The prevalence of obesity, insulin resistance, and diabetes in children has increased substantially over the past three decades. While there are many plausible causes, including changes in diet, food marketing, perinatal exposures, decreased physical activity, and thrifty genes (1), endocrine disrupting environmental chemicals such as bisphenols and phthalates may be another factor driving this epidemic. Given that dietary intake is the chief sources of exposure to bisphenols and phthalates, children and adolescents are uniquely vulnerable to these chemicals as they have greater food consumption per unit body weight and will have prolonged exposure over the course of the lifespan (2).

Bisphenol A (BPA) is a synthetic chemical comprised of two phenol rings connected by a methyl bridge, with two methyl groups attached to the bridge. BPA was initially designed as a synthetic estrogen. It is now widely used for its cross-linking properties in the manufacture of polycarbonate plastics and epoxy resin coatings used in food and beverage containers to prevent metal corrosion. It is present in intravenous tubing, including dialysis circuits. Incomplete polymerization and polymer degradation of BPA causes it to leach out of food and beverage containers and dental sealants. (3). While ingestion of contaminated food is a major source of exposure to bisphenols, dental sealants, and thermal receipt papers represent other sources (4–6). Phthalates are esters of phthalic acid that can be either low-molecular weight (LMW) or high-molecular weight (HMW) (Table 1). LMW phthalates, such as di-butyl phthalate (DBP), are frequently added to cosmetic products such as shampoos, perfume, aftershaves, lotions and other personal hygiene products to preserve scent. HMW phathalates are used to soften and increase the flexibility of a wide range of consumer products such as vinyl plastics used in flooring, toys, plastic bags, food packaging and intravenous tubing. Within the HMW category, di-2-ethylhexylphthalate (DEHP) is commonly found in plastic products used during industrial food production and packaging, and its metabolites are often considered a subcategory of phthalates. (7) Since phthalates are not covalently bound, they can easily leach into their surrounding environment (8). While contaminated food is the major source of exposure to HMW phthalates, they can enter the human body through inhalation and dermal absorption (9).

Cross-sectional studies have associated BPA and phthalates with various cardio-metabolic risk factors including albuminuria, increased systolic blood pressure, increased waist circumference, and insulin resistance in children (10). While the mechanisms underlying these relationships are not completely understood, it is proposed that oxidant stress may be the pathophysiological link between environmental chemical exposures and metabolic dysregulation. Studies in adults have demonstrated an association between BPA, phthalate metabolites and increased systemic oxidant stress markers, including serum C-reactive protein (CRP) and gamma glutamyltransferase (GGT) and urinary malondialdehyde and 8-hydroxydeoxyguanosine (11, 12). Moreover, Kim et al observed that exposure to DEHP in the elderly population was associated with a concomitant increase in oxidative stress and insulin resistance (13). This association is corroborated by in vitro and animal studies that demonstrate BPA and DEHP metabolites increase oxidative stress by increasing the levels of reactive oxidant species, disrupting antioxidant defenses and inducing insulin resistance (14).

In light of these concerns and other adverse health effects, both BPA and DEHP were banned from use on children’s articles of care and toys. Six types of phthalates have been banned for use in children's toys and certain child care articles (15) . In addition, the FDA banned bisphenol A from baby bottles and sippy cups in 2012. To comply with these restrictions, there is a general trend towards reformulating consumer products with newer compounds with similar chemical properties. In fact, the most recent reports from the CDC show a decline in DEHP exposures, while exposure to some other phthalates including di-isononyl phthalate (DINP) and di-isodecyl phthalate (DIDP) have increased (16). Similarly, in the case of bisphenols, with manufacturers forced to respond to consumer concerns about the safety of BPA, Bisphenol S (BPS) has been introduced as a substitute in many plastic products. Although exposure to BPA has declined, exposure to BPS is on the rise (17). Very few studies have investigated these newer chemicals and their toxicity profiles.

