Abstract

1. Introduction

The brain has a protracted period of susceptibility to environmental inputs, which extends well beyond organogenesis up to the second decade of life [1]. Brain development requires a series of sequential and interacting steps, each controlled by intrinsic developmental processes that are modulated by external influences. The primary event in the formation of the central nervous system (CNS) is the appearance of the neural plate from the ectoderm, during the second and the third weeks of gestational age [2], followed by rapid cell proliferation as the plate folds to form the neural tube. Cells formed during this period of intense proliferation are known as neuroblasts and extend in the ventricular zone, where they continue to proliferate and subsequently migrate from this region toward their final destination with the help of the radial glial cells [3], depending on the presence of different molecular gradients in their location [4,5]. In the human fetus, cell migration is nearly complete in the neocortex and in most of the brain by the sixth month of gestation [6].

During the remainder of intrauterine development, neurons differentiate and connect to each other, and these two processes (differentiation and synapse formation) continue for several years after birth. Synapse formation is the final step in the establishment of CNS circuitry: substantially more connections are formed than those that will be eventually retained as many of them will be gradually discarded. The developing brain is characterized by many waves of apoptosis that control the processes of creating, strengthening, and discarding connections among the neurons; these processes continue into early postnatal years in response to the young child’s experiences. The remodeling of synaptic connections to establish functional properties of cortical circuits requires also the glial component of white matter that supports the neural migration, the regulation of extracellular environment and the synaptic connections [7]. The process of synapse elimination is a normal part of development [7,8] and both endogenous (neurotrophic factors, synthesis and release of neurotransmitters, hormones) and environmental factors (i.e., sensorial inputs) concur to influence the fine matchup between pre- and postsynaptic neurons.

Within the constraints posed by the genome, the brain receives information from the environment and uses this information to shape and refine synaptic connections. In such a way, experience adjusts the underlying brain circuitry founded on the distinctive environment in which each individual lives and grows up. This has been clearly demonstrated by pioneering experiments showing the fundamental role of sensory input during critical periods of development for appropriate formation of connections in the visual cortex [9]. Furthermore, the evidence that growing up in enriched environments produces experience-induced alterations in brain morphology in rats (reviewed in [10]) demonstrated that also in the presence of subtle changes in the environment or habits, experience-dependent neural activity plays critical roles in brain development.

Vulnerability to adverse environmental factors represents the drawback of brain plasticity. The dynamic interplay between genes and environment, which forms the basis of typical neurobehavioral maturation, is being also called upon to explain the etiology of complex neurodevelopmental disorders (NDDs) that are characterized by abnormal brain morphology and/or functional activity, even those with a strong genetic component. Diverse environmental stressors—chemical pollutants, drugs, nutritional factors, maternal infection, stress, deprivation—may interfere with typical brain developmental trajectories, eventually increasing the risk of either subclinical neuropsychological alterations or manifest clinical conditions such as learning disabilities, autism spectrum disorders (ASD) and attention deficit/hyperactivity disorder (ADHD).

The present review focuses on early exposure to environmental chemicals. First, we summarize the major lines of evidence derived from epidemiological studies that suggest association between exposure to chemicals and altered neurodevelopment in children, underlying key uncertainties and research needs in the field of environmental origin of major human NDDs. We then introduce the exposome approach, which investigates the relationship between all the exposures of an individual in a lifetime and her/his health outcome, to explain the variability in individual vulnerability and resilience to early chemical exposure. In this framework, we synthetically review the role of variable gene backgrounds, the involvement of epigenetic mechanisms as well as the function played by potential effect modifiers such as socioeconomic status (SES). In the second part of this review, we describe laboratory rodent studies where the neurodevelopmental effects of environmental chemicals are assessed in the presence of either a “vulnerable” gene background or adverse pregnancy conditions (i.e., infective state, maternal stress). Finally, we discuss the need for more descriptive and “lifelike” experimental models of NDDs, in the framework of the new challenges posed by the exposome concept to environmental health research.

2. Environmental Chemicals and Neurobehavioral Toxicity: Key Uncertainties and Research Needs

There is vast literature on the developmental neurotoxicity of environmental chemicals. The hypothesis of a relationship between chemical exposures and neurobehavioral changes was first raised by the evidence that lead exposure was toxic for brain development [11,12,13,14]; similar results were later identified for the exposure to other environmental contaminants in the early phase of neurodevelopment. Significant alterations in neuropsychological maturation in relation to developmental exposure to chemicals have been described for heavy metals [15,16], different classes of pesticides [17,18,19,20], polychlorobiphenyls (PCB) [21], polybrominated diphenyl ethers (PBDE) [22], and phthalates [23]. Chemicals can have an effect at any time in the process of brain development, however, the earlier the stage of brain development the greater will be the impact on brain structure and function [24].

Recently, Grandjean and Landrigan [25] performed an extensive review of studies published from 2006–2012 on the neurotoxic effects of industrial chemicals in human beings. This article updates the previous one published from the same authors in 2006 [26], where a series of 201 chemicals among metals and inorganic compounds, organic solvents, pesticides, and other organic substances were identified as neurotoxic for adult individuals, though, at that moment, only very few of such chemicals had been classified as neurodevelopmental toxicants. In particular, in the 2006 survey, lead, methylmercury, toluene and PCB were implicated in neurobehavioral deficits in children following prenatal exposures at concentrations considered as subtoxic in adults.

