Article pubs.acs.org/est

Heterocyclic Aromatic Hydrocarbons Show Estrogenic Activity upon Metabolization in a Recombinant Transactivation Assay Markus Brinkmann,†,# Sibylle Maletz,†,# Martin Krauss,‡ Kerstin Bluhm,† Sabrina Schiwy,† Jochen Kuckelkorn,† Andreas Tiehm,§ Werner Brack,‡ and Henner Hollert*,†,∥,⊥,∇ †

Institute for Environmental Research, Department of Ecosystem Analysis, ABBtAachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany ‡ Department Effect-Directed Analysis, Helmholtz Centre for Environmental ResearchUFZ, Permoserstraße 15, 04318 Leipzig, Germany § Water Technology Center, Department of Environmental Biotechnology, Karlsruher Strasse 84, 76139 Karlsruhe, Germany ∥ State Key Laboratory of Pollution Control and Resource Reuse, School of Environment, Nanjing University, Xianlin Avenue 163, Nanjing 210023, People’s Republic of China ⊥ College of Resources and Environmental Science, Chongqing University, Tiansheng Road Beibei 1, Chongqing 400030, People’s Republic of China ∇ Key Laboratory of Yangtze Water Environment, Ministry of Education, Tongji University, Siping Road 1239, Shanghai 200092, People’s Republic of China S Supporting Information *

ABSTRACT: Heterocyclic aromatic hydrocarbons (hetero-PAHs) are increasingly studied at contaminated sites; especially at former industrial facilities where coal tar-oil was handled, e.g., wood treatment plants, high concentrations of heteroPAHs are frequently detected in groundwater plumes. In previous studies, fractions of groundwater with high estrogenic activity contained hetero-PAHs and their hydroxylated metabolites. To evaluate this preliminary evidence, selected heteroPAHs were screened for their estrogenic activity in lyticase yeast estrogen screen (LYES) and ER CALUX. All tested substances were inactive in the LYES. HeteroPAHs such as acridine, xanthene, indole, 2-methylbenzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophene, quinoline, and 6-methylquinoline were positive in the ER CALUX, with estradiol equivalence factors (EEFs) from 2.85 × 10−7 to 3.18 × 10−5. The EEF values of these substances were comparable to those of other xenoestrogens (e.g., alkylphenols or bisphenol A) that are sometimes found in surface water. Chemical analyses revealed that T47Dluc cells could metabolize most of the substances. Among the metabolites (tentatively) identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were hydroxides and their keto tautomers, sulfates, sulfoxides, and N-oxides. Because of their high concentrations measured in groundwater, we conclude that hetero-PAHs and metabolites may be a potential risk and should be the subject of further research.

1. INTRODUCTION Heterocyclic aromatic hydrocarbons (hetero-PAHs) containing nitrogen, sulfur, or oxygen heteroatoms represent potential pollutants increasingly studied at contaminated sites. During technical processes, such as tar-oil distillation, wood preservation, or the production of manufactured gas, coke, and dyestuffs, large amounts have been emitted into the environment since the 19th century.1−4 The soil at former industrial sites can still contain high concentrations of heterocyclic PAHs.5,6 In groundwater, long contaminated plumes with concentrations up to the mg L−1 range have been observeda fact that might also imply an elevated risk for human health since groundwater is an important drinking water resource.7,8 While extensive knowledge exists concerning environmental fate, as well as toxicological and ecotoxicological effects of PAHs,9 such knowledge is still limited for their heterocyclic analogues. Just recently, the scientific community began to © 2014 American Chemical Society

comparatively investigate a range of heterocyclic PAHs in different in vitro bioassays: e.g., acute toxicity to Daphnia and growth inhibition of green algae,10 mutagenicity (Ames assay), embryotoxicity in the zebrafish,11 or aryl hydrocarbon12,13 and retinoid receptor14 mediated effects. In most assays, heteroPAHs caused similar or even higher toxicity compared to their homocyclic analogues. However, there are still data gaps, especially with regard to potential endocrine disrupting effects of hetero-PAHs. For homocyclic PAHs, it has already been demonstrated that vertebrates can transform the parent molecules into hydroxylated metabolites [e.g.,ref 15]. In the organisms, this reaction of Received: Revised: Accepted: Published: 5892

April April April April

22, 11, 11, 11,

2013 2014 2014 2014

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Figure 1. Estradiol equivalence factor (EEF) ranges of the investigated heterocyclic compounds relative to E2 as measured in the ER CALUX assay. Bars refer to EEF50s (i.e., the EEFs based on 50% E2-max.), error bars represent the EEF20−80 ranges. Benzofuran, benzothiophene, and carbazole were inactive in the ER CALUX assay, i.e., did not reach 20% induction of the E2 standard.

2. MATERIALS AND METHODS 2.1. Chemicals. Indole (>99%), quinoline (>98%), carbazole (approximately 95%), 6-methylquinoline (>98%), benzothiophene (>98%), and dibenzothiophene (>98%) were obtained from abcr (Karlsruhe, Germany). Acridine (>98%) was supplied by Merck (Darmstadt, Germany). Xanthene (99%), benzofuran (>99%), 2-methylbenzofuran (≥96%), 2,3dimethylbenzofuran (≥97%), and dibenzofuran (approximately 98%) were purchased from Sigma-Aldrich (Deisenhofen, Germany). Stock solutions of the hetero-PAHs investigated were prepared in dimethyl sulfoxide (DMSO). Generalized structures of the investigated compounds are given in Figure 1. 2.2. LYES Assay. Yeasts of the strain Saccharomyces cervisiae have been stably transfected with the human estrogen receptor α (hER α). Additionally, the cells contain an expression plasmid carrying the reporter gene lacZ which encodes for βgalactosidase (β-gal) and the sequences for an estrogen responsive element (ERE). Through binding of a ligand, expression of β-gal is induced and the enzyme secreted into the culture medium, where it converts the chromogenic substrate chlorophenol red-β-D-galactopyranoside (CPRG) to chlorophenol red.21 The LYES was conducted as described by Routledge and Sumpter21 with modifications according to Schultis and Metzger22 and Wagner and Oehlmann.23 Specifically, to increase sensitivity and allow for rapid testing, a digestion step utilizing the enzyme lyticase is added to the protocol. In brief, yeast cells were exposed to a 1:2 dilution series of the heterocyclic PAHs in quadruplicate wells for 24 h. The maximum concentration of the tested chemicals was chosen according to concentrations used in the work of Hinger and Brinkmann et al.12 A dilution series of 17β-estradiol (E2, Sigma-Aldrich, 8 replicate wells) ranging from 1 to 1000 pM and a DMSO solvent control were included in each experiment. Cell density estimation was performed at 595 nm and the exposure was terminated by addition of lacZ solution, containing buffer, CPRG and β-mercaptoethanol. The amount of converted CPRG was measured after 1 h at 540 nm. Absorbance readings were corrected for cell density. 2.3. ER CALUX Assay. T47D human breast adenocarcinoma cells expressing the endogenous estrogen receptors α and β

