicity.
bisphenol A (BPA), a key monomer of polycarbonate plastics and xenoestrogen that is capable of binding to estrogen receptors (ERs) and thus modulating ER target gene expression.2 Because of the production volume, it is not surprising that human biomonitoring confirmed a low but widespread exposure to this substance in the US3 as well as in Europe.4 Examples for BPA containing products include compact discs, beverage containers, plastic dinnerware, impact-resistant safety equipment, automobile parts, and toys.5,6 While BPA has been assessed in a multitude of toxicological studies, the question whether exposure to BPA represents a health risk remains controversial.7 Among the aspects comprehensively addressed are the effects on the reproductive system of laboratory animals,8−10 as well as changes in gene expression.11 Mostly performed under the standardized conditions of single substance exposure, none of these studies provided sufficient evidence for regulatory concern. Yet, due to the ongoing discussions about the potential influence of prolonged (low-dose) exposure or mixture effects regulatory bodies in Canada and the European Union have recently banned the use of BPA in infant feeding bottles as a precautionary measure.12,13 Studies in laboratory animals show that treatment with BPA leads to global changes in the metabolome.11 However, the significance of these findings for potential adversity in humans remains to be addressed as do metabolic and lipidomic effects of BPA and E2 in multicompound exposures. One of the substances relevant in this context is cadmium (actually, that is, Cd2+), a widespread pollutant, well-known for its persistence and tendency to bioaccumulate.14 Apart from being a carcinogen, this heavy metal ion induces oxidative stress, triggers apoptosis, and is suspected to promote estrogenic activity by activation of extracellular signal related kinases 1 and 2 (ERK1/2) and protein kinase B (AKT).15 In addition, it has been reported to activate the ER in breast cancer cells in concentrations as low as 10 picomolar16 and is prone to interact directly with thiol groups of proteins. It is mainly this latter feature which causes most of the acute Cd2+ toxicity due to organelle dysfunction17 and inhibition of cellular protein activity.18 Yet, little is known as to how the multiple effects of Cd2+ translate into metabolomic changes, be it as a consequence of single substance exposures or in combination with other xenoestrogens such as BPA. Metabolomics is ideally suited to address such complex substance interactions as it aims to analyze all metabolites in a biological system at a specified time point. Common targets are particularly the endogenous small molecules (