Since 2006, new data have emerged about the vulnerability of the developing brain and the neurotoxicity of industrial chemicals, and the recent review from the same authors reports new evidence that derives from prospective epidemiological birth cohort studies. Six additional agents have emerged as neurodevelopmental toxicants of concern. In particular, epidemiological data associate manganese, fluoride, chlorpyrifos, dichlorodiphenyltrichloroethane, tetrachloroethylene, and PBDEs with diminished intellectual functioning, learning disabilities, attention problems, aggressiveness, hyperactivity, ADHD and ASD [27,28,29,30,31]. Other suspected developmental neurotoxicants are further indicated: among these, phthalates and bisphenol A, air pollution, and comparable complex emission such as the traffic-related pollution. In particular, carbon monoxide, nitrogen oxides, and polycyclic aromatic hydrocarbons are recently reported to act as neurotoxic agents that can cause cognitive and neurological impairment [20,32], as well as ASD [33], and ADHD [34].

In spite of the impressive number of epidemiological data that indicate an inverse association between chemical exposure and child neurodevelopment (see also [35] for an updated review), there are still important knowledge gaps in this field that hamper the proper evaluation of neurobehavioral effects by risk assessors. In this framework, Bellinger [36] elegantly discussed issues concerning the validity, precision, and interpretation of epidemiologic studies of neurotoxicity. He pointed to the estimation of the dose-effect relationship as a crucial point for establishing the causative role of environmental chemicals in human diseases. Dose-effect estimation requires linking environmental exposure to the biologically effective dose of xenobiotics at the main target tissues, and then relating internal dose at target tissue with the physiological perturbation/health outcome observed. This implies both identification of reliable biomarkers of exposure (i.e., peripheral indicators predictive of concentrations at target tissues) and knowledge of the mechanisms of neurotoxicity for the chemical compounds in study. Indeed, a critical gap relates to the mechanisms by which different classes of chemicals act on behavioral development. Mechanistic studies carried out in animals and in in vitro models point to multiple pathways and targets of toxicity for several established neurotoxicants. Chemicals may directly interfere with formation and closure of the neural tube, cell proliferation, migration, death, or synapse formation [37] inducing changes ranging from overt morphological alterations [38] to subtle dysfunction in synaptic connectivity [39]. The same chemical compound may affect different developmental processes or different cell types, depending on the time window of exposure [40]. Furthermore, compounds belonging to the same chemical class may have dissimilar mechanisms of action, as is the case for different organophosphate insecticides that produce disparate neurotoxic outcomes despite their shared property as cholinesterase inhibitors [41]. To complicate the picture further, the same chemical may have multiple mechanisms of action: agents endowed with endocrine disrupting activity may affect neurobehavioral development by directly interacting with steroid receptors in brain cells and/or in periphery, and at the same time influence the density of synaptic connections in specific brain areas with mechanisms possibly independent from their hormone-like action [42]. Thus, more experimental research is needed to elucidate the causal links between chemical exposure and disease, addressing different levels of biological organization through the combined use of in silico, in vitro and in vivo models. This will support building adverse outcome pathways for neurodevelopmental effects [43,44].

Even if the precision of dose estimates can be improved, a second main issue is how to capture the individual dimension of the exposure history. Different factors may indeed contribute to variability in the measured outcome, including the temporal dimension of the exposure, the co-exposure to other contaminants or stressors, and the existence of genetic vulnerability that may render an individual more susceptible than others. In addition, chemical exposure may be associated with other risk factors that should be precisely measured to prevent underestimation or overestimation of toxicant effects [45]. These factors are usually taken into account as confounders or effect modifiers in the result interpretation: among them, the most frequently considered are child sex and age, and SES indicators, whose association with neurocognitive outcomes (the higher the SES, the better the outcome) has been widely demonstrated (for a review on the effect of SES on the neurocognitive performance see [46]). Together with their role as confounders or effect modifiers with respect to toxicant exposure evaluation, these factors can have a direct beneficial or harmful effect on neurodevelopment that should be studied per se. For example, SES indicators, including education and/or income of either child’s parents or the community have been shown to contribute to ASD prevalence—the higher the “community” SES, the higher the probability of ASD diagnosis for children affected, especially for mild cases [47], and to ASD risk—the lower the family income, the higher the risk of ASD [48].

A third critical question, which is of particular significance for risk assessment, is that of the robustness of the outcome measurements. Developmental neurotoxicity is often evaluated by behavioral symptoms that, by their very nature, are endowed with a wide variability range and high sensitivity to both individual history (family, SES, nutritional factors) and characteristics of the neuropsychological test applied. As a matter of fact, most of prospective epidemiologic studies on neurotoxicity do not report clinically defined conditions (i.e., autism or learning disability) but rather atypical behavioral traits that range from increased/decreased anxiety and aggressiveness, to poorer motor or intellectual development in a significant proportion of exposed infants/children [49]. Whether these sub-clinical behavioral alterations may signal increased risk to develop a frank neuropsychiatric disorder at some point in the individual’s life course remains to be determined. As Bellinger [50] pointed out, the characteristics of the effect outcome in brain/mental diseases has generally led to under-estimation of health risk as far as a disease-oriented approach rather than a population-oriented approach is preferred. Even a small shift in the mean IQ score in a population will result in a substantial increase in the percentage of individuals with extremely low scores, with a significant impact on economic and health costs [51].

