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Ecotoxicology and Human Environmental Health

Phthalates and phthalate alternatives have diverse associations with oxidative stress and inflammation in pregnant women Thomas Joost van 't Erve, Emma Rosen, Emily Barrett, Ruby Nguyen, Sheela Sathyanarayana, Ginger Milne, Antonia M. Calafat, S. H. Swan, and Kelly K. Ferguson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05729 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019

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Environmental Science & Technology

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Phthalates and phthalate alternatives have diverse associations with oxidative stress and inflammation in pregnant women

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Authors: Thomas J. van t Ervea, Emma M. Rosena, Emily S. Barrettbc, Ruby H.N. Nguyend, Sheela Sathyanarayanae, Ginger L. Milnef, Antonia, M. Calafatg, Shanna H. Swanh, Kelly K. Fergusona*

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Affiliations:

7 8

a

Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, 27709, NC, USA

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b

Department of Biostatistics and Epidemiology, Rutgers School of Public Health, Piscataway, NJ, 08901, USA

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c

Department of Obstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, Rochester, NY,14642, USA

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d

Department of Epidemiology and Community Health, University of Minnesota, Minneapolis, MN, 55454, USA

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e

Department of Pediatrics, Seattle Children’s Research Institute, University of Washington, Seattle, WA, 98101, USA

17 18

f

Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, TN 372326602, USA

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g

Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, 30341, USA

21 22

h

Department of Preventive Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

23 24 25 26 27 28

* Correspondence

to: Kelly K. Ferguson. Epidemiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, 27709, NC, USA. Tel: (984)-2873700, Fax: (301)-480-3290, Email: [email protected]

Keywords: 8-iso-PGF2α / PGF2α ratio, F2-isoprostanes, phthalates, phthalate alternatives, oxidative stress biomarkers, inflammation,

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Abstract Exposure to environmental chemicals such as phthalates has been linked to numerous

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adverse pregnancy outcomes, potentially through an oxidative stress mediated mechanism.

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Most research examined urinary 8-iso-prostaglandin F2α (8-iso-PGF2α) as the oxidative stress

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biomarker. However, 8-iso-PGF2α also originates from enzymatic sources linked to

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inflammation. Therefore, associations between phthalates and 8-iso-PGF2α could have been

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misinterpreted. To clarify this, the 8-iso-PGF2α/prostaglandin F2α ratio approach was used to

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quantitatively distinguish between inflammation or oxidative stress derived 8-iso-PGF2α and

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estimate their associations with phthalate metabolites in a cohort of 758 pregnant women from

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The Infant Development and Environment Study (TIDES). Most urinary phthalate metabolites

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were associated with a significant increase in 8-iso-PGF2α. For example, a 22.4% higher 8-iso-

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PGF2α concentration (95% confidence interval = 14.4, 30.9) was observed with an interquartile

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range increase in mono-n-butyl phthalate. For most metabolites, associations were observed

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solely with oxidative stress derived 8-iso-PGF2α. In contrast, monocarboxy-isononyl phthalate

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and mono-isononyl phthalate (MNP) were associated with both sources of 8-iso-PGF2α.

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Metabolites of the phthalate alternative 1,2-cyclohexane dicarboxylic acid, diisononyl ester

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(DINCH), were only associated with inflammation-derived 8-iso-PGF2α, which is interesting

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because DINCH metabolites and MNP have structural similarities. In conclusion, phthalates

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metabolites are not exclusively associated with oxidative stress derived 8-iso-PGF2α. Depending

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on the metabolite structure, some are also associated with inflammation derived sources, which

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provides interesting insights in the toxicology of phthalates.


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Introduction Exposure to some phthalates has been linked to developmental abnormalities as well as

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complications with pregnancy such as preeclampsia, preterm birth, and others.1-5 One

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mechanism behind this association has been hypothesized to involve oxidative stress, as

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numerous studies have shown positive associations between oxidative stress biomarkers levels

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and urinary phthalate metabolite concentrations.6, 7 Most studies investigating the association

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between oxidative stress and phthalates have used 8-iso-prostaglandin F2α (8-iso-PGF2α) as

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their biomarker of oxidative stress. 8-iso-PGF2α is a considered the best marker of oxidative

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stress, can be measured in many biospecimens and is elevated in a wide range of human

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medical conditions as well as in response to environmental exposures.8, 9 However,

