Combined Effect of Per- and Polyfluoroalkyl Substances and Metals on Epigenetic Aging.

Environmental contaminants such as per- and polyfluoroalkyl substances (PFAS) and toxic metals have been implicated in biological aging, yet their combined effects remain poorly understood. This study evaluated the associations of PFAS, lead, and cadmium mixtures with multiple DNA methylation-based measures of epigenetic aging in a nationally representative sample of U.S. adults aged ≥ 50 years. Data were obtained from the 1999-2000 and 2001-2002 National Health and Nutrition Examination Survey (NHANES). The analytic sample included 1119 participants with available data on seven PFAS, blood lead, cadmium, and DNA methylation measures. Epigenetic aging outcomes included HannumAge, HorvathAge, SkinBloodAge, PhenoAge, GrimAge, and DunedinPoAm. Multivariable linear regression and Bayesian Kernel Machine Regression (BKMR) were used to assess individual and joint exposure-response relationships. Cadmium showed the most consistent positive associations with epigenetic aging measures, particularly for the second-generation clocks PhenoAge and GrimAge. Lead was positively associated with GrimAge, while PFAS showed more variable and generally weaker associations, with PFNA demonstrating the most consistent signal. Mixture analyses indicated that higher combined exposure levels were associated with higher DNA methylation age estimates, with stronger patterns observed for second-generation clocks. These findings suggest that combined exposure to PFAS, lead, and cadmium is associated with higher epigenetic aging in older U.S. adults, with cadmium emerging as a key contributor to the observed mixture effects. Evaluating environmental exposures as mixtures may provide important insight into how co-occurring contaminants jointly influence biological aging.