Background
Oxidative stress (or the imbalance between the production of reactive oxygen species and the body’s ability to readily detoxify these reactive intermediates and repair the resulting damageReference Montuschi, Barnes and Roberts 1 ) may provide an underlying pathway linking the observed associations between environmental exposures to phthalates and childhood obesity. Prenatal phthalate exposure has been associated with oxidative stress, measured by concentrations of isoprostane in urine samples of pregnant women,Reference Holland, Huen and Tran 2 , Reference Ferguson, McElrath, Mukherjee, Loch-Caruso and Meeker 3 and by several other biomarkers including 8-hydroxydeoxyguanosine and malondialdehyde.Reference Ferguson, Loch-Caruso and Meeker 4 , Reference Hong, Park and Park 5 8-isoprostanes are prostaglandin-like compounds formed non-enzymatically by free radical catalyzed peroxidation of arachidonic acid.Reference Kaviarasan, Muniandy, Qvist and Ismail 6 Isoprostanes are formed in vivo in the phospholipid domain of cell membranes and circulating lipoproteins. Once they are cleaved by phospholipases, 8-isoprostanes are released extracellularly and are excreted in urine. According to pharmacokinetic and metabolic studies, the half-life of 8-isoprostanes in humans is ~16 min.Reference Kaviarasan, Muniandy, Qvist and Ismail 6 , Reference Basu 7 Previous studies have found that a single urine sample can reflect the levels of 8-isoprostane over a 1-year period [intra-class correlation coefficient for urinary 8-isoprostane is 0.69].Reference Wu, Cai and Xiang 8 Isoprostanes have been used in numerous studies as a reliable biomarker of oxidative stress with low day-to-day variability in healthy subjects.Reference Montuschi, Barnes and Roberts 1 , Reference Morrow and Roberts 9 – Reference Rossner, Rossnerova and Spatova 12 In addition, isoprostane levels have been associated with exposure to environmental pollutants, obesity and other diseases.Reference Rossner, Rossnerova and Spatova 12 – Reference Montuschi, Barnes and Roberts 14 For instance, isoprostane concentrations have been found to be significantly elevated in obese adults.Reference Bloomer and Fisher-Wellman 15 – Reference Il’yasova, Morrow and Wagenknecht 20 However, limited data are available on isoprostane concentrations and body mass in children. In a cross-sectional study of 42 multi-ethnic obese children and adolescents, 8-isoprostane was significantly associated with body mass index (BMI), waist circumference and 24-h systolic blood pressure.Reference Ostrow, Wu and Aguilar 21 In a study of 72 Japanese children and adolescents, increased plasma isoprostane was associated with visceral fat, high molecular weight adiponectin and metabolic complications in obese children.Reference Araki, Dobashi, Yamamoto, Asayama and Kusuhara 22
Phthalates are ubiquitous in the environment; they are widely used as plasticizers, stabilizers and solubilizing agents in numerous consumer and industrial products such as cosmetics, medical devices, building materials, household furnishings and flexible plastics. 23 Detectable levels have been found in various environmental media including indoor air, household dust, food stuffs and drinking water. Di(2-ethylhexyl) phthalate (DEHP) is one of the most commonly used phthalate plasticizer in industrial products, while diethyl phthalate is frequently detected in consumer products such as shampoos, detergents and cosmetics.Reference Guo and Kannan 24 As a result, humans are exposed through multiple routes including ingestion, inhalation, and dermal absorption. Phthalates can affect the hormonal systems and have been linked to numerous health outcomes related to endocrine disruption, such as obesity, asthma, preterm birth and neurobehavioral problems.Reference Braun, Sathyanarayana and Hauser 25 – Reference Ku, Su and Wen 27
