Prenatal Nonylphenol and Bisphenol A Exposures and Inflammation

May 11, 2017 - Air Pollution and Cigarettes Cloud Development. Caleb E. Finch. 2018,131-159. Related Content: Urinary Concentrations of Bisphenols and...
0 downloads 0 Views 367KB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Prenatal nonylphenol and bisphenol A exposures and inflammation are determinants of oxidative/nitrative stress: A Taiwanese cohort study Yu-Fang Huang, Pei-Wei Wang, Li-Wei Huang, Chun-Hao Lai, Winnie Yang, Kuen-Yuh Wu, Chensheng Lu, Hsin-Chang Chen, and Mei-Lien Chen Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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 20

Environmental Science & Technology

Prenatal nonylphenol and bisphenol A exposures and inflammation are determinants of oxidative/nitrative stress: A Taiwanese cohort study

Yu-Fang Huang1,2, Pei-Wei Wang1,3, Li-Wei Huang4, Chun-Hao Lai1, Winnie Yang5, Kuen-Yuh Wu6, Chensheng Alex Lu2, Hsin-Chang Chen6, Mei-Lien Chen1,* 1

Institute of Environmental and Occupational Health Sciences, National Yang Ming University, Taipei, Taiwan

2

Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USA

3

Department of Pediatrics, Taipei City Hospital, Heping Fuyou Branch, Taipei,

Taiwan Department of Obstetrics & Gynecology, Taipei City Hospital, Heping Fuyou Branch, Taipei, Taiwan 5 Division of Pediatrics, Taipei City Hospital, Yangming Branch, Taipei, Taiwan 6 Institute of Occupational Medicine and Industrial Hygiene, National Taiwan University, Taipei, Taiwan 4

Sources of Funding: This work was supported by research grants from National Science Council of the Republic of China, Taiwan (MOST 104-2621-M-010-001 and MOST 105-2621-M-010-001-MY3) and the Taipei City Government Department of Health (102-TPECH11and 105-TPECH-62-023).

No conflicts of interest to disclose.

*

Corresponding author: Mei-Lien Chen

Professor Institute of Environmental and Occupational Health Sciences, National Yang Ming University, No. 155, Sec. 2, Li-Nong St., Beitou, Taipei, Taiwan Tel:+886-22826-7239; Fax: +886-22827-8254 E-mail: [email protected] Yu-Fang Huang and Pei-Wei Wang are co-first authors

1

ACS Paragon Plus Environment

Environmental Science & Technology

1

Abstract

2

Prenatal exposure to nonylphenol (NP) and/or bisphenol A (BPA) has been

3

reported to be associated with adverse birth outcomes; however, the underlying

4

mechanisms remain unclear. The primary mechanism is endocrine disruption of the

5

binding affinity for the estrogen receptor, but oxidative stress and inflammation might

6

also play a contributory role. We aimed to investigate urinary NP and BPA levels in

7

relation to biomarkers of oxidative/nitrative stress and inflammation and to explore

8

whether changes in oxidative/nitrative stress are a function of prenatal exposure to

9

NP/BPA and inflammation in 241 mother-fetus pairs. Third-trimester urinary

10

biomarkers of oxidative/nitrative stress were simultaneously measured, including

11

products of oxidatively and nitratively damaged DNA (8-hydroxy-2’-deoxyguanosine

12

(8-OHdG) and 8-nitroguanine (8-NO2Gua)) as well as products of lipid peroxidation

13

(8-iso-prostaglandin F2α (8-isoPF2α) and 4-hydroxy-2-nonenal-mercapturic acid

14

(HNE-MA)). The antioxidant glutathione peroxidase (GPx) and inflammation

15

biomarkers, including C-reactive protein (CRP) and a panel of cytokines

16

(interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)), were analyzed in maternal

17

and umbilical cord plasma samples. In adjusted models, we observed significant

18

positive associations between NP exposure and 8-OHdG and 8-NO2Gua levels,

19

between BPA and 8-isoPF2α levels, and between maternal CRP levels and HNE-MA

20

levels. Additionally, BPA and TNF-α levels in cord blood were inversely associated

21

with maternal and GPx levels in cord blood as well as maternal TNF-α levels were

22

inversely associated with maternal GPx levels. These results support a role for

23

exposure to NP and BPA and possibly inflammation in increasing oxidative/nitrative

24

stress and decreasing antioxidant activity during pregnancy.

25 26 27

Key words: nonylphenol, bisphenol A, oxidative/nitrative stress, inflammation

2

ACS Paragon Plus Environment

Page 2 of 20

Page 3 of 20

Environmental Science & Technology

28

Introduction

29

The maternal transfer of endocrine disrupting compounds (EDCs) to the fetus

30

has aroused substantial concern in recent years because the estrogenic activities of

31

EDCs could interfere with hormone biosynthesis and metabolism, consequently

32

causing adverse health effects.1 Among these compounds, nonylphenol (NP), a

33

product of the degradation of NP polyethoxylates (NPEOs) and bisphenol A (BPA) are

34

both weakly estrogenic and produced in high volumes worldwide. The annual global

35

production of NPEOs and BPA is approximately 520,0002 and 3.6 million tons,3

36

respectively. The U.S. Environmental Protection Agency (EPA) estimates that over 1

37

million pounds of BPA are released into the environment annually.4 NP is utilized in

38

the chemical industry for the production of surfactants, detergents, emulsifiers,

39

pesticides, lubricants, and oil additives that are used in daily life.5 BPA is widely used

40

in the production of polycarbonate plastics, the epoxy resin linings of beverages and

41

canned foods, dental sealants, and thermal receipt paper.6 Human are exposing to NP

42

and BPA through the environment, dietary intake, and the use of products containing

43

these chemicals via ingestion, dermal absorption, and inhalation. Widespread and

44

continuous exposure to NP and BPA is evidenced by findings of significant NP levels

45

in humans, with 100% of pregnant women and fetuses in Taiwan having measurable

46

NP and approximately 74% exhibiting measurable BPA levels in urine and plasma.7-9

47

NP and BPA have been shown to transfer rapidly across the placenta,9, 10

48

and exposure to these chemicals has been related to adverse birth outcomes, including

49

small for gestational age (SGA), shorter gestational duration, decreased birth length,

50

and low birth weight;7,

51

Although, the etiology remains not yet elucidated, endocrine disruption of the binding

52

affinity for the estrogen receptor and oxidative stress has been implicated in vitro and

