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

Subscriber access provided by The Library | University of Bath

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

Environmental Science & Technology

1

Iodoacetic acid activates Nrf2-mediated antioxidant response in vitro

2

and in vivo

3

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

5

Qu*,†

6 7

† Key Laboratory of the Public Health Safety, Ministry of Education, Department of

8

Environmental Health, School of Public Health, Fudan University Shanghai, 200032,

9

China.

10

Δ

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan Uni-

11

versity, Shanghai 200433, China

12

^ Institutes of Toxicology, School of Public Health, China Medical University Shen-

13

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-

17

cent, Ministry of Education, School of Public Health, Fudan University Shanghai,

18

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

ACS Paragon Plus Environment


Page 2 of 39

23

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

29

or potential competing financial interests.

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 2


Page 3 of 39


45

ABREVIATIONS

46

ARE, antioxidant response element; cytokinesis-block micronucleus (CBMN); DBPs,

47

disinfection byproducts; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's Modified

48

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

50

ECH-associated protein 1; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear

51

factor E2-related factor 2; PBS, phosphate buffer saline; SD, Sprague-Dawley;

52

U.S.EPA, the United States environment protect agency; WHO, world health

53

organization.

54 55 56 57 58 59 60 61 62 63 64 65 66 67 3



Page 4 of 39

68

ABSTRACT

69

Iodoacetic acid (IAA) is an unregulated drinking-water disinfection by-product with

70

potent cytotoxicity, genotoxicity and tumorigenicity in animals. Oxidative stress is

71

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)

74

activity and intracellular glutathione in HepG2 cells. IAA showed significant activa-

75

tion of ARE-luciferase reporter, mRNA and protein expression of Nrf2 and its down-

76

stream genes (GCLC, NQO1 and HO-1). IAA also increased intracellular GSH level

77

in HepG2 cells in a time- and concentration-dependent manner. Moreover, we verified

78

IAA induced Nrf2- mediated antioxidant response in rats. Subsequently, we con-

79

firmed the specific role of Nrf2 in IAA induced toxicity using NRF2-knockdown cells.

80

Deficiency of NRF2 significantly enhanced sensitivity to IAA toxicity and led to an

81

increase of IAA induced micronulei. We also examined the effects of antioxidant on

82

Nrf2-mediated response in IAA treated cells. Pretreatment with curcumin markedly

83

reduced cytotoxicity and genotoxicity (MN formation) IAA in HepG2 cells. Our work

84

here provides direct evidence that IAA activates Nrf2-mediated antioxidant response

85

in vitro and in vivo and that oxidative stress plays a role in IAA toxicity.

86

4


Page 5 of 39

87


INTRODUCTION

88

Iodoacetic acid (IAA) is an emerging disinfection byproduct (DBP) that forms

89

during chloramine-assisted disinfection of water containing naturally occurring io-

90

dide.1 Higher iodide levels occur in source waters from coastal cities (due to salt wa-

91

ter intrusion) and some inland locations, whose surface waters contact natural salt

92

deposits from ancient seas or oil-field brines. Chloramine-disinfection would also

93

form IAA in these locations.2 IAA has potent cytotoxicity,3 genotoxicity,4-5 and in-

94

duces malignant transformation of NIH3T3 cells that then show tumorigenic in nude

95

mice.6 The rank order of cytotoxicity and genotoxicity was IAA > bromoacetic acid >

96

chloroacetic acid in CHO,4 human lymphocytes7 and HepG2 cells.8 Although the ex-

97

act mechanism of IAA toxicity is unclear, IAA increases reactive oxygen species

98

(ROS),9 leading to mitochondrial stress and DNA damage.10 Oxidative stress may also

99

be involved in IAA-mediated genotoxicity and mutagenicity.11

100 101

Oxidative stress controlling pathways are essential for maintaining normal cellu-

102

lar biology. Nuclear factor E2-related factor 2 (Nrf2) is a central regulator of oxida-

103

tive stress from both exogenous and endogenous chemicals. 12 Through binding to the