Several cross-sectional studies have reported associations between higher urinary phthalate metabolite concentrations and increased BMI.Reference Yaghjyan, Sites, Ruan and Chang 28 – Reference Hatch, Nelson and Qureshi 33 However, only a few studies have examined effects of prenatal exposure to phthalates on body size of children, with mixed results.Reference Buckley, Engel and Braun 34 – Reference Agay-Shay, Martinez and Valvi 38 For example, Valvi et al.Reference Valvi, Casas and Romaguera 36 found associations of prenatal phthalate metabolite concentrations with higher BMI Z-scores in girls while Maresca et al.Reference Maresca, Hoepner and Hassoun 37 showed that concentrations of phthalate metabolites (excluding those of DEHP) during pregnancy were inversely associated with BMI Z-scores, fat percentage, and waist circumference in boys. Recently, 8-isoprostane concentrations measured during pregnancy were found to mediate the association between phthalate metabolites and preterm birth.Reference Ferguson, Chen and VanderWeele 39
We previously reported in pregnant women participating in the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) study that urinary concentrations of two metabolites of high molecular weight (HMW) phthalates [monocarboxyoctyl phthalate (MCOP), and monocarboxynonyl phthalate (MCNP)] were associated with isoprostane concentrations at 13 weeks of gestation and all metabolites of low molecular weight (LMW) phthalates and some of the metabolites of HMW phthalates (mono(3-carboxypropyl) phthalate, MCPP; MCNP) were associated with isoprostane concentrations at 26 weeks of gestation.Reference Holland, Huen and Tran 2
The goal of the present study was to characterize isoprostane changes in repeated samples in the CHAMACOS children at 5, 9 and 14 years of age and to explore potential association of in utero exposure to phthalates and isoprostane concentrations in children at these three ages. We also examined the relationship of children 8-isoprostane concentrations on their body mass and cardiometabolic characteristics.
Methods
Subjects and study design
CHAMACOS is a longitudinal birth cohort examining the effects of environmental exposures on neurodevelopment, growth and respiratory disease in pregnant women and their children living in Salinas Valley, California.Reference Eskenazi, Bradman and Gladstone 40 A total of 601 pregnant women were enrolled in the CHAMACOS study between October 1998 and 2000, with 536 live births. Mothers enrolled in the CHAMACOS cohort were primarily young (mean±s.d., 26.3±5.2 years), married, low-income, eligible for low income health insurance (Medi-Cal eligible), Mexican-born, and Spanish-speaking. Most were either farm workers themselves or lived with farm workers at the time of enrollment.
Pregnant women were interviewed at ~13 and 26 weeks of gestation, after delivery, and at the time of each child assessment. At the time of the pregnancy interviews (gestational weeks 13 and 26), maternal phthalate metabolite concentrations were measured in urine to capture early and late pregnancy exposure and to assess variations in phthalate exposure across pregnancy. Detection frequencies of all phthalate metabolites in CHAMACOS participants ranged from 90 to 100%, as described previously in more detail.Reference Holland, Huen and Tran 2 , Reference Huen, Calafat and Bradman 41 We measured 8-isoprostane concentrations in urine from a subset of 5- (n=257), 9- (n=250) and 14- (n=260) year-old CHAMACOS children based on the availability of urine samples at all three time-points. Few of the children born to CHAMACOS mothers were preterm (8%) or had a low birthweight (4%). Their birthweight ranged from 1530 to 4885 g, with an average of 3441 g. This subset was not significantly different from the main CHAMACOS cohort in most demographic and exposure parameters (child sex, maternal country of birth, race, poverty index, education, pre-pregnancy BMI, smoking and alcohol use). However, mothers in this sample tended to be slightly older, lived in the United States longer, and more likely to be multiparous. Study protocols were approved by the University of California, Berkeley and the Centers for Disease Control and Prevention (CDC) Committees for Protection of Human Subjects. Written informed consent was obtained from all mothers, and verbal assent was obtained from children at 5 and 9 and written assent at 14 years of age.
