- Email: [email protected]
Screening of chemicals with binding activities of liver X receptors from reclaimed waters
Journal Pre-proof Screening of chemicals with binding activities of liver X receptors from reclaimed waters
Haifeng Zhang, Yingting Jia, Zhuoheng Tang, Lei Wang, Wenxin Hu, Junmin Gao, Jianying Hu, Min Yang PII:
S0048-9697(20)30080-2
DOI:
https://doi.org/10.1016/j.scitotenv.2020.136570
Reference:
STOTEN 136570
To appear in:
Science of the Total Environment
Received date:
8 November 2019
Revised date:
5 January 2020
Accepted date:
5 January 2020
Please cite this article as: H. Zhang, Y. Jia, Z. Tang, et al., Screening of chemicals with binding activities of liver X receptors from reclaimed waters, Science of the Total Environment (2018), https://doi.org/10.1016/j.scitotenv.2020.136570
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© 2018 Published by Elsevier.
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Screening of chemicals with binding activities of liver X receptors from reclaimed waters
Haifeng Zhang1, Yingting Jia2, Zhuoheng Tang2,3, Lei Wang2, Wenxin Hu2, Junmin Gao3, Jianying Hu2, Min Yang1,4* Key Laboratory of Drinking Water Science and Technology, Research Center for
of
1
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
Sciences, Peking University, China.
Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry
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3
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Laboratory for Earth Surface Processes, College of Urban and Environmental
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2
University of Chinese Academy of Sciences, Beijing 100049, China
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* Corresponding author:
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4
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of Education, Chongqing University, Chongqing 400045, China
Min Yang; Phone: +86-10-62923475; Email: [email protected]
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Abstract Wastewater reclamation and reuse is considered an attractive and practical method for coping with water scarcity. However, the presence of micropollutants in reclaimed water, including endocrine disrupting chemicals (EDCs), is a major public health concern. This study attempted to identify unknown EDCs with liver X receptor
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(LXRα) agonist/antagonist activities in reclaimed wastewater, using nuclear receptors binding extraction coupled with high-resolution mass spectrometry (NRBE-HRMS).
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In total, 105 compounds in the reclaimed wastewater exhibited LXRα-binding
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activity. Among them, two previously unknown LXRα-antagonist compounds,
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catechol and 4-acetamidoantipyrine, were identified, based on authentic standards.
two-hybrid
assay.
Catechol
and
4-acetamidoantipyrine inhibited
the
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yeast
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The two LXRα-antagonist compounds exhibited weak LXRα-antagonist activities in a
β-galactosidase activity induced by 60 nM of TO901317 in an LXRα yeast assay, with
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IC20 values of 79938.9 nM and 6286.4 nM, respectively. To the best of our knowledge, this is the first study to identify EDCs in reclaimed wastewater with LXRα-agonist/antagonist activity using the NRBE-HRMS method.
Keywords: Endocrine disrupting chemicals, Liver X receptors, Reclaimed wastewater, Nuclear receptors binding extraction, High resolution mass spectrometry
1. Introduction Owing to rapid urbanization and economic development, the increasing scarcity of 2
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water is a serious problem in many countries worldwide. Wastewater reclamation and reuse is considered an attractive and practical method for coping with this challenge (1). However, the presence of micropollutants in reclaimed water has been a major concern due to their potential impact on human health and ecosystems (2). To date, more than 100,000 chemicals were synthesized, widely used, and might be released
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into wastewater without considering the potential damage (3). Among these chemicals, endocrine disrupting chemicals (EDCs) have been a particular category of
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pollutants raising major concerns in regards to human health (4, 5) as well as the
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ecosystem (6). EDCs are natural or synthetic chemicals (e.g., estradiol, nonylphenol,
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bisphenol A) that interfere with the normal functions of endogenous hormones,
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causing serious detrimental health effects, such as reproductive health issues,
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developmental deficiencies, cognitive disabilities, and immune disorders (7, 8). EDCs also have adverse effects on wildlife, causing population decline (6).
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The mechanisms of action of EDCs are complex, as they can affect the synthesis, transport, action, metabolism, and/or excretion of hormones (9). Previous evidence shows that EDCs can bind with nuclear receptors, including the estrogen receptor (ER), androgen receptor (AR), and liver X receptor (LXR), affecting the normal signaling processes of organisms (9). To date, most studies have focused on the agonistic/antagonistic activities of known EDCs on the estrogen receptors and androgen receptors (10–12) while the impacts of EDCs on the signaling pathways of other nuclear receptors are not well explored. For example, the mechanisms of EDCs binding with LXR, which are critical modulators of cholesterol homeostasis and 3
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reverse cholesterol transport (13–15), are not well-understood. Recently, Hu et al. (16) developed a novel method using a nuclear receptors binding extraction coupled with high-resolution mass spectrometry (NRBE-HRMS) to identify EDCs with LXR alpha (LXRα)-antagonist activities in complex dust samples. They successfully identified two LXRα-antagonist compounds, flame retardant triphenyl phosphate (TPHP) and
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2-ethylhexyl diphenyl phosphate (EHDPP) and found that exposure to these LXRα-antagonist compounds could cause atherosclerosis by promoting the formation
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of foam cells. Considering the potential human health risks from exposure to the
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LXRs-agonist/antagonist compounds through the reuse of reclaimed wastewater as
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potable water, it is crucial to identify LXRs-agonist/antagonist compounds in
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techniques.
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reclaimed wastewater, to establish regulations for their removal using appropriate
In this study, we aimed to identify unknown EDCs with LXRα-agonist/antagonist
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activities in reclaimed wastewater, using the NRBE-HRMS method. EDCs with LXRα-agonist/antagonist activity were enriched from reclaimed wastewater using solid phase extraction (SPE), extracted using LXRα protein-based affinity binding, and identified using non-target HRMS. Among the compounds with LXRα-binding activities in the reclaimed wastewater, two previously unknown LXRα-antagonist compounds, i.e., catechol and 4-acetamidoantipyrine, were identified based on authentic standards. In addition, we confirmed that the identified LXRα-antagonist compounds showed weak LXRα-antagonist activities in a yeast two-hybrid assay. To the best of our knowledge, this is the first study to identify EDCs with 4
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LXRα-agonist/antagonist activities in reclaimed wastewater using the NRBE-HRMS method.
2. Materials and Methods 2.1 Chemicals
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Ethyl acetate (pesticide grade), methanol (pesticide grade), methyl tert-butyl ether (MTBE, high-performance liquid chromatography (HPLC) grade), and dimethyl
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sulfoxide (DMSO, HPLC grade) were obtained from Fisher Chemicals (New Jersey,
β-D-1-thiogalactopyranoside,
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Isopropyl
-p
USA). Formic acid (HPLC grade) was provided by Dikma Technologies Inc. imidazole,
TO901317,
and
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Tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (St.
na
Louis, MO, USA). Catechol was obtained from Aladdin (Shanghai, China). 4-Acetamidoantipyrine was purchased from Alfa Aesar (Massachusetts, USA).
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Zymolyase 20T was obtained from Nacalai Tesque (Kyoto, Japan). Ultrapure water with a resistivity of 18.2 MΩ·cm was purified using a Milli-Q water purification system
(Millipore,
USA).
Dose
solutions
of
TO901317,
catechol,
and
4-acetamidoantipyrine were prepared in DMSO, based on their molar basis. 2.2 Sampling and sample concentration The effluents of the reclaimed wastewater were collected from the effluent of a reclaimed wastewater treatment plant (RWTP) in Beijing, China. The treatment capacity of this RWTP was 1,000,000 m3/day. The treatment process involved primary settling tanks, secondary treatment by biologically activated sludge, 5
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nitrification, ultrafiltration, ozonation, UV, and disinfection by chlorination. The samples were immediately delivered to a laboratory and filtered through 0.7 μm glass fiber filters (GF/F, Whatman) to eliminate suspended solids, and were then concentrated with SPE. The SPE cartridge (Oasis HLB, 500 mg/6 mL, Waters, USA) was preconditioned with 6 mL of MTBE, 6 mL of methanol, and 6 mL of Milli-Q
of
water. Two-liter effluent samples of the reclaimed water were loaded through the preconditioned cartridge at a constant rate of 10 mL/min. The cartridge was dried
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under a gentle flow of high-purity nitrogen. Then, compounds trapped on the SPE
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cartridge were eluted with 4 mL of methanol/MTBE (1:1, v/v), followed by 4 mL of
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methanol. The eluates from twelve samples were combined and evaporated to
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dryness, under a gentle nitrogen flow at 37 °C. The residue was immediately
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re-dissolved in 1.75 mL of DMSO, and was stored at −20 °C until further analysis. Blank control samples were treated with the same protocol, except that no water
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sample was loaded.
2.3 Screening EDCs with LXRα-binding activities Potential LXRα-agonistic/antagonistic compounds with LXRα-binding activities were extracted by using the NRBE method. The principle of the NRBE method is based on a known concept regarding ligand-receptor binding interaction. The unknown compounds with LXRα-binding activities were extracted from the SPE extracts of the reclaimed water using a freshly purified LXRα receptor protein, as described elsewhere (16). Briefly, human LXRα was expressed via EcoRI, and was purified as described in the supplementary materials. Next, 25 µL of HLB extract was incubated 6
Journal Pre-proof with 2 g of His-pCold-trigger factor(TF)-LXRα (n=6) and 1000 µL of His-select nickel magnetic agarose beads (Beaver Life Science, Suzhou, China) in a 2 mL glass tube at 4 °C under shaking conditions (approximately 170 rpm) for 2.5 h. Then, the supernatant was removed by a magnetic separator, the residues were washed twice with 1 mL buffer (250 mM NaCl and 20 mM Tris, pH 8.0), and the LXRα protein was
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eluted from the His-select nickel magnetic agarose beads with 1 mL buffer (500 mM NaCl, 20 mM Tris, and 10 mM imidazole, pH 8.0) in triplicate. Then, the chemicals
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binding to the LXRα protein were extracted twice with 5 mL ethyl acetate and 60 μL
-p
formic acid under shaking conditions for 20 min on an orbital shaker. After being
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centrifuged at 3800 rpm for 10 min, the extract was transferred to a glass tube and
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was concentrated to near dryness with a gentle high-purity nitrogen flow. The residues
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were reconstituted in methanol for HRMS analysis. A negative control experiment was performed using the same protocol, but with the protein of His-TF instead of
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His-TF-LXRα.