phase I biotransformation has the purpose of increasing the water solubility of a molecule, making it more susceptible to phase II biotransformation reactions; both processes ultimately facilitate the excretion of a compound. In permanent cell-lines, which have been developed for bioanalytical determination of chemicals with mechanism-specific (e.g., dioxin-like or estrogenic) effects these biotransformation reactions could also lead to formation of active metabolites. Consequently, not only direct estrogenic effects of the parent compounds, but also indirect estrogenic effects of the metabolites have to be considered. For PAHs such as benzo[a]pyrene and chrysene, it has been shown that indirect effects can occur in bioanalytical assays for detection of estrogenic substances.16,17 Furthermore, previous bioanalytical studies on the contamination of groundwater with estrogenic substances have shown that fractions with high estrogenic activity contained, among others, hetero-PAHs and their hydroxylated metabolites.18 In another study on endocrine activity of river sediments, a correlation between endocrine activity and fractions containing PAHs was identified.19 To investigate whether heterocyclic PAHs also have the potential to act as indirect estrogenic compounds, i.e., being able to induce estrogenic effects only after biotransformation, we report here on a study of the effects of selected substances in the T47Dluc cell-based estrogen response chemically activated luciferase expression (ER CALUX) assay. The lyticase yeast estrogen screen (LYES) assay was used as a reference that can only detect directly acting estrogenic substances. Most compounds tested here were suggested earlier as being representative for tar-oil contaminated sites by the project framework BMBF KORA [retention and degradation processes to reduce contaminations in groundwater and soil, cf. refs 10,20] and comprised 12 individual compounds: indole, 1-benzothiophene, benzofuran, 2-methylbenzofuran, 2,3-dimethylbenzofuran, quinoline, 6-methylquinoline, carbazole, dibenzothiophene, dibenzofuran, acridine, and xanthene. To assess the loss of parent substance and the formation of metabolites during incubation with T47Dluc cells, supernatants were analyzed by means of liquid chromatography with diode array detection (LC-DAD) and with high resolution tandem mass spectrometry (LC-HRMS/MS). 5893

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Figure 2. Dose−response curves and corresponding 95% confidence bands in the ER CALUX assay for all investigated heterocyclic compounds (closed circles) and the E2 standard curves (open circles) were measured in n = 3 independent assay repetitions. Concentration values on the x-axis refer to nominal medium concentrations of the substances. Dots represent mean values from triplicate measurements in one respective experiment.

PAHs in triplicate for another 24 h. The maximum concentration of the tested chemicals was chosen according to concentrations used in the work of Hinger and Brinkmann et al.12 An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay according to the protocol of Sanderson et al.,27 with modifications, was conducted to ensure that the test items did not cause nonspecific cytotoxic effects. In addition, an E2 (Sigma-Aldrich) dilution series ranging from 0.3 to 30 pM and a DMSO solvent control (0.1%) were included in triplicates on each plate. Medium was removed and luciferase activity was measured after adding 50 μL of PBS and 50 μL of SteadyLite (PerkinElmer, U.S.A.) with a luminescence counter (Infinite 200, Tecan, Crailsheim, Germany).

have been stably transfected with an estrogen-responsive luciferase reporter gene (pEREtata-Luc). This modified cell line (T47Dluc) is sensitive and highly responsible to (anti)estrogenic compounds. In case of binding of an estrogen active agent, the cells produce the enzyme luciferase. The luciferase activity is detected by measurement of light emission after addition of the substrate luciferin.24 The ER CALUX was performed according to Legler et al.24 with minor modifications according to the standard operation procedure (SOP) of BDS25 and detailed in Maletz et al.26 Cells were seeded in a 96-well plate at a density of 1 × 104 cells per well (1 × 105 per mL). After 24 h incubation, the cells were exposed to a 1:2 dilution series of each of the heterocyclic 5894

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Figure 3. Dose−response curves and corresponding 95% confidence bands in the LYES assay for all investigated heterocyclic compounds (closed circles) and the E2 standard curves (open circles) were measured in n = 3 independent assay repetitions. Concentration values on the x-axis refer to nominal medium concentrations of the substances. Dots represent mean values from technical replicates of one respective experiment. All substances were inactive in the LYES assay, i.e., did not reach 20% induction of the E2 standard.

2.4. Data Analysis for Calculation of Estradiol Equivalence Factors. Data analysis of both ER CALUX and LYES were conducted following the recommendations of Villeneuve at al.28 Mean luminescence and absorbance values, respectively, of test items and E2 standards were corrected for the response of the solvent controls. Resulting values were then divided by the maximum induction of the E2 standard (E2max.) to scale all values from 0 (solvent control) to 1 (E2max.). Scaled values from triplicate experiments were plotted using the software GraphPad Prism 5 (GraphPad, San Diego, U.S.A.) and fitted using four-parameter logistic regression with

variable slope, where the top and bottom of the curve was set to 0 and 1, respectively (Figures 1 and 2). EEF20−80 ranges, i.e., multiple estradiol equivalence estimates (EEFs, eq 1) along the concentration−response curves based on a ratio of EC20s, EC50s, and EC80s, were calculated in order to both test the assumptions of parallelism and equal efficacy and give a measure of uncertainty for mass-balance analyses.