53

in utero.13, 17-19 Animal and in vitro studies have shown that increases in NP or/and

54

BPA could induce oxidative/nitrative stress by generating reactive oxygen and

55

nitrogen species (ROS/RNS) and/or by decreasing antioxidant activity,20-22 causing

56

oxidative damage and lipid peroxidation in the livers of rats.20 Free radical-mediated

57

oxidative/nitrative stress products of DNA and lipids, including products of

58

oxidatively and nitratively damaged DNA (8-hydroxy-2’-deoxyguanosine (8-OHdG)

59

and 8-nitroguanine (8-NO2Gua)) and lipid peroxidation products (8-iso-prostaglandin

8, 11-13

however, other reports have found no effects.14-16

3

ACS Paragon Plus Environment

Environmental Science & Technology

60

F2α (8-isoPF2α) and 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA)), are thought to

61

be the key biomarkers of oxidative/nitrative stress.23 We recently reported

62

associations among multiple biomarkers of oxidative/nitrative stress, the antioxidant

63

glutathione peroxidase (GPx), NP exposure, and birth outcomes.13, 24 A few studies to

64

date have examined the relationships between maternal BPA exposure and biomarkers

65

of oxidative stress.19, 25

66

Moreover, inflammation associated with oxidative stress often complicates

67

adverse health effects in pregnant women and neonates, as evidenced by the increased

68

levels of inflammatory biomarkers of C-reactive protein (CRP), interleukin-6 (IL-6),

69

and tumor necrosis factor-α (TNF-α).26-31 A few studies have reported that NP induces

70

inflammation in human cell lines32 and mice,33 whereas BPA has been related to

71

inflammation in post-menopausal and pregnant women.19, 34 Despite those evidences,

72

few studies have explored the associations between oxidative/nitrative stress

73

biomarkers attributed to simultaneous exposure to inflammation and NP (BPA) in

74

human populations. We therefore undertook this cross-sectional cohort study of

75

mother-fetus pairs to examine (a) urinary NP and BPA levels in relation to biomarkers

76

of oxidative/nitrative stress and inflammation, and (b) whether changes in

77

oxidative/nitrative stress are a function of prenatal exposure to NP/BPA and

78

inflammation.

79 80

Materials and methods Recruitment of study population. This analysis was performed on data collected as

81

part of a cross-sectional study design to assess associations among prenatal NP

82

exposure, oxidative/nitrative stress biomarkers, and neonatal birth outcomes. The

83

study design has been described in detail elsewhere.13, 24 The recruitment period was

84

started in March 2014 and ended in March 2016. Eligible mother-fetus pairs were

85

recruited based on the following criteria; (1) mothers were between 18-45 years of age,

86

cancer-free, and at gestational weeks 27-38 with a live singleton pregnancy; and (2)

87

fetuses were healthy, to term (more than 37 weeks of gestation), and with a birth

88

weight of more than 2500 g. Mothers were to be excluded if they smoked and drank

89

during pregnancy or had occupational exposure to NP, as textile workers have been

90

shown to exhibit extremely high NP levels.35 In total, 241 mother-fetus pairs

91

completed the follow-up until delivery. The Ethics Committee of Taipei City Hospital

92

approved the study protocols, and all subjects provided informed consent prior to 4

ACS Paragon Plus Environment

Page 4 of 20

Page 5 of 20

Environmental Science & Technology

93

participation.

94

Questionnaire. After providing informed consent, we collected information on

95

potential routes of NP and BPA exposure, the demographic characteristics (e.g.,

96

maternal age, pre-pregnancy body mass index (BMI), weight gain, education, parity,

97

occupational history, and history of pregnancy), personal lifestyle habits (e.g.,

98

smoking status, alcohol and coffee consumption, and exercise habits), diet and

99

nutrient intake history, disease history, and medical conditions from each mother via

100

the administration of a structured questionnaire.

101

Sampling and storage. Maternal urine samples were collected during a clinic visit at

102

gestational weeks 27-38 and stored at -20 °C until analysis. Maternal venous blood

103

samples were collected upon hospital admission for delivery and umbilical cord blood

104

samples were collected at birth. Plasma was fractioned by centrifugation at 3000 rpm

105

for 15 min, and stored at -80 °C until analysis.

106

Laboratory analysis

107

Urine analyses of NP and BPA.

108

From the 233 urine samples collected, there was a sufficient amount of urine for NP

109

analysis in 232 and for BPA analysis in 230 samples. The levels of 4-n-NP were

110

determined using high-performance liquid chromatography (HPLC) coupled with

111

fluorescence detection (Hitachi, Tokyo), as previously described.13 BPA was analyzed

112

using a time-of-flight (TOF) mass spectrometer (Waters, MA, USA) with an

113

electrospray interface coupled to an Acquity ultra-performance liquid chromatography

114

(UPLC) system (Waters, MA, USA). No plastic products were used during sample

115

pretreatment. The urine samples were processed using Varian PH solid-phase

116

extraction. The average recoveries for NP and BPA were 77-105% (6-235 ng/mL) and

117

93-100% (20-100 ng/mL), respectively. The limits of detection (LODs) for NP and

118

BPA in urine were 0.20 ng/mL and 0.16 ng/mL, respectively, and the regression

119

coefficients (r2) of the standard curves exceeded 0.995. The intra- and inter-day

120

variations, which were expressed as the relative standard deviations (RSDs), were

121

13% and 10% for NP and 0.9% and 13.7% for BPA, respectively, at a level of 30

122

ng/mL.

123

Simultaneous analysis of multiple biomarkers for oxidative/nitrative stress and

124

lipid peroxidation. Products of oxidatively damaged DNA (8-OHdG) and nitratively

125

damaged DNA (8-NO2Gua) as well as products of lipid peroxidation (8-isoPF2α and

126

HNE-MA) were simultaneously analyzed in 233 urine samples using our established 5

ACS Paragon Plus Environment

Environmental Science & Technology

127

HPLC-electrospray ionization (ESI)-MS/MS method.36 The LODs for 8-OHdG,

128

8-NO2Gua, 8-isopPF2α, and HNE-MA in urine were 0.02 ng/mL, 0.03 ng/mL, 0.008

129

ng/mL, and 0.01 ng/mL, respectively. Excellent linearity over the concentration range

130

of 0.1-50 ng/mL was observed, with R2 > 0.9982. To validate the method

131

performance, the urine was spiked with four mixtures of the standards at three levels

132

(0.5, 5, and 25 ng/mL) and then analyzed. The mean accuracy was defined as the

133

percentage ratio of the calculated level of the four standards to the expected spiked

134

concentration and ranged from 97.8 to 102.2%. The intra- and inter-day variations

135

ranged from 3.0 to 8.1% and 3.1 to 9.3%, respectively.