104

antioxidant response elements (AREs), this transcription factor increases levels of cy-

105

toprotective genes that encode phase II detoxifying enzymes and antioxidant proteins

106

13

107

Oxidative stressor or electrophiles, such as carbon tetrachloride,14 arsenic,15 cadmi-

108

um16

109

ECH-associated protein 1 (Keapl), free Nrf2 translocates to the nucleus, where it di-

110

merizes with members of the small Maf family and binds to AREs within regulatory

111

regions. Binding activates transcription of cell defense genes, including antioxidant

112

enzymes catalase, heme oxygenase1(HO-1), sulfiredoxin (SRX), and phase II detoxi-

113

fication enzymes such as glutathione S-transferase (GST), NAD(P)H: quinone oxi-

114

doreductase 1 (NQO-1), glutathione synthetase (GS), glutathione reductase (GR),

115

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



Page 6 of 39

116

modifier subunit (GCLM).17-19 Nrf2-deficient cell and mice show a higher susceptibil-

117

ity to both oxidative damage and chemical carcinogenesis.20-22 In human intestinal ep-

118

ithelial cells (line FHs 74 Int), IAA altered the transcription levels of several oxidative

119

stress responsive genes including thioredoxin reductase 1 (TXNRD1) and sulfiredoxin

120

(SRXN1).23

121 122

Because IAA has potent toxicity in bacterial test system and mammalian cells,3,6

123

it may produce adverse health consequences in exposed human populations though

124

there are few epidemiological studies addressing this question. At present, there are

125

no recommended drinking water criteria for IAA either in WHO or in individual

126

countries. In recent years, alternative toxicity testing strategies based on toxicity

127

mechanisms has gained prominence for assessing the risk of various chemicals using

128

in vitro assays with human cells/cell-lines without resorting to extensive animal test-

129

ing.24 These approaches could greatly assist risk/safety assessments with compounds

130

like IAA.

131 132

While IAA is thought to be a potential oxidative stressor in drinking water, , it is

133

unclear whether IAA exposures activate the Nrf2 pathway. In this study we employed

134

HepG2 cells and intact rats to investigate the effect of exposure to IAA on Nrf2 oxi-

135

dative stress pathway and confirmed the activation of the Nrf2-ARE pathway by IAA.

136

Subsequently, we used NRF2-knockdown HepG2 cells and antioxidant to identify the

137

protective role of Nrf2 in IAA induced cytotoxicity and genotoxicity.

138 139

MATERIALS AND METHODS

140

Reagents. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS),

141

trypsin-EDTA, penicillin, streptomycin, puromycin and TRIzol reagent were from

142

Invitrogen (Carlsbad, CA). Antibodies for NRF2 (sc-13032), KEAP1 (sc-15246) and

143

NQO1 (sc-16463) were from Santa Cruz, Inc. (CA, USA). Antibodies for GCLC 6


Page 7 of 39


144

(ab55435) and HO-1 (ab13243) were from Abcam (MA, USA). Antibody for β-

145

ACTIN (A1978), cytochalasin-B, 5-sulfosalicylic acid, tert-butylhydroquinone

146

(tBHQ), phenylmethanesulfonyl fluoride and curcumin were obtained from Sigma (St

147

Louis, MO). The kits for total glutathione (GSH) quantification were purchased from

148

Dojindo (Kumamoto, Japan).

149 150

Cell Culture.

Human hepatocyte HepG2 cells, purchased from American Type

151

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-

153

midified 5% CO2 atmosphere.

154 155

Animals. Sprague-Dawley (SD) rats were from Shanghai Laboratory Animal Center,

156

Chinese Academy of Science. All rats had free access to food and water under con-

157

trolled conditions (12/12 h light/dark cycle with humidity of 60% ± 5%, 22 ± 2 °C).

158

The animal use was approved by the Animal Ethics Committee of Fudan University.

159

All procedures were conducted following to the National Ethics Committee for Care

160

and Use of Laboratory Animals for Research.

