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Maternal infections during pregnancy and the consequent maternal immune activation (MIA) are the major risk factors for autism spectrum disorder (ASD). Epidemiological evidence is corroborated by the preclinical models in which MIA leads to ASD-like behavioral abnormalities and altered neuroinflammatory profiles, with an increase in pro-inflammatory cytokines and microglial markers. In addition to neuroinflammatory response, an abnormal expression of endogenous retroviruses (ERVs) has been identified in neurodevelopmental disorders and have been found to correlate with disease severity. Our aim was to evaluate the transcriptional profile of several ERV families, ERV-related genes, and inflammatory mediators (by RT real-time PCR) in mouse offspring of both sexes, prenatally exposed to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3 that mimics viral maternal infection during pregnancy. We found that prenatal exposure to Poly I:C deregulated the expression of some ERVs and ERV-related genes both in the prefrontal cortex (PFC) and hippocampus, while no changes were detected in the blood. Interestingly, sex-related differences in the expression levels of some ERVs, ERV-related genes, and inflammatory mediators that were higher in females than in males emerged only in PFC. Our findings support the tissue specificity of ERV and ERV-related transcriptional profiles in MIA mice.

Autism Spectrum Disorder (ASD) is a sex-biased neurodevelopmental disorder with a male to female prevalence of 4:1, characterized by persistent deficits in social communication and interaction and restricted-repetitive patterns of behavior, interests or activities. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune dysregulation in ASD is further supported by evidence suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. The present study was aimed at characterizing the effects of MIA on behavior, gut microbiota and neuroinflammation in the mouse offspring also considering the impact of MIA in the two sexes. MIA offspring exhibited significant ASD-like behavioral alterations (i.e., deficits in sociability and sensorimotor gating, perseverative behaviors). The analysis of microbiota revealed changes in specific microbial taxa that recapitulated those seen in ASD children. In addition, molecular analyses indicated sex-related differences in the neuroinflammatory responses triggered by MIA, with a more prominent effect in the cerebellum. Our data suggest that both sexes should be included in the experimental designs of preclinical studies in order to identify those mechanisms that confer different vulnerability to ASD to males and females.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

Early life low-level lead (Pb) exposure is still an alarming child health issue. To date, animal studies investigating the effects of low doses of Pb since early stages of life to adulthood are scarce. We investigated in a mouse model the behavioral effects of developmental exposure to low-level Pb yielding blood levels similar to those observed in child clinical literature. CD1 outbred mouse dams received Pb (25- or 100-ppm) via drinking water from two weeks pre-mating until the end of lactation. Offspring of both sexes underwent a longitudinal assessment of motor, socio-emotional, and cognitive endpoints from neonatal to adult stage. Pb levels were determined in several matrices (blood, brain and bone) up to six months after the end of exposure. We found that new born pups exposed to Pb have slightly altered motor patterns and reduced preference for the nest odor. Offspring of both sexes exposed to the lowest Pb dose showed diminished interest for social novelty stimuli as adults. Moreover, sex-dependent effects of Pb exposure were observed in the spatial learning and memory task, where males were selectively impaired. Finally, blood, brain and bone Pb levels were elevated in a dose dependent fashion up to six months after termination of exposure. We observed marked accumulation of Pb in bones, with higher Pb levels in 100-ppm exposed females than in males at 7 months of age. In conclusion, developmental Pb exposure caused mild alterations in early- and late-life behavioral domains, particularly involving olfactory and cognitive responses. These findings confirm the importance of animal models to understand how early chronic low-level lead exposure impacts on health in a life-course perspective.

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content—i.e., optimal, sub-optimal, and deficient—and neurodevelopmental, neuroinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in sub-optimal offspring. In addition, sub-optimal, more than deficient supply, reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, deficient and sub-optimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabolism and homeostasis. The finding that the Se sub-optimal was more detrimental than Se deficient diet may suggest that maternal Se deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by sub-optimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development.

Background The unwillingness to share contacts is one of the least explored aspects of the COVID-19 pandemic. Here we report the factors associated with resistance to collaborate on contact tracing, based on the results of a nation-wide survey conducted in Italy in January-March 2021. Methods and findings The repeated cross-sectional on-line survey was conducted among 7,513 respondents (mean age 45.7, 50.4% women) selected to represent the Italian adult population 18–70 years old. Two groups were defined based on the direct question response expressing (1) unwillingness or (2) willingness to share the names of individuals with whom respondents had contact. We selected 70% of participants (training data set) to produce several multivariable binomial generalized linear models and estimated the proportion of variation explained by the model by McFadden R2, and the model’s discriminatory ability by the index of concordance. Then, we have validated the regression models using the remaining 30% of respondents (testing data set), and identified the best performing model by removing the variables based on their impact on the Akaike information criterion and then evaluating the model predictive accuracy. We also performed a sensitivity analysis using principal component analysis. Overall, 5.5% of the respondents indicated that in case of positive SARS-CoV-2 test they would not share contacts. Of note, this percentage varied from 0.8% to 46.5% depending on the answers to other survey questions. From the 139 questions included in the multivariable analysis, the initial model proposed 20 independent factors that were reduced to the 6 factors with only modest changes in the model performance. The 6-variables model demonstrated good performance in the training (c-index 0.85 and McFadden R2 criteria 0.25) and in the testing data set (93.3% accuracy, AUC 0.78, sensitivity 30.4% and specificity 97.4%). The most influential factors related to unwillingness to share contacts were the lack of intention to perform the test in case of contact with a COVID-19 positive individual (OR 5.60, 95% CI 4.14 to 7.58, in a fully adjusted multivariable analysis), disagreement that the government should be allowed to force people into self-isolation (OR 1.79, 95% CI 1.12 to 2.84), disagreement with the national vaccination schedule (OR 2.63, 95% CI 1.86 to 3.69), not following to the preventive anti-COVID measures (OR 3.23, 95% CI 1.85 to 5.59), the absence of people in the immediate social environment who have been infected with COVID-19 (1.66, 95% CI 1.24 to 2.21), as well as difficulties in finding or understanding the information about the infection or related recommendations. A limitation of this study is the under-representation of persons not participating in internet-based surveys and some vulnerable groups like homeless people, persons with disabilities or migrants. Conclusions Our analysis revealed several groups that expressed unwillingness to collaborate on contact tracing. The identified patterns may play a principal role not only in the COVID-19 epidemic but also be important for possible future public health threats, and appropriate interventions for their correction should be developed and ready for the implementation.

Despite the actual availability of COVID-19 vaccines to combat the pandemic, many people are still vacillating in their decision to vaccinate. In this study, we considered the effect of two relevant contextual issues on vaccination intention: the number of people infected with COVID-19 is increasing, and the pace of vaccination is gaining speed. Specifically, we hypothesized that having already contracted SARS-CoV-2 (post-positive reluctance) could lead people to underestimate the importance of vaccination. Moreover, as the number of vaccinated people increases, more hesitant people could fall into the free-riding intention category, benefitting from the immunity provided by others' vaccinations. Vaccine hesitancy becomes more critical as the vaccination campaign proceeds: at one point, it will be inevitable to deal with hesitant people. This study is part of a WHO Regional Office for Europe project and involved a representative sample of 5006 Italians interviewed in January-February 2021. In case of post-positive reluctance, both young age and female gender increase vaccine hesitancy, while a high level of education reduces free-riding intention. Considering post-positive reluctance and free riding, a protective effect on hesitancy is associated with negative affective states, adherence to protective behaviors, trust in health information sources, and resilience. In contrast, increased vaccine hesitancy is associated with a high level of conspiracy-mindedness and trust in media information sources. Recognizing and studying the post-positive reluctance and the phenomenon of free-riding people can help us to become more efficient in combatting the virus.

