Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
1
NJDEP Exposure of Infants to Endocrine Disruptors- Final Report 6-27-2012 NJDEP Contract # SR09-002 Study Investigators: David Q. Rich, ScD, MPH, University of Rochester Stephen W. Marcella, MD, MPH, UMDNJ-School of Public Health Barry Weinberger, MD, UMDNJ- Robert Wood Johnson Medical School Dan Wartenberg, PhD, UMDNJ- Environmental and Occupational Health Science Institute Mark Gregory Robson, PhD, MPH, DrPH (hc), School of Environmental
and Biological Sciences - Rutgers University Anna M. Vetrano, PhD, UMDNJ-Robert Wood Johnson Medical School Faith Archer, BS, UMDNJ-Robert Wood Johnson Medical School
2
INTRODUCTION
Phthalates and bisphenol A (BPA) are used in many products that are ubiquitous in the
urban environment. Phthalates are present in lubricants, cosmetics, construction materials,
wood finishers, adhesives, floorings, and paints. High molecular weight phthalates, such as
di(2-ethyl-hexyl) phthalate (DEHP), are used as plasticizers in the manufacture of polyvinyl
chloride, which is in consumer products such as perfumes, nail polish, automobile interiors, vinyl
shower curtains, wall and floor coverings, food contact applications, and medical devices (1). In
addition to exposure directly via manufactured goods, increasing concentrations of phthalates
have been identified as environmental contaminants. For example, significant quantities have
been documented in snowmelt, wastewater, and household dust (2-4). Moreover, some
phthalate metabolites may persist in the environment due to variable mechanisms and rates of
degradation (5). In its 3rd National Report on Human Exposure to Environmental Chemicals,
the CDC collected urine samples from a randomly selected sample US subjects and found
elevated levels of mono-(2-ethylhexyl) phthalate (MEHP), the primary monoester metabolite of
DEHP in humans. They found a level of 3.5 µg/l urine at the 10th percentile and 13.6 µg/l urine
at the 90th percentile of the population distribution (6). Two oxidative DEHP metabolites,
mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono(2-ethyl-5-oxohexyl) phthalate
(MEOHP), were also present in high concentrations in urine and are indicative of exposure to
DEHP (7). Lower molecular weight phthalates, such as di-n-butyl phthalate (DBP) and diethyl
phthalate (DEP) are also ubiquitous in the environment. They are used as solvents and
plasticizers, and are present in lacquers, varnishes, perfumes, lotions, cosmetics, and coatings.
There is also widespread exposure to BPA and its metabolites in the U.S population (8).
BPA is used in the manufacture of polycarbonate, epoxy resins, and other plastics in printed
circuit boards, composites, and adhesives. Polycarbonates are used for food-contact use, such
3
as in microwave oven-ware, milk and juice containers, baby bottles, and the interior coating of
cans, thus posing a substantial risk for exposure to the public (9). Bisphenol A diglycidyl ether
(BADGE) is the lowest molecular weight oligomer, used in commercial liquid epoxy resins. Like
phthalates, significant levels of BPA have been found in wastewater, drinking water, air, and
dust (10).
The fetal and neonatal periods represent a particularly vulnerable period of susceptibility
to adverse effects of environmental exposures. Therefore, the recent finding that the levels of
several phthalate metabolites are elevated in maternal urine, collected at the time of delivery,
are of concern (11). In our institution, urinary concentrations of MEHP, mono(2-ethyl-5-
hydroxyhexyl) phthalate (MEHHP), and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), three
metabolites of DEHP, were found to be 5-20 times higher in mothers prior to elective cesarean
deliveries than the U.S. general and female population based on NHANES 2001–2002 data.
This suggests either that pregnant females are more exposed to these substances than the
general population, or that the background level of exposure in central New Jersey exceeds that
in the U.S. as a whole (11). Phthalate metabolites in the maternal urine are thought to be more
sensitive biomarkers of exposure than those measured in maternal or cord serum (11-12). Our
group has also found that hospitalized infants excrete much higher concentrations of MEHP,
MEHHP, and MEOHP in their urine than in a US population (NHANES) sample (13) . Similarly,
BPA has been found in follicular and amniotic fluid, umbilical cord blood, and placental tissue
(14-15). Several groups have shown that BPA is present in maternal serum or urine, amniotic
fluid, and/or fetal serum at concentrations comparable to levels known to interfere with normal
development in animals. Mean levels in maternal plasma have been reported from 0.43-3.1
μg/L, with some subjects as high as 22.3 μg/L. Mean amniotic fluid and fetal serum levels are
lower (0.5-8.3 and 0.64-2.3 μg/L, respectively) (15-19). These levels may affect reproductive
and fetal health, constituting an important public health concern.
4
Some previous studies have suggested that phthalates and BPA may exert anti-
androgenic effects and shorten gestation (20-25). For example, males exposed prenatally to
phthalates may have decreased anogenital distance (AGD), penile volume, and scrotal size (20-
21). BPA and its metabolites have a high affinity for the androgen receptor and exhibit marked
anti-androgenic activity (22), and also have been shown to have estrogenic activity in vitro (23).