161 162

Acute Cytotoxicity Assay. Cytotoxicity was evaluated using the crystal violet meth-

163

od.6 About 3×103 cells per well were plated in 96-well plates and the media were re-

164

placed with fresh media containing different concentration of IAA (0−16 μM) 24 h

165

later. Cells were then incubated for an additional 24 h (1.5-2 cell cycles). After incu-

166

bation the medium was aspirated and the cells were fixed in 100 % methanol for 20

167

min and stained with a 1 % crystal violet solution in 50 % methanol for 30 min. The

168

wells were then washed, added 50 μL per well of DMSO and incubated at room tem-

169

perature for 30 min. The absorbance values were measured at 595 nm with a Bio-Rad

170

microplate reader. Each experiment was repeated 3 times, and each treatment group

171

had five replicate wells per experiment. Responses were expressed as a percentage of

172

control cells. The data from the repeated experiments were analyzed using the

173

GraphPad Prism 5 software (San Diego, CA). 7



Page 8 of 39

174 175

Micronuclei Assay. The micronuclei (MNi) analysis was performed using the cyto-

176

kinesis-block micronucleus (CBMN) assay.25 The highest concentration to be used in

177

the CBMN assay was at concentrations giving approximately 50 % toxicity or less,

178

determined by OECD Guideline 487

179

which is defined as:

26

for recommended replicative index (RI),

180 181

(No. binucleated cells + 2 × No. multinucleate cells) / No. total treated cells

182

(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-

185

taining IAA with concentrations selected for genotoxicity assessment and 3 μg/mL

186

cytochalasin-B for 24 h. After washing twice with Hanks’ balanced salt solution, cell

187

cultures were centrifuged and incubated with a pre-warmed (37±2 ◦C) mix of DMEM

188

with water (50:50) to induce a mild hypotonic shock. Then, cells were centrifuged and

189

fixed with carnoy (methanol:acetic acid, 3:1). Drops of the cell suspension were al-

190

lowed to squash onto wet slides. The slides were stained the next day with acridine

191

orange (20 μg/mL) for 10 min. MNi analysis was performed on coded slides by scor-

192

ing 2000 binucleated cells for each sample as OECD Test Guideline 487. Cells con-

193

taining one or more micronuclei were recorded as micronucleated cells. MNi was

194

identified as follows: ① the diameter of the MNi is less than 1/3 of the diameter of

195

the nucleus, ② the MNi boundary is distinguishable from the nuclear boundary with

196

no overlapping, ③ the chromatin of the MNi has the same aspect as the nuclear

197

chromatin. Mitomycin C (1 μM concentration) was used as the positive control.

198 199

Determination of Intracellular ROS. HepG2 cells were incubated with DMEM cul-

200

ture media containing IAA (doses at 0−8 μM). After IAA treatment for 0.5, 1, 2, 4 and

201

6 h, the medium was removed for ROS determination. The intracellular ROS genera-

202

tion in HepG2 cells was measured by a flow cytometer with the oxidation-sensitive

203

fluorescent probes DCFH-DA.27 The treated cells were rinsed three times with Krebs 8


Page 9 of 39


204

Ringer buffer at 37 °C and then incubated in the dark with Krebs Ringer buffer con-

205

taining 10 μM DCFH-DA for 45 min. Fluorescence was measured by FACSCalibur

206

flow cytometry (BD, USA). For each treatment, 10000 cells were counted by

207

CELLQuest software, and the experiment was performed in triplicate.

208 209

Determination of GSH. HepG2 cells were incubated with DMEM culture media

210

containing IAA (0−8 μM). After 6 h IAA treatment, the medium was removed and the

211

cells were collected. The total GSH in treated HepG2 cells was measured by the Total

212

Glutathione Quantification kit according to the manufacturer’s instruction. The assay

213

was designed by using Ellman’s reagent (5, 5’-dithiobis-2-nitrobenzoic acid, DTNB),

214

which reacts with GSH to form 2-nitro-5-thiobenzoic acid, a yellow product with a

215

maximum absorbance at 412 nm. Protein concentrations determined by bicinchoninic

216

acid assay (Beyotime biotechnology, China) were used to normalize the GSH levels.