A non-targeted screen was performed using a Q Exactive high-resolution mass spectrometer. A Dionxe Ultimate 3000 Ultra-HPLC (UHPLC) was used for chromatograph separation. UHPLC separation was achieved on a BEH C18 column (2.1 mm × 100 mm, 1.7 μm; Waters, USA). The mobile phase A was ultrapure water and mobile phase B was methanol. In 0–3 min, mobile phase B increased from 5% to 60%. Then, in 3–13 min, mobile phase B increased from 60% to 100%, and held for 3 min. Finally, mobile phase B returned back to 5%, and held for 4 min for equilibration. The flow rate was 0.30 mL/min. The sample injection volume was 5 μL. 7
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The column temperature and sample compartment temperature were 40 °C and 5 °C, respectively. The HRMS data was acquired in the full-scan MS/ddMS2 mode. The resolution for full scan was set at 70000, and the resolution for ddMS2 was set at 17500. The automatic gain control (AGC) values for the full scan and ddMS2 were 3 × 106 and 5 × 104, respectively, and values of the maximum injection time were set at
of
100 ms and 80 ms, respectively. The isolation window for precursor ions was set to 1.0 m/z. The top 5 most abundant precursor ions were selected for ddMS2. The
ro
collision energy was set to 10, 30, and 50. The electrospray ionization source
-p
parameters were set as follows: spray voltages for the negative and positive modes
re
were set to 3 kV and 3.5 kV, respectively; the auxiliary gas flow and sheath gas flow
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were set to 10 Arb and 35 Arb, respectively; and the capillary temperature was set to
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320 °C. The S-lens rf level was 50.
The data deconvolution and retention alignment were performed using the Progenesis
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QI software (Waters, Milford, U.S.A.). Peaks with a retention time within 0–16 min and a minimum intensity of 5000 (for negative mode) or 10000 (for positive mode) were selected for further analysis. Adduction ions including [M-H]-, [M+FA-H]-, [M-H2O-H]-, and [M+Cl]- in negative mode and [M+H]+, [M+NH4]+, [M+H-H2O]+, [M+Na]+, and [M+K]+ in positive mode were considered for data analysis. Aligned peak lists of the His-TF-LXR group and the His-TF control group were analyzed with principal component analysis (PCA), to reveal the differences between the His-TF-LXR group and the His-TF control group. The fold change for each chemical between the two groups was calculated as the ratios between the areas of each peak in 8
Journal Pre-proof His-TF-LXRα group to those in the control group. Peaks with fold changes >3 (p-value <0.05 with student’s t-test) in the His-TF-LXRα as compared to the control group were considered as potential chemicals exhibiting binding activity to LXRα. The potential LXRα activity compounds were manually checked based on the exact mass and retention time. Reference-accurate mass spectra were used to identify the
of
structures of the LXRα-binding active chemicals, based on high-resolution mass
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spectrometer data provided by the mzCloud database (www.mzcloud.org). 2.4 Assessment of LXRα activity
-p
A yeast two-hybrid assay using human LXRα and coactivator transcription
re
intermediary factor 2 in Saccharomyces cerevisiae Y190 was used to assess the
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LXRα-antagonistic and LXRα-agonistic activities of the identified chemicals. The
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detailed assay procedure has been previously described (17). Briefly, the yeast cells were cultured at 30 °C in a synthetic defined (SD) medium under shaking conditions.
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After 15 h of incubation, 50 μL of cell suspension and 2.5 μL of chemicals (in DMSO) were added to 200 μL of the SD medium, and the culture mixtures were incubated at 30 °C under shaking conditions. After 4 h of exposure, 150 μL of the culture mixtures were removed, for measuring the absorbance at 595 nm by a microplate reader (Thermo Fisher Scientific, CA, USA). Next, 200 μL of Zymolyase 20T buffer was added to the residual cells and incubated at 30 °C for 20 min, to lyse the cell walls. Then, 40 μL of 4 mg/mL 2-nitrophenyl-b-D-galactoside (in water) was added and incubated at 30 °C for 30 min. The reaction was halted by adding 100 μL of 1M Na2CO3. Finally, 150 μL of the mixtures were transferred to a 96-well 9
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microplate, and the absorbances were measured at 414 and 570 nm. A 1% DMSO solution was used as the negative control. Positive controls were performed with the LXRα agonist TO901317. The work solution of TO901317 was prepared by two-fold serially diluting the stock solution with DMSO, and 11 different concentrations (0.05– 3000 nM) were prepared. The half-maximal effective concentration (EC50) values
of
were determined to be 60 nM for LXRα. To evaluate whether the identified chemicals possess LXRα agonistic or antagonistic activities, standards of catechol and
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4-acetamidoantipyrine were tested. The stock solutions were serially diluted by a
-p
two-fold serial dilution with DMSO, respectively, and 10 various concentrations
re
(195.3–100000 nM for catechol and 195.3–100000 nM for 4-acetamidoantipyrine)
lP
were prepared (n=3 per group). To evaluate whether they possess LXRα antagonistic
na
activity, TO901317 at EC50 (60 nM for LXRα) was added to the medium, along with the diluted chemicals. The final concentration of DMSO in the medium was 1%.
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2.5 Statistical Analysis
Using the Statistical Package for the Social Sciences (SPSS) 16.0 (SPSS Inc., Chicago, IL, USA), a one-way analysis of variance test was conducted, followed by a post-hoc Dunnett’s test, to determine the statistical significance of the results. A student’s t-test was used to analyze the data of the NRBE-HRMS method.
3. Results and Discussion 3.1 Screening of LXR ligands in reclaimed water HRMS, as coupled with LC, is increasingly being used to screen and identify 10
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unknown compounds. However, considering the large number (tens of thousands) of compounds that are generated in HRMS analysis, it is still a challenge to identify the unknown compounds with biological activities in complex environmental samples. In this study, a filtering strategy based on receptor protein binding extraction (16, 18) was used to identify the unknown compounds with LXRα-binding activities in the reclaimed water samples. As described previously (16), we used a freshly purified
of
LXRα protein to extract the LXRα ligands from the SPE extracts of the reclaimed
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water. The elution products extracted from the His-TF-LXRα group and the His-TF
-p
control group were analyzed using a non-target HRMS method, to identify potential
re
LXRα ligands. In total, 4743 chromatographic peaks were detected in the positive
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mode, and 3152 peaks were detected in the negative mode. Figure 1 illustrates the
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PCA analysis of the HRMS data. Significant differences are revealed between the His-TF-LXRα groups and His-TF control groups, with the His-TF-LXRα groups
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clustered in the negative PC1 area, and the His-TF control groups clustered in the positive PC1 area (Figure 1). These results indicate that the NRBE method successfully extracted the LXRα ligands from the SPE extracts of the reclaimed water. The volcano plots shown in Figure 2 illustrate the different peaks showing significant differences between the His-TF-LXRα group and the His-TF control group. In total, 537 peaks in the His-TF-LXRα group showed a three-fold higher abundance than those in the control group (p < 0.05) for the positive mode, whereas 553 peaks in the His-TF-LXRα group showed a three-fold higher abundance than those in the control 11
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group (p < 0.05) for the negative mode. Among them, the molecular formulas for 45 peaks in the positive mode and 60 peaks in the negative mode could be assigned with mass error within 5 ppm, and isotopic pattern similarity higher than 80%. The peaks with the assigned formulas are shown in Table 1. For the 105 peaks with assigned formulas, the MS/MS spectra were examined, and were compared with the
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high-resolution reference MS/MS spectra in the mzCloud database. The mass spectra of a negative ion peak at a retention time of 2.66 min and a positive ion peak at a
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retention time of 2.84 min (Figure 3) matched well with the reference MS/MS spectra
-p
of catechol and 4-acetamidoantipyrine, respectively. The presence of these two
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chemicals in the reclaimed water was further confirmed by using authentic standards.
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The measured chromatography and mass spectra of the standards were compared with
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the reclaimed water samples, as shown in Figure 3. It was clear that the two standards matched well with the ion peaks in the chromatograph in terms of the chromatography
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retention time and high-resolution MS/MS spectra, strongly supporting their presence in the reclaimed water.
3.2 LXRα-agonist and antagonist activities of the identified chemicals The LXRα-agonist and LXRα-antagonist activities of the two identified chemicals were evaluated using yeast two-hybrid assays. As shown in Figure 4, the β-galactosidase activities did not increase with increases of the catechol and 4-acetamidoantipyrine
concentrations,
showing
that
they
did
not
exhibit
LXRα-agonist activities. Both of them, however, exhibited weak LXRα-antagonist activities (Figure 5). Catechol inhibited the β-galactosidase activity induced by 60 nM 12
Journal Pre-proof of TO901317 in an LXRα yeast assay, with an IC20 (20% inhibitory concentration) value of 79938.9 nM, whereas the 4-acetamidoantipyrine inhibited the β-galactosidase activity induced by 60 nM of TO901317 with an IC20 value of 6286.4 nM. The IC20 values of catechol and 4-acetamidoantipyrine were 181 and 14 times higher than those of SR9238 (440.8 nM), a positive antagonist of LXRα (16), respectively, indicating
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that catechol and 4-acetamidoantipyrine have only weak LXRα-antagonist activities. The structures of the two compounds are very different from those of classical ligands
ro
(such as SR9238 and SR9243). However, in our recent report, TPHP and EHDPP,
-p
which have structures very different from those of classical ligands, were found to be
re
strong LXRα-antagonist compounds. This suggested that compounds with structures
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different from those of classical LXRα receptor ligands could also exhibit
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LXRα-antagonist activities. Further studies using other evaluation techniques (19, 20) will be performed to confirm the results of this study.
