Article

Perinatal Exposure to an Environmentally Relevant Mixture of Phthalates Results in a Lower Number of Neurons and Synapses in the Medial Prefrontal Cortex and Decreased Cognitive Flexibility in Adult Male and Female Rats

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Abstract

The growth and organization of the developing brain are known to be influenced by hormones, but little is known about whether disruption of hormones affects cortical regions, such as mPFC. This region is particularly important given its involvement in executive functions and implication in the pathology of many neuropsychiatric disorders. Here, we examine the long-term effects of perinatal exposure to endocrine-disrupting compounds, the phthalates, on the mPFC and associated behavior. This investigation is pertinent as humans are ubiquitously exposed to phthalates through a variety of consumer products and phthalates can readily cross the placenta and be delivered to offspring via lactation. Pregnant dams orally consumed an environmentally relevant mixture of phthalates at 0, 200, or 1000 μg/kg/d through pregnancy and for 10 d while lactating. As adults, offspring were tested in an attentional set-shifting task, which assesses cognitive flexibility. Brains were also examined in adulthood for stereological quantification of the number of neurons, glia, and synapses within the mPFC. We found that, independent of sex, perinatal phthalate exposure at either dose resulted in a reduction in neuron number, synapse number, and size of the mPFC and a deficit in cognitive flexibility. Interestingly, the number of synapses was correlated with cognitive flexibility, such that rats with fewer synapses were less cognitively flexible than those with more synapses. These results demonstrate that perinatal phthalate exposure can have long-term effects on the cortex and behavior of both male and female rats.

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... Rat dams daily ate the phthalates on cookies during pregnancy through P10 of lactation. As adults, both male and female offspring were found to have fewer neurons and synapses in the same proportion such that the sex differences in the number of neurons and synapses were the same in all groups ( Fig. 3; Kougias et al. 2018). In addition, performance on an extradimensional set shift task, which is known to involve the mPFC, was similarly disrupted by phthalate exposure in both sexes, and the sex differences in performance persisted in the phthalate-exposed groups (Kougias et al. 2018). ...
... As adults, both male and female offspring were found to have fewer neurons and synapses in the same proportion such that the sex differences in the number of neurons and synapses were the same in all groups ( Fig. 3; Kougias et al. 2018). In addition, performance on an extradimensional set shift task, which is known to involve the mPFC, was similarly disrupted by phthalate exposure in both sexes, and the sex differences in performance persisted in the phthalate-exposed groups (Kougias et al. 2018). More recently, we have found an increase in the number of apoptotic cells in the mPFC at embryonic day 18 and at P10 in both sexes following phthalate exposure . ...
... We also have found that males have more neurons in the adult mPFC than females (Markham et al. 2007;Kougias et al. 2018) as well as a larger dendritic tree in layer five pyramidal neurons (Markham and Juraska 2002). As mentioned previously, males Figure 6. ...
Article
Sex differences occur in the structure and function of the rat cerebral cortex and hippocampus, which can change from the juvenile period through old age. Although the evidence is incomplete, it appears that in at least some portions of the cortex these differences develop due to the rise of ovarian hormones at puberty and are potentially not dependent on the perinatal rise in testosterone, which is essential for sexual differentiation of the hypothalamus and sexual behavior. During aging of female rats, the presence of continued ovarian hormone secretion after cessation of the estrous cycle also influences sex differences in neuroanatomical structure and cognitive behavior, resulting in nullification or reversal of sex differences seen in younger adults. Sex differences can be altered by experience in a stimulating environment during the juvenile/adolescent period, and sex differences in performance even can be affected by the parameters of a task. Thus, broad generalizations about differences such as “spatial ability” are to be avoided. It is clear that to understand how the brain produces behavior, sex and hormones have to be taken into account.
... Four other studies assessed the effects of exposure to lower doses of phthalates with BBP at 10 µg/kg/d, DEHP at 33 mg/kg/d, or a phthalate mixture at 0.2 to 1 mg/kg/d on aversive stimulus associated learning [82], object recognition memory [83], and cognitive flexibility [72,84], respectively. In mice, the novel object recognition test indicated a higher preference for the novel object in mice exposed to 200 mg/kg/d [83]. ...
... Cognitive flexibility tests showed a lower percentage of correct answers and higher percentage of perseveration errors after exposure to a phthalate mixture at 1 mg/kg/d. The cognitive flexibility texts showed a higher percentage of omission errors in rats exposed during the prenatal/postnatal period [84] but not in those exposed during the prepubertal/pubertal period [72]. In particular, the behavioral effects induced by prenatal/postnatal exposure to this phthalate mixture were associated with a lower number of neurons and synapses in the prefrontal cortex and a reduced volume of this brain region in adult females [84]. ...
... The cognitive flexibility texts showed a higher percentage of omission errors in rats exposed during the prenatal/postnatal period [84] but not in those exposed during the prepubertal/pubertal period [72]. In particular, the behavioral effects induced by prenatal/postnatal exposure to this phthalate mixture were associated with a lower number of neurons and synapses in the prefrontal cortex and a reduced volume of this brain region in adult females [84]. This phthalate mixture also induced earlier changes given that reduced neurogenesis and increased apoptosis in the cortex of PND5 and PND10 rats at 200-1000 µg/kg/d [85] and the reduced expression of arginine-vasopressin receptor 1B in the prefrontal cortex of PND10 rats at 0.2 mg/kg/d without changing the expression of other signaling pathways on PND10 or adulthood were reported by this laboratory [60]. ...
Article
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.
... From a translational perspective, it is important to examine the net effects of an environmentally relevant combination of compounds, given their diverse mechanisms of action, along with the reality of human exposure to multiple phthalates. Previously, our laboratory found that low doses of a phthalate mixture reflective of human exposure, administered perinatally, resulted in a decrease in the number of neurons and synapses in the adult medial prefrontal cortex (mPFC) (Kougias et al., 2018b). Perinatal exposure to the mixture also reduced adolescent play behavior in males and decreased performance on an attentional set-shifting task in adults of both sexes (Kougias et al., 2018a, b). ...
... Previous work from our laboratory has shown that perinatal treatment with this phthalate mixture at doses of 0.2 and 1 mg/kg reduces neuron number in the adult mPFC (Kougias et al., 2018b). Addressing the potential role of altered proliferation in producing deficits in neuron number, we first showed both 1 and 5 mg/kg doses of the phthalate mixture led to fewer BrdU positive cells detected on P5. ...
... Action through any of these mechanisms could interfere with developmental processes, including apoptosis. However, given the lack of sex differences in phthalate effects seen in the present study, as well as our previous work (Kougias et al., 2018b), non-steroid hormone related mechanisms must also be considered. For example, phthalates can increase oxidative stress and neuroinflammation (Ma et al., 2015;Win-Shwe et al., 2013), though our past work suggests a minimal inflammatory response after perinatal exposure to this mixture at low levels (Moody et al., 2019). ...
Article
Phthalates are a class of endocrine disruptors found in a variety of consumer goods, and offspring can be exposed to these compounds during gestation and lactation. Our laboratory has found that perinatal exposure to an environmentally relevant mixture of phthalates resulted in a decrease in cognitive flexibility and in neuron number in the adult rat medial prefrontal cortex (mPFC). Here, we examine effects of phthalate treatment on prenatal cellular proliferation and perinatal apoptosis in the mPFC. To examine the phthalate effects on cellular proliferation, dams consumed 0, 1, or 5 mg/kg of the phthalate mixture daily from embryonic day 2 (E2) through the day of birth (P0), and on E16 and E17, they were injected with BrdU. The mPFC of offspring was analyzed on P5 and showed a decrease in labelled cells in the phthalate exposed groups. To examine whether changes in BrdU density observed on P5 were due to altered cell survival, cell death was measured on E18, P0, and P5 using a TUNEL assay in a separate cohort of prenatally exposed offspring. There was an increase in TUNEL labelled cells at E18 in the phthalate exposed groups. In the final experiment, dams consumed the phthalate mixture from E2 through P10, at which time mPFC tissue was stained with TUNEL. Phthalate treated subjects showed a higher density of apoptotic cells at P10. These results indicate both pre- and postnatal phthalate exposure increases apoptosis in the male and female rat mPFC. While the impact of phthalates on proliferation cannot be ruled out, these data do not allow for definitive conclusions.
... Using the elevated plus maze, adult females, not males, exposed to phthalates during adolescence showed indications of reduced anxiety-like behavior while perinatal exposed animals were unaffected. There was no effect of adolescent phthalate exposure on cognitive flexibility using the attentional set shift paradigm in either sex, unlike the impairments we have previously reported following perinatal exposure (Kougias et al., 2018b). Finally, there was no effect of phthalate exposure during either time frame on sensorimotor gating measured using prepulse inhibition. ...
... Previous work in our laboratory has shown exposure to an environmentally relevant mixture of phthalates during the perinatal period disrupts the development of the medial prefrontal cortex (mPFC) resulting in a reduction of neurons and synapses in both adult male and female rats (Kougias et al., 2018b). Additional preliminary work has found that exposure to this phthalate mixture during early development leads to increased postnatal apoptosis within the mPFC in males and females, presumably responsible for the lasting deficit in neuron number (Sellinger, Willing, Drzewiecki, and Juraska, unpublished data). ...
... Cognitive flexibility, a measure of how well subjects adjust their problem-solving strategy to adapt to a changing stimulus, can be assessed in the rat using the attentional set shift paradigm (Birrell and Brown, 2000). Perinatal exposure to the same mixture and doses (200 and 1000 μg/kg/day) of phthalates that led to reduced neuron and synapse number in the mPFC also impaired performance in the attentional set shift task in adult male and female rats (Kougias et al., 2018b). Given the protracted development of the mPFC, adolescence may be an additional period of susceptibility to phthalate action, leading to altered mPFC-related behaviors. ...
Article
Hormones influence neurodevelopment which can result in vulnerability to endocrine disruptors such as phthalates during both the perinatal period and adolescence. Using a rat model, we have previously shown that perinatal exposure to an environmentally relevant phthalate mixture at low doses results in cognitive flexibility deficits in adults and a reduction in neuron and synapse number within the medial prefrontal cortex. Here, we further examined the behavioral effects of exposure to an environmentally relevant mixture of phthalates at low doses during either perinatal development or adolescence. Using the elevated plus maze, adult females, not males, exposed to phthalates during adolescence showed indications of reduced anxiety-like behavior while perinatal exposed animals were unaffected. There was no effect of adolescent phthalate exposure on cognitive flexibility using the attentional set shift paradigm in either sex, unlike the impairments we have previously reported following perinatal exposure (Kougias et al., 2018b). Finally, there was no effect of phthalate exposure during either time frame on sensorimotor gating measured using prepulse inhibition. Environmentally relevant phthalate exposure during the perinatal period or during adolescence did not induce widespread changes in the adult behaviors measured here.
... Rodent studies report that offspring developmentally exposed to DEHP exhibit impaired learning and memory, altered social and anxiety-like behaviors, and changes in hippocampal structure and functional plasticity (Barakat et al., 2018;Hatcher et al., 2019;Holahan and Smith, 2015). Kougias et al. (2018) reported that perinatal administration of an environmentally relevant phthalate mixture mostly comprised of DEP, DEHP, DBP, and di-isononyl phthalate (DINP) reduced cognitive flexibility and altered neuroanatomy of the medial prefrontal cortex. ...
... Findings from epidemiological and animal studies are mixed, but many experimental studies in rodents have demonstrated that prenatal and/or neonatal phthalate exposure alters neural structure and function, resulting in impaired social behaviors and cognition. For example, early life exposure to phthalates decreased brain derived neurotrophic factor (Smith and Holahan, 2014), altered hippocampal or amygdala gene and protein expression (Betz et al., 2013;Hatcher et al., 2019;Xu et al., 2015;You et al., 2018), impaired spatial learning and memory (Li et al., 2013), reduced neurogenesis and synaptogenesis in the prefrontal cortex, and decreased cognitive flexibility (Kougias et al., 2018). Regarding motor skills, phthalates reduced levels of thyroid hormone (Boas et al., 2010;Huang et al., 2007;Meeker and Ferguson, 2011) which is critical for brain development, particularly for the cerebellum that regulates motor learning and coordination (Anderson, 2008;Moog et al., 2017). ...
Article
Phthalates are ubiquitous endocrine-disrupting chemicals, and research indicates that prenatal exposure to some phthalates may affect neurodevelopment. In a prospective birth cohort study, five first-morning urine samples collected across pregnancy were pooled and the following phthalate biomarkers assessed: sum of di-(2-ethylhexyl) phthalate metabolites (ΣDEHP), sum of diisononyl phthalate metabolites (ΣDINP), sum of dibutyl phthalate metabolites (ΣDBP), sum of anti-androgenic metabolites (ΣAA), monoethyl phthalate (MEP), and sum of all phthalate metabolites (ΣAll). The Ages & Stages Questionnaires® (ASQ), a standardized parent-reported, age-adapted screening tool, measured communication, personal-social, problem solving, and motor domains in infants at 4.5 and 7.5 months (n = 123). Adjusting for maternal age, annual household income, gestational age at birth, infant age at assessment, and sex, repeated-measures generalized linear regression models were used to examine associations between prenatal phthalate urine biomarker concentrations and domain scores (assuming a Poisson distribution). Beta estimates were exponentiated back to the domain scale for ease of interpretation. Mothers were mostly white and college-educated, and most reported an annual household income of ≥$60,000. Associations of phthalate concentrations with ASQ outcomes are presented as follows: (1) anti-androgenic phthalate metabolites (ΣDEHP, ΣDINP, ΣDBP, and ΣAA), (2) MEP, which is not anti-androgenic, and (3) ΣAll. Overall, anti-androgenic phthalates were associated with higher (i.e., better) scores. However, there were exceptions, including the finding that a one-unit increase in ΣDBP was associated with a 12% increase in problem solving scores in 4.5-month-old females (β = 1.12; 95% CI: 0.99, 1.28; p = 0.067) but a 85% decrease for 7.5-month-old females (β = 0.54; 95% CI: 0.3, 0.99; p = 0.047). In contrast, MEP was associated with poorer scores on several outcomes. Sex- and timepoint-specific estimates demonstrated a one-unit increase in MEP was associated with: a 52% decrease in personal-social scores in 7.5-month-old males (β = 0.66; 95% CI: 0.46, 0.95; p = 0.02), a 39% decrease in fine motor scores in 7.5-month-old males (β = 0.72; 95% CI: 0.52, 0.98; p = 0.035), and a 6% decrease in fine motor scores in 4.5-month-old females (β = 0.94; 95% CI: 0.88, 0.99; p = 0.03). A one-unit increase in ΣAll was associated with a 4% increase in personal-social scores in 4.5-month-old males (β = 1.04; 95% CI: 0.99, 1.1; p = 0.08) but a 17% decrease in 7.5-month-old males (β = 0.85; 95% CI: 0.73, 0.99; p = 0.03). These data suggest age- and sex-specific associations of prenatal phthalates with infant neurobehavior. The current findings should be confirmed by longitudinal studies with larger sample sizes.