In the present study, we examined the relationship between exposure to bisphenols and phthalates, including the newer molecules, such as bisphenol S, with oxidant stress, insulin resistance, and endothelial dysfunction in healthy children. Given that the endothelium is a major target for oxidant stress leading to arterial stiffness (18), we assessed the state of the vasculature by non-invasive techniques, namely, pulse wave velocity, brachial artery distensibility, and quantification of microparticles (MPs). The latter are small (<1.5 μm) vesicular fragments of endothelial cells released upon cell injury that have been associated with coronary artery disease (CAD) and many of vascular risk factors(19). Our study is one of the first to examine these relationships in children.

A total of 43 participants enrolled in the study. Two participants were excluded because they were taking medications to treat a chronic illness (attention deficit disorder), resulting in a sample size of 41 children. The blood sample for one participant was lost in transit to the laboratory but the participant was retained in the study because all other measurements were completed. Fifty-four percent of the children were female, mean age was 12.1± 1.0 years, and the majority identified as Mexican American or Other Hispanic (Table 2).

The concentrations of the environmental chemicals in the study population are summarized in Table 3. Table 4 reports the results of univariable regression analyses of oxidant stress markers, insulin resistance and endothelial dysfunction markers against potential confounders. Female sex was associated with increased levels of 8-OHdG (42.5 ng/ml, P=0.038, CI 2.4, 82.5), whereas low socioeconomic status showed an association with decreased levels of 8-OHdG (−57.3 ng/ml, P= 0.031, CI −109.1, −5.4). With respect to race/ethnicity, non-Hispanic White showed increased levels of 8-OHdG (100.1 ng/ml, P<0.001, CI 47.9, 152.2), whereas other Hispanics showed increase in the levels of the endothelial-derived microparticles, CD144+63e+ (0.2% increase per ml, P=0.009, CI 0.6, 0.4). Being overweight was associated with increased levels of F2-isoprostane (12.4 ng/ml, P=0.010, CI 3.1, 21.8). Participants in the Other Hispanic category had lower HOMA-IR (−0.5 units, p=0.015, CI −0.9, −0.1) (Table 4). Obesity was also associated with higher HOMA-IR (−0.7 units, p=0.003, CI 0.2, 1.1).

Multivariable linear regression analysis showed that each log unit increase in BPA concentration was associated with higher levels of F2-isoprostane while controlling for confounding factors (P=0.02, CI 1.819, 19.86) (Table 5). A similar association was detected for each log unit increase of DEHP metabolites, although this did not reach statistical significance (P=0.051).

Mean levels of fasting glucose, insulin and HOMA-IR were 81.1 mg/dl, 10.2 μIU/ml and 2.07 respectively. Eight patients had HOMA-IR >3.4 and were categorized as insulin resistant. Linear regressions examining HOMA-IR as a continuous variable showed a positive association between each log unit increase of HMW metabolites and HOMA-IR (p=0.010). In multivariable models, which controlled for age, sex, BMI-z score (except when the outcome examined was BMI), daily caloric intake and physical activity ,each log unit increase of HMW phthalates was associated with an increased likelihood of being overweight (P=0.028) and having a higher BMI (P=0.041) (Table 6). In addition, for each log unit increase in HMW phthalate metabolite levels, we observed a 0.050 increase in HOMA-IR (p=0.019) (Table 6). Bisphenols were not associated with body weight or insulin resistance. No significant associations were detected between bisphenol or phthalate exposure and blood pressure.

The mean level of microalbuminuria in the sample was 0.97±1.96 mg/g creatinine and the value was below 10 mg/g in all participants. Other than a significant relationship between BPS exposure and albumin:creatinine ratio (P=0.040, CI: 0.18, 7.32) (table 6) , there was no association between exposure to any of the environmental chemicals and albuminuria whether considered as a continuous or categorical variable.

