W. Robert Bruce

Epidemiological studies have demonstrated clear associations between specific dietary and environmental risk factors and incidence of colorectal cancer, but the mechanisms responsible for these associations are not known. An animal model could facilitate such an understanding. Both genotoxic and nongenotoxic carcinogens induce aberrant crypt foci (ACF) in the colons of F344 rats. F344 rats were provided with diets that contained putative risk factors for CRC: low calcium and low vitamin D, high iron, high fructose, and decreased light (UV) exposure or a control diet for 14 wk. The rats were then assessed with biochemical measures and by topological examination for evidence of colon abnormalities. Circulating ionized calcium was decreased from 2.85 to 1.69 mmol/L, and ACF were increased from 0.7 to 13.6 lesions/colon (both P < 0.001). Rats exposed to the multiple environmental conditions associated with colon cancer, developed ACF similar to the heterogeneous or ill-defined ACF in the human colon. Heterogeneous ACF are the most frequently seen in humans and are also seen in rats shortly after exposure to the non-genotoxic colon carcinogen, dextransulfate sodium. The rodent model could be used to assess the pathways from diet and environment to colon cancer and to provide guidance for clinical studies.

Colorectal cancer risk is associated with biochemical markers for B-vitamin deficiency, insulin resistance and colonic inflammation, suggesting that these three conditions are each involved in colon carcinogenesis. We expected that dietary supplements of folic acid, n-3 fatty acids and calcium would reduce the markers and thus possibly cancer risk. We therefore randomised 98 participants, with previous colonic polyps or intramucosal carcinomas, to a combined treatment of supplementary folic acid, fish oil and calcium carbonate, or placebos for 28 days. Blood and faecal samples were obtained prior to and at the conclusion of the intervention and analysed for plasma folate, homocysteine, insulin, free fatty acids, triglycerides and faecal calprotectin. In addition, plasma vitamin B₁₂, thiamin, glucose and C-reactive protein were assessed. Our supplemental strategy modestly affected some of the biomarkers associated with folate metabolism and insulin resistance, but had no effect on those associated with colonic inflammation. This pilot study demonstrates the feasibility and practicality of clinical trials aimed at reducing diet-related biochemical risk markers for colon cancer. We suggest that long-term intervention studies with tumour-related end points should be undertaken when the intervention agents used are found effective in short-term biochemical risk marker trials.

Corpet and Pierre (D. E. Corpet and F. Pierre, Cancer Epidemiol. Biomark. Prev., 12: 391-400, 2003) have reviewed the prevention studies made with the azoxymethane rat and Min mouse colon cancer models, and have shown that many agents reduce the numbers of these experimental tumors. They suggest that agents with preventive activity with little or no toxicity should be evaluated in clinical intervention studies without delay. I think that the decision to proceed to a clinical trial is more complex, and involves an understanding of the safety of the agent and of the strength and consistency of the preclinical data. However, I am also impressed by the wide range of agents that have been found to affect the development of colon cancer in animals. This suggests that human colon cancer may be the consequence of many different dietary and lifestyle deficiencies, a view supported by the observation that normal mice develop colon cancer when fed diets deficient in several food components known to prevent tumors with the azoxymethane rat model [Newmark, H. L. et al., Carcinogenesis (Lond.), 22: 1871-1875, 2001]. There is a clear need to evaluate the preventive effects of additional combinations of these agents, identified perhaps from the Corpet and Pierre review (D. E. Corpet and F. Pierre, Cancer Epidemiol. Biomark. Prev., 12: 391-400, 2003), or from actual human high-risk diets. With diets that increase the risk of "spontaneous cancer" in hand, the stage would be set for assessing the most effective ways to reduce colon cancer risk, again first with animal studies, then clinical trials, and then perhaps population studies.

It has recently been reported that 5% polyethylene glycol 8000 (PEG 8000; Mr 8000) in the diet markedly inhibits the development of colonic tumors in carcinogen-treated rats. To assess the possible use of this agent as a preventive or treatment agent for patients with familial adenomatous polyposis, we determined the effect of PEG 8000 on spontaneous carcinogenesis in the Min mouse. PEG at a 5% concentration in the diet of Min mice did not affect the number of small intestinal or cecal tumors but did increase the number of colon tumors and the number of animals with colonic tumors (2 of 18 versus 12 of 22 animals; P < 0.001). Although the chemopreventive effect of PEG 8000 in rats is remarkable, we suggest a cautious approach in long-term testing of PEG as a chemopreventive agent for subjects at risk for colonic neoplasia.