EEFX = 5895

ECXE2 ECX sample

(1)

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Table 1. Comparison of Measured Data with Predicted ER Binding for Parent Compounds and Simulated Metabolites (OECD QSAR toolbox liver metabolism simulator)a maximum concentrationc

measured effects in the ER CALUX

predicted ER binding of parent substanced

metabolism (LC-DAD)c

substance

mg L−1

EEF50 (EEF20−80)

benzofuran 2-methylbenzofuran 2,3dimethylbenzofuran dibenzofuran benzothiophene dibenzothiophene acridine xanthene carbazole indole quinoline 6-methylquinoline

120.9 210.0 110.0

− 2.85 (1.49−5.45) 10−7 1.38 (1.12−1.69) 10−5

− + ++

− − −

n.d. n.d. n.d.

56.9 136.1 40.8 40.9 75.5 25.6 162.7 226.0 528.0

1.82 − 5.53 3.18 3.94 − 5.53 1.25 5.21

(1.74−1.92) 10−6

+ − + ++ + − + + +

− − − − − − − − −

3.3 46.1 86.7 17.0 71.9 24.7 90.0 18.5 −49.3

(5.27−5.80) 10−6 (2.20−4.61) 10−5 (3.94−3.94) 10−6 (5.27−5.80) 10−6 (1.12−1.40) 10−7 (4.45−6.09) 10−7

predicted ER binding of simulated metabolitesd

% − + + ++ + ++ ++ ++ ++ − − +

a

EEF50s and EEF20‑80 ranges are given for use in mass-balance calculations. bn.d, not determinable due to complete loss during incubation without cells. cAccording to the concentration range earlier used by Hinger et al.12 dPredicted estrogen receptor (ER) bindung using the OECD QSAR toolbox.

products, T47luc cells were exposed to 10 mg L−1 of each heterocyclic PAH as for the determination of total metabolism. Instead of using 6-well microplates, cells were seeded in 75 cm2 tissue culture flasks to obtain a higher volume of cell culture supernatant (15 mL). To ensure full compatibility with the LCHRMS/MS method, stock solutions (10 mg mL−1) of the substances were prepared in ethyl acetate instead of DMSO. The final concentration of ethyl acetate in the exposure medium was 0.1%. A solvent control was treated as the dilutions without addition of substance. Following 24 h incubation at 37 °C, cells were detached with a cell scraper and homogenized with the cell culture supernatant using an automatic disperser. The suspension was then centrifuged at 25 °C (4000g), and the supernatant stored at −80 °C until analysis. The samples were analyzed by LC-high resolution mass spectrometry using an Agilent 1200 LC system coupled to a LTQ Orbitrap XL (Thermo Scientific). The LC method is described in ref 30. Full scan chromatograms (m/z 100−1000) were acquired after electrospray ionization in positive and negative ion mode at a nominal resolving power of 100 000 referenced to m/z 400. HRMS product ion spectra were recorded in data-dependent mode using collision-induced fragmentation (CID) and higher-energy collisional dissociation (HCD) at different collision energies. Full scan spectra were manually searched for the masses of human and mammalian metabolites of the studied compounds reported in the literature as well as possible phase I metabolites based on common pathways (e.g., the ion mass of an M+O molecule, corresponding to N-oxides, sulfoxides, hydroxylated metabolites or their corresponding keto tautomers, M+O−2H molecule corresponding to an epoxide, etc.). For detection of unknown and unexpected metabolites we used the software MZmine 2.10.31 Processing of full scan chromatograms was carried out using the settings described in ref 30. The peak lists obtained were searched for peaks newly formed solely by the incubation of each heterocyclic PAH that were absent in all other samples. For these peaks, molecular formulas were determined based on accurate masses and isotope patterns using the Xcalibur software (ThermoScientific) and MS/MS

2.5. Prediction of Estrogen Receptor Binding Affinities. Estrogen receptor (ER) binding affinities were simulated using the (Q)SAR Toolbox 2.3 provided by the Organization for Economic Cooperation and Development (OECD). Simulations were performed for parent compounds, as well as for measured metabolites and metabolites predicted by the liver metabolism simulator of the toolbox. Results of the model were qualitatively compared with experimental data from both bioassays. 2.6. Determination of Total Metabolism by Means of LC-DAD. T47Dluc cells were seeded in 6-well multiplates and incubated in duplicates with the highest concentration used in the assays under the same conditions as in the ER CALUX assay. A control without cells was incubated under the same conditions as the cells to determine substance losses due to, e.g., sorption, precipitation, or volatilization. Subsequently, 2 mL of the cell-culture media were liquid−liquid extracted using ethyl acetate. Extracts were reconstituted in HPLC-grade acetonitrile after reduction under a gentle nitrogen stream. Heterocyclic PAHs and respective metabolites were analyzed according to Mundt and Hollender,29 with slight modifications. Liquid chromatography was performed using a 1200 Series LC chromatograph (Agilent, Waldbronn, Germany) equipped with an UV-diode array detector at 1.0 mL min−1 flow. Briefly, 40 μL of the reconstituted extracts were separated on a Nucleosil C18 prepacked column (250 × 4 mm2, 5 μm particle size; Macherey-Nagel, Düren, Germany) by gradient elution with acetonitrile (solvent A) and 5 mM potassium phosphate buffer at pH 7 (solvent B) using the following program: held 10/90% (v/v) for 2 min, in 2 min up to 50/50% (v/v), up to 60/40% (v/v) in 8 min, up to 75/25% (v/v) in 10 min, to 100/0% (v/ v) in 5 min, back to 10/90% (v/v) in 12 min, and held 6 min for equilibration. The diode array detector signals at 210 and 254 nm, as well as the spectra from 190 to 400 nm were recorded. Acridine was quantified at 254 nm, all other heteroPAHs at 210 nm. Substance losses during incubation with and without cells were compared. 2.7. Identification of Metabolites by LC-High Resolution Tandem Mass Spectrometry (LC-HRMS/MS). To assess the formation of potentially estrogenic transformation 5896

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Table 2. Metabolites of Heterocyclic PAHs Detected and (Tentatively) Identified in the Incubation Medium by High Resolution Tandem Mass Spectrometry (LC-HRMS/MS)a parent compound (RT in min)

metabolite (bold: identified)