136

Creatinine analyses. Urinary creatinine was measured in 233 samples based on the

137

Hinegard and Tiderstrom modification of the Jaff reaction.37 Twenty-six urine

138

samples with creatinine levels that were less than 0.3 g/L or greater than 3.0 g/L were

139

excluded from the data analysis. The urinary NP (BPA), 8-OHdG, 8-NO2Gua,

140

8-isoPF2α, and HNE-MA levels were adjusted with creatinine and expressed as µg/g

141

creatinine.

142

Analysis of the antioxidant enzyme GPx. From the 192 maternal and 154 umbilical

143

cord plasma samples collected, GPx levels were measured in 156 maternal and 123

144

umbilical cord plasma samples with sufficient volume. The activities of GPx in

145

plasma were evaluated by enzyme-linked immunosorbent assays using a Superoxide

146

Dismutase Assay Kit and a Glutathione Peroxidase Assay Kit (Cayman Chemical)

147

according to the manufacturer’s instructions. The activity of GPx was given as

148

nmol/min/mL. All samples were analyzed in duplicate, and the two measurements

149

were averaged for statistical analysis. The LOD of GPx was 10 nmol/min/mL. The

150

intra- and inter-day variations for GPx were 5.7% and 7.2%, respectively.

151

Inflammatory biomarker analysis. Maternal (n=192) and umbilical cord (n=146)

152

plasma CRP was measured using SYNCHRON® Systems reagent with the highly

153

sensitive near-infrared particle immunoassay rate methodology. IL-6 and TNF-α were

154

measured in maternal (n=181) and umbilical cord (n=154) plasma samples using

155

high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits according to the

156

manufacturer’s recommended protocol (R&D Systems, Minneapolis, MN, USA;

157

catalog nos. HS600B and HSTA00C, respectively). The LODs for CRP, IL-6, and

158

TNF-α were 0.2 µg/mL, 0.039 pg/mL, and 0.106 pg/mL, respectively. The intra- and

159

inter-day variations for CRP, IL-6, and TNF-α were 3.7% and 3.5% (13.5 µg/mL),

160

15% and 12% (16 pg/mL), and 15% and 12% (5 pg/mL), respectively. 6

ACS Paragon Plus Environment

Page 6 of 20

Page 7 of 20

Environmental Science & Technology

161

Sample size calculations. G-power 3.1 software was used to calculate the sample size.

162

The F family of tests was used in a linear multiple regression using a fixed model with

163

an a priori power analysis. We assumed a squared multiple correlation of R2=0.1338

164

and then calculated an effect size of f2=0.1494253. We also assumed an alpha

165

error=0.05 and a power=95%, and the number of predictors was six. We calculated a

166

total sample size of 147, considered the loss to follow-up rate (20%), and excluded

167

subjects with outlier creatinine levels (10%). Therefore, at least 210 subjects needed

168

to be recruited.

169

Statistical analysis. All data were analyzed using SPSS 19.0 software (SPSS Inc.,

170

Chicago, IL, USA). NP, BPA, oxidative stress, and inflammatory biomarkers were

171

natural-log transformed to normalize their distributions before statistical analyses.

172

Correlations among NP and BPA exposure, biomarkers of oxidative stress and

173

inflammation, and potential covariates were determined using Spearman’s correlation.

174

Multivariate linear regression models were used to investigate the relationships of

175

biomarkers of oxidative stress and inflammation with NP or BPA exposure. All

176

regression models were adjusted for potential covariates. First, the covariates

177

associated with inflammatory biomarkers, including maternal age, pre-pregnancy

178

BMI, and pregnancy complications and other diseases, were based on statistical

179

consideration and the literature.28 Second, the covariates associated with oxidative

180

stress biomarkers were based on our previous work.13 We considered other potential

181

covariates based on a literature review, and these included gestational age and

182

pregnancy complications and other diseases.39,

183

obtained with the inclusion and exclusion of the covariate of pregnancy complications

184

and other diseases in models. This covariate was included in the final models. We

185

subsequently explored the associations between exposure to NP or BPA and

186

inflammation as determinants and biomarkers of oxidative stress and antioxidant

187

activity. NP or BPA exposure was included as the covariate based on significant

188

relationships between NP or BPA and biomarkers of oxidative stress. All p-values

189

were from two-tailed tests and were considered statistically significant at < 0.05.

190 191 192

Results Participants’

193

characteristics of 241 mother-fetus pairs and maternal urinary NP and BPA levels are

194

presented in Table 1. Most of the pregnant women had at least a bachelor’s degree,

demographic

characteristics

40

Similar analytical results were

and

exposure

7

ACS Paragon Plus Environment

markers.

The

Environmental Science & Technology

195

32% delivered via cesarean section, and 50.2% were primiparous. Maternal urinary

196

NP and BPA levels were detectable in 99.2% and 82.2%, respectively. The geometric

197

mean (GM) of the NP and BPA levels were 3.99 and 2.24 µg/g creatinine,

198

respectively.

199

Oxidative stress, GPx, and inflammatory biomarkers in this cohort. Table 2

200

presents the detection rates, GM, range, and percentiles (5th, 25th, 50th, and 75th) of

201

biomarkers of oxidative stress, GPx and inflammatory profiles in participating

202

mothers and their newborns. An analysis of all samples showed that 8-NO2Gua,

203

8-OHdG, 8-isoPF2α, and HNE-MA were detectable in 80.0%, 99.6%, 100%, and

204

99.6% of urine samples, respectively. Maternal CRP and GPx, TNF-α, and IL-6 levels

205

in mother-fetus pairs were detectable in 98.0%, 100%, 100-98.2%, and 97.9-96.9% of

206

plasma samples, respectively. The LOD for CRP in cord blood was 7.3%, and the GM

207

level was 0.10 µg/mL. This biomarker was excluded from the statistical analysis. The

208

GM of maternal 8-NO2Gua, 8-OHdG, 8-isoPF2α, HNE-MA, and CRP were 10.55,

209

4.91, 88.24, and 17.07 µg/g creatinine and 2.84 µg/mL, respectively. The GM of GPx,

210

TNF-α, and IL-6 for women were 162.82 nmol/min/mL, 2.61 pg/mL, and 5.56 pg/mL,

211

respectively; those for their fetuses were 127.20 nmol/min/mL, 4.31 pg/mL, and 4.14

212

pg/mL, respectively.

