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Ecotoxicology and Human Environmental Health

Personal ozone exposure and respiratory inflammatory response: the role of DNA methylation in the arginase–nitric oxide synthase pathway Yue Niu, Renjie Chen, Yongjie Xia, Jing Cai, Zhijing Lin, Cong Liu, Chen Chen, Li Peng, Zhuohui Zhao, Wen-Hao Zhou, Jianmin Chen, and Haidong Kan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01295 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Environmental Science & Technology

Personal ozone exposure and respiratory inflammatory response: the role of DNA methylation in the arginase–nitric oxide synthase pathway

Yue Niu1,†, Renjie Chen1,2,†, Yongjie Xia1, Jing Cai1, Zhijing Lin1, Cong Liu1, Chen Chen1, Li Peng2, Zhuohui Zhao1, Wenhao Zhou3, Jianmin Chen4, Haidong Kan1,5,*

†

These authors contributed equally to this work.

1

School of Public Health, Key Lab of Public Health Safety of the Ministry of

Education and NHC Key Laboratory of Health Technology Assessment, Fudan University, Shanghai 200032, China; 2

Shanghai Key Laboratory of Meteorology and Health, Shanghai 200030,

China; 3

Department of Neonates, Children's Hospital, Fudan University, Shanghai

201102, China; 4

Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention

(LAP3), Department of Environmental Science and Engineering, Fudan University, Shanghai, 200433, China; 5

Key Laboratory of Reproduction Regulation of National Population and

Family Planning Commission, Shanghai Institute of Planned Parenthood Research, Institute of Reproduction and Development, Fudan University,

ACS Paragon Plus Environment


Shanghai 200032, China.

*Correspondence: Haidong Kan, Department of Environmental Health, School of Public Health, Fudan University, P.O. Box 249, 130 Dong-An Road, Shanghai 200032, China. E-mail: [email protected].


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Abstract (200 words)

2

Little is known regarding the molecular mechanisms behind respiratory

3

inflammatory response induced by ozone. We performed a longitudinal panel

4

study with four repeated measurements among 43 young adults in Shanghai,

5

China from May to October in 2016. We collected buccal samples and

6

measured the fractional exhaled nitric oxide (FeNO) after 3-day personal

7

ozone monitoring. In buccal samples, we measured concentrations of

8

inducible nitric oxide synthase (iNOS) and arginase (ARG), and DNA

9

methylation of NOS2A and ARG2. We used linear mixed-effect models to

10

analyze the effects of ozone on FeNO, two enzymes and their DNA

11

methylation. A 10 ppb increase in ozone (lag 0−8 h) was significantly

12

associated with a 3.89% increase in FeNO, a 36.33% increase in iNOS, and a

13

decrease of 0.36 in the average methylation (%5mC) of NOS2A. Ozone was

14

associated with decreased ARG and elevated ARG2 methylation, but the

15

associations were not significant. These effects were more pronounced among

16

allergic subjects than healthy subjects. The effects were much stronger when

17

using personal exposure monitoring than fixed-site measurements. Our study

18

demonstrated that personal short-term exposure to ozone may result in acute

19

respiratory inflammation, which may be mainly modulated by NOS2A

20

hypomethylation in the arginase–nitric oxide synthase pathway.

21

Key words: ozone; personal exposure; exhaled nitric oxide; respiratory

22

inflammation; DNA methylation



23

1 Introduction

24

Ambient ozone is the main component of photochemical air pollution, and

25

it frequently exceeds the health-based standard worldwide, especially in warm

26

or hot seasons.1 Ozone air pollution arouses increasing public health concerns

27

in recent decades because of accelerated urban motorization. Except for

28

particulate matter with aerodynamic diameter less than 2.5 µm (PM2.5), ozone

29

was the sole air pollutant considered as a risk factor in the Global Burden of

30

Disease Study due to its independent effects on respiratory mortality in the

31

well-known American Cancer Society Cohort Study.2 For the short-term

32

exposure, a large body of epidemiological studies have also shown effects of

33

ozone on various clinical and subclinical outcomes in the respiratory system.3

34

Inflammatory response was central in the pathophysiologic process

35

whereby airborne toxicants damage the respiratory tract.4, 5 Epidemiological

36

studies have associated ozone exposure with elevated levels of the fractional

37

exhaled nitric oxide (FeNO). It is a well-established noninvasive marker of

38

respiratory inflammation,6, 7 and was strongly recommended by the American

39

Thoracic Society Committee.8 However, the underlying mechanisms behind

40

this association remain to be elucidated. The exhaled nitric oxide (NO) is

41

mainly synthesized from L-arginine by inducible nitric oxide synthase (iNOS,

42

mainly encoded by NOS2A in the respiratory tract), which can be inhibited by

43

the arginase (ARG, mainly encoded by ARG2 in the respiratory tract) by

44

competing for a common substrate.9 Consequently, the arginase–nitric oxide


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synthase pathway represents an important mechanism for human respiratory

46

inflammation. Epigenetics, such as DNA methylation, may serve as a bridge

47

between airborne toxicants and adverse health outcomes.10, 11 In most cases,

48

hypermethylation in promoter regions of genes can down-regulate the levels of

49

protein by suppressing gene expression, and hypomethylation can up-regulate

50

the levels.12 However, little is known regarding whether NOS2A and ARG2

51

methylation modulates respiratory inflammatory response induced by ambient

52

ozone pollution.