RT (min)

molec. formula

confirmed by ref std.

predicted ER bindingb

ref

acridine (RT 15.8) acridine acridine acridine quinoline (RT 5.9) quinoline quinoline quinoline 6-methylquinoline (RT 11.9) 6-methylquinoline 6-methylquinoline indole (RT 20.4) indole indole indole carbazole (RT 24.5) xanthene (n.d.)d benzofuran (n.d) benzofuran benzofuran benzofuran 2-methylbenzofuran (n.d.) 2-methylbenzofuran 2-methylbenzofuran 2,3-dimethyl benzofuran (n.d.) 2,3-dimethyl benzofuran 2,3-dimethyl benzofuran dibenzofuran (n.d.) benzothiophene (n.d.) benzothiophene benzothiophene benzothiophene dibenzothiophene (n.d.)

acridone acridine + O acridine + 2O acridine + 2OH 2-hydroxyquinoline quinoline + O quinoline N-oxide quinoline + OH2 6-methylquinoline + O 6-methylquinoline N-oxide 6-methylquinoline + OH2 2-oxindole indole + OH2 indole + OH2 indole − 2H+O carbazole-sulfate xanthone 2-hydroxyphenylacetic acid benzofuran + 2OH benzofuran + CH4O2 benzofuran + CH4O2 2-methylbenzofuran + 2OH 2-methylbenzofuran + 2OH 2-methylbenzofuran − 2H+O 2,3-dimethylbenzofuran + O 2,3-dimethylbenzofuran − 2H+O 2,3-dimethylbenzofuran + 2OH dibenzofuran sulfate benzothiophene + O benzothiophene −S + CH4O3 benzothiophene −S + CH4O3 2-hydroxyphenyl acetic acid dibenzothiophene sulfoxide

21.7 16.9 20.3 9.4 18.2 18.9 14.8 16.9 16.0 18.2 18.0 17.4 14.0 19.1 16.6 21.9 25.3 16.4 17.7 17.8 19.8 17.7 18.5 19.1 16.3 20.2 16.4 23.1 15.5 18.1 18.5 16.4 20.9

C13H9NO C13H9NO C13H9NO2 C13H11NO2 C9H7NO C9H7NO C9H7NO C9H9NO C10H9NO C10H9NO C10H11NO C8H7NO C8H9NO C8H9NO C8H5NO C12H9SO4 C13H8O2 C8H8O3 C8H8O3 C9H10O3 C9H10O3 C9H10O3 C9H10O3 C9H6O2 C10H10O2 C10H8O2 C10H12O3 C12H8SO5 C8H6OS C9H10O3 C9H10O3 C8H8O3 C12H8OS

yes no no no yes no yes no no no no yes no no no no yes yes no no no no no no no no no no no no no yes no

− +c ++c ++c −

61

− − +c − − − +c +c − − − +

62

63 63 64

65

(+) (+) +c +c − +c − ++c − −

+ −

66

a

The (tentatively) identified metabolites are given by name, those not identified by their modification as compared to the parent molecule. The retention times (RT) of the parent compounds are given for reference. bPredicted estrogen receptor (ER) binding using the OECD QSAR toolbox. c Different isomers possible of which some show an estrogenic activity as predicted by the QSAR. dn.d. = parent molecule not detected in ESI.

biotransformation,33 and thus, no hydroxylation of the heterocyclic PAHs by the yeast would be expected. The results from the present study confirmed the expectation that no direct estrogenic effects are caused by heterocyclic PAHs. 3.2. ER CALUX Assay. The EC50 of the E2 standard was 8.1 ± 0.9 pM (mean ± standard error, n = 12) throughout the whole study, which is within the range reported in other studies.24,26,34 Concentration−response curves for the heterocyclic aromatic hydrocarbons are given in Figure 2. Benzofuran, benzothiophene, and carbazole were inactive in the ER CALUX assay, i.e., did not reach 20% induction of the E2 standard. EEF20−80 ranges were calculated for all other tested compounds (Figure 3). The substances 2-methylbenzofuran, quinoline, and 6-methylquinoline, did not reach 80% induction of the E2 standard, making slight extrapolation beyond the measured range of response necessary to establish the EEF20−80 ranges as recommended by Villeneuve at al.28 Because acridine and 2,3dimethylbenzofuran exhibited higher maximum induction compared to the E2 standard, data was normalized to the maximum induction of these substances. In contrast to the LYES assay that utilizes yeast cells, Piao et al.,35 Kuil et al.,36 and Spink et al.37 have shown that the

spectra were visually evaluated. Tentatively identified metabolites were confirmed by reference standards if these were commercially available.

3. RESULTS AND DISCUSSION 3.1. LYES Assay. The EC50 of the E2 standard was 57.7 ± 0.7 pM (mean ± standard error, n = 3) throughout the whole study. Other publications, including studies from our own lab, report lower values in the range of 30−50 pM.22,26 These studies commonly used ethanol as solvent for dosing of the test items. In the present study, however, it was necessary to use another solvent, i.e., DMSO, since the substances did not readily dissolve in ethanol. Concentration−response curves for the tested substances in the LYES assay are given in Figure 3. None of the tested compounds was active in the LYES assay, i.e., reached 20% induction of the E2 standard. No estrogen receptor (ER) binding affinity was predicted from the structural properties of the parent substances by using the OECD (Q)SAR Toolbox (Table 1)especially because the compounds do not contain hydroxy-substituted aromatic rings, which is a prerequisite for binding to the ER.32 The yeast strain used for the LYES assay is not capable of 5897