213

Associations of biomarkers of oxidative stress, GPx, and inflammation with NP

214

and BPA exposure. Table 3 shows that, after controlling for covariates, NP was

215

significantly associated with increases in 8-NO2Gua and 8-OHdG and decreases in

216

TNF-α in the pregnant women in this cross-sectional study. BPA was significantly

217

associated with increased maternal 8-isoPF2α levels and decreased maternal and cord

218

blood GPx levels.

219

Associations of biomarkers of oxidative stress and GPx with exposure to NP

220

(BPA) and inflammation. Table 4 shows that, after controlling for covariates, a

221

significant positive association was found between maternal CRP and HNE-MA

222

levels. We also found inverse associations between (1) maternal IL-6 levels and

223

8-isoPF2α levels, (2) maternal TNF-α levels and 8-NO2Gua, HNE-MA, and maternal

224

GPx levels, (3) cord blood IL-6 levels and 8-OHdG levels, and (4) cord blood TNF-α

225

levels and maternal and cord blood GPx levels.

226 227

Discussion

228

To the best of our knowledge, this is the first study to report prenatal 8

ACS Paragon Plus Environment

Page 8 of 20

Page 9 of 20

Environmental Science & Technology

229

oxidative/nitrative stress levels and GPx activity, which are partly attributable to NP

230

(BPA) exposure and inflammation. In this cross-sectional study, we found that

231

positive associations between NP exposure and 8-OHdG/8-NO2Gua levels, between

232

BPA and 8-isoPF2α levels, and between maternal CRP levels and HNE-MA levels.

233

Additionally, BPA and TNF-α levels in cord blood were inversely associated with

234

maternal and GPx levels in cord blood as well as maternal TNF-α levels were

235

inversely associated with maternal GPx levels. The results reported in this study

236

support a role for NP and BPA, and possibly inflammation, in increasing oxidative

237

stress and decreasing antioxidant activity during pregnancy.

238

Previous studies have verified that NP and BPA are two of the most common

239

contaminants in Taiwan.7, 13, 41 In the present study, the GM of NP in maternal urine

240

was 3.99 µg/g creatinine, which was similar to that previously reported in Taiwanese

241

residents who had not been occupationally exposed to NP8, 11 but higher than those

242

reported in the U.S.,42, 43 Japan,44, 45 and China.46 The high background of NP related

243

to everyday NPEO detergent use and environmental and food contamination via

244

bioaccumulation might contribute to the high levels of NP experienced by Taiwanese

245

residents.47, 48 The GM of maternal BPA was 2.24 µg/g creatinine, which is consistent

246

with levels reported in the literature.7, 12, 14, 49 This finding indicates that exposure to

247

NP and BPA is common among pregnant women in Taiwan.

248

Very few studies have reported findings on the relationship between prenatal

249

NP and BPA exposure and biomarkers of oxidative/nitrative stress and antioxidant

250

activity. Only two studies described preliminary analyses published by our study

251

group have reported the correspondence of NP exposure and maternal 8-OHdG,

252

8-NO2Gua, 8-isoPF2α, and HNE-MA levels. In addition, the previous studies have

253

shown that elevated 8-OHdG levels are significantly associated with decrease of

254

gestational ages.13,

255

significantly associated with increases in 8-isoPF2α levels, a result that is consistent

256

with previous studies.19,

257

between BPA and decreased maternal and cord GPx activity agreed with data

258

collected from an animal study.50 Those results highlight that exposure to NP and BPA

259

might be involved in oxidative/nitrative stress during pregnancy.

24

In this study, we observed that prenatal BPA exposure was 25

Furthermore, our finding of the significant association

260

A few in vitro studies have reported that NP promotes inflammatory responses

261

in the gastrointestinal tract and lung, to induce the expression of the pro-inflammatory

262

genes IL-1ß, IL-8, and IL-23A, and to decrease the expression of the 9

ACS Paragon Plus Environment

Environmental Science & Technology

263

anti-inflammatory genes IL-10 and IL-4 in human cell lines.32 This was the first report

264

of an observed negative association between NP and maternal TNF-α during late

265

pregnancy and presented the contrast results to previous studies. We speculated that

266

differences in the high exposure level of NP and whether the study was performed in

267

vitro may contribute to this discrepancy.

268

Although results from the present study is consistent with the findings of a

269

study of 141 pregnant women in the USA that reported no association between BPA

270

and the levels of CRP, TNF-α, IL-6, IL-10, and IL-1ß,25 we thought the relationships

271

between BPA exposure and biomarkers of inflammation are still inconclusive. This

272

might be explained by the fact that nuanced changes in the inflammatory biomarkers

273

in response to BPA exposure might be detectable when other factors cause greater

274

fluctuations. Regardless, Ferguson et al. showed a significant association between

275

BPA and IL-6 in 482 pregnant women with repeated measurements.19 In another

276

cross-sectional study involving 485 adults, Yang et al. reported a significant

277

association between BPA exposure and CRP levels in post-menopausal Korean

278

women but not in premenopausal women or men.34 The authors hypothesized that the

279

lower estrogen levels in post-menopausal women would increase the availability of

280

estrogen receptors for binding BPA and thereby enhance its adverse effects, including

281

the cellular responses that trigger inflammation.34 Notably, inflammation is a complex

282

process and is implicated in the mediation of the release of a number of cytokines,

283

which might be responsible for the discrepancies.

284

Our results revealed a significant correlation between CRP and IL-6 levels in

285

pregnant women (Supplementary Table S1). CRP is an acute-phase protein that is

286

frequently used as a biomarker of low-grade systemic inflammation because it is

287

synthesized by the liver in response to inflammatory mediators, particularly IL-6.