Exposure to environmental factors, such as neurotoxic metals and micronutrients, during critical periods of development can contribute to long-term consequences in offspring's health, including neurodevelopmental outcomes. The aim of this study was to evaluate the association between simultaneous prenatal exposure to metals [lead (Pb), cadmium (Cd), mercury (Hg)] and micronutrients [selenium (Se), zinc (Zn), copper (Cu)] and neurodevelopmental outcomes in school-age children from the Polish Mother and Child Cohort (REPRO_PL). Metals and micronutrients concentrations were measured in cord blood (Pb, Cd, Se, Zn, Cu) and in maternal hair (Hg) collected during the 3rd trimester of pregnancy. Behavioral and emotional problems, as well as children's cognitive and psychomotor development, were assessed in 436 school-age children using the Strengths and Difficulties Questionnaire (SDQ, filled in by the mothers) and the Polish adaptation of the Intelligence and Development Scales (IDS, administered by trained psychologists). Multivariate regression models were applied after imputation of missing values, using two approaches: (i) a joint analysis taking into account all metals and micronutrients simultaneously, and (ii) an ExWAS study (single-exposure model). In the SDQ, Hyperactivity/Inattention problems and Total difficulties were associated with higher Hg concentrations in maternal hair (0.18, 95% CI: 0.05; 0.3; and 0.14, 95% CI: 0.01; 0.3, respectively), whereas Emotional symptoms were inversely associated with Se and Zn levels in cord blood (−0.13, 95% CI: −0.3; 0.004; and −0.10, 95% CI: −0.2; 0.02, respectively). In the IDS, cord blood Pb levels were found to be negatively associated with Fluid and Crystallized IQ (−0.12, 95% CI: −0.3; 0.02; and −0.14, 95% CI: −0.3; 0.007, respectively) as well as Mathematical skills (−0.15, 95% CI: −0.3; 0.01). The current research has been able to simultaneously assess the exposure to various interacting chemicals during the prenatal period. We demonstrate that prenatal co-exposures to Pb, Hg, Zn and Se have long-term influences on the neuropsychological outcome of school-age children.

Quarantine, loss of routine and social support can negatively impact mothers who have just given birth and their babies, generating concerns and reactions of intense fear. Following the COVID-19 emergency, we described a structured program for screening and treatment of perinatal depression and anxiety as a medium for constant monitoring of perinatal risk factors and early screening, which can also be implemented in emergencies with remote intervention methods, to offer women an appropriate, timely and effective treatment. In this scenario, it is desirable that the monitoring of the psychological well-being of women in postpartum is maintained over time, with the participation of all the professional figures with whom the woman comes into contact, to intercept any forms of psychological distress related to the epidemic and that could occur even after some time.

The study objective was to identify determinants of essential elements and vitamins intake, and microelements and vitamins concentration in blood among pregnant women from Poland. Based on the data from food frequency questionnaires and information about supplements taken (n = 1252), daily supply of six elements (calcium, magnesium, iron, zinc, copper, selenium) and nine vitamins (folate, vitamins A, E, C, B1, B2, B3, B6, B12) was calculated. Zinc, copper, selenium (n = 340), vitamin A and E (n = 358) concentration was determined in blood collected during pregnancy. Most of the women did not meet the demand for essential elements and vitamins with a diet. About 94% of the respondents declared supplements use. The women with higher education, indicating leisure-time, physical activity and multiparity had a higher chance of meeting the average demand for the majority of the analyzed nutrients. On the other hand, factors such as BMI &lt, 18.5kg/m2, a higher level of stress, and late first medical-care visit were associated with a lower chance of meeting the recommendations. Higher socio-economic status was a determinant of a higher selenium concentration in plasma (β = 3.1, 95%CI: 0.2–5.9), whereas BMI ≥ 25 kg/m2, and multiparity of a higher copper concentration in plasma (β = 0.2, 95%CI: 0.03-0.4, β = 0.2, 95%CI: 0.1–0.4). Higher plasma concentration of vitamin E was noted among women older than 30 years of age comparing to those who were 30 or younger (β = 1.5, 95%CI: 0.6–2.4). Although more studies are required, especially such based on laboratory measures, our results indicate target groups for dietary interventions during pregnancy for children’s optimal health and development.

Perinatal hypoxia causes long-term neurobiological consequences, including alterations in mechanisms of activity-dependent synaptic plasticity and cognitive dysfunction. Changes in neurotransmitter receptors have been associated with these alterations, but little is known on how early hypoxia influences the expression and function of metabotropic glutamate (mGlu) receptors in adult life. This is an important issue because mGlu receptors are implicated in mechanisms of synaptic plasticity. Here, we examined the expression of mGlu1, mGlu5, and mGlu2/3 receptor subtypes in the hippocampus, nucleus accumbens, prefrontal cortex, and dorsal striatum in 6-month old Wistar rats (a) born by vaginal delivery; (b) born by caesarean section; and (c) born by caesarean section followed by 20 min of asphyxia. Unexpectedly, we found a large reduction of mGlu1α protein levels in the hippocampus of rats born by caesarean section regardless of the presence of asphyxia. No changes in mGlu1α receptor protein levels were found in the other brain regions. Levels of mGlu5 and mGlu2/3 receptors and levels of GluA2/3 and GluN1 subunits of AMPA and NMDA receptors did not differ among the three groups of rats in any brain region. These results are consistent with previous findings showing that changes in mGlu1 receptors occur within the epigenetic programming caused by early-life events.

Lead (Pb) exposure in early life affects brain development resulting in cognitive and behavioral deficits. Epidemiologic and experimental evidence of sex as an effect modifier of developmental Pb exposure is emerging. In the present study, we investigated Pb effects on behavior and mechanisms of neuroplasticity in the hippocampus and potential sex differences. To this aim, dams were exposed, from one month pre-mating to offspring weaning, to Pb via drinking water at 5 mg/kg body weight per day. In the offspring of both sexes, the longitudinal assessment of motor, emotional, and cognitive end points was performed. We also evaluated the expression and synaptic distribution of N-methyl-D-Aspartate receptor (NMDA) and &alpha, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits at post-natal day (pnd) 23 and 70 in the hippocampus. Neonatal motor patterns and explorative behavior in offspring were affected in both sexes. Pb effects in emotional response and memory retention were observed in adult females only, preceded by increased levels of GluN2A and GluA1 subunits at the post-synapse at pnd 23. These data suggest that Pb exposure during development affects glutamatergic receptors distribution at the post-synaptic spine in females. These effects may contribute to alterations in selected behavioral domains.

Background:The extent to which prenatal low-level mercury (Hg) exposure through maternal fish intake and heavy metals exposure affect children’s neurodevelopment is controversial and may appear in the long term. In 2007, a prospective cohort, the Northern Adriatic Cohort II (NAC-II), was established to investigate the association between prenatal Hg exposure from maternal fish consumption and child neurodevelopment. The study enrolled 900 pregnant women, and 632 and 470 children underwent neurodevelopmental evaluation at 18 and 40 months of age, respectively. The NAC-II cohort is a part of the Mediterranean cohort in the “Public health impact of long-term, low-level, mixed element exposure in susceptible population strata” project. Methods:This protocol describes the follow-up assessment of the effects of prenatal low level Hg and other heavy metals exposure on the developing nervous system of the children born within the NAC-II who reached the age of 7 years. Child diet components are estimated through a Diet Diary. Child hair and urine are collected for determination of Hg level. In addition, levels of other potentially neurotoxic metals, namely Manganese, Cadmium, Lead, Arsenic, and Selenium, are also measured in the same matrices. Discussion:This protocol extends to the first years of schooling age the evaluation of the neurotoxicant effect of Mercury and of the other heavy metals on children’s neurodevelopment, adjusting for the potential confounders, such as the lifestyles and social economic status of children’s families. Longitudinal analysis of neurodevelopment, assessed in different ages (18 months, 40 months, and 7 years), are performed.