... In this study, it was observed that the BBP-exposed offspring had learning and memory impairments, that were accompanied by molecular abnormalities in multiple brain regions (DeBartolo et al., 2016). On the other hand, in order to make an exposure model closer to what happens with the daily use of plastics, Kougias, Sellinger, Willing, and Juraska (2018) performed in rats an experimental approach to evaluate the long-term neurological effects derived from perinatal exposure to an environmentally relevant mixture of phthalates. The results from this study show that adult mice who had a perinatal exposure to phthalates had a decreased number of neurons and synapses in the medial prefrontal cortex and decreased cognitive flexibility (Kougias et al., 2018). ...
... On the other hand, in order to make an exposure model closer to what happens with the daily use of plastics, Kougias, Sellinger, Willing, and Juraska (2018) performed in rats an experimental approach to evaluate the long-term neurological effects derived from perinatal exposure to an environmentally relevant mixture of phthalates. The results from this study show that adult mice who had a perinatal exposure to phthalates had a decreased number of neurons and synapses in the medial prefrontal cortex and decreased cognitive flexibility (Kougias et al., 2018). ...
Article
Full-text available
As a result of human socio‐economic activity, industrial wastes have increased alarmingly. Plastic pollution is globally distributed across the world due to its properties of buoyancy and durability. Two broad classes of plastic‐related chemicals are of critical concern for human health—bisphenol‐A or BPA, and additives used in the synthesis of plastics, which are known as phthalates. Our exposure to them is ubiquitous because they are used in the production of materials that we use daily such as polycarbonate plastics, epoxy resins, flooring, automotive parts, medical devices, dental sealants, and children's toys. Since these compounds are not covalently bound to the products, they easily leach from them, leading to high human exposure. Both, BPA and phthalates, are endocrine‐disruptor compounds (EDCs) with steroidogenic activity, and can bind to different receptors, such as estrogen, androgen, PPAR‐γ, and AhR. These pathways are part of the complex regulatory neuroendocrine network, since its cellular components not only express neuroendocrine receptors, but synthesize and respond to several hormones and other endocrine ligands. On the other hand, the effects of BPA and phthalates on neuroendocrine diseases have been poorly studied and the available data are inconclusive. This can be attributed to the enormous variety of animal models and the different doses used in experiments or levels found in humans. However, what is clear is that exposure to both EDCs during critical life stages induces many changes in the neuroendocrine system of exposed humans that are correlated with different reproductive and neurological diseases.
... Additional experiments using rodents have shown that intrauterine exposure to EDCs is associated with sexual differentiation of the brain (Kubo et al., 2003), which in turn results in sex differences in emotional and behavioral responses (Gioiosa et al., 2007;Palanza et al., 2008). A single investigation has examined the effect of early phthalate exposure on neurodevelopmental outcomes, reporting that perinatal phthalate exposure was related to reduced sized and connectivity of the medial prefrontal cortex, as well as cognitive flexibility deficits in both male and female adult rats (Kougias et al., 2018). However, to our knowledge, no prior study has investigated the associations between prenatal phthalate exposure, brain structural differences, and behavioral outcomes in a human population. ...
... Specifically, our mediation models found evidence for indirect-only mediation (Zhao et al., 2010), which supports the hypothesis that white matter microstructure mediates the association between prenatal phthalate exposure and preschool behavior problems. Although animal studies have shown that prenatal exposure to EDCs affects brain development (Itoh et al., 2012;Kougias et al., 2018;Masuo and Ishido, 2011), to our knowledge, this is the first study to show that prenatal phthalate exposure is related to brain structure in children. ...
Article
Background Previous research reports associations between prenatal exposure to phthalates and childhood behavior problems; however, the neural mechanisms that may underlie these associations are relatively unexplored. Objective This study examined microstructural white matter as a possible mediator of the associations between prenatal phthalate exposure and behavior problems in preschool-aged children. Methods Data are from a subsample of a prospective pregnancy cohort, the Alberta Pregnancy Outcomes and Nutrition (APrON) study (n = 76). Mother-child pairs were included if mothers provided a second trimester urine sample, if the child completed a successful magnetic resonance imaging (MRI) scan at age 3–5 years, and if the Child Behavior Checklist was completed within 6 months of the MRI scan. Molar sums of high (HMWP) and low molecular weight phthalates (LMWP) were calculated from levels in urine samples. Associations between prenatal phthalate concentrations, fractional anisotropy (FA) and mean diffusivity (MD) in 10 major white matter tracts, and preschool behavior problems were investigated. Results Maternal prenatal phthalate concentrations were associated with MD of the right inferior fronto-occipital fasciculus (IFO), right pyramidal fibers, left and right uncinate fasciculus (UF), and FA of the left inferior longitudinal fasciculus (ILF). Mediation analyses showed that prenatal exposure to HMWP was indirectly associated with Internalizing (path ab = 0.09, CI.95 = 0.02, 0.20) and Externalizing Problems (path ab = 0.09, CI.95 = 0.01, 0.19) through MD of the right IFO, and to Internalizing Problems (path ab = 0.11, CI.95 = 0.01, 0.23) through MD of the right pyramidal fibers. Discussion This study provides the first evidence of childhood neural correlates of prenatal phthalate exposure. Results suggest that prenatal phthalate exposure may be related to microstructural white matter in the IFO, pyramidal fibers, UF, and ILF. Further, MD of the right IFO and pyramidal fibers may transmit childhood risk for behavioral problems.
... As far as mechanisms of developmental neurotoxicity, experimental studies have indicated that several pathways relevant to brain development are targeted by phthalates, including dopaminergic neurotransmission (Chen et al., 2011;Dhanya et al., 2003;Tully et al., 2000;Wang et al., 2016), thyroid hormone homeostasis (Liu et al., 2015), and steroids' action through their receptors (Dombret et al., 2017). Very recent data obtained in rats also showed decreased number of neurons and synapses in the medial prefrontal cortex following developmental exposure to an environmentally relevant mixture of major phthalates (Kougias et al., 2018). ...
... A significant effect of environmental relevant exposure to phthalates on the brain is consistently supported by data showing impairment of hippocampal structural and functional plasticity (Holahan and Smith, A. Jankowska, et al. Environmental Research 179 (2019) 108829 2015) and by a very recent study that found decreased number of neurons and synapses in the medial prefrontal cortex following developmental exposure to a mixture of major phthalates (Kougias et al., 2018). Whether these changes occur as a direct neurotoxic effect of phthalates or an indirect effect through disruption of other neurotransmitter or metabolic pathways is not fully understood. ...
Article
Some phthalates are known endocrine disrupting chemicals (EDC). They are widely present in the environment thus their impact on children’s health is of particular scientific interest. The aim of the study was to evaluate the association between phthalate exposure and neurodevelopmental outcomes, in particular behavioral, cognitive and psychomotor development, in 250 early school age children from the Polish Mother and Child Cohort (REPRO_PL). Urine samples were collected at the time of children’s neurodevelopmental assessment and were analysed for 21 metabolites of 11 parent phthalates. Behavioral and emotional problems were assessed by the Strengths and Difficulties Questionnaire (SDQ) filled in by the mothers. To assess children’s cognitive and psychomotor development, Polish adaptation of the Intelligence and Development Scales (IDS) was administered. The examination was performed by trained psychologists. Dimethyl phthalate (DMP) and di-n-butyl phthalate (DnBP) were the two phthalates showing the highest statistically significant associations, with higher total difficulties scores (β=1.5, 95% CI 0.17; 2.7; β=1.5, 95% CI 0.25; 2.8, respectively) as well as emotional symptoms and hyperactivity/inattention problems for DnBP (β=0.46, 95% CI -0.024; 0.94; β=0.72, 95% CI 0.065; 1.4, respectively), and peer relationships problems for DMP (β=0.37, 95% CI -0.013; 0.76). In addition, DnBP and DMP have been found to be negatively associated with fluid IQ (β=−0.14, 95% CI -0.29; 0.0041) and crystallized IQ (β=−0.16, 95% CI -0.29; −0.025), respectively. In the case of mathematical skills, three phthalates, namely DMP (β=−0.17, 95% CI -0.31; −0.033), DEP (β=−0.16, 95% CI -0.29; −0.018) and DnBP (β=−0.14, 95% CI -0.28; 0.0012), have also shown statistically significant associations. This study indicates that exposure to some phthalates seems to be associated with adverse effects on behavioral and cognitive development of early school age children. Further action including legislation, educational and interventional activities to protect this vulnerable population is still needed.
... As far as mechanisms of developmental neurotoxicity, experimental studies have indicated that several pathways relevant to brain development are targeted by phthalates, including dopaminergic neurotransmission (Chen et al., 2011;Dhanya et al., 2003;Tully et al., 2000;Wang et al., 2016), thyroid hormone homeostasis ( Liu et al., 2015), and steroids' action through their receptors (Dombret et al., 2017). Very recent data obtained in rats also showed decreased number of neurons and synapses in the medial prefrontal cortex following developmental exposure to an environmentally relevant mixture of major phthalates ( Kougias et al., 2018). ...
... 2015) and by a very recent study that found decreased number of neurons and synapses in the medial prefrontal cortex following developmental exposure to a mixture of major phthalates ( Kougias et al., 2018). Whether these changes occur as a direct neurotoxic effect of phthalates or an indirect effect through disruption of other neurotransmitter or metabolic pathways is not fully understood. ...
Article
Phthalates are among the most frequently investigated environmental chemicals influencing children's health and particularly their neuropsychological development. However, the reported effects of these compounds on child behavior, cognitive and psychomotor outcomes are not fully consistent. The aim of this study is to evaluate the associations between prenatal and early postnatal phthalate exposures and child neurodevelopment at age of 7 years. A total of 134 mother-child pairs from Polish Mother and Child Cohort (REPRO_PL) constitute the basis for current analysis. Eleven phthalate metabolites were measured in urine samples collected from mothers in the 3rd trimester of pregnancy and from children at the age of 2 years. Child neuropsychological development at early school age (7 years) was assessed by both the Strengths and Difficulties Questionnaire (SDQ) filled by mothers and the Polish adaptation of the Intelligence and Development Scales (IDS) performed by psychologists. Mono-ethyl phthalate (MEP) concentration during pregnancy was significantly associated with increased risk of peer relationship problems in SDQ (OR = 2.7, p = 0.03). The results of the IDS analyses focused on child's cognitive and psychomotor development are not fully conclusive. Negative associations were evident between some phthalates in early childhood period and fluid intelligence and cognition (MEP: β = −5.2; p = 0.006; β = −4.2; p = 0.006; mono-n-butyl phthalate (MnBP): β = −4.9; p = 0.03; β = −4.0; p = 0.03; respectively), while positive associations have been found in the prenatal period (mono-2-ethyl-5-oxo-hexyl phthalate (oxo-MEHP): β = 3.6; p = 0.03 for fluid intelligence; β = 2.9; p = 0.03 for cognition). Further studies are required in order to elucidate which are the most critical periods of phthalate exposure on children's neurodevelopmental outcomes.
... Live cells were counted across both layers as well with a counting frame of 40 × 40 µm with a z-axis 10 µm thick. Protocols for unbiased quantification have been previously described by our laboratory (Kougias et al., 2018;Willing and Juraska, 2015). All quantification was performed by an experimenter blind to the animal's group. ...
Article
Bisphenol A (BPA) is an endocrine disruptor found in polycarbonate plastics and exposure in humans is nearly ubiquitous and it has widespread effects on cognitive, emotional, and reproductive behaviors in both humans and animal models. In our laboratory we previously found that perinatal BPA exposure results in a higher number of neurons in the adult male rat prefrontal cortex (PFC) and less play in adolescents of both sexes. Here we examine changes in the rate of postnatal apoptosis in the rat prefrontal cortex and its timing with brief BPA exposure. Because an increased number of neurons in the PFC is a characteristic of a subtype of autism spectrum disorder, we tested social preference following brief BPA exposure and also expression of a small group of genes. Males and females were exposed to BPA from postnatal days (P) 6 through 8 or from P10 through 12. Both exposures significantly decreased indicators of cell death in the developing medial prefrontal cortex in male subjects only. Additionally, males exposed to BPA from P6 – 8 showed decreased social preference and decreased cortical expression of Shank3 and Homer1, two synaptic scaffolding genes that have been implicated in social deficits. There were no significant effects of BPA in the female subjects. These results draw attention to the negative consequences following brief exposure to BPA during early development.
... Several biological mechanisms may explain the association between phthalate exposure and increased ASD symptoms. Animal studies have shown that phthalate exposure results in a reduction in neuron number, synapse number, and size of the medial prefrontal cortex and a deficit in cognitive function (Kougias et al. 2018). A reduction in neuron and synapse numbers may be associated with ASD symptoms. ...
Article
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Autism spectrum disorder (ASD) comprises a group of neurodevelopmental disorders. The etiology of autism remains unclear, but a growing body of evidence indicates that phthalates play a role in its pathogenesis. The aim of this study was to compare the urine levels of phthalates in children with ASD and healthy children. We also explored whether phthalates have an effect on ASD symptoms. The participants in this study included 101 children with ASD (79 boys and 22 girls) and 101 sex- and age-matched controls. The levels of phthalates were analyzed by gas chromatography–mass spectrometry (GC–MS). We detected significant differences in monoethyl phthalate (MEP) levels between the severe ASD and control groups (p < 0.05). Mono-n-butyl phthalate (MBP) concentration was positively correlated with language skill impairment in ASD (β: 0.387, p = 0.041). MEP levels were associated with the CARS “Imitation” score in all children (OR: 1.470). MBP levels were associated with the “Nonverbal Communication” score among boys (OR: 1.233), and MEP levels were associated with the “Nonverbal Communication” score among girls (OR: 2.648). MEP levels were related to the CARS total score after adjustment for sex (β: 1.524, p = 0.047). Compared with the reference mono(2-ethylhexyl) phthalate (MEHP) group, children with ASD in the medium-exposure group had an OR of 3.370 for aggravating ASD severity. These results suggested that increased exposure to phthalates contributes to more ASD symptoms and that there are potentially sex-specific associations. These findings warrant further confirmation.
