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Peroxisome Proliferator-Activated Receptor # is a Sensitive Target for Oil Sands Process-affected Water: Effects on Adipogenesis and Identification of Ligands Hui Peng, Jianxian Sun, Hattan A. Alharbi, Paul D. Jones, John P. Giesy, and Steve B. Wiseman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01890 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016
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Peroxisome Proliferator-Activated Receptor γ is a Sensitive Target for Oil Sands
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Process-affected Water: Effects on Adipogenesis and Identification of Ligands
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Hui Penga, Jianxian Sun*a, Hattan A. Alharbia, Paul D. Jonesa,b, John. P. Giesy*a, c,d,e,f and Steve Wisemana,g
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Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, Canada, S7N 5B3 b School of Environment and Sustainability, 117 Science Place, Saskatoon, SK, Canada, S7N 5C8 c Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B3 d Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA e State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China f Biology Department, Hong Kong Baptist University, Hong Kong, SAR, China g Department of Biology, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.
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*Corresponding authors: Jianxian Sun, e-mail:
[email protected]; John. P. Giesy,
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Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N5B3, Canada;
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TEL (direct): 306-966-2096; TEL (secretary): 306-966-4680; FAX: 306-966-4796; e-mail:
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ABSTRACT
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Identification of toxic components of complex mixtures is a challenge. Here, oil sands
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process-affected water (OSPW) was used as a case study to identify those toxic components
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with a known protein target. Organic chemicals in OSPW exhibit dose-dependent activation
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of peroxisome proliferator-activated receptor γ (PPARγ) at concentrations less than those
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currently in the environment (0.025× equivalent of full-strength OSPW), by use of a
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luciferase reporter gene assay. Activation of PPARγ-mediated adipogenesis by OSPW was
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confirmed in 3T3L1 preadipocytes, as evidenced by accumulation of lipids and up-regulation
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of AP2, LPL and PPARγ gene expression after exposure to polar fractions of OSPW.
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Unexpectedly, the nonpolar fractions of OSPW inhibited differentiation of preadipocytes via
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activation of the Wnt signaling pathway. Organic chemicals in OSPW that were ligands of
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PPARγ were identified by use of a pull-down system combined with untargeted chemical
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analysis (PUCA), with a recombinant PPARγ protein. Thirty ligands of PPARγ were identified
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by use of the PUCA assay. High resolution MS1 and MS2 spectra were combined to predict
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the formulas or structures of a subset of ligands, and polyoxygenated or heteroatomic
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chemicals, especially hydroxylated carboxylic/sulfonic acids, were the major ligands of
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PPARγ.
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KEYWORDS: OSPW; Untargeted chemical analysis; Pull down; His-tagged recombinant
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protein; Orbitrap ultrahigh resolution mass spectrometry.
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INTRODUCTION
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In the surface mining oil sands industry in Northern Alberta, Canada, extraction of
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bitumen from oil sands results in production of oil sands process-affected water (OSPW).
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Rather than releasing OSPW back to the receiving environment, OSPW is stored in tailings
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ponds and settling basins, and recycled for extraction of bitumen.
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area of approximately 170 km2 and contain greater than 1 billion m3 of OSPW.1 After a
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surface mine is closed or when OSPW is no longer useful for extraction of bitumen, tailings
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ponds must be reclaimed and detoxified, but as yet, no method to achieve this at the scale
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required in this industry has been developed.2
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major strategy for reclamation and detoxification of OSPW.
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constructed in mined-out pits of oil sands mines, will be permanent features of reclaimed
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landscapes, and will be hydraulically connected with the natural environment.3
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30 EPLs have been planned by various companies operating in the surface mining industry,4
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so demonstrating success of this strategy is important. However, because there is concern
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with regard to the rate at which OSPW in EPLs will be detoxified, effectiveness of EPLs for
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eliminating toxicity of OSPW was called into question by a report by the Royal Society of
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Canada.4
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assist industry and regulatory agencies charged with monitoring EPLs.
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include bioassays that allow for monitoring of toxicity or high resolution mass spectrometry
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assays to monitor concentrations of specific chemicals that cause adverse effects.
Tailings ponds cover an
End-pit lakes (EPLs) have been proposed as a In general, EPLs will be
Greater than
Development of tools to monitor detoxification of OSPW in EPLs would greatly These tools could
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Implementation of bioassays or mass spectrometry assays to monitor detoxification of
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EPLs is dependent on a comprehensive knowledge of adverse effects caused by exposure to
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OSPW and identification of specific chemicals that cause these effects.