Anthropometric and cardiometabolic data
Body weight was assessed using an electronic mother-baby scale (Tanita, Model 1582) at age 5 years and a bioimpedance electronic scale (Tanita, Model X) at age 9 and 14 years. Height without shoes was measured using a wall-mounted stadiometer and waist circumference was measured at the iliac crest with a measuring tape. Height and waist circumference measurements were performed in triplicate and then averaged. To calculate BMI, weight in kilograms was divided by height in meters squared. These values were compared to CDC reference data to generate BMI Z-scores standardized by sex and age. Using the age and sex-specific BMI cutoffs from the 2000 CDC child growth charts, children were categorized as normal and overweight (<85th and ⩾85th percentile, respectively). One participant with an extreme BMI (56.6 kg/m2) was excluded from the analyses. Blood pressure measurements were obtained while the participant was at rest, using a Dinamap 9300 sphygmomanometer. Fasting blood samples were collected from children at 9 and 14 years of age, but were not available for 5-year-old children. Serum samples were analyzed at a local laboratory (Quest Diagnostics, San Jose, CA) for glucose and a standard lipid panel including triglycerides, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, non-HDL cholesterol and cholesterol/HDL ratio.
8-Isoprostane and creatinine measurements
Spot urine samples collected from mothers during pregnancy and from children at 5, 9 and 14 years were stored at −80°C at the School of Public Health Biorepository in UC Berkeley until analysis. Concentrations were quantified using a urinary 8-isoprostane competitive enzyme-linked immunosorbent assay (ELISA) kit (Oxford Biomedical Research, Rochester Hills, MI, USA) as previously described.Reference Holland, Huen and Tran 2 Briefly, urine samples were pre-treated with β-glucuronidase (Oxford Biomedical Research) before running the ELISA. The limit of detection (LOD) for 8-isoprostane concentration was 0.08 ng/ml. Undetected oxidative stress measures were replaced with the LOD divided by the square root of 2. Additional quality assurance/quality control provisions included repeats of 5% of samples and blanks, and internal lab controls with good reproducibility of isoprostane (coefficient of variation <7%).
Creatinine levels in children’s samples were analyzed using a urinary creatinine ELISA kit. Samples were randomized across plates and the coefficient of variation was less than 3%. All isoprostane concentrations were adjusted to account for urinary dilution by dividing isoprostane concentrations (ng/ml) by creatinine levels (mg/dl) with results reported in ng/mg creatinine.Reference Holland, Huen and Tran 2
Maternal urinary phthalate metabolite measurements
A total of 11 phthalate metabolite concentrations were measured at the CDC in maternal urinary samples using online solid phase extraction coupled with isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.Reference Silva, Samandar and Preau 42 Briefly, these included metabolites of three LMW phthalates [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP)], four metabolites of DEHP [mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP)], and four metabolites of other HMW phthalates [monobenzyl phthalate (MBzP), MCPP, MCOP, MCNP]. Quality control procedures included laboratory blanks, calibration standards, and spiked controls with high and low concentrations. For concentrations below the LOD, the actual instrument reading value was used, if available, or a measurement below the LOD was imputed from a log-normal distribution using the ‘fill-in’ method described in Lubin et al. (2004), if not.Reference Lubin, Colt and Camann 43 All phthalate metabolite concentrations were corrected for creatinine (µg/g creatinine) by dividing metabolite concentrations (µg/l) by creatinine levels (g/l).
Statistical analysis
Urinary 8-isoprostane and urinary phthalate metabolite concentrations were log10 transformed to normalize their distributions in all statistical analyses, with the exception of summary statistics describing their distributions by age and sex as reported in Tables 1 and 2. In addition, all descriptive statistics and correlation analyses used creatinine-corrected 8-isoprostane concentrations. Regression analyses, however, used uncorrected 8-isoprostane concentrations, instead including creatinine levels as a covariate in the model. Before analysis, two subjects, whose isoprostane concentrations at age 14 years were outliers (>3 s.d. from mean), were dropped. However, the results did not change appreciably when outliers for BMI or isoprostane levels were included in the analysis. Anthropometric and cardiometabolic parameters were log10 transformed to reduce the influence of outliers and to achieve homogeneity of variance.
Population characteristics of CHAMACOS children at ages 5, 9 and 14 years
BMI, body mass index.
a Total number of observations varies due to missing data.
Urinary concentrations of 8-isoprostane (creatinine-adjusted) in 5, 9 and 14-year-old boys and girls (ng/mg creatinine).
P-values are for t-tests comparing isoprostane levels between boys and girls at each age.