... From in vitro tests, it is known that many phthalates have anti-androgenic activity at the cellular level. Many in vivo tests also show that phthalates inhibit fetal testosterone production by disrupting the organization and function of the HPG axis which results in sex-specific neurobehavioral outcomes (Engel et al. 2021;Gore et al. 2019;Kougias et al. 2018). However, researchers have found that DEHP exposure disrupted the courtship behavior of adult male mice by inducing neural AR downregulation in the nucleus and upstream chemosensory regions instead of causing traditional anti-androgenic effects on HPG (Dombret et al. 2017). ...
Chapter
Ambient airborne particular matter (PM) is a major environmental risk to human health in the world. Numerous epidemiological, clinical and experimental studies have demonstrated the association between high exposure levels of PM and an increase in various diseases, including asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, lung cancer, neurodegenerative diseases, heart diseases, and diabetes. The proposed underlying biological and molecular mechanisms whereby PM causes adverse health effects include oxidative stress, inflammation and genotoxicity. This chapter provides an overview of the recent literature reporting new insights about the molecular mechanisms linking ambient PM exposure and health effects.
... From in vitro tests, it is known that many phthalates have anti-androgenic activity at the cellular level. Many in vivo tests also show that phthalates inhibit fetal testosterone production by disrupting the organization and function of the HPG axis which results in sex-specific neurobehavioral outcomes (Engel et al. 2021;Gore et al. 2019;Kougias et al. 2018). However, researchers have found that DEHP exposure disrupted the courtship behavior of adult male mice by inducing neural AR downregulation in the nucleus and upstream chemosensory regions instead of causing traditional anti-androgenic effects on HPG (Dombret et al. 2017). ...
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Pharmaceuticals and personal care products (PPCPs) are emerging contaminants present in the environment. The general population is inevitably exposed to PPCPs in daily life. The PPCP family contains two types of chemicals: pharmaceuticals and personal care products. Pharmaceuticals are used primarily to prevent or treat human and animal diseases, whereas personal care products are used to improve the quality of daily life and include products such as toothpaste, shampoo, lotions, cosmetics, and hair colors. Due to the structural similarity to biologically active compounds, PPCPs have raised public concerns regarding their possible effects on human health and the environment. Over the past two decades, many studies have found that PPCPs are endocrine-disrupting chemicals (EDCs) with profound adverse effects on the endocrine system. Therefore, in this chapter, we discuss several typical PPCPs, such as bisphenol A and its analogues, triclosan, triclocarban, and phthalates, their adverse endocrine-disrupting activities and three typical endocrine system-related modes of action (MOAs) through interaction with estrogen, androgen, and thyroid receptors.
... Similar structural and behavioral defects of the PFC can be observed in kids with prenatal exposure to opioids, cocaine, amphetamines and other drugs-ofabuse [322][323][324][325][326][327][328][329][330]. Similarly, we can find lead and other pollutants to be damaging to the developing brain and PFC [331][332][333][334]. Another field of recent study is the perinatal exposure to pharmaceuticals given to treat the pregnant mother. ...
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During evolution, the cerebral cortex advances by increasing in surface and the introduction of new cytoarchitectonic areas among which the prefrontal cortex (PFC) is considered to be the substrate of highest cognitive functions. Although neurons of the PFC are generated before birth, the differentiation of its neurons and development of synaptic connections in humans extend to the 3rd decade of life. During this period, synapses as well as neurotransmitter systems including their receptors and transporters, are initially overproduced followed by selective elimination. Advanced methods applied to human and animal models, enable investigation of the cellular mechanisms and role of specific genes, non-coding regulatory elements and signaling molecules in control of prefrontal neuronal production and phenotypic fate, as well as neuronal migration to establish layering of the PFC. Likewise, various genetic approaches in combination with functional assays and immunohistochemical and imaging methods reveal roles of neurotransmitter systems during maturation of the PFC. Disruption, or even a slight slowing of the rate of neuronal production, migration and synaptogenesis by genetic or environmental factors, can induce gross as well as subtle changes that eventually can lead to cognitive impairment. An understanding of the development and evolution of the PFC provide insight into the pathogenesis and treatment of congenital neuropsychiatric diseases as well as idiopathic developmental disorders that cause intellectual disabilities.
... In addition to these studies that focus directly on how phthalate exposure may impact both the immune system and development of ASD-like traits in animal models, there are other publications which investigate other aspects of neurodevelopment. Some of the reported effects are quite severe, including a reduction in both brain size and neuron and synapse numbers in rat and zebrafish [52,53]. While the severity of the neurodevelopmental effects are likely dose-dependent observations, there are also instances of more nuanced and translational neurodevelopmental outcomes in addition to ASD. ...
Article
Substances that interfere with the body's hormonal balance or their function are called endocrine disrupting chemicals (EDCs). Many EDCs are ubiquitous in the environment and are an unavoidable aspect of daily life, including during early embryogenesis. Developmental exposure to these chemicals is of critical relevance, as EDCs can permanently alter developmental programs, including those that pattern and wire the brain. Of emerging interest is how these chemicals may also affect the immune response, given the cross-talk between the endocrine and immune systems. As brain development is strongly dependent on hormones including thyroid, androgens, and estrogens, and can also be affected by immunomodulation, this complicated interplay may have long-lasting neurodevelopmental consequences. This review focuses on data available from human cohorts, in vivo models, and in vitro assays regarding the impact of EDCs after a gestational and/or lactational exposure, and how they may impact the immune system and/or neurodevelopment.
... The complexity of fetal brain development requires maturational processes to occur in the right order and at the right time; disruption of these processes could therefore disrupt optimal brain development, resulting in behavioral problems in childhood 56,57 . Animal studies have also shown that prenatal phthalate exposure is associated with a loss of neurons and synapses in the medial prefrontal cortex 58 , a brain area that shows aberrant functional connectivity and structure in children with depression and attention deficit hyperactivity disorder 59,60 . Disruption of fetal brain development by phthalate exposure could create conditions for deviant developmental trajectories, including behavioral problems in childhood 56,57 . ...
Article
Background Phthalate exposure has been associated with increased childhood behavioral problems. Existing studies failed to include phthalate replacements and did not account for high correlations among phthalates. Phthalates’ exposure is higher in Mexico than in U.S. locations, making it an ideal target population for this study. Aim To examine associations between 15 maternal prenatal phthalate metabolite concentrations and children’s behavioral problems. Methods We quantified phthalate metabolites in maternal urine samples from maternal-child dyads (n = 514) enrolled in the Programming Research in Obesity, Growth Environment and Social Stress (PROGRESS) birth cohort in Mexico City. We performed least absolute shrinkage and selection operator (LASSO) regressions to identify associations between specific-gravity adjusted log2-transformed phthalate metabolites and parent-reported 4-6 year old behavior on the Behavior Assessment System for Children (BASC-2), accounting for metabolite correlations. We adjusted for socio-demographic and birth-related factors, and examined associations stratified by sex. Results Higher prenatal mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) urinary concentrations were associated with increased hyperactivity scores in the overall sample (ß = 0.57, 95% CI = 0.17, 1.13) and in girls (ß = 0.54, 95% CI = 0.16, 1.08), overall behavioral problems in boys (ß =0.58, 95% CI = 0.20, 1.15), and depression scores in boys (ß = 0.44, 95% CI = 0.06, 0.88). Higher prenatal monobenzyl phthalate (MBzP) concentrations were associated with reduced hyperactivity scores in girls (ß = -0.54, 95% CI = -1.08, -0.21). Discussion Our findings suggested that prenatal concentrations of phthalates and their replacements altered child neurodevelopment and those associations may be influenced sex.
... Neurons and pyknotic cells within the mPFC were counted using similar methods to those previously described by our laboratory (Drzewiecki et al., 2020;Kougias et al., 2018;Willing and Juraska, 2015) using the StereoInvestigator optical disector, which allows for unbiased stereology. In the mature mPFC, the dorsal border is distinguished by a thinning layer I and increased cell density in layer III while the ventral is marked by a blurring of lamina borders. ...
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Apoptosis, programmed cell death, is a critical component of neurodevelopment occurring in temporal, spatial, and at times, sex-specific, patterns across the cortex during the early postnatal period. During this time, the brain is particularly susceptible to environmental influences that are often used in animal models of neurodevelopmental disorders. In the present study, the timing of peak cell death was assessed by the presence of pyknotic cells in the male and female rat medial prefrontal cortex (mPFC), a cortical region that in humans, is often involved in developmental disorders. One male and one female rat per litter were sacrificed at the following ages: Postnatal day (P)2, 4, 6, 8, 10, 12, 14, 16, 18, and 25. The mPFC was Nissl-stained, the densities of pyknotic cells and live neurons were stereologically collected, and the number of pyknotic cells per 100 live neurons, pyknotic cell density, and neuron density were analyzed. Males and females showed a significant peak in the ratio of pyknotic to live neurons on P8, and in females, this elevation persisted through P12. Likewise, the density of pyknotic cells peaked on P8 in both sexes and persisted through P12 in females. The timing of cell death within the rat mPFC will inform study design in experiments that employ early environmental manipulations that might disrupt this process.
... An additional study reported similar effects on spatial and short-term recognition memory in male mice, but again females were not examined (Barakat et al., 2018). In rats, perinatal exposure to an environmentally relevant mixture of phthalates produced a sex-independent, long-term deficit in cognitive flexibility but not in spatial learning (Kougias et al., 2018b). ...
Article
Sex is a fundamental biological characteristic that influences many aspects of an organism’s phenotype, including neurobiological functions and behavior as a result of species-specific evolutionary pressures. Sex differences have strong implications for vulnerability to disease and susceptibility to environmental perturbations. Endocrine disrupting chemicals (EDCs) have the potential to interfere with sex hormones functioning and influence development in a sex specific manner. Here we present an updated descriptive review of findings from animal models and human studies regarding the current evidence for altered sex-differences in behavioral development in response to early exposure to EDCs, with a focus on bisphenol A and phthalates. Overall, we show that animal and human studies have a good degree of consistency and that there is strong evidence demonstrating that EDCs exposure during critical periods of development affect sex differences in emotional and cognitive behaviors. Results are more heterogeneous when social, sexual and parental behaviors are considered. In order to pinpoint sex differences in environmentally-driven disease vulnerabilities, researchers need to consider sex-biased developmental effects of EDCs.
... Whether neurodevelopmental changes occur as a direct neurotoxic effect of phthalate or an indirect effect through the disruption of other neurotransmitter or metabolic pathways is not fully understood (Jankowska et al., 2019). Experimental evidence has revealed that prenatal phthalate exposure is associated with decreased cognitive flexibility in offspring and that the association is mediated by reduced neuron and synapse numbers (Kougias et al., 2018) and hippocampal neuron apoptosis (Li et al., 2013b). Other studies revealed that phthalates could interfere with brain dopamine receptor D2 protein levels , γ-amino butyric acid (GABA) concentrations (Carbone et al., 2019), thyroid function (Morgenstern et al., 2017;Yao et al., 2016), and placental anthropometry and function (Zhu et al., 2018a;Zong et al., 2015), which may interfere with neurodevelopment to some extent. ...
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Background Phthalates are a group of heavily produced endocrine disruptors that are widely used in personal care products, food packaging, building materials, and medical device. Few epidemiological studies have examined the effect of repeated prenatal exposure to multiple phthalates on preschooler cognitive development. Objectives This study aimed to examine the association between prenatal phthalate exposure measured at multiple time points and the intelligent quotient (IQ) scores of preschoolers, and to further identify the critical windows and specific intelligence domains in which phthalate exposure would affect preschooler cognitive development. Methods The current study was based on the Ma’anshan Birth Cohort (MABC) study. Seven phthalate metabolites were measured in 2128 maternal urine samples collected during the first, second, and third trimesters of pregnancy. The IQ score of preschool-aged children were assessed with the Chinese version of the Wechsler Preschool and Primary Scale of Intelligence, Fourth edition (WPPSI-Ⅳ CN). Linear mixed models (LMMs) were used to assess the longitudinal effects of repeated prenatal phthalate exposure on children’s IQ score. Multiple linear regression models were fitted to determine whether critical window phthalate exposure would affect cognitive development of children. Results Overall, the repeated measures analysis indicated that the verbal comprehension index (VCI), visual space index (VSI) and full-scale intelligence quotient (FSIQ) decreased by 0.30 (95% CI: −0.60, 0; p = 0.05), 0.32 (95% CI: −0.62, −0.01; p = 0.04), and 0.31 (95% CI:-0.57, −0.04; p = 0.02) points, respectively, with each ln-transformed increase in the metabolite concentration of MBP. The fluid reasoning index (FRI) and processing speed index (PSI) increased by 0.30 (95% CI: 0.07, 0.54; p = 0.01) and 0.28 (95% CI: 0.06, 0.51; p = 0.01) points, respectively, with each ln-concentration increase in MEP. Trimester-specific regression models stratified by the sample collection time during pregnancy generated consistent results. In the first trimester, each ln-transformed MBP increase was associated with reductions in VCI, VSI and FSIQ of 0.56 (95% CI:-1.09, −0.02; p = 0.04), 0.60 (95% CI:-1.15, −0.05; p = 0.03) and 0.49 (95% CI:-0.97, −0.01; p = 0.04) points, respectively. In the third trimester, we observed that only MBzP exposure was associated with an increase in VCI (β: 0.48, 95% CI: 0.03, 0.92; p = 0.04). The gender-stratified analyses revealed that boys drove these associations. Conclusions Our results suggest that prenatal phthalate exposure impairs the cognitive development of preschoolers. The first trimester of pregnancy might be the most vulnerable period in terms of neurotoxicitydue to phthalate exposure. These findings warrant further confirmation.
... Currently, there is no report regarding the effect of DEHP on prefrontal working memory using delayed non-match-to-sample tasks. A study regarding the prefrontal function shows that perinatal exposure to a mixture of phthalates results in a deficit in cognitive flexibility [48]. Several studies in rodent models implicate that prenatal DEHP exposure impairs hippocampus-dependent learning and memory by enhanced oxidative damage, decreased N-methyl-d-aspartic acid (NMDA) receptors, reduced neurogenesis, and the impairment of neuronal excitability [3,41,49,50]. ...
