Red Meat Enhances the Colonic Formation of the DNA Adduct O 6 -Carboxymethyl Guanine: Implications for Colorectal Cancer Risk Michelle H. Lewin, 1 Nina Bailey, 1 Tanya Bandaletova, 1 Richard Bowman, 1 Amanda J. Cross, 1 Jim Pollock, 2 David E.G. Shuker, 3 and Sheila A. Bingham 1 1 Diet and Cancer Group, Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Cambridge, United Kingdom; 2 Pollock and Pool Ltd., Reading, Berkshire, United Kingdom; and 3 Department of Chemistry, The Open University, Walton Hall, Milton Keynes, United Kingdom Abstract Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N -nitrosocom- pounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, high- fiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P < 0.0001) increase in endogenous formation of NOC with the red meat diet compared with the vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O 6 -carboxymethyl guanine (O 6 CMG) was significantly (P < 0.001) higher on the high red meat diet. In 13 volunteers, levels were intermediate on the high-fiber, high red meat diet. Fecal ATNC were positively correlated with the percentage of cells staining positive for O 6 CMG (r 2 = 0.56, P = 0.011). The presence of O 6 CMG was also shown in intact small intestine from rats treated with the N -nitrosopeptide N -acetyl-N V -prolyl-N V -nitro- soglycine and in HT-29 cells treated with diazoacetate. This study has shown that fecal NOC arising from red meat include direct acting diazopeptides or N -nitrosopeptides able to form alkylating DNA adducts in the colon. As these O 6 CMG adducts are not repaired, and if other related adducts are formed and not repaired, this may explain the association of red meat with colorectal cancer. (Cancer Res 2006; 66(3): 1859-65) Introduction There is compelling evidence from epidemiologic data that red and processed meat intake is associated with increased risk of colorectal cancer (1). The association with red and processed meat was particularly strong in individuals eating a low-fiber diet (1). The usual explanation for these associations has been that a number of heterocyclic amines (HCA) are formed when meat is cooked and that many HCAs are known carcinogens (2). However, white meat, such as chicken, is not associated with increased colorectal cancer risk, yet chicken meat may contain high levels of HCA (2). Furthermore, variants in the N -acetyl gene required for the activation of HCA are not consistently associated with altered risk of large bowel cancer (3). In 1996, we reported that red but not white meat stimulates endogenous intestinal N -nitrosation in humans (4). The influence of red but not white meat on fecal N -nitrosocompounds (NOC) excretion (measured as apparent total NOC, ATNC) has since been shown in >50 healthy volunteers, all of whom were studied in a metabolic suite where diet could be carefully controlled (4–8). The direction of an increase with increasing red meat is consistent in all individuals and can be attributed to heme iron but not inorganic iron or protein (6). At the higher levels of red meat consumption, concentrations of ATNC are of the same order as the concentration of tobacco-specific NOC in cigarette smoke (4). The majority of NOC investigated are carcinogens, but it has not been established that the NOC formed endogenously in the human gut after eating red meat are genotoxic. Endogenous N -nitrosation occurs because peptides and amino acids are abundant in the intestine and can be nitrosated to form diazopeptides or N -nitrosopeptides (9, 10). Nitrosating agents, primarily from endogenous nitric oxide (NO) production, are also in abundant supply. O 6 -methylguanine (O 6 MeG) is a characteristic promutagenic and toxic adduct formed by many N -methyl-N - nitrosocompounds that either spontaneously decompose or are metabolized to intermediates which are highly reactive methylating agents that react with nucleophilic centers on DNA bases (11). In addition, various nitrosated glycine derivatives react with DNA in vitro to give carboxymethyl adducts [e.g., O 6 -carboxymethyl- deoxyguanosine (O 6 CMG)] and lesser amounts of methyl adducts (e.g., O 6 MeG), as shown in Fig. 1. O 6 CMG has particular potential as a biological marker of DNA carboxymethylation because it does not seem to be repaired by O 6 -alkylguaninealkyltransferase in in vitro assays (11). Nitrosation of glycine by NO under simulated physiologic conditions results in formation of O 6 CMG (12). The use of exfoliated cells as a source of markers for cancer is well established; for example, the Papanicolaou smear is universally used in cervical cancer screening. In colon cancer, the use of exfoliated cells is relatively recent but has a strong biological rationale in that exfoliated cells arise from colonic tissue itself rather than associated manifestations of cancer, such as occult bleeding (13, 14). However, isolation of exfoliated cells from fecal material is difficult, and a reliable method has only recently been developed by us (15). Using this technique, an immunohistochem- ical method for minichromosome maintenance proteins on recovered fecal colonocytes had 92% sensitivity for detecting distal colorectal cancers (16). In the present report, also using immunohistochemistry, we now show that the O 6 CMG DNA Note: A. J. Cross is currently at the Department of Health and Human Services, National Cancer Institute, NIH, 6120 Executive Boulevard, Bethesda, MD 20852. Requests for reprints: Sheila A. Bingham, Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Cambridge, United Kingdom CB2 2XY. Phone: 44-1223-252760; Fax: 44-1223-252765; E-mail: sab@mrc-dunn.cam.ac.uk. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2237 www.aacrjournals.org 1859 Cancer Res 2006; 66: (3). February 1, 2006 Research Article Research. on February 20, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from