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3.3 Environmental implications
To date, few toxicological studies have assessed LXRα-agonist or antagonist activities in environmental water samples, and only two environmental organic chemicals, TPHP and EHDPP, have been reported to have LXR activity. Our results indicated that
some
chemicals
in
the
reclaimed
water
exhibited
dose-dependent
LXRα-antagonist activity. The identification of the specific chemicals responsible for the LXRα-antagonist activity in the reclaimed water is crucial, and challenging. The NRBE-HRMS
method
has
proven
efficient
in
identifying
EDCs
with
LXRα-antagonist activity in complex environmental samples, such as reclaimed water 13
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(this study) and indoor dust samples (16). Thus, we used the NRBE-HRMS method to extract and identify potential LXRα-antagonist activity chemicals which could bind to LXRα from reclaimed water. In total, 105 molecular formulas with LXRα-binding activity were screened from the reclaimed water. Although identification of all of these putative LXRα ligands was hindered by the absence of corresponding reference
of
spectra in the database, two compounds, catechol and 4-acetamidoantipyrine, were identified as novel LXRα antagonists. Catechol (1,2-dihydroxybenzene) is a phenolic
ro
compound, and is a known environmental pollutant. This compound has been
-p
detected in elevated concentrations in the effluents from many industries, such as
re
petroleum refineries, steel mills, and pharmaceuticals (21–23). Concentrations of
lP
catechol in some industrial effluent samples range from a few mg/L to several
na
thousand mg/L (22, 23). For example, catechol has been detected in the effluents from coal carbonization and gasification plants at concentrations as high as 5300 mg/L
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(23). It has adverse effects on the eyes, skin, and respiratory system, and can also lead to a wide range of diseases such as genetic mutations and vascular collapse, or even death in some cases (24–26). In contrast, 4-acetamidoantipyrine is a human metabolite of the analgesic drug metamizole. It is persistent, with a removal rate below 30% during wastewater treatment (27). 4-acetamidoantipyrine concentrations of 1300–2240 ng/L in WWTP effluents have been reported in China (27). Higher 4-acetamidoantipyrine concentrations (from 950 to 6000 ng/L in the effluents) have also been reported for WWTPs in Europe (28, 29). It should be noted that only two compounds were identified from among the 105 14
Journal Pre-proof compounds exhibiting LXRα-binding activity in the reclaimed wastewater. Thus, future efforts should be devoted to identifying the other screened compounds that could bind to LXR, and assessing their potential LXR-agonist/antagonist activities. The potential health impacts of the presence of these EDCs should be evaluated. In addition, by using other nuclear receptors as the affinity protein, the NRBE-HRMS
of
method could be used to reveal novel EDCs that can interact with corresponding
ro
nuclear receptors in various complex environmental samples.
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4. Conclusion
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In this study, we used the NRBE-HRMS method to screen for unknown EDCs with
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LXRα binding activity in reclaimed wastewater. In total, 105 compounds in the
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reclaimed wastewater were found to exhibit LXRα-binding activity. Among them, catechol and 4-acetamidoantipyrine were identified, using authentic standards. Weak
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LXRα-antagonist activities in a yeast two-hybrid assay were observed for the two identified compounds. The results of this study demonstrated the effectiveness of this NRBE-HRMS method in identification of unknown EDCs in complex environmental samples. Acknowledgment This work was funded by the International S&T Cooperation Program of China (2016YFE0117800) and the National Science Foundation of China Grant (Nos. 51420105012 and 51578530).
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nuclear sterol-activated receptors LXR and FXR. Nat. Rev. Mol. Cell. Biol. 2012, 13,
(16) Hu, W.; Jia, Y.; Kang, Q.; Peng, H.; Ma, H.; Zhang, S.; Hiromori, Y.; Kimura, T.; Nakanishi, T.; Zheng, L.; Qiu, Y.; Zhang, Z.; Wan, Y.; Hu, J. Screening of Chemicals for Liver X Receptors Binding Activities from Dust in 11 Chinese Homes and Characterization of Atherosclerotic Activity Using an in vitro Macrophage Cell Line and ApoE-/- Mice. Environ. Health Perspect. 2019, 127, 117003, DOI: 10.1289/EHP5039. (17) Zhang, H.; Zhang, Z.; Nakanishi, T.; Wan, Y.; Hiromori, Y.; Nagase, H.; Hu, J. Structure-dependent activity of phthalate esters and phthalate monoesters binding to human constitutive androstane receptor. Chem Res Toxicol 2015, 28(6): 1196-1204. (18) Creusot, N.; Budzinski, H.; Balaguer, P.; Kinani, S.; Porcher, J.M.; Aït-Aïssa, S. 17
Journal Pre-proof Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors. Anal. Bioanal. Chem. 2013, 405, 2553-66. (19) Sim, W.; Park, S.; Lee, K.; Je, Y.; Yin, H.; Choi, Y.; Sung, S.; Park, S.; Park, H.; Shin, K.; Lee, B. LXR-α antagonist meso-dihydroguaiaretic acid attenuates high-fat diet-induced nonalcoholic fatty liver. Biochemical Pharmacology 2014, 90, 414-424. (20) Liu, X.; Sakai, H.; Nishigori, M.; Suyama, K.; Nawaji, T.; Ikeda, S.; Nishigouchi, M.; Okada, H.; Matsushima, A.; Nose, T., Shimohigashi, M.;
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Shimohigashi, Y. Receptor-binding affinities of bisphenol A and its next-generation
ro
analogs for human nuclear receptors. Toxicology and Applied Pharmacology 2019, 377, 114610.
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(21) Lofrano, G.; Rizzo, L.; Grassi, M.; Belgiorno, V. Advanced oxidation of
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Desalination, 2009, 249, 878-883.
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catechol: a comparison among photocatalysis, Fenton and photo-Fenton processes.
(22) Subramanyam, R.; Mishra, I.M. Biodegradation of catechol (2-hydroxy phenol)
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bearing wastewater in an UASB reactor. Chemosphere, 2007, 69. 816-824. (23) Subramanyam, R.; Mishra, I.M. Treatment of catechol bearing wastewater in an
Jo ur
upflow anaerobic sludge blanket (UASB) reactor: Sludge characteristics. Bioresource Technology, 2008, 99, 8917-8925. (24) Anotai, J.; Su,C.; Tsai, Y.; Lu, M. Effect of hydrogen peroxide on aniline oxidation by electro-Fenton and fluidized-bed Fenton processes. J. Hazard. Mater., 2010, 183, 888-893.
(25) Aghapour, A.A.; Moussavi, G.; Yaghmaeian, K. Degradation and COD removal of catechol in wastewater using the catalytic ozonation process combined with the cyclic rotating-bed biological reactor. J. Environ. Manag., 2015, 157.262-266. (26) Kumar, A.; Kumar, S.; Kumar, S. Adsorption of resorcinol and catechol on granular activated carbon: Equilibrium and kinetics. Carbon 2003, 41, 3015-3025. (27) Qi, W.; Singer, H.; Berg M.; Müller, Beat.; Pernet-Coudrier, B.; Liu, H.; Qu, J. Elimination of polar micropollutants and anthropogenic markers by wastewater 18
Journal Pre-proof treatment in Beijing, China. Chemosphere 2015, 119, 1054-1061. (28) Zühlke, S., 2004. Verhalten von Phenazonderivaten, Carbamazepin und estrogenen Steroiden während verschiedener Verfahren der Wasseraufbereitung. PhD thesis. Technische Universität, Berlin, Germany. (29) Kahle, M.; Buerge, J.I.; Müller, D.M.; Poiger, T. Hydrophilic anthropogenic markers for quantification of wastewater contamination in ground- and surface
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waters. Environ. Toxicol. Chem., 2009, 28, 2528-2536
19
Journal Pre-proof
Figure Caption
Figure 1 Principle component analysis (PCA) plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode.
of
Figure 2 Volcano plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode. The red dots represent a chemical with a fold change of ≥3 and p-value of ≤0.05.
-p
ro
Figure 3 Chromatograph and mass spectra of reclaimed water sample and authentic standards of (a) catechol and (b) 4-acetamidoantipyrine.
lP
re
Figure 4 Dose−response curves for the β-galactosidase activity of catechol and 4-acetamidoantipyrine.
Jo ur
na
Figure 5 Dose−response curves for the relative β-galactosidase activity of catechol and 4-acetamidoantipyrine.
20
Journal Pre-proof
100
pCold-TF-LXRα pCold-TF
(a)
0
of
PC 2 (28.8%)
50
-100 -100
-50
-p
ro
-50
0
50
100
re
PC1 (58.8%) 100
pCold-TF-LXRα pCold-TF
lP
na
Jo ur
PC 2 (10.7%)
50
0
(b)
-50
-100 -100
-50
0
50
100
PC1 (51.9%)
Figure 1 Principle component analysis (PCA) plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode.
21
Journal Pre-proof
12
pCold-TF-LXRα pCold-TF
(a)
8
of
6
4
ro
-Log(p-value)
10
0 -20
20
lP
(b)
na
-Log(p-value)
10
Jo ur
6
0
pCold-TF-LXRα pCold-TF
10
8
-10
re
12
-p
2
4
2
0
-20
-10
0
10
20
Log2 (fold change)
Figure 2 Volcano plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode. The red dots represent a chemical with a fold change of ≥3 and p-value of ≤0.05.
22
Journal Pre-proof
4E+06
2E+06
(a) catechol
(b) 4-acetamidoantipyrine
3E+06
sample
2E+06
sample
1E+06
1E+06 0E+00 2.0
2.5
3.5 Time 4.0
3.0
2.0
standard 2E+06
1E+06
3.0
3.5
Time4.0
standard 2E+07
1E+07
0E+00 2.0
2.5
3.5 Time 4.0
3.0
2.0
109.0294
1.0
1.0
0.5
0.5
94.9805
0.0
3.0
83.0610 104.0499
3.5
Time4.0
228.1131
204.1133 246.1237
-p
76.9700
2.5
ro
0E+00
0.0
2.5
3E+07
of
3E+06
Intensity
Intensity
Relative abundance
0E+00
-0.5
-0.5 -1.0
80
100
120 m/z
50
100
150
200
m/z 250
na
lP
60
re
-1.0
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Figure 3 Chromatograph and mass spectra of reclaimed water sample and authentic standards of (a) catechol and (b) 4-acetamidoantipyrine.