With respect to circulating plasma levels of activated endothelial cell-derived microparticles, each log unit increase of BPS and metabolites of HMW phthalates was associated with decreased levels of the endothelial microparticle CD 144+ 63e+, expressed as a percentage of total microparticles per ml (P=0.021 and P=0.026, respectively) (Table 6).

With the exception of DEHP metabolites, which were associated with decreased brachial artery distensibility (P=0.047, CI: −1.475, −0.010), multivariable regressions indicated that there were no relevant associations between the degree of exposure to any of the other environmental chemicals and brachial artery distensibility or PWV (Table 6).

In this pilot study, we have assessed exposure to a broad range of bisphenols and phthalates in a cohort of healthy, pre-adolescent children living in the metropolitan New York City area. We have confirmed that these molecules are associated with alterations in body weight, insulin resistance, albuminuria, markers of vascular structure and function, and indices of oxidant stress. These findings expand on prior cross sectional data extracted from NHANES surveys of children and adolescents (28, 29). They demonstrate that replacement compounds like BPS are also associated with adverse effects in children.

BPA and DEHP metabolites, which were comparable to levels reported in previous studies (28, 29), were associated with significantly higher urinary levels of F2-isoprostane. Our data are consistent with work by Yang et al in which BPA was associated with increased levels of various oxidant stress markers in women (12). Oxidant stress has been implicated as mechanism of action for various BPA-associated health outcomes in children, including obesity, microalbuminuria, and increased blood pressure (30). Over the past decade, manufacturers have replaced BPA with BPS in some consumer products, resulting in increased BPS exposure. This is a cause for concern as our findings suggest that these newer substitutes may be as harmful as the ones they are replacing (31). Of note, we did not document any adverse effects of BPF exposure; however, this finding should be considered tentative in light of our limited sample size.

8-OHdG and F2-isoprostane are well-studied biomarkers of systemic oxidative stress and have been associated with a range of adverse health outcomes. Urinary levels of 8-OHdG and F2-isoprostane represent two distinct cellular processes. 8-OHdG is DNA adduct formed in the presence of excess reactive oxidative species, such as hydroxyl radicals and is indicative of DNA excision repair (32). F2-isoprostane is formed in a non-enzymatic reaction between reactive oxidative species and arachidonic acid and it is very specific for lipid oxidation. It is not affected by dietary lipid intake and is highly detectable in urine samples (33). The differences in the mechanism of formation of these two molecules may explain why studies, including ours, observe several strong associations with F2-isoprostane, compared to 8-OHdG (11). First, the level of oxidant stress induced by these environmental chemicals in children may not involve DNA damage and therefore, we are unable to identify any significant findings with 8-OHdG. Second, individuals may vary in their DNA repair capacity and this may lead to inter-individual variability in urinary 8-OHdG levels.

We found that levels of HMW phthalate metabolites were associated with an increase in HOMA-IR. This is consistent with previous cross-sectional studies in adolescent NHANES participants (28, 29). In contrast to previous reports, we could not confirm this relationship with insulin resistance when we analyzed DEHP metabolites (13, 34, 35). This may be a consequence of the modest sample size of our sample and may also reflect the fact that DINP is replacing DEHP and, therefore, exposure to DINP is increasing in the general population. Considering that there are few studies investigating the toxicity profiles of these newer chemicals, our results indicate the need to study the potential adverse health effects of DINP.

While the mechanism(s) underlying the association of phthalates with insulin resistance are not well-understood, oxidative stress may be the missing link by inducing alterations in insulin signaling pathways. In preclinical investigations, Rajesh et al showed that DEHP-induced production of reactive oxidative species and lipid peroxidation disrupted insulin-mediated signal transduction. These effects were mitigated if rats were supplemented with Vitamin C and E (36). Although oxidative stress and insulin resistance have been linked and oxidative stress increases with phthalate exposure (36, 37), it was unclear if exposure to environmental phthalates at the community level contributes to the development of insulin resistance by inducing oxidative stress. The findings in our sample suggest that BPA and DEHP metabolites are associated with increased levels of F2-isoprostane, although the latter association fell just short of statistical significance. Metabolites of HMW phthalates are associated with body weight and increased HOMA-IR. This suggests that, at the community level, exposure to bisphenols and phthalates could induce oxidant stress, which may then contribute to development of insulin resistance in otherwise healthy children.

