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Direct and transgenerational effects of low doses of perinatal Di-(2-ethylhexyl) phthalate (DEHP) on social behaviors in mice


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Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disrupting chemical commonly used as a plasticizer in medical equipment, food packaging, flooring, and children’s toys. DEHP exposure during early development has been associated with adverse neurobehavioral outcomes in children. In animal models, early exposure to DEHP results in abnormal development of the reproductive system as well as altered behavior and neurodevelopment. Based on these data, we hypothesized that developmental exposure to DEHP would decrease social interactions and increase anxiety-like behaviors in mice in a dose-dependent manner, and that the effects would persist over generations. C57BL/6J mice consumed one of three DEHP doses (0, 5, 40, and 400 μg/kg body weight) throughout pregnancy and during the first ten days of lactation. The two higher doses yielded detectable levels of DEHP metabolites in serum. Pairs of mice from control, low, and high DEHP doses were bred to create three dose lineages in the third generation (F3). Average anogenital index (AGI: anogenital distance/body weight) was decreased in F1 males exposed to the low dose of DEHP and in F1 females exposed to the highest dose. In F1 mice, juvenile pairs from the two highest DEHP dose groups displayed fewer socially investigative behaviors and more exploratory behaviors as compared with control mice. The effect of DEHP on these behaviors was reversed in F3 mice as compared with F1 mice. F1 mice exposed to low and medium DEHP doses spent more time in the closed arms of the elevated plus maze than controls, indicating increased anxiety-like behavior. The generation-dependent effects on behavior and AGI suggest complex mechanisms by which DEHP directly impacts reproductive and neurobehavioral development and influences germline-inherited traits.
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Direct and transgenerational effects of low
doses of perinatal di-(2-ethylhexyl) phthalate
(DEHP) on social behaviors in mice
Kayla M. Quinnies
, Erin P. Harris
, Rodney W. Snyder
, Susan S. Sumner
, Emilie
F. Rissman
1Department of Biochemistry and Molecular Genetics and Neuroscience Graduate Program, University of
Virginia School of Medicine, Charlottesville, VA, United States of America, 2Discovery Science Technology,
RTI International, Research Triangle Park, NC, United States of America, 3Center for Human Health and the
Environment, North Carolina State University, Raleigh, NC, United States of America
These authors contributed equally to this work.
Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine disrupting chemical commonly used as
a plasticizer in medical equipment, food packaging, flooring, and children’s toys. DEHP
exposure during early development has been associated with adverse neurobehavioral out-
comes in children. In animal models, early exposure to DEHP results in abnormal develop-
ment of the reproductive system as well as altered behavior and neurodevelopment. Based
on these data, we hypothesized that developmental exposure to DEHP would decrease
social interactions and increase anxiety-like behaviors in mice in a dose-dependent manner,
and that the effects would persist over generations. C57BL/6J mice consumed one of three
DEHP doses (0, 5, 40, and 400 μg/kg body weight) throughout pregnancy and during the
first ten days of lactation. The two higher doses yielded detectable levels of DEHP metabo-
lites in serum. Pairs of mice from control, low, and high DEHP doses were bred to create
three dose lineages in the third generation (F3). Average anogenital index (AGI: anogenital
distance/body weight) was decreased in F1 males exposed to the low dose of DEHP and in
F1 females exposed to the highest dose. In F1 mice, juvenile pairs from the two highest
DEHP dose groups displayed fewer socially investigative behaviors and more exploratory
behaviors as compared with control mice. The effect of DEHP on these behaviors was
reversed in F3 mice as compared with F1 mice. F1 mice exposed to low and medium DEHP
doses spent more time in the closed arms of the elevated plus maze than controls, indicating
increased anxiety-like behavior. The generation-dependent effects on behavior and AGI
suggest complex mechanisms by which DEHP directly impacts reproductive and neurobe-
havioral development and influences germline-inherited traits.
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 1 / 19
Citation: Quinnies KM, Harris EP, Snyder RW,
Sumner SS, Rissman EF (2017) Direct and
transgenerational effects of low doses of perinatal
di-(2-ethylhexyl) phthalate (DEHP) on social
behaviors in mice. PLoS ONE 12(2): e0171977.
Editor: Cheryl S. Rosenfeld, University of Missouri
Received: August 2, 2016
Accepted: January 30, 2017
Published: February 15, 2017
Copyright: ©2017 Quinnies et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by NIEHS
R01ES022759 (EFR), NIH Common Fund Grant
U24DK097193 (SSS). EPH was supported in part
by T32GM008328.
Competing interests: The authors have declared
that no competing interests exist.
Di-(2-ethylhexyl) phthalate (DEHP) is a synthesized component of flexible plastics used to
make polyvinyl chloride (PVC), some types of packaging, medical tubing, synthetic flooring
and other products [13]. Concentrations of DEHP in manufactured materials may reach 40%
by weight and over 98% of the US population has detectable levels of its metabolites in urine
[4]. This endocrine disrupting chemical (EDC) crosses the placenta [5], is absorbed through
the skin [6], and its metabolites are found in human breast milk [7]. Daily intake in humans
has been estimated between 6–21 μg/kg, with children at the higher end of this range [8]. Over
85% of the studies included in a recent review reported levels of phthalate exposure in children
above the maximum reference dose set by the United States Environmental Protection Agency
DEHP disrupts endocrine function in various glands and tissues throughout the body, but
is most commonly considered to be anti-androgenic [10]. Maternal levels of the major DEHP
metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are correlated with decreased levels of ste-
roid hormones in human male infants [11], and decreased function of Sertoli and Leydig cells
in adult rodents [12,13]. DEHP has non-monotonic dose-response effects. For example, in
mice, high doses cause a drop in maternal and fetal serum testosterone, whereas lower levels of
DEHP increase testosterone [14]. Anogenital distance (AGD; distance from anus to genitalia)
is determined by androgen action during early development: males have longer AGD than
females, and this can be perturbed by manipulations of androgen levels. Increased gestational
DEHP levels correlate with decreased AGD in male rodents [1416] and humans [1719],
which further indicates its anti-androgenic actions.
Human epidemiological studies have also revealed associations between prenatal phthalate
exposure and adverse neurodevelopmental outcomes in children [2026]. DEHP metabolite
levels in utero are associated with decreased masculine play in boys [27]. Boys with attention
deficit hyperactivity disorder (ADHD) have higher urinary concentrations of DEHP metabo-
lites, which are negatively correlated with cortical thickness [28]. Phthalates have also been
implicated in the pathogenesis of autism spectrum disorder (ASD) [29]. Children with autism
spectrum disorder (ASD) have higher urinary levels of DEHP metabolites [30,31] and show
impaired glucuronidation of DEHP metabolites as compared with typically developing chil-
dren [32]. It is worth noting that DEHP levels are positively correlated with fast food con-
sumption [4]. Thus studies that show correlations between behaviors and current levels of
DEHP in children may reflect food choices.
In animals, DEHP exposure during several developmental periods (gestation and/or suck-
ling, puberty, or adulthood) increases anxiety and depression-like behavior [12,33,34]. DEHP
exposure during puberty decreases adult social interactions in female mice, but enhances inter-
actions in males [33,35]. We recently reported sex-specific effects of DEHP three generations
removed from the initial gestational exposure (F3); juvenile male F3 mice from a DEHP line-
age displayed more digging and less grooming than controls in social interaction tests [36].
While the doses used in this study were relatively high (150 and 200 mg/kg body weight), these
results were the first evidence of a transgenerational effect of DEHP exposure on behavior.
In the present report, we evaluated three low doses of DEHP: 5, 40, and 400 μg/kg, all of
which are below the no observed effect level (NOAEL, 4.8 mg/kg/day) for DEHP and compa-
rable to normal human intake [37]. We detected significant and complex transgenerational
effects of DEHP exposure on AGI, social interactions, as well as a generation-specific effect on
anxiety-like behavior. This is first report of social behavior changes in response to perinatal
low dose DEHP exposure, and the first time that transgenerational effects have been reported
in a low dose exposure lineage.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 2 / 19
Materials and methods
Male and female C57BL/6J mice originally purchased from Jackson Laboratory (Bar Harbor, ME)
were used in our colony at the University of Virginia. All procedures followed were approved by
the University of Virginia Animal Care and Use Committee guidelines. All animals were main-
tained on a 12:12 light/dark cycle (lights on at 1300h), provided with a diet low in phytoestrogens
(Harlan Laboratories, Indianapolis, IN #2918), and water ad libitum. Females were paired with
males shortly before lights off each day and were checked for mating plugs the next day. Dams
were assigned to a dose group at random as they became pregnant, with the goal of keeping
groups as equal in size as possible. Males were assigned at random from a large group of breeding
males. These pairs were the original generation (F0). Pairs were separated and females were indi-
vidually housed on the day the mating plug was observed. On each day of gestation and continu-
ing until the litter was 10 days of age, dams consumed a Cocoa Puff coated in 50 μL of stripped
corn oil containing doses of DEHP equivalent to 0, 5 (“Low dose”), 40 (“Medium dose”), or 400
(“High dose”) μg/kg body weight per day. Doses were presented before lights off and females
were observed to ensure they consumed the entire treat. On postnatal day 1 (P1), we recorded
sex, body weight, and AGD for each pup in all litters. Following these measurements, all litters
were culled to 6 pups with a sex ratio as close as possible to equal; no single sex litters were used.
