Iodoacetic Acid Activates Nrf2-Mediated ... - ACS Publications

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Iodoacetic Acid Activates Nrf2-Mediated Antioxidant Response in Vitro and in Vivo Shu Wang,†,∥ Weiwei Zheng,†,∥ Xiaolin Liu,† Peng Xue,† Songhui Jiang,† Daru Lu,Δ Qiang Zhang,§ Gensheng He,† Jingbo Pi,⊥ Melvin E. Andersen,§ Hui Tan,*,‡ and Weidong Qu*,† †

Key Laboratory of the Public Health Safety, Ministry of Education, Department of Environmental Health, School of Public Health, Fudan University, Yi Xue Yuan Road 138, Shanghai 200032, China Δ State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China ⊥ Institutes of Toxicology, School of Public Health, China Medical University, Shenyang, Liaoning 110013, China § Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709, United States ‡ Key Laboratory of the Public Health Safety, Ministry of Education, Department of Childhood and Adolescent, Fudan University, Shanghai 200032, China S Supporting Information *

ABSTRACT: Iodoacetic acid (IAA) is an unregulated drinking-water disinfection byproduct with potent cytotoxicity, genotoxicity, and tumorigenicity in animals. Oxidative stress is thought to be essential for IAA toxicity, but the exact mechanism remains unknown. Here we evaluated the toxicity of IAA by examining nuclear factor E2-related factor 2 (Nrf2)mediated antioxidant response, luciferase antioxidant response element (ARE) activity, and intracellular glutathione (GSH) in HepG2 cells. IAA showed significant activation of AREluciferase reporter, mRNA, and protein expression of Nrf2 and its downstream genes (GCLC, NQO1, and HO-1). IAA also increased the intracellular GSH level in HepG2 cells in a timeand concentration-dependent manner. Moreover, we verified IAA induced Nrf2-mediated antioxidant response in rats. Subsequently, we confirmed the specific role of Nrf2 in IAA induced toxicity using NRF2-knockdown cells. Deficiency of NRF2 significantly enhanced sensitivity to IAA toxicity and led to an increase of IAA induced micronulei. We also examined the effects of antioxidant on Nrf2-mediated response in IAA treated cells. Pretreatment with curcumin markedly reduced cytotoxicity and genotoxicity (micronuclei formation) IAA in HepG2 cells. Our work here provides direct evidence that IAA activates Nrf2mediated antioxidant response in vitro and in vivo and that oxidative stress plays a role in IAA toxicity.

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INTRODUCTION Iodoacetic acid (IAA) is an emerging disinfection byproduct (DBP) that forms during chloramine-assisted disinfection of water containing naturally occurring iodide.1 Higher iodide levels occur in source waters from coastal cities (due to salt water intrusion) and some inland locations, whose surface waters contact natural salt deposits from ancient seas or oil-field brines. Chloramine-disinfection would also form IAA in these locations.2 IAA has potent cytotoxicity3 and genotoxicity4,5 and induces malignant transformation of NIH3T3 cells that then show tumorigenic in nude mice.6 The rank order of cytotoxicity and genotoxicity was IAA > bromoacetic acid > chloroacetic acid in CHO,4 human lymphocytes,7 and HepG2 cells.8 Although the exact mechanism of IAA toxicity is unclear, IAA increases reactive oxygen species (ROS), 9 leading to mitochondrial stress and DNA damage.10 Oxidative stress may also be involved in IAA-mediated genotoxicity and mutagenicity.11 © 2014 American Chemical Society

Oxidative stress controlling pathways are essential for maintaining normal cellular biology. Nuclear factor E2-related factor 2 (Nrf2) is a central regulator of oxidative stress from both exogenous and endogenous chemicals.12 Through binding to the antioxidant response elements (AREs), this transcription factor increases levels of cytoprotective genes that encode phase II detoxifying enzymes and antioxidant proteins.13 The induction of these genes is a common cellular response to oxidative stressors. Oxidative stressor or electrophiles, such as carbon tetrachloride,14 arsenic,15 cadmium,16 uncouple Nrf2 from its cytoplasmic chaperone protein Kelch-like ECHassociated protein 1 (Keapl); free Nrf2 translocates to the nucleus, where it dimerizes with members of the small Maf Received: Revised: Accepted: Published: 13478

June 11, 2014 October 21, 2014 October 21, 2014 October 21, 2014 dx.doi.org/10.1021/es502855x | Environ. Sci. Technol. 2014, 48, 13478−13488