Oxidant stress plays a pivotal role in endothelial dysfunction and arterial stiffness. Albuminuria is an early manifestation of endothelial dysfunction. Consistent with our earlier studies using NHANES data (28, 29), we documented a relationship between bisphenol (BPS) exposure and the albumin:creatinine ratio. This suggests that childhood exposure to bisphenols may promote endothelial dysfunction, which, contributes to microvascular and atherosclerotic disease burden during adulthood. We did not detect a relationship between exposure to other bisphenols or phthalates and albuminuria, which is likely due to the limited size of our sample.

Oxidant stress-induced injury to the endothelium also contributes to arterial stiffness and subsequently to a higher risk of cardio-metabolic disease (38). We detected a relationship between exposure to DEHP metabolites and reduced brachial artery distensibility. This change may lead to an increased risk of developing large vessel disease in later life. No relationship between exposures and increased pulse wave velocity were noted. This may reflect the young age of our sample and the need for prolonged exposure to the chemicals with increased oxidant stress before there are consistent changes in vascular structure and function. In addition, the environmental chemical exposures may act differently on central, large, more elastic arteries, versus medium-size, muscular arteries that are assessed by the brachial artery distensibility test.

We measured endothelial-derived microparticles to enable a more direct localization of endothelial dysfunction and found an inverse relationship between circulating levels of activated microparticles derived from activated endothelial cells and BPS and HMW phthalates. This observation is at variance with our hypothesis that exposure to environmental chemicals may result in oxidant stress and endothelial dysfunction. The latter state would be characterized by increased release of activated endothelial microparticles. Our data points to the opposite direction but, considering that microparticles are a fairly new method of assessing the state of the endothelium, especially in children, there are gaps in our own knowledge of mechanisms of microparticles. In general, very few studies have investigated microparticles in children, and they have shown increased activation of microparticles to be associated with obesity, chronic kidney disease and Henoch-Schonlein vasculitis (39–41). Our findings need to be confirmed in further studies with larger samples containing a more diverse patient population.

There are several limitations to our study, including the sample size and cross-sectional study design, which prevents us from inferring causality. In addition, reverse causation cannot be ruled out. Thus, an alternative explanation for our findings would be that insulin-resistant children have unhealthy eating behaviors, including more packaged food consumption, and thus have higher urinary levels of phthalates. In addition, we did not collect data on some potential confounders such as exposure to tobacco, which is known to affect the levels of oxidant stress.

In conclusion, our study suggests BPA and DEHP metabolites were associated with increased levels of F2-isoprostane in children, a marker of systemic oxidant stress. Metabolites of HMW phthalates are associated with higher body weight and increased risk of insulin resistance in otherwise healthy children. Exposure to BPS was associated with albuminuria and altered plasma levels of endothelial-derived microparticles. Brachial artery distensibility was reduced in association with DEHP metabolites. The evidence suggesting links between endocrine disruptors and adverse cardiorenal health effects is increasing, and our findings add to the current body of evidence. Bisphenols alter glucose transport, inhibit adiponectin release, stimulate the release of inflammatory cytokines and stimulate lipolysis in human adipose tissue(42). Phthalates simultaneously affects multiple cellular targets, and produce changes in the metabolic and oxidant stress profile of cardiac cells. In light of the emerging evidence that these persistent organic pollutants adversely impact cardiorenal function at a young age, there is a need for increased regulatory consideration and policy initiatives to limit exposure to ubiquitous environmental chemicals with the potential to increase cardiometabolic risk.