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Iodoacetic acid activates Nrf2-mediated antioxidant response in vitro and in vivo Shu Wang, Weiwei Zheng, Xianglin Liu, Peng Xue, Songhui Jiang, Daru Lu, Qiang Zhang, Gengsheng He, Jingbo Pi, Melvin Andersen, Hui Tan, and Weidong Qu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es502855x • Publication Date (Web): 21 Oct 2014 Downloaded from http://pubs.acs.org on October 29, 2014
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Iodoacetic acid activates Nrf2-mediated antioxidant response in vitro
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and in vivo
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Shu Wang,†,|| WeiweiZheng,†,|| Xiaolin Liu,† Peng Xue,† Songhui Jiang,† Daru Lu,Δ Qi-
4
ang Zhang, §Gensheng He, † Jingbo Pi,^ Melvin E. Andersen,§Hui Tan,*, ‡ Weidong
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Qu*,†
6 7
† Key Laboratory of the Public Health Safety, Ministry of Education, Department of
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Environmental Health, School of Public Health, Fudan University Shanghai, 200032,
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China.
10
Δ
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan Uni-
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versity, Shanghai 200433, China
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^ Institutes of Toxicology, School of Public Health, China Medical University Shen-
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yang, 110013, China.
14
§
15
Research Triangle Park, North Carolina 27709, USA;
16
‡
Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences,
Key Laboratory of the Public Health Safety, Department of Childhood and adoles-
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cent, Ministry of Education, School of Public Health, Fudan University Shanghai,
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200032, China.
19 20
||
These authors contributed equally to this work.
21 22
*Corresponding author: Weidong Qu, Yi Xue Yuan Road 138, Shanghai 200032, 1
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China. Tel: 86-21-54237203. Fax: 86-21-64045165. E-mail:
[email protected];
24
[email protected] 25 26
Running title: Iodoacetic acid activates Nrf2-mediated antioxidant response
27 28
Competing financial interests declaration: The authors declare they have no actual
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or potential competing financial interests.
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ABREVIATIONS
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ARE, antioxidant response element; cytokinesis-block micronucleus (CBMN); DBPs,
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disinfection byproducts; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's Modified
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Eagle Medium; FBS, fetal bovine serum; GCLC, catalytic subunit of glutamate
49
cysteine lig-ase; HO-1, heme oxygenase 1; IAA, iodoacetic acid; Keap1, Kelch-like
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ECH-associated protein 1; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear
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factor E2-related factor 2; PBS, phosphate buffer saline; SD, Sprague-Dawley;
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U.S.EPA, the United States environment protect agency; WHO, world health
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organization.
54 55 56 57 58 59 60 61 62 63 64 65 66 67 3
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ABSTRACT
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Iodoacetic acid (IAA) is an unregulated drinking-water disinfection by-product with
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potent cytotoxicity, genotoxicity and tumorigenicity in animals. Oxidative stress is
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thought to be essential for IAA toxicity, but the exact mechanism remains unknown.
72
Here we evaluate the toxicity of IAA by examining nuclear factor E2-related factor 2
73
(Nrf2)-mediated antioxidant response, luciferase antioxidant response element (ARE)
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activity and intracellular glutathione in HepG2 cells. IAA showed significant activa-
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tion of ARE-luciferase reporter, mRNA and protein expression of Nrf2 and its down-
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stream genes (GCLC, NQO1 and HO-1). IAA also increased intracellular GSH level
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in HepG2 cells in a time- and concentration-dependent manner. Moreover, we verified
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IAA induced Nrf2- mediated antioxidant response in rats. Subsequently, we con-
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firmed the specific role of Nrf2 in IAA induced toxicity using NRF2-knockdown cells.
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Deficiency of NRF2 significantly enhanced sensitivity to IAA toxicity and led to an
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increase of IAA induced micronulei. We also examined the effects of antioxidant on
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Nrf2-mediated response in IAA treated cells. Pretreatment with curcumin markedly
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reduced cytotoxicity and genotoxicity (MN formation) IAA in HepG2 cells. Our work
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here provides direct evidence that IAA activates Nrf2-mediated antioxidant response
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in vitro and in vivo and that oxidative stress plays a role in IAA toxicity.