Environmental Science & Technology

Article

Acute Cytotoxicity Assay. Cytotoxicity was evaluated using the crystal violet method.6 About 3 × 103 cells per well were plated in 96-well plates, and the media were replaced with fresh media containing different concentrations of IAA (0−16 μM) 24 h later. Cells were then incubated for an additional 24 h (1.5−2 cell cycles). After incubation the medium was aspirated, and the cells were fixed in 100% methanol for 20 min and stained with a 1% crystal violet solution in 50% methanol for 30 min. The wells were then washed, added to 50 μL per well of DMSO, and incubated at room temperature for 30 min. The absorbance values were measured at 595 nm with a BioRad microplate reader. Each experiment was repeated 3 times, and each treatment group had five replicate wells per experiment. Responses were expressed as a percentage of control cells. The data from the repeated experiments were analyzed using the GraphPad Prism 5 software (San Diego, CA). Micronuclei Assay. The micronuclei (MNi) analysis was performed using the cytokinesis-block micronucleus (CBMN) assay.25 The highest concentration to be used in the CBMN assay was at concentrations giving approximately 50% toxicity or less, determined by OECD Guideline 48726 for recommended replicative index (RI), which is defined as

family and binds to AREs within regulatory regions. Binding activates transcription of cell defense genes, including antioxidant enzymes catalase, heme oxygenase1 (HO-1), sulfiredoxin (SRX), and phase II detoxification enzymes such as glutathione S-transferase (GST), NAD(P)H: quinone oxidoreductase 1 (NQO-1), glutathione synthetase (GS), glutathione reductase (GR), glutamate cysteine ligase catalytic subunit (GCLC), and glutamate cysteine ligase modifier subunit (GCLM).17−19 Nrf2-deficient cells and mice show a higher susceptibility to both oxidative damage and chemical carcinogenesis.20−22 In human intestinal epithelial cells (line FHs 74 Int), IAA altered the transcription levels of several oxidative stress responsive genes including thioredoxin reductase 1 (TXNRD1) and sulfiredoxin (SRXN1).23 Because IAA has potent toxicity in bacterial test system and mammalian cells,3,6 it may produce adverse health consequences in exposed human populations though there are few epidemiological studies addressing this question. At present, there are no recommended drinking water criteria for IAA either in WHO or in individual countries. In recent years, alternative toxicity testing strategies based on toxicity mechanisms have gained prominence for assessing the risk of various chemicals using in vitro assays with human cells/celllines without resorting to extensive animal testing.24 These approaches could greatly assist risk/safety assessments with compounds like IAA. While IAA is thought to be a potential oxidative stressor in drinking water, it is unclear whether IAA exposures activate the Nrf2 pathway. In this study we employed HepG2 cells and intact rats to investigate the effect of exposure to IAA on Nrf2 oxidative stress pathway and confirmed the activation of the Nrf2-ARE pathway by IAA. Subsequently, we used NRF2knockdown HepG2 cells and antioxidant to identify the protective role of Nrf2 in IAA induced cytotoxicity and genotoxicity.

no. binucleated cells + 2 × no. multinucleate cells no. total treated cells no. binucleated cells + 2 × no. multinucleate cells no. total control cells

× 100

Logarithmic growth phase HepG2 cells were incubated with DMEM media containing IAA with concentrations selected for genotoxicity assessment and 3 μg/mL cytochalasin-B for 24 h. After washing twice with Hanks’ balanced salt solution, cell cultures were centrifuged and incubated with a prewarmed (37 ± 2 °C) mix of DMEM with water (50:50) to induce a mild hypotonic shock. Then, cells were centrifuged and fixed with carnoy (methanol:acetic acid, 3:1). Drops of the cell suspension were allowed to squash onto wet slides. The slides were stained the next day with acridine orange (20 μg/mL) for 10 min. MNi analysis was performed on coded slides by scoring 2000 binucleated cells for each sample as recommended by OECD Test Guideline 487. Cells containing one or more micronuclei were recorded as micronucleated cells. MNi was identified as follows: ① the diameter of the MNi is less than 1/3 of the diameter of the nucleus, ② the MNi boundary is distinguishable from the nuclear boundary with no overlapping, and ③ the chromatin of the MNi has the same aspect as the nuclear chromatin. Mitomycin C (1 μM concentration) was used as the positive control. Determination of Intracellular ROS. HepG2 cells were incubated with DMEM culture media containing IAA (doses at 0−8 μM). After IAA treatment for 0.5, 1, 2, 4, and 6 h, the medium was removed for ROS determination. The intracellular ROS generation in HepG2 cells was measured by a flow cytometer with the oxidation-sensitive fluorescent probes DCFH-DA.27 The treated cells were rinsed three times with Krebs Ringer buffer at 37 °C and then incubated in the dark with Krebs Ringer buffer containing 10 μM DCFH-DA for 45 min. Fluorescence was measured by FACSCalibur flow cytometry (BD, USA). For each treatment, 10000 cells were counted by CELLQuest software, and the experiment was performed in triplicate. Determination of GSH. HepG2 cells were incubated with DMEM culture media containing IAA (0−8 μM). After 6 h IAA treatment, the medium was removed, and the cells were

