Prenatal Nonylphenol and Bisphenol A Exposures and Inflammation

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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

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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


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

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Introduction

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

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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

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

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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

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

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

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

50

and low birth weight;7,

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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

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

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

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

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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



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

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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

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Moreover, inflammation associated with oxidative stress often complicates

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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

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

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

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few studies have explored the associations between oxidative/nitrative stress

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

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

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

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

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

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inflammation.

79 80

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

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

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

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

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

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

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

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

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

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

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

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

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


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participation.

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Questionnaire. After providing informed consent, we collected information on

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

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

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

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

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

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the administration of a structured questionnaire.

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

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

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

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

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

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Laboratory analysis

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Urine analyses of NP and BPA.

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

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

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

111

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

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

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electrospray interface coupled to an Acquity ultra-performance liquid chromatography

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

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

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

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

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

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

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

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

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ng/mL.

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Simultaneous analysis of multiple biomarkers for oxidative/nitrative stress and

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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creatinine.

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

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

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

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

146

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

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

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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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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to be recruited.

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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

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

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

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

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Multivariate linear regression models were used to investigate the relationships of

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

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

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

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

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

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

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

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pregnancy complications and other diseases.39,

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

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

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subsequently explored the associations between exposure to NP or BPA and

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inflammation as determinants and biomarkers of oxidative stress and antioxidant

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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

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

190 191 192

Results Participants’

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

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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


markers.

The


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

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

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

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respectively.

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

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

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biomarkers of oxidative stress, GPx and inflammatory profiles in participating

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

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

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

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

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

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

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

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

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

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

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pg/mL, respectively.

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

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

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

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

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

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blood GPx levels.

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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

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

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

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

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

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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


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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

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

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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.

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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

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

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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

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

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

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NP and BPA is common among pregnant women in Taiwan.

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Very few studies have reported findings on the relationship between prenatal

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

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

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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



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

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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

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

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

274

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

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

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

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

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

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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

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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.

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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


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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



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

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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


Page 17 of 20

1 2


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



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


Page 19 of 20


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)

Prenatal Nonylphenol and Bisphenol A Exposures and Inflammation

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