A discussion on the methodological and statistical tools that could improve evaluation of population health burden associated with environmental contamination is beyond the scope of the present review. The harmonization of evaluation tools (i.e., use of validated neuropsychological test batteries to compare the outcomes coming from different cohorts) and proper consideration of confounders would undoubtedly strengthen the robustness of human data in support of a causal association between environmental chemicals and brain diseases. However, the complex etiology of major chronic diseases, including neurodevelopmental and neurodegenerative diseases, requires innovative study paradigms and multifaceted and multidisciplinary approaches, taking into account biological plausibility as supported by experimental data, exposure and health effect modification due to intrinsic (such as genetic susceptibility) and extrinsic (such as socio-economic status) factors.

3. Facing Complexity: The Exposome Concept

According to the need to generate integrative models for the study of the etiology of complex diseases, the concept of the “exposome” represents the actual synthesis of this approach. It was first proposed by Wild [52] to encompass the totality of human environmental exposures from conception onwards and was developed to draw attention to the need for more complete environmental exposure data, with the aim of complementing the genome with a more comprehensive description of lifelong exposure history. Initially conceived to describe the multifactorial origin of cancer and other chronic diseases, the exposome approach may be a powerful tool also to unravel the genetic and environmental contributions to the etiology of NDDs and neurodegenerative disorders.

The exposome paradigm analyzes both endogenous and exogenous sources of non-genetic exposure (Figure 1). In addition to environmental pollutants, the exposome includes diet, life style factors, occupation, pre-existing diseases, infections, psychological stress, and socioeconomic status, together with several endogenous factors—inflammation, hormones, metabolism, and oxidative stress—potentially influencing the response of the organism to the external factors. The overlap and the interplay between the internal and the external domains, together with their relative temporal variation, represent the most challenging features for the exposome characterization. Measuring the exposome requires a many-sided and interdisciplinary approach: the collection of data on several chemical and physical external exposures must be complemented by the characterization of downstream biological events causally linked to exposures by exploiting high-throughput omics technologies [53]. The contributions of omics techniques lie in their potential to measure “signatures” of the biological response to cumulative exposure, thus hopefully allowing a more holistic evaluation of exposure-related health effects [54].

Figure 1

The domains of the exposome: different domains (external and internal) are illustrated. Omic signatures of the exposome can be assessed at different developmental stages in the exposed individuals to fully estimate the health impact.

Altogether, the exposome concept tries to overcome the paradigm of “nature versus nurture” and adopt one defined by complex and dynamic interactions between DNA sequence, epigenetic DNA modifications, gene expression and environmental factors that all combine to influence disease phenotypes. The European Commission launched in 2012 the Exposome European Initiative to support the development of this complex and innovative approach, and three large collaborative projects (HELIX, EXPOsOMICS and HEALS) have been funded and are under way [55,56,57]. HEALS (Health and Environment-wide Associations based on Large population Surveys) in particular comprises re-examination of data from existing large EU cohort studies on chemical exposure and neurodevelopmental disorders, to identify major knowledge gaps and select the most appropriate tools to apply the exposomic approach in a future multi-centered birth cohort study.

4. Multifactorial Origin of NDDs: The ASD and ADHD Examples

NDDs encompass a group of clinical heterogeneous conditions with onset in the developmental period. These disorders typically manifest early in development and are characterized by developmental deficits that produce lifetime impairments of personal, social, academic, or occupational functioning. The range of developmental deficits varies from very specific limitations of learning or control of executive functions to global impairments of social skills or intelligence. Commonly known NDDs include ASD, ADHD, communication, speech and language disorders, and genetic disorders such as Fragile X or Rett syndrome. To date, the etiological bases of the majority of these conditions are still unknown, though a great body of data supports their polygenic and multifactorial etiology [58]. In NDDs, a number of variations (i.e., single nucleotide polymorphisms, mutations, deletions and copy number variants) in different genes may confer higher vulnerability to different kinds of adverse environmental factors (maternal infections, obstetric complication, and exposure to drugs or neurotoxicants during pregnancy) [59].

5. Gene Polymorphisms as a Source of Vulnerability

Among the potential sources of inter-individual variation in susceptibility to chemicals in population studies are genetic polymorphisms. Common gene variants may induce susceptibility to environmental factors by increasing or decreasing physiological responses to common effects from intrauterine infections and cytokines, or from environmental toxins, through the mother’s internal or external environment [114]. Single Nucleotide Polymorphisms (SNPs), namely variations at a single position in a DNA sequence among individuals, are the most common type of human genetic variation. In the past decade, much research has been devoted to find SNPs that may help predict an individual’s response to certain drugs, susceptibility to environmental pollutants, and risk of developing particular diseases [115]. In particular, it is suggested that variations in a group of so called “environmental responsive genes” may confer higher vulnerability to the adverse effects of environmental toxicants [116]. Notably, several studies have identified SNPs in genes involved in the detoxification of environmental pollutants in some individuals with ASD, and it is estimated that more than 100 such genes may contribute to ASD risk [117]. The number of studies investigating the role played by genetic polymorphisms in modifying neurotoxicity has increased rapidly in recent years, but the results of these epidemiologic studies are far from being consistent. In the following two paragraphs, we will review the most recent data investigating common polymorphisms as a source of variation in susceptibility to the neurotoxic effects of Hg and OPs in children.