To make pairwise comparisons of isoprostane concentrations between age groups and with maternal concentrations, Pearson’s correlation coefficients were calculated. Differences in mean isoprostane concentrations by sex were determined by t-tests. To determine cross-sectional associations of isoprostane concentrations at ages 5, 9 and 14 years with physical parameters of obesity at the concurrent age, we calculated Pearson’s correlation coefficients and performed linear regression models adjusting for sex and creatinine.
To model longitudinal changes of 8-isoprostane concentration by age, we performed generalized estimating equations (GEE) with an exchangeable correlation structure and controlled for creatinine, sex and BMI. We also performed mixed effects regression models, including a random effect for CHAMACOS participant, to confirm findings from the GEE models and to assess associations of child sex, age and concurrent BMI with isoprostane concentrations. Mixed effect models account for correlated measures of isoprostanes across the three time points.Reference Laird and Ware 44 Additional covariates, including gestational age and birthweight, were also considered for inclusion in the models based on some previous reports from other studies.Reference Ferguson, McElrath, Ko, Mukherjee and Meeker 26 , Reference Ferguson, McElrath and Meeker 45 , Reference Comporti, Signorini and Leoncini 46 However, these variables were not significantly associated with phthalate or isoprostane levels in CHAMACOS participants. To examine associations of prenatal phthalate metabolite concentrations with isoprostane concentrations in children, separate linear regression models were constructed for each phthalate metabolite (11 individual metabolites and 3 sum measures) at each maternal visit during pregnancy (13 and 26 weeks of gestation) with isoprostane concentrations measured in children at ages 5, 9 and 14 years, controlling for sex and BMI. Linear regression models controlling for BMI, sex and creatinine were used to determine associations of isoprostane concentrations with cardiometabolic parameters (fasting glucose, triglycerides, LDL and HDL cholesterol, HDL cholesterol ratio, non-HDL cholesterol, total cholesterol, and systolic and diastolic blood pressure) at the concurrent age in 9 and 14-year-old children. All statistical analysis was performed in STATA (version 12.1; STATA Corp., College Station, TX). P-values <0.05 were considered significant.
Results
CHAMACOS study participant characteristics
Population characteristics of the CHAMACOS children at ages 5, 9 and 14 years are summarized in Table 1. Among participants in this study, there were similar numbers of boys and girls at each age. At 9 years of age, 36% of girls had entered puberty, compared with 10% of boys. All CHAMACOS participants, with the exception of 9% of males, reached puberty at 14 years of age. As expected, mean body composition measurements including weight, height, waist circumference and BMI increased with age. The prevalence of overweight in CHAMACOS children (⩾85th percentile) was 55, 57 and 57% at ages 5, 9 and 14 years, respectively; most of the children whose BMI was >85th percentile were obese (BMI Z-scores ⩾95th percentile).
Analysis of 8-isoprostane concentrations at different ages
The distribution of 8-isoprostane concentration varied by age, but the medians for 5, 9 and 14-year-old children were similar (3.1, 2.9 and 2.6 ng/mg creatinine, respectively, P=0.73; Table 2). Isoprostane concentrations at 14 years had the greatest variability, ranging from 0.2 to 107.1 ng/mg creatinine when compared with 5- (0.25 to 67.7 ng/mg creatinine) and 9-year-old (0.7–51.1 ng/mg creatinine) children. There was no significant correlation between isoprostane concentrations at each of the three time points (all r<0.10). The relatively large time gaps (4–5 years) between the isoprostane assessments may explain this finding. Although the mean 8-isoprostane concentrations were higher in boys than girls at ages 5, 9 and 14 years, the differences were not statistically significant (Table 2). We did not find any significant associations between age and urinary 8-isoprostane after adjusting for child sex and BMI. We also used mixed effects models, and the results did not change.
Maternal isoprostane urinary concentrations at 13 and 26 weeksReference Holland, Huen and Tran 2 were modestly correlated (all r<0.30) with their children’s concentrations at 5, 9 or 14 years (not shown). Maternal age and parity were not significantly associated with child isoprostane concentrations. Children whose mothers lived in the United States for >11 years (at the time of enrollment) had slightly lower log10 isoprostane concentrations at 9 years of age compared with children whose mothers had lived in the United States for <1 year [β (95% CI): −0.15(−0.27, −0.03)].