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interpretation of elevated 8-iso-PGF2α levels as an indicator solely of oxidative stress is no

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longer appropriate.10, 11 Generation of 8-iso-PGF2α occurs both through chemical free radical

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oxidation as well as enzymatic synthesis via the prostaglandin H synthase pathway, which is a

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hallmark of inflammation.12 Epidemiologic evidence suggests that certain phthalate metabolites

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may be associated with markers of inflammation such as interleukin-6 (IL-6) in addition to 8-iso-

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PGF2α.6, 13 Thus, it is important that association between phthalates and oxidative stress as

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measured by 8-iso-PGF2α be reexamined and the contribution of inflammation be taken into

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account.

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Using a novel measurement strategy, the 8-iso-PGF2α/PGF2α ratio,11 it is possible to

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distinguish the proportions of 8-iso-PGF2α that are generated by inflammation vs. oxidative

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stress. By applying this approach to data from The Infant Development and Environment study

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(TIDES), a cohort of pregnant women, it is now possible to accurately interpret the mechanisms

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behind associations observed in previous studies of phthalates and 8-iso-PGF2α. Additionally, in

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this study we were able to explore new metabolites, including those of di(2-ethylhexyl)



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terephthalate (DEHTP), and 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH, an

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emerging replacement for phthalates in consumer products) in relation to this endpoint.


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Materials and Methods

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Study population

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TIDES is a prospective cohort of pregnant women recruited between 2010-2012 from

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four academic prenatal clinics: University of California, San Francisco; University of Rochester

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Medical Center; University of Minnesota; and University of Washington/Seattle Children’s

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Hospital. The study design and data collection methods have been previously published.14-16

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After meeting the enrollment criteria, at routine prenatal visits, participants completed

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questionnaires and provided spot urine samples. Visit (and sample collection) occurred at the

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participant’s convenience and thus occurred at various times throughout the day. Study

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protocols were approved by the IRBs of each institution as well as the Icahn School of Medicine

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at Mount Sinai. All participants provided signed informed consent before starting any study

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activities.

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Urinary phthalates and phthalate alternatives metabolite measurements

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Urine samples for this part of the study were collected at the third study visit (median

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gestational age 32 weeks) in phthalate-free polypropylene cups.21 Urine samples were shipped

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on dry ice for analysis at the Division of Laboratory Sciences, National Center for Environmental

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Health, Centers for Disease Control and Prevention (CDC).17 The involvement of the CDC did

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not constitute engagement in human subjects research. Total phthalate and phthalate

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alternative metabolite concentrations were measured using automated online solid-phase-

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extraction coupled to isotope-dilution high-performance liquid chromatography tandem mass

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spectrometry, as described in detail elsewhere.18 Quality control samples were analyzed with

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each batch with coefficients of variation within the expected range (CV = 3-7%).

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Participant samples were analyzed in two sets. One subset (n = 169) had been

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previously analyzed in the TIDES study and a second subset (n = 593) was analyzed with an



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expanded phthalate panel for this study in 2017. Depending on the metabolite, the limit of

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detection (LOD) was between 0.2 and 1.2 ng/mL and all blanks were below the LOD. For

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statistical analyses, values below the LOD in the first subset were replaced with the LOD

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divided by the square root of 2, as has been recommended.19 In the second subset, instrument

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reported values were used even when below LOD, per an update in CDC procedure.

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Urinary F2-isoprostane measurements

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F2-isoprostane content was measured in third trimester urine samples by the Eicosanoid

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Core Laboratory at Vanderbilt University Medical Center (Nashville, TN) using established

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methods.20 Specifically, three compounds 8-iso-prostaglandin F2α (8-iso-PGF2α), prostaglandin

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F2α (PGF2α) and 2,3-dinor-5,6-dihydro-15-F2t-isoprostane (F2-IsoP-M), a metabolite of 8-iso-

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PGF2α, were quantified and used as biomarkers in this study. Isotopically labeled standards

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were employed for each compound to provide the most reliable quantitation. Values below the

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LOD (0.101 ng/mL) were assigned the value of the LOD divided by the square root of 2. In

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addition to the measured biomarkers, the origins of 8-iso-PGF2α (chemical and enzymatic lipid

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peroxidation) are needed to conclusively determine oxidative stress as the source of elevated 8-