53

Therefore, we performed a longitudinal panel study to evaluate the roles

54

of NOS2A and ARG2 methylation in respiratory inflammatory response

55

induced by ambient ozone pollution among a group of allergic and healthy

56

subjects. Considering the large spatial-temporal variations in ozone

57

concentrations, its exposure was directly monitored at the individual level.

58

2 Methods

59

2.1 Subjects and Study Design

60

A longitudinal panel study with 4 follow-ups was conducted in Shanghai,

61

China, from May 29 to October 12, 2016. We initially recruited 43 nonsmoking

62

college students from the medical (Fenglin) campus of Fudan University. Three

63

asthmatic patients were excluded to avoid possible influences of asthmatic

64

attacks on our findings. We finally included19 allergic and 21 healthy subjects

65

in this study. The allergic subjects were defined as those who have a history of

66

respiratory allergy but not receive any medication treatments (e.g., intranasal



67

corticosteroids and oral antihistamines) at enrollment. The allergic status was

68

determined by whether having a self-reported physician-diagnosed allergic

69

disease (i.e., allergic rhinitis, atopy, or both) and whether having allergic

70

symptoms according to a modified questionnaire based on the core

71

questionnaires of the International Study of Asthma and Allergies in Childhood.

72

Eligible participants were required to abstain from any alcoholic beverage,

73

medications and dietary supplements during the study period.

74

Four follow-ups were arranged during the periods from May 29 to June 22,

75

June 28 to July 23, July 31 to August 31, and August 31 to October 12,

76

respectively. In each round of follow-ups, we randomly divided these

77

participants into 6 subgroups (6–8 participants for each) and scheduled

78

follow-ups at different days to expand natural variations in the measurements

79

of exposure-response data. For each follow-up, physical examinations were

80

arranged at the same daytime (between 4:00 p.m. and 5:00 p.m.) immediately

81

after a consecutive 3-day (from 8:00 a.m. to 6:00 p.m.) personal ozone

82

monitoring to control for possible circadian rhythms. At baseline, demographic

83

characteristics, including age, gender, height and weight, were collected

84

through a general questionnaire. During the study period, participants were

85

asked to record whether they were experiencing a disease (for example, a

86

cold), and whether they were suffering any allergic symptoms through a

87

symptom questionnaire.

88

The Institutional Review Board of the School of Public Health, Fudan


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University approved the study protocol (NO. 2014-07-0523), and all

90

participants signed the informed consent at the enrollment.

91

2.2 Exposure Measurements

92

Personal exposure to ozone was measured in real time by the Personal

93

Ozone Monitor (POM, 2B Technologies, Boulder, Colorado, USA) base on the

94

method of ultraviolet absorption. This device showed strong inter-unit

95

precisions and good agreements with a fixed-site ozone analyzer (Model 49i,

96

Thermo Fisher Scientific Inc., MA, USA).13 For each round of 3-day exposure

97

measurement, participants were instructed to carry a POM during daytime only

98

(from 8:00 a.m. to 6:00 p.m.). The quality assurance and quality control

99

procedures were strictly followed when conducting personal ozone monitoring.

100

More details about personal monitoring can be found in our recent

101

publication.13 We calculated the maximum 8-h (from 10:00 a.m. to 6:00 p.m.)

102

average concentrations as the daily mean concentrations and numbered them

103

as lag 1 d (the previous day) and lag 2 d (the previous 2 day), respectively. We

104

also averaged the concentrations of 0−2 h, 3−5 h, 6−8 h and 0−8 h (lag 0 d)

105

preceding physical examinations. In addition, we placed a HOBO data logger

106

(Onset Computer Corporation, Pocasset, Massachusetts, USA) to measure

107

personal temperature and relative humidity in real time.

108

To allow for a comparison with measurements from fixed-site monitoring,

109

we obtained hourly ozone concentrations from the nearest state-controlled

110

monitoring station, which is about 3 kilometers away from the campus. They



111

were measured by the method of ultraviolet absorption. To allow for the

112

adjustment of other air pollutants in a sensitivity analysis, we also collected

113

daily concentrations of PM2.5, sulfur dioxide (SO2), nitrogen dioxide (NO2), and

114

carbon monoxide (CO) from the same monitoring station.