Pups selected for culling were randomly chosen within each sex and not based on bodyweight or
AGD measurements. Culled pups were sacrificed by first reducing their body temperatures and
then using cervical dislocation. Mice were weaned on P21, housed in same-sex, same-litter groups
and randomly assigned to behavioral testing or breeding. The mice remained housed in groups
during behavioral testing. Mice were only tested on one task using no more than two mice from
the same litter, one of each sex. F1 pairs were made with dose-matched non-siblings to create F2
offspring. The same breeding protocol was followed with the F2 mice to create F3 mice. Due to
space and cost considerations we conducted the transgenerational work with lineages produced
from the control (0 dose), low (5 μg/kg), and highest dose (400 μg/kg) of DEHP. Observers blind
to the treatments of the mice scored all behaviors.
MEHP analysis
A cohort of females was paired, checked daily for plugs, and given a daily dose of DEHP or
vehicle as previously described. The day we noted the plug was embryonic day 0.5 (E0.5).
These dams were sacrificed at E18.5, no more than three hours after consumption of the daily
DEHP dose. After anesthesia with isoflurane, animals were euthanized by decapitation and
trunk blood was collected from the dams and embryos (pooled by litter). We collected serum
from 4 control dams, 2 control embryo pools, 6 low dose dams, 1 low dose embryo pool, 3
medium dose dams, 1 medium dose embryo pool, 6 high dose dams, and 3 high dose embryo
pools. Serum was frozen on dry ice, stored at -80˚C, and later assayed for DEHP metabolites
using a method modified from [38] as described below.
Standards were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada)
and standard curve spiking solutions were prepared in methanol. The standard curve for LC/
MS/MS analysis consisted of concentrations ranging from 1 to 1000 ng/mL. Internal standard
) was added at 5 μL per sample from a solution in methanol at a concentration of
1μg/mL. Serum (50 μL) was aliquoted into a polypropylene microfuge tube, and 150 μL meth-
anol was added as well as internal standard. Samples were vortexed, centrifuged (13000 rpm
for 10 minutes) and 50 μL of the supernatant was transferred to a glass Agilent LCMS low vol-
ume insert and mixed with 50 μL of 5 mM ammonium acetate. For standards, 50 μL of blank
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 3 / 19
mouse serum was mixed with 5 μL of spiking solution containing each compound and
extracted as per standards. Standard curves were run prior to mouse serum samples and after
mouse serum samples. Quality control samples were included in the middle of the analysis.
Standards were within 15% of nominal or 20% of LOQ. Samples were analyzed using a Waters
Acquity UPLC (Milford, MA, USA) coupled to an Applied Biosystems/Sciex API 5000 Mass
Spectrometer (Concord, Ontario, Canada) with an electrospray ion source.
Body weight and AGD
Prior to culling litters on P1, we recorded sex, body weight, and AGD for each pup in all litters.
For the F1 litters, we recorded measurements from 6 control litters (N = 18 males, N = 17
females), 11 low dose litters (N = 34 males, N = 28 females), 10 medium dose litters (N = 28
males, N = 37 females), and 7 high dose litters (N = 17 males, N = 15 females). For the F3 gen-
eration we took measurements from 9 control litters (N = 19 males, N = 23 females), 7 low
dose lineage litters (N = 28 males, N = 15 females), and 7 high dose lineage litters, (N = 16
males, N = 22 females).
Maternal behaviors
We observed a total of 34 F0 dams: Control N = 10, Low dose N = 7, Medium dose N = 9,
High dose N = 8. We also observed 16 F2 dams with their F3 litters: Control N = 5, Low dose
N = 5, High dose N = 6. Both F0 and F2 dams were observed in their home cages with their lit-
ters for 30 minutes during the dark (under red lights) portion of the light:dark cycle. Observa-
tions were made on postnatal days 2, 4, and 6. Scan-sampling methods were used to record
maternal behaviors: every 15 seconds we noted if the dam was on or off the nest and recorded
her behaviors. Behaviors on the nest were: licking and grooming pups, self-grooming, nursing,
nest building and hovering. Behaviors off the nest were: eating/drinking, self-grooming, and
digging/climbing [39].
Social interaction test
Age-, dose- and sex-matched pairs of non-sibling juvenile mice (between P28-32) were indi-
vidually habituated to novel cages containing only clean bedding for 10 minutes, and recorded
together in another clean cage for 30 minutes. Tests were conducted during the dark portion
of the light cycle under red lights. Behaviors were scored from videos. Scan-sampling was
used: the behavior of each mouse was observed and recorded every 15 seconds. Individuals in
the pair were identified by a Sharpie black stripe on the tail of one of the mice. Each video was
scored twice and the total number of observations was based on individuals in the pair. Inter-
active behaviors recorded included: side-by-side sitting, social grooming, investigation (sniff-
ing), following, crawling, approaching, and circling the partner. Independent behaviors were:
exploring the cage, self-grooming alone, sitting alone, and jumping. In addition, we calculated
total interactive and total independent behaviors by summing the frequencies of each event.
Elevated plus maze
Juvenile (P30-35) F1 and F3 mice were habituated to the behavioral testing room for at least 30
minutes during the dark cycle under red lights, then placed in the center of the elevated plus
maze (Columbus Instruments; wall height: 6“, arm length: 11.75”, arm width: 2”) and recorded
for 5 minutes. The total time spent in the closed and open arms and the numbers of crosses
through the center were scored from the video recordings using Noldus Observer.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 4 / 19
Statistical analysis
All analyses were done using NCSS 2007. Interactions between juvenile pairs and dams with
pups were expressed as a proportion of total activity; we used an arcsine transformation to nor-
malize these data. We did not continue to breed the mice produced from the medium dose
(40 μg/kg) to the third generation, therefore we had 4 dose groups in F0 dams and F1 off-
spring, and 3 doses in F2 dams and their F3 offspring. The generations were analyzed sepa-
rately with two-way ANOVAs (sex and dose). To compare the generations, we used three-way
ANOVAs (sex, dose and generation), only including the 3 doses common to both F1 and F3
(Control, low dose, and high dose groups). Maternal behaviors were analyzed with repeated
measures ANOVAs with pup age as the within subjects factor. All ANOVAs were followed by
Bonferroni-tests corrected multiple comparisons to evaluate pairwise interactions. F statistics
and degrees of freedom for all analyses can be found in S1 and S2 Tables.
Low concentrations of DEHP metabolites in serum
The highest dose, 400 μg/kg, yielded detectable MEHP and 5-OH-MEHP levels in all serum sam-
ples measured. Serum concentrations of MEHP for 400 μg/kg dosed dams averaged 160 ±17.2
ng/ml and 1.6 ±0.4 ng/mL for 5-OH-MEHP. Metabolite concentrations of pooled embryo serum
from 400 μg/kg dosed dams averaged 96.7 ±36.9 ng/ml for MEHP and 1 ±0.20 ng/ml for 5-OH-
MEHP. These values are within the range of levels measured in human blood [40]. Two of the
three serum samples from dams dosed at 40 μg/kg had detectable MEHP levels (mean ±SEM:
14.2 ±12.4 ng/ml) and one had detectable 5-OH-MEHP levels (0.54 ng/ml). Metabolite levels in
pooled serum from embryos of 40 μg/kg-dosed dams were undetectable. DEHP and metabolite
levels in control and low dose pregnant females and their E18.5 embryos were below the detection
limit (LOD) of the assay (LOD for MEHP and 5-OH-MEHP were estimated at 0.1 ng/mL).
Effects of DEHP on AGI and body weights
DEHP exposure decreased AGI in males and females in the F1 generation and in F3 female
mice from the lowest dose lineage had larger AGI than the controls. We evaluated males and
females separately because of the substantial sex difference in AGI measurements. In F1 males,
there was a significant dose effect; AGIs of low dose males were smaller than controls (p<0.05,
Table 1). In F1 females, the high dose (400 μg/kg) group had smaller AGIs than both the con-
trol and low dose groups (p<0.01). Body weights on P1 were not affected by DEHP in either
sex (p>0.05).
In the F3 generation, no differences in AGI were observed in males (p<0.05) and there
were no effects of DEHP lineage on body weights in F3 males (p>0.05). Females in the low
dose lineage had larger AGIs than both controls and high dose lineage females (p<0.01). In
addition, we found an effect of DEHP dose on body weight in females (p<0.001, Table 1) such
that low dose lineage females weighed less than both other groups. It is likely that these body
weight differences produced the effects on AGI. Comparison of F1 and F3 generations
revealed transgenerational effects in females. Dose produced an effect on AGI (p<0.001); high
dose females had smaller AGIs compared with the control and low dose females. We also
found a dose by generation interaction (p<0.05, S1 Table). Additionally, we found effects of
generation (p<0.01), dose, and an interaction for female body weights (p<0.05, S1 Table). F1
females were heavier than F3 females, and the controls and highest dose groups were heavier
than the low dose group. The interactions reveal that the low dose F3 mice were lighter than all
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 5 / 19
other groups, except the F1 controls. In males, body weights and AGI were not affected by
DEHP dose or generation (p>0.05, S1 Table).
Pup age and generation affects maternal behaviors
The original (F0) dams did not display any DEHP-related differences in maternal care. How-
ever, as expected, the age of the litter significantly affected two measures: time in nest and eating
(p<0.05). The direction of the differences was consistent with dams spending more time in the
nest with younger (P2) versus older (P6) pups. The F2 dams did not show any effects of DEHP
dose lineage, but dams spent more time licking and grooming their litters on P2 than on P4
(p<0.05). Comparing maternal behaviors from dams in the three doses represented in both F0
and F2 we noted effects of generation in several measurements of maternal behavior. The effects
of generation suggest a more active phenotype in the F0 dams. In sum, F0 dams spent less time
in the nest (p<0.01), licking and grooming pups (p<0.01), and nursing (p<0.01) as compared
with the F2 dams. F0 dams spent more time digging in the cage (p<0.01) than did the F2 dams.