23
Journal Pre-proof
3500
2500
2000
of
1500
ro
1000
4-acetamidoantipyrine catechol
100
101
Concentration (μM)
102
na
lP
0 10-1
-p
500
re
β-galactosidase activity
3000
Jo ur
Figure 4 Dose−response curves for the β-galactosidase activity of catechol and 4-acetamidoantipyrine.
24
Journal Pre-proof
120%
80%
of
60%
ro
40%
4-acetamidoantipyrine catechol
-p
20%
0% 10-1
100
re
Relative β-galactosidase activity
100%
101
102
na
lP
Concentration (μM)
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Figure 5 Dose−response curves for the relative β-galactosidase activity of catechol and 4-acetamidoantipyrine.
25
Journal Pre-proof Table 1 Formulas of molecules in reclaimed wastewater found to binding LXRα receptor Negative ion peaks
Positive ion peaks
No. Formula
RT1
m/z
1
C14H8O4
1.98
239.0355
2
C6H6O2
2.66
3
C13H12N2O3
3.27
4
C15H18N2O3
5
RT
m/z
2.38
1
C10H13N3O3
2.66
206.0926
0.88
109.0294
-1.27
2
C13H15N3O2
2.84
246.1239
0.73
225.0669
-0.26
3
C7H10N2O
2.89
121.0764
2.39
3.47
319.1296
-1.27
4
C7H12N2O2
3.09
139.0867
0.70
C15H20N2O3
3.49
275.1399
-0.57
5
C16H13NO5
3.33
317.1134
0.68
6
C5H11NO4P
3.55
161.0242
0.00
6
C13H13N3
3.48
212.1183
0.57
7
C3H6N6O4
3.77
235.0431
-0.59
7
C21H36N4O8
3.91
455.2516
3.36
8
C9H19O11P
3.88
379.0642
-1.47
8
C10H10N2O2
4.00
173.0711
0.93
9
C13H14O5
3.92
231.0660
-1.28
9
C16H16N2O5
4.10
299.1027
0.32
10
C11H9N3O4
3.99
228.0415
-0.07
10
C11H10N2O
4.35
169.0761
0.42
11
C8H8O4S
4.00
234.9835
-1.10
11
C12H14N2O
4.42
185.1075
1.01
12
C20H20N6O6S
4.08
471.1104
4.97
12
C10H14N2O
4.50
161.1074
0.60
13
C13H14O3S
4.14
249.0590
-0.41
13
C20H25N3O3
4.56
356.1957
-3.34
14
C8H8O8
4.16
266.9911
-0.73
14
C12H12N2O2
4.72
239.0793
0.92
15
C10H12O6S
4.20
295.0045
-1.58
15
C15H15N3O
4.76
236.1183
0.23
16
C11H9N3O3
4.23
266.0330
-3.56
16
C20H36N2O5
4.95
423.2251
-1.26
17
C12H9N3O
4.41
210.0670
-1.13
18
C16H24O5S
4.42
327.1269
-0.66
19
C10H16N4OS
4.45
275.0747
20
C17H27N3O7
4.57
384.1789
21
C14H31O5P
4.76
355.1886
22
C6H10N6O4
4.81
275.0746
23
C16H16N4O5S
4.87
357.0661
24
C15H16OS
4.96
289.0903
25
C19H25NO5
26
C10H15F3O4
27
Error
re
-p
ro
of
No. Formula
5.04
3.39
19
C24H36O9
5.62
469.2410
-4.61
3.23
20
C12H18O3S
5.79
243.1050
0.14
-1.61
21
C23H32N4O2
7.05
419.2403
-3.67
na
Error
0.19
22
C23H22N6O6
7.71
479.1673
-0.07
-0.46
23
C19H16O
8.28
243.1168
-0.08
-0.42
24
C34H46O6
8.53
573.3189
0.43
C16H21N3O4S
352.1326
0.21
18
C21H31N3O6
5.20
439.2567
3.71
Jo ur
lP
17
5.01
382.1438
3.22
25
C11H10F3N5O4
8.56
356.0577
0.95
5.05
301.0902
-1.15
26
C23H39NO6
8.75
408.2758
3.14
C11H20NO4S
5.13
297.0800
-4.64
27
C14H12F6O3
9.20
381.0322
-0.10
28
C14H22O4S
5.14
267.1060
-0.12
28
C23H24N6O
9.57
423.1907
0.97
29
C8HF15O2
5.72
412.9661
-0.70
29
C20H32O
9.70
306.2791
-0.31
30
C14H20OS
5.85
281.1216
-0.58
30
C20H36O2
9.98
331.2606
-0.05
31
C27H26N6O2
6.33
501.1829
3.74
31
C20H36O6
10.27 395.2396
-2.05
32
C13H12O7S2
6.41
389.0022
4.46
32
C20H27O4P
10.49 385.1538
-0.28
33
C18H20N2O
6.47
315.1271
0.38
33
C20H29F3
10.59 344.2560
0.09
34
C30H41ClN2O5
6.59
543.2625
-1.04
34
C14H24O
10.89 191.1797
1.11
35
C15H24O4S
6.76
299.1320
-0.79
35
C22H38O4
10.96 389.2663
-0.16
36
C10H5F17O3S
7.13
526.9611
-0.90
36
C18H44N6O12
10.96 559.2888
-3.86
37
C24H24O4
7.18
357.1498
0.39
37
C20H43O6P
11.12 411.2869
-0.29
38
C34H33N3O9S
7.27
640.1747
-1.78
38
C20H39NO2
12.01 348.2872
0.00
39
C16H26O3S
7.60
297.1528
-0.61
39
C17H30N2O5
12.07 325.2113
-2.50
40
C18H30O3S
8.19
325.1841
-0.43
40
C17H35NO
12.10 292.2609
-0.27
26
Journal Pre-proof Negative ion peaks
Positive ion peaks
No. Formula
RT1
m/z
41
C18H28O2
8.24
321.2069
42
C17H28O3S
8.31
43
C27H46N6O7
44 45
RT
-0.82
41
C40H74N2O2
12.17 637.5643
0.13
311.1686
-0.15
42
C23H38N2O
12.19 376.3321
-0.43
8.39
601.3133
1.99
43
C21H38N2O
12.66 352.3319
-0.86
C20H39N2O12P3 8.52
627.1432
3.74
44
C40H78N2O2
12.84 641.5956
0.03
C23H26FN6O9P 8.52
625.1462
-0.50
45
C6H7O4P
13.69 175.0154
-0.68
46
C22H36O4
8.63
409.2593
-0.67
47
C20H32O3S
8.90
351.1997
-0.65
48
C20H30OS
8.90
363.1998
-0.32
49
C16H34N4OS
8.90
365.2153
1.58
50
C23H37NO4
9.36
436.2715
2.59
51
C27H38O2
9.54
439.2850
-0.87
52
C17H36N4OS
9.56
379.2310
1.77
53
C15H24O2
9.61
235.1701
-1.10
54
C20H34OS
9.64
367.2310
-0.84
55
C18H30OS
9.72
339.1998
-0.36
56
C19H38O5
10.18 381.2467
0.54
57
C20H43O4P
10.61 377.2825
-0.32
58
C24H38O
11.04 387.2903
-0.54
59
C22H32O2
11.76 327.2327
-0.75
60
C19H36O
12.00 325.2745
lP
re
-p
ro
of
No. Formula
-0.96
Jo ur
na
1. RT: retention time
Error
27
m/z
Error
Journal Pre-proof
Reclaimed wastewater extracts
UPLC-HRMS 4E+06
2E+06
3E+06 2E+06
1E+06
1E+06 0E+00
0E+00 2.0
2.5
3.0
3.5
4.0
2.0
2.5
3.0
3.5
4.0
2.0
2.5
3.0
3.5
4.0
3E+07
Intensity
Intensity
3E+06
2E+06
1E+06
0E+00
2E+07
1E+07
0E+00 2.0
2.5
3.0
3.5
4.0
1.0
1.0
0.5
0.5
0.0
0.0
-0.5
-0.5
-1.0
-1.0 60
80
100
120
50
100
150
200
250
120%
LXRα
100%
LXRα
Wash
of
MB
80%
MB
60%
MB
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na
lP
Liver X receptor binding extraction
-p
LXRα
MB
LXRα-binding chemicals
re
LXRα
ro
Elute
28
40%
20%
0% 10-1
100
101
102
Yeast two-hybrid assay
Journal Pre-proof Highlights • Identification of EDCs with liver X receptor binding activity in reclaimed water • 105 compounds in reclaimed wastewater were found to exhibit LXRα-binding activity
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na
lP
re
-p
ro
of
• Catechol and 4-acetamidoantipyrine were identified as LXRα antagonists
29
Haifeng Zhang, Yingting Jia, Zhuoheng Tang, Lei Wang, Wenxin Hu, Junmin Gao, Jianying Hu, Min Yang PII:
S0048-9697(20)30080-2
DOI:
https://doi.org/10.1016/j.scitotenv.2020.136570
Reference:
STOTEN 136570
To appear in:
Science of the Total Environment
Received date:
8 November 2019
Revised date:
5 January 2020
Accepted date:
5 January 2020
Please cite this article as: H. Zhang, Y. Jia, Z. Tang, et al., Screening of chemicals with binding activities of liver X receptors from reclaimed waters, Science of the Total Environment (2018), https://doi.org/10.1016/j.scitotenv.2020.136570
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© 2018 Published by Elsevier.
Journal Pre-proof
Screening of chemicals with binding activities of liver X receptors from reclaimed waters
Haifeng Zhang1, Yingting Jia2, Zhuoheng Tang2,3, Lei Wang2, Wenxin Hu2, Junmin Gao3, Jianying Hu2, Min Yang1,4* Key Laboratory of Drinking Water Science and Technology, Research Center for
of
1
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
Sciences, Peking University, China.
Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry
re
3
ro
Laboratory for Earth Surface Processes, College of Urban and Environmental
-p
2
University of Chinese Academy of Sciences, Beijing 100049, China
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* Corresponding author:
na
4
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of Education, Chongqing University, Chongqing 400045, China
Min Yang; Phone: +86-10-62923475; Email: [email protected]
1
Journal Pre-proof
Abstract Wastewater reclamation and reuse is considered an attractive and practical method for coping with water scarcity. However, the presence of micropollutants in reclaimed water, including endocrine disrupting chemicals (EDCs), is a major public health concern. This study attempted to identify unknown EDCs with liver X receptor
of
(LXRα) agonist/antagonist activities in reclaimed wastewater, using nuclear receptors binding extraction coupled with high-resolution mass spectrometry (NRBE-HRMS).
ro
In total, 105 compounds in the reclaimed wastewater exhibited LXRα-binding
-p
activity. Among them, two previously unknown LXRα-antagonist compounds,
re
catechol and 4-acetamidoantipyrine, were identified, based on authentic standards.
two-hybrid
assay.
Catechol
and
4-acetamidoantipyrine inhibited
the
na
yeast
lP
The two LXRα-antagonist compounds exhibited weak LXRα-antagonist activities in a
β-galactosidase activity induced by 60 nM of TO901317 in an LXRα yeast assay, with
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IC20 values of 79938.9 nM and 6286.4 nM, respectively. To the best of our knowledge, this is the first study to identify EDCs in reclaimed wastewater with LXRα-agonist/antagonist activity using the NRBE-HRMS method.
Keywords: Endocrine disrupting chemicals, Liver X receptors, Reclaimed wastewater, Nuclear receptors binding extraction, High resolution mass spectrometry
1. Introduction Owing to rapid urbanization and economic development, the increasing scarcity of 2
Journal Pre-proof
water is a serious problem in many countries worldwide. Wastewater reclamation and reuse is considered an attractive and practical method for coping with this challenge (1). However, the presence of micropollutants in reclaimed water has been a major concern due to their potential impact on human health and ecosystems (2). To date, more than 100,000 chemicals were synthesized, widely used, and might be released
of
into wastewater without considering the potential damage (3). Among these chemicals, endocrine disrupting chemicals (EDCs) have been a particular category of
ro
pollutants raising major concerns in regards to human health (4, 5) as well as the
-p
ecosystem (6). EDCs are natural or synthetic chemicals (e.g., estradiol, nonylphenol,
re
bisphenol A) that interfere with the normal functions of endogenous hormones,
lP
causing serious detrimental health effects, such as reproductive health issues,
na
developmental deficiencies, cognitive disabilities, and immune disorders (7, 8). EDCs also have adverse effects on wildlife, causing population decline (6).
Jo ur
The mechanisms of action of EDCs are complex, as they can affect the synthesis, transport, action, metabolism, and/or excretion of hormones (9). Previous evidence shows that EDCs can bind with nuclear receptors, including the estrogen receptor (ER), androgen receptor (AR), and liver X receptor (LXR), affecting the normal signaling processes of organisms (9). To date, most studies have focused on the agonistic/antagonistic activities of known EDCs on the estrogen receptors and androgen receptors (10–12) while the impacts of EDCs on the signaling pathways of other nuclear receptors are not well explored. For example, the mechanisms of EDCs binding with LXR, which are critical modulators of cholesterol homeostasis and 3
Journal Pre-proof
reverse cholesterol transport (13–15), are not well-understood. Recently, Hu et al. (16) developed a novel method using a nuclear receptors binding extraction coupled with high-resolution mass spectrometry (NRBE-HRMS) to identify EDCs with LXR alpha (LXRα)-antagonist activities in complex dust samples. They successfully identified two LXRα-antagonist compounds, flame retardant triphenyl phosphate (TPHP) and
of
2-ethylhexyl diphenyl phosphate (EHDPP) and found that exposure to these LXRα-antagonist compounds could cause atherosclerosis by promoting the formation
ro
of foam cells. Considering the potential human health risks from exposure to the
-p
LXRs-agonist/antagonist compounds through the reuse of reclaimed wastewater as
re
potable water, it is crucial to identify LXRs-agonist/antagonist compounds in
na
techniques.
lP
reclaimed wastewater, to establish regulations for their removal using appropriate
In this study, we aimed to identify unknown EDCs with LXRα-agonist/antagonist
Jo ur
activities in reclaimed wastewater, using the NRBE-HRMS method. EDCs with LXRα-agonist/antagonist activity were enriched from reclaimed wastewater using solid phase extraction (SPE), extracted using LXRα protein-based affinity binding, and identified using non-target HRMS. Among the compounds with LXRα-binding activities in the reclaimed wastewater, two previously unknown LXRα-antagonist compounds, i.e., catechol and 4-acetamidoantipyrine, were identified based on authentic standards. In addition, we confirmed that the identified LXRα-antagonist compounds showed weak LXRα-antagonist activities in a yeast two-hybrid assay. To the best of our knowledge, this is the first study to identify EDCs with 4
Journal Pre-proof
LXRα-agonist/antagonist activities in reclaimed wastewater using the NRBE-HRMS method.
2. Materials and Methods 2.1 Chemicals
of
Ethyl acetate (pesticide grade), methanol (pesticide grade), methyl tert-butyl ether (MTBE, high-performance liquid chromatography (HPLC) grade), and dimethyl
ro
sulfoxide (DMSO, HPLC grade) were obtained from Fisher Chemicals (New Jersey,
β-D-1-thiogalactopyranoside,
re
Isopropyl
-p
USA). Formic acid (HPLC grade) was provided by Dikma Technologies Inc. imidazole,
TO901317,
and
lP
Tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (St.
na
Louis, MO, USA). Catechol was obtained from Aladdin (Shanghai, China). 4-Acetamidoantipyrine was purchased from Alfa Aesar (Massachusetts, USA).
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Zymolyase 20T was obtained from Nacalai Tesque (Kyoto, Japan). Ultrapure water with a resistivity of 18.2 MΩ·cm was purified using a Milli-Q water purification system
(Millipore,
USA).
Dose
solutions
of
TO901317,
catechol,
and
4-acetamidoantipyrine were prepared in DMSO, based on their molar basis. 2.2 Sampling and sample concentration The effluents of the reclaimed wastewater were collected from the effluent of a reclaimed wastewater treatment plant (RWTP) in Beijing, China. The treatment capacity of this RWTP was 1,000,000 m3/day. The treatment process involved primary settling tanks, secondary treatment by biologically activated sludge, 5
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nitrification, ultrafiltration, ozonation, UV, and disinfection by chlorination. The samples were immediately delivered to a laboratory and filtered through 0.7 μm glass fiber filters (GF/F, Whatman) to eliminate suspended solids, and were then concentrated with SPE. The SPE cartridge (Oasis HLB, 500 mg/6 mL, Waters, USA) was preconditioned with 6 mL of MTBE, 6 mL of methanol, and 6 mL of Milli-Q
of
water. Two-liter effluent samples of the reclaimed water were loaded through the preconditioned cartridge at a constant rate of 10 mL/min. The cartridge was dried
ro
under a gentle flow of high-purity nitrogen. Then, compounds trapped on the SPE
-p
cartridge were eluted with 4 mL of methanol/MTBE (1:1, v/v), followed by 4 mL of
re
methanol. The eluates from twelve samples were combined and evaporated to
lP
dryness, under a gentle nitrogen flow at 37 °C. The residue was immediately
na
re-dissolved in 1.75 mL of DMSO, and was stored at −20 °C until further analysis. Blank control samples were treated with the same protocol, except that no water
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sample was loaded.
2.3 Screening EDCs with LXRα-binding activities Potential LXRα-agonistic/antagonistic compounds with LXRα-binding activities were extracted by using the NRBE method. The principle of the NRBE method is based on a known concept regarding ligand-receptor binding interaction. The unknown compounds with LXRα-binding activities were extracted from the SPE extracts of the reclaimed water using a freshly purified LXRα receptor protein, as described elsewhere (16). Briefly, human LXRα was expressed via EcoRI, and was purified as described in the supplementary materials. Next, 25 µL of HLB extract was incubated 6
Journal Pre-proof with 2 g of His-pCold-trigger factor(TF)-LXRα (n=6) and 1000 µL of His-select nickel magnetic agarose beads (Beaver Life Science, Suzhou, China) in a 2 mL glass tube at 4 °C under shaking conditions (approximately 170 rpm) for 2.5 h. Then, the supernatant was removed by a magnetic separator, the residues were washed twice with 1 mL buffer (250 mM NaCl and 20 mM Tris, pH 8.0), and the LXRα protein was
of
eluted from the His-select nickel magnetic agarose beads with 1 mL buffer (500 mM NaCl, 20 mM Tris, and 10 mM imidazole, pH 8.0) in triplicate. Then, the chemicals
ro
binding to the LXRα protein were extracted twice with 5 mL ethyl acetate and 60 μL
-p
formic acid under shaking conditions for 20 min on an orbital shaker. After being
re
centrifuged at 3800 rpm for 10 min, the extract was transferred to a glass tube and
lP
was concentrated to near dryness with a gentle high-purity nitrogen flow. The residues
na
were reconstituted in methanol for HRMS analysis. A negative control experiment was performed using the same protocol, but with the protein of His-TF instead of
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His-TF-LXRα.
A non-targeted screen was performed using a Q Exactive high-resolution mass spectrometer. A Dionxe Ultimate 3000 Ultra-HPLC (UHPLC) was used for chromatograph separation. UHPLC separation was achieved on a BEH C18 column (2.1 mm × 100 mm, 1.7 μm; Waters, USA). The mobile phase A was ultrapure water and mobile phase B was methanol. In 0–3 min, mobile phase B increased from 5% to 60%. Then, in 3–13 min, mobile phase B increased from 60% to 100%, and held for 3 min. Finally, mobile phase B returned back to 5%, and held for 4 min for equilibration. The flow rate was 0.30 mL/min. The sample injection volume was 5 μL. 7
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The column temperature and sample compartment temperature were 40 °C and 5 °C, respectively. The HRMS data was acquired in the full-scan MS/ddMS2 mode. The resolution for full scan was set at 70000, and the resolution for ddMS2 was set at 17500. The automatic gain control (AGC) values for the full scan and ddMS2 were 3 × 106 and 5 × 104, respectively, and values of the maximum injection time were set at
of
100 ms and 80 ms, respectively. The isolation window for precursor ions was set to 1.0 m/z. The top 5 most abundant precursor ions were selected for ddMS2. The
ro
collision energy was set to 10, 30, and 50. The electrospray ionization source
-p
parameters were set as follows: spray voltages for the negative and positive modes
re
were set to 3 kV and 3.5 kV, respectively; the auxiliary gas flow and sheath gas flow
lP
were set to 10 Arb and 35 Arb, respectively; and the capillary temperature was set to
na
320 °C. The S-lens rf level was 50.