Both phthalates and BPA can also induce inflammatory activity, potentially shortening gestation
by triggering parturition (24-25). In our investigation, we quantified maternal exposure to
phthalate and BPA metabolites in a high-risk obstetrical population. In contrast to earlier
studies, these measurements were made at the final prenatal clinic visit, reflecting ongoing
exposure in the home environment, rather than the hospital environment after admission for
delivery of the infant. In addition, multiple urine samples were obtained longitudinally during the
pregnancy at successive clinic visits, and these may be analyzed in future investigations. AGD
measurements were made in the immediate neonatal period, prior to discharge from the
hospital, in order to decrease variability related to postnatal age. We hypothesized that
elevated levels of phthalates and BPA in this population of high-risk pregnancies are associated
with an increased risk for preterm delivery and alterations in genital development.
The incidence rate of prematurity has been increasing in the U.S. during the last two
decades. Given that the complications of even moderate prematurity are associated with
substantial morbidity, mortality, and health care costs, it is important to identify high-prevalence
environmental exposures that may increase the risk of these adverse outcomes.
METHODS
Study population. We recruited a population of 72 mothers, over the age of 18 and
expecting singleton infants, enrolled in the High-Risk Obstetric Clinic at Robert Wood Johnson
University Hospital, a major central New Jersey teaching hospital in New Brunswick. It serves
both urban and suburban populations for a large portion of the state. All subjects provided
5
informed consent prior to participation in the study which was approved by the Institutional
Review Board (IRB) at the University of Medicine of New Jersey-Robert Wood Johnson Medical
School. A detailed medical history, information on household product use, occupation, hobbies,
diet, demographic variables, and ethnicity were collected as a part of the study.
Urinary phthalate and BPA metabolite concentrations. Urine samples were
collected in phthalate and BPA-free containers at the beginning of each prenatal clinic visit,
resulting in 1 to13 successive samples per subject. In the current study, only the samples from
the last prenatal clinic visit prior to delivery were analyzed. Samples were stored in coolers with
ice packs and delivered to the Neonatology Laboratory, located in the same building. No
preservatives were added prior to sampling. All samples were stored at -70oC until transferred
on dry ice to the Analytical Core Facility at the Center for Environmental Exposures and Disease
(CEED) NIEHS Center of Excellence of the Environmental and Occupational Health Sciences
Institute (EOHSI) of Rutgers/UMDNJ. They were maintained at -70oC until analyzed, using a
modification of a high-performance liquid chromatography–atmospheric pressure chemical
ionization tandem mass spectrometry method first described by Silva et al (26). The following
metabolites were quantified: MEHP, monomethyl phthalate (mMP), monoethyl phthalate (mEP),
monobutyl phthalate (mBP), monocyclohexyl phthalate (mCHP), mEHHP, mono-3-methyl-7-
methyloctyl phthalate (isodecyl, mDP), mEOHP, mono-n-octyl phthalate (mOP), mono-3-methyl-
5-dimethylhexyl phthalate (iso-nonyl, mNP), BPA total, BPA sulfate, and BPA glucuronide. The
detection limits for this method range from 0.1 to 0.5 ng/ml, with recovery efficiency of 100 +
12% (26). All LC/MS measurements were made using Thermo Fischer scientific ion trap mass
spectrometers, and normalized to urine specific gravity, measured concurrently. BPA and its
glucuronide and sulfate metabolites were isolated by liquid/liquid extraction using
MTBE/hexane, and quantified by LC/MS/MS using an electrospray interface operated in the
negative ionization mode.
6
Physical examination and anogenital index. Male and female infants were examined
in the nursery at < 72 hrs of age. Anogenital distance (AGD) was measured by tape measure
from the center of the anus to the posterior base of the scrotum in males, and to the posterior
edge of the vagina in females (27). Anogenital index (AGI) (mm/kg) was calculated by
normalizing AGD (mm) to weight (kg). The presence or absence of other genital abnormalities,
including hypospadias or incomplete testicular descent in boys, was also noted. Gestational
age and birth weight were recorded, and data obtained on other medical diagnoses,
medications, and interventions at the time of examination. Gestational age was determined
based on the best obstetric estimate in the medical record, using either sonographic dating or
date of implantation.
Umbilical cord blood inflammatory cytokine levels. Samples of umbilical cord blood
(8-10 ml) were obtained from placentas of study subjects at the time of delivery, when available.
Blood was spun at 3000 x g for 10 min in 1.5 ml Eppendorff tubes in a microcentrifuge. The
serum supernatants were removed, placed in a new tube, and frozen at -70 for batched
analysis. Sera were then incubated with premixed flex set beads coated with antibodies to
macrophage inflammatory protein-1β (MIP-1β), IL-8, IL-6, IL-1β, vascular endothelial growth
factor (VEGF), TNF-α and Regulated upon Activation, Normal T-cell Expressed, and Secreted
protein (RANTES) in 96-well filtration plates. Premixed phycoerythrin-labeled detection reagent
was added to the wells and incubated in the dark for 2 hr. The bead-protein complexes were
analyzed for fluorescence intensity using a BD FACS Array Bioanalyzer. Data were analyzed
using BD FCAP software (v 2.0).