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based on the critical mechanism of toxicity, or that adverse effect that occurs at the least
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concentration of chemicals.
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impaired growth and development,6 endocrine disruption and impairment of reproduction,7, 8
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and immunotoxicity,
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effects.
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complexity of OSPW. Analysis of OSPW by use of ultrahigh-resolution mass spectrometry
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has revealed thousands of chemicals, including species containing oxygen, nitrogen, and
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sulfur.11-13
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that cause acute lethality5, 14, 15 and endocrine disruption.16, 17 However, the EDA strategy has
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some limitations.
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mixture are present in “active” fractions, and each chemical in an “active” fraction might not
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contribute to biological effects.
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fractionation of organic chemicals in the aqueous phase of OSPW, two fractions that caused
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acute lethality were produced, but several classes of chemicals were present in each fraction,
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and it is not known if each of the classes of chemicals was responsible for acute lethality.
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Alternatively, when the mode of toxic action involves binding of chemicals to a protein, it is
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possible to use the protein target as an affinity matrix to specifically capture bioactive
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metabolites or drugs.18 Using this strategy, physically-interacted ligands can be identified with
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a lesser false positive rate, and laborious fractionation of samples is not necessary. However,
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because the high background interferences produced from the process decreases sensitivity
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and specificity of the method, application of the strategy to environmental matrices is a great
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Bioassays should be
Adverse effects of exposure to OSPW include acute lethality,5
but little is known about specific toxic components that cause these
One major impediment to identification of toxic components is the chemical
Effects directed analysis (EDA) has been used to identify chemicals in OSPW
For example, it is not known if all the causative chemicals in a complex
In the study by Morandi et al,14 after three rounds of
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challenge because of the complexity and low abundance of ligands in environmental samples.
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Therefore, it is necessary to develop a more robust strategy to improve identification of toxic
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components in OSPW that cause adverse effects.
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Naphthenic acids (NAs), which are a major chemical constituent of OSPW, are a group of
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cyclic and acyclic, alkyl-substituted carboxylic acids with the general formula of CnH2n+ZO2.19
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It has been suggested that some NAs are structurally similar to fatty acids.19, 20
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acids are ligands of peroxisome proliferator-activated receptor γ (PPARγ),21,
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receptor that is a ligand activated transcription factor and important regulator of adipogenesis,
Because fatty 22
a nuclear
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PPARγ might be an important target of OSPW.
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contaminants, such as tributyltin (TBT) and triphenyl phosphate (TPP), causes adverse effects
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such as promotion of adipogenesis resulting in weight gain and obesity.23-26 Therefore,
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disruption of PPARγ signaling might be an important mechanism of toxicity of OSPW.
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Activation of PPARγ by environmental
The goal of the current study was to test the hypothesis that ligands of PPARγ are present
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in OSPW and to identify these ligands.
Activation of PPARγ signaling was determined by
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use of a reporter assay and promotion of adipogenesis was evaluated by use of 3T3L1 cells.
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Finally, a novel method to identify ligands of PPARγ was developed in which a pull-down
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assay with recombinant PPARγ protein was combined with untargeted chemical analysis
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(PUCA) by use of Orbitrap ultrahigh-resolution mass spectrometry.
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Materials and Methods
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Chemicals and Reagents. Details are provided in Supporting Information.
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OSPW Sample Collection and Extraction. Samples of OSPW from two sources were
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investigated in the present study. Fresh OPSW was collected from the West-In-Pit tailings
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pond (Syncrude Canada Ltd., Fort McMurray, AB, Canada). The West-in-Pit settling basin
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was commissioned as Base Mine Lake (BML-OSPW), which is the first end pit lake in the oil
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sands industry, in December 2012. A sample of aged OSPW also was collected from an
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experimental reclamation pond called Pond 9 (P9- OSPW) that was constructed in 1993 and
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has not received input of OSPW since that time. All samples were collected in September of
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2012, shipped to the University of Saskatchewan (Saskatoon, SK, Canada), and stored in the
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dark, and used for fractionation immediately upon arrival.14, 27 Fractionation of BML-OSPW
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samples (n=3) or P9-OSPW (n=1) was conducted by use of EVO-LUTE® ABN SPE
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cartridges (Biotage, Charlotte, NC, USA) because of their ability to extract a broad range of
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chemicals, as demonstrated in our previous study.27 Preliminary data also showed that PPARγ
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activity of BML-OSPW extracted by EVO-LUTE® ABN SPE cartridges was greater than
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activity of the same sample of OSPW extracted by HLB cartridges. A procedure blank was
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conducted using ultrapure water, and no significant activity of PPARγ was detected. All the
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extracts were dissolved in ethanol and stored at -20 oC. Details of the sample pretreatment are
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provided in Supporting Information.
