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 (PM2.5, PM10, NO2, 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 NO2 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 PM2.5 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 NO2, lipid peroxidation for ozone, energy-metabolism perturbations for PM2.5, potential endocrine activity for chlorothalonil metabolites, and possible obesogenic effects of 2,4-di-tert-butylphenol.

The authors have declared no competing interest.

This work was supported by the EXPANSE and EXPOSOME-NL projects. The EXPANSE project is funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No 874627. The EXPOSOME-NL project is funded through the Gravitation program of the Dutch Ministry of Education, Culture, and Science and the Netherlands Organization for Scientific Research (NWO grant number 024.004.017). J.K, L.H and T.E were supported by the Estonian Research Council grant PRG1291. J.K and L.H were supported by the Estonian Research Council grant PRG3105. The research was conducted using the Estonian Center of Genomics/Roadmap II funded by the Estonian Research Council (project number TT17). Data analysis was carried out in part in the High-Performance Computing Center of University of Tartu.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Individual level data analysis in the Estonian Biobank participants was carried out under ethical approval 1.1-12/1468 from the Estonian Committee on Bioethics and Human Research (Estonian Ministry of Social Affairs), using data according to release application 3-10/GI/31961 from the Estonian Biobank.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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↵+ Estonian Biobank research team: Andres Metspalu, Lili Milani, Tõnu Esko, Mait Metspalu

Data used is person-level data of Estonian Biobank participants, which cannot be published by the Estonian law. All data access to the Estonian Biobank’s data must adhere to the informed consent regulations established by the Estonian Committee on Bioethics and Human Research. Access to the datasets analysed in the current study can be arranged on a reasonable request to the Estonian Biobank. To initiate a request for phenotype data, it is necessary to submit a preliminary request to the Estonian Biobank releasesut.ee. Information about data access, including necessary steps required to access data on the University of Tartu servers can be found at https://genomics.ut.ee/en/content/estonian-biobank.