Statistical Analysis. We calculated descriptive statistics for gestational age and AGI,
as well as each urinary phthalate metabolite, BPA sulfate, and total BPA concentration. We
7
used separate linear regression models to estimate the change in gestational age associated
with each interquartile range (IQR) increase in the metabolite (BPA sulfate, MEP, MEHHP,
MEOHP, MCHP, MEHP, and MBP) concentration. We first fit unadjusted bivariate linear
regression models with each of the metabolites, as well as parity, race, and other predictors of
gestational age including maternal education, maternal race, gestational age group, parity,
gravid, maternal employment, paternal employment, fast food consumption, maternal age, birth
country (US vs. outside of US). We then adjusted the models of individual metabolites by
adding parity and maternal race, those variables that were significant in univariate models (here
defined as p<0.15). Finally, we re-ran the same models after stratifying by gender (n= 40 for
boys and 32 for girls). We used a similar set of models with AGI as the outcome, but included
gestational age (preterm birth [<37 weeks] versus term births [≥ 37 weeks]) and maternal birth
country (outside US versus US) in the adjusted models.
To evaluate our assumption of a linear relationship between our continuous,
independent variables (the metabolites) and the response variables, we log transformed each
outcome and classified the metabolite concentrations into quartiles with the first quartile as the
reference and then re-ran the regression models. We separately tested for trend by including
an ordinal quartile term (0, 1, 2 or 3) in the model, instead of the indicator variables. All
statistical analyses were done using SAS V.9.2 (© SAS Institute, Inc., Cary, NC).
Separate exploratory analyses regressing gestational age on the two metabolites,
MEHHP and total BPA, that showed some significant association in the above models, were
performed with and without individual umbilical cord blood cytokine measurements to determine
if the relationship of the metabolite and gestational age was mediated by the cytokine. This was
repeated individually for each cytokine. Due to the small sample size of subjects with umbilical
blood specimens for cytokines (N=38) and the exploratory nature of this analysis, we performed
these regressions without the other independent covariates that were used to adjust for
modeling the effects of the metabolites on gestational age without cytokine data.
8
RESULTS
The characteristics of the 72 pregnant woman enrolled in the study are shown in Table
1. Indications for enrollment in the High-Risk Obstetric Clinic included previous preterm birth
(36%), febrile illness (18%), hypertension (19%), diabetes (14%), urinary tract infection (10%),
and preterm labor (6%). Forty infants were male (56%), and 32 female (44%). Of these 72
infants, 7 were preterm (32-33 weeks gestation), 14 late preterm (34-36 weeks), and 51 term
(37-42 weeks). Table 2 summarizes newborn measurements and gestational age at birth.
Gestational age was 37.47 +/- 2.41 weeks (mean +/- SD), with birth weights 3073.35 g +/-
754.91 g. The distribution of phthalate and BPA metabolite concentrations in maternal urine,
standardized to specific gravity, is shown in Table 3. The DEHP metabolites MEHP, MEHHP,
and MEOHP, as well as mEP, mCHP and mBP were present in measurable quantities, while
mMP, mOP, mNP, and mDP were present at low concentrations or were not detectible.
Therefore, we did not further analyze urinary mMP, mOP, mNP, or mDP. BPA and BPA sulfate
were also detected in maternal urine. Table 4 shows the correlation matrix among metabolites:
MEOHP correlated highly with mEHP (r=0.80) and MEHHP was moderately correlated with
mEOHP (r= 0.76) and MEHP (r= 0.50). Moderate correlations were also noted for mBP with
mCHP and mEHP (r=0.46 and 0.31 respectively), for BPA with mEHHP and mEOHP (r=0.34
and 0.32 respectively), and for BPA with BPA sulfate (r= 0.30).
After adjusting for parity and maternal race, each interquartile range increase in urinary
MEHHP concentration was associated with a significant 4.2 day decrease in gestation (95%
confidence interval = -7.9, -0.4) (Table 5). Similarly, each interquartile range increase in total
BPA was associated with a significant 1.1 day decrease in gestation (95% CI = -2.0, -0.1).
Although not statistically significant, we observed decreased gestation associated with each
interquartile range increase in MEOHP (-0.6 days), MCHP (-0.9 days), MBP (-2.1 days) and
BPA sulfate (-0.5 days), but not with MEP or MEHP (Table 5).