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Early-life exposure to di-(2-ethylhexyl)-phthalate (DEHP) has been suggested to relate to hyperactivity, lack of attention, and working memory deficits in school-age children. Brain-derived neurotrophic factor (BDNF) and endocannabinoids are induced by aerobic exercises to provide beneficial effects on brain functions. This study investigated the mechanisms underlying working memory impairment and the protective role of exercise in prenatal DEHP-exposed male rats. Sprague Dawley dams were fed with vehicle or DEHP during gestation. The male offspring were trained to exercise on a treadmill for 5 weeks, which was followed by an assessment of their working memory with a T-maze delayed non-match-to-sample task. The expressions of BDNF, dopamine D1 receptor (D1R), cannabinoid receptor 1 (CB1R), and fatty acid amide hydrolase (FAAH) in the prefrontal cortex were detected by Western blot. The results showed that DEHP-exposed rats exhibited working memory impairments without significant alterations in locomotor activities. The reduced expressions of prefrontal BDNF and CB1R were obtained in the DEHP-exposed rats, while D1R and FAAH were barely affected. Importantly, aerobic exercise during childhood-adolescence prevented the impairment of working memory in the DEHP-exposed rats by recovering the BDNF and CB1R expressions in the prefrontal cortex. These findings suggest that exercise may provide beneficial effects in ameliorating the impairment of working memory in the prenatal DEHP-exposed male rats at late adolescence.
... It is a trait that has been the focus of much recent research. Recent studies on it have spanned an array of animal species including mice (Marwari & Dawe, 2018;Morris-Schaffer, et al., 2018;Piantadosi et al., 2019), rats (Dhawan et al., 2019;Kougias et al., 2018;Zhang et al., 2017), rhesus monkeys (Shnitko et al., 2019), chimpanzees (Lacreuse et al., 2018), horses (Fortin et al., 2018), parrots (van Horik & Emery, 2018), pigeons (Darby et al., 2018), and bees (Loukola et al., 2017). Increasingly, scientists have begun to realize that cognitive flexibility may be crucial in shaping certain higher order thought processes in humans, and that those processes help to shape personal characteristics and worldviews. ...
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Religious ideology and extremism have had an increasing influence on political agendas in the United States and much of the developed world in the past 60 years, with right-wing ideology becoming more prevalent this decade. This article serves as a review of studies investigating the correlations between political ideology, religiosity, right-wing authoritarianism, ingroups/outgroups, and prejudice in an attempt to describe and understand the well-established links between these dimensions. We discuss several group-level theories including Terror Management Theory, Social Identity Theory, Realistic Group Conflict Theory among others to frame the intercorrelations of these constructs in an effort to better understand the underlying mechanisms that drive individuals to embody religious and political beliefs. We then discuss individual-level cognitive and psychological differences such as intelligence, cognitive flexibility, and specific biological and neurological limitations of brain function that may influence people to adopt certain religious and political beliefs. Through a more comprehensive understanding of the underlying mechanisms of religious and political extremism, we may be better equipped to assuage the fear and denigration that is associated with many of these beliefs.
Article
We previously observed that developmental marginal zinc deficiency affects neurogenesis. Maternal phthalate exposure could disrupt fetal zinc homeostasis by triggering an acute phase response, causing maternal liver zinc retention that limits zinc availability to the fetus. Thus, we currently investigated whether exposure to di-2-ethylhexyl phthalate (DEHP) during gestation in rats alters fetal brain neurogenesis by impairing zinc homeostasis. Dams consumed an adequate (25 μg zinc/g diet) (C) or a marginal zinc deficient (MZD) (10 μg zinc/g diet) diet, without or with DEHP (300 mg/kg BW) (C + DEHP, MZD + DEHP) from embryonic day (E) 0 to E19. To evaluate neurogenesis we measured parameters of neural progenitor cells (NPC) proliferation and differentiation. Maternal exposure to DEHP and/or zinc deficiency lowered fetal brain cortical tissue (CT) zinc concentrations. Transcription factors involved in NPC proliferation (PAX6, SOX2, EMX1), differentiation (TBR2, TBR1) and mature neurons (NeuN) were lower in MZD, MZD + DEHP and C + DEHP than in C E19 brain CT, being the lowest in the MZD + DEHP group. VGLUT1 levels, a marker of glutamatergic neurons, showed a similar pattern. Levels of a marker of GABAergic neurons, GAD65, did not vary among groups. Phosphorylated ERK1/2 levels were reduced by both MZD and DEHP, and particularly in the MZD + DEHP group. MEHP-treated human neuroblastoma IMR-32 cells and E19 brains from DEHP-treated dams showed that the zinc-regulated phosphatase PP2A can be in part responsible for DEHP-mediated ERK1/2 downregulation and impaired neurogenesis. Overall, gestational exposure to DEHP caused secondary zinc deficiency and impaired neurogenesis. These harmful effects could have long-term consequences on the adult offspring brain structure and function.
Article
Rodents used for research can be humanely housed in a variety of ways. As such, a vast number of different housing environments are used, but are often not described in research publications. However, many elements of housing environments, including bedding, diet, water bottles, and cage material, can expose rodents to natural and synthetic compounds that can have lasting effects on the body, brain, and behavior. Some environmental items contain endocrine-disrupting compounds (EDCs), which can affect many commonly assessed physiological and behavioral endpoints in rodents. Here, we compare the effects of 2 commonly used housing environments for male and female Long Evans rats on body weight, pubertal onset, and a battery of behavioral tests measuring activity, anxiety-like behavior, and cognition. One standard environment was comparatively high in EDCs (standard rodent chow, plastic cages, plastic water bottles, and corncob bedding), while the other was a relatively low-EDC environment (phytoestrogen-free chow, polysulfone cages, glass water bottles, and wood-chip bedding). As compared with the Standard group, rats raised in the Low-EDC environment reached puberty earlier, displayed less anxiety-like behavior in the elevated plus maze and open field test, and showed less overall object exploration in the novel object recognition task. These effects occurred only if rats had been raised in these conditions since conception. An acute change from one environment to the other in adulthood did not yield these same effects. These results provide further evidence for the effects of common housing environments on development and behavior and highlight the importance of reporting environmental conditions in the literature to promote reproducibility in research using animal subjects.
Thesis
Adult male mice exposure to low doses of ubiquitous endocrine disruptors of phthalate family, DEHP, induces alteration of neural function and of behaviors due to down-regulation of androgen receptors (AR). The link between reduced androgen sensitivity, neurovascular unit (NVU) dysfunction and neuroinflammation has been demonstrated. Whether phthalates exposure affects the integrity and function of the NVU remained to be explored. Exposure to low doses of DEHP alone or in an environmental phthalate mixture, increased blood-brain barrier (BBB) permeability, affecting the accessory tight junction protein Zona Occludens-1 and caveolae protein Cav-1 in capillaries of the hypothalamus mPOA and the hippocampal CA1 and CA3 areas, key cerebral areas involved in the sexual and cognitive behaviors in male, respectively. This was associated with an inflammatory profile including an astrocyte activation and iNOS expression in the mPOA, and a microglial activation in the mPOA and the hippocampal CA1 and CA3 areas. The protein levels of the inflammatory molecule, COX-2 were increased in activated microglial cells in the mPOA. None of the major effects induced by DEHP alone or in a mixture was detected in the hippocampal dendate gyrus. DEHP decreased AR protein levels in cerebral capillaries of the mPOA. Such exposure reduced the protein levels of basement membrane components and receptors involved in cell-matrix interactions while MMP-2 and MMP-9 activity was enhanced. These data point out a vulnerability of the NVU to exposure to DEHP alone or in a phthalate mixture, with similarities and differences in the induced effects between the studied androgen-sensitive brain regions.
Article
Phthalates are chemicals widely used in packaging and consumer products, which have been shown to interfere with normal hormonal function and development in some human and animal studies. In recent decades, pregnant women's exposure to phthalates has been shown to alter the cognitive outcomes of their babies, and some studies have found delays in motor development. Methods: electronic databases including PubMed/MEDLINE and Scopus were searched from their inception to March 2021, using the keywords "phthalate", "cognitive" and "motor". Results: most studies find statistically significant inverse relationships between maternal urinary phthalate concentration during pregnancy and subsequent outcomes in children's cognitive and motor scales, especially in boys rather than girls. However, many associations are not significant, and there were even positive associations, especially in the third trimester. Conclusion: the relationship between exposure to phthalates during pregnancy and low results on neurocognitive scales is sufficiently clear to adopt policies to reduce exposure. Further studies are needed to analyze sex differences, coordination and motor scales, and phthalate levels during breastfeeding.
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Plasticizers, used to increase the flexibility of plastic materials, can leach into the environment and the human body. Various adverse health effects are attributed to exposure to plasticizers, particularly phthalates. Premature newborns admitted to a neonatal intensive care unit (NICU) are exposed to many indwelling plastic devices containing plasticizers while in a developmentally vulnerable period. This is the first comprehensive review of its kind, providing an overview of DEHP and alternative plasticizers (APs), their sources and degree of exposure in the NICU, and the resulting health risks in (premature) infants. Despite the 2017 EU Medical Devices Regulation (2017/745), di-(2-ethylhexyl) phthalate (DEHP) is still a commonly present plasticizer in plastic medical devices in the NICU, with current labeling being insufficient to guarantee absence. Estimated NICU exposures remain elevated above the tolerable daily intake. Medical procedures leading to the highest exposure of phthalates and APs, are extracorporeal membrane oxygenation, blood transfusion, parenteral nutrition and respiratory support. As traditional matrices, such as blood and urine, are not always readily available in neonates, the accumulation of plasticizers in alternative matrices, such as hair and nails, provides an opportunity to study long-term accumulation of toxic chemicals. Impaired respiratory and neurodevelopment correlate with phthalate exposure at both biological and epidemiological levels in childhood, yet knowledge gaps about the effects in neonates prevail. Some APs provide interesting opportunities to reduce toxicity, but human data regarding health effects remain limited. Although toxicologists and regulators have addressed the problem for some time, awareness is lacking mainly among healthcare professionals.
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Phthalates are chemical substances that are widely used to provide flexibility and durability to plastic materials. They leach from products in which they are mixed and reach living organisms. Results from experimental studies suggest that exposure to phthalates can have a negative impact on an individual's neuronal system and behavior. In this regard, exposure during early ontogenesis seems to be particularly dangerous due to the extensive growth and development of body structures and functions. Disruption during this critical time can result in alterations of behavior and the emergence of neurodevelopmental disorders, such as autism spectrum disorder (ASD). Various animal models have been used to elucidate the pathogenesis of this disease. They are fundamental for research, and although the translation of results to humans is difficult, new animal models are being developed. The aim of this review is to summarize laboratory rodent studies in which early developmental phthalate exposure resulted in brain alterations and autistic-like behavioral traits. We also discuss the possibility of using early developmental phthalate exposure in rodents to create a new animal model of autism.
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Exposure to stress during adolescence is a risk factor for developing several psychiatric disorders, many of which involve prefrontal cortex (PFC) dysfunction. The human PFC and analogous rodent medial prefrontal cortex (mPFC) continue to mature functionally and anatomically during adolescence, and some of these maturational events coincide with pubertal onset. As developing brain regions are more susceptible to the negative effects of stress, this may make puberty especially vulnerable. To test this, we exposed male and female rats to isolation and restraint stress during the onset of puberty or during the post‐pubertal period of adolescence. In young adulthood, both stressed groups and an unstressed control group underwent testing on a battery of tasks to assess emotional and cognitive behaviors, and the volume of the mPFC was quantified postmortem. Factor analysis revealed only subjects stressed peri‐pubertally showed a long‐term deficiency compared to controls in prepulse inhibition. Additionally, both sexes showed volumetric mPFC decreases following adolescent stress, and these losses were most pronounced in females. Our findings suggest that pubertal onset may be a vulnerable window wherein adolescents are most susceptible to the negative consequences of stress exposure. Furthermore, it highlights the importance of accounting for pubertal status when studying adolescents.
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Robust data from longitudinal birth cohort studies and experimental studies of perinatally exposed animals indicate that exposure to ortho-phthalates can impair brain development and increase risks for learning, attention, and behavioral disorders in childhood. This growing body of evidence, along with known adverse effects on male reproductive tract development, calls for immediate action. Exposures are ubiquitous; the majority of people are exposed to multiple ortho-phthalates simultaneously. We thus recommend that a class approach be used in assessing health impacts as has been done with other chemical classes. We propose critically needed policy reforms to eliminate ortho-phthalates from products that lead to exposure of pregnant women, women of reproductive age, infants, and children. Specific attention should be focused on reducing exposures among socially vulnerable populations such as communities of color, who frequently experience higher exposures. Ortho-phthalates are used in a vast array of products and elimination will thus necessitate a multipronged regulatory approach at federal and state levels. The fact that manufacturers and retailers have already voluntarily removed ortho-phthalates from a wide range of products indicates that this goal is feasible. (Am J Public Health. Published online ahead of print February 18, 2021: e1–e9. https://doi.org/10.2105/AJPH.2020.306014 )
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Dihexyl phthalate (DHP) is one of the most commonly used phthalate esters in various plastic and consumer products. Human are inevitably exposed to DHPs. Although several animal and human experiments have revealed that DHP can cause multiple toxicities, few studies have previously assessed the effects of DHP exposure by liquid chromatography mass spectrometry (LC‐MS) analysis combine with molecular biology methods on human cells. Therefore, the purpose of our study was to investigate the effect of DHP on human cell metabolism by systems biology methods. In this study, U2OS cancer cells were treated with 10 μM DHP for metabolomics analysis and apoptosis analysis at indicate time. Metabolomic study of the metabolic changes caused by DHP in U2OS cells was performed for the first time using integrative liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (LC‐Q‐TOF‐MS). To investigate the possible reason of fatty acids level altered by DHP, we measured some key fatty acid synthesis and oxidation‐related enzyme expression levels by quantitative real‐time PCR (Q‐PCR). Apoptotic cells were analyzed by flow cytometry and apoptosis‐related gene expressions were measured by Q‐PCR. 2′,7′‐Dichlorofluorescein diacetate (DCFH‐DA) staining was used to evaluate ROS content. Partial least squares‐discriminate analysis (PLS‐DA) clearly showed that significant differences in metabolic profiles were observed in U2OS cells exposed to DHP compared with controls. A total of 58 putative metabolites in electrospray ionization source (ESI) + mode and 32 putative metabolites in ESI‐mode were detected, the majority of the differential metabolites being lipids and lipid‐like molecules. Among them, the altered fatty acids level corresponded to expression levels of genes encoding enzymes related to fatty acids synthesis and oxidation. Moreover, DHP induced reactive oxygen species (ROS) accumulation, promoted cell apoptosis and inflammation, and resulted in a significant increase in apoptosis and inflammation‐related gene expression levels compared with controls. In summary, our results suggested that metabolomics combined with molecular bioanalysis methods could be an efficient tool to assess toxic effects, which contribute to explore the possible cytotoxicity mechanisms of DHP, and provide a basis for further research.