5.1. Gene Polymorphisms Increase Susceptibility to Mercury

Hg is one of the most well studied environmental pollutants. Methylmercury (MeHg) originates from methylation of inorganic Hg by bacteria in aquatic systems [118], it is absorbed from the human gastrointestinal tract and readily crosses the placenta and blood-brain barrier [119]. The intake of seafood is the main route of human exposure to MeHg [120]; seafood contains many important nutrients for brain maturation such as long chain polyunsatured fatty acids (PUFAs) which are the major benefits of fish-derived lipids [121]. However several studies have demonstrated that the increase of fish consumption during pregnancy is responsible of several adverse effects of prenatal exposure to MeHg [122], but the results of the epidemiological studies conducted among fish-consumer populations remain controversial. In a cohort of the Faroe Islands, elevated cord blood Hg levels were significantly associated with adverse effects on motor, attention and language functions in children at seven and 14 years of age [123,124]. On the contrary, three cohort studies conducted in the Seychelles Islands, where prenatal exposure to MeHg occurs at one of the highest levels in the world [125,126,127], did not find an association between elevated Hg levels and neurodevelopment functions. A population-based birth cohort from Spain was studied at four years of age, showing that Hg levels in hair and children’s motor and cognitive abilities were inversely related [128]. In contrast, the results of a prospective birth cohort study in Northern Italy did not indicate any correlation between prenatal Hg exposure and child neurodevelopment [129]. These conflicting findings have been related to variations in genes implicated in MeHg uptake, distribution and excretion [130,131] and/or to the beneficial effects of PUFAs present in fish, which play a protective role in brain development [132,133].

The hypothesis that genetic factors may increase susceptibility to MeHg toxicity has been addressed by several epidemiological studies, by considering gene mutations that affect the absorption, distribution, metabolism and elimination of elemental Hg and MeHg in the body. Echevarria and coworkers [134] identified a SNP in the exon 4 of the gene encoding the heme biosynthetic pathway enzyme coproporphyrinase oxidase (CPOX), which may be responsible for susceptibility to the neurobehavioral effects associated to elemental Hg exposure in humans. Woods and coworkers then analyzed the interaction between this same SNP and Hg exposure through dental amalgam tooth filling on neurobehavioral functions of children aged eight to 12 years. The allele frequencies was equally distributed among males and females (about 15%), but Hg exposure was strongly associated with a reduction in cognitive performance in males carrying the CPOX4 allele [135]. The mechanisms by which COPX4 allele confers higher vulnerability to Hg exposure are not clear: the heme biosynthetic pathway plays a regulatory role in neural and synaptic development, and it is possible that both Hg and the genetic variant impact on this same pathway with additive effects on neurodevelopment [136]. Notably, children with ASD have higher mean urinary porphyrin concentrations, suggesting that the processes that change porphyrin excretion might be related with processes involved in ASD pathogenesis [81]. Other studies have described Hg effects on neurodevelopment in boys with genetic variants of metallothionein (MTs) proteins that play a crucial role in both dispersal and storage of metals such as Hg in the body [137]. The MTs display two different isoforms in humans: MT1M and MT2A, which can modify the Hg toxicokinetics in adults [138], and possibly enhance the adverse neurobehavioral effects of MeHg exposure in children. Woods and coworkers [139] showed that both variants exacerbated the Hg effects on neurological functions in children, in particular for verbal learning, memory and executive functions. These findings are consistent with those of a mouse study where the deletions of MT1/2 genes worsen learning and memory impairments associated with Hg exposure [140].

In a prospective study, Ng and coworkers [141] examined the interaction between different alleles of the apolipoprotein E and neurodevelopment in two-year-old children exposed to Hg as measured in cord blood. The gene product, apolipoprotein E, is a protein transporter expressed in the brain; the Epsilon4 allele, considered a risk factor for Alzheimer disease, is associated with poor neural repair function and it has also been shown to modulate the neurobehavioral toxicity of lead in adults [142]. The authors reported that the individuals carrying the Epsilon4 allele are more vulnerable to the adverse effects of Hg on neurodevelopment than individuals carrying other alleles [141].

Another line of evidence reported variations in genes implicated in the placental transfer of xenobiotics: MeHg is able to cross the placenta through specific transporter proteins, the superfamily of ATP binding cassette (ABC) transporters, a widely expressed protein family responsible for the active transport of various xenobiotics across the cell membranes. Llop and coworkers [143] evaluated the relationship between some polymorphisms in ABC genes (ABCB1, ABCC1 and ABCC2) and prenatal exposure to MeHg in two Mediterranean birth cohorts. The results showed that the association between maternal fish intake and mercury in cord blood depends on the child’s genotype for ABC transporters, as risk alleles cause increased accumulation of MeHg during early development.

