Mapping the Cellular Injury Landscape of Acute Ozone Exposure: A Plasma Cell-Free DNA Profiling Study in Humans

Ozone pollution is a growing global health threat, yet current evidence lacks cellular-level resolution to pinpoint its systemic damage. This randomized controlled crossover trial introduces plasma cell-free DNA (cfDNA) as a novel, sensitive biosensor for ozone-induced injury. In 29 healthy adults, acute ozone exposure (282.2 ± 44 ppb) significantly altered cfDNA characteristics: it reduced the proportion of 150–165 bp fragments and increased cfDNA derived from megakaryocytes (21.35%), vascular endothelia (135.10%), and B/NK/T cells (174.69%), revealing these as key cellular targets. Epigenetic analysis revealed increased perplexity at transcription start sites, indicating enhanced entropy and potential transcriptional dysregulation. These cfDNA signatures correlated with systemic physiological changes, including elevated inflammatory markers (interleukin-6, tumor necrosis factor-alpha), oxidative stress (8-hydroxy-2′-deoxyguanosine), endothelial dysfunction (endothelin-1), and lung epithelial injury (Clara cell secretory protein 16). Our findings demonstrate that cfDNA profiling can dynamically reflect ozone-induced cellular damage in the vascular, hematopoietic, and immune systems, moving beyond organ-level assessments. This approach provides a powerful and noninvasive tool for monitoring early systemic effects of air pollution, with implications for understanding mechanistic pathways and developing targeted interventions.