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Identification of Chemicals in Oil Sands ProcessAffected Water That Cause Oxidative Stress Jianxian Sun, Hui Peng, Hattan A. Alharbi, Paul D. Jones, John P. Giesy, and Steve B. Wiseman Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01987 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on July 2, 2017
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Identification of Chemicals in Oil Sands Process-Affected Water That Cause Oxidative
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Stress
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Jianxian Suna, Hui Peng*a, Hattan A. Alharbia, Paul D. Jonesa,b, John. P. Giesya,b,c,d,e,f,g, and
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Steve B. Wiseman*h
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Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, Canada School of Environment and Sustainability, 117 Science Place, Saskatoon, SK, Canada c Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada d Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA e School of Biological Sciences, University of Hong Kong, Hong Kong Special Administrative Region, Peoples republic of China f State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China g Biology Department, Hong Kong Baptist University, Hong Kong, SAR, China h Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada
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*Corresponding author: Hui Peng,
[email protected]; Toxicology Centre, University of
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Saskatchewan, Saskatoon, SK, Canada; Tel.: +1-306-966-8733; Fax: +1-306 966 4796
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Steve Wiseman,
[email protected], Department of Biological Sciences, University of
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Lethbridge, Lethbridge, AB, Canada: Tel +1-403-329-2320
b
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ABSTRACT
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Oil sands process-affected water (OSPW) has been reported to cause oxidative stress in
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organisms, yet the causative agents remain unknown. In this study, a high-throughput in vitro
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Nrf2 reporter system was used, to determine chemicals in OSPW that cause oxidative stress.
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Five fractions, with increasing polarity, of the dissolved organic phase of OSPW were
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generated by use of solid phase extraction cartridges. The greatest response of Nrf2 was
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elicited by F2 (2.7 ± 0.1-fold), consistent with greater hydroperoxidation of lipids in embryos
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of Japanese medaka (Oryzias latipes) exposed to F2. Classic naphthenic acids were mainly
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eluted in F1, and should not be causative chemicals. When F2 was fractionated into 60
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sub-fractions by use of HPLC, significant activation of Nrf2 was observed in three grouped
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fractions: F2.8 (1.30 ± 0.01-fold), F2.16 (1.34 ± 0.05-fold), and F2.25 (1.28 ± 0.15-fold). 54
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compounds were predicted to be potential chemicals causing Nrf2 response, predominated by
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SO3+ and O3+ species. By use of high-resolution MS2 spectra, these SO3+ and O3+ species
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were identified as hydroxylated aldehydes. This study demonstrated that polyoxygenated
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chemicals, rather than classic NAs, were the major chemicals responsible for oxidative stress
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in the aqueous phase of OSPW.
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KEYWORDS:
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Untargeted chemical analysis; Orbitrap; Ultra-high resolution mass spectrometry; EDA; SPE
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INTRODUCTION
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Extraction of bitumen from the oil sands of Alberta, Canada has increased rapidly with
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projected output ranging from 2.0 to 2.9 million barrels per day by 2020.1, 2 In surface mining
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operations, extraction of bitumen from oil sands with hot water results in production of oil
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sands process-affected water (OSPW). Development of oil sands and leakage from tailings
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ponds has raised concerns about potential pollution and ecological/health risks posed by
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exposure to OSPW.3, 4 Specifically, there is concern about the rate at which OSPW in end pit
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lakes (EPLs) will be detoxified.5
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high-resolution mass spectrometry analysis of specific chemicals that cause toxicity, to
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monitor detoxification of OSPW in EPLs would greatly assist industry and regulatory
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agencies charged with monitoring EPLs.
Development of tools, such as bioassays and
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OSPW has been reported to cause multiple chronic toxicities,6-12 but the mechanism(s) of
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these toxicities are not well understood. Numerous studies have suggested that oxidative
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stress, which can lead to damage to nucleotide acids, proteins, and lipids, and thereby causing
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cellular dysfunction and ultimately cause phenotypic adverse effects, might be an important
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mechanism of toxicity of OSPW.7, 9, 13 In hepatocytes of rainbow trout (Oncorhynchus mykiss),
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expression of genes related to oxidative stress was significantly up-regulated after exposure to
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OSPW,14 and oxidative stress and apoptosis were induced in embryos of fathead minnows
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exposed to OSPW.7 Cytotoxicity in strains of yeast exposed to OSPW has also been reported
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to be mediated by pathways related to oxidative stress.13 Results of both in vitro and in vivo
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experiments indicated the significance of oxidative stress as mechanism of toxicity of OSPW,
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but identities of those chemicals in OSPW that cause oxidative stress are not known.
