Methodology is proposed to enhance the usefulness of Randerath's successful 32P post labeling procedure for detecting DNA adducts. This enhancement will consist of a technique to shortcut the identification of unknown adducts encountered by the 32P post labeling procedure. The steps in this proposed new identification technique are as follows: (1) excise the unknown spot from the autoradiographic TLC sheet, (2) extract the unknown adduct, (3) chemically digest the adduct to release the foreign chemical, (4) derivatize this chemical with an electrophore, and (5) obtain some structural information about the resultant derivative by gas chromatography/mass spectrometry (GC/MS) techniques. In particular, negative chemical ionization mass spectrometry (NCI-MS) and electron capture detection (ECD) will be used to provide high sensitivity. Current detection limits by GC-NCI-MS/ECD reach amounts near 10-18 mole, providing greater than 10-3 sensitivity over the greater than 50 fmol amounts per TLC spot of many unknown aromatic DNA adducts currently encountered by Randerath's technique. Initial adducts to be investigated are those from benzo(a)pyrene, DMBA, MBA, AAF and 2-nitrotoluene. Also this new methodology will be applied to aliphatic adducts arising from exposure to methylating agents, ethylating agents, and ethylene oxide. For both aromatic and aliphatic adducts, there is a long term potential for the methodology to define the total dose of a nonradioactive chemical to human DNA in a single procedure, and perhaps even give an ultrasensitive fingerprint of damage to human DNA from many chemicals. The proposed work is intended to speed up advances in quantitative risk assessment. Kurt Randerath at Baylor, and Douglas Rickert and James Swenberg at CIIT, are the collaborators on this project.
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