(720b) Mechanistic Assessment of the Effect of Phthalates and Heavy Metals on Neurodevelopment

Authors: 
Sarigiannis, D., Aristotle University of Thessaloniki
Papaioannou, N., Aristotle University of Thessaloniki
Fafouti, M., Aristotle University of Thessaloniki
Dickinson, M., Fera Science Ltd
Polanska, K., Nofer Institute for Occupational Medicine
Gabriel, A., Aristotle University of Thessaloniki
Karakitsios, S., Aristotle University of Thessaloniki
Exposome studies will require novel tools to address the complexity of emerging environmental health issues. In this sense, they have to bring together existing geospatial, environmental, health and socioeconomic data, as well as collection of new high-resolution data. Toward this aim, several technological advances have to be employed, including innovative environmental micro-sensors, remote sensing or other community and omics/systems biology based approaches to describe the exposome for e.g. endocrine disruption-related syndromes and sex-related changes (menopause), neurodevelopment, neurodegenerative or respiratory diseases. Mapping the entire lifecycle of an individual may not be necessary if critical lifetime events where an individual’s geospatial lifeline crosses a noteworthy environmental event are recognized and understood. Whereas exposure during all life stages may entail adverse effects, children, pregnant women and the elderly are particularly susceptible. Thus, focus will have to be on susceptibility windows during growth (including pregnancy) and development, and on the unequal distribution of the burden of disease associated with dietary and environmental stressors to vulnerable populations such as the young, elderly, socio-economic disadvantaged, gender and ethnic minorities; thus these population sub-groups have been the focus of this study.

Large-scale exposome projects like HEALS (Health and Environment-wide associations via Large population Studies) try to reverse 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. Data collected in on-going epidemiological studies across the world involving mother/infant pairs, children, or adults including the elderly have been analysed to evidence relevant environmental exposure/health outcome associations. These associations aid in designing pilot surveys using an integrated approach, where the combined set of selected biomarkers of exposure, effects and individual susceptibility results in integrated risk assessment. Environmental conditions that can affect the epigenome of an individual include both external and internal factors. Individual behaviors such as smoking and alcohol consumption, physical activity, dietary intake, temperature changes and stress are external factors that have been proposed to have a long-term influence on epigenetic modifications. However, it is possible that small defects in transmitting epigenetic information through successive cell divisions, or maintaining it in differentiated cells, accumulate in a process that could be considered as an ‘‘epigenetic drift’’ associated with aging.

In the frame of HEALS we have been applying the exposome connectivity paradigm on several pre-existing cohorts re-analysing biobanked samples using a full multi-omics platform coupling transcriptomics and metabolomics and applying integrative bioinformatics and exposome-wide association algorithms to draw links between combined exposures to metals and endocrine disrupters and metabolic pathway dysregulation, as well as between metabolic pathway perturbations and clinically observed phenotypes of neurodevelopmental disorders such as problems in linguistic, motor development and cognitive capacity. Within this study, results from two cohorts (ReproPL and PHIME) relevant to the study of neurodevelopmental disorders are presented.

In the ReproPL cohort study, the neurodevelopmental progress of 148 children in Lodz, Poland was assessed at year 1 and 2 of their life using the Bailey battery of neurodevelopmental testing. Their mothers exposure to heavy metals and endocrine disrupting compounds has been assessed during the 2nd and 3rd trimester of pregnancy. Biobanked plasma and urine samples from the mothers were analysed using a combination of LC-MS/MS ToF and NMR.

In Phime cohort study, the neurodevelopmental exposome paradigm was applied on a 178 mother-infant pairs cohort. Heavy metals prenatal exposure was determined by measuringmercury in hair samples collected at birth, while cord blood and breast milk samples were analysed for mercury, cadmium, lead, and arsenic, as well as for essentialelements (selenium, zinc, copper). Cognitive function, language, andmotor development were assessed in children at the age of 18 months by the Bayley Scale for Infant Development (Bayley-III) development tool.