Recently, the Avon Longitudinal Study of Parents and Children (Bristol, UK) analyzed the association between prenatal MeHg exposure and IQ scores at eight years in 1135 children for whom data on 247 SNPs within relevant genes were available. Among 40 SNPs showing nominally significant main effects, MeHg interactions with IQ scores were detected for paraoxonase 1 (PON1), progesterone receptor, transferrin and brain-derived neurotrophic factor. Thus, heterogeneities in several relevant genes not specifically linked to Hg uptake or excretion indicate possible genetic predisposition to Hg neurotoxicity in a substantial proportion of a population with a low level of Hg exposure [45]. Finally, a very recent study carried out by Woods and coworkers [144] chose 13 candidate genes identified as associated with various neuropsychiatric disorders and also with alterations in Hg toxicokinetics and tissue distributions [144]. In particular, these genes present different variants that modify the effects of Hg exposure on a broad or limited range of neurobehavioral functions in children. Of the 13 genes evaluated, only four of them showed variants that significantly alter the effects of Hg exposure on neuropsychological domains in children: the gene encoding the heme pathway enzyme, CPOX4, the MTs with their two isoforms MT1M and MT2A and the cathecol-O-methyltransferase gene (COMT) that has been linked to diverse neuropsychiatric conditions such as schizophrenia and ADHD.

In conclusion, the observations raised by different epidemiological studies seem to support the hypothesis that some genetic variants may be in part responsible for the increased susceptibility of the negative effects of Hg exposure on neurobehavior in children (Table 1). Regression coefficients β of the most relevant effects reported in the table provide information on the effect size of the studies. It should be kept in mind that regression coefficients β represent the change in the dependent variable (e.g., neuropsychological outcome) for any unitary increase of the independent variable (e.g., blood levels of Hg). As an example we refer to paper [44]: in subjects bearing TT allele in TF gene, an increase of one unit in the Log₁₀ cord blood Hg (i.e., a ten-fold increase in the cord blood Hg value) yields a mean decrease of 22.7 points in the WISC-III Performance IQ score, that can be relevant since the WISC-III Performance IQ score in typical developing children has a mean value of 100, and borderline functioning can be suspected below the score of 84.

Table 1

Genetic polymorphisms and Hg exposure in humans.

Besides the genetic polymorphisms modulating Hg toxicity, the social environment, the potential for MeHg to interact with other chemicals present in marine food and the protective effects of some nutrients present in fish (e.g., PUFA, selenium) might also significantly contribute to the final behavioral outcome [145]. In addition, we must consider that the clinical significance of a gene-environment interaction at the population level depends not only on the size of the effect of a genetic variant on toxicity, but also on the frequency of that variant in the population [146]. Hence, the relevance of a gene-environment interaction on vulnerability must be always evaluated with respect to the specific population under study, taking into account the size of the genetic effect on environmental toxicity together with the frequency of that genetic subgroup in that population. Distribution of genetic variants is known only for the populations where specific epidemiological studies have been conducted, and differs across populations: for this reason, the information on the distribution of a genetic trait available in one population cannot be translated to others, making the plain extrapolation of toxicity effects due to gene-environment interaction across populations questionable.

6. Susceptibility to Environmental Chemicals: A Key Role for Epigenetics?

Mutations or SNPs in several genes may explain differential vulnerabilities, but genes are not the only actors playing a role in health and disease. Increasing data shed light on the complex relation between genes and their expression by means of epigenetic mechanisms, and identify a further level of vulnerability for chemical exposure during neurodevelopment [174].

As described in Section 4, the numerous gene polymorphisms identified in specific NDDs may possibly contribute to their pathogenesis. However, monozygotic twins share nearly 100% of their genetic polymorphisms, and there is no NDD for which concordance rates between monozygotic twins reach the estimated heritability. Epigenetic mechanisms may provide a molecular link between environmental and genetic factors in the development of psychopathology [175]. As epigenetic programming determines the state of expression of genes, epigenetic differences could have the same consequences as genetic polymorphisms [176]. Moreover, there is experimental evidence that exposures during the prenatal window can influence disease risk transgenerationally through epimutations in the germline [174]. Therefore, epigenetic mechanisms may account for much of the discrepancies between monozygotic discordance rates and heritability estimates found in major NDDs [177].

Epigenetic mechanisms are related to relevant modifications in the genome that influence gene expression without changes in nucleotide sequence. These modifications alter how the histone proteins are complexed with DNA to form the chromatin, and are stable across generations [178]. The epigenetic modifications include several processes, as DNA methylation, histone modifications, different mechanisms of chromatin organizations and microRNAs [179], which have the potential to influence the long-term effects of environmental exposure to neural gene expression and therefore the risk of developing NDDs [180]. Some epidemiological studies suggest that environmental exposure to various neurotoxic chemicals, such as metals, pesticides and endocrine disrupters induce aberrant changes in epigenetic pathways with consequent adverse effects on human neurodevelopment [181].