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iso-PGF2α. The sources of 8-iso-PGF2α are derived from the 8-iso-PGF2α / PGF2α ratio as

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described by van ‘t Erve et al.11 Briefly, when the source of 8-iso-PGF2α is solely from oxidative

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stress, the urine 8-iso-PGF2α/PGF2α would be near 1; if the source was predominantly

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enzymatic, the ratio would be near 0. The exact distribution of 8-iso-PGF2α originating from the

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two pathways (ng/mL 8-iso-PGF2α by chemical lipid peroxidation vs. ng/mL 8-iso-PGF2α by

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enzymatic lipid peroxidation) is calculated with the custom interface for the R package

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“Constrained Linear Mixed Effects (CLME)”21 using formulas determined by van ‘t Erve et al.11

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Statistics

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All measured concentrations of biomarkers of phthalates, phthalate alternatives, and

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oxidative stress were adjusted for urine dilution by correcting for urinary specific gravity (SG) Page 6 of 27 ACS Paragon Plus Environment

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which was measured within 30 minutes of urine collection using a handheld refractometer.22

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Concentrations were corrected using the formula: Mc=M[(1.014-1)/(SG-1)], where 1.014 is the

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median SG in the TIDES population, M is the biomarker concentration as measured, and Mc is

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the SG-corrected marker.6

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Frequencies and counts were used to describe demographic characteristics of the study

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population. Distributions of urinary biomarkers were examined using geometric means,

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geometric standard deviations, and selected percentiles. For all analyses, urinary biomarkers

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were natural log transformed. Biomarkers detected in $25,000 per year (73.1%). Samples were collected

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at median 32.7 weeks gestation. Most participants reported no smoking (88%) or alcohol use

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(84.3%) as well as frequent consumption of a prenatal vitamin (86.3%).

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Structures and names for each phthalate and DINCH metabolite measured are

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presented in Table 2 in order from lowest to highest molecular weight of the parent compound.

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Distributions of urinary phthalate and DINCH metabolites in this population are shown in Table

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3. Correlation between the measured metabolites is shown in Supplemental Table 1 and is

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highest among metabolites that share a common parent phthalate. Most metabolites had a

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relatively high detection rate except for the DINCH metabolites, one DEHTP metabolite, and

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mono-isononyl phthalate (MNP), which were detected in less than 25% of samples. Typically,

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metabolites of lower molecular weight phthalates were present in higher concentrations than

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those of higher molecular weight phthalates. Highest concentrations were for mono-ethyl

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phthalate (MEP), monocarboxyoctyl phthalate (MCOP), and mono-n-butyl phthalate (MBP), with

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geometric means of 41.7, 15.9 and 9.8 ng/mL, respectively. The two DINCH metabolites

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(monohydroxy-isononyl ester, 1,2-cyclohexane dicarboxylic acid [MHiNCH] and monocarboxy-

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isooctyl ester, 1,2-cyclohexane-dicarboxylic acid [MCOCH]) were detected at the lowest

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frequency and had geometric mean (geometric standard deviation) values of 0.3 ± 2.5 and 0.4 ±

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2.0 ng/mL, respectively. The terephthalate metabolite mono(2-ethyl-5-carboxypentyl)

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terephthalate (MECPTP) had a high detection rate and a geometric mean concentration of 3.2

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ng/mL. Concentrations of each phthalate and phthalate alternative metabolite by demographic Page 8 of 27 ACS Paragon Plus Environment

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factors are presented in Supplemental Table 2. In general, higher phthalate metabolite

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concentrations were observed in urine of women who were African American compared to

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White or Other race/ethnicity, had a lower education or income level, who smoked or consumed

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alcohol in pregnancy, and who had a higher BMI. Interestingly, for the DINCH metabolites, the

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reverse was observed.

