Urinary organophosphate metabolites, DNA methylation aging, and heart disease mortality in middle-aged and older adults: an exploratory cohort study with in vitro evidence of biological plausibility

Abstract

Background Chronic low-dose exposure to organophosphorus pesticides (OPPs) is widespread in the general population and has been linked to heart disease mortality, yet the biological pathways connecting such exposure to fatal outcomes remain poorly defined. We examined whether urinary OPP metabolites are associated with heart disease mortality and whether DNA methylation biomarkers might contribute to this association. Methods We studied 502 adults aged ≥50 years from the National Health and Nutrition Examination Survey (NHANES) 1999–2002 cycles with urinary OPP metabolite measurements, DNA methylation profiles, and linked mortality follow-up. Survey-weighted Cox models were used to relate baseline metabolites [per 1-standard-deviation (SD) increase] to heart disease mortality, with sensitivity analyses for exposure definition and confounding. DNA methylation biomarkers that were significant after false discovery rate (FDR) correction were examined in exploratory single-mediator models, with Benjamini–Hochberg correction applied across the five indirect-effect tests, and in a secondary survival random forest. THP-1 monocytes were exposed to chlorpyrifos as a mechanistic probe of pathways suggested by the epidemiological findings. Results Among four OPP metabolites, only diethylthiophosphate (DETP) was associated with heart disease mortality after multivariable adjustment [hazard ratio 1.33, 95% confidence interval (CI) 1.11–1.61 per 1-SD increase, FDR = 0.010], with a positive association across quartiles. After Benjamini–Hochberg correction, DETP showed FDR-significant linear associations with five DNA methylation biomarkers. In separate single-mediator models, MonoPP and ZhangAge showed nominal indirect effects at raw p < 0.05, with estimated proportions mediated of approximately 9.2 and 8.4%, respectively; however, neither remained statistically significant after Benjamini–Hochberg correction across the five mediation models. A survival random forest combining DETP with the five markers achieved a 5-fold cross-validated 20-year area under the curve (AUC) of 0.71 (95% CI 0.66–0.76). In vitro, sub-cytotoxic chlorpyrifos raised pro-inflammatory cytokines, increased DNMT1, suppressed TET2, and induced global DNA hypermethylation. Conclusion In this exploratory analysis, higher urinary DETP was associated with greater heart disease mortality in middle-aged and older adults. MonoPP and ZhangAge showed suggestive indirect effects in separate exploratory models, each accounting for an estimated 8–9% of the association, but neither effect remained significant after FDR correction. Most of the DETP-related risk, therefore, remained unexplained by the measured methylation markers. The in vitro experiments provided hypothesis-generating support—most directly for inflammatory signaling—rather than confirmation of the epidemiological pathway.