288

Both IL-6 and TNF-α are pro-inflammatory cytokines secreted by adipose tissue. The

289

former is a potent inducer of the acute-phase response, which is characterized by the

290

hepatic release of CRP,51 and the latter is involved in systemic inflammation. The

291

mechanism through which inflammation induces oxidative stress and vice versa is

292

unknown but is of great importance because these factors are apparently associated

293

with pregnancy complications and adverse birth outcomes.13, 26-31 To date, two studies

294

have reported associations between phenols (e.g., BPA and triclosan) and paraben

295

levels and between biomarkers of inflammation and oxidative stress during

296

pregnancy.19, 25 Another study simultaneously examined the inflammatory process and 10

ACS Paragon Plus Environment

Page 10 of 20

Page 11 of 20

Environmental Science & Technology

297

oxidative stress associated with delivery in 56 healthy women and their neonates

298

throughout parturition.52 We observed a positive association between maternal CRP

299

levels and HNE-MA and found inverse associations between maternal TNF-α levels

300

and maternal GPx, and between cord blood TNF-α levels and maternal and cord blood

301

GPx. These results suggest that inflammation may be related to the induction of

302

oxidative stress.

303

Before extrapolating our study results, it should be cautious of the study

304

limitations. First, the sample sizes was discrepant among maternal (n=192), cord

305

blood (n=154), and maternal urine samples (n=233) because of invasive blood

306

samples collection and loss of cord blood samples. Second, the assessments of NP or

307

BPA exposure and biomarkers of oxidative stress were performed during the third

308

trimester of pregnancy, but inflammation biomarkers in mother-fetus pairs were

309

determined upon admission for delivery and after delivery. This was due to oxidative

310

stress increase during pregnancy39 and previous study designs assessing the

311

relationships among exposure to NP, oxidative/nitrative stress and birth outcomes.13

312

During pregnancy, NP levels have been shown to remain constant,8 but BPA levels

313

vary, as evidenced by reports of intraclass correlation coefficients in the range of

314

0.12-0.23.49, 53 Because of the short half-life and high degree of variability of BPA,

315

serial urine samples should be considered to avoid exposure misclassification. Third,

316

this cross-sectional study could not establish causality. Finally, we were unable to

317

adjust for the residual confounding by other environmental EDCs, such as phthalates

318

or parabens, or other medical conditions that may have effects on the outcomes that

319

we measured in this study. Regardless of the above limitations, the strength of the

320

study is that we have measured multiple biomarkers of oxidative stress and

321

inflammation in relation to NP and BPA exposure, which can aid to the identification

322

of biological pathways.

323

In conclusion, results from this study suggest that prenatal exposures to NP

324

and BPA and inflammation might be involved in mediating oxidative stress. Further

325

studies are necessary to elucidate and confirm the mechanisms by which NP or BPA

326

exposure and inflammation affect oxidative stress and their effects on birth outcomes

327

in this vulnerable sub-population.

328 329 330

Declaration of interest The authors are solely responsible for the content and writing of the manuscript. The 11

ACS Paragon Plus Environment

Environmental Science & Technology

331

authors report no conflict of interest.

332

Acknowledgments

333

The authors would like to thank the National Science Council of the Republic of

334

China, Taiwan (MOST 104-2621-M-010-001 and MOST 105-2621-M-010-001-MY3)

335

and the Taipei City Government Department of Health (102-TPECH11and

336

105-TPECH-62-023) for financially supporting this research. We acknowledge

337

American Journal Experts for their editorial assistance.

338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377

References 1. Evanthia Diamanti-Kandarakis, J.-P. B., Linda C. Giudice, Russ Hauser, Gail S. Prins, Ana M. Soto, R. Thomas Zoeller, and Andrea C. Gore, Endocrine-Disrupting Chemicals: An Endocrine Society Scientific Statement. Endocr Rev. Jun 2009, 30, (4), 293-342. 2. White, R.; Jobling, S.; Hoare, S.; Sumpter, J.; Parker, M., Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 1994, 135, (1), 175-182. 3. Geens, T.; Aerts, D.; Berthot, C.; Bourguignon, J.-P.; Goeyens, L.; Lecomte, P.; Maghuin-Rogister, G.; Pironnet, A.-M.; Pussemier, L.; Scippo, M.-L.; Van Loco, J.; Covaci, A., A review of dietary and non-dietary exposure to bisphenol-A. Food and Chemical Toxicology 2012, 50, (10), 3725-3740. 4. EPA, U. E. P., Bisphenol A (BPA) Action Plan. In 2016. 5. Ying, G.-G.; Williams, B.; Kookana, R., Environmental fate of alkylphenols and alkylphenol ethoxylates—a review. Environment International 2002, 28, (3), 215-226. 6. CDC, C. f. D. C. a. P., Fourth National Reporton Human Exposure to Environmental Chemicals. In 2017, July. ed.; 2014. 7. Chou, W.-C.; Chen, J.-L.; Lin, C.-F.; Chen, Y.-C.; Shih, F.-C.; Chuang, C.-Y., Biomonitoring of bisphenol A concentrations in maternal and umbilical cord blood in regard to birth outcomes and adipokine expression: a birth cohort study in Taiwan. Environmental Health 2011, 10, (1), 1. 8. Tsai, M. S.; Chang, C. H.; Tsai, Y. A.; Liao, K. W.; Mao, I.; Wang, T. H.; Hwang, S. M.; Chang, Y. J.; Chen, M. L., Neonatal outcomes of intrauterine nonylphenol exposure A longitudinal cohort study in Taiwan. Science of The Total Environment 2013, 458, 367-373. 9. Huang, Y.-F.; Wang, P.-W.; Huang, L.-W.; Yang, W.; Yu, C.-J.; Yang, S.-H.; Chiu, H.-H.; Chen, M.-L., Nonylphenol in pregnant women and their matching fetuses: Placental transfer and potential risks of infants. Environmental research 2014, 134, 143-148. 10. Corbel, T.; Gayrard, V.; Puel, S.; Lacroix, M. Z.; Berrebi, A.; Gil, S.; Viguié, C.; Toutain, P. L.; Picard-Hagen, N., Bidirectional placental transfer of Bisphenol A and its main metabolite, Bisphenol A-Glucuronide, in the isolated perfused human placenta. Reproductive Toxicology 2014, 47, 51-58. 11. Chang, C.-H.; Chen, M.-L.; Liao, K.-W.; Tsai, Y.-A.; Mao, I.; Wang, T.-H.; Hwang, S.-M.; Chang, Y.-J.; Tsai, M.-S., The association between maternal nonylphenol exposure and parity on neonatal birth weight: A cohort study in Taiwan. Chemosphere 2013, 93, (6), 1145-1152. 12. Huo, W.; Xia, W.; Wan, Y.; Zhang, B.; Zhou, A.; Zhang, Y.; Huang, K.; Zhu, Y.; 12