115

2.3 Physical Examinations and Lab Analyses

116

FeNO levels were tested by a trained staff using a portable NIOX MINO

117

machine (Aerocrine AB, Solna, Sweden) according to the standardized

118

procedures proposed by the American Thoracic Society and the European

119

Respiratory Society.14 Briefly, all subjects inhaled NO-free air through the filter

120

(built-in NO scrubbers) to close to total lung capacity and exhaled air at a flow

121

rate of 50 ± 5 ml/s into the measurement chamber containing the sensor.

122

Foods, beverages and intense exercises were not allowed at least within 1 h

123

before the test.

124

Buccal samples were collected after FeNO tests using Isohelix

125

SK-1S/MS-01 Buccal Swabs (Cell Projects, Kent, UK). Specifically, subjects

126

were requested to rinse the mouth using purified water before sampling. Then,

127

they were instructed to rub firmly against the inside of their cheeks with sterile

128

swabs for 1 minute. Upon completion, the swabs were immediately placed into

129

microcentrifuge tubes containing 1200 µL of phosphate buffer solution and the

130

buccal-cell suspensions were frozen at -80℃ till use.

131

The levels of iNOS and ARG protein in buccal samples were detected by

132

the method of simple Western Blot. All procedures were performed with


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manufacturers reagents (Proteinsimplem, San Jose, California, USA)

134

according to the user manual. Briefly, buccal samples were mixed with

135

fluorescent master mix prior to tests. Then, prepared samples, blocking

136

reagent,

137

substrate and wash buffer were dispensed into designated wells in the

138

manufacturer-provided microplate. The separation and immunodetection were

139

performed automatically using default settings. The level of protein was

140

analyzed by the inbuilt Compass software and was expressed as a ratio

141

relative to a reference protein (β-actin).

primary

antibodies,

secondary

antibodies,

chemiluminescent

142

Genomic DNA was extracted from buccal samples using the QIAmp DNA

143

Mini Kit (Qiagen, Hilden, Germany) and was quantified using a NanoDrop

144

spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). The

145

extracted DNA (2 µg) were bisulfite converted and purified using the EpiTect

146

Fast DNA Bisulfite Kit (Qiagen, Hilden, Germany) according to manufacturer’s

147

recommended protocol. The purified DNA were eluted in Buffer EB solution

148

and stored at -80℃ for DNA methylation analysis. We selected 3 CpG loci in

149

the promoter region of NOS2A gene and 3 CpG loci in the promoter region of

150

ARG2 gene according to a previous study (see Table S1).9 Polymerase chain

151

reaction (PCR) primers were designed for the targeted gene regions using the

152

Pyromark Assay Design SW 2.0 Software (Qiagen, Hilden, Germany).

153

Methylation levels of each targeted CpG were examined by the methods of

154

bisulfite-PCR

pyrosequencing

assay

using

the


Pyromark

Q96

MD


155

pyrosequencing instrument (Qiagen, Hilden, Germany) following standard

156

protocols. The output from pyrosequencing was expressed as the proportion of

157

methylated cytosines over the sum of methylated and unmethylated cytosines,

158

that is, the percentage of 5-methylcytosine (%5mC).

159

2.4 Statistical Analyses

160

Exposure and health data were merged by the time of physical

161

examinations (rounded to the integer hour). We applied the linear mixed-effect

162

(LME) model to analyze the associations of ozone exposure with FeNO, two

163

enzymes and their DNA methylation. This model automatically allows each

164

subject to serve as his or her own control over time by introducing a

165

random-effect intercept, which reflect each individual's baseline.15 The levels

166

of FeNO, iNOS and ARG were natural log-transformed before statistical

167

analyses because of their almost log-normal distributions, while methylation

168

data was directly entered the models because they were normally distributed.

169

In the main model, personal ozone concentration was introduced as a

170

fixed-effect term and an identity number for each subject was added as a

171

random-effect intercept to account for correlations among multiple repeated

172

measurements per person. We also included several covariates in the main

173

models: (1) a natural cubic smooth function of the day within the study with 3

174

degrees of freedom to account for any unmeasured time trends; (2) personal

175

temperature and relative humidity that were simultaneously measured to

176

control for the potential confounding effects of weather conditions;16 (3)


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individual characteristics, including age, gender, body mass index (BMI),

178

allergic status (as a binary variable) and whether having a cold during 3-day

179

follow-up; and (4) the plate number (only for the methylation data). Various

180

lags of exposure were introduced into the main model one at a time to

181

investigate the lag pattern in the effects of ozone. We further performed

182

stratification analysis by allergic status using the afore-mentioned main models

183

to compare respiratory response to ozone in allergic and healthy adults.