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INTRODUCTION
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Iodoacetic acid (IAA) is an emerging disinfection byproduct (DBP) that forms
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during chloramine-assisted disinfection of water containing naturally occurring io-
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dide.1 Higher iodide levels occur in source waters from coastal cities (due to salt wa-
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ter intrusion) and some inland locations, whose surface waters contact natural salt
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deposits from ancient seas or oil-field brines. Chloramine-disinfection would also
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form IAA in these locations.2 IAA has potent cytotoxicity,3 genotoxicity,4-5 and in-
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duces malignant transformation of NIH3T3 cells that then show tumorigenic in nude
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mice.6 The rank order of cytotoxicity and genotoxicity was IAA > bromoacetic acid >
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chloroacetic acid in CHO,4 human lymphocytes7 and HepG2 cells.8 Although the ex-
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act mechanism of IAA toxicity is unclear, IAA increases reactive oxygen species
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(ROS),9 leading to mitochondrial stress and DNA damage.10 Oxidative stress may also
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be involved in IAA-mediated genotoxicity and mutagenicity.11
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Oxidative stress controlling pathways are essential for maintaining normal cellu-
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lar biology. Nuclear factor E2-related factor 2 (Nrf2) is a central regulator of oxida-
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tive stress from both exogenous and endogenous chemicals. 12 Through binding to the
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antioxidant response elements (AREs), this transcription factor increases levels of cy-
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toprotective genes that encode phase II detoxifying enzymes and antioxidant proteins
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13
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Oxidative stressor or electrophiles, such as carbon tetrachloride,14 arsenic,15 cadmi-
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um16
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ECH-associated protein 1 (Keapl), free Nrf2 translocates to the nucleus, where it di-
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merizes with members of the small Maf family and binds to AREs within regulatory
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regions. Binding activates transcription of cell defense genes, including antioxidant
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enzymes catalase, heme oxygenase1(HO-1), sulfiredoxin (SRX), and phase II detoxi-
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fication enzymes such as glutathione S-transferase (GST), NAD(P)H: quinone oxi-
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doreductase 1 (NQO-1), glutathione synthetase (GS), glutathione reductase (GR),
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glutamate cysteine ligase catalytic subunit (GCLC), and glutamate cysteine ligase
The induction of these genes is a common cellular response to oxidative stressors.
uncouple
Nrf2
from
its
cytoplasmic
chaperone
protein
Kelch-like
5
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modifier subunit (GCLM).17-19 Nrf2-deficient cell and mice show a higher susceptibil-
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ity to both oxidative damage and chemical carcinogenesis.20-22 In human intestinal ep-
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ithelial cells (line FHs 74 Int), IAA altered the transcription levels of several oxidative
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stress responsive genes including thioredoxin reductase 1 (TXNRD1) and sulfiredoxin
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(SRXN1).23
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Because IAA has potent toxicity in bacterial test system and mammalian cells,3,6
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it may produce adverse health consequences in exposed human populations though
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there are few epidemiological studies addressing this question. At present, there are
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no recommended drinking water criteria for IAA either in WHO or in individual
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countries. In recent years, alternative toxicity testing strategies based on toxicity
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mechanisms has gained prominence for assessing the risk of various chemicals using
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in vitro assays with human cells/cell-lines without resorting to extensive animal test-
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ing.24 These approaches could greatly assist risk/safety assessments with compounds
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like IAA.
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While IAA is thought to be a potential oxidative stressor in drinking water, , it is
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unclear whether IAA exposures activate the Nrf2 pathway. In this study we employed
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HepG2 cells and intact rats to investigate the effect of exposure to IAA on Nrf2 oxi-
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dative stress pathway and confirmed the activation of the Nrf2-ARE pathway by IAA.
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Subsequently, we used NRF2-knockdown HepG2 cells and antioxidant to identify the
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protective role of Nrf2 in IAA induced cytotoxicity and genotoxicity.
