Research Article Inhibition by Resistant Starch of Red Meat–Induced Promutagenic Adducts in Mouse Colon Jean Winter 1 , Laura Nyskohus 1 , Graeme P. Young 1 , Ying Hu 1 , Michael A. Conlon 2 , Anthony R. Bird 2 , David L. Topping 2 , and Richard K. Le Leu 1,2 Abstract Population studies have shown that high red meat intake may increase colorectal cancer risk. Our aim was to examine the effect of different amounts and sources of dietary protein on induction of the promutagenic adduct O 6 -methyl-2-deoxyguanosine (O 6 MeG) in colonocytes, to relate these to markers of large bowel protein fermentation and ascertain whether increasing colonic carbohydrate fermentation modified these effects. Mice (n ¼ 72) were fed 15% or 30% protein as casein or red meat or 30% protein with 10% high amylose maize starch as the source of resistant starch. Genetic damage in distal colonocytes was detected by immunohistochemical staining for O 6 MeG and apoptosis. Feces were collected for measurement of pH, ammonia, phenols, p-cresol, and short-chain fatty acids (SCFA). O 6 MeG and fecal p-cresol concentrations were significantly higher with red meat than with casein (P < 0.018), with adducts accumulating in cells at the crypt apex. DNA adducts (P < 0.01) and apoptosis (P < 0.001) were lower and protein fermentation products (fecal ammonia, P < 0.05; phenol, P < 0.0001) higher in mice fed resistant starch. Fecal SCFA levels were also higher in mice fed resistant starch (P < 0.0001). This is the first demonstration that high protein diets increase promutagenic adducts (O 6 MeG) in the colon and dietary protein type seems to be the critical factor. The delivery of fermentable carbohydrate to the colon (as resistant starch) seems to switch from fermentation of protein to that of carbohydrate and a reduction in adduct formation, supporting previous observations that dietary resistant starch opposes the mutagenic effects of dietary red meat. Cancer Prev Res; 4(11); 1920–8. Ó2011 AACR. Introduction Colorectal cancer (CRC) incidence is high in developed countries, and dietary habits have been implicated as the main factor in determining CRC risk. The World Cancer Research Fund recently identified red meat as a convincing cause of CRC, with the suggestion that intake more than approximately 500 g of cooked weight per week is associ- ated with significantly increased risk of CRC (1). Prospective human studies have shown that increasing red or processed meat intake significantly increases the risk of CRC, partic- ularly in the distal colon (2–5). However, greater consump- tion of protein intake as white meat, such as fish or chicken, is not associated with increased CRC risk (2, 6). Recent studies from animal models in vivo have shown that high dietary protein can increase DNA damage as single- and double-strand breaks, measured by the comet assay, par- ticularly with red (but not white) meat intake (7, 8). These data support the findings of prospective population studies, but the mechanisms by which increasing red meat intake can increase CRC risk is not completely understood. A number of mechanisms have been proposed. These include increased N-nitroso compounds (NOC) forming DNA adducts due to excess amino acids and peptides in the colon (9), increased dietary heme from hemoglobin in red meat forming reactive oxygen species (10), and increased muta- genic compounds such as heterocyclic amines from cooking red meat at high temperatures (11). Furthermore, it is broadly implied that high protein diets increase protein bacterial fermentation of red meat in the colon, which might play a pivotal role in altering the biomarkers asso- ciated with CRC formation (12). Dietary fiber is thought to exert a protective effect in the colon. In 1971, Burkitt proposed that dietary fiber decreases the incidence of CRC. This was based on comparisons of apparent differences in fiber intake between low- and high- risk populations. However, subsequent epidemiologic stud- ies have given conflicting results (13–15). One possible reason is a misunderstanding of the relative contribution of the polysaccharides that contribute to total dietary fiber. Nonstarch polysaccharides (NSP) resist small intestinal digestion completely and enter the large bowel. Resistant starch is the component of dietary starch that escapes Authors' Affiliations: 1 Flinders Centre for Cancer Prevention and Control, Flinders University of South Australia, Bedford Park; and 2 Preventative Health National Research Flagship, CSIRO, and CSIRO Food and Nutri- tional Sciences, Adelaide, South Australia, Australia Corresponding Author: Richard K. Le Leu, Department of Gastroenter- ology, Flinders University, Flinders Medical Centre, Bedford Park 5042, South Australia, Australia. Phone: 618-204-5170; Fax: 618-8204-3943; E-mail: richard.leleu@flinders.edu.au doi: 10.1158/1940-6207.CAPR-11-0176 Ó2011 American Association for Cancer Research. Cancer Prevention Research Cancer Prev Res; 4(11) November 2011 1920 Downloaded from http://aacrjournals.org/cancerpreventionresearch/article-pdf/4/11/1920/2250087/1920.pdf by guest on 20 June 2022