9
Next, we stratified by gender, and found that larger and more consistent reductions in
gestation were associated with phthalate metabolites in male infants. Gestational effects for
BPA total were nearly identical in both genders although statistically significant only for the
males. We observed significant reductions in gestation associated with IQR increases in
MEHHP (-5.1 days, 95% CI = -9.6, -0.6) and BPA total concentrations (-1.1 days, 95% CI = -2.1,
-0.1), and non-significant reductions in all other phthalates and metabolites. Conversely, for
female infants, we observed generally smaller and non-significant decreases in gestation
associated with MEHHP (-1.4 days, 95% CI = -8.4, 5.7), MCHP (-0.4 days, 95% CI = -7.4, 6.7),
MBP (-0.5, 95% CI = -6.2, 5.1), and BPA total (-1.6, 95% CI = -4.1, 1.0), and no such decreases
in gestation associated with MECP, MEOHP, MEHP, or BPA sulfate.
For AGI, after adjusting for parity and maternal race, we found no statistically significant
associations with interquartile range increase in urinary concentration any of the metabolites for
all infants combined as well as for female only infants. For boys, there was one statistically
significant association of MEP with AGI, but this was of borderline clinical significance (-.0.2
mm, 95% CI = 0.0, -0.4) (Table 6).
Next, we evaluated whether our inference would change if we repeated our analyses
after log-transforming gestational age and using indicator variables for the quartiles of
phthalate/BPA metabolite in our models, instead of a continuous phthalate/BPA metabolite
concentration variable. Generally, gestation decreased with increasing quartile of MEHHP
(Figure 1) and BPA total (Figure 2). For boys only, gestation decreased with each increasing
quartile of maternal urinary MEHHP concentration (Figure 3) and BPA total concentration
(Figure 4).
MIP-1β, IL-8, IL-6, IL-1β, VEGF, TNF-α and RANTES were all detected in measurable
quantities in umbilical cord blood sera (Table 7). The cytokines themselves did not appear to
have any significant effect on gestational age with the exception of RANTES (beta = -.0002,
p=.05 and .06) when modeled with MEHHP and total BPA respectively. IL-1β had an
10
association of increasing gestational age (beta = 0.05, p=.36 and beta= 0.07, p= 0.21) when
modeled with MEHHP and total BPA, respectively. However, there were no changes in the
effect estimates of MEHHP or total BPA on gestational age when the regressions were done
with or without the addition of the individual cytokines (Table 8).
DISCUSSION
We found that each IQR increase in maternal urinary MEHHP was associated with a 4.2
day shorter gestational age. This effect was observed primarily in males (5.1 days/21.9 ng/mL)
but not in female infants. This is consistent with some previous animal studies demonstrating
spontaneous abortion and preterm delivery in animals exposed to phthalates (ATSDR 2001). In
humans, one recent study suggested that MEHP-positive newborns had lower mean gestational
age than controls (28). In another study in an inner-city population, gestational age was shorter
by 1.1 days (95% CI: 0.2-1.8 days) for each 1-logarithmic unit increase in specific gravity-
adjusted MEHP concentrations, and averaged 5.0 days (95% CI: 2.1-8.0 days) less among
subjects with the highest versus lowest quartile concentrations. Results were similar and
statistically significant for the other DEHP metabolites (25). MEHHP is of particular interest
because it is one of the most abundant of DEHP metabolites. In one recent cohort, it was found
in measurable quantities in the urine of 100% of women and was associated with exposure to
body lotions and bottled water (29).
Biologically plausible mechanisms for the induction of shorter gestation by phthalates
include the induction of inflammation, which can trigger parturition. Several phthalate
metabolites, that are present in a high proportion of urine samples from the general U.S.
population, are associated with increased serum C-reactive protein and γ-glutamyltransferase,
which are markers of inflammation and oxidative stress, respectively (30). Both DEHP and
MEHP have been shown to bind the transcription factor PPAR-γ (31-34), potentially blocking
important anti-inflammatory pathways and resulting in inflammatory signaling and possible
11
triggering of labor. In the current studies, we could not demonstrate a change of effect of the
metabolites by adding key inflammatory cytokines into the models suggesting the absence of
either a confounding or mediating effect. However, the sample size of available cytokines was
small for this analysis. There was some evidence of an independent association of RANTES
blood levels with a decrease in gestational age. In the range of the values seen in our 38
subjects, a change in 10,000 units of this cytokine with a beta of .0002 (change in gestational
age in weeks per unit increase of RANTES) translates to a decrease in gestational age of 14
days, a very significant clinical effect. An association of high RANTES levels with shortened
gestation is biologically plausible because RANTES, also known as CCL5, is chemotactic for T
cells, eosinophils, and basophils, and plays an active role in recruiting leukocytes into
inflammatory sites. It is produced “late” (3-5 days) after T-lymphocyte activation, consistent with
a possible contribution to sustained inflammatory stimuli triggering labor. However, this should
be considered a very preliminary finding since it was not an a priori hypothesis. This finding
could be a type 1 error or it could be due to a combination effect of other cytokines that are
correlated with RANTES. This does indicate the need for further research in this area. An
Expert Panel Update from the Center for the Evaluation of Risks to Human Reproduction has
confirmed that there is an urgent public health need for more data on the toxicity of phthalates in
human neonates, including the potential roles of PPAR signaling and preterm labor on these
outcomes (35).
We also found that total BPA was associated with shortened gestational period (by 1.1
days/180.1 ng/mL), and that this effect was also stronger in males. This is consistent with
several recent studies showing that BPA and BADGE exert pro-inflammatory effects. Serum
levels as low as 2.59 + 5.23 μg/L have been associated with recurrent miscarriage (36).
Recently, studies in Mexico and China have suggested dose-related effects on the risk of
delivery at less than 37 weeks of gestation (24, 37) and low birth weight (38). BADGE is a
competitive inhibitor of PPAR-γ signaling in vitro (39). Thus, BADGE abrogates the induction of
12
mRNA for the adipose triglyceride lipase by rosiglitazone (40) and blocks the activation of
PPAR-γ by ω-3 PUFAs (41). In vivo, BADGE blocks the protective effects of rosiglitazone in
carageenan-induced edema and pleurisy, resulting in increased histologic indicators of
inflammatory injury (42). BADGE also reverses the protective effects of the PPAR-γ agonists,
ciglitazone and PGJ2, in experimental allergic encephalomyelitis (43). Prenatal exposure of
animals to BPA results in decreased immune tolerance to protein allergens (44), as well as
reduced numbers of regulatory T cells in mice, promoting increased Th1 responses (45).
BADGE has been shown to have direct cytotoxic effects, including alterations in cell
morphology, adhesion, and F-actin depolymerization in cultured cells in vitro (46).
The increased susceptibility of males to the induction of prematurity by phthalates is
consistent with epidemiologic data suggesting that males are more likely to deliver prematurely,
in general (47). In one study, there was a 7.2% excess of males among white singleton preterm
births, distributed uniformly from 20-37 weeks of gestation (48). Several plausible biologic
mechanisms have been proposed for the higher incidence of preterm labor by male infants.
These include the action of androgen precursors, which are involved in the production of
estrogen and are elevated in male relative to female fetuses (49). Alternatively, induction of
labor may be promoted by interleukin (IL)-1 in males, who have lower levels of IL-1 receptor
antagonist in amniotic fluid than females (50-51).
Currently, only limited or inadequate data exist on the relationship between phthalate
exposure and developmental anomalies in humans. In our investigation we did not identify any
association between phthalate levels and AGI. Some studies have previously suggested that
phthalates may act as developmental and reproductive toxicants in experimental animals and
humans. The commonly observed anomalies include reduction in androgen-dependent tissue
weights, and increased incidence of malformations of the external genitalia such as
hypospadias. Males exposed prenatally to phthalates may have decreased AGD, penile
volume, and scrotal size, as well as increased incidence of incomplete testicular descent (20-
13
21). Significantly, the median exposures to DEHP and other phthalate metabolites associated
with reduced anogenital distance were lower than the current U.S. EPA reference doses (RfD)
for these chemicals, which are based on increased relative liver weight and therefore not
directly applicable to their endocrine disruption effects (21). In one recent study, exposure to
phthalates was associated with shortened AGD, but frank genital anomalies were not seen (52).
In that data set, MEP, MBP, and three DEHP metabolites (MEHP, MEOHP, and MEHHP) were
inversely related to AGD (1). We only observed a significant decrease in AGI for MEP, but not
other metabolites, in boys. The imprecision of these outcomes and their inconsistency may be
partially explained by inaccurate measurements and non-differential misclassification as well as
small sample size. This imprecision does not rule out clinically significant effects as some of the
confidence interval boundaries are consistent with clinically important effects.
Like phthalates, elevated urinary BPA has been linked to reduced levels of
gonadotrophic hormones in males (53), and BPA and its metabolites have estrogenic activity in
vitro (23). These compounds also have a high affinity for the androgen receptor and exhibit
marked anti-androgenic activity (22). BPA initiates rapid alterations in cell membranes via
estrogen receptors at concentrations that are below those demonstrated in maternal and fetal
tissues (54-55). In vivo exposure of pregnant rats to BPA results in decreased spermatogenesis
and lower plasma testosterone levels in male offspring (56). BPA may also interfere with
normal development of the prostate gland, increasing the susceptibility to carcinogenesis.
Alterations in sexually dimorphic patterns of behavior after low doses of BPA have been noted
(57-58). However, no previous studies have demonstrated developmental effects of BPA in
humans (16), and in our study we also did not observe any alterations in AGD.
Our study has several limitations, most notable being the small sample size. We also
examined multiple metabolites, so it is possible that some of our associations occurred by
chance. Our tool for measuring AGI was crude (tape measure of length between genitalia and
center of the anus), thereby increasing the chance of nondifferential outcome misclassification
14
resulting in a bias towards the null. We were limited to one urine sample per mother, and thus
we were only able to obtain 1 narrow, point-in-time measurement of metabolites. Therefore, we
were unable to assess the variability of each metabolite across the pregnancy. The one-time
urine measurement does not reflect the variable and overall, cumulative exposure to these
metabolites in utero. It is not known at what point(s) in gestation an infant is most potentially
vulnerable to these compounds. Analyzing the other urine samples from earlier time points in
the pregnancy in further studies may be helpful in addressing this question. This would help to
provide a measure of the variability of exposure to these compounds in the mother’s
environment, as well as a more longitudinal and nuanced characterization of the exposure to the
developing fetus. Finally, the analytic technique was not sensitive enough to measure several
of these phthalate metabolites at the low end of the level found in urine (mMP, mOP, mNP, and
mDP).
Our study focused on high-risk pregnancies so it is possible that our results may not be
directly generalizable to a more general population of pregnant women. For example, our mean
gestational age and birth weight was 38.5 weeks and 3255 grams respectively, as compared to
that of New Jersey with means of 37.5 weeks and 3073 grams. However, we do not feel that
these differences are significant enough to consider that the associations we describe would not
also be seen in the more general population. The strengths of this study are that the study
group was a very diverse population of high-risk pregnancies, and our measurements of BPA
and phthalates were obtained prospectively and blindly with respect to anatomic measurements.
Gestational age was established by sonographic dating or date of implantation, thus providing
an accurate accounting of this outcome.
15
REFERENCES
1. Swan SH. Environmental phthalate exposure in relation to reproductive outcomes and
other health endpoints in humans. Environmental research. 2008;108(2):177-84. PMCID:
2775531.
2. Bjorklund K, Stromvall AM, Malmqvist PA. Screening of organic contaminants in urban
snow. Water Sci Technol. 2011;64(1):206-13.
3. Butte W, Heinzow B. Pollutants in house dust as indicators of indoor contamination. Rev
Environ Contam Toxicol. 2002;175:1-46.
4. Deblonde T, Cossu-Leguille C, Hartemann P. Emerging pollutants in wastewater: a
review of the literature. Int J Hyg Environ Health. 2011;214(6):442-8.
5. Liang DW, Zhang T, Fang HH, He J. Phthalates biodegradation in the environment. Appl
Microbiol Biotechnol. 2008;80(2):183-98.
6. CDC. Third National Report on Human Exposure to Environmental Chemicals. Atlanta,
GA: Centers for Disease Control and Prevention, US Department of Health and Human
Services.; 2005 Contract No.: Document Number|.
7. Kato K, Silva MJ, Reidy JA, Hurtz D, 3rd, Malek NA, Needham LL, et al. Mono(2-ethyl-5-
hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate as biomarkers for human
exposure assessment to di-(2-ethylhexyl) phthalate. Environ Health Perspect. 2004;112(3):327-
30. PMCID: 1241862.
8. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. Exposure of the U.S. population to
bisphenol A and 4-tertiary-octylphenol: 2003-2004. Environ Health Perspect. 2008;116(1):39-44.
PMCID: 2199288.
9. Poole A, van Herwijnen P, Weideli H, Thomas MC, Ransbotyn G, Vance C. Review of
the toxicology, human exposure and safety assessment for bisphenol A diglycidylether
(BADGE). Food additives and contaminants. 2004;21(9):905-19.
16
10. Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure to
bisphenol A (BPA). Reprod Toxicol. 2007;24(2):139-77.
11. Yan X, Calafat A, Lashley S, Smulian J, Ananth C, Barr D, et al. Phthalates Biomarker
Identification and Exposure Estimates in a Population of Pregnant Women. Hum Ecol Risk
Assess. 2009;15(3):565-78. PMCID: 2913903.
12. Sathyanarayana S, Calafat AM, Liu F, Swan SH. Maternal and infant urinary phthalate
metabolite concentrations: are they related? Environmental research. 2008;108(3):413-8.
PMCID: 2651957.
13. Calafat AM, Needham LL, Silva MJ, Lambert G. Exposure to di-(2-ethylhexyl) phthalate
among premature neonates in a neonatal intensive care unit. Pediatrics. 2004;113(5):e429-34.
14. Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y. Determination of bisphenol A
concentrations in human biological fluids reveals significant early prenatal exposure. Human
reproduction (Oxford, England). 2002;17(11):2839-41.
15. Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, Chahoud I. Parent bisphenol
A accumulation in the human maternal-fetal-placental unit. Environ Health Perspect.
2002;110(11):A703-7.
16. Chapin RE, Adams J, Boekelheide K, Gray LE, Jr., Hayward SW, Lees PS, et al. NTP-
CERHR expert panel report on the reproductive and developmental toxicity of bisphenol A. Birth
defects research. 2008;83(3):157-395.
17. Padmanabhan V, Siefert K, Ransom S, Johnson T, Pinkerton J, Anderson L, et al.
Maternal bisphenol-A levels at delivery: a looming problem? J Perinatol. 2008;28(4):258-63.
18. Yamada H, Furuta I, Kato EH, Kataoka S, Usuki Y, Kobashi G, et al. Maternal serum
and amniotic fluid bisphenol A concentrations in the early second trimester. Reprod Toxicol.
2002;16(6):735-9.