184

In addition, we reran the main models using the ozone concentrations

185

measured by the nearest fixed-site monitor in place of personal exposure. We

186

calculated the marginal R square (RM2) and the Akaike Information Criterion

187

(AIC) values to compare the model fit. Higher RM2 and lower AIC mean better

188

model fit.

189

At last, we performed a sensitivity analysis to evaluate whether our results

190

were confounded by the simultaneous exposure to other air pollutants. We fit

191

multiple two-pollutant models by introducing the present-day concentrations of

192

PM2.5, SO2, NO2 and CO into the main model one at a time.

193

All models were ﬁtted in R software (V.3.4.0, R Foundation for Statistical

194

Computing, Vienna, Austria) with the LME using “lme4” package. p-values less

195

than 0.05 were considered statistically signiﬁcant. The estimated effects on

196

FeNO, iNOS and ARG were presented as the percent changes and their 95%

197

confidence intervals (CIs) associated with a 10 ppb increase in ozone

198

concentrations. The estimated effects on NOS2A and ARG2 methylation were



199

presented as the absolute changes and their 95% CIs in %5mC associated

200

with a 10 ppb increase in ozone concentrations.

201 202

3 Results

203

3.1 Descriptive statistics

204

We finally included 40 participants (30 females and 10 males) into analyses,

205

with a mean age of 24 years and an average BMI of 21 kg/m2. Twenty-seven

206

participants completed scheduled 4 rounds of follow-ups. Eleven participants

207

missed one follow-up due to personal reasons (e.g., schedule conflict). Two

208

participants missed 2 rounds of follow-ups because they declined frequent

209

physical examinations. They all declared no use of alcohol, medications and

210

dietary supplements and no suffering from apparent allergic symptoms during

211

the study period.

212

As shown in Table 1, during the study period, the average ozone exposure

213

at the individual level varied from 18 ± 20 ppb to 23 ± 17 ppb by different lag

214

periods. Meanwhile, ozone concentrations measured by the nearest fixed-site

215

monitor were almost twice as high as personal measurements, with an

216

average of 46 ± 18 ppb, which is close to the guideline recommended by the

217

World Health Organization (100 µg/m3, 47 ppb), but is below National Ambient

218

Air Quality Standard of the United States (70 ppb). The correlations between

219

personal ozone exposures and ambient ozone concentrations as well as the

220

distributions of correlations have been described in detail in our recent


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publication.13

222

In total, we collected 145 FeNO measurements and 145 buccal samples.

223

As summarized in Table 1, FeNO measurements varied appreciably with a

224

mean of 11 ± 7 ppb. The levels of iNOS and ARG relative to β-actin varied

225

considerably and the averages were 0.3 ± 0.8 and 20.6 ± 32.5, respectively.

226

Because methylation levels at 3 loci in both genes were highly correlated, we

227

averaged them before entering statistical analyses. In general, the NOS2A

228

was heavily methylated [73.83 ± 11.51 on average (%5mC)] and ARG2 was

229

mildly methylated [2.88 ± 1.95 on average (%5mC)].

230

3.2 Regression results

231

Table 2 shows the changes in FeNO, iNOS protein, ARG protein, and

232

DNA methylation of NOS2A and ARG2 associated with ozone exposure at

233

different lag periods. We observed that FeNO increased immediately after two

234

hours of ozone exposure, then attenuated at longer lag hours. iNOS

235

significantly increased from lags 2 h to 8 h. Similarly, NOS2A methylation

236

showed a significantly inverse association with ozone from lags 2 h to 8 h.

237

Ozone exposure was inversely associated with ARG and positively associated

238

with ARG2 methylation, but the associations were not significant. The

239

associations are somewhat larger when using lag 0-8 h than lag 1 d and 2 d.

240

Therefore, we used the lags 0 to 8 h in subsequent analyses. At this specific

241

lag period, an increase of 10 ppb in ozone was associated with a 3.89% (95%

242

CI: 0.45%, 7.46%) increase in FeNO, a 36.33% (95% CI: 11.04%, 67.37%)



243

increase in iNOS, a 7.15% (95% CI: -9.43%, 21.22%) decrease in ARG, a

244

decrease of 0.36 (95% CI: 0.06, 0.67) in the average methylation (%5mC) of

245

NOS2A gene, and an increase of 0.18 (95% CI: -0.11, 0.48) in the average

246

methylation (%5mC) of ARG2 gene.