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The urinary oxidative stress biomarkers were detectable in all study participants (Table

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3). In this population of pregnant women, specific gravity corrected PGF2α levels were on

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average twice as high as the corrected 8-iso-PGF2α levels. The average ratio of 8-iso-PGF2α /

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PGF2α was 0.47 ± 1.89, which indicates the majority of 8-iso-PGF2α in the urine samples was

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formed chemically. Associations between the oxidative stress biomarkers and demographic

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characteristics are presented in Supplemental Table 3 and have been previously discussed for

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this study population.23

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Adjusted associations between the three measured urinary oxidative stress biomarkers

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and urinary phthalate and DINCH metabolites are presented in Table 4. All phthalates

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metabolites except for MEP had significant positive associations with 8-iso-PGF2α. Among

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these, the associations that were greatest in magnitude were observed with mono(2-ethyl-5-

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hydroxyhexyl) phthalate (MEHHP), mono-isobutyl phthalate (MiBP), and MBP, where an IQR

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difference in urinary concentration was associated with a 21.5, 17.8 and 15.3% respective

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elevation in 8-iso-PGF2α levels. Among the phthalates treated dichotomously, the largest

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magnitude association was observed for MNP with a 19.6% difference in 8-iso-PGF2α levels

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between detect vs. non-detect individuals. Certain phthalate metabolites were positively

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associated with PGF2α, specifically MNP (20.3% higher levels with detection) and monocarboxy-

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isononyl phthalate (MCNP; 13.5% higher levels with an IQR difference). Smaller but still

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significant associations were observed for MiBP and mono(2-ethylhexyl) phthalate (MEHP). All

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phthalates metabolites were also associated with an elevation in the 8-iso-PGF2α metabolite (F2Page 9 of 27 ACS Paragon Plus Environment


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IsoP-M) levels. In comparison to the parent 8-iso-PGF2α, associations between phthalate

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metabolites and F2-IsoP-M were smaller in magnitude, with larger associations being observed

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only for MEP and the non-specific phthalate metabolite mono(3-carboxypropyl) phthalate

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(MCPP). For metabolites of the phthalate alternatives, detection of the two DINCH metabolites

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(MHiNCH and MCOCH) had large positive associations with PGF2α and F2-IsoP-M. However, no

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association with 8-iso-PGF2α was observed. For the two DEHTP metabolites, positive

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associations were observed with all measured biomarkers except between MECPTP and F2-

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IsoP-M. All unadjusted associations were similar and are presented in Supplemental Table 4.

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With the significant positive associations observed for certain metabolites with 8-iso-

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PGF2α as well as PGF2α, it is important to investigate the source of 8-iso-PGF2α generation to

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mechanistically interpret the findings. The 8-iso-PGF2α / PGF2α ratio was used to distinguish

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between enzymatic (i.e., PGHS) and chemical (i.e. oxidative stress) sources of 8-iso-PGF2α.

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Contribution from the chemical source represents traditional oxidation of arachidonic acid by

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free radicals and/or reactive oxygen species, often referred to as oxidative damage, whereas

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enzymatic sources are through direct synthesis by PGHS which is induced during inflammation.

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Distinction between the two sources was determined for only those metabolites which had

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significant associations with 8-iso-PGF2α in the adjusted linear model, therefore DINCH

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metabolites and MEP were excluded. Generally, phthalates and DEHTP metabolites had

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positive associations only with the chemical pathway (6.9 – 23.8% change with an IQR increase

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in metabolite concentration, Figure 1). Exceptions to this rule were the two phthalate

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metabolites MCNP and MNP which were associated only with the enzymatic pathway. MNP

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was only statistically significantly associated with the enzymatic pathway in the unadjusted

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linear model, and MCNP was significant in both models. No other phthalate or DINCH

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metabolites were associated with changes in 8-iso-PGF2α derived from the enzymatic pathway.

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Effect estimates for adjusted and unadjusted associations for all metabolites and the enzymatic


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and chemical sources of 8-iso-PGF2α are presented in Supplemental Tables 5 and 6,

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respectively. Lastly, results from models of raw concentrations with the inclusion of specific

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gravity as a covariate in the model produced similar results (Supplemental Tables 7 and 8).

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Effect modification of additional variables on the association between phthalate or

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DINCH metabolites and biomarker concentrations were examined. Analyses were limited to

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investigating effects on 8-iso-PGF2α and those phthalates or DINCH metabolites that previously

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showed a significant association. Modification by maternal race/ethnicity was investigated, as

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levels of urinary oxidative stress biomarkers are notably different in White mothers compared to

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those of other races/ethnicities. Additionally, consumption of fish oil supplements was

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investigated as there had been previous reports of potential effects supplementation on 8-iso-

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PGF2α levels.24 Fish oil supplementation would specifically diminish associations with chemically

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generated 8-iso-PGF2α through its potential to act as an antioxidant. No effect modification by

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maternal race/ethnicity was observed (data not shown). However, among the 172 women self-

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reporting fish oil supplementation, we did note large changes in the associations observed

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between 8-iso-PGF2α and five phthalate metabolites: MBP, mono(3-hydroxybutyl) phthalate

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(MHBP), monobenzyl phthalate (MBzP), MiBP, and mono-hydroxyisobutyl phthalate (MHiBP)

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(Figure 2).



