Nitrite-preserved ("processed") meat is a reported cause of colon cancer. This may occur because processed meat contains N-nitroso compounds (NOC), which induce the cancer. Previously, the PI's group demonstrated that addition of ascorbate greatly reduced NOC formation in a chemical system. On this basis, the meat industry now adds 500 mg/kg of sodium erythorbate (an ascorbate isomer) together with 120 mg/kg of sodium nitrite (NaNO2) in processed meat to reduce NOC formation. We recently reported that hot dogs contain 5.5 nmol/g of total non-volatile apparent NOC (ANC). The ANC are mutagenic by the Ames test and induced aberrant crypts (a premalignant lesion) in the mouse colon. We wish to understand the role played by these ANC in colon cancer etiology and wish to learn how to reduce exposure to these ANC, Therefore, we now propose 5 Specific Aims. Aim 1: Effect of varying erythorbate and nitrite levels on ANC content of hot dog patties prepared here: We will test effects of added NaNO2 and Na erythorbate on ANC in hot-dog patties prepared here when we raise erythorbate stepwise from 500 to 7,500 mg/kg, lower NaNO2 from 120 to 60 and 0 mg/kg, or omit both additives. If our views are correct, our findings in this Aim could produce changes in the meat industry that would prevent thousands of deaths each year from colon cancer. Aim 2: Identification of hot dog ANC precursor (ANCP): We will purify ANCP from standard hot dog paties by adsorption-desorption and various HPLC systems, and try to identify the ANCP by mass spectrometry. We will synthesize and characterize the ANCP, N-1-glucosyl glycine, its N-nitroso derivative and analogs prepared from other sugars and amino acids. Aim 3: Mutagenesis tests: We will use the Ames test to examine the mutagenicity of nitrosated ANC prepared from hot dog patties manufactured under standard conditions and those giving minimum ANC levels, from feces of rats fed NaNO2 or hot dogs, and from the reaction of glucose with glycine and other amino acids. Aim 4: Permeation studies: To test whether ANC in the colonic lumen can be absorbed by the colonic mucosa, we will examine permeation of purified hot-dog-derived ANC through the mouse colon wall using side-by-side diffusion chambers. Aim 5: Induction of aberrant crypts by NaNO2 and hemin: Like hot dogs, dietary NaNO2, especially together with hemin, increased fecal ANC excretion in mice, but NaNO2 is not a colon carcinogen. Because hot-dog-derived ANC induced colonic aberrant crypts (a precursor lesion for colon cancer) and this induction was probably caused by the raised ANC levels in the colon, we will test whether NaNO2 and hemin, which also increased fecal ANC levels, induce colonic aberrant crypts in mice. PUBLIC HEALTH RELEVANCE: Project narrative Apparent N-nitroso compounds (ANC) derived from processed (nitrite-preserved) meat are a likely cause of colon cancer associated with processed meat such as hot dogs. We propose to test whether alterations in the curing mixture used to prepare hot dogs will reduce the ANC content of this food. This alteration could significantly reduce the incidence of colon cancer. We also plan to chemically identify components of these ANC, determine mutagenicity of the hot dog ANC fraction and its components, and establish whether hot dog ANC can penetrate the mouse colon wall. We will test whether sodium nitrite which, like hot dog ANC, increased fecal ANC levels in mice, behaves like hot dog ANC in inducing aberrant crypts (a precancer lesion for colon cancer) in the mouse colon.