Carcinogens generated in tobacco smoke (TS) are able to cause DNA damage and mutations that may initiate lung carcinogenesis. Intriguingly, despite the presence of substantial amounts of DNA damaging agents in TS, only 10-15% lifetime tobacco smokers develop lung cancer. Although TS has been found to cause mutations in many genes related to lung cancer, a definable cause-effect relationship has not been established. The p53 and K-ras genes are two frequently mutated genes in TS-related lung cancers. Mutational features in these two genes in the lung cancers of smokers and non-smokers are different. Using a cultured human lung cell system as a model, we have made three discoveries: TS carcinogens preferentially form DNA adducts at p53 mutational hotspots and at codon 12 of the K-ras gene, adducts formed at these sequences are poorly repaired, and most of p53 mutational hotspots occur at sites that contain a CpG sequence and C5 cytosine methylation causes preferential adduct formation at these sites. These findings led us to hypothesize that TS-induced DNA damage plays a central role in lung carcinogenesis. In lung cells, both the cytosine methylation status in the p53 gene and an unknown epigenetic factor in the K-ras gene may determine an individual's susceptibility to TS-induced DNA damage. We recently found that nickel, chromium, and lipid peroxidation metabolites can greatly reduce cellular DNA repair capacity. Since TS contains significant amounts of these heavy metals and also induces excessive oxidative stress, it is possible that TS may cause inhibition of DNA repair. We propose that variations in every individual's response contribute to the differences in susceptibility to TS-induced lung cancer. TS may induce different levels of one, DNA damage at genes crucial for developing lung cancer, such as p53and K-ras genes, and two, inhibition of DNA repair capacity among different individuals. To test these hypotheses we proposed to determine three factors in the lung cells of tobacco smokers with and without lung cancer: one, DNA damage distribution in the p53 and K-ras genes, two, C5 cytosine methylation status in the p53 gene, and three, the repair capacity. We will also determine the mutations in genes related to lung cancer in both tumor and "normal" cells in these lung tissue samples. Finally, we will determine the epigenetic modification in the K-ras gene that causes preferential carcinogen binding and poor repair at codon12 of this gene. Results from these studies will enhance our understanding of lung cancer susceptibility and DNA damage-induced carcinogenesis in humans, and enable us to develop biomarkers.