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Discussion In this study, the association between urinary concentrations of metabolites of phthalates

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and DINCH in pregnant women and urinary F2-isoprostane levels were reinterpreted using the

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novel 8-iso-PGF2α/PGF2α ratio approach. The results show that most phthalates are indeed

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associated with the chemical pathways specifically reported before. However, certain phthalate

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and phthalate alternative metabolites with a specific structure were associated with the

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enzymatic pathway and not the chemical as had been previously assumed. This suggests that

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there is a structure-dependent relationship driving different mechanisms of toxicity for phthalates

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and phthalate alternative metabolites, and there is not simply a general rule for this class of

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compounds.

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The distribution of phthalate and DINCH metabolites in this population were overall

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similar to previously reported values in pregnant as well as non-pregnant women. For the most

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concentrated metabolites MEP and MCOP, levels were higher than the National Health and

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Nutrition Examination Survey (NHANES) reported values and levels of MBP were slightly lower

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(geometric means of 41.7, 15.9 and 9.8 ng/mL compared to 56.7, 6.3, and 11.1 ng/mL,

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respectively).25 The two DINCH metabolites, MHINCH and MCOCH, were detected at the

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lowest frequency (36% and 32%, respectively) and had geometric mean values of 0.3 and 0.4

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ng/mL, respectively. Low detection rates (56% and 30%, respectively) as well as low mean

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values (0.4 and 2.7 ng/mL, respectively) were similarly reported previously in NHANES.25

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In previous reports, most urinary phthalate metabolites, except for metabolites of low

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molecular weight phthalates like MEP, have been associated with higher levels of 8-iso-PGF2α

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in both pregnant and non-pregnant women as well as in men and children.6, 7, 26-28 The

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associations with 8-iso-PGF2α reported in this work are comparable to other studies in pregnant

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women, although slightly smaller in magnitude.6, 7, 26 This is most likely due to the change in


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analytical technique used (GC/MS in the present study compared to enzyme linked

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immunosorbent assay [ELISA]). Due to antibody cross-reactivity with PGF2α, the ELISA assay

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tends to overestimate the amount of 8-iso-PGF2α, and if the urinary phthalate metabolite is

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associated with both 8-iso-PGF2α and PGF2α then the association would consequently be

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overestimated. This does not happen when 8-iso-PGF2α is measured by GC/MS and is able to

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precisely identify 8-iso-PGF2α.

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The phthalate and DINCH metabolites MNP, MHBP, MHiBP, MHiNCH, and MCOCH

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have only been studied in relation to 8-iso-PGF2α in a small study of non-pregnant women.28 Wu

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et al. observed associations between MHiBP and MNP and elevated 8-iso-PGF2α in that study,

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but no associations were observed for MHiNCH, MHBP, or MCOCH. Similarly, we also

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observed positive associations for MHiBP and MNP but not for MHiNCH and MCOCH.

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However, we did observe a positive association with MHBP. The differences between the two

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studies likely arise from using different methodologies and sample sizes but could also be

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attributed to the difference in pregnancy status.

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This study is the first to report on associations with MECPTP and mono-2-ethyl-5-

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hydroxyhexyl terephthalate (MEHHTP), metabolites of DEHTP, which is used as a replacement

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for di(2-ethylhexyl) phthalate (DEHP). Much like DEHP metabolites, the DEHTP metabolites are

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also associated with 8-iso-PGF2α as well as the 8-iso-PGF2α metabolite. This study is also the

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first to report on the associations between phthalates metabolites and PGF2α which

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independently is an indicator of inflammation.