Examination of the relationship between isoprostane with body mass
After adjusting for sex, we did not observe significant associations between current isoprostane concentrations and children’s BMI, BMI Z-score or waist circumference (Supplementary Table 1). Isoprostane concentrations did not differ significantly by weight categories (normal v. overweight/obese) within each age group. For example, at age 5 years, mean isoprostane concentrations were 0.49 ng/mg creatinine in normal weight children and 0.50 ng/mg creatinine in overweight/obese group (P=0.72); at 9 and 14 years, isoprostane values were also similar between two BMI groups (P=0.06 and 0.3, respectively).
Isoprostane and cardiometabolic parameters in 9- and 14-year-old children
Cardiometabolic parameters in 9 and 14-year-old children are summarized in Table 3. Among 9-year olds, no children had elevated over the reference range fasting glucose levels (⩾110 mg/dl), 25% had high triglyceride levels (⩾110 mg/dl), 15% had low HDL levels (⩽40 mg/dl), and 7.5% had high blood pressure (⩾90th percentile for age and sex). These numbers increased at age 14 with 0.4% of the subjects having high fasting glucose levels, 35% with high triglycerides, 23% with low HDL and 18% with high blood pressure. We found no significant associations of isoprostane with cardiometabolic parameters at 9 years of age. However, at 14 years, isoprostane urinary concentrations were positively and significantly associated with concurrent measures of total cholesterol (β: 95% CI=0.88: 0.20–1.57, P=0.01), systolic blood pressure (β: 95% CI=0.01: 0.002–0.015, P=0.02) and diastolic blood pressure (β: 95% CI=0.011: 0.001–0.020, P=0.02) (Table 3).
Cardiometabolic parameters and their cross sectional associations with 8-isoprostane urinary concentrations in CHAMACOS children at 9 and 14 years
LDL, low-density lipoprotein; HDL, high-density lipoprotein; BMI, body mass index.
β coefficients, 95% confidence intervals and P-values on the right side of the table represent results of each individual linear regression model of isoprostane concentrations (dependent variable) with cardiometabolic parameters (independent variable) at the concurrent age in 9 and 14-year-old children. All models controlled for BMI, sex and creatinine. Triglycerides, cholesterol HDL ratio and total cholesterol are log transformed.
Bolded values are significant, P<0.05.
a Total number of observations varies due to missing data.
b Values are significantly different from 9 and 14 years, P<0.05.
Relationship between urinary prenatal phthalate metabolite concentrations and isoprostane concentrations
There were no significant associations between phthalate metabolite concentrations at 13 weeks of gestation and isoprostane concentrations in 5-year-old children, after adjusting for sex and BMI (Table 4). Associations between prenatal maternal phthalate metabolite concentrations and isoprostane concentrations in 9-year olds were also not statistically significant with the exception of three metabolites of HMW phthalates – MBzP, MCOP and MCNP, which were negatively associated with isoprostane (Table 4). Isoprostane concentrations in 14-year-old children were positively associated with concentrations of maternal MEHP, MECPP and ∑DEHP metabolites during pregnancy [β: 95% CI=0.10 (0.001, 0.20), 0.14 (0.001, 0.29), 0.14 (0.001, 0.28), respectively] and borderline significantly associated with MEHHP and MEOHP concentrations [β: 95% CI=0.12 (−0.01, 0.25), 0.10 (−0.01, 0.22), respectively]. None of the prenatal phthalate metabolite concentrations at 26 weeks of gestation were associated with children’s isoprostane concentrations at either 5, 9 or 14 years of age.