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MATERIALS AND METHODS
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Reagents. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS),
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trypsin-EDTA, penicillin, streptomycin, puromycin and TRIzol reagent were from
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Invitrogen (Carlsbad, CA). Antibodies for NRF2 (sc-13032), KEAP1 (sc-15246) and
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NQO1 (sc-16463) were from Santa Cruz, Inc. (CA, USA). Antibodies for GCLC 6
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(ab55435) and HO-1 (ab13243) were from Abcam (MA, USA). Antibody for β-
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ACTIN (A1978), cytochalasin-B, 5-sulfosalicylic acid, tert-butylhydroquinone
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(tBHQ), phenylmethanesulfonyl fluoride and curcumin were obtained from Sigma (St
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Louis, MO). The kits for total glutathione (GSH) quantification were purchased from
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Dojindo (Kumamoto, Japan).
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Cell Culture.
Human hepatocyte HepG2 cells, purchased from American Type
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Culture Collection (ATCC, Manassas, VA), were cultured in DMEM supplemented
152
with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a hu-
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midified 5% CO2 atmosphere.
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Animals. Sprague-Dawley (SD) rats were from Shanghai Laboratory Animal Center,
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Chinese Academy of Science. All rats had free access to food and water under con-
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trolled conditions (12/12 h light/dark cycle with humidity of 60% ± 5%, 22 ± 2 °C).
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The animal use was approved by the Animal Ethics Committee of Fudan University.
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All procedures were conducted following to the National Ethics Committee for Care
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and Use of Laboratory Animals for Research.
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Acute Cytotoxicity Assay. Cytotoxicity was evaluated using the crystal violet meth-
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od.6 About 3×103 cells per well were plated in 96-well plates and the media were re-
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placed with fresh media containing different concentration of IAA (0−16 μM) 24 h
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later. Cells were then incubated for an additional 24 h (1.5-2 cell cycles). After incu-
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bation the medium was aspirated and the cells were fixed in 100 % methanol for 20
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min and stained with a 1 % crystal violet solution in 50 % methanol for 30 min. The
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wells were then washed, added 50 μL per well of DMSO and incubated at room tem-
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perature for 30 min. The absorbance values were measured at 595 nm with a Bio-Rad
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microplate reader. Each experiment was repeated 3 times, and each treatment group
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had five replicate wells per experiment. Responses were expressed as a percentage of
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control cells. The data from the repeated experiments were analyzed using the
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GraphPad Prism 5 software (San Diego, CA). 7
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Micronuclei Assay. The micronuclei (MNi) analysis was performed using the cyto-
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kinesis-block micronucleus (CBMN) assay.25 The highest concentration to be used in
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the CBMN assay was at concentrations giving approximately 50 % toxicity or less,
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determined by OECD Guideline 487
179
which is defined as:
26
for recommended replicative index (RI),
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(No. binucleated cells + 2 × No. multinucleate cells) / No. total treated cells
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(No. binucleated cells + 2 × No. multinucleate cells) / No. total control cells
×100 183 184
Logarithmic growth phase HepG2 cells were incubated with DMEM media con-
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taining IAA with concentrations selected for genotoxicity assessment and 3 μg/mL
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cytochalasin-B for 24 h. After washing twice with Hanks’ balanced salt solution, cell
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cultures were centrifuged and incubated with a pre-warmed (37±2 ◦C) mix of DMEM
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with water (50:50) to induce a mild hypotonic shock. Then, cells were centrifuged and
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fixed with carnoy (methanol:acetic acid, 3:1). Drops of the cell suspension were al-
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lowed to squash onto wet slides. The slides were stained the next day with acridine
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orange (20 μg/mL) for 10 min. MNi analysis was performed on coded slides by scor-
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ing 2000 binucleated cells for each sample as OECD Test Guideline 487. Cells con-
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taining one or more micronuclei were recorded as micronucleated cells. MNi was
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identified as follows: ① the diameter of the MNi is less than 1/3 of the diameter of
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the nucleus, ② the MNi boundary is distinguishable from the nuclear boundary with
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no overlapping, ③ the chromatin of the MNi has the same aspect as the nuclear
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chromatin. Mitomycin C (1 μM concentration) was used as the positive control.