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Phthalates are found in different plastic materials, such as packaging, toys and medical devices. Some of these compounds are endocrine disruptors, substances that are able to induce multiple hormonal disturbances and downstream developmental effects, including disruption of androgen‐dependent differentiation of male reproductive tract and changes in pathways that regulate hormone‐dependent behaviors. In a previous study, metabolites of diisopentyl phthalate (DiPeP), a potent antiandrogenic phthalate, were found in the urine of Brazilian pregnant women. Therefore, the aim of this study was to evaluate the effects of DiPeP exposure during critical developmental periods on behaviors controlled by sex hormones in rats. Pregnant Wistar rats were treated with DiPeP (1, 10 or 100 mg/kg/day) or canola oil by oral gavage between gestational day (GD) 10 and post‐natal day (PND) 21. Male offspring were tested in a behavioral battery, including the elevated plus maze task, play behavior, partner preference and sexual behavior. After the behavioral tests, the hypothalamus and pituitary of these animals were removed on PND 60‐65 and PND 145‐160 to quantify gene expression for aromatase, androgen receptor (Ar) and estrogen receptors α (Esr1) and β (Esr2). Male rats exposed to 1 and 10 mg/kg/day DiPeP displayed no preference for the female stimulus rat in the partner preference test and 1 mg/kg/day DiPeP rats also showed a significant increase in mount and penetration latencies when mated with receptive females. A decrease in pituitary Esr1 expression was observed in all DiPeP treated groups regardless of age. A reduction in hypothalamic Esr1 expression in rats exposed to 10 mg/kg/day DiPeP was also observed. No significant changes in Ar, Esr2 and aromatase expression in the hypothalamus were found. These results suggest that DiPeP exposure during critical windows of development in rats may induce changes in behaviors related to mating and sexual motivation of males.
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Background: Early-life phthalate exposures may adversely affect children's neurodevelopment by disrupting thyroid function, reducing gonadal hormone levels, or altering fatty acid concentrations in the brain. This study aimed to identify periods of heightened susceptibility during gestation, infancy, and childhood to the impact of phthalates on children's cognitive abilities. Methods: We used data from 253 mother-child pairs in the Health Outcomes and Measures of the Environment (HOME) Study (Cincinnati, Ohio), a longitudinal pregnancy and birth cohort. We quantified urinary concentrations of 11 phthalate metabolites in samples collected twice during gestation and 6 times during study visits when children were aged 1-8 years using a modified method of on-line solid phase extraction coupled with isotope dilution-high performance liquid chromatography-tandem mass spectrometry. We assessed children's intelligence (IQ) at ages 5 and 8 years using the Wechsler Preschool and Primary Scale of Intelligence-III and Wechsler Intelligence Scale for Children-IV, respectively. We estimated covariate-adjusted associations between a 1-standard deviation increase in log10-transformed urinary phthalate metabolite concentrations at each visit and children's IQ, adjusting for demographic, perinatal, and child factors; we tested for differences in these associations across visits using multiple informant models. Results: Associations between some phthalate metabolites and IQ varied by visit (phthalate x visit interaction p-values<0.20). The sum of di(2-ethylhexyl) phthalate metabolites (ΣDEHP), mono(3-carboxypropyl) phthalate, and monoethyl phthalate at age 3 years, and monobenzyl phthalate at 16 weeks gestation and child ages 3, 5, and 8 years were inversely associated with children's full-scale IQ. For example, each 1-standard deviation increase in ΣDEHP at age 3 was associated with a 1.9-point decrease in full-scale IQ (95% confidence interval: -3.7, -0.2). Mono-n-butyl phthalate and mono-isobutyl phthalate at age 4 years were positively associated with children's full-scale IQ. Conclusion: Urinary concentrations of several phthalate metabolites at age 3 years, compared to other time periods, were more strongly associated with decreased cognitive abilities in these children.
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Microglia play key roles in brain development, homeostasis, and function, and it is widely assumed that the adult population is long lived and maintained by self-renewal. However, the precise temporal and spatial dynamics of the microglial population are unknown. We show in mice and humans that the turnover of microglia is remarkably fast, allowing the whole population to be renewed several times during a lifetime. The number of microglial cells remains steady from late postnatal stages until aging and is maintained by the spatial and temporal coupling of proliferation and apoptosis, as shown by pulse-chase studies, chronic in vivo imaging of microglia, and the use of mouse models of dysregulated apoptosis. Our results reveal that the microglial population is constantly and rapidly remodeled, expanding our understanding of its role in the maintenance of brain homeostasis.
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Dibutyl phthalate (di-n-butyl phthalate, DBP) is one of the most commonly used phthalate esters. DBP is widely used as a plasticizer in a variety of household industries and consumer products. Because phthalates are not chemically bound to products, they can easily leak out to enter the environment. DBP can pass through the placental and blood-brain barriers due to its chemical structure, but little is known about its mechanism of action in neuronal cells. This study demonstrated the toxic and apoptotic effects of DBP in mouse neocortical neurons in primary cultures. DBP stimulated caspase-3 and LDH activities as well as ROS formation in a concentration (10 nM-100 µM) and time-dependent (3-48 h) manner. DBP induced ROS formation at nanomolar concentrations, while it activated caspase-3 and LDH activities at micromolar concentrations. The biochemical effects of DBP were accompanied by decreased cell viability and induction of apoptotic bodies. Exposure to DBP reduced Erα and Pparγ mRNA expression levels, which were inversely correlated with protein expression of the receptors. Treatment with DBP enhanced Ahr mRNA expression, which was reflected by the increased AhR protein level observed at 3 h after exposure. ERα, ERβ, and PPARγ antagonists stimulated DBP-induced caspase-3 and LDH activities. AhR silencing demonstrated that DBP-induced apoptosis and neurotoxicity are mediated by AhR, which is consistent with the results from DBP-induced enhancement of AhR mRNA and protein expression. Our study showed that AhR is involved in DBP-induced apoptosis and neurotoxicity, while the ERs and PPARγ signaling pathways are impaired by the phthalate.
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The human prefrontal cortex, important for executive functions, loses gray matter throughout the adolescent period. In rats, our laboratory demonstrated that a loss of neurons between adolescence and adulthood partially underlies the loss of volume, and this loss is greater in females than males. Here, we examine whether being deprived of gonadal hormones before puberty through adulthood influences the number of neurons in the medial prefrontal cortex (mPFC). Prior to puberty, the testes or ovaries were removed in male and female rats. In adulthood, the number of neurons and glia in the mPFC were quantified using unbiased stereology, and the volume of the frontal white matter was measured. Prepubertal ovariectomy resulted in a higher number of neurons and glia and a larger volume of white matter compared to sham control littermates. Castrated males were not different from sham males on any measure. Thus ovarian hormones secreted after puberty influence the cellular composition of the medial prefrontal cortex. © 2015 Wiley Periodicals, Inc. Dev Psychobiol. © 2015 Wiley Periodicals, Inc.
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During the perinatal period, male mice are exposed to higher levels of testosterone (T) than females, which promotes sexual dimorphism in their brain structures and behaviors. In addition to acting via estrogen receptors after being locally converted into estradiol by aromatase, T also acts directly through androgen receptor (AR) in the brain. Therefore, we hypothesized that AR expression in the developing mouse cortex and hippocampus was sexually dimorphic. To test our hypothesis, we measured and determined AR mRNA and protein levels in mouse cortex/hippocampus collected on the day of birth (PN0) and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. We demonstrated that, as age advanced, AR mRNA levels increased in the cortex/hippocampus of both sexes but showed no sex difference. Two AR proteins, the full-length (110 kDa) and a smaller isoform (70 kDa), were detected in the developing mouse cortex/hippocampus with an age-dependent increase in protein levels of both AR isoforms at PN21 and a transient masculine increase in expression of the full-length AR protein on PN7. Thus, we conclude that the postnatal age and sex differences in AR protein expression in combination with the sex differences in circulating T may cause sexual differentiation of the mouse cortex/hippocampus.
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Objectives Phthalates might be implicated with obesity and insulin sensitivity. We evaluated the levels of primary and secondary metabolites of Di-(2-ethylhexyl) phthalate (DEHP) in urine in obese and normal-weight subjects both before and during puberty, and investigated their relationships with auxological parameters and indexes of insulin sensitivity. Design and Methods DEHP metabolites (MEHP, 6-OH-MEHP, 5-oxo-MEHP, 5-OH-MEHP, and 5-CX-MEHP), were measured in urine by RP-HPLC-ESI-MS. Traditional statistical analysis and a data mining analysis using the Auto-CM analysis were able to offer an insight into the complex biological connections between the studied variables. Results The data showed changes in DEHP metabolites in urine related with obesity, puberty, and presence of insulin resistance. Changes in urine metabolites were related with age, height and weight, waist circumference and waist to height ratio, thus to fat distribution. In addition, clear relationships in both obese and normal-weight subjects were detected among MEHP, its products of oxidation and measurements of insulin sensitivity. Conclusion It remains to be elucidated whether exposure to phthalates per se is actually the risk factor or if the ability of the body to metabolize phthalates is actually the key point. Further studies that span from conception to elderly subjects besides further understanding of DEHP metabolism are warranted to clarify these aspects.
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We review the plastic changes of the prefrontal cortex of the rat in response to a wide range of experiences including sensory and motor experience, gonadal hormones, psychoactive drugs, learning tasks, stress, social experience, metaplastic experiences, and brain injury. Our focus is on synaptic changes (dendritic morphology and spine density) in pyramidal neurons and the relationship to behavioral changes. The most general conclusion we can reach is that the prefrontal cortex is extremely plastic and that the medial and orbital prefrontal regions frequently respond very differently to the same experience in the same brain and the rules that govern prefrontal plasticity appear to differ for those of other cortical regions.
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Background: Few studies have shown an association between prenatal phthalate exposure and adverse effects on neurodevelopment and behavior in young children. Objectives: We aimed to assess the relationship between prenatal exposure to phthalate esters and behavior syndromes in children at 8 years of age. Methods: A total of 122 mother-child pairs from the general population in central Taiwan were studied from 2000 to 2009. Mono-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), and three di-(2-ethylhexyl) phthalate (DEHP) metabolites-mono-2-ethylhexyl, mono-2-ethyl-5-hydroxyhexyl, and mono-2-ethyl-5-oxohexyl phthalates (MEHP, MEHHP, and MEOHP)--were measured in maternal urine collected during the third trimester of pregnancy using liquid chromatography-electrospray ionization-tandem mass spectrometry. Behavioral syndromes of children at 8 years of age were evaluated using the Child Behavior Checklist (CBCL). Associations between log10-transformed creatinine-corrected phthalate concentrations and standardized scores of the CBCL were estimated using linear regression models or multinomial logistic regressions with adjustments for potential confounders. Results: Externalizing problem scores were significantly higher in association with a 1-unit increase in log10-transformed creatinine-corrected concentrations of maternal MBP (β = 4.29; 95% CI: 0.59, 7.99), MEOHP (β = 3.74; 95% CI: 1.33, 6.15), and MEHP (β = 4.28 ; 95% CI: 0.03, 8.26) after adjusting for the child's sex, intelligence, and family income. Meanwhile, MBP and MEOHP were significantly associated with Delinquent Behavior and Aggressive Behavior scores. The same pattern was found for borderline and/or clinical ranges. Conclusions: Our findings suggest positive associations between maternal DEHP and dibutyl phthalate (DBP) exposure and externalizing domain behavior problems in 8-year-old children.
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Phthalates are ubiquitous endocrine disrupting chemicals associated with diabetes. Although women and minorities are more likely to be exposed to phthalates, no prior studies have examined phthalate exposure and markers of diabetes risk evaluating effect modification by gender and race/ethnicity. We analyzed CDC data for 8 urinary phthalate metabolites from 3,083 non-diabetic, non-pregnant participants aged 12- < 80 years in the National Health and Nutrition Examination Survey (NHANES) 2001-2008. We used median regression to assess the associations between urinary phthalate metabolites and fasting blood glucose (FBG), fasting insulin and Homeostatic Model Assessment of insulin resistance (HOMA-IR), controlling for urinary creatinine as well as several sociodemographic and behavioral factors. Stratified analyses were conducted to compare the gender- and race/ethnicity-specific patterns for the associations. Urinary levels of several phthalate metabolites, including MBzP, MnBP, MiBP, MCPP and [n-ary summation]DEHP showed significant positive associations with FBG, fasting insulin and HOMA-IR. No clear difference was noted between men and women. Mexican-Americans and non-Hispanic blacks had stronger dose-response relationships for MnBP, MiBP, MCPP and [n-ary summation]DEHP compared to non-Hispanic whites. For example, the highest quartile of MiBP relative to its lowest quartile showed a median FBG increase of 5.82 mg/dL (95% CI: 3.77, 7.87) in Mexican-Americans, 3.63 mg/dL (95% CI: 1.23, 6.03) in blacks and 1.79 mg/dL (95% CI: -0.29, 3.87) in whites. The findings suggest that certain populations may be more vulnerable to phthalates with respect to disturbances in glucose homeostasis. Whether endocrine disrupting chemicals contribute to gender and racial/ethnic differences in diabetes risk will be an important area for further study.
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Longitudinal studies of a binary outcome are common in the health, social, and behavioral sciences. In general, a feature of random effects logistic regression models for longitudinal binary data is that the marginal functional form, when integrated over the distribution of the random effects, is no longer of logistic form. Recently, Wang and Louis (2003) proposed a random intercept model in the clustered binary data setting where the marginal model has a logistic form. An acknowledged limitation of their model is that it allows only a single random effect that varies from cluster to cluster. In this paper, we propose a modification of their model to handle longitudinal data, allowing separate, but correlated, random intercepts at each measurement occasion. The proposed model allows for a flexible correlation structure among the random intercepts, where the correlations can be interpreted in terms of Kendall's τ. For example, the marginal correlations among the repeated binary outcomes can decline with increasing time separation, while the model retains the property of having matching conditional and marginal logit link functions. Finally, the proposed method is used to analyze data from a longitudinal study designed to monitor cardiac abnormalities in children born to HIV-infected women.