19. Ye X, Pierik FH, Hauser R, Duty S, Angerer J, Park MM, et al. Urinary metabolite
concentrations of organophosphorous pesticides, bisphenol A, and phthalates among pregnant
17
women in Rotterdam, the Netherlands: the Generation R study. Environmental research.
2008;108(2):260-7.
20. Ema M, Miyawaki E, Hirose A, Kamata E. Decreased anogenital distance and increased
incidence of undescended testes in fetuses of rats given monobenzyl phthalate, a major
metabolite of butyl benzyl phthalate. Reprod Toxicol. 2003;17(4):407-12.
21. Marsee K, Woodruff TJ, Axelrad DA, Calafat AM, Swan SH. Estimated daily phthalate
exposures in a population of mothers of male infants exhibiting reduced anogenital distance.
Environ Health Perspect. 2006;114(6):805-9.
22. Satoh K, Ohyama K, Aoki N, Iida M, Nagai F. Study on anti-androgenic effects of
bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their
derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen. Food
Chem Toxicol. 2004;42(6):983-93.
23. Nakazawa H, Yamaguchi A, Inoue K, Yamazaki T, Kato K, Yoshimura Y, et al. In vitro
assay of hydrolysis and chlorohydroxy derivatives of bisphenol A diglycidyl ether for estrogenic
activity. Food Chem Toxicol. 2002;40(12):1827-32.
24. Cantonwine D, Meeker JD, Hu H, Sanchez BN, Lamadrid-Figueroa H, Mercado-Garcia
A, et al. Bisphenol a exposure in Mexico City and risk of prematurity: a pilot nested case control
study. Environ Health. 2010;9:62. PMCID: 2965706.
25. Whyatt RM, Adibi JJ, Calafat AM, Camann DE, Rauh V, Bhat HK, et al. Prenatal di(2-
ethylhexyl)phthalate exposure and length of gestation among an inner-city cohort. Pediatrics.
2009;124(6):e1213-20. PMCID: 3137456.
26. Silva MJ, Samandar E, Preau JL, Jr., Reidy JA, Needham LL, Calafat A. Automated
solid-phase extraction and quantitative analysis of 14 phthalate metabolites in human serum
using isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. J
Anal Toxicol. 2005;29(8):819-24.
18
27. Salazar-Martinez E, Romano-Riquer P, Yanez-Marquez E, Longnecker MP, Hernandez-
Avila M. Anogenital distance in human male and female newborns: a descriptive, cross-
sectional study. Environ Health. 2004;3(1):8. PMCID: 521084.
28. Latini G, De Felice C, Presta G, Del Vecchio A, Paris I, Ruggieri F, et al. In utero
exposure to di-(2-ethylhexyl)phthalate and duration of human pregnancy. Environ Health
Perspect. 2003;111(14):1783-5.
29. Romero-Franco M, Hernandez-Ramirez RU, Calafat AM, Cebrian ME, Needham LL,
Teitelbaum S, et al. Personal care product use and urinary levels of phthalate metabolites in
Mexican women. Environment international. 2011;37(5):867-71.
30. Ferguson KK, Loch-Caruso R, Meeker JD. Urinary phthalate metabolites in relation to
biomarkers of inflammation and oxidative stress: NHANES 1999-2006. Environmental research.
2011;111(5):718-26. PMCID: 3110976.
31. Hurst CH, Waxman DJ. Activation of PPARalpha and PPARgamma by environmental
phthalate monoesters. Toxicol Sci. 2003;74(2):297-308.
32. Kaya T, Mohr SC, Waxman DJ, Vajda S. Computational screening of phthalate
monoesters for binding to PPARgamma. Chemical research in toxicology. 2006;19(8):999-1009.
33. Hansen JS, Larsen ST, Poulsen LK, Nielsen GD. Adjuvant effects of inhaled mono-2-
ethylhexyl phthalate in BALB/cJ mice. Toxicology. 2007;232(1-2):79-88.
34. Gourlay T, Samartzis I, Stefanou D, Taylor K. Inflammatory response of rat and human
neutrophils exposed to di-(2-ethyl-hexyl)-phthalate-plasticized polyvinyl chloride. Artif Organs.
2003;27(3):256-60.
35. NTP-CERHR expert panel update on the reproductive and developmental toxicity of
di(2-ethylhexyl) phthalate. US Department of Health and Human Services National Toxicology
Program, Center for the Evaluation of Risks to Human Reproduction. 2005.
19
36. Sugiura-Ogasawara M, Ozaki Y, Sonta S, Makino T, Suzumori K. Exposure to bisphenol
A is associated with recurrent miscarriage. Human reproduction (Oxford, England).
2005;20(8):2325-9.
37. Meeker JD, Hu H, Cantonwine DE, Lamadrid-Figueroa H, Calafat AM, Ettinger AS, et al.
Urinary phthalate metabolites in relation to preterm birth in Mexico city. Environ Health
Perspect. 2009;117(10):1587-92. PMCID: 2790514.