It is known that epigenetic modifications are important tools by which neurons modify their transcriptional response to developmental and environmental factors [182] but the mechanisms by which early exposure to environmental toxins induce permanent epigenetic changes able to interfere with typical brain development are far from understood. Variations in genes encoding epigenetic regulators may play a critical role, as they hamper the capacity of neurons to respond appropriately to environmental signals in critical developmental phases [183]. Of particular interest are mutations which involve many chromatin regulators, as proteins with methylbinding domain such as the methyl CpG binding protein 2 (MeCP2) that causes Rett Syndrome. MeCP2 is a DNA binding protein that shows high affinity for methylated cytosine and acts either as a positive or negative modulator of gene expression [184]. MeCP2 exerts an important role in the regulation of synaptic plasticity [182,185]; there are many examples of epigenetic alterations linked to reduced expression of MeCP2 and other epigenetically regulated genes such as FMR1, which is associated to the Fragile X syndrome, idiopathic autism, and intellectual disabilities [186]. Thus environmental toxins and genetic vulnerability (i.e., variations/mutations in epigenetic regulators genes) may converge on the same epigenetic pathways, leading to phenotypic changes [187]. Environmental toxins may also directly interfere with modulation of DNA expression, as in the case of the antiepileptic agent valproic acid (VPA). As previously mentioned, VPA is one of the most studied chemical agents linked to autism, as maternal use of VPA during pregnancy is associated with a significantly increased risk of ASD and other developmental disabilities [188]. Multiple mechanisms are called upon to explain both the therapeutic and neurotoxic effects of VPA: direct interference with GABAergic neurotransmission, with folate metabolism and increased production of free radicals. The role of VPA in epigenetic regulation has only recently come to light. In particular VPA is a non-selective inhibitor of histone deacetylase of class I and II (HDAC1 and HDAC2) expressed in the brain [189]. Inhibition of HDACs is thought to occur through direct binding of VPA to the active site of the enzyme, preventing the hydrolytic removal of acetyl groups from DNA. The uncondensed chromatin may be exposed to several transcription factors that can induce excessive transcription of potentially damaging genes, such as Bcl-2 and Hoxa1, thus interfering with cell proliferation, morphogenesis, programmed cell death and/or synaptogenesis depending on the time window of exposure [190].

7. Not in Our Genes: Socioeconomic Factors Modulate Environmental Chemicals’ Toxicity

Genes are only one among many factors modulating the response of an organism to adverse environmental stressors. Adverse maternal conditions, such as stress, infections, and malnutrition can profoundly interfere with fetal brain development through a plethora of mechanisms, including abnormal activation of the hypothalamic-pituitary-adrenal axis, neuroinflammation, alteration of the hormonal milieu, and dysfunction in the immune responses.

The observation that health and chemical burden are sustained at higher level by low SES populations informs on the potential risk for human health in those population in which cumulative risk factors, such as psychological stress due to socioeconomic deprivation and chemical exposure could occur [191,192]. In this respect, recommendations from the US National Academy of Science to the US Environmental Protection Agency stated that nonchemical stressors and other important aspects of vulnerability should be adequately incorporated into the risk assessment process [193,194]. Cumulative risk assessment represents a procedure for processing relevant information to analyze, characterize, and possibly quantify the combined harmful effects from exposure to a mixture of both chemical and nonchemical stressors. Overall, sufficient attention has been kept in considering cumulative risk which derives from exposure to chemical mixtures, though several methodological limitations have so far allowed just an estimation of the cumulative risk [195]. Few studies, however, used the “allostatic load model”, which take into account the cumulative biological burden triggered by responses to a specific “ecological niche”, and potentially includes several categories of stressors. In the allostatic load model, it is implicit that complex biological mechanisms are involved in the adaptive process defined as allostasis, which maintain homeostasis by producing multiple physiological mediators (e.g., activation of the HPA axis, sympathetic and parasympathetic regulations, and inflammatory and oxidative stress molecules). Chronic allostatic load could result in over-activity and dysregulation of allostasis mediators and determine co-occurring risk across multiple physiological systems and accumulation of such risk across time at the individual level [196]. The individual vulnerability rising from allostatic load can in turn influence responses to chemical exposure modulating response and resilience, with health effects which appear dependent on geographical, race and social factors [197].

Both epidemiological and experimental studies have explored hypotheses on the interaction between allostatic load and neurotoxicant exposure during development suggesting common target physiological systems and pathways for both toxicants and stress. In a very recent study conducted on the US cohort of women in reproductive age enrolled inside the National Health and Nutrition Examination Survey (NHANES) program, chronic stress was found to modify the association between elevated lead/methylmercury exposure and race/ethnicity [198], highlighting the importance of evaluating chemical and nonchemical stressor exposures. Previous studies had showed controversial relationships between higher environmental chemicals’ exposure and lower social economic status, suggesting that more epidemiological research is needed to clarify which variables (chemical and nonchemical) are really involved in the potential interaction between chronic stress and chemical hazard [199,200].