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The prefrontal cortex (PFC) is critical for decision making, and it becomes dysfunctional in many neuropsychiatric disorders. Studies in schizophrenia patients and relevant animal models suggest loss of PFC inhibitory interneuron function. For instance, rats with a neonatal ventral hippocampal lesion (NVHL) show a deficient modulation of PFC interneurons by dopamine (DA). Whether the PFC becomes disinhibited in this model and alters decision making remains to be determined. Here, we recorded neural activity in the medial PFC of NVHL rats during a reward-discounting choice task that activated DA systems. Rats were trained to sample odors that instructed them to select one of two feeders that delivered unequal amounts of liquid. Putative pyramidal neurons in the PFC were hyperactive whereas task-related field potential oscillations were significantly reduced in NVHL rats, consistent with impaired interneuron activation by DA during odor sampling leading to disorganized processing. Cognitive flexibility was tested by examining response bias and errors after reversing reward outcomes. NVHL rats demonstrated impaired flexibility as they were less able to track changes in reward outcome and made more response errors than controls did. Reducing cortical excitability with the metabotropic glutamate receptor 2/3 agonist LY379268 (1 mg/kg, i.p.) improved behavioral flexibility in NVHL rats but not controls. Furthermore, D2 dopamine receptors were involved, as the antagonist eticlopride (0.02 mg/kg, i.p.) reduced the ability to switch only in control animals. We conclude that NVHL rats present PFC disinhibition, which affects neural information processing and the selection of appropriate behavioral responses.
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Phthalates are widely used chemicals, and human exposure is extensive. Recent studies have indicated that phthalates may have thyroid-disrupting properties. We aimed to assess concentrations of phthalate metabolites in urine samples from Danish children and to investigate the associations with thyroid function, insulin-like growth factor I (IGF-I), and growth. In 845 children 4-9 years of age, we determined urinary concentrations of 12 phthalate metabolites and serum levels of thyroid-stimulating hormone, thyroid hormones, and IGF-I. Phthalate metabolites were detected in all urine samples, of which monobutyl phthalate was present in highest concentration. Phthalate metabolites were negatively associated with serum levels of free and total triiodothyronine, although statistically significant primarily in girls. Metabolites of di(2-ethylhexyl) phthalate and diisononyl phthalate were negatively associated with IGF-I in boys. Most phthalate metabolites were negatively associated with height, weight, body surface, and height gain in both sexes. Our study showed negative associations between urinary phthalate concentrations and thyroid hormones, IGF-I, and growth in children. Although our study was not designed to reveal the mechanism of action, the overall coherent negative associations between urine phthalate and thyroid and growth parameters may suggest causative negative roles of phthalate exposures for child health.
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Experimental and observational studies have reported biological consequences of phthalate exposure relevant to neurodevelopment. Our goal was to examine the association of prenatal phthalate exposure with behavior and executive functioning at 4-9 years of age. The Mount Sinai Children's Environmental Health Study enrolled a multiethnic prenatal population in New York City between 1998 and 2002 (n = 404). Third-trimester maternal urines were collected and analyzed for phthalate metabolites. Children (n = 188, n = 365 visits) were assessed for cognitive and behavioral development between the ages of 4 and 9 years. In multivariate adjusted models, increased loge concentrations of low molecular weight (LMW) phthalate metabolites were associated with poorer scores on the aggression [beta = 1.24; 95% confidence interval (CI), 0.15- 2.34], conduct problems (beta = 2.40; 95% CI, 1.34-3.46), attention problems (beta = 1.29; 95% CI, 0.16- 2.41), and depression (beta = 1.18; 95% CI, 0.11-2.24) clinical scales; and externalizing problems (beta = 1.75; 95% CI, 0.61-2.88) and behavioral symptom index (beta = 1.55; 95% CI, 0.39-2.71) composite scales. Increased loge concentrations of LMW phthalates were also associated with poorer scores on the global executive composite index (beta = 1.23; 95% CI, 0.09-2.36) and the emotional control scale (beta = 1.33; 95% CI, 0.18- 2.49). Behavioral domains adversely associated with prenatal exposure to LMW phthalates in our study are commonly found to be affected in children clinically diagnosed with conduct or attention deficit hyperactivity disorders.
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Endocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose-response character, and cellular target(s) within the fetal testis are not known. In this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP), and several of their metabolites on the development of organo-cultured testes from rat fetus. We removed testes from 14.5-day-old rat fetuses and cultured them for 1-3 days with or without DEHP, MEHP, and the metabolites. DEHP (10(-5) M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro. We hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects.
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This paper gives an account of the cytoarchitectonic characteristics that make it possible to delineate, from as early as day 6, different subareas of the prefrontal cortex of the rat. Three phases can be distinguished during postnatal development. The first phase (from day 1 until day 18) is dominated by differentiation of the neurons within the cortical plate and by the formation of the cortical layers. At day 1, regional differences are observed in the cytoarchitecture of the cortical plate which correspond to the future subareas of the prefrontal cortex. The formation of layer IV occurs in the dorsolateral cortex around day 6, and from this age the agranular prefrontal cortex is well demarcated from the other parts of the frontal cortex. Between day 6 and day 10, the cortical plate has disappeared and all cortical layers can be recognized in the prefrontal cortex. Differentiation of the cells within the cortical layers changes the cytoarchitectonic character of the layers through day 18. During the second phase (from day 18 until day 30) little change occurs in the cytoarchitectonic characteristics of the prefrontal subareas. During the third phase (from day 30 until day 90) the delineation of the cortical layers becomes less clear in Nissl-stained sections, and the individual cytoarchitectonic variance increases. On the basis of cytoarchitectonic criteria it can be concluded that the orbital prefrontal cortex develops earlier than does the medial prefrontal cortex.
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The present experiments investigated the role of the prelimbic-infralimbic areas in behavioral flexibility using a place-response learning paradigm. All rats received a bilateral cannula implant aimed at the prelimbic-infralimbic areas. To examine the role of the prelimbic-infralimbic areas in shifting strategies, rats were tested on a place and a response discrimination in a cross-maze. Some rats were tested on the place version first followed by the response version. The procedure for the other rats was reversed. Infusions of 2% tetracaine into the prelimbic-infralimbic areas did not impair acquisition of the place or response discriminations. Prelimbic-infralimbic inactivation did impair learning when rats were switched from one discrimination to the other (cross-modal shift). To investigate the role of the prelimbic-infralimbic areas in intramodal shifts (reversal learning), one group of rats was tested on a place reversal and another group tested on a response reversal. Prelimbic-infralimbic inactivation did not impair place or response intramodal shifts. Some rats that completed testing on a particular version in the cross-modal and intramodal experiments were tested on the same version in a new room for 3 d. The transfer tests revealed that rats use a spatial strategy on the place version and an egocentric response strategy on the response version. Overall, these results suggest that the prelimbic-infralimbic areas are important for behavioral flexibility involving cross-modal but not intramodal shifts.
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Humans are ubiquitously exposed to many phthalates, a class of endocrine-disrupting chemicals commonly used in many consumer goods, and diet, especially fatty foods, is presumed to be a major source of exposure. Here, we use a rat model of human prenatal exposure to investigate the potential interactive effects of an environmentally relevant mixture of phthalates and a maternal high-fat diet (HFD). From gestation through postnatal day (P)10, dams consumed the mixture of phthalates (0, 200, or 1000 μg/kg/day) and were fed a control or HFD. In males, perinatal exposure to the mixture of phthalates decreased prepubertal body weight and, in a dose-specific manner, periadolescent social play behavior. A dose-specific effect from phthalates with HFD was also seen in increased time alone in females during social play. HFD resulted in dams consuming more calories, having greater gestational weight gain, and licking and nursing their pups more, such that an early postnatal HFD generally increased pup body weight. There also was a tendency for increased oxidative stress markers at P10 within the medial prefrontal cortex of males exposed to the relatively high dose of phthalates and HFD. Effects on gene expression were inconsistent at P10 and P90 in both the medial prefrontal cortex and hypothalamus. Overall, this study demonstrates that phthalates and a maternal HFD only rarely interacted, except in oxidative stress markers in males. Additionally, perinatal exposure to an environmentally relevant mixture of phthalates can have a modest, but lasting, impact on social behaviors in both males and females.
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Existing literature on the profile of executive dysfunction in autism spectrum disorder showed inconsistent results. Age, comorbid attention-deficit/hyperactivity disorder (ADHD) and cognitive abilities appeared to play a role in confounding the picture. Previous meta-analyses have focused on a few components of executive functions. This meta-analysis attempted to delineate the profile of deficit in several components of executive functioning in children and adolescents with high-functioning autism spectrum disorder (HFASD). Ninety-eight English published case-control studies comparing children and adolescents with HFASD with typically developing controls using well-known neuropsychological measures to assess executive functions were included. Results showed that children and adolescents with HFASD were moderately impaired in verbal working memory (g = 0.67), spatial working memory (g = 0.58), flexibility (g = 0.59), planning (g = 0.62), and generativity (g = 0.60) except for inhibition (g = 0.41). Subgroup analysis showed that impairments were still significant for flexibility (g = 0.57–0.61), generativity (g = 0.52–0.68), and working memory (g = 0.49–0.56) in a sample of autism spectrum disorder (ASD) subjects without comorbid ADHD or when the cognitive abilities of the ASD group and the control group were comparable. This meta-analysis confirmed the presence of executive dysfunction in children and adolescents with HFASD. These deficits are not solely accounted for by the effect of comorbid ADHD and the general cognitive abilities. Our results support the executive dysfunction hypothesis and contribute to the clinical understanding and possible development of interventions to alleviate these deficits in children and adolescents with HFASD. Autism Res 2016. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.
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Toxicological studies of defined chemical mixtures assist human health risk assessment by establishing how chemicals interact with one another to induce an effect. This paper reviews how antiandrogenic chemical mixtures can alter reproductive tract development in rats with a focus on the reproductive toxicant phthalates. The reviewed studies compare observed mixture data to mathematical mixture model predictions based on dose addition or response addition to determine how the individual chemicals in a mixture interact (e.g., additive, greater, or less than additive). Phthalate mixtures were observed to act in a dose additive manner based on the relative potency of the individual phthalates to suppress fetal testosterone production. Similar dose additive effects have been reported for mixtures of phthalates with antiandrogenic pesticides of differing mechanisms of action. Overall, data from these phthalate experiments in rats can be used in conjunction with human biomonitoring data to determine individual hazard indices, and recent cumulative risk assessments in humans indicate an excess risk to antiandrogenic chemical mixtures that include phthalates only or phthalates in combination with other antiandrogenic chemicals.
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Background: Exposure to bisphenol-A (BPA) and phthalates is highly prevalent. Prior studies have not assessed associations between urinary levels of BPA and phthalate metabolites and body composition. Methods: National Health and Nutrition Examination Survey (NHANES) data from 1999 to 2006 on adults aged ≥20 were analyzed by linear regression for associations between urinary BPA, monoethyl phthalate, monobutyl phthalate (MBP), monoethylhexyl phthalate (MEHP), and monobenzyl phthalate (MBzP) and lean mass, fat mass, and percent body fat. Results: BPA and phthalate metabolites were not independently associated with fat mass or percent body fat. Significant inverse associations were observed with lean mass, with the strongest association observed for BPA in men (mean lean mass 1.39 kg lower for quartile 4 vs. quartile 1, p trend = 0.02). Conclusions: BPA and some phthalates could have important, negative effects on muscle and may affect conditions related to deficits in lean mass, though additional research is needed.
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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.
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Di(2-ethylhexyl) phthalate (DEHP) is currently the most commonly used phthalate for the production of flexible polyvinyl chloride. Phthalates including DEHP have been labeled as potential endocrine disruptors The effect on the development of the neocortex, however, is unknown. To evaluate the neurodevelopmental effects of prenatal DEHP exposure at 1 and 100mg/kg/day or 100 and 500mg/kg/day in fetal and newborn mice, we performed a detailed histologic analysis of the developing dorsal telencephalon and neocortex. The observation of fetuses exposed to DEHP revealed reductions of proliferation and neurogenesis (1 and 100mg/kg) and an increase in cell death (500mg/kg). In addition, the newborns prenatally exposed to DEHP showed an abnormal neuronal distribution and a decrease in neurons. These findings suggest that prenatal DEHP exposure induces neurodevelopmental toxicity associated with the neural stem cell niche and corticogenesis.
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Adolescence is a unique period of development, marked by maturation of the prefrontal cortex (PFC), a region important for executive functioning. During this time, the human PFC decreases in overall volume and thickness. Likewise in adolescent rodents, losses of neurons, dendrites, dendritic spines and neurotransmitter receptors have been documented within the medial prefrontal cortex (mPFC), sometimes with sex and layer specificity. However, changes in the number of synapses during this time have not been examined. In the present study, we stereologically quantified the number of synaptophysin-immunoreactive boutons in the male and female rat mPFC across multiple time points from the juvenile period through adulthood (postnatal days (P) 25, 35, 45, 60 and 90). In females, there was a significant decrease in synaptophysin boutons between P35 and P45, coinciding with the onset of puberty. In males, there was no significant main effect of age on synaptophysin boutons; however, in both males and females, pubertal onset was associated with significant synaptic losses. These results suggest that puberty is a critical period for synaptic pruning within the rat mPFC, potentially contributing to maturation of adolescent executive function. This article is protected by copyright. All rights reserved.
Article
Cognitive flexibility is the ability to switch between different rules or concepts and behavioral flexibility is the overt physical manifestation of these shifts. Behavioral flexibility is essential for adaptive responses and commonly measured by reversal learning and set-shifting performance in rodents. Both tasks have demonstrated vulnerability to stress with effects dependent upon stressor type and number of repetitions. This review compares the effects of stress on reversal learning and set-shifting to provide insight into the differential effect of stress on cognition. Acute and short-term repetition of stress appears to facilitate reversal learning whereas the longer-term repetition of stress impair reversal learning. Stress facilitated intradimensional set-shifting within a single, short-term stress protocol but otherwise generally impaired set-shifting performance in acute and repeated stress paradigms. Chronic unpredictable stress impairs reversal learning and set-shifting whereas repeated cold intermittent stress selectively impairs reversal learning and has no effect on set-shifting. In considering the mechanisms underlying the effects of stress on behavioral flexibility, pharmacological manipulations performed in conjunction with stress are also reviewed. Blocking corticosterone receptors does not affect the facilitation of reversal learning following acute stress but the prevention of corticosterone synthesis rescues repeated stress-induced set-shifting impairment. Enhancing post-synaptic norepinephrine function, serotonin availability, and dopamine receptor activation rescue behavioral flexibility performance following stress. While this review highlights a lack of a standardization of stress paradigms, some consistent effects are apparent. Future studies are necessary to specify the mechanisms underlying the stress-induced impairments of behavioral flexibility, which will aid in alleviating these symptoms in patients with some psychiatric disorders.
