Methods will be developed and validated fore quantifying human exposure to potent dietary mutagens. We will focus on amino- imidazoazaarenes (AIAs), chemical mutagens formed in meats by moderate temperature cooking and therefore widely consumed in the American diet. These chemicals are potent mutagens in the Ames/Salmonella bacterial mutagenesis assay and, where they have been tested in long-term bioassays, are carcinogens. It is only in the past 5-6 years that a subset of these compounds have been isolated, identified and synthesized in sufficient quantities for studies on their mechanisms of action. The next logical step in determining the human health risk posed by these chemicals requires quantification of their levels in the diet. In many ways the exposure to cooked-meat mutagens is prototypic of environmental and occupational exposures to aromatic amines. There is chronic low level exposure to low doses of a complex mixture of chemicals for which only a subset of the biologically active ones have been identified structurally. Our studies will provide a comprehensive approach for using immunological methods for chemical dosimetry in a complex mixture where the biologically active molecules are present in the part per billion level. We already have monoclonal antibodies (isolated and partially characterized during the first 2 years of the grant) that recognize specifically 3 of the metagenic AIA compounds and a set of others that are class specific. Interestingly, all three of the specific antibodies see cross-reacting material in the meat fractions following HPLC separations, but they do not see any of the structurally related isomeric standards. In this proposal we will validate the immunochemical methods previously developed to quantify the AIA mutagens, IQ, MeIQx, PhIP, DiMeIQx, and MeIQ in cooked-ground beef (specific aim 1). Once validated, we will use the immunoassay to examine the AIA content office additional cooked foods (specific aim 2) Immunoassay will be performed after bulk clean up and the first preparative HPLC run. The quantitative results obtained will be compared to those recorded for fried -ground beef and those obtained by conventional analytical means for the other foods. Unknown mutagens and major cross-reacting chemicals will be purified using affinity chromatography and identified using MS and NMR analysis (specific aim 4). The most mass abundant mutagens will be synthesized and further characterized (specific aim 5). Finally, with the understand of the types and quantities of these potent mutagens/carcinogens and major cooked foods in our diet, we can make a better estimate of the risk to human of the risk to humans of their daily consumption.
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