Associations of prenatal maternal urinary phthalate metabolite concentrations at 13 weeks with isoprostane urinary concentrations in their children at 5, 9 and 14 years
MEP, monoethyl phthalate; MBP, mono-n-butyl phthalate; MiBP, mono-isobutyl phthalate; LMW, low molecular weight; MEHP, mono(2-ethylhexyl) phthalate; MEHHP, mono(2-ethyl-5-hydroxyhexyl) phthalate; MEOHP, mono(2-ethyl-5-oxohexyl) phthalate; MECPP, mono(2-ethyl-5-carboxypentyl) phthalate; DEHP, di-2-ethylhexyl phthalate; MBzP, monobenzyl phthalate; MCPP, mono(3-carboxypropyl) phthalate; MCOP, monocarboxyoctyl phthalate; MCNP, monocarboxynonyl phthalate; HMW, high molecular weight.
Separate linear regression models were performed for each phthalate metabolite (11 individual metabolites and 3 sum measures) at 13 weeks of gestation with isoprostane concentrations measured in children at ages 5, 9 and 14 years, controlling for sex and body mass index.
Bolded values are significant, P<0.05.
We used PARAMED, a STATA package, to run mediation analysis testing the hypothesis that oxidative stress mediates the effect of phthalate exposure on cardiometabolic parameters. We investigated the effect of MEOHP and MEHHP on 14-year diastolic blood pressure, as mediated by 14-year isoprostane levels. These parameters were chosen as the only significant results after tests for associations between phthalate metabolite levels in maternal urine samples and cardiometabolic parameters in 14-year-old children. Although we observed a marginally significant natural indirect effect for MEOHP and MEHHP (P=0.08 and 0.06, respectively), they were no longer significant after accounting for child sex and BMI at 14 years of age.
Discussion
In the CHAMACOS longitudinal birth cohort study, we found no differences in isoprostane concentrations by children’s sex or age (ages 5, 9 or 14 years). Maternal concentrations of DEHP metabolites (MEHP, MECPP, ∑DEHP) at 13 weeks of gestation (but not at 26 weeks) were positively associated with isoprostane concentrations in the 14-year-old children and these isoprostane concentrations were positively associated with concurrent measures of total cholesterol and blood pressure, but not associated with BMI, BMI Z-score, and waist circumference (at 5, 9 and 14 years). In contrast, other high molecular weight metabolites (MBzP, MCOP, MCNP) at 13 weeks were negatively associated with isoprostane concentrations at 9 years.
In a prospective study of children at risk for type 1 diabetes, isoprostane concentrations were found to be highest in infancy, decreased nonlinearly until the children reached 6 years of age and then increased until 7 years of age.Reference Kauffman, Sokol and Jones 47 Our results showing no association with age may have differed because our study population was older and more overweight than the other study population. We did not find that isoprostane concentrations were associated with concurrent measures of BMI, BMI Z-score or waist circumference. These findings were unexpected based on available cross-sectional reports showing a positive relationship of isoprostane concentrations and BMI, waist circumference and percent body fat in adolescent children.Reference Warolin, Coenen and Kantor 18 , Reference Ostrow, Wu and Aguilar 21 , Reference Dennis, Ergul, Gower, Allison and Davis 48 One possible explanation for differences in study results is that in our relatively overweight/obese population there is a compensatory mechanism involved in the maintenance of energy homeostasis related to the children’s body mass.Reference Weyer, Pratley and Salbe 49 – Reference Rosenbaum, Leibel and Hirsch 52 Fat oxidation plays an important role in metabolic adaptation because it helps regulate the physiological response to weight gain or loss. Prospective studies indicate that lower baseline levels of fat oxidation (lower half) predict weight gain and individuals are also more likely to sustain weight gain if their metabolic adaptation is weak.Reference Schutz, Tremblay, Weinsier and Nelson 50 , Reference Tremblay and Doucet 51 , Reference Leibel, Rosenbaum and Hirsch 53
Previous studies have found positive relationships between biomarkers of oxidative stress, specifically 8-isoprostane, with important metabolic parameters in both children and adults.Reference Otani 54 , Reference Touyz 55 Our results suggest that isoprostane concentrations were significantly and positively correlated with total cholesterol, systolic and diastolic blood pressure in 14-year children. This is in agreement with similar findings of higher urinary isoprostane concentrations in children with elevated 24-h ambulatory blood pressure.Reference Ostrow, Wu and Aguilar 21 Similarly, another study found that levels of peroxy radicals in urine of obese children and adolescents were positively correlated with total cholesterol, systolic and diastolic blood pressure.Reference Atabek, Vatansev and Erkul 56 These findings suggest that an oxidative stress measure is associated with increased blood pressure and total cholesterol; however, it is not clear whether changes in blood pressure and cholesterol precede changes in oxidative stress or vice versa.