8. Unraveling Complexity: The Need for Experimental Models

The epidemiological and clinical data collected so far, while supporting the hypothesis of a substantial contribution of environmental factors to NDDs, highlight the difficulty of establishing causative links between each of these factors and the health outcome. The simultaneous exposure of an individual to multiple risk factors, which may interact in an additive, synergistic, or even antagonistic way, remains to be explained and defined, especially in a mechanistic perspective. The exposome approach has the ambition of unfolding such complexity by means of several omic signatures possibly describing exposure, effects, individual vulnerability and their dynamic interplay [53].

However, when one tries to apply the exposome concept to quantify the effects of environmental exposures on neurodevelopment, a major critical issue dampens enthusiasm: the brain, the target organ of neurotoxicants, is not accessible unless highly invasive or extremely costly (e.g., neuroimaging) methods are used. Peripheral biomarkers of exposure so far available for many environmental chemicals are indeed poor predictors of effects on the CNS [201] and estimating the concentration of a toxic compound or metabolite in the brain on the basis of concentrations found in other matrices may lead to exposure misclassification [36]. The same holds true for biomarkers of effect, which should measure early biological changes related to exposure and also predict health effects. To date no available biomarker is a clear and validated indicator of typical brain development: recent studies suggest that levels of inflammatory cytokines in amniotic fluid could predict ASD risk [202]; placental miRNA expression profiles and DNA methylation of specific genes are associated with measures of neurobehavioral outcome in the infants as well as with increased risks of neurological and neurodegenerative diseases [203]; altered amyloid-beta protein in plasma is related with neurodegenerative risk after prenatal Pb exposure [204]. It is widely acknowledged that much research is needed to identify omic biomarkers in peripheral tissues that can reliably inform on typical and “abnormal” brain development. Even if animal models can be extremely useful to validate peripheral biomarkers as informative measures also of CNS status (e.g., for oxidative stress biomarkers [205,206]) evidence is still scant on this topic.

In this framework, experimental in vivo models may be extremely helpful to generate mechanistic hypotheses and identify robust biomarkers of exposure, effects and susceptibility, to be subsequently verified in large prospective cohort studies. Studies with laboratory rodents permit intensive and methodological evaluation of important parameters, such as dose-response relationships, critical periods of susceptibility, and the relative contribution of genetic, epigenetic and environmental factors. Since the mid-seventies of the past century, when “behavioral teratology” originated as a sub-speciality of teratology to explain the behavioral anomalies induced by many teratogens in infants, the use of animal models has been of paramount importance to investigate the mechanisms by which chemicals influence brain development in humans [207]. In particular, animal models are a powerful tool to test what gene-environment combination or what combination of adverse environmental factors may produce significant disruption of neurobehavioral development that lead to clinically-relevant outcomes. Notably, this kind of information can only be achieved by testing the living organism as the face validity of an animal model of NDD consists primarily in a robust behavioral phenotype [208].

However, to date, the neurobehavioral toxicity of environmental pollutants and the contribution of candidate genes or adverse environmental factors to NDDs have typically been tested in vivo independently, with very few attempts to identify the effects of their interaction. In the following sections, we will review the in vivo laboratory studies analyzing the neurobehavioral effects of different environmental chemicals in combination with (i) mutations or variants in NDD candidate genes or (ii) maternal stress in pregnancy.

9. Designing in Vivo Experiments to Model Complexity

One of the aims of laboratory research in behavioral toxicology should be that of identifying and validating biomarkers, to empower the exposome approach with a set of peripheral accessible markers of susceptibility, vulnerability and effect, able to predict the neuropsychological outcome.

To this aim, experiments performed in animals should consider different exposure factors, modeling human exposure scenarios. Together with the toxicant under study, factors mimicking demographic and socio-economic variables usually considered in humans should be included (e.g., sex, age, maternal stress, rearing conditions, environmental enrichment). The inclusion of these factors implies a greater complexity of the experimental design and statistical analysis, and it can produce higher variability of the outcome measures, rendering it more difficult to highlight the effect of the toxicant in study. However, it would increase generalizability of the results and thus give greater translational value to the study performed.

One important opportunity of modeling developmental neurotoxicity is the possibility to discriminate different size effects in association with different dosages. This allows to describe more than one scenario of human exposure and to detect dose-effect response: from the dosage that triggers very subtle effects to the limit dose which still allows reproduction and viability of individuals though in the presence of impairment in other domains.

Use of genetically-modified mouse lines carrying common and potentially functional polymorphisms is recommended to verify the impact of contaminants, always considering the matter of validity when a particular genetic model is chosen. Especially with the aim of testing susceptibility and in particular its gene-environment basis, the use of genetic model often makes disease phenotypes stable and well defined, which offer low resolution for the effects of environmental factors added in the experimental setting to study interaction. The use of heterozygous lines is more promising in this perspective as well as natural strains harboring specific polymorphism. They model a human genetic condition that is more frequent and more difficult to be detected (being often asymptomatic) than homozygosity, but still may result in increased vulnerability to toxicant effects with respect to wild-type strains.

Dealing with experimental models of NDDs the “time” factor should be considered twice, both in terms of critical time windows of susceptibility and in terms of age of the subjects involved in the study. Indeed, there are many cases of critical periods of vulnerability of developing organisms (during prenatal phase, infancy, adolescence) to so called “sub toxic” doses of environmental contaminants, chlorpyrifos being a paradigmatic example in this context [240].

