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The long term goal of this project is to set up a novel, practical, omic-type assay for food carcinogens that becomes widely employed. By "omic-type assay" we mean that this assay detects a diversity of carcinogens, both known and unknown, in a single procedure. In this assay, a bait molecule is employed to trap food carcinogens, in order to make them very detectable by mass spectrometry. The Supporting Objectives for ths project are as follows. Objective 1.Prepare an easily-harvested, response-normalizing, highly-sensitive, modified DNA nucleobase to function as a bait for carcinogens. Objective 2. Set up a sample preparation procedure permitting the detection of a model, spiked, pre-adducted nucleobase bait in overcooked meat. Objective 3. Apply the method to overcooked meat and detect multiple carcinogens.
Methods and Feasibility.
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To achieve Objective 1: A guanine-bait compound will be synthesized. Purification of the intermediate compounds and final product will be accomplished by crystallization, precipitation, or chromatography (TLC, flash, or preparative HPLC). Structures will be confirmed by mass spectrometry or NMR. The conditions for the synthesis will be adopted from those used in the literature to convert guanine to N2-acylguanine products. We will take advantage of our experience in progressively replacing the bromo groups on α,α´-dibromo-o-xylene with triethylamine and a nucleomonomer.
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Feasibility goal for Objective 1: The quanine-bait compound is synthesized and readily detected (< fmol level) by MALDI-TOF-MS.To achieve Objective 2, we will form a pre-adducted guanine bait by reacting our guanine-bait compound with methyl iodide-d3 to methylate the guanine moiety (no doubt mostly on the N7 and N9 positions). A hamburger will be fried in canola oil until well done (noting the time and heat setting), cut into pieces, transferred to a blender with a buffer that is compatible with S9, spiked with the pre-adducted guanine-bait, and placed on a shaker. This will be followed by addition of magnetic streptavidin beads (from Thermo-Pierce) and further shaking. The streptavidin beads will be harvested with a magnet, washed with buffer (initial buffer may contain Tween to reduce nonspecific binding) and then water. After drying, the pre-adducted guanine-bait will be eluted from the beads with warm aqueous acetonitrile. An aliquot will be combined with CCA matrix for detection by MALDI-TOF/TOF-MS.
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Feasibility goal for Objective 2: the pre-adducted guanine-bait is readily detected (< pmol level) after spiking into a homogenized, cooked hamburger.
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To achieve Objective 3, we will take advantage of the conditions that emerge from our work on Objective 2. The same kind of assay will be performed, including the addition of the above pre-adducted guanine-bait as one internal standard, and the proximate carcinogen benzo[a]pyrene-d12 as model carcinogen. Guanine-bait will be added as well, along with human liver S9 microsomes (obtained from Life Technologies).
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Feasibility goal for Objective 3: Multiple (n > 5) carcinogens (defined here as genotoxic chemicals) are detected in cooked hamburger in a single procedure.