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MATERIALS AND METHODS Reagents. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, penicillin, streptomycin, puromycin, and TRIzol reagent were from Invitrogen (Carlsbad, CA). Antibodies for NRF2 (sc-13032), KEAP1 (sc15246), and NQO1 (sc-16463) were from Santa Cruz, Inc. (CA, USA). Antibodies for GCLC (ab55435) and HO-1 (ab13243) were from Abcam (MA, USA). Antibody for β-actin (A1978), cytochalasin-B, 5-sulfosalicylic acid, tert-butylhydroquinone (tBHQ), phenylmethanesulfonyl fluoride, and curcumin were obtained from Sigma (St. Louis, MO). The kits for total glutathione (GSH) quantification were purchased from Dojindo (Kumamoto, Japan). Cell Culture. Human hepatocyte HepG2 cells, purchased from American Type Culture Collection (ATCC, Manassas, VA), were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Animals. Sprague−Dawley (SD) rats were from Shanghai Laboratory Animal Center, Chinese Academy of Science. All rats had free access to food and water under controlled conditions (12/12 h light/dark cycle with humidity of 60% ± 5%, 22 ± 2 °C). The animal use was approved by the Animal Ethics Committee of Fudan University. All procedures were conducted following to the National Ethics Committee for Care and Use of Laboratory Animals for Research. 13479

dx.doi.org/10.1021/es502855x | Environ. Sci. Technol. 2014, 48, 13478−13488

Environmental Science & Technology

Article

Figure 1. (A) Dose−response curve of cytotoxicity induced by IAA in HepG2 cells. Acute (24 h) effects of IAA exposure on HepG2 cells were measured using crystal violet staining method. (B) IAA (0−8 μM) and Cd (10 μM) was loaded for 1 h prior to cell harvest and ROS detection. The DCF mean fluorescent intensity in IAA treated cells was substantially increased. (C) Observation of the time course for ROS in cells treated with IAA. HepG2 cells were incubated with DMEM culture media containing IAA (6 μM). After toxin treatment for 0.5, 1, 2, 4, and 6 h, the medium was removed, and intracellular ROS levels in HepG2 cells were stained with DCFH-DA and analyzed by flow cytometry. Data are shown as mean ± SE, n = 3. *, P < 0.05 vs control.

CA). The resultant DNA-free RNA samples were stored at −80 °C until use. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) was performed as previously described.28 RNA from each sample was diluted in RNase-free H2O and quantified by Nanodrop (Thermo, Wilmington, DE) at 260 nm and reverse-transcribed with Fast Quant RT Kit (TIANGEN Biotech Co., Ltd., China). The SYBR Green PCR Kit (Applied Biosystems) was used for qPCR analysis. The primers (sequences are shown in Tables S1 and S2 of the SI) were designed using Primer Express software 3 (Applied Biosystems, Carlsbad, CA) and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Real-time fluorescence detection was carried out using an Eppendorf qPCR System (Hamburg, Germany). Relative differences in gene expression between groups were determined from cycle threshold (Ct) values. These values were first normalized to 18 rRNA (18S) in the same sample (ΔCt) and expressed as the fold-change over control (2−ΔΔCt). Preparation of Protein Extracts and Western Blot. Isolation of cell fractions and Western blotting were performed as previously detailed.30 Cells for protein immunoblot were treated with IAA (0−8 μM) for 6 h. Cells were washed 3 times with ice-cold phosphate buffer saline, and whole cell extracts were obtained by using cell lysis buffer for Western and IP (Beyotime, Inc., China) with 1 mM phenylmethanesulfonyl fluoride (Sigma). All of the protein fractions were stored at −80 °C until use. Proteins were separated by 4−10% tris-glycine gel (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membranes. Upon blocking with 5% albumin from bovine serum (BSA, Sigma) for 1 h, the blots were probed with the primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Antibody incubations were performed in Tris-buffered saline containing 0.05% Tween 20 (Sigma). Immunoreactive proteins were detected by chemiluminescence using ECL reagent (Amersham Pharmacia, Piscataway, NJ) and subsequently visualized by auto radiography with a Las3000 Luminescent Image Analyzer (Fujifilm, Japan). Confirmation of IAA Induced Nrf2-Mediated Antioxidant Response in Rats. Eight-weeks-old male SD rats were quarantined and acclimatized for an additional week prior to initiation of the experiments. Rats were randomly divided into 4 groups of 10 rats each (between group variance