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Determination of Intracellular ROS. HepG2 cells were incubated with DMEM cul-
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ture media containing IAA (doses at 0−8 μM). After IAA treatment for 0.5, 1, 2, 4 and
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6 h, the medium was removed for ROS determination. The intracellular ROS genera-
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tion in HepG2 cells was measured by a flow cytometer with the oxidation-sensitive
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fluorescent probes DCFH-DA.27 The treated cells were rinsed three times with Krebs 8
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Ringer buffer at 37 °C and then incubated in the dark with Krebs Ringer buffer con-
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taining 10 μM DCFH-DA for 45 min. Fluorescence was measured by FACSCalibur
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flow cytometry (BD, USA). For each treatment, 10000 cells were counted by
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CELLQuest software, and the experiment was performed in triplicate.
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Determination of GSH. HepG2 cells were incubated with DMEM culture media
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containing IAA (0−8 μM). After 6 h IAA treatment, the medium was removed and the
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cells were collected. The total GSH in treated HepG2 cells was measured by the Total
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Glutathione Quantification kit according to the manufacturer’s instruction. The assay
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was designed by using Ellman’s reagent (5, 5’-dithiobis-2-nitrobenzoic acid, DTNB),
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which reacts with GSH to form 2-nitro-5-thiobenzoic acid, a yellow product with a
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maximum absorbance at 412 nm. Protein concentrations determined by bicinchoninic
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acid assay (Beyotime biotechnology, China) were used to normalize the GSH levels.
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ARE-Luciferease Reporter Assay. HepG2 cells were transfected with Cignal Lenti
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ARE reporter lentiviral particles (SABiosciences Frederick, MD, U.S.), expressing a
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luciferase gene driven by multiple ARE (TCACAGTGACTCAGCAAAATT) repeats,
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and this transfection was performed as previously described.28 Cells were grown to
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90 % confluency and subcultured in medium containing 3.5 μg/mL of puromycin. In
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time-response relationship study, HepG2 cells were treated with 6 μM IAA for 0, 2, 6,
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12, 24 h, while in the dose-response relationship study, HepG2 cells were treated with
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different concentration of IAA (0−8 μM) for 6 h. Treatment with 100 μM tBHQ was
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the positive control. The activity of ARE-luciferase reporter was measured by Lucif-
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erase Reporter Assay System (Promega, Madison, WI) according to the manufactur-
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er’s protocol and normalized with cell viability.
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Lentiviral-based shRNA transduction. The NRF2-knockdown HepG2 cells were a
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gift from Jingbo Pi of the Hamner Institutes for Health Sciences. MISSION shRNA
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lentiviral particles were obtained from Sigma. Transduction of HepG2 cells with len-
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tiviral-based shRNAs targeting NRF2 (SHVRS-NM_006164) or scrambled non-target 9
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negative control (SHC002V) was performed as described previously.29 Cells were
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maintained in medium containing 1.0 μg/mL of puromycin.
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To confirm the specific role of Nrf2 in antioxidant response in cellular defense
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against IAA, NRF2-KD and SCR cells were treated with IAA (0−8 μM) for 6 h and
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the expression of NRF2 and ARE-dependent genes, cytotoxicity and genotoxicity
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were compared. In addition, HepG2 cells were pretreated with curcurmin 1 h before
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IAA exposure to observe the effect of pre-activation of Nrf2 on IAA induced cytotox-
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icity and genotoxicity.
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RNA Extraction and RT-qPCR. Total RNA was isolated with TRIzol reagent and
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then subjected to cleanup using RNase-Free DNase Set and RNeasy Mini kit (Qiagen,
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Valencia, CA). The resultant DNA-free RNA samples were stored at −80 °C until use.
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Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) was
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performed as previously described.28 RNA from each sample was diluted in
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RNase-free H2O and quantified by Nanodrop (Thermo, Wilmington, DE) at 260 nm
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and reverse-transcribed with Fast Quant RT Kit (TIANGEN Biotech Co., Ltd, China).