There were effects of the age of the litter for several measures. Dams spent more time on their
nests (p<0.01), licking and grooming pups (p<0.01), and less time eating (p<0.001) when the
pups were P2 as compared with P6 (Table 2,S1 Table).
Social interactions during pair-tests
F1 generation. In general, F1 juvenile mice exposed in utero to the higher doses of DEHP
(400 and 40 μg/kg) were less interactive than low dose (5 μg/kg) and control mice. We noted
an effect of DEHP dose (p<0.001) on frequency of side-by-side sitting. Mice exposed to the
medium and high doses exhibited less sitting side-by-side than mice in the low dose and con-
trol groups (Fig 1A). The effect of DEHP was limited to males of each dose group, creating a
significant interaction between sex and dose (p<0.01). DEHP dose also affected the frequency
of partner sniffing (p<0.001). F1 mice exposed to the medium dose sniffed their test partner
more frequently than mice in the low dose and control groups (Fig 1B). Medium dose females
had a higher sniffing frequency than low dose females and controls of both sexes, resulting in
an interaction between sex and dose (p<0.05). Increased sniffing by low dose males led to a
sex difference (males sniffing more than females) in mice exposed to the lowest dose. Finally,
when we summed the frequencies of all interactive behaviors we noted an effect of sex; males
Table 1. Effects on AGI and body weights in F1 and F3 generations.
Generation Males AGI (mm/g) Body Weight (g) Females AGI (mm/g) Body Weight (g)
F1 Control (18) 1.17 ±0.05 1.34 ±0.05 Control (17) 0.73 ±0.03 1.30 ±0.03
5μg/kg (34) 1.03 ±0.02
1.37 ±0.02 5μg/kg (28) 0.72 ±0.02 1.31 ±0.02
40 μg/kg (28) 1.03 ±0.03 1.38 ±0.02 40 μg/kg (37) 0.65 ±0.03 1.34 ±0.02
400 μg/kg (17) 1.07 ±0.03 1.31 ±0.03 400 μg/kg (15) 0.61 ±0.05*1.37 ±0.03
F3 Control (19) 1.07 ±0.03 1.31 ±0.03 Control (23) 0.63 ±0.02 1.32 ±0.02
5μg/kg (28) 1.08 ±0.02 1.29 ±0.02 5μg/kg (15) 0.71 ±0.02***1.19 ±0.03***
400 μg/kg (16) 1.02 ±0.03 1.35 ±0.03 400 μg/kg (22) 0.64 ±0.009 1.30 ±0.02
Mean ±SEM. Number of pups per group in (). Data were collected on postnatal day 1.
*Significantly different from same-sex F1 control and 5 μg/kg groups, p<0.05.
**Significantly different from same-sex F1 control group, p<0.05.
***Significantly different from all other same-sex F3 groups, p<0.05.
Number of F1 litters per group: Control N = 6 litters, 5 μg/kg N = 10 litters, 40 μg/kg N = 10 litters, 400 μg/kg N = 6 litters. Number of F3 litters per group:
Control lineage N = 9 litters, 5 μg/kg lineage N = 7 litters, 400 μg/kg lineage N = 6 litters.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 6 / 19
were more interactive than females (p<0.01, S1 Table). However, there were no effects of dose,
nor an interaction of dose and sex (p>0.05). The same statistical effects apply to the inverse
measurement, summed independent behaviors.
In F1 mice, two independent behaviors were affected by DEHP dose. Control and low dose
mice sat alone more frequently than either of the two higher DEHP dose groups (p<0.001, Fig
1C). For exploring the cage alone, we found effects of sex (p<0.05) and dose (p<0.05). Females
explored more than males, and mice exposed to the highest dose of DEHP explored more fre-
quently than both the control and low dose groups (Fig 1D).
F3 generation. Several behavioral differences were related to DEHP lineage in the F3 ani-
mals. In general, F1 and F3 effects of DEHP were reversed. In contrast to high dose (400 ug/
kg) F1 groups, F3 males from the highest DEHP lineage in particular, were highly interactive.
We found effects of sex (p<0.05), dose lineage (p<0.05), and an interaction (p<0.01) on fre-
quencies of sitting together where males exhibited more side-by-side sitting than females and
high dose lineage animals sat side-by-side with their partners more often than controls. The
frequency of side-by-side sitting for the high dose male group was greater than all groups
except low dose females (Fig 2A). There was no effect of ancestral DEHP exposure or sex on
total sniffing frequencies (Fig 2B). However, the total frequency of all interactive behaviors was
affected by sex (p<0.05), DEHP dose (p<0.001), and there was an interaction (p<0.05, S1
Table). Males interacted more frequently with their test partners than females, high dose ani-
mals displayed more interactive behaviors, and the high dose males drove both effects. The
inverse was true for total independent behaviors: we observed fewer independent behaviors
overall in high dose F3 males. For exploration, the most frequently displayed independent
behavior; a dose lineage effect was revealed (p<0.001). F3 mice in the high dose lineage mice
explored the cage alone less than control and low dose lineage groups (Fig 2D). For sitting
Table 2. Maternal behavior in F0 and F2 dams.
F1 F3
Control (10) 5 μg/kg (7) 40 μg/kg (9) 400 μg/kg (8) Control (5) 5 μg/kg (5) 400 μg/kg (6)
Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM
On Nest^ P2*0.47 0.13 0.44 0.14 0.44 0.10 0.51 0.11 P2 0.73 0.14 0.78 0.20 0.62 0.11
P4*0.33 0.12 0.37 0.17 0.41 0.14 0.33 0.08 P4 0.65 0.11 0.46 0.15 0.41 0.17
P6*0.26 0.09 0.31 0.12 0.21 0.12 0.13 0.05 P6 0.66 0.10 0.58 0.16 0.31 0.09
Total nursing^ P2 0.22 0.10 0.21 0.13 0.09 0.06 0.22 0.11 P2 0.34 0.12 0.53 0.22 0.19 0.09
P4 0.13 0.09 0.19 0.15 0.12 0.08 0.07 0.05 P4 0.42 0.16 0.23 0.19 0.27 0.17
P6 0.12 0.06 0.20 0.10 0.11 0.09 0.00 0.00 P6 0.47 0.12 0.29 0.16 0.09 0.06
Licking P2 0.13 0.04 0.10 0.03 0.15 0.04 0.11 0.03 P2*0.20 0.04 0.17 0.08 0.24 0.05
and grooming^ P4 0.09 0.04 0.06 0.03 0.16 0.06 0.06 0.02 P4*0.12 0.02 0.15 0.08 0.04 0.02
P6 0.05 0.03 0.06 0.01 0.06 0.03 0.04 0.02 P6*0.11 0.01 0.15 0.07 0.11 0.03
Digging P2 0.29 0.07 0.35 0.09 0.34 0.07 0.33 0.09 P2 0.14 0.09 0.11 0.10 0.13 0.04
P4 0.32 0.07 0.43 0.13 0.35 0.10 0.41 0.07 P4 0.09 0.03 0.18 0.09 0.18 0.07
P6 0.31 0.05 0.26 0.06 0.38 0.08 0.56 0.10 P6 0.11 0.04 0.20 0.09 0.32 0.09
Eating P2*0.20 0.06 0.15 0.05 0.17 0.03 0.10 0.03 P2 0.12 0.05 0.11 0.10 0.20 0.09
P4*0.29 0.06 0.18 0.06 0.20 0.07 0.23 0.04 P4 0.26 0.09 0.35 0.12 0.36 0.12
P6*0.40 0.08 0.34 0.06 0.37 0.08 0.28 0.07 P6 0.22 0.09 0.20 0.09 0.33 0.07
Mean proportion of maternal behaviors on each day measured: postnatal days 2 (P2), 4 (P4), and 6 (P6). Number of dams per group in ().
*Significant effect of litter age within generation, p<0.05.
^Significant effect of generation, p<0.01.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 7 / 19
alone, a less frequently displayed independent behavior, there was an effect of DEHP dose line-
age (p<0.05). Mice in the high dose lineage sat alone more frequently than controls (Fig 2C).
Comparison of F1 and F3 behaviors. To assess generational differences directly we com-
pared F1 versus F3 mice in the three doses studied in both generations. Comparing F1 and F3
mice we found an effect of sex (p<0.01) and generation (p<0.001) for side-by-side sitting.
Males were sat side-by-side more than females and F3 mice displayed this behavior more fre-
quently than F1 mice. We also found interaction effects between dose and generation
(p<0.001), sex and generation (p<0.05), and all three variables (p<0.001, S1 Table). Control
and low dose mice in the F1 generation displayed more sitting side-by-side than F3 control
and low dose mice. Additionally, F3 females displayed significantly less side-by-side sitting
than F3 males and F1 mice of both sexes. For partner sniffing, there was a dose by sex interac-
tion (p<0.01, S1 Table). Regardless of generation, control females sniffed their partners less
often than low dose females and high dose males. For the total frequency of interactive behav-
iors, we found an effect of dose (p<0.001), a two-way interaction of dose and generation
(p<0.001), and a three-way interaction of dose, sex, and generation (p<0.01, Fig 3A). Mice in
the highest dose group were more interactive, particularly the F3 high dose males. Addition-
ally, there were effects of sex (p<0.001) and generation (p<0.05) whereby males were more
interactive than females, and F1 mice were less interactive than F3 mice. The statistical
Fig 1. Effects of DEHP on juvenile pairs social behaviors tests in F1 mice. Mean ±SEM proportion of
time spent A) sitting side-by-side, B) sniffing partner, C) sitting alone, and D) exploring the cage alone during a
30-min. test. White bars represent data from control mice, light grey bars show data from the lowest dose
(5 μg/kg), dark grey bars show data from the medium dose (40 μg/kg), and black bars represent data from the
highest dose (400 μg/kg). *Dose(s) significantly different from control and 5 μg/kg groups, p<0.05. &&
Significantly different from same-sex control and 5 μg/kg groups, p<0.05. #Significantly different from males,
p<0.05. ##Significantly different from females of the same dose group, p<0.05. Numbers of F1 mice per
group: Control male N = 18, Control female N = 14, 5 μg/kg Male N = 14, 5 μg/kg Female N = 14, 40 μg/kg
Male N = 6, 40 μg/kg Female N = 12, 400 μg/kg Male N = 8, 400 μg/kg Female N = 6.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 8 / 19
significance for total interactive behaviors is the same for the inverse measure, total indepen-
dent behaviors (Fig 3B).