247

In stratification analysis (see Table 3), the increment in FeNO was larger

248

among allergic subjects than among healthy subjects. Accordingly, the positive

249

association with iNOS and the negative association with NOS2A methylation

250

remained significant among allergic subgroup. Notably, there was a positive

251

but non-significant association with iNOS and a negative but non-significant

252

association with NOS2A methylation in the healthy subgroup. The effects of

253

ozone on ARG and ARG2 methylation were not statistically significant in both

254

subgroups.

255

As shown in Table 4, the use of personal measurements yielded much

256

larger effect estimates than the use of fixed-site measurements at the lag 0−8

257

h. Meanwhile, the models using personal measurements had an average of

258

9.6% higher RM2 and 4.6% lower AIC values than the models using fixed-site

259

measurements.

260

In the sensitivity analysis, the associations of ozone with FeNO, iNOS and

261

NOS2A methylation were not appreciably changed after controlling for other

262

air pollutants in two-pollutant models (see Table S2–S5).

263 264

4 Discussion


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The present investigation has the advantage of using personal real-time

266

monitoring of ozone in a repeated panel study, which allows for an accurate

267

assessment on its respiratory effects. We found that a short-term exposure to

268

ozone could induce an acute increase in FeNO levels. This increment was

269

accompanied by alterations in DNA methylation in the arginase–nitric oxide

270

synthase pathway that leads to the production of NO. We further found much

271

stronger effects of ozone using personal than fixed-site measurements. To our

272

knowledge, this is the first epidemiological study to explore the potential

273

molecular biological mechanisms underlying the respiratory inflammation

274

induced by ambient ozone exposure.

275

In the present study, we found acute respiratory inflammatory response

276

represented by increased FeNO immediately after hours’ exposure to low-level

277

ozone, which was consistent with the broad literatures. For example, a panel

278

study in Changsha, China, estimated that 24-h ozone exposure levels for

279

white-collar workers ranged from 1.4 ppb to 19.4 ppb. Within this range, they

280

found per 10 ppb increase in ozone was associated with a 18.1% (95% CI:

281

4.5%, 33.5%) increase in FeNO.17 A panel study in Greece also showed that a

282

very low personal exposure to ozone (5–11 µg/m3) may result in a significant

283

increase in FeNO.18 Likewise, a longitudinal study based on fixed-site

284

monitoring observed a significant increase in FeNO following exposure to low

285

levels of ambient ozone (31.6 ppb).19

286

The molecular biological pathway whereby an inhalation of ozone induces



287

the production of NO remained unknown. The arginase–nitric oxide synthase

288

pathway was considered biologically responsible for the NO production, and

289

there was a potential that DNA methylation may modulate this pathway.20 Our

290

results indicated that a short-term exposure to ozone resulted in an immediate

291

decrease in NOS2A methylation and an increase in iNOS protein. The

292

hypothesized pathway is that oxidative stress induced by ozone inhalation may

293

cause DNA lesions, subsequently lead to NOS2A hypomethylation by

294

interfering DNA methyltransferases binding to damaged DNA,21 and further

295

enhance the expression of iNOS.12 Although no studies to date have directly

296

evaluated the influences of ozone on NOS2A methylation, similar associations

297

have been established between particulate air pollution and NOS2A

298

hypomethylation.10, 11, 22 These findings provided the biological plausibility that

299

NOS2A hypomethylation may modulate the process of NO production

300

stimulated by an acute ozone exposure.

301

ARG is another important enzyme that inhibits the function of iNOS in the

302

arginase–nitric oxide synthase pathway. Theoretically, low expression of ARG

303

protein caused by hypermethylation of ARG2 may ensure a sufficient

304

L-arginine supply by reducing the hydrolysis process, resulting in accelerated

305

production of NO.20 Few studies have explored the role of ARG or its DNA

306

methylation in elevated FeNO levels by air pollutants. In the present study, we

307

observed inverse associations of ozone with ARG and positive associations

308

with ARG2 methylation, but all the associations were not statistically significant.


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Likewise, Jung et al found positive but non-significant associations of PM2.5

310

with ARG2 methylation among a group of urban children.23 Paradoxically,

311

another human-based study associated ARG2 hypermethylation with

312

decreased FeNO,9 revealing the complexity inherent in the arginase–nitric

313

oxide synthase pathway. There exist other unknown regulating factors in NO

314

production.

315

It is noteworthy that the inflammation-related response to ozone differed

316

greatly by the allergic status. We observed significantly elevated FeNO levels,

317

increased iNOS concentrations and decreased NOS2A methylation following

318

ozone exposure in allergic subjects rather than in healthy subjects. This finding

319

indicated that people with pre-existing respiratory allergic diseases were more

320

susceptible to the airway inflammatory response induced by ozone exposure.