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Because the aqueous phase of OSPW is a complex mixture of dissolved organic
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compounds it is difficult to identify those chemicals that cause toxicities. Advances in
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ultrahigh-resolution mass spectrometry have led to understanding that the aqueous phase of 4
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OSPW contains not only naphthenic acids (NAs; CnH2n+ZO2),15 but also mono- and
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polyoxygenated compounds, many unidentified species of acids containing sulfur and
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nitrogen atoms, and a variety of polar neutral substances containing oxygen, sulfur, and
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nitrogen.16-18 Initial studies of the toxicity of OSPW suggested that toxicity of OSPW was
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caused by NAs.19 However, it has been argued that these studies implicate a broader group
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of polar organic acids as causative of the toxicity of OSPW.15, 20 Recently, NAs and several
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species of polar neutral compounds containing oxygen and sulfur were identified as causing
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acute lethality.11,12 In another study, a pull-down assay, combined with untargeted chemical
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analysis (termed PUCA) was used to identify ligands of peroxisome proliferator-activated
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receptor γ (PPARγ) from extracts of OSPW.21 However, because oxidative stress could be
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mediated by multiple pathways, rather than one known target, targeted enrichment of
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corresponding toxic components using the PUCA assay is not available.
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Effects-directed analysis (EDA) has been used extensively to identify toxic components
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of complex mixtures.6, 22, 23 In the current study, a high-throughput and reproducible in vitro
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assay and high-resolution EDA were recruited for identification of causative chemicals in
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OSPW. The transcription factor NF-E2-related factor 2 (Nrf2) has been identified as a general
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regulator of cellular defense against oxidative stress through binding to the antioxidant
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response element (ARE) in the upstream promoter region of many genes that are important
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for defense against oxidative stress.24, 25 Elevated levels of ROS or electrophilic species that
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resulted in an altered redox status in cells could trigger the transcriptional response mediated
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by Nrf2.24,
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pTA-NRF2-luciferase reporter vector was used as in vitro monitoring system, in combination
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with in vivo Medaka exposure, and untargeted chemical analysis conducted by Q Exactive
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quadrupole, Orbitrap, to identify chemicals in OSPW that cause oxidative stress.
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In this study, a stable cell line that has been stably transfected with a
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Materials and Methods
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Chemicals and OSPW. Hexane, dichloromethane (DCM), and methanol (MeOH), each of
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HPLC grade, were purchased from Fisher Scientific (Ottawa, ON, Canada). Anhydrous
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ethanol was obtained from GreenField Ethanol Inc (Brampton, ON, Canada). OSPW was
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collected during September of 2012, from Base Mine Lake, which is the first end-pit-lake in
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the surface mining oil sands industry and was constructed from the West-In-Pit settling basin
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that received water from the main extraction facility on the site of Syncrude Canada, Ltd.
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(Fort McMurray, AB, Canada). The OSPW was shipped to the University of Saskatchewan
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(Saskatoon, SK, Canada), stored in the dark, and used for fractionation immediately upon
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arrival.21, 27
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OSPW Fractionation. A 500 mg EVO-LUTE ABN solid phase extraction (SPE) cartridge
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(Biotage, Charlotte, NC, USA) was used for initial extraction and fractionation of organic
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chemicals in the aqueous phase of OSPW. This adsorbent material was used because initial
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experiments showed that this cartridge could capture more constituents of OSPW than did
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HLB cartridges.19 A procedure blank was conducted using ultrapure water, and no significant
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Nrf2 activity was detected. Prior to fractionation, to remove particulate matter, 1 L of OSPW
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was passed through a glass microfiber filter (GF/D 0.47 mm, Whatman) then pH was adjusted
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to 2 by use of concentrated HCl (37%). Cartridges were conditioned with 6 mL of methanol
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and 6 mL of pure water and a 1 L aliquot of filtered and acidified OSPW was passed through
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cartridges, which were subsequently washed with water and allowed to dry under vacuum for
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30 min. Fractionation, based on polarity, was used to separate constituents of OSPW into five
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fractions isolated with successive mixtures of solvents of increasing polarity. Fractions were
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eluted with 6 mL of solvents (volume percentage, v/v) in sequence as follows (Figure S1): F1)
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100% Hexane; F2) 20% DCM in hexane; F3) 50% DCM in hexane; F4) 100% DCM; and F5)
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100% MeOH. Fractions were dried under nitrogen and re-dissolved in 500 µl of ethanol to get
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a final 2000× nominal concentration of original OSPW sample. The distribution of each
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chemical specie across SPE fractions has been described in our previous study.21 Equal
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volumes of the five fractions were pooled to make a reconstituted total extract (TE) of
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dissolved organic chemicals from OSPW. Recoveries of OSPW compounds were determined
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by comparing the relative abundance of species in original OSPW water and OSPW extracts
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eluted from SPE cartridges.
