Detection of DNA damage derived from a direct acting ethylating agent present in cigarette smoke by use of liquid chromatography-tandem mass spectrometry.

Cigarette smoke contains a complex mixture of chemicals, including some that are genotoxic. A number of epidemiological and clinical studies have reported the association of increased DNA adduct levels with the development of lung cancer in smokers. The majority of chemicals present in cigarette smoke require cytochrome P450-mediated metabolic activation to form the ultimate reactive species that covalently binds with DNA. We have investigated the presence of a direct-acting ethylating agent present in cigarette smoke by studying the formation of N-7 ethylguanine (N-7EtG) following exposure of DNA to cigarette smoke in vitro. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) was developed for the detection of N-7EtG in DNA. DNA samples were subjected to thermal hydrolysis to selectively release the N-7EtG, which was then quantified by LC-MS/MS MRM using a stable isotope internal standard [15N5]N-7EtG. The limit of detection of the method for N-7EtG was 2.0 fmol injected on column with 100 microg of calf thymus DNA as the matrix (0.6 N-7EtG adducts per 10(8) nucleotides). A linear dose-response was observed for the formation of N-7EtG in calf thymus DNA treated with diethyl sulfate at concentrations ranging from 1 to 1000 microM. Calf thymus DNA treated with smoke generated from 1, 5, and 10 commercially available cigarettes resulted in the formation of 1.3, 3.6, and 8.4 N-7EtG adducts per 10(8) nucleotides, respectively. There was a positive correlation between the formation of N-7EtG and the number of cigarettes (r = 0.9938). These results confirm the presence of an as yet unidentified direct acting ethylating agent in cigarette smoke, which is present at levels that can produce DNA damage that could ultimately have adverse implications for human health, particularly in the case of the development of lung cancer.