Prenatal treatment with the antiepileptic drug valproic acid (VPA) is associated with a significant risk of somatic anomalies, neurodevelopmental delays, and 7-10× increase in the incidence of autism spectrum disorders (ASD) in children. Rodents exposed to VPA in pregnancy show birth defects, deficits in neurodevelopment, and cognitive/social anomalies resembling those of ASD children. Mechanisms of VPA neurobehavioral toxicity are still unclear but as VPA is a non-selective inhibitor of histone deacetylases, epigenetic modifications are likely involved. This study was aimed to evaluate the transgenerational impact of prenatal VPA exposure on mouse early behavioral development, studying F1, F2, and F3 generations after VPA challenge on gestational day (GD) 10.5. We also analyzed in brain and in peripheral blood mononuclear cells the expression levels of different endogenous retrovirus (ERV) families, potential biomarkers of derailed brain development, since human ERVs have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs) such as ASD. Somatic effects of VPA were evident only in F1 generation and more markedly in the female sex. Across F1 and F2 generations, VPA delayed righting reflex, increased motor activity, and reduced ultrasonic vocalizations. The behavioral changes in F3 are milder though in the same direction. VPA increased expression of most ERVs across the three generations in brain and blood. In utero VPA induced neurodevelopmental alterations more marked in the maternal lineage that persisted also in F3, suggesting ERVs as possible downstream effectors of the VPA epigenetic alterations.

Prenatal viral/bacterial infections are considered risk factors for autism spectrum disorders (ASD) and rodent models of maternal immune activation (MIA) have been developed and extensively used in preclinical studies. Poly inosinic-cytidylic acid (Poly I:C) was injected in C57BL6/J dams to mimic a viral infection on gestational day 12.5; the experimental design includes 10/12 litters in each treatment group and data were analysed always considering the litter-effect; neonatal (spontaneous motor behaviour and ultrasonic vocalizations) and adult [open field, marble burying, social approach, fear conditioning, prepulse inhibition (PPI)] offspring of both sexes were tested. In vivo magnetic resonance imaging/spectroscopy (MRI-MRS) and high-performance liquid chromatography (HPLC) to quantify both aminoacid and/or neurotransmitter concentration in cortical and striatal regions were also carried out. In both sexes high levels of repetitive motor responses and sensory gating deficits in PPI were the more striking effects of Poly I:C, whereas no alteration of social responses were evidenced. Poly I:C treatment did not affect mean values, but, intriguingly, increased variability in the levels of four aminoacids (aspartate glycine and GABA) selectively in males. As a whole prenatal Poly I:C induced relevant long-term alterations in explorative-stereotyped motor responses and in sensory gating, sparing cognitive and social competences. When systematically assessing differences between male and female siblings within each litter, no significant sex differences were evident except for increased variability of four aminoacid levels in male brains. As a whole, prenatal Poly I:C paradigms appear to be a useful tool to investigate the profound and translationally-relevant effects of developmental immune activation on brain and behavioural development, not necessarily recapitulating the full ASD symptomatology.

Human endogenous retroviruses (HERVs) are genetic elements resulting from relics of ancestral infection of germline cells, now recognized as cofactors in the etiology of several complex diseases. Here we present a review of findings supporting the role of the abnormal HERVs activity in neurodevelopmental disorders. The derailment of brain development underlies numerous neuropsychiatric conditions, likely starting during prenatal life and carrying on during subsequent maturation of the brain. Autism spectrum disorders, attention deficit hyperactivity disorders, and schizophrenia are neurodevelopmental disorders that arise clinically during early childhood or adolescence, currently attributed to the interplay among genetic vulnerability, environmental risk factors, and maternal immune activation. The role of HERVs in human embryogenesis, their intrinsic responsiveness to external stimuli, and the interaction with the immune system support the involvement of HERVs in the derailed neurodevelopmental process. Although definitive proofs that HERVs are involved in neurobehavioral alterations are still lacking, both preclinical models and human studies indicate that the abnormal expression of ERVs could represent a neurodevelopmental disorders-associated biological trait in affected individuals and their parents.

The first Italian human biomonitoring survey (PROBE - PROgramme for Biomonitoring general population Exposure) considered a reference population of adolescents, aged 13-15 years, living in urban and rural areas and investigated their exposure to metals. The study was expanded up to 453 adolescents living in the same areas of Latium Region (Italy) and blood samples were analyzed for 19 metals (As, Be, Cd, Co, Cr, Hg, Ir, Mn, Mo, Ni, Pb, Pd, Pt, Rh, Sb, Sn, Tl, V, and W) by sector field inductively coupled plasma mass spectrometry. The exposure assessment was contextualized following an exposome approach that considered several determinants related to the subjects, available environmental parameters and geo-coding of residence address. To assess the influence of exposure determinants and modifiers on children biomarkers levels we used two independent methodologies. The first makes use of the so-called Environment-Wide Association Study (EWAS) methodology while the second was based on the application of a Generalized Liner Model (GLM) capturing co-exposures to pairs of key determinants. Based on our analysis, Hg and As were positively associated with dietary pathways (primarily linked to fish and to a lesser extent to milk consumption) while Cr showed a more complex interaction between co-exposure to different dietary pathways (milk and fish) coupled to proximity of residence to industrial activities. In addition to diet, socio-economic status of the mother revealed robust statistical associations with Cd, Ni and W biomonitoring levels in the respective children.

BTBR is an inbred mouse strain that displays several behavioral alterations resembling the core symptoms of Autism Spectrum Disorder, including deficit in sociability. In the present study, we investigated whether the pup-induced maternal behavior in virgin female mice, a naturally rewarding behavior, is impaired in this strain similarly to social interaction with adult conspecifics. We firstly assessed the maternal responsiveness towards newly born pups expressed by either virgin female mice of the BTBR strain or of the normo-social B6 strain. Next, we examined in both strains the expression of c-Fos as a marker of neuronal activity in selected brain areas involved in the regulation of maternal behavior in rodents including the olfactory bulb, the medial preoptic area and the paraventricular nucleus (PVN). We also examined the effects of pup presentation on oxytocinergic neurons of the PVN, the major brain site of synthesis of oxytocin, which has a pivotal role in facilitation of maternal response and social responsiveness in general. As a final step, we assessed the c-Fos expression pattern comparing the effect of exposure to pups with that induced by exposure to another social stimulus, focusing on other areas implicated in maternal responsiveness as well as in the affective component of social behavior such as pyriform cortex and central and basolateral amygdala. Our data showed that BTBR virgin females are less responsive to presentation of pups in comparison to B6, in parallel with lower activation of brain areas implicated in the maternal and social responsiveness.

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.

Characterization of the exposome, the totality of all environmental factors that one is exposed to from conception onwards, has been recommended to better evaluate the role of environmental influences on developmental programming and life- course vulnerability to major chronic diseases. In the framework of the Health and Environment- wide Associations based on Large population Surveys (HEALS) project we considered the pregnancy exposome exploiting two databases (PRIME and REPRO PL) that include birth cohorts from three EU countries (Croatia, Slovenia and Poland). The databases contained information on several chemical exposures, socio-demographic, lifestyle and health related factors from conception to child birth, and neuropsychological scores assessed by the Bayley Scales of Infant and Toddler Development in the first two years of life. Our main goal was to assess consistency of environmental influences on neurodevelopment, if any, across European countries differing for geographical, socio-demographic characteristics and levels of chemical exposures to metals such as lead (Pb), mercury (Hg), cadmium (Cd) and trace elements, including micronutrients such as zinc (Zn) and selenium (Se). To this aim, we first selected variables common to the different databases, then applied univariate and multivariate regression analyses to identify factors linked to neurodevelopment, and finally performed meta-analysis to detect potential heterogeneity among cohorts and pooled estimates. Significant differences in exposure levels among the three sub-cohorts were observed as for Hg and Se ; exposure levels under study were relatively low and within the range described in existing EU biomonitoring studies. The univariate analyses did not show any common pattern of association as only in the Polish cohort chemical exposure had an impact on neuropsychological outcome. In the meta-analysis, some consistent trends were evident, relative to the adverse influence of Pb on children's language and cognition and the positive influence of Se on language abilities. The effects of the neurotoxic metal Hg positively influenced the motor scores in the Polish cohorts, while it decreased the motor scores in the Slovenia and Croatian sub-cohorts. The only socio-demographic factor consistently associated to the outcome among cohorts was child's sex, with females performing better than males on cognitive and language scores. These findings point to the need of harmonizing existing cohorts or creating prospective study designs that facilitate comparisons in the exposome over time, places and kind of environmental exposures.