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In addition, we explored the sources of the higher levels of 8-iso-PGF2α by employing the

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new ratio approach to quantify the chemical vs. enzymatic (i.e., oxidative stress vs. inflammation

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derived) sources of 8-iso-PGF2α. It is important to know the origins of 8-iso-PGF2α because each

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distinct pathway is associated with different secondary health outcomes. In addition,



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misclassifying sources can lead to the design of ineffective strategies to diminish toxicities

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associated with high exposure to phthalates. Even though the equations designed to quantify

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the amounts of chemical and enzymatic lipid peroxidation were derived from in vitro

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experiments and later confirmed in animal and human studies,10, 11, 29 there is no reason to

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expect that the underlying chemistry and enzymes operate differently in pregnant women than

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in other human or animal populations. In addition, the chemistry is not altered for different

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biospecimens with the exception of serum, in which enzymatic F2-isoprostanes are formed ex

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vivo, tainting the results.30 For most phthalates, associations in this population were only

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observed with chemically derived 8-iso-PGF2α suggesting an association between phthalate

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concentration and oxidative stress. Notable exceptions are MCNP and MNP which both are only

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association with the prostaglandin synthase derived 8-iso-PGF2α, i.e., inflammation-derived.

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Both are high molecular weight isomeric phthalates. The association between MNP and

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inflammation can be substantiated by its structural similarities to DINCH which can be

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synthesized by hydrogenation of DiNP (the parent compound of MNP). The two DINCH

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metabolites are strongly associated with PGF2α and therefore it is plausible that a structure-

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dependent relationship exists where the isononyl group of both compounds could be inducing

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inflammation. The higher levels of inflammatory markers may furthermore be linked to

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hypertrophy of the thyroid observed in general toxicity studies on DINCH;31-33 thyroid

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hypertrophy has been shown to increase prostaglandin H synthase activity.34

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There is experimental evidence behind our findings of diverse structure-dependent

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effect. Specific phthalates have been shown to induce oxidative stress by activating Kupffer

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cells, increasing the production of free radicals in vivo.35 However, there is also evidence that

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certain phthalates metabolites, such as MCNP and some of the DEHP metabolites, are

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associated with increases in inflammation markers such as IL-6 in pregnant women.6 The ability

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to differentially induce these two enzymes through a structure activity / receptor driven


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mechanism would explain the diverse effects of the phthalate and phthalate alternative

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metabolites. It is therefore important that the sources of 8-iso-PGF2α be distinguished before

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changes in 8-iso-PGF2α can be mechanistically interpreted.

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Lastly, we explored effect modification of the association between urinary phthalate

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metabolites and 8-iso-PGF2α by fish oil supplementation. Fish oil supplements are commonly

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taken during pregnancy as a source of omega-3 fatty acids such as docosahexaenoic acid and

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eicosapentaenoic acid to support fetal brain development.36 In addition to being essential for

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healthy development, omega-3 fatty acids have also been shown, in some cases, to inhibit 8-

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iso-PGF2α formation, presumably by scavenging free radicals.37 A marked reduction in the

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association between 8-iso-PGF2α and metabolites of three parent compounds (di-n-butyl

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phthalate, di-isobutyl phthalate, and benzylbutyl phthalate) was observed for pregnant women

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supplementing with fish oil. Interestingly, all metabolites from these three parent compounds

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were associated with higher levels of 8-iso-PGF2α through the chemical pathway specifically.

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Fish oil contains high concentrations of potential anti-oxidants which would decrease 8-iso-

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PGF2α formed through the chemical pathway only.24 The effect modification of fish oil

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supplementation altering the association with the chemical derived 8-iso-PGF2α strengthens the

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conclusions of the involvement of oxidative stress in the toxicity mechanism of specific

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phthalates.

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There are some limitations to the analysis in this study. Biomarkers were measured in a

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single urine specimen collected in the third trimester of pregnancy. Given the quick metabolism

336

of phthalates and likely episodic nature of the exposures, concentrations of metabolites in urine

337

represent only recent exposures, whereas urinary 8-iso-PGF2α represents a compounded effect

338

of a much longer duration relative to phthalate metabolites. This could have led to measurement

339

error. Additionally, these findings may not be generalizable outside of this specific population of

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pregnant women (i.e., to other pregnant women, non-pregnant women, or men). Finally, we did Page 15 of 27 ACS Paragon Plus Environment


Page 16 of 30

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not have complete information on time of day of urine sample collection, which could have led to

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residual confounding. Despite these limitations, this study has numerous strengths. In addition

