W. Robert Bruce

Diet, aberrant crypt foci and colorectal cancer

We have used the aberrant crypt focus (ACF) assay to test and develop hypotheses linking diet and colon cancer. The hypotheses were suggested by epidemiological studies that identified possible dietary factors associated with colorectal cancer risk. The ACF assay was used to quantitate the effect of the dietary factors on the initiation and growth of these putative precursors of colon cancers in experimental animals. Using this approach we have developed 3 new hypotheses for the role of diet in colorectal cancer. These are (1) a risk associated with 5-hydroxymethyl-2-furaldehyde in caramelized sugar, (2) a risk associated with some factor in thermolyzed casein, and (3) a risk associated with single nutrient boluses of sucrose and fructose. The importance of these hypotheses has still to be tested in long term carcinogenesis experiments, in analytic epidemiology studies and then, perhaps, in intervention trials.

Characterization of cytotoxic steroids in human faeces and their putative role in the etiology of human colonic cancer

It has been shown previously that chemically induced nuclear abeerrations in the murine colon are correlated with the carcinogenicity of the respective chemicals. Consequently, the nuclear aberration assay was utilized for the identification of putatife carcinogens in human faeces. Human fecal samples were fractionated by several chromatographic methods, and the assay led to the isolation of two substances. A combination of spectroscopic (mass, nuclear magnetic resonance, ultraviolet, and infrared) and chromatographic (HPLC and GLC) methods showed that they are 5-alpha- cholestan-3-one (I) and cholest-4-en-3-one (II). A number of C-27-C-30 steroids isolated from closely related fractions of feces were inactive in this assay. Thus I and II could play a role in etiology of large bowel cancer in humans.

On distribution of different fecapentaenes, the fecal mutagens, in the human population

It has been shown by an HPLC analysis using a quarternary solvent mixture in an isocratic mode that human excretors of these fecal mutagens excrete both fecapentaene -12 and -14 but the ratios vary greatly between individuals. Since these mutagens are produced by the bacterial flora of the colon, this may indicate differences in the flora between these individuals or differences in the availability of different precursor molecules in their colons. Any relationship of these findings to the etiology of colonic cancer is not clear.

The effects of partial thiamin deficiency and oxidative stress (i.e., glyoxal and methylglyoxal) on the levels of α-oxoaldehyde plasma protein adducts in Fischer 344 rats

We hypothesized that in marginal thiamin deficiency intracellular α‐oxoaldehydes form macromolecular adducts that could possibly be genotoxic in colon cells; and that in the presence of oxidative stress these effects are augmented because of decreased detoxification of these aldehydes. We have demonstrated that reduced dietary thiamin in F344 rats decreased transketolase activity and increased α‐oxoaldehyde adduct levels. The methylglyoxal protein adduct level was not affected by oral glyoxal or methylglyoxal in the animals receiving thiamin at the control levels but was markedly increased in the animals on a thiamin‐reduced diet. These observations are consistent with our suggestion that the induction of aberrant crypt foci with marginally thiamin‐deficient diets may be a consequence of the formation of methylglyoxal adducts.

A western-style diet reduces bone mass and biomechanical bone strength to a greater extent in male compared with female rats during development.

Evidence from epidemiological and animal-feeding trials suggests that a western-style diet that is high in fat, and low in Ca, vitamin D and folic acid may result in low bone mass and poor bone quality: this leads to an increased risk of fragility fracture. The overall objective of the present study was to determine the effect of feeding a western-style diet (low in Ca (0.4 g/kg diet, Ca:P ratio 1:10), cholecalciferol (3 microg/kg diet), folic acid (0.23 mg/kg diet) and fibre (20 g/kg diet), and high in fat (200 g/kg diet)) for 17 weeks on bone mineral content (BMC) and the biomechanical bone strength of rat femurs. A secondary objective was to determine whether femurs from male and female rats (seven to eight rats per group) respond differently to the western-style diet. Male and female rats weighing 150-180 g were fed a western-style diet or a control diet for 17 weeks. At the end of the feeding trial, femur BMC was measured by ashing, and biomechanical properties were determined by three-point bending. Femur BMC and the majority of biomechanical properties measured were lower (P<0.05) among male and female rats fed a western-style diet compared with a control diet, despite similar weight gain and final body weight within genders. However, the western-style diet had a greater negative effect on femur BMC and biomechanical strength properties among male rats compared with females. This may be because male rats experienced greater overall body growth, as assessed by weight gain, than female rats, and suggests that the nutrient composition of the western-style diet did not support the development of strong femurs.

The Association between Glyceraldehyde-Derived Advanced Glycation End-Products and Colorectal Cancer Risk