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estrogenicity of different complex samples [e.g., refs 49−52]. Previous research on contamination of groundwater with estrogenic compounds identified polycyclic aromatic hydrocarbons, including hydroxylated and heterocyclic analogues, in those fractions with high estrogenic activity when applying effect-directed analyses using the E-Screen assay.18 Further studies53,54 found several heterocyclic PAHs, including quinoline, carbazole, acridine, benzofuran, and benzothiophene, in concentrations up to 4 mg L−1 in groundwater samples from tar-oil contaminated sites in Germany. Acridine, which showed a higher induction in the ER CALUX than 17β-estradiol, was found in concentrations up to 3 μg L−1. Regarding its high potential to activate the ER in mammalian cells, acridine should possibly be included in the screening battery of chemical analyses of estrogenically active water samples in the catchment area of former tar-oil sites. Additionally, acridine and some of its transformation products are among the major photodegradation products of the antiepileptic pharmaceutical carbamazepine,55,56 which is regularly found in surface, as well as drinking water.57,58 In summary, our study demonstrates that some heterocyclic PAHs have comparable estrogenicity to some nonsteroidal EDCs of relevance and are consequently of highest relevance with respect to the estrogenicity of water resources as well as possibly to human health. Overall, this substance class has to be taken into account regarding the estimation of endocrine activity in surface and groundwater.59,60 Since groundwater is an important drinking water resource the hetero-PAHs investigated could imply an increased risk for human health. Furthermore, we could show that test systems without the ability for biotransformation, such as the YES assay, may be susceptible to underestimation of the estrogenic potential of organic compounds in water samples. Consequently, a metabolic activation step should be included in the bioanalytical identification of potential indirect estrogenic substances.

T47Dluc cells express a number of cytochrome P450 (CYP) isoforms as well as 17β-HSD, and thus are readily capable of biotransformation and conversion of steroids. It has been demonstrated by van Lipzig et al.16 that the homocyclic PAHs benzo[a]pyrene (BaP) and chrysene were transformed into several hydroxylated metabolites during incubation with βnaphtoflavone-induced rat-liver S9. Furthermore, BaP itself caused a significant induction of luciferase in the ER CALUX assay that was completely inhibited by the CYP inhibitor 3′-,4′dimethoxyflavone. Similar results have been shown previously in a transfected human breast cancer cell-line, MCF-7.38,39 By use of a gastrointestinal simulator, Van de Wiele et al.40 have demonstrated that ingested PAHs can be transformed into estrogenic metabolites by colon microbiota and may thus pose a higher risk to human health after absorption via food or drinking water than would be expected from the parent substances. We hypothesize that heterocyclic PAHs are comparable with their homocyclic analogues a class of indirect estrogenic compounds that are transformed into the active form (hydroxylated and other) by (mammalian) biotransformation enzymes. The observed EEFs were relatively high and within the range of other nonsteroidal EDCs of high concern commonly found in surface water, e.g. alkylphenols,41 bisphenol A, phthalates or pesticides.42,43 3.3. Identification of Metabolites. To test the hypothesis that heterocyclic PAHs are transformed into more reactive metabolites, we first investigated the losses of parent substances from the incubation medium by means of LC-DAD. It was shown that T47Dluc cells were able to metabolize most of the substances to a significant extent (Table 1). In a next step, metabolites were detected and (tentatively) identified using LC-HRMS/MS. We were able to detect one to four individual metabolites for each of the parent substances investigated (Table 2). For six of these compounds the identity was confirmed by authentic reference compounds (acridone, 2hydroxyquinoline, quinoline N-oxide, oxindole, xanthone, and 2-hydroxyphenylacetic acid formed from both benzofuran and benzothiophen). In four cases a tentative structural assignment was possible based on MS/MS fragmentation behavior and analogy to spectra of reference standards of other compounds (Table 2). Details are given in the Supporting Information, SI. For the other detected metabolites an assignment based on MS/MS spectra, retention times and ionization behavior were considered too speculative. Among the identified metabolites, estrogenic activity was only predicted for 2-hydroxyphenylacetic acid by the OECD QSAR toolbox. However, MS/MS spectra showed neutral losses of H2O in many cases (see SI), suggesting that a range of hydroxylated and thus phenolic compounds are formed, of which at least some isomers show an estrogenic activity as predicted by the QSAR. 3.4. Possible Contribution to Estrogenic Activity in Water Resources. The main sources of anthropogenic estrogenic substances in freshwater aquatic environments are probably from municipal sewage and agriculture. The overall activity, however, is a result of complex substance mixtures from many different sources.44,45 These include high amounts of pharmaceuticals in hospital sewage46 and diverse point sources, such as the tar-oil contaminated sites investigated herein. Most attempts to explain the overall estrogenic activity of water samples by mass-balance studies have not been conclusive, and detection of target analytes often underestimates the response in the bioassays.47,48 A number of studies confirmed that hydroxylated PAHs contributed to the

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information on tentatively identified and confirmed metabolites including extracted ion chromatograms and MS/ MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 (0)241−80/26678; fax: +49 (0)241−80/22182; e-mail: [email protected]. Author Contributions #

Both authors contributed equally to the manuscript and share first authorship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support by a Seed funds project of RWTH Aachen University supported by the German Excellence Initiative and the project framework Tox-Box supported by the German Federal Ministry of Education and Research (BMBF) (funding number 02WRS1282I). Tox-Box is a constitutive part of the BMBF action plan “Sustainable water management (NaWaM)”. It is part of the funding scheme “Risk Management of Emerging Compounds and Pathogens in the 5898