217 218

ARE-Luciferease Reporter Assay. HepG2 cells were transfected with Cignal Lenti

219

ARE reporter lentiviral particles (SABiosciences Frederick, MD, U.S.), expressing a

220

luciferase gene driven by multiple ARE (TCACAGTGACTCAGCAAAATT) repeats,

221

and this transfection was performed as previously described.28 Cells were grown to

222

90 % confluency and subcultured in medium containing 3.5 μg/mL of puromycin. In

223

time-response relationship study, HepG2 cells were treated with 6 μM IAA for 0, 2, 6,

224

12, 24 h, while in the dose-response relationship study, HepG2 cells were treated with

225

different concentration of IAA (0−8 μM) for 6 h. Treatment with 100 μM tBHQ was

226

the positive control. The activity of ARE-luciferase reporter was measured by Lucif-

227

erase Reporter Assay System (Promega, Madison, WI) according to the manufactur-

228

er’s protocol and normalized with cell viability.

229 230

Lentiviral-based shRNA transduction. The NRF2-knockdown HepG2 cells were a

231

gift from Jingbo Pi of the Hamner Institutes for Health Sciences. MISSION shRNA

232

lentiviral particles were obtained from Sigma. Transduction of HepG2 cells with len-

233

tiviral-based shRNAs targeting NRF2 (SHVRS-NM_006164) or scrambled non-target 9



Page 10 of 39

234

negative control (SHC002V) was performed as described previously.29 Cells were

235

maintained in medium containing 1.0 μg/mL of puromycin.

236 237

To confirm the specific role of Nrf2 in antioxidant response in cellular defense

238

against IAA, NRF2-KD and SCR cells were treated with IAA (0−8 μM) for 6 h and

239

the expression of NRF2 and ARE-dependent genes, cytotoxicity and genotoxicity

240

were compared. In addition, HepG2 cells were pretreated with curcurmin 1 h before

241

IAA exposure to observe the effect of pre-activation of Nrf2 on IAA induced cytotox-

242

icity and genotoxicity.

243 244

RNA Extraction and RT-qPCR. Total RNA was isolated with TRIzol reagent and

245

then subjected to cleanup using RNase-Free DNase Set and RNeasy Mini kit (Qiagen,

246

Valencia, CA). The resultant DNA-free RNA samples were stored at −80 °C until use.

247

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) was

248

performed as previously described.28 RNA from each sample was diluted in

249

RNase-free H2O and quantified by Nanodrop (Thermo, Wilmington, DE) at 260 nm

250

and reverse-transcribed with Fast Quant RT Kit (TIANGEN Biotech Co., Ltd, China).

251

The SYBR Green PCR Kit (Applied Biosystems) was used for qPCR analysis. The

252

primers (sequences are shown in Table S1, S2 of the SI) were designed using Primer

253

Express software 3 (Applied Biosystems, Carlsbad, CA) and synthesized by Sangon

254

Biotech Co.,Ltd. (Shanghai, China). Real-time fluorescence detection was carried out

255

using an Eppendorf qPCR System (Hamburg, Germany). Relative differences in gene

256

expression between groups were determined from cycle threshold (Ct) values. These

257

values were first normalized to 18 rRNA (18S) in the same sample (ΔCt) and ex-

258

pressed as the fold-change over control (2−ΔΔCt).

259 260

Preparation of Protein Extracts and Western Blot. Isolation of cell fractions and

261

Western blotting were performed as previously detailed.30 Cells for protein im-

262

munoblot were treated with IAA (0−8 μM) for 6 h. Cells were washed 3 times with

263

ice-cold phosphate buffer saline and whole cell extracts were obtained by using cell 10


Page 11 of 39


264

lysis buffer for Western and IP (Beyotime, Inc., China) with 1 mM phenylme-

265

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

267

onto polyvinylidene fluoride (PVDF) membranes. Upon blocking with 5 % albumin

268

from bovine serum (BSA, Sigma) for 1 h, the blots were probed with the primary an-

269

tibodies followed by incubation with horseradish peroxidase-conjugated secondary

270

antibodies. Antibody incubations were performed in Tris-buffered saline containing

271

0.05 % Tween 20 (Sigma). Immunoreactive proteins were detected by chemilumines-

272

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).