For independent behaviors, sitting alone was affected by generation (p<0.001): F1 mice sat
alone significantly more often than F3 mice. Specifically, an interaction between generation
and dose showed that control and low dose F1 mice sat alone more than mice in any other
group (p<0.001, S1 Table). The data also revealed an effect of dose for exploring where high
dose mice explored the cage less than control and low dose groups (p<0.01). An interaction
between dose and generation (p<0.001, S1 Table) for exploring was caused by the high dose
F3 mice, which explored less than any other groups. Additional statistics for social grooming,
crawling, approach, circling, self-grooming alone, and jumping are listed in S2 Table.
Sex differences in elevated plus maze behavior. We found a sex difference on the EPM
in juvenile mice. F1 juvenile males spent more time in the closed arms than females, resulting
in an effect of sex (p<0.05). DEHP dose affected the amount of time spent in the closed arms
(p<0.05) where low and medium dose groups spent more time in the closed arms than con-
trols (Fig 4A). There was a trend for an effect of sex in the number of crosses through the cen-
ter of the maze, an indirect measure of activity (p = 0.06), where females tended to be more
active than males on the maze. There were no effects of DEHP dose, nor was there an interac-
tion of sex and dose on crosses through the center (p>0.05, Fig 4B). There were no significant
effects of DEHP on time spent in the open arms or in the center portion of the maze (p>0.05,
Fig 2. Effects of DEHP on juvenile pairs social behaviors tests in F3 mice. Mean ±SEM proportion of
time spent A) sitting side-by-side, B) sniffing partner, C) sitting alone, and D) exploring the cage alone during a
30-min. test. White bars represent data from control mice; light grey bars show data from the lowest dose
(5 μg/kg), and black bars represent data from the highest dose (400 μg/kg). *Significantly different from other
groups, p<0.05. **Significantly different from same-sex controls, p<0.05. & Significantly different from all
other groups except low DEHP dose females, p<0.05. Numbers of F3 mice per group: Control lineage male
N = 10, Control lineage female N = 6, 5 μg/kg Lineage Male N = 8, 5 μg/kg Lineage Female N = 12, 400 μg/kg
Lineage Male N = 4, 400 μg/kg Lineage Female N = 6.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 9 / 19
S1 Table). In F3 mice, we also observed that males spent more time in the closed arms than
females (p<0.05, Fig 4C). There was no significant dose effect, nor was there an interaction
between dose and sex (p>0.05). Center crosses in F3 mice were not affected by sex, dose, nor
was an interaction present (p>0.05; Fig 4D). DEHP lineage did not affect time spent in the
open arms or center of the maze in the F3 generation (p>0.05, S1 Table).
A between-generation comparison of the doses common to all groups yielded similar statis-
tical results. Males spent more time in the closed arms than females (p<0.01; Fig 4A and 4C).
There was no effect of generation or dose (p>0.05) for closed time. Sex and generation both
affected the number of crosses through the center of the maze (p<0.05, S1 Table). Overall,
Fig 3. Comparison of total interactive and independent behaviors between generations. Mean ±SEM
proportion of time spent engaging in A) all interactive behaviors (side-by-side sitting, social grooming, sniffing,
following, approaching, crawling, and circling the partner). B) All independent behaviors (exploring the cage,
self-grooming alone, sitting alone, and jumping). White bars represent data from control groups, light grey
bars show data from the low dose lineage (5 μg/kg), and black bars represent data from the highest dose
lineage (400 μg/kg). ^Significantly different from other generation, same group(s), p<0.05. ***Significantly
different from all other groups (sexes and generations), p<0.05. M = Male, F = Female
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 10 / 19
females crossed through the center of the maze more than males (p<0.05) and F3 mice made
more crosses through the center than F1 mice (p<0.05, Fig 4B and 4D). Across F1 and F3 gen-
erations, females spent more time in the center of the maze than males (p<0.01), but there was
no significant sex difference in time spent in the open arms (p>0.05). Likewise, there were no
effects of dose or generation for either measure (p>0.05, S1 Table).
Here we report that neonatal exposure to DEHP has dose-, sex-, and generation-dependent
effects on social behavior, anxiety-like behavior, and AGI in mice. While some of the measures
we examined have been reported on previously, here, we used very low doses, on the order of
500-fold lower than used in other animal studies [12,34,35]. Daily oral DEHP exposure to
pregnant females resulted in serum metabolite levels within the range of human exposure.
Serum MEHP levels in embryos from the highest dose averaged 96.7 ±36.9 ng/mL, much
lower than levels in human cord blood serum which at birth average 0.52 ±0.61 μg/mL [41].
We found no significant effect of DEHP exposure on maternal behavior in the F1 or F3 gener-
ations, indicating that the transgenerational effects of DEHP are likely not socially transmitted
via differences in maternal care of offspring. However, we recognize that maternal behavior
Fig 4. Elevated Plus Maze Behavior. Mean ±SEM for each group A) Time (sec) spent in the closed arm of
the elevated plus maze (EPM) in F1 mice. B) Total number of crosses through the center of the EPM in F1
mice. C) Time (in seconds) spent in the closed arm of the EPM in F3 mice. D) Total number of crosses into the
center of the EPM in F3 mice. White bars represent data from control mice, light grey bars show data from the
lowest dose (5 μg/kg), dark grey bars show data from the medium dose (40 μg/kg), and black bars represent
data from the highest dose (400 μg/kg). **Doses significantly different from controls, p<0.05. #Significantly
different from other sex, p<0.05. Numbers of F1 mice per group: Control male N = 8, Control female N = 9,
5μg/kg Male N = 8, 5 μg/kg Female N = 11, 40 μg/kg Male N = 5, 40 μg/kg Female N = 9, 400 μg/kg Male
N = 5, 400 μg/kg Female N = 4. Numbers of F3 mice per group: Control male N = 7, Control female N = 7,
5μg/kg Lineage Male N = 13, 5 μg/kg Lineage Female N = 9, 400 μg/kg Lineage Male N = 8, 400 μg/kg
Lineage Female N = 12.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 11 / 19
was only measured during the first week, and that future studies could evaluate this effect
throughout the entire lactation period.
Our social behavior results are consistent with epidemiological studies in human popula-
tions indicating significant impacts of early life exposure to phthalates on neurobehavioral out-
comes [2026]. Several of the individual behavioral measures we observed were modified by
gestational and lactational exposure to DEHP, and some behavioral differences persisted to the
F3 generation. F1 generation mice exposed to the two highest doses (40 and 400 μg/kg) dis-
played less frequent side-by-side sitting and sitting alone than control animals. High dose ani-
mals also explored the cage more than controls, which could indicate a difference in overall
activity. However, our indirect activity data from the EPM do not support this interpretation.
Additionally, we noted an inverted “U”-shaped response for males in the frequency of sniffing
their partners, which is suggestive of a non-monotonic dose response.
The only existing data comparable to ours are from research conducted in ICR mice that
received doses of DEHP given by gavage during puberty (P28-P42). In this study, males
exposed to 50 mg/kg DEHP engaged in more “social play” and spent more time in social inves-
tigation time than controls [35]. Females in the same study exposed to several doses of DEHP
(1, 10, 50 or 200 mg/kg) displayed less investigation than controls [33,35]. In contrast, in this
study social sniffing frequency was increased by the medium dose (40 ug/kg, a greater than
1000-fold lower dose) in both sexes. It is difficult to directly compare the results of these stud-
ies as the age of subjects, time of exposure, doses, route of administration, and mouse strain
are different. Exposure to DEHP during puberty cannot affect early neurodevelopmental pro-
cesses occurring during the critical period of gestation, but it may affect the maturation of
reproductive system and levels of circulating sex steroids, which could alter social behaviors
[42]. Additionally, there are documented strain-dependent differences in social interaction
behaviors as well as sensitivity to EDCs, including DEHP [4345]. Despite these differences,
both studies demonstrate significant effects of DEHP exposure during critical periods on social
behaviors in mice.
We found transgenerational effects of DEHP on several specific behaviors; side-by-side sit-
ting, sitting alone, and exploring. The mice in the high dose lineage group were primarily
responsible for these effects. High dose lineage mice of both sexes sat alone more frequently
and explored less during the test compared to controls, whereas side-by-side sitting was
increased in high dose lineage males only. When all measurements for interactive and inde-
pendent behaviors were combined, we found a transgenerational enhancement in social inter-
actions and a reduction in independent behaviors in the high dose lineage males. The only
other study on transgenerational behaviors in DEHP lineages is from our group [36]. In that
study, a higher (150 mg/kg) dose was given by gavage to F0 dams and the exposure was
restricted to gestational days 7–14. Juvenile F3 DEHP lineage males displayed more digging
and less self-grooming than controls in a similar social interaction test.