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Newborn mice were continuously given 120 injections of [3H]thymidine at 6 h intervals for 30 days, by which all the proliferated cells in the CNS (central nervous system) until the mice became adult could be labeled, and loss of the labeled CNS cells were studied in the pulse labeling experiment after the continuous labeling. At 30 days of age, about 89% of the glial cells, 88% of the meningeal cells, 4.4% of the ependymal cells, and 3.9% of the choroid-plexus cells, were labeled, respectively. This indicates that nine-tenth of glial cells in the adult mouse CNS arise in postnatal days, whereas most of ependymal and choroid-plexus cells arise in the embryonic stage. By the pulse labeling, it was found that the labeled glial cells decreased in number; the labeled cells died off, and the surviving glial cells happened to divide by mitosis, or precursor cells for glia proliferated to cover the cell loss. A half life of the glial cells was 140 days in the corpus callosum, 160 days in the cerebral cortex, 230 days in the cerebellar molecular layer and the white matter, and 150 days in the spinal white matter, respectively. Although the glial cells were not subdivided into astrocytes and oligodendrocytes, the present study successfully revealed that both glial cell types are renewed. On the other hand, only a few labeled cells were lost in the meningeal, ependymal and choroid-plexus cells even after 250 days of the pulse labeling. These cell types are renewed much more slowly than the glial cells.
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Phthalate exposure has recently been associated with behavioral actions that are linked to its endocrine-disrupting properties. The purpose of this study was to investigate the molecular, anatomical, and behavioral effects of indirect perinatal benzyl butyl phthalate (BBP) exposure in offspring of BBP-treated pregnant dams. In two separate experiments, we administered BBP (10.0 μg/ml) on food pellets to pregnant dams and examined the offspring. The first experiment revealed reproductive anatomical abnormalities linked to BBP's endocrine-disrupting properties, whereas histological analysis revealed preserved hippocampal neuronal migration. The second experiment demonstrated learning and memory impairments accompanied by molecular abnormalities in multiple brain regions. Offspring from BBP-treated dams had altered levels of several proteins important for neuronal circuitry formation, tissue development, and maturation. We suggest that BBP administration disrupts normal tlearning and that these effects could be related to alterations in brain development and result in a phenotype similar to that observed in neurodevelopmental disorders.
Article
Emerging evidence from observational studies suggests that prenatal exposure to phthalates affects neurodevelopment in children. To conduct a systematic review of the existing literature on the association between urinary phthalate concentrations and children's neurodevelopment. We searched electronic bibliographic databases (MEDLINE, PubMed, EMBASE, PsycINFO, CINAHL, Global Health, CAB abstracts, and ERIC) (1910 to February 21st, 2014); reference lists of included articles, and conference abstracts (American Psychiatric Association, American Academy of Neurology, and Pediatric Academic Societies). Two independent reviewers screened abstracts and extracted data. We included original studies reporting on the association between prenatal or childhood urinary phthalate metabolites, and cognitive and behavioral outcomes (e.g., IQ scores, BASC-2 scores or equivalent) in children 0-12 years of age. Of 2804 abstracts screened, 11 original articles met our criteria for inclusion. A systematic review of the literature supports the contention that prenatal exposure phthalates is associated with adverse cognitive and behavioral outcomes in children, including lower IQ, and problems with attention, hyperactivity, and poorer social communication. Further research characterizing the associations between specific phthalate metabolites and children's neurodevelopmental outcomes is needed to support the development of mitigation strategies and enhance the development of appropriate health policy. Copyright © 2015 Elsevier Inc. All rights reserved.
Article
Numerous psychiatric and behavioral disorders such as Autism, Attention Deficit Disorder and Schizophrenia may involve disruptions in the development of the mesocortical dopamine pathway, consisting of dopaminergic projections from the midbrain ventral tegmental area (VTA) to the medial prefrontal cortex (mPFC). Nuclear steroid hormone receptors are powerful transcription factors and can profoundly and permanently alter fundamental processes of neural development. Nuclear progesterone receptor (PR) is transiently expressed in both the VTA and the PFC of rodents during perinatal life, suggesting that PR may regulate the normal development of this important behavioral circuit. Here, we demonstrate that virtually all PR immunoreactive (PRir) cells in the VTA also express tyrosine hydroxylase-ir (THir). In addition, retrograde tract tracing reveals that many PRir cells in the VTA project to the mPFC. Administration of a PR antagonist to rats during the neonatal period decreased THir fiber density in prelimbic mPFC of juveniles (P25) and decreased levels of THir in the VTA of adults. Neonatal treatment with a PR antagonist impaired adult performance on a passive inhibitory avoidance task and an attentional set shift task, measures of behavioral inhibition/impulsivity and cognitive flexibility, respectively. THir levels in VTA were reduced and cognitive flexibility was impaired in PR knockout mice as well. These findings provide novel insights into a potential role for PR in the developmental etiology of behavioral disorders that involve impairments in complex cognitive behaviors and have implications for the use of synthetic progestins in humans during critical neurodevelopmental periods. © 2015 S. Karger AG, Basel.
Article
Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disrupter. Currently, little is known about neurodevelopmental toxicity of DEHP in wildlife and humans. The present study investigated the effects of DEHP, focusing on the changes in the behavior of offspring mice at the ages of 6 and 12 w, respectively, following utero and lactational exposure to DEHP (10, 50, and 200 mg/kg/d) from gestation day 7 through postnatal day 21. The results of open field tasks showed that DEHP increased the grooming of males at age 6 w and females at age 12 w but decreased the frequency of rearing of 6-w-old females and the number of grid crossings of 12-w-old females. In the Morris water maze task, 50 and 200 mg/kg/d DEHP significantly prolonged the time of searching the hidden platform in water maze and reduced the time staying in the target quadrant during a probe trial of 6-w-old male mice, but not of 6-w-old females nor 12-w-old mice of both sexes, suggesting an impaired spatial learning and memory among younger males after perinatal exposure to DEHP. Western blot analyses further showed that DEHP at 50 and 200 mg/kg/d decreased the levels of the N-methyl-D-aspartic acid (NMDA) receptor subunits NR1 and NR2B in the hippocampus of 6-w-old males. These results suggest that uterine and lactational exposure to low doses of DEHP sex-specifically impacted behaviors, including locomotion activity and spatial memory, via the concomitant inhibition of the NMDA receptor of the hippocampus in offspring mice. Copyright © 2015. Published by Elsevier Inc.
Article
Phthalates are synthetically derived chemicals used as plasticizers in a variety of common household products. They are not chemically bound to plastic polymers and over time, easily migrate out of these products and into the environment. Experimental investigations evaluating the biological impact of phthalate exposure on developing organisms are critical given that estimates of phthalate exposure are considerably higher in infants and children compared to adults. Extensive growth and re-organization of neurocircuitry occurs during development leaving the brain highly susceptible to environmental insults. This review summarizes the effects of phthalate exposure on brain structure and function with particular emphasis on developmental aspects of hippocampal structural and functional plasticity. In general, it appears that widespread disruptions in hippocampal functional and structural plasticity occur following developmental (pre-, peri- and post-natal) exposure to phthalates. Whether these changes occur as a direct neurotoxic effect of phthalates or an indirect effect through disruption of endogenous endocrine functions is not fully understood. Comprehensive investigations that simultaneously assess the neurodevelopmental, neurotoxic, neuroendocrine and behavioural correlates of phthalate exposure are needed to provide an opportunity to thoroughly evaluate the neurotoxic potential of phthalates throughout the lifespan. Copyright © 2015. Published by Elsevier B.V.
Article
Background: Previous studies have implicated the relationship between environmental phthalate exposure and attention deficit hyperactivity disorder (ADHD) symptoms of childhood, but no studies have been conducted in children who have a confirmed diagnosis of ADHD obtained through meticulous diagnostic testing. We aimed to determine whether phthalate metabolites in urine would be higher in children with ADHD than in those without ADHD and would correlate with symptom severity and cortical thickness in ADHD children. Method: A cross-sectional examination of urine phthalate metabolite concentrations was performed; scores for ADHD symptoms, externalizing problems, and continuous performance tests were obtained from 180 children with ADHD, and brain-imaging data were obtained from 115 participants. For the control group, children without ADHD (N = 438) were recruited. Correlations between phthalate metabolite concentrations and clinical measures and brain cortical thickness were investigated. Results: Concentrations of phthalate metabolites, particularly the di(2-ethylhexyl) phthalate (DEHP) metabolite, were significantly higher in boys with ADHD than in boys without ADHD. Concentrations of the di-n-butyl phthalate (DBP) metabolite were significantly higher in the combined or hyperactive-impulsive subtypes compared to the inattentive subtype, and the metabolite was positively correlated with the severity of externalizing symptoms. Concentrations of the DEHP metabolite were negatively correlated with cortical thickness in the right middle and superior temporal gyri. Conclusions: The results of this study suggest an association between phthalate concentrations and both the diagnosis and symptom severity of ADHD. Imaging findings suggest a negative impact of phthalates on regional cortical maturation in children with ADHD.
Article
Introduction Acute or prolonged dietary restriction has been shown to have significant effects on learning and memory, and also on the food seeking behaviour in animals. Aim This study investigated whether or not there are sex differences in spatial learning and memory following short-term dietary restriction in the rat. Methods Two month-old male (n = 24) and female (n = 24) Sprague-Dawley rats were randomly assigned to either (1) male or female control (i.e. normal diet regimen); (2) male or female 40% dietary restriction for either 2 hours (2h-DR) or 2 weeks (2wks-DR) duration. Following the restriction paradigm (i.e. either 2 h or 2 weeks), animals were weighed, and learning and memory was assessed daily for a total of 5 days by Morris Water Maze. Results Dietary restriction for 2 h provoked high speed swimming in female rats compared to controls. However the females performance in water maze was inferior to the control animals. Two weeks after 40% DR resulted in reduction of male's body weight by 20% compared to their control group. However, both males and females showed difficulties in water maze learning and memory test after the two weeks 40% DR. The animals swam longer distance and took longer time to reach the platform when compared to their age-matched controls. Conclusion In fully developed brain, adult animal's cognitive performance is shown to be affected by acute and prolonged stress in the form of food restriction. Interestingly, the impact of this stress was different according to the sex of the animals. In female rats, dietary restriction has a negative effect on learning and memory after 2 h and 2 weeks intervals. In male rats, 2 h of DR has a positive effect on learning and memory; however this effect is not maintained and by 2 weeks there is a negative effect similar to that seen in female animals.
Article
Dibutyl-phthalate (DBP) is a ubiquitous environmental contaminant. However, its neurotoxic effects on neonatal, immature or mature brains remain unclear. Here, we aimed to investigate the neurotoxicity of perinatal exposure of DBP on rodent offspring animals. Pregnant rats received intragastric DBP (500mg/kg body weight) daily from gestational day (GD) 6 to postnatal day (PND) 21. Animals in the control group received the same volume of edible corn oil. Brain sections or tissues from offspring rats on PND5, PND21 and PND60 were collected for analysis. Histological examination demonstrated that perinatal exposure of DBP resulted in hippocampal neuron loss and structural alternation in neonatal and immature offspring rats (PND5 and PND21), while no significant change was found in mature rats (PND60). DBP exposure induced cell apoptosis in hippocampal neurons of these neonatal and immature animals, as evidenced by the increased number of TUNEL-positive and Annexin V-propidium iodide (PI) positive cells and up-regulated caspase-3 activity. Moreover, DBP exposure decreased the expression of synaptophysin in the hippocampus and reduced both the slope and amplitude of field excitatory postsynaptic potentials (fEPSPs). DBP also impaired the spatial learning and memory of offspring rats. However, no significant difference in the susceptibility to DBP-induced neurotoxicity was found between male and female offspring rats. Our findings indicated that perinatal exposure of DBP could induce neurotoxicity in neonatal and immature offspring animals, but had no influence on mature animals after DBP withdrawal. These results may provide basic experimental evidence for better understanding the neurotoxic effects of DBP on neonatal, immature and mature brains.
Article
Estrogens have many functions in the developing rodent brain, and most of these depend on the presence of estrogen receptors. Understanding how expression of these receptors are regulated is crucial for understanding the roles of estradiol in the male and female brain during development In rodents, the prefrontal cortex (PFC) has been shown to be involved in working memory, attention, and behavioral inhibition. Many studies have demonstrated an effect of estradiol on sex difference in these functions attributed to differences in the PFC. We have previously demonstrated that estrogen receptor alpha (ERα) expression decreases in the isocortex across early postnatal development. This decrease corresponds with an increase in methylation of many sites along the ERα promoter. Here we have examined both ERα and ERβ mRNA expression in the PFC to determine if methylation also plays a role in this important brain region. We investigated expression of alternate promoters for ERα and methylation of CpG sites along two of these promoters. We found that the pattern of ERα mRNA expression in PFC was similar to the pattern of ERα expression in the isocortex and that there were no sex differences in the level of expression across development. We did, however, find subtle differences in promoter expression and methylation that may indicate a sex-specific difference in PFC during development resulting in a difference in adult response.
Article
We have previously shown that males have more neurons than females in the primary visual cortex, and neonatal androgens play an important role in this difference. Also, we have found that females experience more cell death during development in this region than in males. Therefore, we hypothesized that the neonatal hormone environment directly influences the amount of cell death. In the present experiment, female rats were implanted with dihydrotestosterone (DHT) or estradiol at postnatal day 1. These animals, along with control males and females, were sacrificed on postnatal days 6, 11 and 25. Using unbiased stereology to quantify neuronal and pyknotic cell density, we observed that females implanted with DHT had a similar pattern and proportion of cells dying as control males. Additionally, developmental cell death in females implanted with estradiol was not significantly different than control females. Thus, neonatal androgens have an inhibitory effect on developmental cell death in the rat primary visual cortex.
Article
Phthalates and bisphenol A have very widespread use leading to significant exposure of humans. They are suspected to interfere with the endocrine system, including the androgen, estrogen and the thyroid hormone system. Here we analyzed the antiandrogenic activity of six binary, and one ternary mixture of phthalates exhibiting complete antiandrogenic dose-response curves, and binary mixtures of phthalates and bisphenol A at equi-effective concentrations of EC(10), EC(25) and EC(50) in MDA-kb2 cells. Mixture activity followed the concentration addition (CA) model with a tendency to synergism at high and antagonism at low concentrations. Isoboles and the toxic unit approach (TUA) confirmed the additive to synergistic activity of the binary mixtures BBP+DBP, DBP+DEP and DEP+BPA at high concentrations. Both methods indicate a tendency to antagonism for the EC(10) mixtures BBP+DBP, BBP+DEP and DBP+DEP, and the EC(25) mixture of DBP+BPA. A ternary mixture revealed synergism at the EC(50), and weak antagonistic activity at the EC(25) level by the TUA. A mixture of five phthalates representing a human urine composition and reflecting exposure to corresponding parent compounds showed no antiandrogenic activity. Our study demonstrates that CA is an appropriate concept to account for mixture effects of antiandrogenic phthalates and bisphenol A. The interaction indicates a departure from additivity to antagonism at low concentrations, probably due to interaction with the androgen receptor and/or cofactors. This study emphasizes that a risk assessment of phthalates should account for mixture effects by applying the CA concept.