Age of the subject is also a critical factor, and since by definition NDDs refer to developing individuals, the neurobehavioral outcome should be assessed at different life stages, obviously including neonatal and/or juvenile period to identify early alterations potentially predictive of later behavioral deficit.

In our experience, the selection of behavioral tests to be used in such studies should include different domains (cognitive, emotional, social profiles) to increase both the total informative value of such analysis (generalized vs. more selective effects) and level of comparability with the neuropsychological test batteries used to assess infants/children. Again in this framework the requirement of early behavioral assessment is mandatory, to fully integrate the results of in vivo studies in animal models with those of epidemiological birth cohort studies on the role of neurotoxicant in NDDs.

In parallel with the characterization of the behavioral effects of the chemical in study, omic biomarkers (microRNAs, DNA methylation, and protein and metabolite levels) should be measured in peripheral organ/tissues such as the blood and in the brain at different time points, to evidence parallel changes and possible differences, eventually to validate the peripheral biomarkers as indexes of a CNS disorder. Once the effect of the contaminant in the genetic model has been characterized in multidose experiments, one may verify the effects on the behavioral outcome of adding to the chemical in study a “non-chemical” stressor such as mild stress in pregnancy or maternal inflammation.

A last methodological caveat concerns the experimental design and related statistical methods: in experiments on developmental toxicology, pups are usually exposed to toxicants through the mother either prenatally (in utero) or postnatally (via lactation). In both cases, neurotoxicants, that are primarily administered to mothers, can affect the offspring neurodevelopment not only directly by reaching the target systems (nervous or neuroendocrine), but also indirectly, interfering with dam’s maternal care in both qualitative and quantitative terms [241]. Early experiences of pups have been shown to influence epigenetically the nervous and endocrine development, and hence must be taken into appropriate account to better understand causes and pathways of toxicant effects on developmental outcomes [242]. Experimental design and the statistical analysis of data can address this issue. As for the experimental design, the sample size must be calculated with respect to the litters and not to the individual pups (since littermates that are reared by the same dam cannot be considered as independent statistical units, their response to stimuli being strongly influenced by the maternal environment). In addition, littermates must be balanced as well as possible for all within-litter factors under study (e.g., sex and repeated testing). With respect to the statistical analysis, the litter must be included as a statistical unit for all between-subject factors (such as prenatal treatment(s) and rearing conditions), and as random blocking factor for all within-litter factors (such as sex, and age in case of repeated testing on the same statistical units). Statistical analyses that do not take into appropriate account the litter effect can be strongly biased in whatever direction, potentially leading to either an overestimation or an underestimation of treatment effects: this has been repeatedly pointed out in the past [243,244], but these methodological warnings keep on being often neglected in current literature of NDD mouse models as recently illustrated for prenatal VPA studies [245].

10. Conclusions

Strong evidence exists that environmentally relevant exposure to chemical pollutants at critical developmental stages affects neural and behavioral development in children. Recent advances in research offer important clues into pathogenetic mechanisms of autism and other NDDs, indicating that environmental risk factors cannot be ruled out. Specifically, in NDDs, variations in several candidate genes may confer higher vulnerability to different kinds of adverse environmental stressors, including early exposure to chemicals. Although there is a lot of information derived by epidemiological data that support the adverse effects of many chemicals on child neurodevelopment there are still large gaps in knowledge in this field, as for the mechanisms of toxicity, the estimation of the dose-response relationship and the individual dimension of the exposure history. Furthermore, the large number of studies investigating the role of gene polymorphisms, epigenetic mechanisms, nutritional and socioeconomic factors in the determination of individual vulnerability to chemical exposure has not substantially advanced our knowledge of the complex etiology of NDDs and brain disorders in general.

All this considered, there is an overall need to invest more in discovery research able to fill out the knowledge gaps which lay beneath neurotoxicological hypothesis for NDDs. Along this line, the exposomic approach is aimed at characterizing and quantifying the exogenous and endogenous exposures and modifiable risk factors that predispose to and predict NDDs. A critical issue in such context is the identification and validation of peripheral biomarkers of effects that can inform on typical and atypical brain development, and help to establish biologically plausible links between chemical exposure and health effects. This requires a trans-disciplinary approach bridging several scientific fields such as epidemiology, environmental and exposure science and toxicology, to collect data from different biological sources and organizational levels.

Behavioral toxicology studies might significantly contribute to this innovative research approach, by modeling in “simpler” living organisms the complexity of the human exposure scenarios. Studies with laboratory rodents allow assessment of dose-response relationships, critical periods of susceptibility, and the relative contribution of genetic, epigenetic and environmental factors. More importantly in the neuropsychiatric disease field, the use of the in vivo models permits the selection of omic biomarkers anchored to the behavioral phenotype, which increases their translational value. Thus, in synergy with mechanistic in vitro studies [246], physiologically-based toxicokinetic (PBTK) models [247] and human observational and biomonitoring data, in vivo models may be pivotal to identify candidate biomarkers and pinpoint susceptible groups or lifestages to be translated to large prospective studies within the exposome context.

Acknowledgments

Author Contributions

All authors worked collaboratively on all aspects of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References