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PPARγ Assay. Activation of PPARγ was determined by use of a human PPARγ reporter assay,
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according to the protocol provided by the manufacturer (Cayman Chemical, Ann Arbor, MI,
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USA), which is described in Supporting Information. Antagonism of PPARγ was determined
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by mixing 50 nM of rosiglitazone (~EC50), which is an agonist of PPARγ, with samples of
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BML-OSPW or T0070907 (antagonist of PPARγ). Antagonistic activity was determined
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using the same method used to determine activation of PPARγ. All exposures were conducted
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in triplicate (n=3).
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Differentiation of 3T3-L1 Cells. Adipogenesis was assessed using 3T3L1 preadipocytes
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(ATCC® CL-173™, Manassas, VA) as described previously28 and in the Supporting
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Information. Expression of genes that are regulated by PPARγ (PPARγ, AP2, LPL and WISP2)
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were quantified by use of quantitative real-time PCR (qPCR). Expression of target genes was
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normalized to expression of 36B4.28, 29 Details of the qPCR protocol are provided in the
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Supporting Information.
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His-PPARγ Pull-Down Assay. Details of the pull-down experiments to identify ligands of
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PPARγ are described in Supporting Information.
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Mass Spectrometry and Untargeted Data Processing. Aliquots of extracts were analyzed
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using a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a Dionex™
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UltiMate 3000 UHPLC system.
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accomplished with an in-house R program as described in the Supporting Information.
Analysis of untargeted mass spectrometry data was
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Results and Discussion
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PPARγ Agonistic Activity of OSPW. The total extract (TE) of organic chemicals from the
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aqueous phase of BML-OSPW significantly activated PPARγ-driven reporter activity and the
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effect was dose-dependent (Figure 1A). Maximum activation was 14.6±0.19-fold (relative to
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vehicle control; corresponding to 21.8% of the maximal activity of rosiglitazone at 625 nM)
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in response to a 1× equivalent of the TE.
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activity (1.3±0.12-fold, p=0.037) was observed at concentrations as small as 0.025×, which
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represents a 40-fold dilution of dissolved organic chemicals in BML-OSPW.
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bioassay-derived rosiglitazone equivalent (REQ) of BML-OSPW was calculated to be 55.7
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nM (Figure S1A, Supporting Information). Dose-dependent activation of PPARγ also was
Significant activation of PPARγ-driven reporter
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detected in two other replicate samples of BML-OSPW (Figure S1B). Previous studies have
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reported nuclear receptor activities or other toxicities of OSPW, but no other study has
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reported an effect of OSPW at such a small concentration (40× dilution).
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the EPL strategy, OSPW will be diluted by input of waters from natural surface and
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groundwater, runoff and precipitation.3
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ensure that dilution of OSPW is adequate to ameliorate effects on PPARγ.
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of OSPW from tailings ponds to surface water has been reported,30 it would be appropriate to
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determine if these surface waters have greater potential to activate PPARγ than natural surface
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waters not impacted by seepage from tailings ponds. Activation of PPARγ by P9-OSPW was
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less compared to activation by BML-OSPW (Figure S1B), and the response at 1× was only
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5.9±0.4% of the maximal activity of rosiglitazone at 625 nM. These results suggest that aging
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of OSPW is effective to detoxify OSPW.
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According to
These results suggest that it will be important to Because seepage
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To further investigate the PPARγ activity of BML-OSPW, the replicate causing greatest
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activation (replicate-1) was selected for further fractionation by use of SPE cartridges.
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Consistent with effects of the TE, each of the five fractions of BML-OSPW activated
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PPARγ-driven reporter activity (Figure 1B).
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DCM in hexane as the elution solvent) and F5 (methanol as the elution solvent), with
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induction of 9.6±0.55-fold (p