321

These findings were biologically plausible because FeNO is inherently a

322

biomarker of eosinophilic inflammation and allergic subjects (allergic rhinitis

323

and atopy in this study) were characterized by eosinophilic inflammation in the

324

airway. Nevertheless, it should be noted that a prior study still reported evident

325

associations between ozone and FeNO in non-asthmatic children.19 This

326

inconsistency may be caused by different study designs, sample sizes and

327

populations.

328

We found much stronger associations of ozone using personal than

329

fixed-site

330

misclassification leads to substantial attenuation of the associations. The

measurements,

suggesting

that

nondifferential


exposure


331

better model fit for personal-exposure models than for ambient-exposure

332

models further revealed that the use of direct personal measurements could

333

more effectively predict human health response. This reflects the fact that

334

ozone is highly spatially heterogenous due to its chemical reactions with NO,24

335

as well as the fact that its concentrations can differ much between outdoors

336

and indoors due to the rapid surface adsorption in indoor environments.25, 26

337

Therefore, our findings demonstrated that ambient ozone measurements from

338

fixed-site monitors was not a perfect surrogate of personal exposure to ozone

339

in epidemiological studies. Nevertheless, it should be acknowledged that most

340

previous epidemiological studies have associated health outcomes with ozone

341

measured based on fixed-site monitoring or residential modelling, which may

342

have led to considerable underestimation of its health effects and

343

corresponding disease burden.

344

Our study has some limitations. First, the relatively small sample size

345

limited the power and precision of this study, and consequently some important

346

associations might have been missed. Second, the enrollment of college

347

students reduced the potential of uncontrolled confounding (such as smoking,

348

alcohol drinking, indoor air pollution, and dietary structures), but may limit the

349

generalizability to other populations and contexts. Third, we were unable to

350

evaluate causality and temporal associations from ozone exposure to NO

351

production through DNA methylation and protein expression because buccal

352

sample collection and FeNO tests were simultaneously conducted. Fourth,


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although our results were robust to the adjustment of other air pollutants

354

measured by a fixed-site station, their confounding effects cannot be excluded

355

because of the lack of personal monitoring data.

356

In summary, our study demonstrated that short-term exposure to ozone

357

may result in acute respiratory inflammation. Our results further offered a new

358

pathophysiologic insight that this inflammatory response may be mainly

359

modulated by NOS2A hypomethylation in the arginase–nitric oxide synthase

360

pathway. Further research with larger sample size and controlled-exposure

361

design in various subpopulations is warranted to confirm our findings.

362



363

Acknowledgments: We appreciate the contributions of all volunteers in this

364

study. This work was funded by the National Natural Science Foundation of

365

China (91643205 and 91743111), China Medical Board Collaborating Program

366

(16-250), the Research Program of Shanghai Environmental Protection

367

Bureau (2016-11), and Shanghai Key Laboratory of Meteorology and Health

368

(QXJK201702).

369 370

Supporting Information

371

This information is available free of charge via the Internet at

372

http://pubs.acs.org/. Table S1. Primer sequences for NOS2A and ARG2 genes.

373

Table S2–S5. Changes in FeNO, iNOS, ARG, NOS2A and ARG2 methylation

374

associated with a 10 ppb increase in personal ozone levels at different lag

375

periods, adjusting for other air pollutants.

376 377

Notes: The authors declared no conflicts of interests.

378 379


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380

References:

381

(1) Environmental Protection Agency, U. S., Integrated Science Assessment

382

for Ozone and Related Photochemical Oxidants. In Research Triangle Park, U.

383

S., Ed. 2013.

384

(2) Turner, M. C.; Jerrett, M.; Pope, C. A.; Krewski, D.; Gapstur, S. M.; Diver,

385

W. R.; Beckerman, B. S.; Marshall, J. D.; Su, J.; Crouse, D. L.; Burnett, R. T.

386

Long-Term Ozone Exposure and Mortality in a Large Prospective Study. Am. J.

387

Respir. Crit. Care.Med. 2016, 193 (10), 1134-1142.

388

(3) Khaniabadi, Y. O.; Hopke, P. K.; Goudarzi, G.; Daryanoosh, S. M.;

389

Jourvand, M.; Basiri, H. Cardiopulmonary mortality and COPD attributed to

390

ambient ozone. Environ. Res. 2017, 152, 336-341.

391

(4) Kim, C. S.; Alexis, N. E.; Rappold, A. G.; Kehrl, H.; Hazucha, M. J.; Lay, J.

392

C.; Schmitt, M. T.; Case, M.; Devlin, R. B.; Peden, D. B.; Diaz-Sanchez, D.

393

Lung function and inflammatory responses in healthy young adults exposed to

394

0.06 ppm ozone for 6.6 hours. Am. J. Respir. Crit. Care.Med. 2011, 183 (9),

395

1215-1221.