ACS Paragon Plus Environment

Page 12 of 20

Page 13 of 20

Environmental Science & Technology

378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

Wu, C.; Peng, Y., Maternal urinary bisphenol A levels and infant low birth weight: A nested case–control study of the Health Baby Cohort in China. Environment international 2015, 85, 96-103. 13. Wang, P. W.; Chen, M. L.; Huang, L. W.; Yang, W.; Wu, K. Y.; Huang, Y. F., Prenatal nonylphenol exposure, oxidative and nitrative stress, and birth outcomes: A cohort study in Taiwan. Environmental pollution (Barking, Essex : 1987) 2015, 207, 145-51. 14. Cantonwine, D.; Meeker, J. D.; Hu, H.; Sánchez, B. N.; Lamadrid-Figueroa, H.; Mercado-García, A.; Fortenberry, G. Z.; Calafat, A. M.; Téllez-Rojo, M. M., Bisphenol a exposure in Mexico City and risk of prematurity: a pilot nested case control study. Environmental Health 2010, 9, (1), 1. 15. Philippat, C.; Mortamais, M.; Chevrier, C.; Petit, C.; Calafat, A. M.; Ye, X.; Silva, M. J.; Brambilla, C.; Pin, I.; Charles, M.-A., Exposure to phthalates and phenols during pregnancy and offspring size at birth. Environmental health perspectives 2012, 120, (3), 464-70. 16. Wolff, M. S.; Engel, S. M.; Berkowitz, G. S.; Ye, X.; Silva, M. J.; Zhu, C.; Wetmur, J.; Calafat, A. M., Prenatal phenol and phthalate exposures and birth outcomes. Environmental health perspectives 2008, 116, (8), 1092. 17. Soto, A. M.; Justicia, H.; Wray, J. W.; Sonnenschein, C., p-Nonyl-phenol: an estrogenic xenobiotic released from" modified" polystyrene. Environmental health perspectives 1991, 92, 167. 18. Takayanagi, S.; Tokunaga, T.; Liu, X.; Okada, H.; Matsushima, A.; Shimohigashi, Y., Endocrine disruptor bisphenol A strongly binds to human estrogen-related receptor γ (ERRγ) with high constitutive activity. Toxicology letters 2006, 167, (2), 95-105. 19. Ferguson, K. K.; Cantonwine, D. E.; McElrath, T. F.; Mukherjee, B.; Meeker, J. D., Repeated measures analysis of associations between urinary bisphenol-A concentrations and biomarkers of inflammation and oxidative stress in pregnancy. Reproductive Toxicology 2016, 66, 93-98. 20. Bindhumol, V.; Chitra, K.; Mathur, P., Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology 2003, 188, (2), 117-124. 21. Zhang, Y.-Q.; Mao, Z.; Zheng, Y.-L.; Han, B.-P.; Chen, L.-T.; Li, J.; Li, F., Elevation of inducible nitric oxide synthase and cyclooxygenase-2 expression in the mouse brain after chronic nonylphenol exposure. International journal of molecular sciences 2008, 9, (10), 1977-1988. 22. Wu, M., Oxidative stress in zebrafish embryos induced by short term exposure to bisphenol A, nonylphenol, and their mixture. Environmental Toxicology and Chemistry 2011. 23. Wu, C.; Chen, S.-T.; Peng, K.-H.; Cheng, T.-J.; Wu, K.-Y., Concurrent quantification of multiple biomarkers indicative of oxidative stress status using liquid chromatography-tandem mass spectrometry. Analytical Biochemistry 2016, 512, 26-35. 24. Wang, P. W.; Chen, M. L.; Huang, L. W.; Yang, W.; Wu, K. Y.; Huang, Y. F., Nonylphenol exposure is associated with oxidative and nitrative stress in pregnant women. Free Radic Res 2015, 49, (12), 1469-78. 25. Watkins, D. J.; Ferguson, K. K.; Del Toro, L. V. A.; Alshawabkeh, A. N.; Cordero, J. F.; Meeker, J. D., Associations between urinary phenol and paraben concentrations and markers of oxidative stress and inflammation among pregnant women in Puerto Rico. International journal of hygiene and environmental health 2015, 218, (2), 212-219. 26. Amarilyo, G.; Oren, A.; Mimouni, F.; Ochshorn, Y.; Deutsch, V.; Mandel, D., 13

