Mass specific discovery of DNA adducts.

B24

Up until the present time, unknown DNA adducts in biosamples generally have been detected with low specificity, impeding their development as biomarkers for cancer, for example to help individualize cancer prevention. We are reporting a new method (IMI-MS) that broadly discovers unknown DNA adducts with high specificity by mass spectrometry. The sensitivity is 1 in 10e8 for an authentic, digestion-spiked dGMP adduct of acetylaminofluorene. In our method, chromatographically-enriched adducts from a nuclease digest of DNA (50-100 µg) are phosphate-specifically tagged with an isotopic pair of benzoyl-histamines and detected, after additional chromatography, by MALDI-TOF-MS. Discovered peak pairs providing accurate adduct masses are confirmed to be modified deoxynucleotides by fragmentation. Examples of adduct data from the method are as follows. Calf thymus DNA exposed in a test tube to cigarette smoke and DNA from smoker lung share the following adducts: 355.067 (etheno-dAMP), 387.093, 401.110 and 441.105. The latter might be N7-phenylfapydGMP (exact mass 441.105) derived from exposure to benzene oxide. Adduct 359.064, that is observed in many DNA samples, may be formyldAMP (exact mass 359.064), a previously undetected lesion that might arise from the reported oxidative intermediate formylphosphate on DNA. In a test tube exposure, p-benzoquinone, a metabolite of the ubiquitous carcinogen benzene, is observed for the first time (perhaps with some propensity) to adduct onto 5-methylcytosine in DNA, in addition to forming known C, A and G adducts. Especially if this BQ-MeC lesion, by perturbing local DNA structure, enhances damage in one way or another to a diagonal MeC deoxynucleotide, then exposure to benzene could erase epigenetic sites. The first direct detection by the method of N⁶-methyladenine in mammalian DNA samples (e.g. human leukocytes) remains to be interpreted as bacterial contamination (uninteresting result), or as further evidence for this compound as the sixth base of DNA (interesting observation). The method is a new tool for studies of both simple and complex (e.g. human) exposures, and encourages the development of a mass registry of DNA adducts. It will help to determine unknown DNA adduct structures, to make precise adduct connections between different exposures, and to formulate and test adduct-related hypotheses. (Supported by CA84641, CA106006, and the Environmental Cancer Research Program. Tissue samples were from NDRI.)