The goal of this project is to quantitate DNA adducts in human samples by gas chromatography with electron capture detection (GC-ECD) and with detection by negative chemical ionization mass spectrometry (GC-NCI-MS). High sensitivity is obtained by labeling the adducts with electrophores. For standards of pyrimidine bases and nucleosides, detection limits at the low fg level are currently observed. Overall the methodology consists of the following steps: (1) purification of the DNA; (2) hydrolysis (acid or enzymes) of the DNA down to DNA monomers; (3) separation of lesion (damage) from normal DNA monomers by high performance liquid chromatography (HPLC); and either (4a) chemical labeling of the lesion monomers with """"""""direct electrophores"""""""", followed by characterization and quantitation of these electrophore-labeled monomers by GC-ECD/GC-NICI-MS, or (4b) chemical labeling of the lesion monomers with """"""""release tag electrophores"""""""", followed by characterization using HPLC and quantitation by GC-ECD. First the methodology will be applied, including ongoing work, to several alkyl adducts: O6-alkyl-guanine, O4-alkyl-thymine, O2-alkyl-thymine, and O2-alkyl-cytosine. Next the corresponding hydroxyethyl adducts anticipated to arise from exposure to ethylene oxide will be determined. Finally N6-hydroxymethyl-adenine and protein-(lysyl)-nucleoside will be determined as potential adducts caused by exposure to formaldehyde. This research is needed to help determine the carcinogenic and mutagenic risks of human exposure to chemicals.