ACS Paragon Plus Environment

Environmental Science & Technology

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

Increased cord serum inflammatory markers in small-for-gestational-age neonates. Journal of Perinatology 2011, 31, (1), 30-32. 27. Ernst, G. D.; de Jonge, L. L.; Hofman, A.; Lindemans, J.; Russcher, H.; Steegers, E. A.; Jaddoe, V. W., C-reactive protein levels in early pregnancy, fetal growth patterns, and the risk for neonatal complications: the Generation R Study. American journal of obstetrics and gynecology 2011, 205, (2), 132. e1-132. e12. 28. Catarino, C.; Santos-Silva, A.; Belo, L.; Rocha-Pereira, P.; Rocha, S.; Patrício, B.; Quintanilha, A.; Rebelo, I., Inflammatory disturbances in preeclampsia: relationship between maternal and umbilical cord blood. Journal of pregnancy 2012, 2012. 29. Lausten-Thomsen, U.; Olsen, M.; Greisen, G.; Schmiegelow, K., Inflammatory Markers in Umbilical Cord Blood from Small-For-Gestational-Age Newborns. Fetal and Pediatric Pathology 2014, 33, (2), 114-118. 30. Sorokin, Y.; Romero, R.; Mele, L.; Iams, J. D.; Peaceman, A. M.; Leveno, K. J.; Harper, M.; Caritis, S. N.; Mercer, B. M.; Thorp, J. M., Umbilical cord serum interleukin-6, C-reactive protein, and myeloperoxidase concentrations at birth and association with neonatal morbidities and long-term neurodevelopmental outcomes. American journal of perinatology 2014, 31, (08), 717-726. 31. de Sousa Rocha, V.; Della Rosa, F. B.; Ruano, R.; Zugaib, M.; Colli, C., Association between magnesium status, oxidative stress and inflammation in preeclampsia: A case–control study. Clinical Nutrition 2015, 34, (6), 1166-1171. 32. Kim, A.; Jung, B. H.; Cadet, P., A novel pathway by which the environmental toxin 4-Nonylphenol may promote an inflammatory response in inflammatory bowel disease. Medical Science Monitor Basic Research 2014, 20, 47-54. 33. Suen, J. L.; Hsu, S. H.; Hung, C. H.; Chao, Y. S.; Lee, C. L.; Lin, C. Y.; Weng, T. H.; Yu, H. S.; Huang, S. K., A common environmental pollutant, 4-nonylphenol, promotes allergic lung inflammation in a murine model of asthma. Allergy 2013, 68, (6), 780-787. 34. Yang, Y. J.; Hong, Y.-C.; Oh, S.-Y.; Park, M.-S.; Kim, H.; Leem, J.-H.; Ha, E.-H., Bisphenol A exposure is associated with oxidative stress and inflammation in postmenopausal women. Environmental research 2009, 109, (6), 797-801. 35. Chen, M. L.; Lee, W. P.; Chung, H. Y.; Guo, B. R.; Mao, I. F., Biomonitoring of alkylphenols exposure for textile and housekeeping workers. International Journal of Environmental Analytical Chemistry 2005, 85, (4-5), 335-347. 36. Chen, S.-T.; Hsin-Chang, C.; Tsun-Jen, C.; Kuen-Yun, W., Simultaneous Analysis of Multi-Biomarkers for Oxidative and Nitrative Stress and Lipid Peroxidation with Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry Mass Spectrometry. Revised in Bioanalysis 2015. 37. Jaffe, M., Uber den niederschlag, welchen pikriksaure in normalen harn erzeugt und uber eine neue reaction des kreatinins. Z. Physiol. Chem. 1886, 10, 391. 38. Cohen, J., Statistical Power Analysis for the Behavioral Sciences. Routledge Academic: New York, 1998. 39. Palm, M.; Axelsson, O.; Wernroth, L.; Basu, S., F2-Isoprostanes, tocopherols and normal pregnancy. Free radical research 2009, 43, (6), 546-552. 40. Negi, R.; Pande, D.; Karki, K.; Kumar, A.; Khanna, R. S.; Khanna, H. D., Association of oxidative DNA damage, protein oxidation and antioxidant function with oxidative stress induced cellular injury in pre-eclamptic/eclamptic mothers during fetal circulation. Chemico-biological interactions 2014, 208, 77-83. 41. Lu, Y. Y.; Chen, M. L.; Sung, F. C.; Wang, P. S.-G.; Mao, I., Daily intake of 4-nonylphenol in Taiwanese. Environment international 2007, 33, (7), 903-910. 42. Kuklenyik, Z.; Ekong, J.; Cutchins, C. D.; Needham, L. L.; Calafat, A. M., 14

ACS Paragon Plus Environment

Page 14 of 20

Page 15 of 20

Environmental Science & Technology

478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

Simultaneous measurement of urinary bisphenol A and alkylphenols by automated solid-phase extractive derivatization gas chromatography/mass spectrometry. Analytical chemistry 2003, 75, (24), 6820-6825. 43. Calafat, A. M.; Kuklenyik, Z.; Reidy, J. A.; Caudill, S. P.; Ekong, J.; Needham, L. L., Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. Environmental health perspectives 2005, 113, (4), 391. 44. Inoue, K.; Kawaguchi, M.; Okada, F.; Takai, N.; Yoshimura, Y.; Horie, M.; Izumi, S. i.; Makino, T.; Nakazawa, H., Measurement of 4-nonylphenol and 4-tert-octylphenol in human urine by column-switching liquid chromatography-Mass spectrometry. Analytica Chimica Acta 2003, 486, (1), 41-50. 45. Kawaguchi, M.; Sakui, N.; Okanouchi, N.; Ito, R.; Saito, K.; Izumi, S.-i.; Makino, T.; Nakazawa, H., Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography–mass spectrometry for measurement of phenolic xenoestrogens in human urine samples. Journal of Chromatography B 2005, 820, (1), 49-57. 46. Tang, R.; Chen, M.-j.; Ding, G.-d.; Chen, X.-j.; Han, X.-m.; Zhou, K.; Chen, L.-m.; Xia, Y.-k.; Tian, Y.; Wang, X.-r., Associations of prenatal exposure to phenols with birth outcomes. Environmental Pollution 2013, 178, (0), 115-120. 47. Pan, Y.-P.; Tsai, S.-W., Determination and residual characteristic of alkylphenols in household food detergents of Taiwan. Chemosphere 2009, 76, (3), 381-386. 48. Chen, H.; Liang, C.; Wu, Z.; Chang, E.; Lin, T.; Chiang, P.; Wang, G., Occurrence and assessment of treatment efficiency of nonylphenol, octylphenol and bisphenol-A in drinking water in Taiwan. Science of The Total Environment 2013, 449, 20-28. 49. Braun, J. M.; Kalkbrenner, A. E.; Calafat, A. M.; Bernert, J. T.; Ye, X.; Silva, M. J.; Barr, D. B.; Sathyanarayana, S.; Lanphear, B. P., Variability and predictors of urinary bisphenol A concentrations during pregnancy. Environmental health perspectives 2011, 119, (1), 131. 50. Chen, M.; Xu, B.; Ji, W.; Qiao, S.; Hu, N.; Hu, Y.; Wu, W.; Qiu, L.; Zhang, R.; Wang, Y.; Wang, S.; Zhou, Z.; Xia, Y.; Wang, X., Bisphenol A Alters n-6 Fatty Acid Composition and Decreases Antioxidant Enzyme Levels in Rat Testes: A LC-QTOF-Based Metabolomics Study. PLOS ONE 2012, 7, (9), e44754. 51. Heinrich, P. C.; Castell, J. V.; Andus, T., Interleukin-6 and the acute phase response. Biochemical journal 1990, 265, (3), 621. 52. Díaz-Castro, J.; Florido, J.; Kajarabille, N.; Prados, S.; de Paco, C.; Ocon, O.; Pulido-Moran, M.; Ochoa, J. J., A new approach to oxidative stress and inflammatory signaling during labour in healthy mothers and neonates. Oxidative medicine and cellular longevity 2015, 2015. 53. Braun, J. M.; Smith, K. W.; Williams, P. L.; Calafat, A. M.; Berry, K.; Ehrlich, S.; Hauser, R., Variability of Urinary Phthalate Metabolite and Bisphenol A Concentrations before and during Pregnancy. Environmental Health Perspectives 2012, 120, (5), 739-745.

