Epidemiologic studies have linked exposure to ambient particulate matter (PM) air pollution with sub-clinical and clinical cardiovascular disease (CVD). Although PM inhalation also has been linked with increases in inflammatory, oxidative, endothelial, metabolic, and coagulation biomarkers in blood, biomarker discovery and understanding of the molecular mechanisms producing such effects in human populations remain incomplete. We and others have consistently found that PM exposure is associated with altered global and gene-specific methylation measured in peripheral leukocyte DNA, an environmentally inducible and dynamic epigenetic mechanism that controls gene expression. DNA methylation in leukocytes and other tissues also has been associated with CVD, implicating it as a primary molecular mechanism mediating the cardiovascular effects of environmental exposures. However, few studies have examined the epigenetics of PM air pollution. Those that have tend to be underpowered, unreplicated, cross-sectional analyses of candidate gene DNA methylation conducted within environmentally homogeneous, single-city populations of white men, without attention to other factors capable of affecting methylation and in turn, CVD risk. We therefore propose to conduct a two- stage, longitudinal study of associations between PM air pollution, DNA methylation, and CVD risk factors among independent subsets of the exam site- and race-stratified, randomly selected 6% minority oversample of approximately 4,300 Women's Health Initiative clinical trial (WHI CT) participants who had fasting blood draws and resting, standard, twelve-lead electrocardiograms (ECGs) repeated at three-year intervals from 1993 to 2004. Stage 1 will focus on the interrogation, discovery and ranking of >450,000 DNA methylation sites potentially sensitive to PM in 1999-2001 blood samples from 800 of the participants. In up to three blood samples collected serially from the remaining 3,500 participants in 1993-2004, Stage 2 will focus on the longitudinal validation of the ten most PM-sensitive DNA methylation sites identified by Stage 1, the temporal relationship between PM and DNA methylation at those sites, and that between site-specific DNA methylation, CVD risk factors, and CVD. The proposed epigenetic data analyses will be conducted within a phenomics framework, well-powered and appropriately adjusted for both ancestral admixture and multiple comparisons. Findings will be externally validated in the ARIC and NAS cohorts. Generalizable findings will advance understanding of epigenetic mechanisms underlying, and biomarkers identifying susceptibility to PM-mediated CVD risk in pre- and post-menopausal women, younger black and older white men. At the same time, they will support inference to the larger, dynamic population of WHI CT participants from which the study's minority oversample was drawn, one living in U.S. Environmental Protection Agency (EPA) Regions 1-10 and potentially benefitted by the science-based establishment of and compliance with stricter National Ambient Air Quality Standards. PUBLIC HEALTH RELEVANCE: The proposed study will advance understanding of epigenetic mechanisms underlying, and biomarkers identifying susceptibility to PM-mediated CVD risk in a large population of post-menopausal women living in U.S. Environmental Protection Agency (EPA) Regions 1-10, one potentially benefitted by the science-based establishment of and compliance with stricter National Ambient Air Quality Standards. To do so, it will use DNA from peripheral blood leukocytes, a readily collected cell population with an established role in the etiology of CVD and the highest potential for clinical use in identifying epigenetic biomarkers that may ultimately affect preventive or clinical management strategies.