In both cohorts, the individual-level biological profiles were characterized using both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) for the untargeted urinary and plasma metabolomics analysis. Integrated pathway analysis and exposome-wide association algorithms were used for the evaluation of the associations between in utero exposure to metals and metabolic pathway dysregulation, as well as between metabolic pathway perturbations and neurodevelopment.

In order to generate a list of candidate biomarkers for the stressors of interest, which were identified by untargeted analysis of metabolic profile, a literature search was performed including combinations of the following terms: ‘’co-exposure’’, ‘’phthalates’’, ‘’metals’’, ‘’neurodevelopment disorders’’, ‘’biomarkers’’ and ‘’underlying mechanisms’’. The query was perform for both levels of analysis (molecular biomarkers and pathway analysis) for all the experiments. It was designed not only to determine candidate biomarkers based on their known biological function, but also to avoid the exclusion of a biomarker, which is associated with the occurrence of a stressor, even though there is yet paucity of knowledge regarding its exact biological function.

The imbalance between the cellular reactive oxygen species (ROS), which may be an effect of exposure to phthalates and metals, and the unability of the cell to detoxify them, leads to oxidative stress. According to literature, abnormalities in the citric acid cycle, urea cycle, and amino acid metabolism play a key role in the pathogenesis of oxidative stress. Glutathione, methionine, cysteine, pyruvate, N-acetylglutamic acid, β-alanine, serine, and arginine, which have been detected in samples from ReproPLand PHIME cohort studies, could play the role of candidate biomarkers for the neurodevelopmental disorders due to oxidative stress. In addition, the perturbations of the identified pathways, for the homeostatic operation of which the presence of the above biomarkers is crucial must be examined as a putative underlying mechanism. Some of these identified pathways are: S-methyl-5-thio-Alpha-D-ribose 1-phosphate degradation, folate metabolism, serotonin degradation, taurine biosynthesis, citrulline-nitric oxide cycle etc.

Our results corroborate by previous studies that have shown changes in lipid concentration in the brain after exposure to phthalates, when the former are linked to oxidative stress. Additional metabolic pathways the induction of which during the third trimester of pregnancy is statistically associated with clinically observed neurodevelopmental disorders in the first two years of the child’s life based on our results include the following:

  • citrulline nitric oxide cycle,
  • urea cycle,
  • cysteine biosynthesis/homocysteine degradation,
  • lysine degradation II and
  • purine ribonucleoside degradation to ribose 1 phosphate

So far no experimental validation of the induction of these specific pathways and neurotoxicity exists in vitroor in vivoin the literature. Our current results indicate that in vitrotesting coupled to targeted metabolomics on metabolically active relevant cell lines could be a viable way forward towards providing the mechanistic evidence of the observed exposome-wide associations.

Two are the main characteristics that stand out in this first-of-a-kind study:

(a) The fact that we have chosen to consider factors such as smoking behavior or socio-economic status as additional parameters associated with child neurodevelopment. Moreover, we were particularly interested in capturing the overall metabolome perturbation and associate it with (i) the totality of environmental exposure factors we could quantify; and (ii) clinically observed neurodevelopmental disorders. The scope of the study was to investigate how the perinatal and early-life exposome affected child neurodevelopment.

(b) Exposome-based associations of clinical or sub-clinical health outcomes with the early-life external and internal exposome supports the elucidation of the mechanisms through which xenobiotics interact with and eventually perturb cell metabolism to induce specific pathways of toxicity in infants and young children.

The connectivity approach to environmental health, builds upon the connectivity across different biological scales in a systems biology approach to elucidate the mechanisms underlying the environmental burden of disease. Adopting a data-driven paradigm that is guided by systems biology principles this approach couples comprehensiveness in exposome and health associations and biological plausibility. Thus, it can unravel causal links between environmental exposures, genetic predisposition and early biological markers of adverse health outcomes without needing to revert to very large population sizes.