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(14) Benisek, M.; Kubincova, P.; Blaha, L.; Hilscherova, K. The effects of PAHs and N-PAHs on retinoid signaling and Oct-4 expression in vitro. Toxicol. Lett. 2011, 200 (3), 169−175. (15) Varanasi, U.; Stein, J. E.; Nishimoto, M., Biotransformation and Disposition of PAH in Fish. In Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment; Varanasi, U., Ed.; CRC Press Inc.: Boca Raton, FL, 1989; pp 93−150. (16) van Lipzig, M. M. H.; Vermeulen, N. P. E.; Gusinu, R.; Legler, J.; Frank, H.; Seidel, A.; Meerman, J. H. N. Formation of estrogenic metabolites of benzo[a]pyrene and chrysene by cytochrome P450 activity and their combined and supra-maximal estrogenic activity. Environ. Toxicol. Pharmacol. 2005, 19 (1), 41−55. (17) Santodonato, J. Review of the estrogenic and antiestrogenic activity of polycyclic aromatic hydrocarbons: Relationship to carcinogenicity. Chemosphere 1997, 34 (4), 835−848. (18) Kuch, B.; Kern, F.; Metzger, J.; Trenck, K. Effect-related monitoring: Estrogen-like substances in groundwater. Environ. Sci. Pollut. Res. 2010, 17 (2), 250−260. (19) Higley, E.; Grund, S.; Jones, P. D.; Schulze, T.; Seiler, T.-B.; Varel, U. L.-v.; Brack, W.; Wölz, J.; Zielke, H.; Giesy, J. P.; Hollert, H.; Hecker, M. Endocrine disrupting, mutagenic, and teratogenic effects of upper Danube River sediments using effect-directed analysis. Environ. Toxicol. Chem. 2012, 31 (5), 1053−1062. (20) Blotevogel, J.; Reineke, A. K.; Hollender, J.; Held, T. Identification of NSO-heterocyclic priority substances for investigating and monitoring creosote-contaminated sites. Grundwasser 2008, 13 (3), 147−157. (21) Routledge, E. J.; Sumpter, J. P. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 1996, 15 (3), 241−248. (22) Schultis, T.; Metzger, J. W. Determination of estrogenic activity by LYES-assay (yeast estrogen screen-assay assisted by enzymatic digestion with lyticase). Chemosphere 2004, 57 (11), 1649−1655. (23) Wagner, M.; Oehlmann, J. Endocrine disruptors in bottled mineral water: total estrogenic burden and migration from plastic bottles. Environ. Sci. Pollut. Res. 2009, 16 (3), 278−286. (24) Legler, J.; van den Brink, C. E.; Brouwer, A.; Murk, A. J.; van der Saag, P. T.; Vethaak, A. D.; van der Burg, P. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicol. Sci. 1999, 48 (1), 55−66. (25) BDS. Analysis of estrogen receptor mediated luciferase activity in ER CALUX cells. 2007, P-BDS-011, (H). (26) Maletz, S.; Floehr, T.; Beier, S.; Klümper, C.; Brouwer, A.; Behnisch, P.; Higley, E.; Giesy, J. P.; Hecker, M.; Gebhardt, W.; Linnemann, V.; Pinnekamp, J.; Hollert, H. In vitro characterization of the effectiveness of enhanced sewage treatment processes to eliminate endocrine activity of hospital effluents. Water Res. 2013, 47 (4), 1545− 1557. (27) Sanderson, J. T.; Slobbe, L.; Lansbergen, G. W. A.; Safe, S.; van den Berg, M. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 2001, 61 (1), 40−48. (28) Villeneuve, D. L.; Blankenship, A. L.; Giesy, J. P. Derivation and application of relative potency estimates based on in vitro bioassay results. Environ. Toxicol. Chem. 2000, 19 (11), 2835−2843. (29) Mundt, M.; Hollender, J. Simultaneous determination of NSOheterocycles, homocycles and their metabolites in groundwater of tar oil contaminated sites using LC with diode array UV and fluorescence detection. J. Chromatogr. A 2005, 1065 (2), 211−218. (30) Hug, C.; Ulrich, N.; Schulze, T.; Brack, W.; Krauss, M. Identification of novel micropollutants in wastewater by a combination of suspect and nontarget screening. Environ. Pollut. 2014, 184 (0), 25− 32. (31) Pluskal, T.; Castillo, S.; Villar-Briones, A.; Oresic, M. MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 2010, 11 (1), 395.

Water Cycle (RiSKWa)”. M.B. received a personal stipend by the German National Academic Foundation (Studienstiftung des deutschen Volkes). The study sponsors had no influence on study design, collection, analysis, or interpretation of data, or in writing of or the decision to submit the manuscript for publication. We want to express our gratitude to Mrs. Simone Hotz, for technical assistance during the experiments. The authors would like to thank Prof. J.P. Sumpter of Brunel University, United Kingdom, for supplying the yeast cells for the YES assay, Martin Wagner (University of Frankfurt) for introduction into the modified YES protocol, and Claus Bornemann (Eurofins) for supplying some metabolite reference standards.

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REFERENCES

(1) Dyreborg, S.; Arvin, E.; Broholm, K. Biodegradation of NSOcompounds under different redox-conditions. J. Contam. Hydrol. 1997, 25, 177−197. (2) Alinsafi, A.; Motta, M. d.; Bonte, S. L.; Pons, M. N.; Benhammou, A. Effect of variability on the treatment of textile dyeing wastewater by activated sludge. Dyes Pigm. 2006, 69, 31−39. (3) Becker, L.; Matuschek, G.; Lenoir, D.; Kettrup, A. Leaching behaviour of wood treated with creosote. Chemosphere 2001, 42 (3), 301−308. (4) Thomas, D.; Crain, S. M.; Sim, P. G.; Benoit, F. M. Application of reversed-phase liquid-chromatography with atmospheric-pressure chemical-ionization tandem mass-spectrometry to the determination of polycyclic aromatic sulfur heterocycles in environmental-samples. J. Mass Spectrom. 1995, 30 (7), 1034−1040. (5) Meyer, S.; Steinhart, H. Effects of heterocyclic PAHs (N, S, O) on the biodegradation of typical tar oil PAHs in a soil/compost mixture. Chemosphere 2000, 40 (4), 359−367. (6) Meyer, S.; Steinhart, H. Fate of PAHs and hetero-PAHs during biodegradation in a model soil/compost-system: Formation of extractable metabolites. Water Air Soil Pollut. 2001, 132 (3−4), 215−231. (7) Reineke, A.-K.; Göen, T.; Preiss, A.; Hollender, J. Quinoline and derivatives at a tar oil contaminated site: Hydroxylated products as indicator for natural attenuation? Environ. Sci. Technol. 2007, 41 (15), 5314−5322. (8) Tiehm, A.; Müller, A.; Alt, S.; Jacob, H.; Schad, H.; Weingran, C. Development of a groundwater biobarrier for the removal of PAH, BTEX, and heterocyclic hydrocarbons. Water Sci. Technol. 2008, 58 (7), 1349−1355. (9) Douben, P. E. T. PAHs: An Ecotoxicological Perspective; John Wiley & Sons Ltd.: Chichester, England, 2003; p 36. (10) Eisentraeger, A.; Brinkmann, C.; Hollert, H.; Sagner, A.; Tiehm, A.; Neuwoehner, J. Heterocyclic compounds: Toxic effects using algae, daphnids, and the Salmonella/microsome test taking methodical quantitative aspects into account. Environ. Toxicol. Chem. 2008, 27 (7), 1590−1596. (11) Peddinghaus, S.; Brinkmann, M.; Bluhm, K.; Sagner, A.; Hinger, G.; Braunbeck, T.; Eisenträger, A.; Tiehm, A.; Hollert, H.; Keiter, S. H. Quantitative assessment of the embryotoxic potential of NSOheterocyclic compounds using zebrafish (Danio rerio). Reprod Toxicol 2012, 33 (2), 224−232. (12) Hinger, G.; Brinkmann, M.; Bluhm, K.; Sagner, A.; Takner, H.; Eisenträger, A.; Braunbeck, T.; Engwall, M.; Tiehm, A.; Hollert, H. Some heterocyclic aromatic compounds are Ah receptor agonists in the DR-Calux and the EROD assay with RTL-W1 cells. Environ. Sci. Pollut Res. 2011, 18, 1297−1304. (13) Sovadinová, I.; Bláha, L.; Janoscaronek, J.; Hilscherová, K.; Giesy, J. P.; Jones, P. D.; Holoubek, I. Cytotoxicity and aryl hydrocarbon receptor-mediated activity of N-heterocyclic polycyclic aromatic hydrocarbons: Structure-activity relationships. Environ. Toxicol. Chem. 2006, 25 (5), 1291−1297. 5899