Previously, we observed positive relationships between phthalate metabolite urinary concentrations and isoprostane concentrations in pregnant women in our cohort.Reference Holland, Huen and Tran 2 In the present study, we did not find any significant associations of isoprostane concentrations in the 5-year-old children from the same cohort with either early or late prenatal phthalate exposure nor their average. However, we observed negative associations with MBzP, MCOP and MCNP, all metabolites of HMW phthalates, measured in maternal urine at 13 weeks of gestation with isoprostane concentrations in older CHAMACOS children at age 9 years. In addition, DEHP metabolite concentrations at 13 weeks (MEHP, MECPP, MEHHP and MEOHP) were either significantly or borderline significantly positively associated with isoprostane in 14-year-old children. As no associations between isoprostane concentrations in each of these three childhood time points were observed for prenatal phthalate metabolite concentrations at 26 weeks, our results suggests that 13 weeks of gestation may coincide with a critical window of in utero phthalate exposure that may affect childhood growth and development later in life.
The observed associations between phthalate metabolite concentrations at 13 weeks of gestation and isoprostane concentrations at 9 and 14 years represent novel findings. The negative associations of early pregnancy HMW metabolites with 9-year isoprostanes, as well as the positive associations of DEHP metabolites with 14-year isoprostanes, may indicate potential remodeling of associations during hormonal shifts that occur as youth enter puberty at varying time points. It may also be related to a difference in the effects of these two phthalates during in utero exposure on the developmental processes during childhood and adolescence. For instance, endocrine disrupting chemicals (EDC), including some phthalates, have been linked to puberty onsetReference Fisher and Eugster 57 – Reference Herman-Giddens, Wang and Koch 59 and prenatal EDC exposure has been associated with changes in hormone levels in boys from the CHAMACOS cohort.Reference Eskenazi, Rauch and Tenerelli 60 We found that maternal blood concentrations of brominated diphenyl ether (BDE)-100 and −153 during pregnancy were associated with an increase of the leutenizing hormone levels, and BDE-153 was associated with an increase in testosterone in 12-year-old CHAMACOS boys; while dichlorodiphenyltrichloroethane (DDT), after accounting for Tanner stage, was associated with decreases in the hormone levels. In animal studies, high levels of phthalates have been associated with decreased production of both estrogen and testosterone. 23 In a Japanese youth cohort, grade in school (used as a surrogate of age) was negatively associated with markers of oxidation, potentially mediated by sex hormones.Reference Kogawa, Nishimura, Kurauchi and Kashiwakura 61 Although we have not yet examined the relationships between prenatal phthalate exposure and hormone levels in the CHAMACOS youth, we hypothesize that phthalate exposure sets off a cascade of effects, including hormone and oxidation shifts, which could explain the contrasting relationships between different phthalates in utero, and isoprostanes at 9 and 14 years. It is possible that endocrine disruption caused by phthalate exposure with potential shifts in the hypothalamic–pituitary–gonadal axis could also alter oxidative stress biomarkers.
Although we found significant associations between maternal urinary concentrations of phthalate metabolites at 13 weeks of gestation and isoprostane concentrations in 9 and 14-year-old children, additional research to validate the current findings and examine whether childhood phthalate metabolite concentrations (as yet not available for our cohort) are also associated with isoprostane concentrations would be of interest.