38. Miao M, Yuan W, Zhu G, He X, Li DK. In utero exposure to bisphenol-A and its effect on
birth weight of offspring. Reprod Toxicol. 2011;32(1):64-8.
39. Wright HM, Clish CB, Mikami T, Hauser S, Yanagi K, Hiramatsu R, et al. A synthetic
antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte
differentiation. J Biol Chem. 2000;275(3):1873-7.
40. Liu LF, Purushotham A, Wendel AA, Koba K, DeIuliis J, Lee K, et al. Regulation of
adipose triglyceride lipase by rosiglitazone. Diabetes, obesity & metabolism. 2009;11(2):131-42.
41. Li H, Ruan XZ, Powis SH, Fernando R, Mon WY, Wheeler DC, et al. EPA and DHA
reduce LPS-induced inflammation responses in HK-2 cells: evidence for a PPAR-gamma-
dependent mechanism. Kidney international. 2005;67(3):867-74.
42. Cuzzocrea S, Pisano B, Dugo L, Ianaro A, Maffia P, Patel NS, et al. Rosiglitazone, a
ligand of the peroxisome proliferator-activated receptor-gamma, reduces acute inflammation.
Eur J Pharmacol. 2004;483(1):79-93.
43. Raikwar HP, Muthian G, Rajasingh J, Johnson CN, Bright JJ. PPARgamma antagonists
reverse the inhibition of neural antigen-specific Th1 response and experimental allergic
encephalomyelitis by Ciglitazone and 15-deoxy-Delta12,14-prostaglandin J2. Journal of
neuroimmunology. 2006;178(1-2):76-86.
44. Ohshima Y, Yamada A, Tokuriki S, Yasutomi M, Omata N, Mayumi M. Transmaternal
exposure to bisphenol a modulates the development of oral tolerance. Pediatr Res.
2007;62(1):60-4.
20
45. Yan H, Takamoto M, Sugane K. Exposure to Bisphenol A prenatally or in adulthood
promotes T(H)2 cytokine production associated with reduction of CD4CD25 regulatory T cells.
Environ Health Perspect. 2008;116(4):514-9.
46. Ramilo G, Valverde I, Lago J, Vieites JM, Cabado AG. Cytotoxic effects of BADGE
(bisphenol A diglycidyl ether) and BFDGE (bisphenol F diglycidyl ether) on Caco-2 cells in vitro.
Arch Toxicol. 2006;80(11):748-55.
47. Ingemarsson I. Gender aspects of preterm birth. BJOG. 2003;110 Suppl 20:34-8.
48. Cooperstock M, Campbell J. Excess males in preterm birth: interactions with gestational
age, race, and multiple birth. Obstet Gynecol. 1996;88(2):189-93.
49. Romero R, Scoccia B, Mazor M, Wu YK, Benveniste R. Evidence for a local change in
the progesterone/estrogen ratio in human parturition at term. American journal of obstetrics and
gynecology. 1988;159(3):657-60.
50. Bry K, Teramo K, Lappalainen U, Waffarn F, Hallman M. Interleukin-1 receptor
antagonist in the fetomaternal compartment. Acta Paediatr. 1995;84(3):233-6.
51. Romero R, Mazor M, Tartakovsky B. Systemic administration of interleukin-1 induces
preterm parturition in mice. American journal of obstetrics and gynecology. 1991;165(4 Pt
1):969-71.
52. Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, et al. Decrease in
anogenital distance among male infants with prenatal phthalate exposure. Environ Health
Perspect. 2005;113(8):1056-61.
53. Hanaoka T, Kawamura N, Hara K, Tsugane S. Urinary bisphenol A and plasma hormone
concentrations in male workers exposed to bisphenol A diglycidyl ether and mixed organic
solvents. Occupational and environmental medicine. 2002;59(9):625-8.
54. vom Saal FS, Hughes C. An extensive new literature concerning low-dose effects of
bisphenol A shows the need for a new risk assessment. Environ Health Perspect.
2005;113(8):926-33.
21
55. Welshons WV, Nagel SC, vom Saal FS. Large effects from small exposures. III.
Endocrine mechanisms mediating effects of bisphenol A at levels of human exposure.
Endocrinology. 2006;147(6 Suppl):S56-69.
56. Hyoung UJ, Yang YJ, Kwon SK, Yoo JH, Myoung SC, Kim SC, et al. Developmental
toxicity by exposure to bisphenol A diglycidyl ether during gestation and lactation period in
Sprague-Dawley male rats. Journal of preventive medicine and public health = Yebang
Uihakhoe chi. 2007;40(2):155-61.
57. Farabollini F, Porrini S, Dessi-Fulgherit F. Perinatal exposure to the estrogenic pollutant
bisphenol A affects behavior in male and female rats. Pharmacology, biochemistry, and
behavior. 1999;64(4):687-94.
58. Patisaul HB, Polston EK. Influence of endocrine active compounds on the developing
rodent brain. Brain research reviews. 2008;57(2):352-62.