Two recent developments in cancer epidemiology and experimental carcinogenesis provide the basis for two possible mechanisms relating diet and colon cancer risk. The first development is the accumulating epidemiological evidence for an association between insulin resistance and colonic adenomas and cancers. This evidence suggests the following mechanism: the consumption of excess dietary energy results in the development of insulin resistance with increased circulating levels of insulin, triglycerides, and non-esterified fatty acids. These circulating factors subject colonic epithelial cells to a proliferative stimulus and also expose them to reactive oxygen intermediates. These long-term exposures result in the promotion of colon cancer. The second development is the continuing identification of agents that significantly inhibit experimental colon carcinogenesis. These observations suggest the following mechanism: focal loss of epithelial barrier function resulting from a failure of terminal differentiation results in the "leak" of a presently undefined toxin and a focal inflammatory response characterized by evidence of the activation of the COX-2 enzyme and an oxidative stress with the release of reactive oxygen intermediates. The resulting focal proliferation and mutagenesis give rise to aberrant crypt foci and adenomas. The process is inhibited by: (a) demulcents confined to the colonic lumen that "repair" the surface; (b) anti-inflammatory agents; or (c) antioxidants. The two mechanisms, i.e., insulin resistance acting throughout the body and focal epithelial barrier failure acting locally, can describe most of the known relationships between diet and colon cancer risk.

Diet is clearly implicated in the origin of colorectal cancer, with risk factors for the disease including reduced consumption of vegetables, fiber, and starch and increased consumption of red meat and animal fat. Several hypotheses have been developed to explain these associations. Most recently, McKeown-Eyssen and Giovannucci noted the similarity of the risk factors for colorectal cancer and those for insulin resistance and suggested that insulin resistance leads to colorectal cancer through the growth-promoting effect of elevated levels of insulin, glucose, or triglycerides. We briefly review the evidence from observational, epidemiological, and experimental animal studies linking diet with insulin resistance and colorectal cancer. The evidence suggests that diets high in energy and saturated fat and with high glycemic index carbohydrate and low levels of fiber and n-3 fatty acids lead to insulin resistance with hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. We then consider how insulin, the related insulin-like growth factors, triglycerides, and nonesterified fatty acids could lead to increased growth of colon cancer precursor lesions and the development of colorectal cancer. Finally, we consider the implications of this scheme on possible future research directions, including studies of satiety and clinical tests of the importance of insulin resistance in the colon carcinogenesis process.

Thermally oxidized animal fat (beef tallow) was assessed for colon cancer‐promoting and ‐initiating activity in F‐344 rats and CF‐1 mice with the use of the aberrant crypt focus (A CF) assay. In two promotion studies, extensively oxidized beef tallow (110°C for 144–168 h, peroxide value approx 200 meq/kg, with >80% loss of allylic and olefinic protons) had relatively little effect on the growth of ACF in F‐344 rats. The multiplication constant for treatment/control of ACF size in aberrant crypts per ACF at 100 days was 1.07 (95% confidence interval = 1.01–1.14) and 0.98 (95% confidence interval = 0.91–1.06). ACF size was not affected by less extensively oxidized beef tallow or by a 10‐fold reduction of dietary α‐tocopherol during the growth of the ACF. In initiation studies, extensively oxidized beef tallow administered by gavage increased the number of animals with ACF and the number of ACF per colon (11 of 23 and 5 of 29 animals with ACF; 1.09 ± 0.29 and 0.21 ± 0.09 ACF/colon, respectively). Less severely oxidized beef tallow was without effect. Further studies with CF‐1 mice confirmed that extensively oxidized beef tallow increased numbers of animals with ACF and average ACF per colon. The unsaturated aldehyde acrolein was without effect in the ACF assay. These data suggest that highly thermolyzed beef tallow contains an uncharacterized initiator or leads to conditions in which spontaneously initiated ACF are increased.

This work describes the application of high‐resolution proton nuclear magnetic resonance (¹H‐NMR) spectroscopy to the study of the thermal peroxidation of beef tallow and corn oil under standardized conditions. The approach provides a rapid, quantitative method for determining the degree of oxidation of unsaturated fatty acids in animal and vegetable fats and oils by quantitating the decreasing intensities of ¹H‐NMR peaks for allylic and olefinic protons in unsaturated fatty acid chains of triglycerides and the increasing peak intensities of hydroperoxide and saturated and α,β‐unsaturated aldehydic protons in relation to the less labile protons in the triglyceride molecule. Two‐dimensional correlation spectroscopy analysis of highly oxidized beef tallow (180°C for 24 h) suggested that the unsaturated aldehydes that persisted were apparently associated with carboxy groups.