Exposures to toxic elements or deficiencies of essential elements during pregnancy may be associated to various birth complications or even diseases in early life. The aim of this paper was to assess the concentrations of selected toxic (As, Cd, Cr, Hg, Ni, Pb) and essential trace elements (Co, Cu, Mn, Se and Zn) in blood and urine samples of delivering women at different periods of gestation and cord blood, as well as to evaluate the placental permeability for these elements. A total of 53 women participating in the HEALS-EXHES study were enrolled. In particular, 48 blood samples from 1st trimester of pregnancy, 40 blood samples at delivery, and 31 cord blood at delivery were collected. Moreover, mothers' urine were sampled at the 1st (53 samples), 2nd (53 samples) and 3rd trimester (49 samples) of pregnancy. Results showed that Hg and Mn levels in cord blood were about 2.0 times higher than in maternal blood, suggesting that these elements may be transferred from mother to fetus. The cord blood levels of As and Pb were lower (ca. the 65%) than those in maternal blood, showing that the placenta modulates the rate of transfer for these elements. Essential elements as Cu and Zn showed significantly lower levels in cord than in maternal blood suggesting that the transplacental transfer of these nutrients was very limited. In addition, correlation between paired maternal and cord blood samples for As, Hg and Pb was statistically significant indicating that the fetal body burden may reflect the maternal exposure. Cadmium, Co, Cr, Ni and Se levels did not show significant correlations between maternal and cord blood. Maternal urinary concentrations of trace elements, including As, Cr, Cu, Hg, Se and Zn decreased along pregnancy, which may cause variations in fetal exposure. The levels of toxic and essential elements in maternal blood and urine, as well as in cord blood, were for most elements at the lower end of the ranges found in the scientific literature not being of special concern for pregnant women and the unborn.

Background: Characterization of dietary patterns represents a valid and meaningful measure of overall diet quality and nutrient intake. The study aims at evaluating the sociodemographic, lifestyle, environmental, and pregnancy-related determinants of maternal dietary patterns during pregnancy. Methods: The analysis was conducted on a group of pregnant women from the Polish Mother and Child Cohort (REPRO_PL). During the second trimester of pregnancy, 1306 women filled in a modified version of the validated food frequency questionnaire (FFQ). Dietary patterns were estimated using an exploratory factor analysis. Potential dietary determinants were assessed via a questionnaire or biomarker measurements (saliva cotinine level). Results: Two dietary patterns were identified. The Prudent dietary pattern was characterized by high consumption of fruit, vegetables, legumes, whole grains, poultry, and low-fat dairy products, while the Western dietary pattern included high intake of refined grains, processed meat, potatoes, and very low intake of whole grains. Clear sociodemographic, environmental, lifestyle, and pregnancy-related determinants of diet quality were established. Older age (&beta, = 0.2, p &lt, 0.01), higher educational level (&beta, = 0.3, 0.01), and socioeconomic status (SES) (&beta, 0.01), overweight/obesity before (&beta, 0.01), as well as physical activity during pregnancy (&beta, p = 0.01) were positive determinants of a healthier diet (increasing Prudent&ndash, Western difference in dietary pattern scores). On the other hand, parity (&beta, = &minus, 0.2, p = 0.04) and living in small cities (&beta, 0.3, p = 0.02) were significantly related to a rather Western dietary pattern. Conclusions: The current study presents evidence on specific factors influencing dietary patterns. They need to be accounted for in educational programs and interventions focused on healthy diet recommendations during pregnancy.

Impairments in social interaction and verbal and non verbal communication are among the main features of Autism Spectrum Disorder (ASD). The causes of ASD are still unknown but the research efforts of the last decade have identified a number of factors (rare gene mutations, gene variations and adverse environmental events) that, interacting in complex ways, affect early brain development. The clinical evidence that prenatal exposure to the antiepileptic drug valproate (VPA) is associated with increased risk of neurodevelopmental delay, cognitive deficits and autism in children, has drawn the attention of scientists on VPA as a tool to unravel the environment contribution to ASD risk in children. In agreement with the clinical evidence, rodents prenatally exposed to VPA display behavioral anomalies resembling ASD symptoms. The mechanisms by which administration of VPA in pregnancy increases the risk of autism are still far to be clear as are still undetermined the specific targets of VPA in the developing brain both in humans and rodents. However, the robustness of the behavioral alterations, mainly in the social domain, and the neural/molecular changes revealed so far support the VPA model as a reliable instrument to investigate the neural underpinnings of social impairment. Here we provide an update of preclinical studies on prenatal exposure to VPA in rodents with a focus on the social and communication deficits induced by VPA, discussing potential pitfalls and future directions in this research field and corroborating the potential of the VPA model to identify new pharmacological targets for ASD. This article is part of the Special Issue entitled ‘The neuropharmacology of social behavior: from bench to bedside’.

Urinary metabolites of organophosphate (OP) and pyrethroid (PYR) pesticides from seven years old children of a birth cohort study (n=199; PHIME cohort of Trieste, Italy) have been measured. Six OP and two PYR metabolites have been investigated, 2-diethylamino-6-methylpyrimidin-4-ol (DEAMPY, pirimiphos metabolite) was the one found at higher concentrations, median 3.4 ng/mL specific gravity adjusted (SG adjusted), followed by 4-nitrophenol (PNP, median 1.4 ng/mL SG adjusted) and 3,5,6-trichloro-2-pyridinol (TCPY, median 0.36 ng/mL SG adjusted), parathion and chlorpyriphos metabolites, respectively. TCPY concentrations were low in comparison to other distributions of OP metabolites in children from other studies. Accordingly, the PHIME cohort showed a distinct OP metabolite distribution with high concentrations of pirimiphos and parathion. Another specific characteristic of this cohort was the high concentration of 3-phenoxybenzoic acid (3-BPA, median 0.36 ng/mL SG adjusted), a general metabolite of PYR pesticides. Evaluation of anthropometric and socio-demographic characteristics of children and families only showed a positive association between family educational level and urinary concentrations of DEAMPY metabolite (p, Authors are grateful to the women who participated in our study as well as the people who helped in the recruitment in the different countries. This work was supported by the EU through its Sixth Framework Programme for RTD project PHIME (contract no FOOD-CT-2006- 016253), by GRANT LIFE 2012 ENV/GR/001040 "Crome Life - Cross Mediterranean Environment and Health Network”, by European Union projects HEALS (FP7-ENV-2013- 603946) and Neurosome (H2020-MSCA-ITN-2017 SEP-210411486), by RC 12/12 of Institute for Maternal and Child Health -IRCCS “Burlo Garofolo” – Trieste – Italy funded by the Italian Ministry of Health.