To explore the nature of biological relationships between isoprostanes, phthalates and the cardiometabolic parameters assessed in the current study, we rely on several lines of evidence. Using mediation analysis, we observed a marginal indirect effect for two of the 11 phthalate metabolites on cardiometabolic parameters in 14-year-old children. However, it was abated after accounting for child sex and BMI. It is known that phthalate exposure in utero can potentially disrupt developmental processes that may lead to an increased risk of cardiometabolic diseases later in life.Reference Valvi, Casas and Romaguera 36 Phthalates are agonists for peroxisome proliferator-activated receptors, which play a key role in adipogenesis and lipid accumulation.Reference Desvergne, Feige and Casals-Casas 62 In addition, a relationship between obesity, oxidative stress and an increased risk of cardiometabolic disease is demonstrated in adults and children.Reference Block, Dietrich and Norkus 63 , Reference Lammi, Moltchanova and Blomstedt 64 Phthalate metabolites have been also shown to be associated with markers of inflammation and oxidative stress.Reference Ferguson, Loch-Caruso and Meeker 4 , Reference Jepsen, Abildtrup and Larsen 65
We note several limitations of the current study. Given the high prevalence of overweight and obesity in this Mexican-American cohort, its low socioeconomic status and exposure to other factors typical for an agricultural setting, our results may not be generalizable to other ethnic groups or populations where overweight/obesity rates are lower. The overall percentage of overweight and obese children in our cohort was consistently above 55% at all ages from 5 to 14 years, which is higher than the National Health and Nutrition Examination Survey prevalence of obesity (overweight and obese combined) in Hispanic youth (38.9%) or all U.S. youth aged 2–19 years (31.8%).Reference Ogden, Carroll, Kit and Flegal 66 Our study used indirect measurements for generalized adiposity and central adiposity, which may be less accurate, compared with other direct measurements, such as dual-energy X-ray absorptiometry images. In addition, maternal phthalate metabolites and isoprostane concentrations were measured using spot urine samples. These concentrations may be subject to temporal changes and high within-person variability over time and thus may not accurately reflect average exposures over pregnancy, leading to non-differential misclassification and bias toward the null. However, stable measurements of phthalate metabolites in spot urine samples were reported over relatively long periods of time.Reference Ferguson, McElrath, Ko, Mukherjee and Meeker 26 , Reference Cantonwine, Cordero and Rivera-Gonzalez 67 – Reference Adibi, Whyatt and Williams 69
In conclusion, oxidative stress levels, assessed by average urinary concentrations of 8-isoprostane, in a cohort of mainly obese or overweight Mexican-American children did not appreciably change over ages 5, 9 and 14 years. These concentrations did not differ by sex, BMI, BMI Z-score or waist circumference, however the relationship of 8-isoprostane with three cardiometabolic parameters was statistically significant in 14-year-old children. Total cholesterol, systolic and diastolic blood pressure were positively associated with isoprostane concentrations in 14-year-old children, supporting the hypothesis that oxidative stress may be related to certain cardiometabolic risk factors. The significant relationships between prenatal phthalate metabolite concentrations at 13 weeks (but not at 26 weeks) of gestation with isoprostane concentrations in children at 9 and 14 years suggests that 13 weeks may be a critical period when the fetus is more vulnerable to phthalate exposure.
Acknowledgments
The authors are grateful to the laboratory and field staff and participants of the CHAMACOS study for their contributions. Authors’ contributions: Dr Holland and Ms Vy Tran conceived and designed the study. Ms Tran and Mr Nguyen performed the experiments. Ms Tran and Ms Tindula performed the data analysis with contribution from Dr Huen and prepared the manuscript with important intellectual contribution from Drs Holland, Bradman and Eskenazi. Dr Harley, Ms Kogut and Ms Parra were involved in collection of biological samples and characterization of the health of CHAMACOS children. Dr Calafat and Ms Ye quantified phthalate metabolite concentrations, and contributed to the manuscript. All authors approved the final manuscript.
Financial Support
This publication was made possible by grants R826886 and R82670901 from the U.S. Environmental Protection Agency (EPA) and PO1 ES009605 and R01ES021369 from the National Institute of Environmental Health Sciences (NIEHS). The findings and conclusions in this report are those of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention (CDC), NIEHS and the EPA. Use of trade names is for identification only and does not imply endorsement by the CDC, the Public Health Service, or the U.S. Department of Health and Human Services.
Conflicts of Interest
None.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S2040174416000763