275 276

Confirmation of IAA induced Nrf2-mediated antioxidant response in rats.

277

Eight-weeks-old male SD rats were quarantined and acclimatized for an additional

278

week prior to initiation of the experiments. Rats were randomly divided into 4 groups

279

of 10 rats each (between group variance < 5 %; within-group variance < 10%). IAA

280

was administered by gavage with the volume of 0.5 ml/100g body weight after fasting

281

overnight, giving doses equivalent to 0, 1, 100, 1000-fold those found in drinking wa-

282

ter (2 μg/L31,32). The maximum dose was 0.067 mg/kg body weight/day, equivalent to

283

a 60 kg person drinking 2 L water/day. After 24 h, rats were euthanized. Body and

284

liver weights were measured and an portion of and liver tissue was harvested for RNA

285

extraction and RT-qPCR.

286 287

Statistical Analysis. Data were analyzed using the GraphPad Prism 5 software (San

288

Diego, CA). Multiple comparisons with a specific control were assessed by the use of

289

one-way analysis of variance (ANOVA) followed by the Bonferroni-t test. Interac-

290

tions between IAA and curcumin on HepG2 cells were examined by two-way

291

ANOVA. The statistical tests were two-tailed with significance levels of 0.05. Results

292

were expressed as mean ±standard error (S.E.). 11



Page 12 of 39

293 294

RESULTS

295

1. IAA induced cytotoxicity and ROS formation in HepG2 cells.

296 297

Fig. 1 inserted here.

298 299

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

303

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).

306 307

2. IAA induced Nrf2-mediated antioxidant response in vitro.

308 309


310 311

Time-effect The time-response relationship of IAA induced Nrf2-mediated an-

312

tioxidant was examined in HepG2 cells incubated with DMEM culture media con-

313

taining 6 μM IAA (ensuring the survival rate was more than 75%). IAA increased the

314

Nrf2-mediated antioxidant response effect (Fig. 2). Exposure to 6 μM IAA resulted in

315

an initial increase of NRF2 protein expression in HepG2 cells at the first 2−6 h with

316

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-

318

stream target genes in a time-dependent manner. The expression of NRF2, KEAP1,

319

HO-1 and GCLC increased gradually after IAA exposure, peaked at 6 h, and thereaf-

320

ter decreased (Fig. 2 C−F), except for gene NQO1 peaked at 12 h (Fig. 2G). All these

321

initial results showed that exposure of IAA for 6 h produced maximal responses for

322

ARE activation and expression of Nrf2 and its target genes. Thus, the HepG2 cells 12


Page 13 of 39

323


were treated with IAA for 6 h in subsequent experiments.

324 325

We also measured the effect of IAA treatment on GSH in HepG2 cells. IAA in-

326

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


332 333

Dose-response There were clear concentration-dependent changes in all meas-

334

ured proteins – NRF2, KEAP1, HO-1, NQO1, and GCLC at the 6 h time point (Fig.

335

3A). IAA significantly increased NRF2 and KEAP1 protein expression at concentra-

336

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

338

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

346

these HepG2 cells.

347 348

When HepG2 cells were treated with different concentrations of IAA for 6 h, in-

349

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-

351

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



Page 14 of 39

353 354

3. IAA induced Nrf2-mediated antioxidant response in primary rat hepatocytes

355

and in vivo.

356

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

359

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). .

361 362

Next, we measured the effect of IAA on the expression of Nrf2 and its down-

363

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,

367

Fig. 4B, E), and IAA at 1000 times of levels in drinking water significantly increased

368

expression of Nrf2，Keap1，Ho-1，Nqo1 and Gclc genes compared with control (P

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

Recommend Documents