Perhaps the most interesting aspect of our data is the shift in behavioral patterns between
F1 and F3 offspring. In general, in the highest dose group, F1 mice were less interactive and
more independent while F3 mice had the reversed pattern. It is not unusual for transgenera-
tional effects of EDCs to be enhanced or even reversed over the generations [4648]. For
example, in our studies on the transgenerational actions of bisphenol A (BPA) we noted a
behavioral shift between generations exposed to BPA versus control diets in social interactions,
using a similar behavioral testing paradigm [48]. Like F1 DEHP mice, in F1 juveniles exposed
to BPA during gestation we note fewer interactive and more independent behaviors than con-
trols. However, over the generations we found a shift. In the F2 and F4 generations, the BPA
lineage mice were more interactive and less independent than controls. We have since
reported a transgenerational effect of BPA on social recognition. Juvenile mice exposed during
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 12 / 19
gestation to BPA exhibit delayed habituation to the familiar stimulus animal in a social recog-
nition task. F3 offspring show this same delay, but in addition, they failed to recognize the
novel stimulus animal in the last trial. Additionally, F3 BPA lineage mice were more active in
the open field test than controls, but there were no significant effects on activity in the F1 gen-
eration [49]. Unlike the BPA data set, transgenerational actions of DEHP were largely
restricted to males. These data illustrate that EDC actions on the developing brain (in F1) ver-
sus long-term effects on germ cells (F3 and beyond) can result in different behavioral
Transgenerational effects of low doses of EDCs are mainly attributed to epigenetic changes,
presumably at the level of the germ cell. Exposure to DEHP in utero produced increased global
DNA methylation and increased expression of DNA methyltransferases (DNMTs) in fetal tes-
tes [50]. In a different study DEHP exposure during gestation increased DNMT expression in
adult testes of F1 and F2 rat offspring [51]. Additionally, gestational exposure to 40 μg/kg
DEHP (our medium dose) in mice resulted in heritable changes in DNA methylation of
imprinted genes in primordial germ cells [52]. Prenatal phthalate exposure is also associated
with altered DNA methylation patterns in human placenta [5355] and cord blood samples
[56]. Studies of other EDCs have characterized multigenerational and transgenerational mech-
anisms [5761], but in general more research on different aspects of epigenetic regulation (i.e.
miRNA, histone modifications, long non-coding RNAs, etc.) is necessary to determine how
changes in behavior are inherited over generations.
DEHP has documented anti-androgenic actions. In humans, maternal phthalate metabo-
lites in the first trimester of pregnancy are negatively correlated with AGD in infant boys [17,
19] and with human chorionic gonadotropins, which in turn correlated with shorter AGD in
boys and longer AGD in girls [62]. In this study, we observed effects on AGI in both sexes.
Based on dose-response studies conducted in rats and mice [14,16], we hypothesized that we
would observe a non-monotonic dose-response for AGI: the higher doses (40 and 400 μg/kg)
would exert anti-androgenic actions whereas the lower dose (5 μg/kg) would have an andro-
genic effect. However, both low and high doses of DEHP had an anti-androgenic effect in F1
generation males and females. The AGIs in low dose males and high dose females were signifi-
cantly shorter than in the same-sex controls. These findings suggest sex differences in the sen-
sitivity of the developing anogenital region to DEHP in utero. In a similar study in CD-1 mice,
AGI was increased in male fetuses exposed to 5 μg/kg DEHP from gestational days 9–18, while
a larger dose had no effect [14]. Several differences in experimental design may explain the dis-
crepancy between this result and ours. In addition to using a different strain of mouse and a
shorter exposure window, only male fetuses adjacent to one other male fetus (1M) in the
uterus were measured, whereas we could not account for variability due to intrauterine posi-
tion. For females of the F3 generation, ancestral exposure to low doses of DEHP was associated
with increased AGI and decreased body weight compared with control females. It is unclear
whether the dose effect on AGI in low dose F3 females is a secondary effect of decreased body
weight or a genuine effect of dose lineage on reproductive development. In a previous study,
higher doses of DEHP (150 or 200 mg/kg) were given during a more limited exposure period
(gestational days 7–14). Transgenerationally, F3 males from the 150 mg/kg lineage had signifi-
cantly larger AGI and F3 200 mg/kg lineage males had increased body weights, whereas nei-
ther measure was significantly affected by DEHP lineage in F3 females [36].
Lastly, we recorded an increase in anxiety-like behavior in both sexes of the low and
medium doses specifically in the F1 generation. This is the first example of DEHP affecting
anxiety-like behavior at doses this low. To date, there are several reports of increased anxiety-
like behavior in mice directly exposed to higher doses of DEHP (10–540 mg/kg) [12,34,63,
64]. The mechanism underlying the enhanced anxiety-like behavior is not clear. One way
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 13 / 19
DEHP may influence anxiety-like behavior is by disturbing the hypothalamic-pituitary-adrenal
(HPA) axis. MEHP regulates glucocorticoid metabolism by inhibiting 11 beta-hydroxysteroid
dehydrogenase type 2 (11β-HSD2), the enzyme responsible for inactivating the stress hormone
corticosterone [65,66]. 11β-HSD2 is highly expressed in the placenta and embryonic brain and
serves as a barrier to protect developing tissues from excess maternal corticosterone [67]. Rats
exposed to high levels of corticosterone during gestation and early life spend more time in the
closed arms of the elevated plus maze as adults [68]. Dexamethasone, a synthetic glucocorticoid,
amplifies the effect of prenatal phthalate exposure on reproductive outcomes (decreased testos-
terone production, shortened AGD, etc.) in mice [69]. Similarly, maternal stress levels and
DEHP exposure during pregnancy can interact in complex ways to affect AGD in human male
infants [70,71]. These data support the hypothesis that excess gestational corticosterone may
potentiate the effects of DEHP on anxiety-like behavior in the elevated plus maze. We have pre-
viously reported that ancestral exposure to a high dose of DEHP (150 mg/kg/day) is associated
with decreased serum corticosterone at baseline and following restraint stress in DEHP lineage
females as compared with controls, despite observing no significant anxiety-like phenotype on
the elevated plus maze [36].
Some of these changes in behavior may be explained by the impact of DEHP and its metab-
olites on neurodevelopment, of which there are several examples. Dopaminergic neuron num-
bers and tyrosine hydroxylase (TH) immunoreactivity in the midbrain is decreased in 6-week-
old mice following 1 mg/kg daily gestational and neonatal administration of DEHP [72]. Fur-
thermore, pubertal exposure to DEHP at doses ranging from 1–200 mg/kg decreased dopa-
mine receptor D2 protein in the striatum of adult female mice, accompanied by significant
alterations of social and anxiety-like behavior [33]. Additionally, mouse neurons co-cultured
with astrocytes have increased oxidative stress markers and increased gliosis following expo-
sure to biologically relevant DEHP levels [73]. DEHP affects the brain in widespread ways that
may have extensive effects on neurodevelopment and behavior, but the mechanisms for effects
must be more thoroughly explored.
This dose-response response study identified transgenerational effects of perinatal DEHP
exposure on social behaviors in juvenile mice. We also showed increased anxiety-like behavior
and decreased AGI in mice directly exposed to DEHP. This is the first study to report effects of
DEHP exposure on social behavior in F1 and F3 generations at doses this low. Now that behav-
ioral changes in each dose, sex, and generation have been identified, future studies will focus
more directly on mRNA and protein changes in the brain in response to specific doses within
this exposure model. This will provide insight into the impact of DEHP on humans, especially
during critical periods of development.
Supporting information
S1 Table. Supplemental F Statistics. F statistics, degrees of freedom, and symbolic representa-
tions of p values for each factor analyzed in the results section. p<0.05; p<0.01; 
p<0.001; ^ p = 0.06
S2 Table. Statistical values for additional social behavior measures. F statistics, degrees of
freedom, and p values for each effect for the analyses of F1, F3, and F1 vs. F3 for social groom-
ing, crawl, approach, circling, self-grooming alone, and jumping.
DEHP transgenerational social behavior
PLOS ONE | DOI:10.1371/journal.pone.0171977 February 15, 2017 14 / 19
We thank Aileen Ryalls for her technical assistance.
Author Contributions
Conceptualization: EFR EPH KMQ.
Formal analysis: EFR EPH KMQ.
Funding acquisition: SSS EFR.
Investigation: EPH KMQ RWS.
Methodology: EFR EPH KMQ.
Project administration: EFR.
Resources: SSS EFR.
Supervision: EFR.
Visualization: EPH KMQ.
Writing – original draft: EPH KMQ.
Writing – review & editing: EFR EPH KMQ SSS RWS.
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... Animal models of the "developmental origins of health and disease" (DOHaD) hypothesis show cause-and-effect relationships between early life exposures to individual EDCs and abnormalities in cognitive, affective, and social behaviors later in life [1][2][3][4][5][6][7][8][9]. Epidemiological evidence in humans suggests that those with higher body burdens of EDCs may have a greater propensity for aberrations in these same behaviors [10][11][12][13][14]. Specific to anxiety-like disorders, these relationships have been shown, to date, for bisphenol A (BPA) [15][16][17][18], phthalates such as DEHP [19,20], and persistent organic pollutants such as polychlorinated biphenyls (PCBs) [21,22] in both rodent and human studies. ...
... In the current study, we designed a mixture comprising bisphenols, phthalates, perfluorinated, polybrominated and polychlorinated compounds, and vinclozolin. Their selection for the Neuro-Mix was based on evidence showing that each chemical individually causes deficits in behaviors and/or changes in neurobiological markers in animals [5,6,19,[63][64][65][66] and is detectable in humans [30,32,[67][68][69][70][71][72]. This current study adds to the literature by using these chemicals in combination at human-relevant dosages and administered by the most relevant (oral) route. ...
... In the current study, we designed a mixture comprising bisphenols, phthalates, perfluorinated, polybrominated and polychlorinated compounds, and vinclozolin. Their selection for the NeuroMix was based on evidence showing that each chemical individually causes deficits in behaviors and/or changes in neurobiological markers in animals [5,6,19,[63][64][65][66] and is detectable in humans [30,32,[67][68][69][70][71][72]. This current study adds to the literature by using these chemicals in combination at human-relevant dosages and administered by the most relevant (oral) route. ...
