ABSTRACT Aging is the main risk factor for many age-related lung diseases, including COPD and pulmonary fibrosis. It has been speculated that this aging-disease relationship is due to the complex interaction of multiple genetic, epigenetic and transcriptomic alterations that occur in normal human lung over time. In turn, these age-related changes are impacted by environmental factors, such as cigarette smoking. Unfortunately, little is known about age-related molecular alterations in human lung. To some extent this is due to the lack of methods to analyze human tissues for changes occurring stochastically, i.e., affecting individual cells or groups of cells in different ways. The best example is genome instability, one of the hallmarks of aging. Genome instability can be defined as changes in the genetic code, varying from large chromosomal aberrations, to smaller deletions, insertions and copy number variation, and base substitution mutations. The underlying causes of genome instability are errors during cell division, DNA replication or DNA repair of DNA damage. Such changes are irreversible and are in striking contrast to DNA damage, which can be repaired. DNA damage involves physical alterations in DNA structure, e.g., breaks, depurination, depyrimidination, crosslinks, modified bases. Unfortunately, mutations in normal tissues are different from cell to cell and very difficult to study, except using single cell approaches as we will do in the proposed project. However, DNA damage induced in the lung during aging also evokes a DNA damage response, which can lead to multiple epigenetic alterations that are consistent from cell to cell and can be detected in bulk DNA. In addition, a stochastic pattern of age-related changes is likely to occur during aging, the detection of which requires a single cell approach. Here we propose to comprehensively analyze human lung bronchial epithelium for both stochastic and adaptive changes in the genome, epigenome and transcriptome. This project will integrate these comprehensive spectra of genomic and epigenomic change of normal human bronchial epithelium in relation to age and tobacco smoke exposure. This will provide reference data for future comparisons of lung disease states, enhance our understanding of age-and smoking-related mechanisms of lung disease, and thereby facilitate the development of early detection, prevention and therapeutic strategies.