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Identification of putative steroid receptor antagonists in bottled water: combining bioassays and high-resolution mass spectrometry.

Wagner M, Schl├╝sener MP, Ternes TA, Oehlmann J - PLoS ONE (2013)

Bottom Line: We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%.Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water.This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.

View Article: PubMed Central - PubMed

Affiliation: Department Aquatic Ecotoxicology, Faculty of Biological Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany.

ABSTRACT
Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MS(n) experiments elucidated the molecule's structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MS(n) data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably, dioctyl fumarates and maleates have been overlooked by science and regulation to date. This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.

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Related in: MedlinePlus

Correlation of the peak areas (Z-transform) of the final candidate (exact mass 363.25047) with the antiandrogenicity.Data from Orbitrap experiment 1 is shown here exemplarily. Triangles indicate outliers, the linear regression (with 95% confidence bands) is shown in grey.
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pone-0072472-g002: Correlation of the peak areas (Z-transform) of the final candidate (exact mass 363.25047) with the antiandrogenicity.Data from Orbitrap experiment 1 is shown here exemplarily. Triangles indicate outliers, the linear regression (with 95% confidence bands) is shown in grey.

Mentions: From the raw data we manually reanalyzed the exact masses (m/z), retention times, and peak areas of the three remaining candidates (m/z 229.14103, 352.09008, and 363.25047). Correlation of the recalculated peak areas with the biological activity indicated that two candidates (229.14 and 325.09) were only loosely and in some cases not significantly correlated in the individual experiments (Figure S4). In addition, these candidates correlated negatively with the antagonistic activity rendering them biologically implausible. In contrast, the candidate with the mass 363.25 Da correlated positively with the antiestrogenic and antiandrogenic activity consistently throughout all experiments (p<0.05, see example in Figure 2). However, we identified three samples that did not conform with this correlation: samples 7 and 11 as well as 8 and 11 induced potent antagonistic activity but the mass 363.25 was detected in low concentrations in those samples in LTQ-Orbitrap experiments 1 and 2, respectively. In general, the correlation with antiandrogenicity was more pronounced than with the antiestrogenic activity because bottled water inhibited androgen receptor more potently (Figure S3 and S4).


Identification of putative steroid receptor antagonists in bottled water: combining bioassays and high-resolution mass spectrometry.

Wagner M, Schl├╝sener MP, Ternes TA, Oehlmann J - PLoS ONE (2013)

Correlation of the peak areas (Z-transform) of the final candidate (exact mass 363.25047) with the antiandrogenicity.Data from Orbitrap experiment 1 is shown here exemplarily. Triangles indicate outliers, the linear regression (with 95% confidence bands) is shown in grey.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756062&req=5

pone-0072472-g002: Correlation of the peak areas (Z-transform) of the final candidate (exact mass 363.25047) with the antiandrogenicity.Data from Orbitrap experiment 1 is shown here exemplarily. Triangles indicate outliers, the linear regression (with 95% confidence bands) is shown in grey.
Mentions: From the raw data we manually reanalyzed the exact masses (m/z), retention times, and peak areas of the three remaining candidates (m/z 229.14103, 352.09008, and 363.25047). Correlation of the recalculated peak areas with the biological activity indicated that two candidates (229.14 and 325.09) were only loosely and in some cases not significantly correlated in the individual experiments (Figure S4). In addition, these candidates correlated negatively with the antagonistic activity rendering them biologically implausible. In contrast, the candidate with the mass 363.25 Da correlated positively with the antiestrogenic and antiandrogenic activity consistently throughout all experiments (p<0.05, see example in Figure 2). However, we identified three samples that did not conform with this correlation: samples 7 and 11 as well as 8 and 11 induced potent antagonistic activity but the mass 363.25 was detected in low concentrations in those samples in LTQ-Orbitrap experiments 1 and 2, respectively. In general, the correlation with antiandrogenicity was more pronounced than with the antiestrogenic activity because bottled water inhibited androgen receptor more potently (Figure S3 and S4).

Bottom Line: We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%.Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water.This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.

View Article: PubMed Central - PubMed

Affiliation: Department Aquatic Ecotoxicology, Faculty of Biological Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany.

ABSTRACT
Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MS(n) experiments elucidated the molecule's structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MS(n) data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably, dioctyl fumarates and maleates have been overlooked by science and regulation to date. This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.

Show MeSH
Related in: MedlinePlus