(37h) Pathway Analysis of Combined in Utero exposure to Heavy Metals and Phthalates and Its Association with Child Psychomotor Development

The exposome represents the totality of exposures from conception onwards, simultaneously identifying, characterizing and quantifying the exogenous and endogenous exposures and modifiable risk factors that predispose to and predict diseases throughout a person’s life span. Exposome came as a complement to the human genome; although decoding the human genome increased our understanding of the underlying causes of disease, the genome explains only a percentage of the population burden. Current evidence indicates that environmental factors are equally or even more important than genetic predisposition alone. What is actually critical is the interaction of environmental factors with biological systems. Towards understanding better the causal links among the genome, the environment and disease, unraveling the exposome implies that both environmental exposures and genetic variation are reliably measured simultaneously.

Exposome studies will require novel tools to address the complexity of emerging environmental health issues. Critical for success will be the ability to bring together existing geospatial, environmental, health and socioeconomic data, and to collect new high resolution data using 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 epigenetically active food and environment-related disease 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 associtations 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.

The children (n=148) who are subjects of this study were tested at year 1 and 2 of their life using the Bailey battery of neurodevelopmental testing. Their mothers had been exposed to metals and/or endocrine disrupting compounds during the 2nd and 3rd trimester of pregnancy in Lodz, Poland. Biobanked plasma and urine samples from the mothers were analysed using a combination of LC-MS/MS ToF and NMR. The metabolomics data were further analysed using the MZMine and Genespring bioinformatics platforms to identify the metabolic pathways that seemed to be the most perturbed from the exposure to the mixture of phthalates and metals that was the primary objective of this study. The results were summarized in correlation globe that revealed the most statistically significant associations between the exposure variables as measured using human biomonitoring (in this case, exposure to metals such as Pb, Se, Cu, and tobacco smoke measured by the cotinine levels in blood).

A second correlation globe analysis provided an overview of the observed statistically significant associations between neurodevelopmental health outcomes as measured in terms of the Bayley test scores for different types of neurodevelopmental endpoints (motor development, cognitive function, linguistic skills, verbal expression) and the main metabolic pathways that are perturbed in the sampled population. Coupling the outcomes of the two correlation globes, several key metabolic pathways that are linked to exposure to metals and phthalates on the one hand and to perturbations in normal child neurodevelopment at 1 and 2 years of age on the other have been identified. Exposome-wide associations of metabolic pathway perturbations with adverse outcomes regarding neurological development during early life revealed two main mechanisms through which co-exposure to phthalates and metals during pregnancy (in particular during the second and third trimester) may hamper the normal neurological development of newborns and infants.

One key mechanism is perturbation of oxidative phosphorylation leading to disruption in mitochondrial respiration. The latter has been clinically associated with problems in motor development. ATP overproduction inhibits the activity of one of the enzymes involved in glycolysis, thus blocking sugar catabolism. Disruption in oxidative phosphorylation in the mitochondria is also observed after exposure to metals such as Pb or Hg. This could lead to reduction in mitochondrial energy production during fetal development, affecting negatively its neurodevelopment. Moreover, the overproduction of reactive oxygen species (ROS) due to the presence of metals inside the cell induces oxidative stress. The latter favors the production of pro-oxidant species that may lead to oxidative damage. Redox homeostasis may be disrupted due to exposure to xenobiotics. Overexpression of superoxide dismutase (SOD)-coding genes will result in increased quantities of SOD; the latter is linked to neurodevelopmental disorders due to the overproduction of ROS. Verbal development is linked to the presence of glutathione peroxidase (GPx3) during pregnancy and the presence of GPx1 in the umbilical cord. Finally, motor development is associated with the presence of Se at birth. A possible putative mechanism that explains this is the fact that Se is bound to selenoproteins, which protect the cell from ROS-induced destruction.

Our results are corroborated 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 vitro or in vivo in the literature. Our current results indicate that in vitro testing 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 not to consider factors such as smoking behavior or socio-economic status as confounders. Rather, we were 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.