Metabolomic Responses to External and Internal Environmental Exposures: Evidence of Lipid and Energy Metabolism Disruption in the Estonian Biobank

ABSTRACT Objective To investigate associations between long-term environmental exposures, both external (ambient air pollution and built environment) and internal (circulating anthropogenic chemicals), and the human plasma metabolome, with the aim of generating biologically plausible hypotheses about affected metabolic pathways. Methods We analyzed plasma from 989 Estonian Biobank participants using untargeted LC-HRMS (Metabolon HD4). External exposures ( PM 2.5 , PM 10 , NO 2 , ozone and built-environment metrics) were assigned using spatiotemporally resolved models developed in the EXPANSE project. Internal exposures were defined as ubiquitous anthropogenic compounds detected in the same metabolomics dataset. Associations between exposures and individual metabolites were quantified using left-censored regression models and then mapped to metabolite classes (Metabolon) and KEGG pathways. For enrichment analyses, one-sided Kolmogorov-Smirnov tests were applied to external exposures and Fisher’s exact tests to internal exposures. False discovery rate was controlled at 1% per exposure and database. Results External air pollutants exhibited distinct metabolic patterns: Higher NO 2 exposure was associated with enrichment of metabolites involved in tyrosine metabolism; higher ozone with monohydroxy and dicarboxylate fatty acids (consistent with lipid peroxidation); and higher PM 2.5 with acyl-carnitine subclasses and carbohydrate metabolism (glycolysis / gluconeogenesis / pyruvate). Built-environment associations were heterogeneous across metabolites and pathways. Internal anthropogenic chemicals showed broader metabolic associations than external exposures, involving a larger number of metabolites and metabolic classes. PFAS (PFOA, PFOS) were associated with long-chain polyunsaturated fatty acids (n3/n6) and lysophospho-lipids. Associations with 4-hydroxychlorothalonil, a fungicide, pointed to androgenic steroid metabolites and alpha-linolenic acid metabolism. The phenolic 2,4-di-tert-butylphenol, a plastic associated chemical, showed widespread associations with lipid classes, suggesting disruption of membrane remodeling and fatty acid handling. Conclusion Long-term environmental exposures, both external and internal, are measurably reflected in the human plasma metabolome. Across exposure domains, recurrent signals involved lipid metabolism, membrane composition, and oxidative stress–related pathways, highlighting these as common biological targets of environmental exposures. The findings generate testable hypotheses, including nitrosative stress–related alterations for NO 2 , lipid peroxidation for ozone, energy-metabolism perturbations for PM 2.5 , potential endocrine activity for chlorothalonil metabolites, and possible obesogenic effects of 2,4-di-tert-butylphenol.