343

to a larger sample size compared to previous studies, we measured multiple F2-isoprostane

344

biomarkers and used the ratio approach to better understand the biological mechanisms driving

345

our associations. This approach is currently the most accurate for determining oxidative stress

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and its origins.11

347

In summary, we observed that phthalate and phthalate alternative metabolites have

348

associations 8-iso-PGF2α that arise from different pathways, including chemical oxidative stress

349

as well as inflammation. Accurately determining the mechanism behind elevated 8-iso-PGF2α

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can give us more information to determine the overall toxicological effects of phthalate exposure

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on human health and provide insights into the design of future intervention strategies to reduce

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or alleviate toxicity and human health hazards.


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Acknowledgements

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The author gratefully acknowledges the study coordinators: Garry Alcedo, Sarah

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Caveglia, Alana Cordeiro, and Stacey Moe. We also acknowledge Drs. Stephanie London and

356

Maria Kadiiska for their scientific input and feedback, and Dr. Manori Silva, Ella Samanda, Jim

357

Preau, and Tao Jia for the quantification of the phthalates and DINCH metabolites. This work

358

was supported by the Intramural Research Program, National Institutes of Health, National

359

Institute of Environmental Health Sciences (ZIA103313) and the extramural NIEHS grants R01

360

ES016863-04 and P30 ES005022.

361

Conflict of Interest

362

The authors have declared that no conflict of interest exists. The findings and

363

conclusions in this report are those of the authors and do not necessarily represent the official

364

position of the Centers for Disease Control and Prevention. Use of trade names is for

365

identification only and does not imply endorsement by the CDC, the Public Health Service, or

366

the US Department of Health and Human Services.

367 368 369 370 371 372

Abbreviations:

8-iso-PGF2α: 8-iso-prostaglandin F2α PGF2α: prostaglandin F2α F2-IsoP-M: 2,3-dinor-5,6-dihydro-15-F2t-isoprostane

Supporting information

373

Spearman correlation matrix of specific gravity-corrected urinary phthalate and phthalate

374

alternative metabolites; Urinary phthalate and phthalate alternative metabolite geometric means

375

by demographics; Urinary oxidative stress biomarker geometric means by demographics;

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Unadjusted associations between urinary phthalate and phthalate alternative metabolites and

377

measured oxidative stress biomarkers; Adjusted associations between urinary phthalate and

378

phthalate alternative metabolites and derived oxidative stress biomarkers; Unadjusted



Page 18 of 30

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associations between urinary phthalate and phthalate alternative metabolites and derived

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oxidative stress biomarkers; Adjusted association between uncorrected urinary phthalate and

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phthalate alternative metabolites and measured oxidative stress biomarkers with specific gravity

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in the model; Adjusted association between uncorrected urinary phthalate and phthalate

383

alternative metabolites and derived oxidative stress biomarkers with specific gravity in the

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model.

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Figure Legends

518 519

Figure 1: Percent change (95% confidence interval) in chemical and enzymatic derived 8-

520

iso-PGF2α and PGF2α in association with an interquartile range increase in urinary

521

phthalate metabolite concentration. Results from linear model adjusted for maternal

522

Race/Ethnicity, education, income, and body mass index. *Denotes significant association after

523

adjustment, p < 0.05. $Denotes significant association only in unadjusted model, p < 0.05

524

Figure 2: Percent change (95% confidence interval) in 8-iso-PGF2α in association with an

525

interquartile range increase in urinary phthalate metabolite concentration from models

526

stratified by fish oil supplementation. Results from linear model adjusted for maternal

527

Race/Ethnicity, education, income, and body mass index. *Denotes significant difference

528

between groups, p < 0.05, from linear adjusted model with an interaction term between fish oil

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supplementation and exposure.

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Page 23 of 30


Table 1: Study population demographic characteristics. Characteristic Continuous variables Maternal age (years) Pre-pregnancy BMI (kg/m2) Gestational age at urine collection (weeks) Categorical variables Race/Ethnicity White African-American Other Study Center University of California San Francisco University of Minnesota University of Rochester Medical Center University of Washington Education =College graduate Missing Income $75k per year Missing Current smoking No Yes Missing Current alcohol use No Yes Missing Prenatal vitamins No Yes Missing Body mass index at study visit

Phthalates and phthalate alternatives have diverse ... - ACS Publications

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