This proposal seeks to utilize state-of-the-art techniques in molecular cytogenetics to measure the accumulation and persistence of stable chromosome rearrangements in mice chronically exposed to heterocyclic amine carcinogens. The chronic effects of at least two such compounds will be examined. These are 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (MelQx). Beginning at 8 weeks of age, mice will be fed a diet containing 0, 100, 250, or 400 ppm of these compounds. At times corresponding to 1, 2, 4, and 6 months later, 4 mice per treatment group will be euthanized and peripheral blood and spleen lymphocytes will be obtained. These cells will be cultured and metaphase preparations examined for stable chromosome aberrations (translocations) using chromosome-specific composite DNA probes detected with fluorescence in situ hybridization. This procedure is known as """"""""chromosome painting"""""""" and is a rapid method of quantifying stable aberrations. Painting is currently used in only a few laboratories, and then only with human, not mouse, material. We will also evaluate cells from parallel cultures for sister chromatid exchanges (SCEs). Finally, we will score micronuclei in normochromatic erythrocytes. The endpoints will provide essential information concerning the accumulation and persistence of stable and unstable cytogenetic damage in multiple cell types. We will relate these results a) to metabolism and DNA adduct measurements made by other portions of this Program Project using these same animals, and b) to tissue-specific tumor formation data available in the literature. The second portion of this project will utilize matched DNA-repair deficient/proficient Chinese hamster ovary (CHO cell lines into which functional copies of the cytochrome P450 cDNAs have been transfected. The cell lines will be exposed to heterocyclic amine carcinogens, including PhlP and MelQx, and examined for chromosomal aberrations, SCEs, and micronuclei in cytokinesis-blocked cells. These cytogenetic results will be related to the formation of DNA adducts using parallel cultures. We will also determine which of the various other heterocyclic amines are metabolized by these cell lines in sufficient amounts to induce measurable cytogenetic damage, and will compare the spectrum of chromosomal aberrations in these matched cell lines to the DNA adducts formed. These studies should help establish a relationship between chromosomal alterations, DNA adduct formation, mutation, and cancer following exposure to dietary heterocyclic amine carcinogens.
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