Objectives:
The overall aim of this WP is to quantify the link between exposures to metals, pesticides and chemical compounds with endocrine disrupting activity (i.e. polyhalogenated aromatic hydrocarbons, phthalates and PCB/PBDE) and neurodevelopmental disorders, by applying the HEALS methodological framework. Specific objectives are to:
- Perform data analysis, harmonisation and identification of major gaps in knowledge using the cohorts available in the HEALS consortium that focus on environmental exposures and their effects on neurodevelopment;
- Assess the spatial relationships between environmental exposure and health data from the different cohorts;
- Assess internal exposure, investigate mechanistic hypotheses (through omics and other methodologies from Stream 2) and identify robust biomarkers of exposure, effect and susceptibility;
- Apply the HEALS methodology to validate a model strategy for establishment of causal links between exposure and health effects as for neurodevelopmental disorders in general population and twins studies.
Description of work and role of partners:
Concern is increasing on the impact that prolonged exposure to apparently non-toxic doses of neurotoxicants during early development may have on children’s health, possibly representing a major risk factor for neurodevelopmental disorders (NDs). These conditions range from clinical overt cognitive and behavioural abnormalities, such as Autism Spectrum Disorders (ASD) and Attention Deficit Hyperactivity Disorders (ADHD), to subtle neuropsychological deficits. The aetiology of most of NDs is unknown, but there is general agreement that they result from complex interaction between multiple genes conferring vulnerability (i.e. polymorphisms or defect in genes involved in metabolism of environmental toxins) and adverse environmental factors. Exposures, whether chemical, physical, or microbiological, may act directly upon neural cells, interfering with critical processes of proliferation, migration, differentiation, synaptogenesis, programmed apoptosis and myelination, or may affect the expression of genes that regulate such embryonic/fetal/infant processes. They operate during sensitive periods of gestation by disrupting endocrine systems and altering the hormonal milieu necessary for fetal brain development. Moreover, environmental exposures could contribute to a dysregulated immune system that interacts at the molecular level with glial cells and astrocytes, creating a neuroinflammatory condition.
Several epidemiological studies mainly carried out in the USA have identified a number of chemical compounds that might affect neuropsychological development in exposed infants.They include heavy metals, organophosphate pesticides, organohalogens, and organic compounds with endocrine disrupting activity. In vivo and in vitro experimental studies have confirmed that many of these compounds have significant developmental neurotoxicity at low, environmentally relevant doses, but so far experimental data need to be confirmed in focused clinical studies. Large prospective studies beginning before or during early pregnancy are needed to assess the relative importance of parental and childhood exposure. In addition large case-control studies may help to assess the role of environmental exposure and their interaction with genetic vulnerability in relatively uncommon outcomes such as ASD: in the USA, federally-funded trials are currently carried out to evaluate the genetic and environmental contributions to ASD etiology taking into account environmental exposure, internal exposure, genetic background and biomarkers potentially associated to CNS functioning (beincharge.ucdavis.edu).
The review of existing literature will help identifying gaps in the knowledge of link between exposures to metals, pesticides and chemical compounds with endocrine disrupting activity and neurodevelopmental disorders. Additional inputs that will be of help also in the implementation of WP15 will result from the European commission-funded project DENAMIC “Developmental Neurotoxicity Assessment of Mixtures in Children” that is investigating neurotoxic effects of low-concentration mixtures of pesticides and a number of common environmental pollutants in children on (subclinical) effects on learning (cognitive skills) and developmental disorders in children (e.g. ADHD, autism spectrum disorders and anxiety disorders). Of interest for HEALS will be the results of two distinct DENAMIC pillars:
– The first pillar involves hazard characterisation for neurotoxic chemicals and (environmentally relevant) mixtures thereof. Novel tools, testing methods and procedures for screening (mixtures of) chemicals for neurotoxicity are developed, together with improved assessment methods for exposure and effects.
– In the second pillar (exposure/epidemiology), perinatal and early-childhood exposure will be studied in maternal urine and cord blood, as well as breast milk and urine of the child. In the epidemiological part these developed integrated tools are tested in a tailor made cohort of mother/child pairs focusing on learning and developmental disorders, including the onset of ADHD. In both the experimental and epidemiological part of DENAMIC, biomarkers for developmental neurotoxicity will be developed and/or validated by using innovative biotechnology tools.
An important aspect of DENAMIC that will be of utility in HEALS is the development of biomarkers for (developmental) neurotoxicity in animal models using (epi-)genomics, proteomics and metabolomics as HEALS is not going to conduct ex vivo and in vivo studies in mouse and zebra-fishes. The links with DENAMIC will be established through collaborations, exchanges, publications and other initiatives including common initiatives like a workshop. The fact that two of HEALS partners are also DENAMIC partners will help in liaising.