Full-text available
Humans and wildlife are exposed to endocrine-disrupting chemicals (EDCs) throughout their lives. Environmental EDCs are implicated in a range of diseases/disorders with developmental origins, including neurodevelopment and behavior. EDCs are most often studied one by one; here, we assessed outcomes induced by a mixture designed to represent the real-world situation of multiple simultaneous exposures. The choice of EDCs, which we refer to as “NeuroMix,” was informed by evidence for neurobiological effects in single-compound studies and included bisphenols, phthalates, vinclozolin, and perfluorinated, polybrominated, and polychlorinated compounds. Pregnant Sprague Dawley rats were fed the NeuroMix or vehicle, and then offspring of both sexes were assessed for effects on postnatal development and behaviors and gene expression in the brain in adulthood. In order to determine whether early-life EDCs predisposed to subsequent vulnerability to postnatal life challenges, a subset of rats were also given a stress challenge in adolescence. Prenatal NeuroMix exposure decreased body weight and delayed puberty in males but not females. In adulthood, NeuroMix caused changes in anxiety-like, social, and mate preference behaviors only in females. Effects of stress were predominantly observed in males. Several interactions of NeuroMix and stress were found, especially for the mate preference behavior and gene expression in the brain. These findings provide novel insights into how two realistic environmental challenges lead to developmental and neurobehavioral deficits, both alone and in combination, in a sex-specific manner.
... Adult exposure. Four studies investigated the effects of adult phthalate exposure on maternal behavior, with exposure beginning during gestation and lasting until the lactation period [60,[68][69][70]. Lee et al. [70] reported that dams exposed to DBP (50 or 100 mg/kg/d) exhibited a higher latency to retrieve the second pup and created a poor-quality nest in the DBP-50 group. In contrast, no effects of exposure to high doses of DINP (300-900 mg/kg/d) or to a phthalate mixture (0.2 to 1 mg/kg/d) were found on pup retrieval or spontaneous maternal behavior in rats [60,68]. ...
... In contrast, no effects of exposure to high doses of DINP (300-900 mg/kg/d) or to a phthalate mixture (0.2 to 1 mg/kg/d) were found on pup retrieval or spontaneous maternal behavior in rats [60,68]. Similarly, exposure to DEHP (5 to 400 µg/kg/d) had no effect on the spontaneous maternal behavior of F0 or F2 mouse dams [69]. ...
... Eight studies (five on rats, three on mice) investigated the effects of prenatal/postnatal or prepubertal/pubertal exposure to DEHP or a phthalate mixture on anxiety-like behavior of F1 offspring, using mainly the elevated plus maze alone or in combination with the open-field (Table 2). Among these studies, seven analyzed these effects on cyclic females [69,[71][72][73][74][75][76] and one in postpartum females [67]. Four out of seven studies found increased anxiety-state levels in PND30-35 mice exposed to 5 or 40 µg/kg/d of DEHP [69], in PND42 and adult females at the diestrus or estrus stage exposed to DEHP above 10 mg/kg/d [74,75], and in adult females of unknown estrous stage exposed to DEHP from 1 mg/kg/d [73]. ...
Full-text available
Phthalates have been widely studied for their reprotoxic effects in male rodents and in particular on testosterone production, for which reference doses were established. The female rodent brain can also represent a target for exposure to these environmental endocrine disruptors. Indeed, a large range of behaviors including reproductive behaviors, mood-related behaviors, and learning and memory are regulated by sex steroid hormones. Here we review the experimental studies addressing the effects and mechanisms of phthalate exposure on these behaviors in female rodents, paying particular attention to the experimental conditions (period of exposure, doses, estrous stage of analyses etc.). The objective of this review is to provide a clear picture of the consistent effects that can occur in female rodents and the gaps that still need to be filled in terms of effects and mode(s) of action for a better risk assessment for human health.
... Known as the Developmental Origins of Health and Disease or DOHaD [3], this phenomenon has been well studied for a variety of health outcomes in individuals who experienced direct exposure early in life (F1 generation). Regarding neuroendocrine functions and hormonedependent behaviors, the focus of this study, exposures to EDCs including bisphenol A (BPA), phthalates, and persistent organic pollutants such as polychlorinated biphenyls (PCBs) induce adverse phenotypic outcomes in animal studies [4][5][6][7][8][9][10][11][12][13][14][15][16][17], and are associated with increased prevalence of neurobehavioral disorders in epidemiological studies in humans [18][19][20][21][22]. ...
... The F3 generations and beyond can exhibit phenotypic changes in the absence of direct exposure, presumably through germline epigenetic inheritance [23,24]. Although few in number, studies on inter-and transgenerational effects of EDCs have reported sexually dimorphic effects on behaviors, especially those influenced by early life endogenous hormones ( [16,[25][26][27][28][29][30][31][32]; reviewed in [33]). More research comparing generational effects is needed to better understand how legacy chemicals that are no longer actively manufactured but are still persistent in the environment, such as PCBs, may lead to heritable effects generations later. ...
Full-text available
Endocrine-disrupting chemicals (EDCs) lead to endocrine and neurobehavioral changes, particularly due to developmental exposures during gestation and early life. Moreover, intergenerational and transgenerational phenotypic changes may be induced by germline exposure (F2) and epigenetic germline transmission (F3) generation, respectively. Here, we assessed reproductive and sociosexual behavioral outcomes of prenatal Aroclor 1221 (A1221), a lightly chlorinated mix of PCBs known to have weakly estrogenic mechanisms of action; estradiol benzoate (EB), a positive control; or vehicle (3% DMSO in sesame oil) in F1-, F2-, and F3-generation male and female rats. Treatment with EDCs was given on embryonic day (E) 16 and 18, and F1 offspring monitored for development and adult behavior. F2 offspring were generated by breeding with untreated rats, phenotyping of F2s was performed in adulthood, and the F3 generation were similarly produced and phenotyped. Although no effects of treatment were found on F1 or F3 development and physiology, in the F2 generation, body weight in males and uterine weight in females were increased by A1221. Mating behavior results in F1 and F2 generations showed that F1 A1221 females had a longer latency to lordosis. In males, the F2 generation showed decreased mount frequency in the EB group. In the F3 generation, numbers of ultrasonic vocalizations were decreased by EB in males, and by EB and A1221 when the sexes were combined. Finally, partner preference tests in the F3 generation revealed that naïve females preferred F3-EB over untreated males, and that naïve males preferred untreated over F3-EB or F3-A1221 males. As a whole, these results show that each generation has a unique, sex-specific behavioral phenotype due to direct or ancestral EDC exposure.
... Compared with the control group, F1 mice in the 40 and 400 μg/kg bw/d dose group showed less social investigation behavior and more exploration behavior. In F3 mice, DEHP has the opposite effect on behavior as F1 (Quinnies et al., 2017). Another study by Quinnies et al. also confirmed the cross-generational behavior effect of DEHP. ...
Di-2-Ethylhexyl Phthalate (DEHP) is often used as an additive in polyvinyl chloride (PVC) to give plastics flexibility, which makes DEHP widely used in food packaging, daily necessities, medical equipment, and other products. However, due to the unstable combination of DEHP and polymer, it will migrate to the environment in the materials and eventually contact the human body. It has been recorded that low-dose DEHP will increase neurotoxicity in the nervous system, and the human health effects of DEHP have been paid attention to because of the extensive exposure to DEHP and its high absorption during brain development. In this study, we review the evidence that DEHP exposure is associated with neurodevelopmental abnormalities and neurological diseases based on human epidemiological and animal behavioral studies. Besides, we also summarized the oxidative damage, apoptosis, and signal transduction disorder related to neurobehavioral abnormalities and nerve injury, and described the potential mechanisms of neurotoxicity caused by DEHP. Overall, we found exposure to DEHP during the critical developmental period will increase the risk of neurobehavioral abnormalities, depression, and autism spectrum disorders. This effect is sex-specific and will continue to adulthood and even have an intergenerational effect. However, the research results on the sex-dependence of DEHP neurotoxicity are inconsistent, and there is a lack of systematic mechanisms research as theoretical support. Future investigations need to be carried out in a large-scale population and model organisms to produce more consistent and convincing results. And we emphasize the importance of mechanism research, which can enhance the understanding of the environmental and human health risks of DEHP exposure.
... DEHP exposure during childhood development is linked to negative neurobehavioral outcomes . Early DEHP exposure causes aberrant reproductive development as well as behavioral and neurological abnormalities in animal models (Crobeddu et al. 2019;Quinnies et al. 2017;Zhang et al. 2022). ...