dx.doi.org/10.1021/es405731j | Environ. Sci. Technol. 2014, 48, 5892−5901

Environmental Science & Technology

Article

(48) Leusch, F. D. L. Tools to Detect Estrogenic Activity in Environmental Waters; Global Water Research Coalition: 2008. (49) Wenger, D.; Gerecke, A. C.; Heeb, N. V.; Schmid, P.; Hueglin, C.; Naegeli, H.; Zenobi, R. In vitro estrogenicity of ambient particulate matter: contribution of hydroxylated polycyclic aromatic hydrocarbons. J. Appl. Toxicol. 2009, 29 (3), 223−232. (50) Kamiya, M.; Toriba, A.; Onoda, Y.; Kizu, R.; Hayakawa, K. Evaluation of estrogenic activities of hydroxylated polycyclic aromatic hydrocarbons in cigarette smoke condensate. Food Chem. Toxicol. 2005, 43 (7), 1017−1027. (51) Muthumbi, W.; De Boever, P.; Pieters, J. G.; Siciliano, S.; D’Hooge, W.; Verstraete, W. Polycyclic aromatic hydrocarbons (PAHs) and estrogenic compounds in experimental flue gas streams. J. Environ. Qual. 2003, 32 (2), 417−422. (52) Machala, M.; Ciganek, M.; Bláha, L.; Minksová, K.; Vondrácǩ , J. Aryl hydrocarbon receptor-mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments. Environ. Toxicol. Chem. 2001, 20 (12), 2736−2743. (53) Blum, P.; Sagner, A.; Tiehm, A.; Martus, P.; Wendel, T.; Grathwohl, P. Importance of heterocylic aromatic compounds in monitored natural attenuation for coal tar contaminated aquifers: A review. J. Contam. Hydrol. 2011, 126 (3−4), 181−194. (54) Schlanges, I.; Meyer, D.; Palm, W.-U.; Ruck, W. Identification, quantification and distribution of PAC-metabolites, heterocyclic PAC and substituted PAC in groundwater samples of tar-contaminated sites from Germany. Polycycl. Aromat. Compd. 2008, 28 (4−5), 320−338. (55) Kosjek, T.; Andersen, H. R.; Kompare, B.; Ledin, A.; Heath, E. Fate of Carbamazepine during Water Treatment. Environ. Sci. Technol. 2009, 43 (16), 6256−6261. (56) Calisto, V.; Domingues, M. R. M.; Erny, G. L.; Esteves, V. I. Direct photodegradation of carbamazepine followed by micellar electrokinetic chromatography and mass spectrometry. Water Res. 2011, 45 (3), 1095−1104. (57) Heberer, T. Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicol. Lett. 2002, 131 (1−2), 5−17. (58) Benotti, M. J.; Trenholm, R. A.; Vanderford, B. J.; Holady, J. C.; Stanford, B. D.; Snyder, S. A. Pharmaceuticals and endocrine disrupting compounds in U.S. drinking water. Environ. Sci. Technol. 2008, 43 (3), 597−603. (59) Brack, W.; Dulio, V.; Slobodnik, J. The NORMAN Network and its activities on emerging environmental substances with a focus on effect-directed analysis of complex environmental contamination. Environ. Sci. Eur. 2012, 24 (1), 29. (60) Grummt, T.; Kuckelkorn, J.; Bahlmann, A.; Baumstark-Khan, C.; Brack, W.; Braunbeck, T.; Feles, S.; Gartiser, S.; Glatt, H.; Heinze, R.; Hellweg, C.; Hollert, H.; Junek, R.; Knauer, M.; Kneib-Kissinger, B.; Kramer, M.; Krauss, M.; Kuster, E.; Maletz, S.; Meinl, W.; Noman, A.; Prantl, E.-M.; Rabbow, E.; Redelstein, R.; Rettberg, P.; Schadenboeck, W.; Schmidt, C.; Schulze, T.; Seiler, T.-B.; Spitta, L.; Stengel, D.; Waldmann, P.; Eckhardt, A. Tox-Box: securing drops of lifeAn enhanced health-related approach for risk assessment of drinking water in Germany. Environ. Sci. Eur. 2013, 25 (1), 27. (61) Thiebaud, N.; Veloso Da Silva, S.; Jakob, I.; Sicard, G.; Chevalier, J.; Ménétrier, F.; Berdeaux, O.; Artur, Y.; Heydel, J.-M.; Le Bon, A.-M. Odorant metabolism catalyzed by olfactory mucosal enzymes influences peripheral olfactory responses in rats. PLoS One 2013, 8 (3), e59547. (62) LaVoie, E. J.; Adams, E. A.; Shigematsu, A.; Hoffman, D. On the metabolism of quinoline and isoquinoline: possible molecular basis for differences in biological activities. Carcinogenesis 1983, 4 (9), 1169− 1173. (63) Scharping, C. E.; Duke, C. C.; Holder, G. M.; Larden, D. The hepatic metabolism of two methylquinolines. Carcinogenesis 1993, 14 (4), 1041−1047. (64) Skordos, K. W.; Skiles, G. L.; Laycock, J. D.; Lanza, D. L.; Yost, G. S. Evidence supporting the formation of 2,3-epoxy-3-methylindo-