Article
In recent years, many studies of thyroid-disrupting effects of environmental chemicals have been published. Of special concern is the exposure of pregnant women and infants, as thyroid disruption of the developing organism may have deleterious effects on neurological outcome. Chemicals may exert thyroid effects through a variety of mechanisms of action, and some animal experiments and in vitro studies have focused on elucidating the mode of action of specific chemical compounds. Long-term human studies on effects of environmental chemicals on thyroid related outcomes such as growth and development are still lacking. The human exposure scenario with life long exposure to a vast mixture of chemicals in low doses and the large physiological variation in thyroid hormone levels between individuals render human studies very difficult. However, there is now reasonably firm evidence that PCBs have thyroid-disrupting effects, and there is emerging evidence that also phthalates, bisphenol A, brominated flame retardants and perfluorinated chemicals may have thyroid disrupting properties.
Article
Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in plastics. Its reproductive toxicity and teratogenic effects are well known. DEHP can cause liver damage and peroxisome proliferation, as well as carcinogenesis. Animal study has shown that DEHP causes neurodegeneration in rat brain. Prenatal exposure to DEHP disrupts brain development and decreases brain weight in rats. But its mechanism of action in the brain is not clear. This study used a neuroblastoma cell line, Neuro-2a cells, to investigate the toxic effect of DEHP. The results revealed that DEHP inhibits cell proliferation, activate caspase-3, induce apoptosis in a dose and time dependent manner, and activate expression of the PPARγ and Trim17 protein. Administration of the PPARγ agonist (troglitazone) enhanced DEHP-induced Trim17 protein expression and this enhancement could be reversed by the PPARγ antagonist (GW9662). These results suggest that DEHP activates the Trim17 protein via PPARγ leading to cleavage pro-caspase-3 and apoptosis. This finding may account for the central nervous system toxicity of DEHP and implies DEHP can impair fetal brain development.
Article
The purpose of this study was to evaluate male reproductive-organ development in early postnatal male rats following neonatal exposure to di(n-butyl) phthalate (DBP) and identify a mechanism of action. Neonatal male rats were injected subcutaneously from d 5 to 14 after birth with corn oil (control) and DBP (5, 10, or 20 mg/animal). Animals were killed at postnatal day (PND) 31 and PND 42, respectively, and testes, epididymis, seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles (LABC), and Cowper's glands were weighed. In addition, the expressions of androgen receptor (AR), estrogen receptors (ERs), and steroidogenic factor-1 (SF-1) were also examined in the testes. Total body weights gains were significantly reduced at PND 29-31, but gradually recovered on PND 42. However, DBP (20 mg/animal) significantly reduced the weights of testes and accessory sex organs (seminal vesicles, LABC, and Cowper's glands), but not of the epididymis. These adverse effects persisted through puberty at PND 42. Serum testosterone levels did not show any significant changes in the control and DBP treatment groups. Histomorphological examination showed mild diffuse Leydig-cell hyperplasia in the interstitium of severely affected tubules on PND 31. Only a few multinuclear germ cells were observed. DBP (20 mg/animal) significantly decreased the expression of AR, whereas ER expression and SF-1 expression were increased in a dose-dependent manner on PND 31 in the rat testes. On PND 42, DBP (20 mg/animal) significantly inhibited ER expression in the testes, but not AR, ER, and SF-1. These results demonstrate that neonatal exposure to DBP produces permanent changes in the endocrine system and leads to abnormal male reproductive-tract development until puberty. Thus our data suggest that DBP is likely to exert its antiandrogenic actions through disruption of AR or ER expression during the early neonatal stage.
Article
Di-(2-ethylhexyl)-phthalate (DEHP) is the most widely used plasticizers in daily-life products. In this study we evaluated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, on the neurodevelopment in vitro. After neuronotypic PC12 cells were exposed to MEHP (0.25, 2.5, 25, and 250 μM), the effects of which on cell proliferation and differentiation were investigated. In undifferentiated PC12 cells, MEHP inhibited cell proliferation in a dose-dependent manner. After 24h of MEHP treatment, there was a dose-dependent G2/M cell cycle arrest as well as a sharp drop of DNA synthesis. During the process of NGF-induced differentiation of the cell line, 4 days of MEHP exposure (2.5-250 μM) increased membrane and cytoskeletal protein contents, enhanced NGF-induced neurite outgrowth, up-regulated the choline acetyl transferase (ChAT) mRNA and down-regulated tyrosine hydroxylase (TH) mRNA levels, which suggested the promoted differentiation towards the acetylcholine (ACh) phenotype at the expense of the dopamine (DA) phenotype. Take together, our results indicate that MEHP has a potential to disturb neurodevelopment by suppressing cell proliferation and promote cell differentiation in neurocytes.
Article
The purposes of this review are to (1) evaluate human and experimental evidence for adverse effects on reproduction and development in humans, produced by exposure to phthalates, and (2) identify knowledge gaps as for future studies. The widespread use of phthalates in consumer products leads to ubiquitous and constant exposure of humans to these chemicals. Phthalates were postulated to produce endocrine-disrupting effects in rodents, where fetal exposure to these compounds was found to induce developmental and reproductive toxicity. The adverse effects observed in rodent models raised concerns as to whether exposure to phthalates represents a potential health risk to humans. At present, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) have been demonstrated to produce reproductive and developmental toxicity; thus, this review focuses on these chemicals. For the general population, DEHP exposure is predominantly via food. The average concentrations of phthalates are highest in children and decrease with age. At present, DEHP exposures in the general population appear to be close to the tolerable daily intake (TDI), suggesting that at least some individuals exceed the TDI. In addition, specific high-risk groups exist with internal levels that are several orders of magnitude above average. Urinary metabolites used as biomarkers for the internal levels provide additional means to determine more specifically phthalate exposure levels in both general and high-risk populations. However, exposure data are not consistent and there are indications that secondary metabolites may be more accurate indicators of the internal exposure compared to primary metabolites. The present human toxicity data are not sufficient for evaluating the occurrence of reproductive effects following phthalate exposure in humans, based on existing relevant animal data. This is especially the case for data on female reproductive toxicity, which are scarce. Therefore, future research needs to focus on developmental and reproductive endpoints in humans. It should be noted that phthalates occur in mixtures but most toxicological information is based on single compounds. Thus, it is concluded that it is important to improve the knowledge of toxic interactions among the different chemicals and to develop measures for combined exposure to various groups of phthalates.
Article
Phthalates, such as butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), and their metabolites (mono-buyl phthalate [MBP], mono-benzyl phthalate [MBzP] and phthalic acid [PA]), are known to obstruct normal development in mammals. BBP and DBP have been reported to have estrogenic activity, while MBP and MBzP exhibit weak or no estrogenic effects. We previously showed that BBP and DBP have inhibitory roles on nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors are widely distributed and have roles in developmental processes. In this study, the effects of BBP, DBP, MBP, MBzP and PA on calcium signaling coupled to nicotinic acetylcholine receptors was investigated in bovine adrenal chromaffin cells and human neuroblastoma SH-SY5Y cells. Following an epibatidine-induced [Ca2+](c) increase, the IC(50)s of BBP, DBP, MBzP and MBP were 3.41, 5.01, 432 and 695 microM in bovine adrenal chromaffin cells and 0.28, 0.44, 58 and 116 microM in human SH-SY5Y cells, respectively. Although PA suppressed the epibatidine-induced [Ca2+](c) increase, the suppression was less than with MBP. The suppression potency of phthalates was related to their chemical structures. The suppression effects of BBP, DBP, MBP and MBzP remained similar potency under chronic treatments. This study demonstrated that MBP, MBzP and PA, the metabolites of BBP and DBP, had suppressor roles on the calcium signaling pathway coupled to nicotinic receptors.
Article
The hormonal control of cell death is currently the best-established mechanism for creating sex differences in cell number in the brain and spinal cord. For example, males have more cells than do females in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) and spinal nucleus of the bulbocavernosus (SNB), whereas females have a cell number advantage in the anteroventral periventricular nucleus (AVPV). In each case, the difference in cell number in adulthood correlates with a sex difference in the number of dying cells at some point in development. Mice with over- or under-expression of cell death genes have been used to test more directly the contribution of cell death to neural sex differences, to identify molecular mechanisms involved, and to determine the behavioural consequences of suppressing developmental cell death. Bax is a pro-death gene of the Bcl-2 family that is singularly important for apoptosis in neural development. In mice lacking bax, the number of cells in the BNSTp, SNB and AVPV are significantly increased, and sex differences in total cell number in each of these regions are eliminated. Cells rescued by bax gene deletion in the BNSTp express markers of differentiated neurones and the androgen receptor. On the other hand, sex differences in other phenotypically identified populations, such as vasopressin-expressing neurones in the BNSTp or dopaminergic neurones in AVPV, are not affected by either bax deletion or bcl-2 over-expression. Possible mechanisms by which testosterone may regulate cell death in the nervous system are discussed, as are the behavioural effects of eliminating sex differences in neuronal cell number.
Article
Plasma membranes of the hypothalamic arcuate neurons of the rat show a sexually dimorphic phenotype: the numerical density of intramembrane protein particles is greater in females. Male and female Sprague-Dawley rats, 10, 20 and 100 days old, were studied in order to determine whether sexual differentiation of the neuronal plasma membrane in the soma of arcuate neurons is associated with the establishment of sex differences in the pattern of axo-somatic synaptic contacts. Axo-somatic synapses were counted in thin sections of the arcuate nucleus and intramembrane particles were assessed in freeze-fracture replicas of the neuronal membrane. The number of synapses per length of perikaryal membrane increased from day 10 to day 20 in both sexes, reaching by 20 days values similar to those found on day 100. A sex difference in the number of synapses was observed only in 20-day-old and 100-day-old rats: neurons from females showed a greater number of presynaptic inputs than males (P less than 0.05). This sex difference was abolished by administration of testosterone propionate to 5-day-old females. Quantitative evaluation of freeze-fracture replicas of the arcuate neuronal perikarya revealed sex differences in the numerical density of intramembrane particles at all time points studied: neurons from females contained significantly more particles in their plasma membranes than neurons from males or androgenized females of the same age (P less than 0.001). These results indicate that sexual differentiation of the plasma membrane in neuronal somas precedes the establishment of sex differences in axo-somatic synapses. The results are compatible with a possible role of neuronal membranes in the sexual differentiation of synaptic connectivity.
Article
Five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (2 g/kg) given to rats on Days 2-6, 6-10, or 14-18 of lactation caused significant decreases in body weight and increases in hepatic peroxisomal enzymes palmitoyl CoA oxidase and carnitine acetyltransferase in the dams and their suckling pups. Plasma cholesterol and triglyceride levels were decreased in the lactating dams. Decreased food consumption, as indicated by pair-fed rats, accounted for the decreased body weight in the pups but not the increases in enzyme activities. To determine whether DEHP and mono(2-ethylhexyl) phthalate (MEHP) were transferred through the milk, milk and plasma were collected from lactating rats 6 hr after the third dose of DEHP. The milk contained 216 +/- 23 micrograms/ml DEHP and 25 +/- 6 micrograms/ml MEHP (mean +/- SE), while the plasma contained less than 0.5 micrograms/ml DEHP and 75 +/- 12 micrograms/ml MEHP. The high milk/plasma ratio for DEHP (greater than 200) indicates efficient extraction of DEHP from the plasma into the milk. DEHP dosing during lactation also caused a decrease in mammary gland weight and a decrease in mammary gland RNA content which reflects synthetic activity. The water content of the milk was reduced, which probably accounted for the increase in lipid in the milk. Milk lactose was decreased in DEHP-treated and pair-fed rats, consistent with the decrease in milk production. The results show that exposure to high doses of DEHP during lactation in rats can result in changes in milk quality and quantity and can lead to DEHP and MEHP exposure in the suckling rat pups.
Article
Estrogen markedly promotes the specific formation of dendritic shaft synapses in the medial amygdaloid nucleus (AMN) of the rat when given in early postnatal days. The high incidence of this type of synapse permanently persists until sacrifice at adult ages. The data provide evidence for the synaptic plasticity of the developing amygdala to estrogen and suggest a possible morphological mechanism for sexual differentiation of the brain.
Article
Mammalian reproduction depends on the coordinated expression of behavior with precisely timed physiological events that are fundamentally different in males and females. An improved understanding of the neuroanatomical relationships between sexually dimorphic parts of the forebrain has contributed to a significant paradigm shift in how functional neural systems are approached experimentally. This review focuses on the organization of interconnected limbic-hypothalamic pathways that participate in the neural control of reproduction and summarizes what is known about the developmental neurobiology of these pathways. Sex steroid hormones such as estrogen and testosterone have much in common with neurotrophins and regulate cell death, neuronal migration, neurogenesis, and neurotransmitter plasticity. In addition, these hormones direct formation of sexually dimorphic circuits by influencing axonal guidance and synaptogenesis. The signaling events underlying the developmental activities of sex steroids involve interactions between nuclear hormone receptors and other transcriptional regulators, as well as interactions at multiple levels with neurotrophin and neurotransmitter signal transduction pathways.
Article
The present study was conducted to identify the localization and possible contribution of the two estrogen receptor (ER) subtypes in the rat brain at postnatal (P) days 3, 7 and 14. Evaluation of the distribution of ERalpha and ERbeta immunoreactive (ir) nuclei did not reveal gender differences at the developmental point times examined. With the exception of the cerebral cortex, the pattern of staining for these ERs was unchanged across the postnatal ages examined. The distribution of ERalpha-ir nuclei was wider than ERbeta-ir during brain development. From P3, ERbeta and ERalpha-ir nuclei were found in different regions of the cerebral cortex, basal forebrain, amygdala, thalamus, hypothalamus, mesencephalon, pons, cerebellum and medulla oblongata. In addition, ERalpha-ir nuclei were exclusively detected in the hippocampal subfields, epithalamus and in several circumventricular organs. ERalpha and ERbeta dual immunofluorescence revealed positive nuclei in the medial part of the bed nucleus of the stria terminalis, periventricular preoptic nucleus and in caudal aspects of the ventrolateral part of the ventromedial hypothalamic nucleus. Although the functional significance of the dual expression of both ERs within the same nuclei remains unknown, it is possible that ERs play different roles in gene regulation within the same cell. The presence of ERs in diverse brain