The impact of exposure to lead on child neurodevelopment has been well established. However, sex differences in vulnerability are still not fully explained. We aimed at evaluating the effect of a low-level lead exposure, as measured between 20 to 24 weeks of pregnancy and in cord blood, on developmental scores up to 24 months of age in 402 children from the Polish Mother and Child Cohort (REPRO_PL). Additionally, sex-dependent susceptibility to lead at this very early stage of psychomotor development was assessed. The blood lead levels were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). In order to estimate the children&rsquo, s neurodevelopment, the Bayley Scales of Infant and Toddler Development was applied. The geometric mean (GM) for blood lead level during 20&ndash, 24 weeks of pregnancy was 0.99 &plusmn, 0.15 &micro, g/dL and, in the cord blood, it was 0.96 &plusmn, 0.16 &micro, g/dL. There was no statistically significant impact of lead exposure during prenatal period on the girls&rsquo, psychomotor abilities. Among the boys, we observed lower scores for cognitive functions, along with increasing cord blood lead levels (&beta, = &minus, 2.07, p = 0.04), whereas the results for the language and motor abilities were not statistically significant (p &gt, 0.05). Our findings show that fetal exposure to very low lead levels might affect early cognitive domain, with boys being more susceptible than girls. Education on health, higher public awareness, as well as intervention programs, along with relevant regulations, are still needed to reduce risks for the vulnerable population subgroups.

BACKGROUND: The studies on the impact of selenium (Se) levels in different pregnancy periods on child psychomotor functions are limited. The aim of this study was to evaluate the impact of prenatal Se on child neurodevelopment. METHODS: The study population consisted of 410 mother-child pairs from Polish Mother and Child Cohort. Se levels were measured in each trimester of pregnancy, at delivery, and in cord blood by graphite furnace atomic absorption spectrometry. Psychomotor development was assessed in children at the age of 1 and 2 y using the Bayley Scales of Infant and Toddler Development. RESULTS: Plasma Se levels decreased through pregnancy (from 48.3 ± 10.6 µg/l in the first trimester to 38.4 ± 11.8 µg/l at delivery; P < 0.05). A statistically significant positive association between Se levels in the first trimester of pregnancy and motor development (β = 0.2, P = 0.002) at 1 y of age, and language development (β = 0.2, P = 0.03) at 2 y of age was observed. The positive effect of Se levels on cognitive score at 2 y of age was of borderline significance (β = 0.2, P = 0.05). CONCLUSION: Prenatal selenium status was associated with child psychomotor abilities within the first years of life. Further epidemiological and preclinical studies are needed to confirm the association and elucidate the underlying mechanisms of these effects.

Maternal exposure to toxic and essential trace elements represents a surrogate of exposure to the unborn child. Variables of exposure as sociodemographic, lifestyles and diet may contribute to different exposure of pregnant women to specific trace elements. Blood, urine and cord blood samples of 53 pregnant women of the HEALS-EXHES cohort, recruited in Reus (Catalonia, Spain) between 2016 and 2017, were analysed for the concentrations of As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se and Zn. Univariate and multivariate models were built in order to assess associations between element concentrations in each matrix, and variables obtained by questionnaires on mothers' characteristics and dietary habits. Results showed several significant associations between various variables and essential trace and toxic elements. Age was associated with higher levels of Cd and Pb in cord blood samples. Multiparous women showed lower levels of Cd in maternal blood and Pb in both maternal and cord blood than nulliparous women. Hispanic mothers presented higher levels of blood As and lower levels of blood Se compared to mothers of different ethnicity. Higher education level was associated with higher As and Hg concentrations in both maternal and cord blood samples. Higher annual income diminished the level of Pb in maternal blood. Smoking in pregnancy incremented the levels of Cd in mothers' blood. Alcohol consumption may affect the absorption of Cu, Mn and Zn. Supplementations with multivitamins, folic acid and iron showed effects on elements as Cr, Mn, Se and Zn. Regarding food group intake, bluefish incremented Pb levels, while canned fish and seafood affected levels of some elements as As, Hg, Cu and Se. Other elements such as Mn and Pb were influenced by the intake of different kinds of foods. The present results showed that some modifiable lifestyles and food intakes could be the target of interventions to help pregnant women to maintain suitable concentrations of essential elements and lower levels of toxic ones, and to improve consequently neonatal health outcomes.

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.

Studies on the impact of micronutrient levels during different pregnancy periods on child psychomotor functions are limited. The aim of this study was to evaluate the association between maternal plasma concentrations of selected micronutrients, such as: copper (Cu), zinc (Zn), selenium (Se), and child neuropsychological development. The study population consisted of 539 mother-child pairs from Polish Mother and Child Cohort (REPRO_PL). The micronutrient levels were measured in each trimester of pregnancy, at delivery and in the cord blood. Psychomotor development was assessed in children at the age of 1 and 2 years using the Bayley Scales of Infant and Toddler Development. The mean plasma Zn, Cu and Se concentrations in the 1st trimester of pregnancy were 0.91±0.27mg/l, 1.98±0.57mg/l and 48.35±10.54μg/l, respectively. There were no statistically significant associations between Cu levels and any of the analyzed domains of child development. A positive association was observed between Se level in the 1st trimester of pregnancy and child language and motor skills (β=0.18, p=0.03 and β=0.25, p=0.005, respectively) at one year of age. Motor score among one-year-old children decreased along with increasing Zn levels in the 1st trimester of pregnancy and in the cord blood (β=-12.07, p=0.003 and β=-6.51, p=0.03, respectively). A similar pattern was observed for the association between Zn level in the 1st trimester of pregnancy and language abilities at one year of age (β=-7.37, p=0.05). Prenatal Zn and Se status was associated with lower and higher child psychomotor abilities, respectively, within the first year of life. Further epidemiological and preclinical studies are necessary to confirm the associations between micronutrient levels and child development as well as to elucidate the underlying mechanisms of their effects.

The developing fetus is especially vulnerable to environmental toxicants, including tobacco constituents. The aim of this study was to assess the impact of environmental tobacco smoke (ETS) exposure during pregnancy on child neurodevelopment within the first two years of life. The study population consisted of 461 non-smoking pregnant women (saliva cotinine level

The complexity of the neuroendocrine level of investigation requires the assessment of behavioral patterns that extend beyond the reproductive functions, which are age- and sex-specific in rodents, described by defined clusters of behavioral items regulated by genetic, hormonal, and epigenetic factors. The study of social behavior in laboratory rodents reveals sex-dimorphic effects of environmental chemicals that may be undetected either by a traditional neurotoxicological approach or referring to the classical definition of endocrine disrupting chemicals. Here we review data on the neurobehavioral effects of developmental exposure to the non-persistent organophosphorus insecticide chlorpyrifos, whose neurotoxic activity at low doses is currently a matter of concern for children's health. In mice exposed to chlorpyrifos in utero and/or in early development social/emotional responses are differently affected in the two sexes in parallel with sex-dependent interference on hypothalamic neuroendocrine pathways regulating social behaviors (vasopressin, oxytocin, and steroid regulated systems). Through the analysis of complex sex-dimorphic behavioral patterns we show that neurotoxic and endocrine disrupting activities of CPF overlap. This widely diffused organophosphorus pesticide might thus be considered as a neuroendocrine disruptor possibly representing a risk factor for sex-biased neurodevelopmental disorders in children.

The developmental origin of health and disease hypothesis states that adverse fetal and early childhood exposures can predispose to obesity, cardiovascular, and neurodegenerative diseases (NDDs) in adult life. Early exposure to environmental chemicals interferes with developmental programming and induces subclinical alterations that may hesitate in pathophysiology and behavioral deficits at a later life stage. The mechanisms by which perinatal insults lead to altered programming and to disease later in life are still undefined. The long latency between exposure and onset of disease, the difficulty of reconstructing early exposures, and the wealth of factors which the individual is exposed to during the life course make extremely difficult to prove the developmental origin of NDDs in clinical and epidemiological studies. An overview of animal studies assessing the long-term effects of perinatal exposure to different chemicals (heavy metals and pesticides) supports the link between exposure and hallmarks of neurodegeneration at the adult stage. Furthermore, models of maternal immune activation show that brain inflammation in early life may enhance adult vulnerability to environmental toxins, thus supporting the multiple hit hypothesis for NDDs' etiology. The study of prospective animal cohorts may help to unraveling the complex pathophysiology of sporadic NDDs. In vivo models could be a powerful tool to clarify the mechanisms through which different kinds of insults predispose to cell loss in the adult age, to establish a cause-effect relationship between "omic" signatures and disease/dysfunction later in life, and to identify peripheral biomarkers of exposure, effects, and susceptibility, for translation to prospective epidemiological studies.