The data deconvolution and retention alignment were performed using the Progenesis
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QI software (Waters, Milford, U.S.A.). Peaks with a retention time within 0–16 min and a minimum intensity of 5000 (for negative mode) or 10000 (for positive mode) were selected for further analysis. Adduction ions including [M-H]-, [M+FA-H]-, [M-H2O-H]-, and [M+Cl]- in negative mode and [M+H]+, [M+NH4]+, [M+H-H2O]+, [M+Na]+, and [M+K]+ in positive mode were considered for data analysis. Aligned peak lists of the His-TF-LXR group and the His-TF control group were analyzed with principal component analysis (PCA), to reveal the differences between the His-TF-LXR group and the His-TF control group. The fold change for each chemical between the two groups was calculated as the ratios between the areas of each peak in 8
Journal Pre-proof His-TF-LXRα group to those in the control group. Peaks with fold changes >3 (p-value <0.05 with student’s t-test) in the His-TF-LXRα as compared to the control group were considered as potential chemicals exhibiting binding activity to LXRα. The potential LXRα activity compounds were manually checked based on the exact mass and retention time. Reference-accurate mass spectra were used to identify the
of
structures of the LXRα-binding active chemicals, based on high-resolution mass
ro
spectrometer data provided by the mzCloud database (www.mzcloud.org). 2.4 Assessment of LXRα activity
-p
A yeast two-hybrid assay using human LXRα and coactivator transcription
re
intermediary factor 2 in Saccharomyces cerevisiae Y190 was used to assess the
lP
LXRα-antagonistic and LXRα-agonistic activities of the identified chemicals. The
na
detailed assay procedure has been previously described (17). Briefly, the yeast cells were cultured at 30 °C in a synthetic defined (SD) medium under shaking conditions.
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After 15 h of incubation, 50 μL of cell suspension and 2.5 μL of chemicals (in DMSO) were added to 200 μL of the SD medium, and the culture mixtures were incubated at 30 °C under shaking conditions. After 4 h of exposure, 150 μL of the culture mixtures were removed, for measuring the absorbance at 595 nm by a microplate reader (Thermo Fisher Scientific, CA, USA). Next, 200 μL of Zymolyase 20T buffer was added to the residual cells and incubated at 30 °C for 20 min, to lyse the cell walls. Then, 40 μL of 4 mg/mL 2-nitrophenyl-b-D-galactoside (in water) was added and incubated at 30 °C for 30 min. The reaction was halted by adding 100 μL of 1M Na2CO3. Finally, 150 μL of the mixtures were transferred to a 96-well 9
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microplate, and the absorbances were measured at 414 and 570 nm. A 1% DMSO solution was used as the negative control. Positive controls were performed with the LXRα agonist TO901317. The work solution of TO901317 was prepared by two-fold serially diluting the stock solution with DMSO, and 11 different concentrations (0.05– 3000 nM) were prepared. The half-maximal effective concentration (EC50) values
of
were determined to be 60 nM for LXRα. To evaluate whether the identified chemicals possess LXRα agonistic or antagonistic activities, standards of catechol and
ro
4-acetamidoantipyrine were tested. The stock solutions were serially diluted by a
-p
two-fold serial dilution with DMSO, respectively, and 10 various concentrations
re
(195.3–100000 nM for catechol and 195.3–100000 nM for 4-acetamidoantipyrine)
lP
were prepared (n=3 per group). To evaluate whether they possess LXRα antagonistic
na
activity, TO901317 at EC50 (60 nM for LXRα) was added to the medium, along with the diluted chemicals. The final concentration of DMSO in the medium was 1%.
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2.5 Statistical Analysis
Using the Statistical Package for the Social Sciences (SPSS) 16.0 (SPSS Inc., Chicago, IL, USA), a one-way analysis of variance test was conducted, followed by a post-hoc Dunnett’s test, to determine the statistical significance of the results. A student’s t-test was used to analyze the data of the NRBE-HRMS method.
3. Results and Discussion 3.1 Screening of LXR ligands in reclaimed water HRMS, as coupled with LC, is increasingly being used to screen and identify 10
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unknown compounds. However, considering the large number (tens of thousands) of compounds that are generated in HRMS analysis, it is still a challenge to identify the unknown compounds with biological activities in complex environmental samples. In this study, a filtering strategy based on receptor protein binding extraction (16, 18) was used to identify the unknown compounds with LXRα-binding activities in the reclaimed water samples. As described previously (16), we used a freshly purified
of
LXRα protein to extract the LXRα ligands from the SPE extracts of the reclaimed
ro
water. The elution products extracted from the His-TF-LXRα group and the His-TF
-p
control group were analyzed using a non-target HRMS method, to identify potential
re
LXRα ligands. In total, 4743 chromatographic peaks were detected in the positive
lP
mode, and 3152 peaks were detected in the negative mode. Figure 1 illustrates the
na
PCA analysis of the HRMS data. Significant differences are revealed between the His-TF-LXRα groups and His-TF control groups, with the His-TF-LXRα groups
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clustered in the negative PC1 area, and the His-TF control groups clustered in the positive PC1 area (Figure 1). These results indicate that the NRBE method successfully extracted the LXRα ligands from the SPE extracts of the reclaimed water. The volcano plots shown in Figure 2 illustrate the different peaks showing significant differences between the His-TF-LXRα group and the His-TF control group. In total, 537 peaks in the His-TF-LXRα group showed a three-fold higher abundance than those in the control group (p < 0.05) for the positive mode, whereas 553 peaks in the His-TF-LXRα group showed a three-fold higher abundance than those in the control 11
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group (p < 0.05) for the negative mode. Among them, the molecular formulas for 45 peaks in the positive mode and 60 peaks in the negative mode could be assigned with mass error within 5 ppm, and isotopic pattern similarity higher than 80%. The peaks with the assigned formulas are shown in Table 1. For the 105 peaks with assigned formulas, the MS/MS spectra were examined, and were compared with the
of
high-resolution reference MS/MS spectra in the mzCloud database. The mass spectra of a negative ion peak at a retention time of 2.66 min and a positive ion peak at a
ro
retention time of 2.84 min (Figure 3) matched well with the reference MS/MS spectra
-p
of catechol and 4-acetamidoantipyrine, respectively. The presence of these two
re
chemicals in the reclaimed water was further confirmed by using authentic standards.
lP
The measured chromatography and mass spectra of the standards were compared with
na
the reclaimed water samples, as shown in Figure 3. It was clear that the two standards matched well with the ion peaks in the chromatograph in terms of the chromatography
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retention time and high-resolution MS/MS spectra, strongly supporting their presence in the reclaimed water.
3.2 LXRα-agonist and antagonist activities of the identified chemicals The LXRα-agonist and LXRα-antagonist activities of the two identified chemicals were evaluated using yeast two-hybrid assays. As shown in Figure 4, the β-galactosidase activities did not increase with increases of the catechol and 4-acetamidoantipyrine
concentrations,
showing
that
they
did
not
exhibit
LXRα-agonist activities. Both of them, however, exhibited weak LXRα-antagonist activities (Figure 5). Catechol inhibited the β-galactosidase activity induced by 60 nM 12
Journal Pre-proof of TO901317 in an LXRα yeast assay, with an IC20 (20% inhibitory concentration) value of 79938.9 nM, whereas the 4-acetamidoantipyrine inhibited the β-galactosidase activity induced by 60 nM of TO901317 with an IC20 value of 6286.4 nM. The IC20 values of catechol and 4-acetamidoantipyrine were 181 and 14 times higher than those of SR9238 (440.8 nM), a positive antagonist of LXRα (16), respectively, indicating
of
that catechol and 4-acetamidoantipyrine have only weak LXRα-antagonist activities. The structures of the two compounds are very different from those of classical ligands
ro
(such as SR9238 and SR9243). However, in our recent report, TPHP and EHDPP,
-p
which have structures very different from those of classical ligands, were found to be
re
strong LXRα-antagonist compounds. This suggested that compounds with structures
lP
different from those of classical LXRα receptor ligands could also exhibit
na
LXRα-antagonist activities. Further studies using other evaluation techniques (19, 20) will be performed to confirm the results of this study.
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3.3 Environmental implications
To date, few toxicological studies have assessed LXRα-agonist or antagonist activities in environmental water samples, and only two environmental organic chemicals, TPHP and EHDPP, have been reported to have LXR activity. Our results indicated that
some
chemicals
in
the
reclaimed
water
exhibited
dose-dependent
LXRα-antagonist activity. The identification of the specific chemicals responsible for the LXRα-antagonist activity in the reclaimed water is crucial, and challenging. The NRBE-HRMS
method
has
proven
efficient
in
identifying
EDCs
with
LXRα-antagonist activity in complex environmental samples, such as reclaimed water 13
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(this study) and indoor dust samples (16). Thus, we used the NRBE-HRMS method to extract and identify potential LXRα-antagonist activity chemicals which could bind to LXRα from reclaimed water. In total, 105 molecular formulas with LXRα-binding activity were screened from the reclaimed water. Although identification of all of these putative LXRα ligands was hindered by the absence of corresponding reference
of
spectra in the database, two compounds, catechol and 4-acetamidoantipyrine, were identified as novel LXRα antagonists. Catechol (1,2-dihydroxybenzene) is a phenolic
ro
compound, and is a known environmental pollutant. This compound has been
-p
detected in elevated concentrations in the effluents from many industries, such as
re
petroleum refineries, steel mills, and pharmaceuticals (21–23). Concentrations of
lP
catechol in some industrial effluent samples range from a few mg/L to several
na
thousand mg/L (22, 23). For example, catechol has been detected in the effluents from coal carbonization and gasification plants at concentrations as high as 5300 mg/L
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(23). It has adverse effects on the eyes, skin, and respiratory system, and can also lead to a wide range of diseases such as genetic mutations and vascular collapse, or even death in some cases (24–26). In contrast, 4-acetamidoantipyrine is a human metabolite of the analgesic drug metamizole. It is persistent, with a removal rate below 30% during wastewater treatment (27). 4-acetamidoantipyrine concentrations of 1300–2240 ng/L in WWTP effluents have been reported in China (27). Higher 4-acetamidoantipyrine concentrations (from 950 to 6000 ng/L in the effluents) have also been reported for WWTPs in Europe (28, 29). It should be noted that only two compounds were identified from among the 105 14
Journal Pre-proof compounds exhibiting LXRα-binding activity in the reclaimed wastewater. Thus, future efforts should be devoted to identifying the other screened compounds that could bind to LXR, and assessing their potential LXR-agonist/antagonist activities. The potential health impacts of the presence of these EDCs should be evaluated. In addition, by using other nuclear receptors as the affinity protein, the NRBE-HRMS
of
method could be used to reveal novel EDCs that can interact with corresponding
ro
nuclear receptors in various complex environmental samples.