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The purpose of this study was to evaluate at the link between gastrointestinal illness and urine phthalate metabolite concentrations in children and adolescents in the United States between 2005 and 2016. A total of 4008 National Health and Nutrition Examination Survey (NHANES) participants had urine samples obtained during the survey and self-reported their gastrointestinal functional status over the previous week. High performance liquid chromatography/tandem mass spectrometry (HPLC/MS–MS) was used to identify twelve phthalate metabolites in urine samples. The link between PAE concentrations and gastrointestinal illnesses was investigated using logistic regression, which was controlled for possible confounders. The combined and independent effects of PAEs on gastrointestinal illnesses were investigated using Bayesian Kernel Machine Regression (BKMR) and quantile-based g-computation (qgcomp). In children and adolescents, the prevalence of gastrointestinal infection was 9.0%. One log-unit increase in urinary concentrations was associated with an increased risk of gastrointestinal infection for monocarboxyoctyl phthalate (MCOP) (adjusted odd ratio (aOR) = 1.36, 95 percent confidence interval (95%ci): 1.08, 1.62), mono(2-ethylhexyl) phthalate (MEHP) (aOR = 1.18, 95 percent CI: 1.05, 1.32) and mono(2-eth The mixed exposure model findings revealed that the combined effect of PAEs was substantially linked with gastrointestinal infection; exposure to the combination of PAEs was positively associated with the risk of gastrointestinal infection. In the BKMR model, the exposure to the mixture of PAEs was positively associated with the risk of gastrointestinal infection. In qgcomp, a substantial positive correlation between PAEs and gastrointestinal illnesses was identified (OR = 1.16, 95 percent CI: 1.05, 1.28). MCOP and MEHP may be the major contributors after controlling for other PAE homologs. These associations were more pronounced in overweight and obese children and adolescents. Mixed exposure to phthalates (PAEs) in children and adolescents was significantly associated with gastrointestinal infections, with MCOP and MEHP accounting for the major proportions. These associations were more pronounced in overweight and obese children and adolescents.
... In our study, the sociability and social novelty of pups were tested, and not surprisingly, the pups in the VPA group faced obvious communication barriers, which is consistent with previous studies (Qin et al., 2016;Schneider and Przewłocki, 2005). Quinnies et al. found that perinatal exposure to 0.04 mg/kg/day DEHP would affect the social behaviors to some degree of mice offspring (Quinnies et al., 2017). We found that perinatal DEHP exposure at 10 mg/kg/day and 100 mg/kg/day significantly affected the behavior of the rat offspring, presenting as the reduced ability to socially interact with strange rats. ...
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Autism spectrum disorders (ASD), also known as childhood autism, is a common neurological developmental disorder. Although it is generally believed that genetic factors are a primary cause for ASD development, more and more studies show that an increasing number of ASD diagnoses are related to environmental exposure. Epidemiological studies indicated that perinatal exposure to endocrine disruptors might cause neurodevelopmental disorders in children. Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in many products. To explore the neurodevelopmental effect induced by perinatal exposure to DEHP on rat offspring, and the potential mechanisms, female Wistar rats were exposed to 1, 10, and 100 mg/kg/day DEHP during pregnancy and lactation, while valproic acid (VPA) was used as a positive control. The behavior tests showed that rat pups exposed to VPA and 100 mg/kg/day DEHP were not good as those from the control group in both their socialability and social novelty. Expression of mTOR pathway-related components increased while the number of autophagosomes decreased in the brain tissue of the rat offspring exposed to 100 mg/kg/day DEHP. In addition, perinatal exposure to DEHP at all dosages decreased the level of autophagy proteins LC3II and Beclin1 in the brain tissue of rat pups. Our results indicated that perinatal DEHP exposure would induce ASD-like behavioral changes in rat offspring, which might be mediated by activation of the mTOR signaling pathway, and inhibition of autophagy in the brain.
... Although many epidemiological studies have shown associations between neurodevelopmental outcomes and prenatal exposure to EDCs, at present the epidemiological literature remains inconclusive due to the heterogeneity and inherent limitations of such studies. However, the epidemiological evidence is supported by data obtained from animal studies showing that in utero/perinatal exposure to EDCs can lead to effects on behavior, memory, and motor activity (reviewed in [31][32][33][34]). ...
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Introduction: Brain development is highly dependent on hormonal regulation. Exposure to chemicals disrupting endocrine signaling has been associated with neurodevelopmental impairment. This raises concern about exposure to the suspected thousands of endocrine disruptors, and has resulted in efforts to improve regulation of these chemicals. Yet, the causal links between endocrine disruption and developmental neurotoxicity, which would be required for regulatory action, are still largely missing. Areas covered: In this review, we illustrate the importance of two endocrine systems, thyroid hormone and retinoic acid pathways, for neurodevelopment. We place special emphasis on TH and RA synthesis, metabolism, and how endocrine disrupting chemicals known or suspected to affect these systems are associated with developmental neurotoxicity. Expert opinion: While it is clear that neurodevelopment is dependent on proper hormonal functioning, and evidence is increasing for developmental neurotoxicity induced by endocrine disrupting chemicals, this is not grasped by current chemical testing. Thus, there is an urgent need to develop test methods detecting endocrine disruption in the context of neurodevelopment. Key to this development is further mechanistic insights on the involvement of endocrine signaling in neurodevelopment as well as increased support to develop and validate new test methods for the regulatory context.
By interfering with sex hormone signaling, endocrine-disrupting compounds (EDCs) may affect several levels of reproduction, such as the development of reproductive organs and secondary sexual characteristics, sexual function, and sexual orientation/core gender identity. Results from the studies conducted on animals provide insights into the potential mechanisms of EDCs on the sexual differentiation and function and point to the differential sensitivity, timing, and type of exposure to these compounds across the species. Luckily, reports on the environmental or domestic EDCs’ exposure and impaired sexual differentiation or function in humans are scarce. The evidence that EDCs can also influence the developing brain in a way that interferes with sexual behaviors has considerably grown in the last decade. Data from recent rodent studies point to a possible etiological link between prenatal exposure to EDCs and the development of sexual orientation and core gender identity. Nevertheless, most of the data come from animal experiments and therefore further studies are warranted.
Phthalates are one of the ubiquitous chemicals found in day-to-day products like food packaging, children's toys, and other consumer commodities. There is rising concern that repeated exposure to phthalates during pregnancy and lactation could have long-term effects on maternal and fetal health. We hypothesize that exposure to DEHP during the developmental windows might affect the expression of molecules that regulate uterine function and that this effect would be passed on to further generations. Rat dams were treated with olive oil (vehicle) or DEHP (100 mg/kg b.wt./day) orally from gestational day 9 (GD 9) to the end of lactation (PND 21). F0 maternal DEHP exposure resulted in multigenerational (F1 and F2) reproductive toxicity, as evidenced by an extended estrous cycle, decreased mating, fertility, and fecundity indices. Serum progesterone and estradiol levels were decreased and their cognate receptors (PR and ERα) in the uterus were decreased in the DEHP exposed offspring rats. Further analysis of the expression of estrogen and progesterone regulatory genes such as Hox A11, VEGF A, Ihh, LIFR, EP4, PTCH, NR2F2, BMP2, and Wnt4 were reduced in the uteri of adult F1 and F2 generation rats born from DEHP-exposed F0 dams. Decreased expression of these crucial proteins due to DEHP exposure may lead to defects in epithelial proliferation and secretion, uterine receptivity, and decidualization in the uteri of successive generations. This study showed that maternal DEHP exposure impairs the expression of molecules that regulate uterine function and this multigenerational effect is transmitted via maternal lineage.
Phthalates are organic pollutants frequently detected in the environment. The effects of these substances on male reproduction have been extensively studied but their potential impact on female reproductive behaviors in particular at environmental doses still remains to be documented. In the present study, we examined the effects of chronic exposure to di (2-ethylhexyl) phthalate (DEHP) alone at 5 or 50 μg/kg/d, or in an environmental phthalate mixture on maternal behavior of lactating female mice after a first (primiparous) and a second gestation (multiparous). Exposure of DEHP alone or in a phthalate mixture reduced pup-directed behaviors, increased self-care and forced nursing behaviors and altered nest quality for both primiparous and multiparous dams. In pup-retrieval test, primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture retrieved their pups more rapidly, probably due to a higher emission of ultrasonic vocalizations by the pups. At lactational day 2 following the third and last gestational period, the neural circuitry of maternal behavior was analyzed. A lower number of oxytocin-immunoreactive neurons in the paraventricular and anterior commissural nuclei was found in dams exposed to DEHP alone or in a phthalate mixture, while no changes were observed in the number of arginine-vasopressin immunoreactive cells. In the medial preoptic area, exposure to DEHP alone or in a phthalate mixture reduced ERα-immunoreactive cell number. Dendritic spine density assessed for DEHP at 5 μg/kg/d was also reduced. Thus, exposure to DEHP alone or in a phthalate mixture altered maternal behavior probably through a neuroendocrine mode of action involving oxytocin and estrogen through ERα, key pathways necessary for neuroplasticity and behavioral processing.
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Strong evidence implicates maternal phthalate exposure during pregnancy in contributing to adverse birth outcomes. Recent research suggests these effects might be mediated through the improper regulation of DNA methylation in offspring tissue. In this study, we examined associations between prenatal phthalate exposure and DNA methylation in human placenta. We recruited 181 mother-newborn pairs (80 fetal growth restriction newborns, 101 normal newborns) in Wenzhou, China and measured third trimester urinary phthalate metabolite concentrations and placental DNA methylation levels of IGF2 and AHRR. We found urinary concentrations of mono (2-ethyl-5- hydroxyhexyl) phthalate (MEHHP), and mono (2-ethyl-5-oxohexyl) phthalate (MEOHP) were significantly inversely associated with placental IGF2 DNA methylation. The associations were much more evident in fetal growth restriction (FGR) newborns than those in normal newborns. These findings suggest that changes in placental DNA methylation might be part of the underlying biological pathway between prenatal phthalate exposure and adverse fetal growth.