The organophosphate chlorpyrifos (CPF) is a pesticide largely used worldwide. Studies from animal models indicate that CPF exposure during development at low doses can target different neurotransmitter systems in the absence of overt cholinergic effects.Late gestational exposure (gestational days 14-17) to CPF at the dose of 6 mg/kg was evaluated in CD-1 mice at adulthood. Neurobehavioural effects likely involving serotonin (5-hydroxytryptamine, 5HT) transmission were assessed both in males and females, through the light-dark exploration test to assess CPF effects on anxiety profiles and the forced swimming test to evaluate the response to the 5HT transporter (5HTT) inhibitor fluvoxamine (30 mg/kg). In females only, we evaluated the effects of gestational exposure to CPF on maternal aggression, under basal condition or after injection of fluvoxamine.Gestational CPF exposure increased anxiety levels only in female mice, as shown by the augmented thigmotaxis behaviour and the lower latency to enter in the dark compartment. In the forced swimming test, no differences between CPF and control mice were found when assessed under basal condition (saline administration), but both male and female CPF mice missed to show the typical behavioural effects of the 5HTT inhibitor fluvoxamine. During maternal aggression, CPF females showed lower propensity to and intensity of aggressive behaviour, together with mild decreased responsiveness to fluvoxamine administration.Overall, the present results confirm a specific and sex-dependent vulnerability of affective/emotional domains to developmental CPF exposure. Furthermore, data provide clear indication on the disrupting effects of prenatal CPF on serotoninergic transmission.

Organophosphorus insecticides, as Chlorpyrifos (CPF), are widely used in agriculture and against household pests; these compounds receive an increasing consideration as potential endocrine disrupters. The aim of the present study was to examine the potential short- and long-term effects of CPF on thyroid and adrenal glands in CD1 mice following exposure at dose levels not inducing brain acetyl cholinesterase (AchE) inhibition, during gestational and/or postnatal vulnerable phases. Pregnant dams were treated with 0, 3, 6 mg/kg bw/day of CPF on gestational days 15-18. After delivery, pups were treated subcutaneously on postnatal days (PND) 11-14 with: 0, 1, 3 mg/kg bw/day of CPF. Serum thyroxin (T4), thyroid and adrenals histology and histomorphometry were evaluated in dams and in F1 mice. In dams at 6 mg/kg, decreased T4 levels and increased cell height in thyroid were observed, and adrenal histology showed a slightly increased vacuolization in the X-zone. In the F1, short-term morphological modifications (reduced follicular size at PND 2) and long-term morphological (increased necrotic follicular cells) and biochemical alterations (reduced serum T4 levels) were found at PND 150 with an apparent higher vulnerability of males. For the first time these results indicate that CPF exposure at dose levels not inducing brain AchE inhibition causes thyroid alterations in dams and in F1 CD1 mice. Thyroid may be a sensitive target to CPF developmental exposure possibly leading to long-term effects on thyroid function. Because thyroid plays a pivotal role in mammalian development, these findings can be relevant to humans.

Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods, in bleaching steps and flour treatment. Experimental data showed SEM acting as osteolathyrogen agent, but few toxicological data are available in susceptible life-stages. This study aimed to evaluate effects of SEM oral administration for 28 days at 0, 40, 75, 140 mg/kg bw day during the juvenile period in Sprague-Dawley rats. Histopatological examinations of: epiphyseal cartilage - potential target of SEM lathyrogen action - testes, ovary, uterus, thyroid, thymus, spleen, adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were performed. Mortality at high and mid dose levels and significantly decreased body weight gain were observed in males even at the lowest dose. Lack of mineralization in cartilage at all dose levels was present. Marked alterations of spontaneous motor and exploratory behaviours were evident even at 40 mg/kg. Histological alterations were observed in all tissues; thyroid and ovary effects were present also at 40 mg/kg. The present study indicate that the NOAEL in juvenile rats is lower than 40 mg/kg for SEM oral administration. SEM administration during juvenile period exerted pleiotropic effects and further studies are suggested to elucidate mechanisms.

CD-1 mice were exposed to the organophosphate pesticide chlorpyrifos (CPF) throughout postnatal days (PND) 11-14 at the subtoxic dose of 3 mg/kg. At adolescent age, females and males underwent a sociability test in which level of sociability and social preference were measured. At adulthood only females' behavior was analyzed. Maternal behavior of CPF-exposed females was assessed on postpartum day 1 after removal of the pups for 1 h, while anxiety levels were measured in a 5 min dark-light test on postpartum day 2. Nest defense response to an unfamiliar male intruder was assessed on postpartum day 7. In addition, from birth to postpartum day 7 a detailed analysis of nest building activity was carried out. Neonatal CPF exposure does not interfere with social behavior and social preferences at adolescence, whereas at adulthood it induces significant behavioral alterations in lactating females. Motivation to build and defend the nest was decreased in CPF females that were also less anxious than controls in the dark-light paradigm. These results confirm that developmental exposure to CPF induces long-lasting alterations in selected sexual-dimorphic responses of the adult social repertoire, and suggest that early exposure to CPF might interfere with hypothalamic neuroendocrine mechanisms regulating social responses. © 2008 Elsevier Inc. All rights reserved.

BACKGROUND: Increasing evidence from animal and human studies indicates that chlorpyrifos (CPF), similar to other organophosphorus insecticides still widely used, is a developmental neurotoxicant. Developmental exposure to CPF in rodents induces sex-dimorphic behavioral changes at adulthood, including social and agonistic responses, which suggests that CPF may interfere with maturation of neuroendocrine mechanisms. OBJECTIVES: We assessed the hypothesis that CPF affects the levels of neurohypophyseal hormones acting as modulators of social behavior in mammals, such as oxytocin (OT), arginine vasopressin (AVP), and prolactin (PRL). METHODS: Pregnant female mice were orally administered with either vehicle (peanut oil) or 3 or 6 mg/kg CPF on gestational day (GD) 15 to GD18, and offspring were treated subcutaneously with either vehicle or 1 or 3 mg/kg CPF on postnatal days (PNDs) 11 to PND14. Dose levels were chosen to avoid systemic toxicity and inhibition of brain acetylcholinesterase. Offspring were sacrificed at 5 months of age, and expression of OT, AVP, and PRL was analyzed in the hypothalamus by Western blot or enzyme-linked immunosorbent assay (ELISA) analysis. RESULTS: Both male and female mice showed dose-related enhancement of OT expression, with males presenting the more intense effect. AVP expression was significantly reduced in male mice at the higher prenatal and postnatal dose. We observed no significant effect on PRL expression in either sex. Overall, outcomes were mainly attributable to fetal exposure, whereas postnatal doses appeared to potentiate the prenatal effects. CONCLUSIONS: Our data indicate that developmental exposure to CPF may permanently interfere with specific key signaling proteins of the hypothalamic peptidergic system, with time-, dose-, and sex-related effects still evident at adulthood.

The present study investigated the effects of intracebroventricular injections of 192 IgG-saporin in 7-day-old rats on (i) ultrasound vocalizations (USVs) on postnatal day (pnd) 13 following isolation and reunion with the mother and (ii) fear conditioning on pnd 18-19 recording both freezing and other behavioural responses as well as USVs. On pnd 13 lesioned and control pups showed comparable USV baseline values; a brief reunion with the mother induced a significant increase in USVs in all rats (maternal potentiation). On pnd 18, during the fear conditioning training, 192 IgG-saporin rats emitted a lower number of USVs. On pnd 19 all rats showed a stronger conditioned response (with full inhibition of locomotion) to auditory than to contextual cues. Surprisingly, lesioned rats showed a stronger fear-conditioned response to contextual cues than controls. These results suggest that early selective removal of the cholinergic basal forebrain paradoxically enhances hippocampally dependent fear-conditioned responses on pnd 19.