-p
4. Conclusion
re
In this study, we used the NRBE-HRMS method to screen for unknown EDCs with
lP
LXRα binding activity in reclaimed wastewater. In total, 105 compounds in the
na
reclaimed wastewater were found to exhibit LXRα-binding activity. Among them, catechol and 4-acetamidoantipyrine were identified, using authentic standards. Weak
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LXRα-antagonist activities in a yeast two-hybrid assay were observed for the two identified compounds. The results of this study demonstrated the effectiveness of this NRBE-HRMS method in identification of unknown EDCs in complex environmental samples. Acknowledgment This work was funded by the International S&T Cooperation Program of China (2016YFE0117800) and the National Science Foundation of China Grant (Nos. 51420105012 and 51578530).
15
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Reference (1) U.S. Environmental Protection Agency. Guidelines for Water Reuse; EPA/625/R-04/108; EPA: Washington, DC, 2004. (2) Eggen, R.; Hollender, J.; Joss, A.; Schärer, M.; Stamm, C. Reducing the discharge of micropollutants in the aquatic environment: The benefits of upgrading wastewater treatment plants. Environ. Sci. Technol., 2014, 48, 7683-7689. (3) UNEP. Global Chemical Outlook; Towards Sound Management of Chemicals;
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(6) Skakkebaek, N. E.; Rajpert-De Meyts, E.; Buck Louis, G. M.; Toppari, J.; Andersson, A. M.; Eisenberg, M. L.; Jensen, T. K.; Jørgensen, N.; Swan, S. H.; Sapra, K. J.; Ziebe, S.; Priskorn, L. Male Reproductive Disorders and Fertility Trends: Influences of Environment and Genetic Susceptibility. Physiol. Rev. 2016, 96, 55– 97. (7) De Coster, S.; van Larebeke, N. Endocrine-Disrupting Chemicals: Associated Disorders and Mechanisms of Action. J. Environ. Public Health 2012, 2012, 713696 (8) Fucic, A.; Gamulin, M.; Ferencic, Z.; Katic, J.; Krayer von Krauss, M.; Bartonova, A.; Merlo, D. F. Environmental Exposure to Xenoestrogens and Oestrogen Related Cancers: Reproductive System, Breast, Lung, Kidney, Pancreas, and Brain. Environ. Health 2012, 11, S8 (9) Balaguer, P., Delfosse, V.; Bourguet, W.; Mechanisms of endocrine disruption 16
Journal Pre-proof through nuclear receptors and related pathways. Current Opinion in Endocrine and Metabolic Research 2019, 7:1–8 (10) Vajda, A. M.; Barber, L. B.; Gray, J. L.; Lopez, E. M.; Woodling, J. D.; Norris, D. O. Reproductive disruption in fish downstream from an estrogenic wastewater effluent. Environ. Sci. Technol., 2008, 42, 3407-3414 (11) Wu, Q.; Hu, H.; Zhao, X.; Sun, Y. Effect of chlorination on the estrogenic/antiestrogenic activities of biologically treated wastewater. Environ. Sci. Technol. 2009, 43, 4940-4945.
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(12) Rostkowski, P.; Horwood, J.; Shears, J. A.; Lange, A.; Oladapo, F. O.; Besselink
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H. T.; Tyler, C. R.; Hill, E. M. Bioassay-directed identification of novel
Sci. Technol. 2011, 45, 10660-10667.
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antiandrogenic compounds in bile of fish exposed to wastewater effluents. Environ.
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(13) Beaven, S. W.; Tontonoz, P. Nuclear receptors in lipid metabolism: Targeting the
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heart of dyslipidemia. Annu. Rev. Med. 2006, 57, 313−329. (14) Kalaany, N. Y.; Mangelsdorf, D. J. LXRs and FXR: The yin and yang of
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cholesterol and fat metabolism. Annu. Rev. Med. 2006, 68, 159−191. (15) Calkin, A. C.; Tontonoz, P. Transcriptional integration of metabolism by the
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nuclear sterol-activated receptors LXR and FXR. Nat. Rev. Mol. Cell. Biol. 2012, 13,
(16) Hu, W.; Jia, Y.; Kang, Q.; Peng, H.; Ma, H.; Zhang, S.; Hiromori, Y.; Kimura, T.; Nakanishi, T.; Zheng, L.; Qiu, Y.; Zhang, Z.; Wan, Y.; Hu, J. Screening of Chemicals for Liver X Receptors Binding Activities from Dust in 11 Chinese Homes and Characterization of Atherosclerotic Activity Using an in vitro Macrophage Cell Line and ApoE-/- Mice. Environ. Health Perspect. 2019, 127, 117003, DOI: 10.1289/EHP5039. (17) Zhang, H.; Zhang, Z.; Nakanishi, T.; Wan, Y.; Hiromori, Y.; Nagase, H.; Hu, J. Structure-dependent activity of phthalate esters and phthalate monoesters binding to human constitutive androstane receptor. Chem Res Toxicol 2015, 28(6): 1196-1204. (18) Creusot, N.; Budzinski, H.; Balaguer, P.; Kinani, S.; Porcher, J.M.; Aït-Aïssa, S. 17
Journal Pre-proof Effect-directed analysis of endocrine-disrupting compounds in multi-contaminated sediment: identification of novel ligands of estrogen and pregnane X receptors. Anal. Bioanal. Chem. 2013, 405, 2553-66. (19) Sim, W.; Park, S.; Lee, K.; Je, Y.; Yin, H.; Choi, Y.; Sung, S.; Park, S.; Park, H.; Shin, K.; Lee, B. LXR-α antagonist meso-dihydroguaiaretic acid attenuates high-fat diet-induced nonalcoholic fatty liver. Biochemical Pharmacology 2014, 90, 414-424. (20) Liu, X.; Sakai, H.; Nishigori, M.; Suyama, K.; Nawaji, T.; Ikeda, S.; Nishigouchi, M.; Okada, H.; Matsushima, A.; Nose, T., Shimohigashi, M.;
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analogs for human nuclear receptors. Toxicology and Applied Pharmacology 2019, 377, 114610.
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(21) Lofrano, G.; Rizzo, L.; Grassi, M.; Belgiorno, V. Advanced oxidation of
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Desalination, 2009, 249, 878-883.
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catechol: a comparison among photocatalysis, Fenton and photo-Fenton processes.
(22) Subramanyam, R.; Mishra, I.M. Biodegradation of catechol (2-hydroxy phenol)
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bearing wastewater in an UASB reactor. Chemosphere, 2007, 69. 816-824. (23) Subramanyam, R.; Mishra, I.M. Treatment of catechol bearing wastewater in an
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upflow anaerobic sludge blanket (UASB) reactor: Sludge characteristics. Bioresource Technology, 2008, 99, 8917-8925. (24) Anotai, J.; Su,C.; Tsai, Y.; Lu, M. Effect of hydrogen peroxide on aniline oxidation by electro-Fenton and fluidized-bed Fenton processes. J. Hazard. Mater., 2010, 183, 888-893.
(25) Aghapour, A.A.; Moussavi, G.; Yaghmaeian, K. Degradation and COD removal of catechol in wastewater using the catalytic ozonation process combined with the cyclic rotating-bed biological reactor. J. Environ. Manag., 2015, 157.262-266. (26) Kumar, A.; Kumar, S.; Kumar, S. Adsorption of resorcinol and catechol on granular activated carbon: Equilibrium and kinetics. Carbon 2003, 41, 3015-3025. (27) Qi, W.; Singer, H.; Berg M.; Müller, Beat.; Pernet-Coudrier, B.; Liu, H.; Qu, J. Elimination of polar micropollutants and anthropogenic markers by wastewater 18
Journal Pre-proof treatment in Beijing, China. Chemosphere 2015, 119, 1054-1061. (28) Zühlke, S., 2004. Verhalten von Phenazonderivaten, Carbamazepin und estrogenen Steroiden während verschiedener Verfahren der Wasseraufbereitung. PhD thesis. Technische Universität, Berlin, Germany. (29) Kahle, M.; Buerge, J.I.; Müller, D.M.; Poiger, T. Hydrophilic anthropogenic markers for quantification of wastewater contamination in ground- and surface
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na
lP
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waters. Environ. Toxicol. Chem., 2009, 28, 2528-2536
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Figure Caption
Figure 1 Principle component analysis (PCA) plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode.
of
Figure 2 Volcano plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode. The red dots represent a chemical with a fold change of ≥3 and p-value of ≤0.05.
-p
ro
Figure 3 Chromatograph and mass spectra of reclaimed water sample and authentic standards of (a) catechol and (b) 4-acetamidoantipyrine.
lP
re
Figure 4 Dose−response curves for the β-galactosidase activity of catechol and 4-acetamidoantipyrine.
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na
Figure 5 Dose−response curves for the relative β-galactosidase activity of catechol and 4-acetamidoantipyrine.
20
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100
pCold-TF-LXRα pCold-TF
(a)
0
of
PC 2 (28.8%)
50
-100 -100
-50
-p
ro
-50
0
50
100
re
PC1 (58.8%) 100
pCold-TF-LXRα pCold-TF
lP
na
Jo ur
PC 2 (10.7%)
50
0
(b)
-50
-100 -100
-50
0
50
100
PC1 (51.9%)
Figure 1 Principle component analysis (PCA) plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode.
