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

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

Schicksal, Wege und Methoden zur Bestimmung von ausgewählten Antibiotika und Steroid Hormonen in der Umwelt

Pharmaceuticals are mainly introduced into the environment via two pathways. The first path is the application of veterinary drugs to animals, as they are excreted, high concentration are found in manure. As this contaminated manure is utilised for fertilising the fields a soil issue arises. The second path is the treatment of infections in the human medicine. When these residues are excreted they are transported through the sewers to sewage treatment plants and are then discharged with the treated wastewater into the aquatic environment. In this thesis the enviromental fate of the antibiotics erythromycin, roxithromycin, clarithromycin, tylosin, oleandomycin, tiamulin, salinomycin, the steroid hormones 17 β-estradiol, estrone, estriol, 16 α-hydroxyestrone and β-estradiol 17-acetate, the hormone-conjugates β-estradiol 3-sulfate and estrone 3-sulfate, the oral contraceptives 17 α-ethinylestradiol and mestranol were studied. To assess the fate of the macrolide antibiotics, ionophores, pleuromutilins, steroid hormones, oral contraceptives and hormone-conjugates, three new analytical methods were developed in respect of the different matrices manure, soil and wastewater. These analytical methods are based on the extraction methods liquid liquid extraction (LLE), solid phase extraction (SPE) and accelerated solvent extraction (ASE) in combination with the clean-up steps SPE, size exclusion chromatography (SEC) and the detection in different ionisation modes of highperformance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Isotopic labelled internal standards were used to account for matrix-effects in the HPLC-MS/MS analysis. As the availability of deuterated macrolide antibiotics standards is poor, a new macrolide internal standard was synthesised. For the determination of veterinary pharmaceuticals the limit of quantifications (LOQ) were determined. They were between 1.4 and 11 ng/g for manure and 0.6 to 30 ng/g in soil, whereas the LOQ in wastewater ranged from 0.6 to 35 ng/L . For the analysis of the steroid hormones and macrolide antibiotics in wastewater compliance within EU decision 657/2002/EC was achieved. The fate of the veterinary used antibiotics was studied with degradation experiments under anaerobic and aerobic conditions in manure and soil. Half-lives for antibiotics in manure ranged from 5 days up to >200 days and half-lives in soil ranged from 5 days up to >120 days. Additionally new metabolites of the antibiotic salinomycin in manure were identified by means of high performance liquid chromatography with electrospray ionisation coupled with high resolution time of flight mass spectrometry (HPLC-ESI-HR-TOF-MS) and different tandem mass spectrometric techniques. The fate of steroid hormones and macrolide antibiotics during wastewater treatment was researched by testing the elimination efficiencies of three different concepts of STPs over four weeks at different weather conditions. While larger STPs eliminated hormones more constantly than smaller STPs, heavy rainfall events led to a collapse of the biological treatment step. By using trickling filter techniques for the treatment of wastewater an elimination of the steroid hormones could not be observed. Also no significant elimination of macrolide antibiotics during wastewater treatment could be detected in all three STPs

DNA-Aptamere als eine neuartige biologisch aktive Komponente in Biosensoren

Die Erkennung von Molekülen mittels selektiver Bindung von Enzymen, Antikörpern oder selektiver Chelatoren ist die Grundlage zur Entwicklung von Biosensoren. Systematic Evolution of Ligands by EXponential enrichment (SELEX) ist eine kombinatorisch chemische Methode, welche die Eigenschaft von einzelsträngigen Nukleinsäuren (RNA, ssDNA) nutzt zu stabilen 3-dim. Strukturen zu falten und somit – in Analogie zu Antikörpern (Abs)– eine selektive und hochaffine Bindung von Zielmolekülen ermöglicht. Der Einsatz von Oligonukleotidliganden (Aptamere) als biologisch aktiver Teil von Biosensoren bietet gegenüber Abs eine Reihe potentieller Vorteile. Aptamere können mit hoher Genauigkeit und Reproduzierbarkeit durch automatisierte Prozesse synthetisiert werden und ermöglichen eine relativ einfache, kovalente Bindung von Reportermolekülen und/oder Transducern an definierten Stellen. Immobilisierte Aptamere können recycled werden und gegen jegliche Art von Zielmolekülen gerichtet sein (auch gegen toxische oder nicht immunogene Proteine oder kleine organische Moleküle). Als Modellsystem haben wir eine auf Fluoreszenzdetektion basierende Selektionsmethode von endothelzellbindenden ssDNA-Aptameren entwickelt und zeigen daß fluoreszenzkonjugierte Aptamere in Analogie zu Antikörpern als diagnostische Werkzeuge zur Targetidentifizierung genutzt werden können, somit ideale Voraussetzungen bieten als biologisch aktiver Teil in Mikrobiosensoren Verwendung zu finden. Der Einsatz von fluoreszenzkonjugierten DNA-Aptameren zeigte, daß diese neuartigen Sonden ohne Einbuße ihrer Bindungseigenschaften an Reportereinheiten gekoppelt bzw. auf Oberflächen immobilisiert werden können. DNA-Aptamer-Sonden – gekoppelt an einen Transducer –bergen somit ein enormes Potential als sensible und flexible biologisch aktive Komponente in Biosensoren zur Detektion jeglicher Art von Zielmolekül (Proteine - auch toxisch oder nicht immunogen, Peptide oder kleine organische Moleküle) Verwendung zu finden

Fate of conjugated natural and synthetic steroid estrogens in crude sewage and activated sludge batch studies

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Environmental Science & Technology, copyright © American Chemical Society after peer review. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/es801952h.Steroids are excreted from the human body in the conjugated form but are present in sewage influent and effluent as the free steroid, the major source of estrogenic activity observed in water courses. The fate of sulfate and glucuronide conjugated steroid estrogens was investigated in batch studies using activated sludge grown on synthetic sewage in a laboratory-scale Husmann simulation and crude sewage from the field. A clear distinction between the fate of sulfate and glucuronide conjugates was observed in both matrices, with sulfated conjugates proving more recalcitrant and glucuronide deconjugation preferential in crude sewage. For each conjugate, the free steroid was observed in the biotic samples. The degree of free steroid formation was dependent on the conjugate moiety, favoring the glucuronide. Subsequent degradation of the free steroid (and sorption to the activated sludge solid phase) was evaluated. Deconjugation followed the first order reaction rate with rate constants for 17α-ethinylestradiol 3-glucuronide, estriol 16α-glucuronide, and estrone 3-glucuronide determined as 0.32, 0.24, and 0.35 h respectively. The activated sludge solid retention time over the range of 3−9 days had 74 to 94% of sulfate conjugates remaining after 8 h. In contrast, a correlation between increasing temperature and decreasing 17α-ethinylestradiol 3-glucuronide concentrations in the activated sludge observed no conjugate present in the AS following 8 h at 22 °C Based on these batch studies and literature excretion profiles, a hypothesis is presented on which steroids and what form (glucuronide, sulfate, or free) will likely enter the sewage treatment plant.EPSR

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]+ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MSn analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well