BACKGROUND: Chlorpyrifos (CPF) is one of the most widely used organophosphate pesticides worldwide. Epidemiological studies on pregnant women and their children suggest a link between in utero CPF exposure and delay in psychomotor and cognitive maturation. A large number of studies in animal models have shown adverse effects of CPF on developing brain and more recently on endocrine targets. Our aim was to determine if developmental exposure to CPF affects social responsiveness and associated molecular neuroendocrine markers at adulthood. METHOD: Pregnant CD1 outbred mice were fed from gestational day 15 to lactation day 14 with either a CPF-added (equivalent to 6 mg/kg/bw/day during pregnancy) or a standard diet. We then assessed in the offspring the long-term effects of CPF exposure on locomotion, social recognition performances and gene expression levels of selected neurondocrine markers in amygdala and hypothalamus. RESULTS: No sign of CPF systemic toxicity was detected. CPF induced behavioral alterations in adult offspring of both sexes: CPF-exposed males displayed enhanced investigative response to unfamiliar social stimuli, whereas CPF-exposed females showed a delayed onset of social investigation and lack of reaction to social novelty. In parallel, molecular effects of CPF were sex dimorphic: in males CPF increased expression of estrogen receptor beta in hypothalamus and decreased oxytocin expression in amygdala; CPF increased vasopressin 1a receptor expression in amygdala in both sexes. CONCLUSIONS: These data indicate that developmental CPF affects mouse social behavior and interferes with development of sex-dimorphic neuroendocrine pathways with potential disruptive effects on neuroendocrine axes homeostasis. The route of exposure selected in our study corresponds to relevant human exposure scenarios, our data thus supports the view that neuroendocrine effects, especially in susceptible time windows, should deserve more attention in risk assessment of OP insecticides.

Autism spectrum disorders are characterized by impaired social and communicative skills and repetitive behaviors. Emerging evidence supported the hypothesis that these neurodevelopmental disorders may result from a combination of genetic susceptibility and exposure to environmental toxins in early developmental phases. This study assessed the effects of prenatal exposure to chlorpyrifos (CPF), a widely diffused organophosphate insecticide endowed with developmental neurotoxicity at sub-toxic doses, in the BTBR T+tf/J mouse strain, a validated model of idiopathic autism that displays several behavioral traits relevant to the autism spectrum. To this aim, pregnant BTBR mice were administered from gestational day 14 to 17 with either vehicle or CPF at a dose of 6 mg/kg/bw by oral gavages. Offspring of both sexes underwent assessment of early developmental milestones, including somatic growth, motor behavior and ultrasound vocalization. To evaluate the potential long-term effects of CPF, two different social behavior patterns typically altered in the BTBR strain (free social interaction with a same-sex companion in females, or interaction with a sexually receptive female in males) were also examined in the two sexes at adulthood. Our findings indicate significant effects of CPF on somatic growth and neonatal motor patterns. CPF treated pups showed reduced weight gain, delayed motor maturation (i.e., persistency of immature patterns such as pivoting at the expenses of coordinated locomotion) and a trend to enhanced ultrasound vocalization. At adulthood, CPF associated alterations were found in males only: the altered pattern of investigation of a sexual partner, previously described in BTBR mice, was enhanced in CPF males, and associated to increased ultrasonic vocalization rate. These findings strengthen the need of future studies to evaluate the role of environmental chemicals in the etiology of neurodevelopment disorders.

Perinatal asphyxia may induce major neurological deficits shortly after birth as well as neurological/behavioural disorders later in development. We used a rat model of global perinatal asphyxia to model acute intrauterine asphyxia around the time of birth. Caesarean section was performed in rats and their pups, still in uterus horns, were placed into a water bath at 37 °C for periods of 0, 10 or 20 min. Pups were then given to surrogate mothers, and examined for long-term behavioural effects of the perinatal asphyctic insult. Behavioural assessment included analysis of novelty seeking behaviour at adolescence, while spatial discrimination abilities, response to both an acute and a chronic stress, and the effects of the full D1 receptor agonist SKF 82958 on open field behaviour were assessed at adulthood. Overall, no marked abnormalities were found in the novelty seeking test, in the ability to discriminate spatial changes in the test environment and in physiological response to stress. However, adult rats subjected to severe perinatal asphyxia (20 min) showed lower activity level and lower stereotyped behaviour after the administration of SKF 82958 in an open field test. These results support the observations from human and animal studies that perinatal insult can produce long-term dysfunction of dopaminergic neurotransmission, and points to the need of more thorough examination of the potential effects of perinatal asphyxia on hypothalamic–pituitary–adrenal (HPA) axis. Altogether, the present findings suggest that the present 20 min perinatal asphyxia model might serve for the study of neurodevelopmental disorders associated with perinatal insults.

We investigated the effects of Nerve Growth Factor (NGF) administration on the maturation of reactivity to spatial and non-spatial novelty in developing mice. CD-1 mice of both sexes received intracerebral administration of NGF on postnatal day (pnd) 15, and their response to object displacement (spatial novelty) and object substitution (object novelty) were assessed in a spatial open-field with four objects on pnd 18 or 28. On pnd 18, NGF induced only in males precocious appearance of spatial novelty discrimination, while increasing choline acetyltransferase activity in neocortex and hippocampus of both sexes. The behavioral and neurochemical effects disappeared by pnd 28. NGF triggers adult-like responding to spatial novelty in developing mice and such effect is gender-specific.

Long-term changes in exploratory, social and agonistic behavior have been reported in rodents following developmental exposure to zidovudine (AZT), an agent commonly administered to pregnant seropositive women and their neonates to prevent HIV-1 transmission. The present study evaluates the effects of either prenatal or prolonged AZT treatment on spatial and nonspatial novelty discrimination in mice, using an open-field test with four objects, in which responses to both spatial rearrangement of familiar objects and object novelty are assessed. AZT (160 mg/kg) or Saline was given orally twice daily to pregnant mice from gestational days (GD) 10 to 19 (Experiment 1) or from GD 10 to lactation day 10 (Experiment 2). Offspring of both sexes were tested on postnatal day (PND) 28, 45 or 70. Depending on treatment schedule, AZT altered different behavioral responses, males being more affected than females. The prenatal treatment (Experiment 1) reduced exploration of the objects at all ages considered and increased wall and top rearing at ages 45 and 70. Following prolonged treatment (Experiment 2), AZT offspring were markedly more active than controls and displayed more wall rearing at age 70 while showing lower grooming frequency at all ages. Both AZT and control mice failed to respond to object rearrangement at adulthood, a discrepancy from previous data, which is discussed in relation to perinatal stress effects.

Several pieces of evidence from animal and human studies indicate that the organophosphate insecticide chlorpyrifos (CPF) acts as a developmental neurotoxicant at environmentally relevant doses, and it is possibly endowed with endocrine-disrupting activity. Data collected in rodent models show that developmental exposure to CPF at sub-toxic doses induces long-lasting and sex-dimorphic changes in social and investigative responses in exposed offspring. The aim of this study was to evaluate the effects of gestational CPF treatment on social and olfactory discrimination in adult mice of both sexes. Pregnant CD1 out-bred mice were exposed to CPF per os on gestational days (GD) 14–17 at the sub-toxic dose of 6 mg/kg/bw. At adulthood, male and female offspring underwent the same experimental paradigms, namely i) a social discrimination test where mice were presented with a simultaneous binary choice between a novel conspecific and a familiar one, and ii) an olfactory habituation/dishabituation test to evaluate their capability to discriminate between odors with different eco-ethological salience (non-social vs. social odors). Results showed that in the social discrimination test prenatal CPF primarily affected the female sex by raising the investigation time in females to the same levels as found in vehicle- and CPF-exposed males. The ability to discriminate between a familiar and a novel social mate was not affected by CPF in either sex. In the olfactory habituation/dishabituation test, mice of both sexes successfully discriminated non-social from social odors regardless of the prenatal treatment received. These results confirm previous evidence indicating that developmental exposure to CPF causes long-lasting and sex-dimorphic changes in responsiveness to social cues, in the absence of significant impairment of social and olfactory discrimination capacity. These findings are discussed within the framework of recent data pointing to the limbic/hypothalamic circuitry and steroid hormonal regulations as possible targets for CPF neurotoxicity.