In HEALS, the neurodevelopmental toxicity of metals/metalloids, pesticides and organic compounds with EDC activity will be studied on mother-child cohorts where both exposure (fetal and/or neonatal) and neuropsychological outcomes have been measured. They include the EDEN cohort (France, n= 1,200), the ReproPL cohort (Poland, n = 400), the PHIME cohort (Slovenia and Croatia, n = 675), and the JSI cohort (Slovenia, n = 600) for a total of 2,875 children in whom exposure assessments have been or will be performed. Exposure assessment will include biomarkers of exposure as well as exposure to environmental pollutants (e.g. flame retardants, plasticizers, perfluorinated compounds, metals) or their sources that are known neurotoxicants. As both pesticides as well as some of these environmental contaminants may very well have a similar mechanism in common, possible mixture effects via human exposure will also be included in HEALS. Neurodevelopmental disorders and related environmental stressors will be also investigated in pre-existing twins registries when appropriated data will be available.
We will start with a review of the cohorts, to identify significant gaps in knowledge with reference to the framework established in Stream 1 and with methodologies and criteria developed in Stream 3 and 4. On this basis, we will then select one or more cohorts in order to build a model strategy to clarify the link between the chemicals in study and the increased risk of neurodevelopmental disorders in general population. The results of large biomonitoring studies performed in children will be also consulted to obtain EU reference values for the compounds in study. In this work, as previously said, results from the EU DENAMIC project will be taken into account.
Methods perfected in the previous WPs (-omics; multi-media, pharmacokinetics and systems biology modelling) will be used to define mechanistic associations between environmental stressors and adverse effects on neuropsychological functions, as assessed by validated test batteries (mental and psychomotor scales).
The work in WP15 will be decomposed in the following four tasks:
Task 15.1 Data analysis, harmonisation and identification of major gaps in knowledge (ISS, JSI, UPMC, NIOM, FERA, CSIC, URV)
Revision of the data available in the cohorts, considering strengths and weaknesses of each epidemiological study (sample size, quality of exposure assessment, control of confounders, methods to evaluate outcomes, etc.) also according to the recent by White et al. that provides an up-to-date strategy for the assessment of brain function in longitudinal cohort studies of children.
Harmonisation in descriptions and formats will be necessary, while accounting for the ethical aspects of sample and data sharing according to informed consent, local ethical approval and the governance structures of each HEALS partner. Identification of major gaps in knowledge and acquisition of missing data (i.e. georeferencing of the place of residence if not performed yet) will be carried out for all cohort participants.
Task 15.2 External exposure assessment (USTUTT, VTT, TNO, FERA, CSIC, OIKON, URV, KCL, NIPH, SDU, AUTH, UPMC)
The spatial relationships between environmental and health data will be analysed. In close connection with Stream 3 partners, the availability of time-related and cumulative environmental exposures (air, soil, water) to metals/pesticides/EDC will be checked. If necessary (time mismatch, coarse spatial scale, etc.), new modelling will be envisaged. Wherever they exist, reference to national and/or regional registries of neurodevelopmental disorders (autism, ADHD, learning disabilities) may help to establish a link between prevalence of specific disorders and exposure data. The organisation (fusion/integration) and easy updating of these complex space/time environmental and health databases will be made possible through the HEALS database platform developed in Stream 4.
Task 15.3 Internal exposure assessment (ISS, AUTH, TNO, KCL, SDU, NIPH, URV, UPMV)
Critical data analysis and -omics analyses in existing samples. With the support of Stream 2 partners, available biological samples will be selected for omics and biomarker analyses where possible for those cohorts presenting the most robust measures of health outcome (or: for those where testing is still on-going or has to be replicated, as to obtain the most reliable indication of children’s recent exposure profile). Attempts will also be made to reconstruct exposure concentration through pharmacokinetic modelling (reverse dosimetry), to understand the effects of mixtures in in vitro and possibly in vivo models, and to identify biomarkers of effects linked to nervous system dysfunction (DNA methylation, metabolomic, lipidomic). Levels of internal exposure reported in the examined cohorts will be compared with existing data inventories as well as with the results of previous biomonitoring studies performed in EU for the chemicals in study. For each of the chemicals to which mothers and their children are exposed, exposure concentration will be reconstructed using PBBK models using the method developed in WP6 for better estimation of toxicant levels at critical brain sites. Analysis of cohort data should identify exposure biomarkers able to capture the temporal dimension of the exposure (e.g., prenatal versus postnatal and/or acute versus chronic). Biomarkers of effects (clinical parameters, -omics, etc.) indicative of significant neurobehavioural alterations may help to bridge the gap between internal dose and altered structure/function. The problem of co-exposures will be also properly addressed in data re-analysis and as much information as possible will be extracted to derive hypothesis as for co-exposures and synergic/multiple health effects. Finally, cohorts where both measures of gene polymorphisms and environmental factors have been already obtained for a high proportion of study subjects, such as ReproPl and PHIME (with similar clinical parameters and outcomes obtained) will allow assessment of impact of genetic susceptibility in vulnerability to environmental risk.
Task 15.4 Application of the HEALS methodology to population studies (ISS, UPMC, KCL, NIPH, SDU, OIKON, CSIC, URV, FERA, UM, NIOM, AUTH, USTUTT)
Multi-level multi-exposure analyses will be carried out to model the relative influence of internal and external exposures on neuropsychological outcome. Thus, we will draw upon the two exposome components (internal and external exposures) to predict biomarker levels from environmental and domestic exposure sources, and to establish a causal link between biomarkers and disease risk. The resulting association will be applied in the pilot EXHES study performed in WP17.