The recoveries were ranged from 30.9% (SO4+) to 143% (O5+).
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Fraction F2 exhibited the greatest potency to cause oxidative stress, so it was selected for
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further fractionation using reversed-phase, high-performance liquid chromatography
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(RP-HPLC). 50 µL of F2 were injected, and fractionation was accomplished by use of a
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Betasil C18 column (5 µm; 2.1 mm×100 mm; Thermo Fisher Scientific). Ultrapure water (A)
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and methanol (B) were used as mobile phases. Initially, 5% B was increased to 30% over 5
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min, then increased to 100% at 20 min and held static for 8 min, followed by a decrease to
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initial conditions of 5% B and held for 2.5 min to allow for column re-equilibration. The flow
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rate was 0.3 mL/min. The column and sample chamber temperatures were maintained at 30
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and 10 oC, respectively. Fractions were collected at 0.5 min intervals obtaining a total of 60
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sub-fractions.
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Nrf2 Bioassay. The Nrf2 Luciferase reporter cell line (Signosis, Santa Clara, CA, USA) was
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stably transfected with the pTA-NRF2-luciferase reporter vector. The amount of luciferase
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produced is directly proportional to the activation of Nrf2 signaling, and thus also
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proportional to the amount of oxidative stress present. The culture medium was Dulbecco’s
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Modified Medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% (v/v) of
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fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 75 mg/L of G418 (Life
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Technologies, Burlington, ON, Canada). The amount of FBS was reduced to 0.1% (v/v) in
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exposure media. Cells were incubated at 37 °C, in a 5% humidified CO2 incubator. Fourth
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generation Nrf2 cells were used for assessing potency of extracts to activate Nrf2. Cells were 7
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seeded to 96-well flat bottom microplates with 5 × 104 in 100 µL per well, and were dosed
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after incubation overnight in exposure medium. A 5-fold dilution of TE or each fraction
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starting from 5× was added to exposure medium to yield a final concentration of solvent of
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0.1% (v/v). Cells were dosed with 0.1% (v/v) of ethanol as the solvent control. Cells were
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exposed to the 60 sub-fractions of F2 at 1× without serial dilution because responses of Nrf2
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were relatively small and the dose-response was linear. To confirm oxidative stress responses
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of exposed cells, rescue experiments were conducted by co-exposing cells to 10 mM of
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reduced glutathione (GSH) (Sigma) with different concentrations of active fractions. Each
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concentration of each sample was conducted with four replicates. Cells were exposed to
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tBHQ (tert-butylhydroquinone, Sigma), which is an reference chemical known to activate
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Nrf2 through two-electron oxidation to form an electrophilic quinone in cells,26, 28 with 3-fold
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serial concentrations ranging from 50 to 0.6 µM. Exposure of 5.6 µM tBHQ was used as
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positive control in each plate. Response of Nrf2 cells to hydrogen peroxide (H2O2, Sigma), a
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well-known reactive oxidant, was also conducted (10 mM – 13.7 µM, with 3-fold dilutions).
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After 16 h of exposure, activity of luciferase was detected by measurement of light produced
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by use of the SteadylitePlus Kit (PerkinElmer, MA, USA).
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Cytotoxicity
of
fractions
was
measured bromide
by
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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Hayward, CA, USA). Cells were plated and exposed the same as in measurements of potency.
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An aliquot of 10 µL of MTT was added per well and incubated for 4 h at 37 °C. Afterward,
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200 µL of DMSO was added to each well for 30 min to dissolve crystals of formazan. The
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OD570 of the supernatant was measured and corrected for background absorbance at 690 nm
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using a POLARStar OPTIMA microplate reader (BMG Labtech, Offenburg, Germany). If
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viability of cells was
