The objective of this project is to develop ultrasensitive methodology for measuring damage to DNA caused by environmental carcinogens. Gas phase electrophores as labeling agents for the damaged DNA will provide the ultrasensitivity when measured by gas chromatography with electron capture detection (GC-ECD) or gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Preliminary results suggest, at least for standards, that damaged DNA monomers (bases, nucleosides, or nucleotides) potentially can be quantitated at the low 10-16 mole level, which would allow only 100 modifications of DNA per cell to be measured in human microbiopsy samples. 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 (damaged) 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.
The specific aims are to apply this new methodology first to model lesion DNA monomers involving methyl/ethyl damage, then to selected benzo(a)pyrene and acetylaminofluorene adducts of DNA monomers, and finally to corresponding damaged DNA polymers. Analysis of DNA extracted from cells treated in tissue culture also will be undertaken. Ultrasensitive analysis is needed to determine the importance of human exposure to low levels of carcinogens. The project involves the scientific disciplines of analytical chemistry and biochemical pharmacology.