15

ACS Paragon Plus Environment

Environmental Science & Technology

521 522

523 524 525 526

Page 16 of 20

Table 1 Demographic characteristic and exposure markers of study population (n=241) Characteristic Percent (%) or mean ± SD Mother Age (year) 33.0 ± 3.5 2 Pre-pregnancy BMI (kg/m ) 21.7 ± 3.3 Weight gain (kg) 12.1 ± 4.0 Education(year) LOD Analyte N GM Min 5th 25th 50th 75th 95th Max Urine in ng/mL NP 232 99.2 2.81 0.35 0.75 1.86 2.98 4.02 9.42 38.88 BPA 230 82.2 1.63 0.35 0.35 1.00 1.77 3.00 7.60 29.81 Creatinine-adjusted (µg/g creatinine)& NP 206 99.2 3.99 0.16 1.06 2.37 4.05 7.03 13.85 40.54 BPA 205 82.2 2.24 0.16 0.30 1.19 2.53 4.70 11.41 46.58 # Total =19: nine with gestational diabetes, five with gestational hypertension, one with gestational diabetes and hypertension, one with thalassemia, one atopic disorder and two asthma.; LOD= limit of detection; &urine samples with creatinine less than 0.3 g/L or greater than 3.0 g/L were excluded.

16

ACS Paragon Plus Environment

Page 17 of 20

1 2

Environmental Science & Technology

Table 2 Distribution of oxidative stress, GPx and inflammatory biomarkers in this cohort Percentiles n

%>LOD GM

Min

5th

7.17

0.20

0.20 1 1.30

3.34 55.62

0.05 2.00

0.60 5.40

2.20 3.70 21.25 66.40

5.90 12.90 27.10 152.70 385.70 997.50

8-isoPF2α 233 99.6 Maternal urine (µg/g creatinine)&

11.07

0.10

1.80

5.85

11.70

20.35

68.13

8-NO2Gua 8-OHdG HNE-MA 8-isoPF2α Maternal blood GPx (nmol/min/mL) CRP (µg/mL) IL-6 (pg/mL) TNF-α (pg/mL) Cord blood# GPx(nmol/min/mL) IL-6 (pg/mL) TNF-α (pg/mL)

Maternal urine (ng/mL) 8-NO2Gua 233 80.0 8-OHdG HNE-MA

3 4 5 6 7

233 99.6 233 100.0

25th

50th

75th

95th

Max

14.60

35.50

136.78 243.50

181.40

207 207 207 207

80.0 99.6 100 99.6

10.55 4.91 88.24 17.07

0.09 0.30 1.80 0.37

0.17 1.00 8.88 3.89

2.25 2.93 41.93 9.75

18.75 5.15 99.60 16.65

45.58 8.90 212.83 28.95

184.08 730.10 19.68 62.70 459.18 1455.1 74.60 171.80

156 192 181 181

100 98.0 97.9 100

162.82 2.84 5.56 2.61

11.46 0.10 0.02 0.20

51.89 0.50 0.75 0.53

77.68 1.50 3.01 1.07

213.94 2.60 6.26 3.65

297.35 5.40 13.10 6.50

424.70 519.57 24.50 133.80 36.08 70.29 10.70 24.12

123 100 154 96.9 154 98.2

127.20 56.03 65.46 94.24 117.16 165.55 322.95 379.49 4.14 0.02 0.41 2.03 3.70 8.02 55.91 147.17 4.31 0.05 1.04 3.82 5.47 7.13 10.10 15.61

Abbreviations: LOD= limit of detection; GM= geometric mean; Min: minimum; Max: maximum & Urine samples with creatinine less than 0.3 g/L or greater than 3.0 g/L were excluded. # The LOD for CRP in cord blood was 7.3%, and the GM level was 0.10 µg/mL. This biomarker was excluded from the statistical analysis.

8

17

ACS Paragon Plus Environment

Environmental Science & Technology

Page 18 of 20

1

Table 3

2

Multivariate linear regression for the associations of ln-transformed NP and BPA levels

3

with oxidative stress, antioxidant and inflammatory biomarkers Third-trimester NP

Third-trimester BPA

Beta (SE)

Beta (SE)

Maternal oxidative stress biomarkersa (µg/g creatinine) 31.48 (10.30) 8-NO2Gua

p

p

0.003

-2.04 (8.51)

0.81

8-OHdG

1.54 (0.75)

0.04

0.59 (0.61)

0.33

HNE-MA

4.90 (21.18)

0.82

10.25 (16.92)

0.55

8-isoPF2α

-4.09 (2.81)

0.15

4.50 (2.23)

0.05

-30.98 (14.61)

0.04

Antioxidanta GPx (nmol/min/mL)

-37.66 (19.91) 0.06

Inflammatory biomarkersb CRP (µg/mL)

0.05 (0.13)

0.69

-0.06 (0.10)

0.57

IL-6 (pg/mL)

1.35 (1.30)

0.30

-0.82 (0.98)

0.40

TNF-α (pg/mL)

-0.90 (0.42)

0.03

-0.16 (0.32)

0.62

-6.19 (13.11)

0.64

-29.40 (10.30)

0.01

-2.43 (2.88)

0.40

-0.74 (2.30)

0.75

0.10 (0.32) 0.76 -0.14 (0.26) Adjusted covariates: maternal age, pre-pregnancy BMI, gestational age, pregnancy complications and other diseases, regular exercise, and vitamin supplements (n=200, 135 and 106 for the associations between NP (BPA) and oxidative stress, between NP (BPA) and maternal GPx, and between NP (BPA) and cord GPx, respectively). b Adjusted covariates: maternal age, pre-pregnancy BMI, and pregnancy complications and other diseases. (n=161, 156 and 127 for the associations between NP (BPA) and maternal CRP, between NP (BPA) and IL-6 and between TNF-α in maternal and cord plasma, respectively).

0.59

Cord

a

Antioxidant

GPx (nmol/min/mL) b

Inflammatory biomarkers (pg/mL) IL-6 TNF-α

4 5 6 7 8 9 10 11 12

a

18

ACS Paragon Plus Environment

Page 19 of 20

Environmental Science & Technology

1

Table 4

2

Multivariate linear regression for associations between oxidative stress biomarkers and antioxidant with ln-transformed inflammation Maternal inflammation biomarkers CRP (mg/dL)

Variables

Beta (SE)

IL-6 (pg/mL)

p

Beta (SE)

Oxidative stress biomarkers adjusted in creatinine (µg/g cre) 0.26 (0.17) 0.13 0.23 (0.13) 8-NO2Guaa a

TNF-α (pg/mL)

IL-6 (pg/mL)

p

Beta (SE)

Beta (SE)

0.07

-0.65 (0.17)