396

(5) Lin, W.; Huang, W.; Zhu, T.; Hu, M.; Brunekreef, B.; Zhang, Y.; Liu, X.;

397

Cheng, H.; Gehring, U.; Li, C.; Tang, X. Acute respiratory inflammation in

398

children and black carbon in ambient air before and during the 2008 Beijing

399

Olympics. Environ. Health Perspect. 2011, 119 (10), 1507-1512.

400

(6) Li, H.; Wu, S.; Pan, L.; Xu, J.; Shan, J.; Yang, X.; Dong, W.; Deng, F.; Chen,

401

Y.; Shima, M.; Guo, X. Short-term effects of various ozone metrics on



402

cardiopulmonary function in chronic obstructive pulmonary disease patients:

403

Results from a panel study in Beijing, China. Environ. Pollut. 2018, 232,

404

358-366.

405

(7) Modig, L.; Dahgam, S.; Olsson, D.; Nyberg, F.; Wass, K.; Forsberg, B.;

406

Olin, A. C. Short-term exposure to ozone and levels of exhaled nitric oxide.

407

Epidemiology 2014, 25 (1), 79-87.

408

(8) Dweik, R.; Boggs, P.; Erzurum, S.; Irvin, C.; Leigh, M.; Lundberg, J.; Olin,

409

A.; Plummer, A.; Taylor, D. An official ATS clinical practice guideline:

410

interpretation of exhaled nitric oxide levels (FENO) for clinical applications. Am.

411

J. Respir. Crit. Care.Med. 2011, 184 (5), 602-615.

412

(9) Breton, C. V.; Byun, H. M.; Wang, X.; Salam, M. T.; Siegmund, K.; Gilliland,

413

F. D. DNA methylation in the arginase-nitric oxide synthase pathway is

414

associated with exhaled nitric oxide in children with asthma. Am. J. Respir. Crit.

415

Care.Med. 2011, 184 (2), 191-197.

416

(10)

417

H.; Zhao, Z.; Xu, X.; Hu, H.; Kan, H. Fine Particulate Matter Constituents, Nitric

418

Oxide Synthase DNA Methylation and Exhaled Nitric Oxide. Environ. Sci.

419

Technol. 2015, 49 (19), 11859-11865.

420

(11) Salam, M. T.; Byun, H. M.; Lurmann, F.; Breton, C. V.; Wang, X.; Eckel, S.

421

P.; Gilliland, F. D. Genetic and epigenetic variations in inducible nitric oxide

422

synthase promoter, particulate pollution, and exhaled nitric oxide levels in

423

children. J. Allergy Clin. Immunol. 2012, 129 (1), 232-9 e1-7.

Chen, R.; Qiao, L.; Li, H.; Zhao, Y.; Zhang, Y.; Xu, W.; Wang, C.; Wang,


Page 22 of 31

Page 23 of 31


424

(12)

425

Marsden, P. A. Epigenetic Basis for the Transcriptional Hyporesponsiveness of

426

the Human Inducible Nitric Oxide Synthase Gene in Vascular Endothelial Cells.

427

The J. Immunol. 2005, 175 (6), 3846-3861.

428

(13)

429

Wang, W.; Peng, L.; Xia, X.; Fu, Q.; Kan, H. Estimation of personal ozone

430

exposure using ambient concentrations and influencing factors. Environ. Int.

431

2018, 117, 237-242.

432

(14)

433

recommendations for standardized procedures for the online and offline

434

measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide,

435

2005. Am. J. Respir. Crit. Care.Med. 2005, 171 (8), 912-930.

436

(15)

437

R.; Alzheimer's Disease Neuroimaging, I. Statistical analysis of longitudinal

438

neuroimage data with Linear Mixed Effects models. NeuroImage. 2013, 66,

439

249-60.

440

(16)

441

D.; Staimer, N.; Delfino, R. J. Personal and Ambient Air Pollution is Associated

442

with Increased Exhaled Nitric Oxide in Children with Asthma. Environ. Health

443

Perspect. 2006, 114 (11), 1736-1743.

444

(17)

445

J.; Ohman-Strickland, P. A.; Sundell, J.; Weng, W.; Zhang, Y.; Zhang, J. J.

Chan, G. C.; Fish, J. E.; Mawji, I. A.; Leung, D. D.; Rachlis, A. C.;

Niu, Y.; Cai, J.; Xia, Y.; Yu, H.; Chen, R.; Lin, Z.; Liu, C.; Chen, C.;

American Thoracic Society; European Respiratory Society. ATS/ERS

Bernal-Rusiel, J. L.; Greve, D. N.; Reuter, M.; Fischl, B.; Sabuncu, M.

Kleinman, M. T.; George, S. C.; Fung, K.; Sioutas, C.; Tjoa, T.; Gillen,

Day, D. B.; Xiang, J.; Mo, J.; Li, F.; Chung, M.; Gong, J.; Weschler, C.