Background The new antiretroviral treatments that combine the zidovudine (AZT) regimen with lamivudine (3TC) appear as a cost-effective alternative to the current AZT monotherapy to prevent mother-to-fetus transmission of the HIV-1 virus. Recent evidence in uninfected children raised concern about the long-term effects of perinatal exposure to AZT and 3TC, especially when used in combination. Animal studies indicated behavioral changes in offspring exposed perinatally to both AZT and 3TC, whereas no animal data are available on the effects of the perinatal exposure to the AZT + 3TC combination on neurodevelopment. Methods Pregnant CD-1 mice received p.o. AZT + 3TC (160 and 500 mg/kg, respectively) or vehicle solution (NaCl 0.9%) twice daily from gestational day 10 to delivery. Maternal reproductive endpoints such as pregnancy length, abortion, litter size, sex ratio, and offspring viability were assessed. Pups were scored for different somatic and behavioral endpoints, including sensorimotor development, homing performance on postnatal day (PND) 10, passive-avoidance testing (PND 22–23), locomotor activity (PND 23), and social interaction (PND 35). Results While no effects were observed on maternal reproductive endpoints, treated pups showed a long-lasting reduction of body weight and a slightly delayed maturation of placing and grasping reflexes and pole grasping. No effects on passive-avoidance or locomotor activity were found. AZT + 3TC-treated mice showed selective alterations in the social interaction test; the treated female offspring also displayed a significant reduction of affiliative interactions. Conclusions The combination of AZT and 3TC (1) induced small, but more marked, effects on somatic and sensorimotor development than either of these drugs administered separately; and (2) affected juvenile social behavior. Teratology 63:26–37, 2001. © 2001 Wiley-Liss, Inc.

Rationale: AZT is commonly administered to seropositive women and their neonates to prevent mother-to-child transmission of HIV. Recently, animal studies performed in monkeys and rodents have revealed that pre- and/or perinatal exposure to AZT induces age- and sex-dependent behavioural alterations in the offspring, possibly resulting from an action of this drug on CNS targets. Long-term effects of prenatal AZT treatment on social/aggressive behaviour of adult male mice have been previously described. Specifically, AZT has been shown to induce selective changes in the offensive components of agonistic interactions. Objective: The aim of the present study was to extend previous findings, analysing the long-term effects of a more prolonged AZT exposure on intraspecific male mice agonistic behaviour. Methods: AZT was given orally twice daily to pregnant CD-1 mice. The dosage selected for AZT was 160 mg/kg. Saline solution (0.9% NaCl) was used as vehicle. Starting on postnatal day (PND) 60 isolated males underwent five 15-min repeated encounters with an opponent of the same age and strain isolated for the same amount of time. Furthermore, a locomotor activity test (PND 67) and a hot-plate test (52±0.1°C) (PND 74) were performed to assess AZT effects on, respectively, general activity and pain sensitivity. Results: AZT perinatal exposure reduced attack behaviour of adult mice, while increasing the likelihood of them behaving as subordinates. Furthermore, long-term effects of AZT treatment on pain sensitivity were found in the hot-plate test, with AZT mice showing higher pain thresholds than controls. Conclusions: Overall, these data indicate that perinatal exposure to drugs such as AZT exerts selective effects on the developing CNS, resulting in long-term behavioural disturbances. Future studies will need to address the issue of the specific mechanisms underlying these effects.

Recent evidence has shown that perinatal administration of zidovudine (AZT) to HIV-infected mothers reduces the risk of maternal-infant transmission of the virus. Treatment of pregnant seropositive women with AZT is becoming a common medical practice, despite the paucity of information about the potential neurotoxic/behavioral-teratogenic effects of AZT on the developing organism. The aim of the present study is to evaluate in mice the short-, medium-, and long-term effects of prenatal exposure to AZT on neurobehavioral development. Pregnant mice were given 0.2, 0.4, and 2.0 mg/ml AZT in drinking water from day 10 of gestation to delivery. Offspring's viability was severely affected in the 2.0 mg/ml AZT group. Thus, behavioral analysis was carried out in offspring of 0.2 and 0.4 mg/ml AZT-treated females only. Some limited but significant alterations were found, such as stunted body weight, delayed appearance of the pole-grasping reflex, and a slight impairment in the acquisition phase of a passive avoidance response. Moreover, sexual differences in some items of the social behavior repertoire appeared to be affected by AZT treatment.

Summary The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.

Maternal-fetal HIV-1 transmission can be prevented by administration of AZT, alone or in combination with other antiretroviral drugs to pregnant HIV-1-infected women and their newborns. In spite of the benefits deriving from this life-saving prophylactic therapy, there is still considerable uncertainty on the potential long-term adverse effects of antiretroviral drugs on exposed children. Clinical and experimental studies have consistently shown the occurrence of mitochondrial dysfunction and increased oxidative stress following prenatal treatment with antiretroviral drugs, and clinical evidence suggests that the developing brain is one of the targets of the toxic action of these compounds possibly resulting in behavioral problems. We intended to verify the effects on brain and behavior of mice exposed during gestation to AZT, the backbone of antiretroviral therapy during human pregnancy. We hypothesized that glutamate, a neurotransmitter involved in excitotoxicity and behavioral plasticity, could be one of the major actors in AZT-induced neurochemical and behavioral alterations. We also assessed the antioxidant and neuroprotective effect of L-acetylcarnitine, a compound that improves mitochondrial function and is successfully used to treat antiretroviral-induced polyneuropathy in HIV-1 patients. We found that transplacental exposure to AZT given per os to pregnant mice from day 10 of pregnancy to delivery impaired in the adult offspring spatial learning and memory, enhanced corticosterone release in response to acute stress, increased brain oxidative stress also at birth and markedly reduced expression of mGluR1 and mGluR5 subtypes and GluR1 subunit of AMPA receptors in the hippocampus. Notably, administration during the entire pregnancy of L-acetylcarnitine was effective in preventing/ameliorating the neurochemical, neuroendocrine and behavioral adverse effects induced by AZT in the offspring. The present preclinical findings provide a mechanistic hypothesis for the neurobehavioral effects of AZT and strongly suggest that preventive administration of L-acetylcarnitine might be effective in reducing the neurological side-effects of antiretroviral therapy in fetus/newborn.

The aim of this study was to assess in developing mice whether the neutralization of endogenous NGF following ICV administration of anti-NGF antibodies (50 micrograms/2 microliters) on postnatal days 3, 6, 9, and 12 affected locomotor activity, exploratory behavior, and response to the cholinergic blocker scopolamine. In Experiments 1 and 2 activity and age-typical scopolamine effects were evaluated on PND 13 or 17 in an automated apparatus. No significant main effect of anti-NGF treatment was found at either age. On day 13 scopolamine (0.2, 1, or 2 mg/kg) decreased locomotion in both anti-NGF and control animals. In Experiment 3, locomotion and exploratory behavior were analyzed in an open field arena or in a hole board apparatus on PND 16. No significant effects of anti-NGF treatment on general motor activity and investigation of a novel object in the open field was found, though anti-NGF animals tended to be less active than controls. In the hole board anti-NGF pups showed a different pattern of head dipping behavior from controls, exploring mainly the holes located in the periphery of the apparatus.