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The SYBR Green PCR Kit (Applied Biosystems) was used for qPCR analysis. The
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primers (sequences are shown in Table S1, S2 of the SI) were designed using Primer
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Express software 3 (Applied Biosystems, Carlsbad, CA) and synthesized by Sangon
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Biotech Co.,Ltd. (Shanghai, China). Real-time fluorescence detection was carried out
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using an Eppendorf qPCR System (Hamburg, Germany). Relative differences in gene
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expression between groups were determined from cycle threshold (Ct) values. These
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values were first normalized to 18 rRNA (18S) in the same sample (ΔCt) and ex-
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pressed as the fold-change over control (2−ΔΔCt).
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Preparation of Protein Extracts and Western Blot. Isolation of cell fractions and
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Western blotting were performed as previously detailed.30 Cells for protein im-
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munoblot were treated with IAA (0−8 μM) for 6 h. Cells were washed 3 times with
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ice-cold phosphate buffer saline and whole cell extracts were obtained by using cell 10
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lysis buffer for Western and IP (Beyotime, Inc., China) with 1 mM phenylme-
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thanesulfonyl fluoride (Sigma). All of the protein fractions were stored at −80 °C until
266
use. Proteins were separated by 4−10 % tris-glycine gel (Invitrogen) and transferred
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onto polyvinylidene fluoride (PVDF) membranes. Upon blocking with 5 % albumin
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from bovine serum (BSA, Sigma) for 1 h, the blots were probed with the primary an-
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tibodies followed by incubation with horseradish peroxidase-conjugated secondary
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antibodies. Antibody incubations were performed in Tris-buffered saline containing
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0.05 % Tween 20 (Sigma). Immunoreactive proteins were detected by chemilumines-
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cence using ECL reagent (Amersham Pharmacia, Piscataway, NJ) and subsequently
273
visualized by auto radiography with a Las3000 Luminescent Image Analyzer (Fuji-
274
film, Japan).
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Confirmation of IAA induced Nrf2-mediated antioxidant response in rats.
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Eight-weeks-old male SD rats were quarantined and acclimatized for an additional
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week prior to initiation of the experiments. Rats were randomly divided into 4 groups
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of 10 rats each (between group variance < 5 %; within-group variance < 10%). IAA
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was administered by gavage with the volume of 0.5 ml/100g body weight after fasting
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overnight, giving doses equivalent to 0, 1, 100, 1000-fold those found in drinking wa-
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ter (2 μg/L31,32). The maximum dose was 0.067 mg/kg body weight/day, equivalent to
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a 60 kg person drinking 2 L water/day. After 24 h, rats were euthanized. Body and
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liver weights were measured and an portion of and liver tissue was harvested for RNA
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extraction and RT-qPCR.
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Statistical Analysis. Data were analyzed using the GraphPad Prism 5 software (San
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Diego, CA). Multiple comparisons with a specific control were assessed by the use of
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one-way analysis of variance (ANOVA) followed by the Bonferroni-t test. Interac-
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tions between IAA and curcumin on HepG2 cells were examined by two-way
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ANOVA. The statistical tests were two-tailed with significance levels of 0.05. Results
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were expressed as mean ±standard error (S.E.). 11
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RESULTS
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1. IAA induced cytotoxicity and ROS formation in HepG2 cells.
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Fig. 1 inserted here.
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IAA significantly reduced the survival rate of HepG2 cells (P < 0.05) in a con-
300
centration dependent manner (Fig. 1A). To ensure that IAA could generate oxidative
301
stress in HepG2 cells, we examined the level of ROS. Exposure to IAA significantly
302
increased fluorescence intensity compared with control when the concentration of
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IAA was greater than 2 μM (Figure 1B). We also examined the time course for ROS
304
in cells treated with 6 μM IAA. IAA initially increased at 0.5 h, peaked at 1 h, with
305
subsequent decreases thereafter (Figure 1C).
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2. IAA induced Nrf2-mediated antioxidant response in vitro.
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Fig. 2 inserted here.