Page 24 of 31

446

Association of Ozone Exposure With Cardiorespiratory Pathophysiologic

447

Mechanisms in Healthy Adults. JAMA Intern. Med. 2017, 177 (9), 1344-1353.

448

(18)

449

Papakosta, D.; Spyratos, D.; Grivas, G.; Tasi, S.; Angelis, N.; Thirios, A.;

450

Tsiotsios, A.; Katsouyanni, K. Weekly Personal Ozone Exposure and

451

Respiratory Health in a Panel of Greek Schoolchildren. Environ. Health

452

Perspect. 2017, 125 (7), 077017.

453

(19)

454

Escamilla-Nunez,

455

Cortez-Lugo, M.; Holguin, F.; Diaz-Sanchez, D.; Olin, A. C.; Romieu, I. Air

456

pollution, airway inflammation, and lung function in a cohort study of Mexico

457

City schoolchildren. Environ. Health Perspect. 2008, 116 (6), 832-838.

458

(20)

459

Wenzel, S. E. Nitric oxide and related enzymes in asthma: relation to severity,

460

enzyme function and inflammation. Clin. Exp. Allergy. 2012, 42 (5), 760-768.

461

(21)

462

species (ROS)--induced genetic and epigenetic alterations in human

463

carcinogenesis. Mutat. Res. 2011, 711 (1-2), 167-173.

464

(22)

465

Gilliland, F. D. Particulate matter, DNA methylation in nitric oxide synthase, and

466

childhood respiratory disease. Environ. Health Perspect. 2012, 120 (9),

467

1320-1326.

Karakatsani, A.; Samoli, E.; Rodopoulou, S.; Dimakopoulou, K.;

Barraza-Villarreal, M.

A.;

C.;

Sunyer,

Sienra-Monge,

J.; J.

Hernandez-Cadena, J.;

Ramirez-Aguilar,

L.; M.;

Yamamoto, M.; Tochino, Y.; Chibana, K.; Trudeau, J. B.; Holguin, F.;

Ziech, D.; Franco, R.; Pappa, A.; Panayiotidis, M. I. Reactive oxygen

Breton, C. V.; Salam, M. T.; Wang, X.; Byun, H. M.; Siegmund, K. D.;


Page 25 of 31


468

(23)

469

J.; Jezioro, J. R.; Hoepner, L.; Ross, J.; Perera, F. P.; Chillrud, S. N.; Miller, R. L.

470

Short-term exposure to PM2.5 and vanadium and changes in asthma gene

471

DNA methylation and lung function decrements among urban children. Respir.

472

Res. 2017, 18 (1), 63.

473

(24)

474

C. Characterizations of ozone, NOx, and VOCs measured in Shanghai, China.

475

Atmos. Environ. 2008, 42 (29), 6873-6883.

476

(25)

477

exposure to "outdoor ozone" on the relationship between ozone and

478

short-term mortality in U.S. communities. Environ. Health Perspect. 2012, 120

479

(2), 235-240.

480

(26)

481

factor in residences under infiltration conditions: application in a multifamily

482

apartment unit. Indoor Air 2016, 26 (4), 571-581.

Jung, K. H.; Torrone, D.; Lovinsky-Desir, S.; Perzanowski, M.; Bautista,

Geng, F.; Tie, X.; Xu, J.; Zhou, G.; Peng, L.; Gao, W.; Tang, X.; Zhao,

Chen, C.; Zhao, B.; Weschler, C. J. Assessing the influence of indoor

Zhao, H.; Stephens, B. A method to measure the ozone penetration

483



Table of Contents (TOC):


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Table 1. Descriptive statistics of personal and ambient ozone concentrations at different lag periods, FeNO, iNOS, ARG, and methylation levels of NOS2A and ARG2 genes. Lag period

Mean

SD

Min

P25

Median

P75

Max

0−2 h

22.23

14.16

4.50

12.28

19.82

28.13

80.27

3−5 h

22.70

17.36

4.50

11.18

18.17

30.31

103.76

6−8 h

17.97

20.45

4.50

7.58

13.32

21.29

201.95

0−8 h

21.01

14.71

4.50

11.54

17.43

26.14

104.27

1d

19.42

11.24

4.50

10.93

18.38

25.94

76.19

2d

19.57

11.53

4.50

10.67

17.26

25.65

67.94

Ambient ozone (ppb) a

0−8 h

46.15

17.62

11.63

36.40

47.20

58.41

86.45

FeNO (ppb)

-

11.34

7.16

4.00

7.00

9.00

12.00

43.00

iNOS / β-actin

-

0.32

0.78

Personal ozone exposure and respiratory ... - ACS Publications

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