21
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12
pCold-TF-LXRα pCold-TF
(a)
8
of
6
4
ro
-Log(p-value)
10
0 -20
20
lP
(b)
na
-Log(p-value)
10
Jo ur
6
0
pCold-TF-LXRα pCold-TF
10
8
-10
re
12
-p
2
4
2
0
-20
-10
0
10
20
Log2 (fold change)
Figure 2 Volcano plots of reclaimed water sample and control sample data set: (a) negative mode and (b) positive mode. The red dots represent a chemical with a fold change of ≥3 and p-value of ≤0.05.
22
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4E+06
2E+06
(a) catechol
(b) 4-acetamidoantipyrine
3E+06
sample
2E+06
sample
1E+06
1E+06 0E+00 2.0
2.5
3.5 Time 4.0
3.0
2.0
standard 2E+06
1E+06
3.0
3.5
Time4.0
standard 2E+07
1E+07
0E+00 2.0
2.5
3.5 Time 4.0
3.0
2.0
109.0294
1.0
1.0
0.5
0.5
94.9805
0.0
3.0
83.0610 104.0499
3.5
Time4.0
228.1131
204.1133 246.1237
-p
76.9700
2.5
ro
0E+00
0.0
2.5
3E+07
of
3E+06
Intensity
Intensity
Relative abundance
0E+00
-0.5
-0.5 -1.0
80
100
120 m/z
50
100
150
200
m/z 250
na
lP
60
re
-1.0
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Figure 3 Chromatograph and mass spectra of reclaimed water sample and authentic standards of (a) catechol and (b) 4-acetamidoantipyrine.
23
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3500
2500
2000
of
1500
ro
1000
4-acetamidoantipyrine catechol
100
101
Concentration (μM)
102
na
lP
0 10-1
-p
500
re
β-galactosidase activity
3000
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Figure 4 Dose−response curves for the β-galactosidase activity of catechol and 4-acetamidoantipyrine.
24
Journal Pre-proof
120%
80%
of
60%
ro
40%
4-acetamidoantipyrine catechol
-p
20%
0% 10-1
100
re
Relative β-galactosidase activity
100%
101
102
na
lP
Concentration (μM)
Jo ur
Figure 5 Dose−response curves for the relative β-galactosidase activity of catechol and 4-acetamidoantipyrine.
25
Journal Pre-proof Table 1 Formulas of molecules in reclaimed wastewater found to binding LXRα receptor Negative ion peaks
Positive ion peaks
No. Formula
RT1
m/z
1
C14H8O4
1.98
239.0355
2
C6H6O2
2.66
3
C13H12N2O3
3.27
4
C15H18N2O3
5
RT
m/z
2.38
1
C10H13N3O3
2.66
206.0926
0.88
109.0294
-1.27
2
C13H15N3O2
2.84
246.1239
0.73
225.0669
-0.26
3
C7H10N2O
2.89
121.0764
2.39
3.47
319.1296
-1.27
4
C7H12N2O2
3.09
139.0867
0.70
C15H20N2O3
3.49
275.1399
-0.57
5
C16H13NO5
3.33
317.1134
0.68
6
C5H11NO4P
3.55
161.0242
0.00
6
C13H13N3
3.48
212.1183
0.57
7
C3H6N6O4
3.77
235.0431
-0.59
7
C21H36N4O8
3.91
455.2516
3.36
8
C9H19O11P
3.88
379.0642
-1.47
8
C10H10N2O2
4.00
173.0711
0.93
9
C13H14O5
3.92
231.0660
-1.28
9
C16H16N2O5
4.10
299.1027
0.32
10
C11H9N3O4
3.99
228.0415
-0.07
10
C11H10N2O
4.35
169.0761
0.42
11
C8H8O4S
4.00
234.9835
-1.10
11
C12H14N2O
4.42
185.1075
1.01
12
C20H20N6O6S
4.08
471.1104
4.97
12
C10H14N2O
4.50
161.1074
0.60
13
C13H14O3S
4.14
249.0590
-0.41
13
C20H25N3O3
4.56
356.1957
-3.34
14
C8H8O8
4.16
266.9911
-0.73
14
C12H12N2O2
4.72
239.0793
0.92
15
C10H12O6S
4.20
295.0045
-1.58
15
C15H15N3O
4.76
236.1183
0.23
16
C11H9N3O3
4.23
266.0330
-3.56
16
C20H36N2O5
4.95
423.2251
-1.26
17
C12H9N3O
4.41
210.0670
-1.13
18
C16H24O5S
4.42
327.1269
-0.66
19
C10H16N4OS
4.45
275.0747
20
C17H27N3O7
4.57
384.1789
21
C14H31O5P
4.76
355.1886
22
C6H10N6O4
4.81
275.0746
23
C16H16N4O5S
4.87
357.0661
24
C15H16OS
4.96
289.0903
25
C19H25NO5
26
C10H15F3O4
27
Error
re
-p
ro
of
No. Formula
5.04
3.39
19
C24H36O9
5.62
469.2410
-4.61
3.23
20
C12H18O3S
5.79
243.1050
0.14
-1.61
21
C23H32N4O2
7.05
419.2403
-3.67
na
Error
0.19
22
C23H22N6O6
7.71
479.1673
-0.07
-0.46
23
C19H16O
8.28
243.1168
-0.08
-0.42
24
C34H46O6
8.53
573.3189
0.43
C16H21N3O4S
352.1326
0.21
18
C21H31N3O6
5.20
439.2567
3.71
Jo ur
lP
17
5.01
382.1438
3.22
25
C11H10F3N5O4
8.56
356.0577
0.95
5.05
301.0902
-1.15
26
C23H39NO6
8.75
408.2758
3.14
C11H20NO4S
5.13
297.0800
-4.64
27
C14H12F6O3
9.20
381.0322
-0.10
28
C14H22O4S
5.14
267.1060
-0.12
28
C23H24N6O
9.57
423.1907
0.97
29
C8HF15O2
5.72
412.9661
-0.70
29
C20H32O
9.70
306.2791
-0.31
30
C14H20OS
5.85
281.1216
-0.58
30
C20H36O2
9.98
331.2606
-0.05
31
C27H26N6O2
6.33
501.1829
3.74
31
C20H36O6
10.27 395.2396
-2.05
32
C13H12O7S2
6.41
389.0022
4.46
32
C20H27O4P
10.49 385.1538
-0.28
33
C18H20N2O
6.47
315.1271
0.38
33
C20H29F3
10.59 344.2560
0.09
34
C30H41ClN2O5
6.59
543.2625
-1.04
34
C14H24O
10.89 191.1797
1.11
35
C15H24O4S
6.76
299.1320
-0.79
35
C22H38O4
10.96 389.2663
-0.16
36
C10H5F17O3S
7.13
526.9611
-0.90
36
C18H44N6O12
10.96 559.2888
-3.86
37
C24H24O4
7.18
357.1498
0.39
37
C20H43O6P
11.12 411.2869
-0.29
38
C34H33N3O9S
7.27
640.1747
-1.78
38
C20H39NO2
12.01 348.2872
0.00
39
C16H26O3S
7.60
297.1528
-0.61
39
C17H30N2O5
12.07 325.2113
-2.50
40
C18H30O3S
8.19
325.1841
-0.43
40
C17H35NO
12.10 292.2609
-0.27
26
Journal Pre-proof Negative ion peaks
Positive ion peaks
No. Formula
RT1
m/z
41
C18H28O2
8.24
321.2069
42
C17H28O3S
8.31
43
C27H46N6O7
44 45
RT
-0.82
41
C40H74N2O2
12.17 637.5643
0.13
311.1686
-0.15
42
C23H38N2O
12.19 376.3321
-0.43
8.39
601.3133
1.99
43
C21H38N2O
12.66 352.3319
-0.86
C20H39N2O12P3 8.52
627.1432
3.74
44
C40H78N2O2
12.84 641.5956
0.03
C23H26FN6O9P 8.52
625.1462
-0.50
45
C6H7O4P
13.69 175.0154
-0.68
46
C22H36O4
8.63
409.2593
-0.67
47
C20H32O3S
8.90
351.1997
-0.65
48
C20H30OS
8.90
363.1998
-0.32
49
C16H34N4OS
8.90
365.2153
1.58
50
C23H37NO4
9.36
436.2715
2.59
51
C27H38O2
9.54
439.2850
-0.87
52
C17H36N4OS
9.56
379.2310
1.77
53
C15H24O2
9.61
235.1701
-1.10
54
C20H34OS
9.64
367.2310
-0.84
55
C18H30OS
9.72
339.1998
-0.36
56
C19H38O5
10.18 381.2467
0.54
57
C20H43O4P
10.61 377.2825
-0.32
58
C24H38O
11.04 387.2903
-0.54
59
C22H32O2
11.76 327.2327
-0.75
60
C19H36O
12.00 325.2745
lP
re
-p
ro
of
No. Formula
-0.96
Jo ur
na
1. RT: retention time
Error
27
m/z
Error
Journal Pre-proof
Reclaimed wastewater extracts
UPLC-HRMS 4E+06
2E+06
3E+06 2E+06
1E+06
1E+06 0E+00
0E+00 2.0
2.5
3.0
3.5
4.0
2.0
2.5
3.0
3.5
4.0
2.0
2.5
3.0
3.5
4.0
3E+07
Intensity
Intensity
3E+06
2E+06
1E+06
0E+00
2E+07
1E+07
0E+00 2.0
2.5
3.0
3.5
4.0
1.0
1.0
0.5
0.5
0.0
0.0
-0.5
-0.5
-1.0
-1.0 60
80
100
120
50
100
150
200
250
120%
LXRα
100%
LXRα
Wash
of
MB
80%
MB
60%
MB
Jo ur
na
lP
Liver X receptor binding extraction
-p
LXRα
MB
LXRα-binding chemicals
re
LXRα
ro
Elute
28
40%
20%
0% 10-1
100
101
102
Yeast two-hybrid assay
Journal Pre-proof Highlights • Identification of EDCs with liver X receptor binding activity in reclaimed water • 105 compounds in reclaimed wastewater were found to exhibit LXRα-binding activity
Jo ur
na
lP
re
-p
ro
of
• Catechol and 4-acetamidoantipyrine were identified as LXRα antagonists
29