310 311
Time-effect The time-response relationship of IAA induced Nrf2-mediated an-
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tioxidant was examined in HepG2 cells incubated with DMEM culture media con-
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taining 6 μM IAA (ensuring the survival rate was more than 75%). IAA increased the
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Nrf2-mediated antioxidant response effect (Fig. 2). Exposure to 6 μM IAA resulted in
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an initial increase of NRF2 protein expression in HepG2 cells at the first 2−6 h with
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subsequent decrease at 12 h and 24 h (Fig. 2A). A similar time-course was seen for
317
the ARE-luciferase reporter assay (Fig. 2B). IAA also increased NRF2 and its down-
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stream target genes in a time-dependent manner. The expression of NRF2, KEAP1,
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HO-1 and GCLC increased gradually after IAA exposure, peaked at 6 h, and thereaf-
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ter decreased (Fig. 2 C−F), except for gene NQO1 peaked at 12 h (Fig. 2G). All these
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initial results showed that exposure of IAA for 6 h produced maximal responses for
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ARE activation and expression of Nrf2 and its target genes. Thus, the HepG2 cells 12
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were treated with IAA for 6 h in subsequent experiments.
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We also measured the effect of IAA treatment on GSH in HepG2 cells. IAA in-
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creased intracellular GSH level in HepG2 cells in a time-dependent manner. Intracel-
327
lular GSH was significantly greater than control at 2 h, peaking at 12 h. Overall, GSH
328
increased to 1.8 times above background levels, and returned to control by 24 h (Fig.
329
2H).
330 331
Fig. 3 inserted here.
332 333
Dose-response There were clear concentration-dependent changes in all meas-
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ured proteins – NRF2, KEAP1, HO-1, NQO1, and GCLC at the 6 h time point (Fig.
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3A). IAA significantly increased NRF2 and KEAP1 protein expression at concentra-
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tions greater than 2 μM and significantly augmented ARE activity at concentrations
337
greater than 4 μM (Fig. 3B). The positive control tBHQ33 also significantly increased
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the activity of ARE-luciferase reporter. In agreement with the ARE activation, the 6 h
339
exposure to IAA significantly increased mRNA expression of NRF2, KEAP1, and
340
Nrf2 downstream genes, including HO-1, NQO1, and GCLC (Fig. 3 C−G). The low-
341
est concentration of IAA inducing a significant increase of gene expression differed
342
for the various genes – 2 μM for KEAP1 and GCLC, 4 μM for NRF2, HO-1, and 6 μM
343
for NQO1. The high degree of consistency between the Nrf2 activation measured by
344
target gene and protein expression and the ARE-luciferase reporter assay indicated
345
that Nrf2-mediated antioxidant response becomes activated with IAA treatment in
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these HepG2 cells.
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When HepG2 cells were treated with different concentrations of IAA for 6 h, in-
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tracellular GSH levels increased. Four μM IAA increased GSH levels by 1.61-fold (P
350
< 0.05) and 8 μM IAA increased GSH to 1.95-fold (Fig. 3H). In addition, the increas-
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es in GSH induced by different concentrations of IAA were consistent with the ex-
352
pression of GCLC, a key enzyme regulating GSH synthesis and regeneration. 13
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3. IAA induced Nrf2-mediated antioxidant response in primary rat hepatocytes
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and in vivo.
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We also used freshly isolated primary hepatocytes from rat to test the toxicity of
357
IAA (Fig. S1). The results showed that IAA significantly increased ROS levels com-
358
pared with control in a time- and dose-dependent manner, just as observed in the
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HepG2 cells (Fig. S1 C-E). The 6 h exposure to IAA significantly increased protein
360
expression of Nrf2 in primary rat hepatocytes
(Fig. S1 F, G). .
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Next, we measured the effect of IAA on the expression of Nrf2 and its down-
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stream target genes in the liver of SD rats 24-h after dosing with IAA at 0−1000 times
364
of the concentrations found in drinking water. Dosing with IAA in vivo increased
365
protein expression of Nrf2 in the liver of rats (Fig.4A). IAA at 100 times the envi-
366
ronmental levels significantly enhanced gene expression of Nrf2 and Gclc (P < 0.05,
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Fig. 4B, E), and IAA at 1000 times of levels in drinking water significantly increased
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expression of Nrf2,Keap1,Ho-1,Nqo1 and Gclc genes compared with control (P