(175bd) An Interdisciplinary Framework Based on High-Dimensional Biology for a Comprehensive Understanding of the Effects of Environmental Stressors on Non-Communicable Diseases

This study aimed to establish an interdisciplinary framework for a comprehensive understanding of the effects of environmental stressors on chronic non-communicable diseases, including neurodevelopmental disorders. It highlights the significance of systems biology approaches for characterizing the internal exposome. The developed framework was employed across three birth cohorts to investigate the impacts of prenatal exposure to real-life mixtures of metals, phthalates/DINCH, organophosphates, and organochlorines on fetal and infant development, with a specific focus on developmental neurotoxicity. The selected chemicals were chosen based on their scientific and regulatory significance, given their potential adverse effects on early childhood development and their ability to cross the placental barrier.

The first step was to optimized untargeted LC-MS and NMR metabolomics protocols for preparing and acquiring data from both human and in vitro samples within exposome research, accommodating the use of various instruments like Orbitrap and Q-TOF. This was followed by an exhaustive account of a robust bioinformatics workflow tailored for the data pre-processing and processing of untargeted MS and NMR metabolomics data, utilizing R packages. Subsequently, a workflow for the integration of multi-omics data, encompassing transcriptomics, proteomics, and metabolomics, employing R packages and online platforms was introduced. This sets the stage for exposome data analysis aimed at establishing connections between human biomonitoring, sociodemographic and lifestyle variables, metabolic pathways, and clinical phenotypes. To achieve this, advanced mathematical approaches are employed, including Bayesian kernel machine regression (BKMR) and Environment-wide association studies (EWAS), highlighting the study's innovative approach to comprehensively understanding the impact of environmental exposures on human health.

The methodologies outlined were employed across three distinct birth cohorts: PHIME (Slovenia, n=180), REPRO_PL (Poland, n=148), and EXHES Tarragona (Spain, n=35). These cohorts were integral components of the Health and Environment-wide Associations based on Large Population Surveys (HEALS) project (http://heals-eu.eu/). The HEALS project aimed to formulate an integrated methodology conducive to the thorough investigation of exposure-health associations. This goal was pursued by systematically organizing environmental, socioeconomic, exposure, biomarker, and health effect data, coupled with providing the requisite analytical and computational frameworks to facilitate comprehensive studies.

The study elucidated the intricate relationships between prenatal exposure to a concoction of metals, phthalates/DINCH, and pesticide metabolites and their consequential effects on cognitive, language, and motor developmental scores within the initial two years of life. Results from BKMR analysis notably underscored the pronounced effect of mono-(2-ethylhexyl) phthalate (MEHP) amidst a blend of metals, phthalates/DINCH, organophosphates, and organochlorines on birth height, marked by a Posterior Inclusion Probability (PIP) of 0.86. Further, EWAS analysis shed light on the detrimental influence of prenatal exposure to organophosphates (IMPY) and organochlorine pesticides such as DDD, β-HCH, DDT, and pirimiphos-methyl on birth height. The study also unveiled correlations among various factors including birth weight, height, gestational age, preterm delivery, socioeconomic status, and paternal education, along with intriguing associations between exposure or neurodevelopmental parameters and the metabolism of amino acids and lipids across different metabolic pathways.

To gain deeper insights into the mechanisms underpinning the adverse effects of prenatal exposure to metals and phthalates, a comprehensive multi-omics analysis was undertaken using the HepaRG cell line exposed to mixtures of phthalates (DEHP, DiNP, and BBzP) and heavy metals (Pb, Cd, and Hg), derived from human biomonitoring data from the PHIME and REPRO_PL studies. Utilizing the INTEGRA computational platform, effective concentrations of these chemicals in the liver were extrapolated from human biomonitoring data and applied to the hepatocyte model at both the actual dose and a ten-fold higher concentration. This multi-omics study revealed significant differential expressions in 29 genes, 230 proteins, and 157 metabolites, linking these differentially expressed biomarkers to numerous metabolic pathways relevant to neurodevelopment, such as the TCA cycle, arginine metabolism, and glycolysis. These findings offer valuable insights into the potential mechanisms driving the impact of these chemical mixtures on developmental processes, highlighting the complex interplay between chemical exposures and child development.

Overal, the study conclusively indicates that simultaneous exposure to metals, phthalates, organophosphates, and organochlorines has a profound effect on the Tricarboxylic Acid (TCA) cycle, causing disruptions in mitochondrial function that may lead to developmental neurotoxicity. This interference with the TCA cycle due to the combined impact of metals and phthalates can be traced back to two interconnected mechanisms. Firstly, the combined exposure alters glutamine levels, which in turn affects glutamate expression, ornithine levels, and arginine production. This sequence of changes compromises arginine's essential functions in urea cycle catabolism and nitric oxide (NO) production, ultimately influencing oxygen delivery and adenosine triphosphate (ATP) synthesis in the TCA cycle. Secondly, exposure to specific agents such as DEHP, DiNP, BBzP, Pb, Cd, and Hg leads to a reduction in lactate levels, causing a consequential drop in pyruvate levels. As pyruvate serves as a critical precursor for Acetyl-CoA, its reduction directly affects the availability of Acetyl-CoA in the mitochondria. The identification of certain genes and metabolites, including CPS1, HSD17B6, ADH1A, lactate, arginine, glutamate, glutamine, ATP, and Acetyl-CoA, as potential biomarkers highlights the intricate relationship between exposure to environmental toxins and its implications on cellular and developmental processes.

In conclusion, the integration of methods for characterizing the external exposome into the high-dimensional biology paradigm underscores the importance of a comprehensive approach to elucidate the complex relationships between the exposome and human health. This holistic understanding is pivotal for devising effective disease prevention strategies, performing accurate risk assessments, and developing personalized interventions. Such an approach not only enhances our grasp of the environmental determinants of health but also paves the way for tailored health solutions that can mitigate the impact of harmful exposures and promote overall well-being.