Phthalates are widely present in human environment. Widespead exposure to those agents, which are compounds of numerous daily use products, is unavoidable. In the current paper following phthalates benzylbutyl phthalate (BBP), di- n-butyl phthalate (DBP), di(2-ethylhexyl)phthalate (DEHP), diethyl phthalate (DEP), di-isononyl phthalate (DINP) are described. Phthalates mainly enter to the composition of plastic goods, like boxes and containers for storage of foods, toys, medical devices, and also cosmetics, personal care products, as well as paints, vanishes, printing inks. This paper describes the occurence of individual phthalates in the environment (water, air) and in different products. During production, transportation, manufacturing of goods and improper disposal, phthalates released into soil, water and air. For example indoor air included 13 mg/m3 phthalates, where 72 % of all constitutes DEP (2.29 mg/m(3)), BBP (3.97 mg/m(3)) and DEHP (2.43 mg/m(3)). Exposure to phthalates take place mainly by ingestion or inhalation air or through the skin. Presence of phthalates were observed in numerous food products and is connected with migration of those compounds from food storage containers to preserved food. They could mirgate to salivia during sucking and chewing of toys and this way increased exposure to of children. The results of studies regarding to concentration of phthalates in human tissues and excretions are also described. The level of phthalates were measured in numerous of human biological samples. For example, DEHP, DEP and DBP were detected at levels of 5.71 mg/L in blood serum, of 0.30 mg/L in semen and of 0.72 mg/kg in fat samples.
Phthalates are ubiquitous environmental contaminants which are used in industry as plasticizers and additives in cosmetics. They are classified as Endocrine Disrupting Chemicals (EDCs) which impair the human endocrine system inducing fertility problems, respiratory diseases, childhood obesity and neuropsychological disorders. The aim of this review is to summarize the current state of knowledge on the toxicity that phthalates pose in humans based on human biomonitoring studies conducted over the last decade. Except for conventional biological matrices (such as urine and serum), amniotic fluid, human milk, semen, saliva, sweat, meconium and human hair are also employed for the estimation of exposure and distribution of pollutants in the human body, although data are not enough yet. Children are highly exposed to phthalates relative to adults and in most studies children's daily intake surpasses the maximum reference dose (RfD) set from US Environmental Protection Agency (US EPA). However, the global trend is that human exposure to phthalates is decreasing annually as a result of the strict regulations applied to phthalates.
Background: Available evidence implicates environmental factors in the pathogenesis of autism spectrum disorders (ASD). However, the role of specific environmental chemicals such as phthalate esters that influence ASD risk remains elusive. This paper systematically reviews published evidences on association between prenatal and/or childhood exposure to phthalate and ASD. Methods: Studies pertaining to systematic literature search from Scopus, PubMed, PsycInfo and Web of Science prior to December 2015 were identified. The authors included studies which assessed the effect of exposure to phthalates on occurrence of ASD. This comprehensive bibliographic search identified five independent studies. Each eligible paper was summarized with respect to its methods and results with particular attention to study design and exposure assessment. Because of the heterogeneity in the type of included studies, different methods of assessing exposure to phthalates and the use of different statistics for summarizing the results, meta-analysis could not be used to combine the results of included studies. Results: The results of this systematic review have revealed the limited number of studies conducted and assessed phthalate exposure. Seven studies were regarded as relevant to the objectives of this review. Two of them did not measure phthalate exposure directly and did not result in quantitative results. Out of the five studies in which phthalate exposure was mainly measured by the examining biomarkers in biological samples, two were cohort studies (one with positive results and another one with not clear association). Among the three case control studies, two of them showed a significant relation between exposure to phthalate and ASD and the last case control study had negative results. Indeed, this case control studies showed a compromised phthalate metabolite glucuronidation pathway, as a probable explanation of mechanism of the relation between phthalate exposure and ASD. Conclusions: This review reveals evidence showing a connection between exposure to phthalates and ASD. Nevertheless, further research is needed with appropriate attention to exposure assessment and relevant pre and post-natal cofounders.
Endocrine disruptors (ED) are environmental pollutants that mimic endogenous hormonal signals. Exposure to EDs during fetal and early life is a public health concern because these are periods characterized by high cellular plasticity that influence the physiology and development of disease later in life. Phthalates are plasticizers used in the industry to manufacture polyvinyl chloride products and several consumer products. Di(2-ethylhexyl) phthalate (DEHP) is one of the most produced plasticizers; it is ubiquitously found contaminating the environment, and has been shown to be an ED. Human exposure to phthalates starts during fetal development and continues after birth through contact of the newborn with the environment and contaminated food sources. We used a rat model in which pregnant dams are gavaged with DEHP from gestational day 14 until birth to study the long-term effects of DEHP. This window of fetal exposure results in decreased circulating testosterone and aldosterone levels in adult male offspring and estradiol in the female. The observed steroid changes are likely of an epigenetic origin as DEHP is rapidly cleared after birth. Here, we review the long-term effects of fetal exposure to DEHP with a focus on the molecular and epigenetic changes, including DNA methylation, which may mediate long-term endocrine dysfunction.
Phthalates are frequently used in personal care products and plasticizers and phthalate exposure is ubiquitous in the US population. Exposure to phthalates during critical periods in utero has been associated with a variety of adverse health outcomes but the biological mechanisms linking these exposures with disease are not well characterized. In this study, we examined the relationship of in utero phthalate exposure with repetitive element DNA methylation, an epigenetic marker of genome instability, in children from the longitudinal birth cohort CHAMACOS. Methylation of Alu and long interspersed nucleotide elements (LINE-1) was determined using pyrosequencing of bisulfite-treated DNA isolated from whole blood samples collected from newborns and 9 year old children (n=355). Concentrations of eleven phthalate metabolites were measured in urine collected from pregnant mothers at 13 and 26 weeks gestation. We found a consistent inverse association between prenatal concentrations of monoethyl phthalate, the most frequently detected urinary metabolite, with cord blood methylation of Alu repeats (β(95%CI): -0.14 (-0.28,0.00) and -0.16 (-0.31, -0.02)) for early and late pregnancy, respectively, and a similar but weaker association with LINE-1 methylation. Additionally, increases in urinary concentrations of di-(2-ethylhexyl) phthalate metabolites during late pregnancy were associated with lower levels of methylation of Alu repeats in 9 year old blood (significant p-values ranged from 0.003 to 0.03). Our findings suggest that prenatal exposure to some phthalates may influence differences in repetitive element methylation, highlighting epigenetics as a plausible biological mechanism through which phthalates may affect health.
Di-(2-ethylhexyl) phthalate (DEHP), a main member of phthalates used as plasticizer in PVC plastics, is an environmental endocrine disrupter. The present study investigated the effect of DEHP on social behavior of mice following pubertal exposure (1, 10, 50, and 200 mg/kg/d) from postnatal day 28 through postnatal day 42. The results showed that, in pubertal females, DEHP reduced the time spent in social play and social investigation and inhibited sociability, but a contrary effect was found in pubertal males, suggesting that the effect of DEHP on pubertal social behavior displays sex differences. In adults, DEHP reduced sociability in females and inhibited social play and social investigation in males, suggesting that early pubertal exposure to DEHP not only plays a significant role in puberty but also alters social behavior in adults. In addition, the present study showed that the higher dose of DEHP (50, 200 mg/kg/d) reduced the relative weight of bilateral testis and anogenital distance of pubertal or adult males, suggesting an anti-androgenic activity of DEHP. These results suggest that early pubertal exposure to DEHP sex- and age- specifically affected the social behaviors of pubertal and even adult mice.
Background: Prenatal phthalate exposure is associated with altered male reproductive tract development, and in particular, shorter anogenital distance (AGD). AGD, a sexually dimorphic index of prenatal androgen exposure, may also be altered by prenatal stress. How these exposures interact to impact AGD is unknown. Here, we examine the extent to which associations between prenatal phthalate exposure and infant AGD are modified by prenatal exposure to stressful life events (SLEs). Methods: Phthalate metabolites [including those of diethylhexyl phthalate (DEHP) and their molar sum (ΣDEHP)] were measured in first trimester urine from 738 pregnant women participating in The Infant Development and the Environment Study (TIDES). Women completed questionnaires on SLEs, and permitted infant AGD measurements at birth. Subjects were classified as 'lower' and 'higher' stress (0 first trimester SLEs vs. 1+).We estimated relationships between phthalate concentrations and AGD (by infant sex and stress group) using adjusted multiple regression interaction models. Results: In the lower stress group, first trimester ΣDEHP was inversely associated with two measures of male AGD: anoscrotal distance (AGD-AS; β = -1.78; 95% CI -2.97, -0.59) and anopenile distance (AGD-AP; β = -1.61; 95% CI -3.01, -0.22). By contrast, associations in the higher stress group were mostly positive and non-significant in male infants. No associations were observed in girls. Conclusions: Associations between prenatal phthalate exposure and altered genital development were only apparent in sons of mothers who reported no SLEs during pregnancy. Prenatal stress and phthalates may interact to shape fetal development in ways that have not been previously explored.
DEHP, one of the most commonly phthalates used in plastics and many other products, is an environmental endocrine disruptor (EED). Puberty is another critical period for the brain development besides the neonatal period and is sensitive to EEDs. Social behavior is organized during puberty, so the present study is to investigate whether pubertal exposure to DEHP influenced social behavior of adult female mice. The results showed that pubertal exposure to DEHP for 2 weeks did not change the serum level of 17β-estradiol and the weight of uterus of adult females, but decreased the number of grid crossings and the frequency of rearing, and increased grooming in open field. DEHP reduced the open arm entries and the time spent in open arms in the elevated plus maze. DEHP reduced mutual sniffing and grooming between unfamiliar conspecifics in social play task and reduced the right chamber (containing unfamiliar female mouse) entries and the frequency of sniffing unfamiliar female mouse. DEHP at 1 mg kg(-1) d(-1) reduced the time spent in right chamber. Furthermore, Western blot analyses showed that DEHP decreased the levels of estrogen receptor β (ERβ), dopamine receptor D2, and the phosphorylation of ERKs in striatum. These results suggest that pubertal exposure to DEHP impaired social investigation and sociability and influenced anxiety-like state of adult female mice. The decreased activity of ERK1/2, and the down-regulated D2 and ERβ in striatum may be associated with the DEHP-induced changes of emotional and social behavior in mice.