(32) Blair, R. M.; Fang, H.; Branham, W. S.; Hass, B. S.; Dial, S. L.; Moland, C. L.; Tong, W.; Shi, L.; Perkins, R.; Sheehan, D. M. The estrogen receptor relative binding affinities of 188 natural and xenochemicals: Structural diversity of ligands. Toxicol. Sci. 2000, 54 (1), 138−153. (33) Connolly, L.; Ropstad, E.; Verhaegen, S. In vitro bioassays for the study of endocrine-disrupting food additives and contaminants. TrAC Trends Anal. Chem. 2011, 30 (2), 227−238. (34) Legler, J.; Dennekamp, M.; Vethaak, A. D.; Brouwer, A.; Koeman, J. H.; van der Burg, B.; Murk, A. J. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays. Sci. Total Environ. 2002, 293 (1−3), 69−83. (35) Piao, Y.-s.; Peltoketo, H.; Jouppila, A.; Vihko, R. Retinoic acids increase 17β-hydroxysteroid dehydrogenase type 1 expression in JEG3 and T47D cells, but the stimulation is potentiated by epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, and cyclic adenosine 3′,5′-monophosphate only in JEG-3 cells. Endocrinology 1997, 138 (3), 898−904. (36) Kuil, C. W.; Brouwer, A.; van der Saag, P. T.; van der Burg, B. Interference between Progesterone and Dioxin Signal Transduction Pathways: Different mechanisms are involved in repression by the progesterone receptor a and b isoforms. J. Biol. Chem. 1998, 273 (15), 8829−8834. (37) Spink, D. C.; Spink, B. C.; Cao, J. Q.; DePasquale, J. A.; Pentecost, B. T.; Fasco, M. J.; Li, Y.; Sutter, T. R. Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 1998, 19 (2), 291−298. (38) Charles, G. D.; Bartels, M. J.; Zacharewski, T. R.; Gollapudi, B. B.; Freshour, N. L.; Carney, E. W. Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-α reporter gene assay. Toxicol. Sci. 2000, 55 (2), 320−326. (39) Fertuck, K. C.; Matthews, J. B.; Zacharewski, T. R. Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo. Toxicol. Sci. 2001, 59 (2), 231− 240. (40) Van de Wiele, T.; Vanhaecke, L.; Boeckaert, C.; Peru, K.; Headley, J.; Verstraete, W.; Siciliano, S. Human colon microbiota transform polycyclic aromatic hydrocarbons to estrogenic metabolites. Environ. Health Perspect. 2005, 113 (1), 6−10. (41) Preuss, T. G.; Gehrhardt, J.; Schirmer, K.; Coors, A.; Rubach, M.; Russ, A.; Jones, P. D.; Giesy, J. P.; Ratte, H. T. Nonylphenol isomers differ in estrogenic activity. Environ. Sci. Technol. 2006, 40 (16), 5147−5153. (42) Houtman, C. J.; van Houten, Y. K.; Leonards, P. E. G.; Brouwer, A.; Lamoree, M. H.; Legler, J. Biological validation of a sample preparation method for ER-CALUX bioanalysis of estrogenic activity in sediment using mixtures of xeno-estrogens. Environ. Sci. Technol. 2006, 40 (7), 2455−2461. (43) Legler, J.; Dennekamp, M.; Vethaak, A. D.; Brouwer, A.; Koeman, J. H.; van der Burg, B.; Murk, A. J. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays. Sci. Total Environ. 2002, 293 (1−3), 69−83. (44) Moltmann, J. F.; Liebig, M.; Knacker, T.; Keller, M.; Scheurer, M.; Ternes, T., Relevance of Ondocrine Disrupting Substances and Pharmaceuticals in Surface Waters; Umweltbundesamt: 2007; pp 1− 129. (45) Hecker, M.; Hollert, H. Endocrine disruptor screening: regulatory perspectives and needs. Environ. Sci. Eur. 2011, 23 (1), 1−14. (46) Verlicchi, P.; Galletti, A.; Petrovic, M.; Barceló, D. Hospital effluents as a source of emerging pollutants: An overview of micropollutants and sustainable treatment options. J. Hydrol. 2010, 389 (3−4), 416−428. (47) Di Dea Bergamasco, A. M.; Eldridge, M.; Sanseverino, J.; Sodre, F. F.; Montagner, C. C.; Pescara, I. C.; Jardim, W. F.; Umbuzeiro, G. d. A. Bioluminescent yeast estrogen assay (BLYES) as a sensitive tool to monitor surface and drinking water for estrogenicity. J. Environ. Monit. 2011, 13 (11), 3288−3293. 5900

dx.doi.org/10.1021/es405731j | Environ. Sci. Technol. 2014, 48, 5892−5901

Environmental Science & Technology

Article

line: A reactive intermediate of the pneumotoxin 3-methylindole. Chem. Res. Toxicol. 1998, 11 (7), 741−749. (65) Connelly, J. C.; Connor, S. C.; Monte, S.; Bailey, N. J. C.; Borgeaud, N.; Holmes, E.; Troke, J.; Nicholson, J. K.; Gavaghan, C. L. Application of directly coupled high performance liquid chromatography-NMR-mass spectometry and 1H NMR spectroscopic studies to the investigation of 2,3-benzofuran metabolism in Sprague-Dawley rats. Drug Metab. Dispos. 2002, 30 (12), 1357−1363. (66) Vignier, V.; Berthou, F.; Dreano, Y.; Floch, H. H. Dibenzothiophene sulphoxidation: a new and fast high-performance liquid chromatographic assay of mixed-function oxidation. Xenobiotica 1985, 15 (12), 991−999.

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dx.doi.org/10.1021/es405731j | Environ. Sci. Technol. 2014, 48, 5892−5901