Background: A large proportion of colorectal cancers are thought to be associated with unhealthy dietary and lifestyle exposures, particularly energy excess, obesity, hyperinsulinemia, and hyperglycemia. It has been suggested that these processes stimulate the production of toxic reactive carbonyls from sugars such as glyceraldehyde. Glyceraldehyde contributes to the production of a group of compounds known as glyceraldehyde-derived advanced glycation end-products (glycer-AGEs), which may promote colorectal cancer through their proinflammatory and pro-oxidative properties. The objective of this study nested within a prospective cohort was to explore the association of circulating glycer-AGEs with risk of colorectal cancer. Methods: A total of 1,055 colorectal cancer cases (colon n = 659; rectal n = 396) were matchced (1:1) to control subjects. Circulating glycer-AGEs were measured by a competitive ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (95% CI), adjusting for potential confounding factors, including smoking, alcohol, physical activity, body mass index, and diabetes status. Results: Elevated glycer-AGEs levels were not associated with colorectal cancer risk (highest vs. lowest quartile, 1.10; 95% CI, 0.82–1.49). Subgroup analyses showed possible divergence by anatomical subsites (OR for colon cancer, 0.83; 95% CI, 0.57–1.22; OR for rectal cancer, 1.90; 95% CI, 1.14–3.19; Pheterogeneity = 0.14). Conclusions: In this prospective study, circulating glycer-AGEs were not associated with risk of colon cancer, but showed a positive association with the risk of rectal cancer. Impact: Further research is needed to clarify the role of toxic products of carbohydrate metabolism and energy excess in colorectal cancer development. Cancer Epidemiol Biomarkers Prev; 24(12); 1855–63. ©2015 AACR.

Dietary cholesterol inhibits the development of aberrant crypt foci in the colon

We evaluated the effect of dietary cholesterol and oxidized cholesterol on the promotion of aberrant crypt foci (ACF), which are putative precancerous lesions in the colon. Sixty female C57BL/6J mice were given four weekly injections (ip) of azoxymethane (AOM) then fed either a control AIN-76 diet or the control diet supplemented with 0.3% cholesterol or 0.3% oxidized cholesterol for 100 days. The oxidized cholesterol was prepared by heating cholesterol at 110 degrees C for 48 hours. Gas chromatographic analysis of the oxidized cholesterol showed that 96% of the cholesterol was unchanged and less than 2% of the cholesterol was oxidized. The remaining 2% impurities were unidentified and present in both the cholesterol and heated cholesterol. The number of ACF in the group fed cholesterol was significantly lower than the control group (7.9 +/- 1.0 vs. 12.5 +/- 1.2, p < 0.01). The number of ACF in the group fed oxidized cholesterol (10.1 +/- 1.1) was not different from the control or cholesterol groups. The size of the ACF (no. of crypts per focus) did not differ between the three dietary groups. Serum low-density lipoprotein (LDL) cholesterol was greater in the cholesterol-fed group than the control group (40.5 +/- 4.6 vs. 24.3 +/- 3.6 mg/dl, p < 0.05). LDL cholesterol from the animals fed oxidized cholesterol (37.7 +/- 4.7 mg/dl) was not different from the control or cholesterol-fed animals. Total and high-density lipoprotein (HDL) cholesterol did not differ between the groups. The results show that dietary cholesterol significantly inhibits the promotion of ACF in the colon. The elevated LDL cholesterol may inhibit de novo cholesterol synthesis in the preneoplastic colonic epithelial cells, thereby inhibiting DNA synthesis and cell proliferation.

Manipulation of fecal pH by dietary means

Epidemiological and animal studies suggest that colonic fermentation and fecal pH may be risk factors for colorectal cancer. To modify these factors, we sought to develop a simple instrument for use in intervention studies. Three 14-day studies with 32, 40, and 30 healthy volunteers maintained on their regular diet were carried out to evaluate the effect of various food supplements on fecal pH. The interventions tested included supplementary lactulose, Metamucil, oat bran, wheat bran, or no supplement. The results showed that it is possible to provide a simple intervention to reduce fecal pH by 0.4 unit with oat bran administered at 75-100 g/day over a 14-day period, using a wheat combination, equivalent in macronutrients and fiber, as a control.

Serum Endotoxins and Flagellin and Risk of Colorectal Cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC) Cohort

Background: Chronic inflammation and oxidative stress are thought to be involved in colorectal cancer development. These processes may contribute to leakage of bacterial products, such as lipopolysaccharide (LPS) and flagellin, across the gut barrier. The objective of this study, nested within a prospective cohort, was to examine associations between circulating LPS and flagellin serum antibody levels and colorectal cancer risk. Methods: A total of 1,065 incident colorectal cancer cases (colon, n = 667; rectal, n = 398) were matched (1:1) to control subjects. Serum flagellin- and LPS-specific IgA and IgG levels were quantitated by ELISA. Multivariable conditional logistic regression models were used to calculate ORs and 95% confidence intervals (CI), adjusting for multiple relevant confouding factors. Results: Overall, elevated anti-LPS and anti-flagellin biomarker levels were not associated with colorectal cancer risk. After testing potential interactions by various factors relevant for colorectal cancer risk and anti-LPS and anti-flagellin, sex was identified as a statistically significant interaction factor (Pinteraction < 0.05 for all the biomarkers). Analyses stratified by sex showed a statistically significant positive colorectal cancer risk association for men (fully-adjusted OR for highest vs. lowest quartile for total anti-LPS + flagellin, 1.66; 95% CI, 1.10–2.51; Ptrend, 0.049), whereas a borderline statistically significant inverse association was observed for women (fully-adjusted OR, 0.70; 95% CI, 0.47–1.02; Ptrend, 0.18). Conclusion: In this prospective study on European populations, we found bacterial exposure levels to be positively associated to colorectal cancer risk among men, whereas in women, a possible inverse association may exist. Impact: Further studies are warranted to better clarify these preliminary observations. Cancer Epidemiol Biomarkers Prev; 25(2); 291–301. ©2016 AACR.

Dehydroalanine and lysinoalanine in thermolyzed casein do not promote colon cancer in the rat

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydroalanine, which have been considered possible health hazards. We observed that thermolyzed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolyzed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity.