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Sulforaphane (SFN) is a potent and promising naturally occurring dietary cancer chemopreventive compound that exerts its cancer protective effects by the induction of genes including cellular defensive genes such as phase II detoxifying and antioxidant enzymes. This gene induction primarily occurs via the transcription factor Nrf2 acting on the antioxidant response element (ARE) located at the 5'-flanking region of these genes. In the present study, transcriptional expression profiles of the livers obtained from the nrf2 wild-type mice and the nrf2 knockout (-/-) mice after treatments with vehicle or SFN at 3 and 12h were generated using the Affymetrix 39K oligonucleotide microarray. The Nrf2-dependent, SFN-inducible genes were identified which include detoxification phase I, II drug metabolizing enzymes and phase III transporters. Unexpected clusters of genes include genes for heat shock proteins (HSP), ubiquitin/26S proteasome subunits, and lipid metabolism genes. Collectively, SFN increases the expression of genes through the Nrf2 signaling pathway that directly detoxify exogenous toxins/carcinogens or endogenous reactive oxygen species, and genes involved in the recognition and repair/removal of damaged proteins, which could provide secondary protection against DNA or protein damage that enhance cell survival.

Electrophiles generated during metabolic activation of carcinogens and reactive oxygen species formed from endogenous and exogenous sources might play a significant role in carcinogenesis. Cancer chemoprevention by induction of phase II detoxifying enzymes to counteract the insults of these reactive intermediates is under intensive investigation. Nrf2, a bZIP transcription factor, plays a central role in the regulation of phase II genes by binding to the antioxidant response element (ARE) in their promoters. Identification of novel Nrf2-regulated genes is likely to provide insight into cellular defense systems against the toxicities of electrophiles and oxidants and may define effective targets for achieving cancer chemoprevention. Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent that exerts its effects by induction of phase II enzymes via activation of Nrf2. In the present study, a transcriptional profile of liver of the wild-type (Nrf2+/+) and knock-out (Nrf2-/-) mice after treatments with vehicle or PEITC at 3 h and at 12 h was generated using the Affymetrix Mouse Genome 430 2.0 Array. Comparative analysis of gene expression changes between different treatment groups of wild-type and Nrf2-deficient mice facilitated identification of numerous genes regulated by Nrf2. These Nrf2-dependent and PEITC-inducible genes include known detoxication enzymes, as well as novel xenobiotic-metabolizing genes regulated by Nrf2 such as CYP 2c55, CYP 2u1 and aldehyde oxidase. Unexpected clusters included genes for heat shock proteins, ubiquitin/26 S proteasome subunits, and lipid metabolism molecules. Collectively, the identification of these genes not only provides novel insight into the effect of PEITC on global gene expression and chemoprevention, but also reveals the role of Nrf2 in those processes, which would confer cancer chemopreventive future.

Oligofructose and inulin, selective fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria, which are regarded as beneficial strains in the colon. Studies were designed to evaluate inulin (Raftiline) and oligofructose (Raftilose) for their potential inhibitory properties against the development of colonic aberrant crypt foci (ACF) in rats. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop in the colon. The results of this study indicate that dietary administration of oligofructose and inulin inhibits the development of ACF in the colon, suggesting the potential colon tumor inhibitory properties of chicory fructans. The degree of ACF inhibition was more pronounced in animals given inulin than in those fed oligofructose. Because these prebiotics selectively stimulate the growth of bifidobacteria, ornithine decarboxylase (ODC) activities, ras-p21 ontoprotein expressions and tumor inhibitory activity of lyophilized cultures of Bifidobacterium longum against chemically induced colon and mammary carcinogenesis and against colonic tumor cell proliferation were examined. Dietary administration of lyophilized cultures of B. longum strongly suppressed colon and mammary tumor development and tumor burden. Inhibition of colon carcinogenesis was associated with a decrease in colonic mucosal cell proliferation and activities of colonic mucosal and tumor ornithine decarboxylase and ras-p21. Human clinical trials are likely to broaden our insight into the importance of the pre- and probiotics in health and disease.

Studies have demonstrated a reduced risk of colon cancer when populations with diets high in total fat switched to a diet high in total fiber and certain whole-grain foods. Case-control studies have shown convincingly the relationship between dietary fiber and colon cancer. Studies in animal models have demonstrated that the inhibitory effects of dietary fiber on the development of colonic neoplasms depend on the nature and source of the fiber; that wheat bran appears to inhibit colon tumor development more consistently than do other dietary sources of fiber, such as oat and corn bran; and that dietary administration of phytic acid, high levels of which are present in wheat bran, inhibits colon carcinogenesis. Human dietary intervention studies have also indicated that the modifying effect of dietary fiber on bacterial enzymes involved in the production of putative colon tumor promoters depends on the type of fiber consumed. Dietary wheat bran, but not oat or corn bran, significantly decreased the levels of several tumor promoters in the colon, independent of stool bulk.

In studies of various human diseases, it is critical that reliable animal models are developed that demonstrate similarity to the human disease. The relative rarity of spontaneous epithelial tumors of the colon in experimental animals and the absence of evidence of virally induced large bowel tumors provided rationale to develop chemically induced experimental models for colon carcinogenesis. Such animal models have been developed to study the multiple environmental factors involved in the pathogenesis of cancer of the colon. These animal models are the following: (1) induction of colon tumors in rats through aromatic amines such as 3,2′-dimethyl-4-aminobiphenyl (DMAB); (2) derivatives and analogues of cycacin such as methylazoxymethanol (MAM), 1,2-dimethylhydrazine (DMH), and azoxymethane (AOM) in rats and mice of selected strains; (3) direct-acting carcinogens of the type of alkylureas, such as methylnitrosourea (MNU) or N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG); and (4) heterocyclic amines such as 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP). The spectrum of epithelial lesions induced in the colon by these chemical carcinogens is similar to various types of neoplastic lesions observed in the colorectum of humans. Some of the earlier studies tested the carcinogenicity of the agents. Subsequent studies were of histogenesis, cell proliferation kinetics, genetic susceptibilities, effects of dietary components, effect of fecal stream, bile acids, and bacterial flora, to cite a few. Some recent studies were directed toward inhibition of colon carcinogenesis by nutritional factors and chemopreventive agents. This brief review discusses the strengths and weaknesses of various animal models to study the relationship between the nutritional factors and chemopreventive agents and colon carcinogenesis. Animal models are extremely valuable in understanding human disease, but one must use extreme care in selecting realistic models. Indeed, one must be familiar with the limitations of the model system that is being used to study the human disease. Based on the analysis of weaknesses and strengths of several models, we found that the AOM model system appears to be appropriate, because of the similarities in histopathology of adenomas and adenocarcinomas induced in the colon and regional distribution of colon tumors with human large bowel tumors. Also, the ongoing nutritional and chemoprevention trials in humans are, in part, based on the results generated using this model system.

BACKGROUND & AIMS: Multiple studies show that continuous use of nonsteroidal anti-inflammatory drugs (NSAIDs) lowers the risk of colon cancer in humans and carcinogen-treated rodents. One target for NSAIDs is cyclooxygenase (COX), and two isoforms of this enzyme have been identified: COX-1 and COX-2. The present study was undertaken to determine if there is differential expression of COX in colonic tumors in azoxymethane-treated rats. METHODS: COX-1 and COX-2 messenger RNA levels were determined by Northern blot analysis of total RNA isolated from colonic tumors and normal adjacent mucosa. COX-2 protein levels were determined by Western blotting analysis. Quantitation of relative band densities was performed using standard densitometry scanning techniques. RESULTS: There was a marked increase in COX-2 RNA levels in six of six colonic tumors compared with paired normal mucosa. In contrast, there was equivalent intensity of the COX-1 RNA transcript between the normal mucosa and tumor in all of the specimens examined. Western blotting analysis showed an increase in the level of the COX-2 protein in four of five of the colonic tumor samples. CONCLUSIONS: COX- 2 but not COX-1 gene expression is markedly elevated in most colonic tumors examined in azoxymethane-treated rodents. COX-2 may provide a target for chemopreventive strategies for colorectal cancer. (Gastroenterology 1996 Apr;110(4):1259-62)

Background & Aims: Rat colon neoplasms are distributed 60% in the distal colon (DC) and 40% in the proximal colon (PC), similar to distribution of colon cancers in the industrialized world. The effects of chemopreventive agents that affect colon tumor incidence on the distribution of colon tumors were studied. Methods: Colon tumor distribution, numbers, and volumes were measured in the DC and PC of rats administered azoxymethane (15 mg/kg subcutaneously 2×) as an initiating agent and fed diets containing various chemopreventive agents. Results: In control rats, azoxymethane-induced tumor incidence in the DC exceeded that in the PC, but tumor volume was greater in the PC than the DC. Ellagic acid showed no chemopreventive effect and maintained the PC-DC colon tumor gradient. Oltipraz, a modestly effective chemopreventive agent, principally reduced the incidence of DC tumors. dl- d -difluoromethylornithine also greatly altered tumor number in the DC compared with the PC. In contrast, piroxicam (400 ppm) reduced PC tumors by 82% but DC tumors only by 57%. With all regimens, tumor volume remained greater in the PC than the DC. Conclusions: Chemopreventive agents have a selective regional effect on colon tumorigenesis in the rat. Elucidation of the mechanism for these differences may help clarify the modes of action of chemopreventive agents in colon cancer.

Experimental studies have indicated dietary fish oil as a protective agent in colon carcinogenesis. Prostaglandins have been suggested to be involved in this process. In the present study, the effects of fish oil on rectal cell proliferation (i.e., intermediate biomarker of cancer risk), mucosal membrane fatty acids, and prostaglandin E2 (PGE2) release were investigated in 12 healthy volunteers.In addition to a controlled basal diet, the test subjects received either fish oil (4.4 g omega-3 fatty acids/day) or corn oil supplements for two 4-week periods in a double-blind, crossover trial. Rectal cell proliferation was determined by bromodeoxyuridine immunohistochemistry and ornithine decarboxylase activity. After 2-hour incubation with bromodeoxyuridine, PGE2 concentration in the incubation medium was measured by radioimmunoassay. Mucosal membrane fatty acids were analyzed by gas chromatography.Bromodeoxyuridine labeling index (9.2% vs. 10.9%; P0.05), ornithine decarboxylase activity (19.7 vs. 36.4 pmol.mg protein-1.h-1; P0.005), and PGE2 release from rectal biopsy specimens (435.5 vs. 671.5 pg/mg wet tissue; P0.05) were significantly lower during the fish oil than the corn oil period, whereas membrane fatty acids were not statistically different.The results support the hypothesis that dietary fish oil may protect against colon cancer.

The modifying effect of 30% caloric restriction on the occurrence of azoxymethane (AOM)-induced glutathione S-transferase placental form (GST-P) positive hepatocellular foci was investigated in male F344 rats. Starting at 5 weeks of age, groups of animals were fed ad libitum a high-fat (23.5%) semipurified diet. At 7 weeks of age, all animals except the vehicle-treated groups were s.c. injected with AOM (15 mg/kg body wt., once weekly for 2 weeks). Four days after the second injection, groups of animals were continued on high-fat diet and fed ad libitum (ad libitum group) whereas other groups were restricted no 70% of total calories (calorie-restricted group) consumed by the ad libitum group, but received the same amounts of fiber, vitamins and minerals. Thirty-two weeks after AOM injections, all animals were necropsied and livers were sectioned and stained for GST-P by a immunohistochemical technique for quantitative analysis of enzyme altered foci of the liver. Comparing AOM treated groups. The density and the unit area of enzyme altered foci were significantly lower in the calorie-restricted group (3.84 ± 1.55/cm 2 , 7.96 ± 5.43%) than in the ad libitum group (10.14 ± 3.62/cm 2 , 28.11 ± 12.33%). The size of foci was also reduced in the calorie-restricted group (17.15 × 10 −3 mm 2 vs. 32.36 × 10 −3 mm 2 ). The incidence and density of hepatocellular foci in rats fed calorie restricted diet were significantly lower than those in rats fed ad libitum, comparing vehicle-treated groups. These results indicate that calorie restriction inhibited the occurrence of both of spontaneous and AOM induced GST-P positive foci in rats.

Because of the potential significance of colonic bacteria and secondary bile acids in the pathogenesis of colon cancer, the present study investigated the effect of different types of dietary fiber on fecal bacterial enzymes, namely, beta-glucuronidase, 7 alpha-dehydroxylase, nitroreductase, and azoreductase, and on bile acids and neutral sterols in premenopausal women. The subjects consumed 13-15 g of wheat, oat, or corn bran daily for 8 weeks in addition to their normal diet. Stools collected during the normal and fiber diet periods were analyzed for the above constituents. Dietary wheat bran decreased the concentrations of fecal deoxycholic acid, lithocholic acid, 12-ketolithocholic acid, and neutral sterols and the activities of all bacterial enzymes. Oat bran had no effect on secondary bile acids and 7 alpha-dehydroxylase but decreased beta-glucuronidase, nitroreductase, and azoreductase levels. Dietary corn bran increased 7 alpha-dehydroxylase, lithocholic acid, and cholesterol levels and decreased deoxycholic acid coprostanol, cholestenone, nitroreductase, and azoreductase levels. These results show that the modifying effect of dietary fiber on secondary bile acids and bacterial enzymes that may play a role in carcinogenesis depends on the type of fiber consumed.

The effect of voluntary exercise on azoxymethane-induced hepatocarcinogenesis was investigated in male F344 rats. Beginning at 5 weeks of age, all animals were divided into two groups (sedentary and exercise) and fed AIN-76A semipurified diet ad libitum. At 7 weeks of age, animals were given azoxymethane (AOM) s.c. at a dose level of 15 mg/kg of body weight, once weekly for 2 weeks. Four days after the second dose of AOM, all animals in the exercise group were housed in individual wheel-cage units and the animals in the sedentary group were housed in plastic cages. The experiment was terminated at 38 weeks post-AOM treatment. Body weights of animals in the exercise and sedentary groups were comparable. Immunohistochemical staining of glutathione S-transferase placental form (GST-P) was performed in the liver and measured GST-P positive foci. Density (number of GST-P positive foci/cm2 area of liver section), average area of foci and unit area of foci were significantly inhibited in the exercise group, although the incidence of neoplastic nodules and GST-P positive foci were unaffected by the exercise. Thus, energy expenditure due to exercise may reduce hepatocarcinogenesis in a laboratory animal model.

Quantitative food frequency questionnaires and 5-day food records were used to explore relationships between food consumption and nutrient intake among 65 healthy volunteers who were willing to participate in a dietary fiber intervention study. Spearman's correlation coefficient was calculated between the nutrient intake and the frequency of consumption of each food item, as well as the amount consumed per month. Percentage of calories from fat was related to frequency of consumption and amount of consumption, respectively, of bacon (r = .48, .49), frankfurters and sausage (r = .45, .45), and french fries and fried potatoes (r = .43, .39). Frequency and amount, respectively, of consumption of fruits were most highly correlated with intake of vitamin A (r = .45, .46), vitamin C (r = .44, .48), and dietary fiber (r = .43, .43). We conclude that specific food consumption amounts and/or frequency of eating foods such as legumes, fruits, and whole-grain or bran-rich cereals should be recommended to assist individuals in meeting dietary goals.

Large bowel cancer is one of the leading causes of cancer deaths in the United States and other Western countries. Based on epidemiologic and mechanistic studies, preclinical studies were designed to evaluate the chemopreventive efficacy of several nonsteroidal antiinflammatory drugs (NSAIDs) and phytochemicals and their synthetic analogues in colon carcinogenesis. These studies demonstrated that NSAIDs such as piroxicam, ibuprofen, aspirin and sulindac, d,l -α-difluoromethylornithine, oltipraz, anethole trithione, and diallyl disulfide inhibited colon adenocarcinomas. These results provide compelling rationale to pursue clinical trials in humans.

Fecal mutagenic activity and dietary pattern of rural and urban Finnish population groups with distinct risk for the development of colon cancer were studied in a low-risk population in rural Kuopio and an intermediate-risk population in urban Helsinkii. The average daily intake of protein and fat was the same in the two groups but the frequency of consumption of whole-grain cereals and whole-grain bread, as well as the amount of fiber from the bread were higher in Kuopio as compared to Helsinki. Fecal samples collected for 2 days were incubated under anaerobic conditions at 37°C for 96 h, extracted with hexane: peroxide-free diethyl ether, partially purified on a silica Sep-Pak cartridge, and assayed for mutagenic activity using the Salmonella typhimurium /mammalian microsome system. The fecal mutagenic activity was observed with the tester strains TA98 and TA100 with and without microsomal activation in both the population groups. The percentage of samples showing fecal mutagenic ratio greater than 3 with TA98 and TA100 with microsomal activation, was higher in volunteers from Helsinki than in Kuopio.

In this study, the amount and type of dietary fat were adjusted to determine the effect on cholesterol metabolism in free-living men.

Administration of butylated hydroxyanisole (BHA), a widely used food additive, has been found to inhibit the carcinogenic and toxic effects of various chemicals in animal models. To study the relationship of dietary BHA to the acute toxicity of methylazoxymethanol (MAM) acetate, a colonspecific carcinogenic compound, groups of female CF1 mice were fed NIN-07 diet containing 0, 300, 1000, 3000 or 6000 ppm BHA or a semipurified diet containing 0 or 6000 ppm BHA for 4 wk, and were injected ip with MAM acetate (20 mg/kg body weight) at the end of the first 2 wk and again 4 days later. At levels of 300–6000 ppm. BHA was found to protect against death caused by MAM acetate. The mortality rates in MAM-treated mice were 80 and 92% in those fed the diets with no BHA and 0 and 1% in those fed 6000 ppm BHA, and were inversely related to the amount of BHA in the diet. The protection was associated with increased levels of hepatic cytochrome P-450 and b5 and with a reduction in necrotic changes in the liver.

The purpose of the present investigation was to determine whether altering the format of a food frequency questionnaire affected its accuracy and reproducibility. Three questionnaires were tested at one-month intervals. Questionnaire I contained 39 food items listed according to food categories. Questionnaire II contained 55 food items, presented according to the meal in which they were consumed. Questionnaire III contained 31 food items presented in a similar order to that in Questionnaire II. To determine the accuracy of the food frequency questionnaire, results were compared to those obtained from a simultaneously administered seven-day diet diary. Results from this study showed that food frequency questionnaires containing broad categories of food items presented in order of the meals in which they are consumed are the most reproducible. Highly reproducible results also can be obtained from seven-day diet diary, if data are calculated in terms of nutrients (i.e. carbohydrates, fats, proteins, etc.) and not specific food items. Finally, this study suggests that individuals more closely maintain their intake of nutrients, rather than specific food items.

The effect of dietary wheat bran and citrus pulp on hepatic, small intestinal and colonic HMG-CoA reductase activity, cytochrome P450 and cytochrome b5 levels was studied in F344 rats. Colonic and small intestinal HMG-CoA reductase activity was significantly lower in the wheat bran group than the control group. No differences in hepatic HMG-CoA reductase activity occurred between the wheat bran, citrus pulp and control groups. A direct relationship between hepatic cholesterol levels and hepatic Cytochrome P450 levels was demonstrated. A similar trend was exhibited between fecal cholesterol and colonic cytochrome P450 levels in the colon. These results suggest that the effect of certain types of dietary fiber in small intestinal and colonic cholesterogenesis is mediated through alterations in small intestinal and colonic mixed function oxidase activity.

It has been postulated that reflux of bile into the stomach promotes gastric carcinogenesis. Bilestained aspirates from 50 asymptomatic patients, partially gastrectomized more than 10 years earlier, were examined bacteriologically and with regard to conjugated and deconjugated bile acids. Endoscopic biopsies showed atrophic gastritis in all patients, cancer in two and severe dysplasia in another two. pH in the reflux aspirates was 7.3 ± 0.4 (mean ± standard deviation). Bacterial cultures were positive in all patients studied. Fecal type flora, mostly E. coli, klebsiella and Clostridium perfringens, was found in 85 percent of the patients. Total bile acids were found to be 2.6 ± 2.0 mg/ml, 23 percent of which were deconjugated. Deoxycholic acid, known to promote carcinogenesis in animals, amounted to 27 percent of total bile acids and deconjugated deoxycholic acid was 5 percent of total bile acids. The mostly anaerobic microflora and the presence of mainly free secondary and primary bile acids may contribute to the high incidence of cancer in the gastric remnant observed after Billroth I or II operations.

The effect of a high-fat, high-beef diet and of method of preparation of beef in the diet on the fecal bile acids and neutral sterols and on the activities of fecal bacterial beta-glucuronidase, cholesterol dehydrogenase and 7 alpha-dehydroxylase was studied in healthy men and women, 24-41 years old, who were consuming a customary mixed-western diet. The experimental diets were high in fat and beef, which was cooked rare, medium or well-done. The sequence of dietary regimen was selected at random and each diet phase lasted for 4 weeks. Individual 24-hour fecal specimens were collected from each volunteer for the final 3 days during each dietary period, including pre- and post-experimental periods while they were consuming a customary mixed-western diet. The mode of cooking beef in the high-fat, high-beef experimental diets had no influence on the fat and protein content of the diets but the fat content of experimental diets was high compared to customary mixed-western diet. Fecal bacterial beta-glucuronidase activity and fecal secondary bile acid and cholesterol metabolite levels were significantly higher during the experimental diet periods but the fecal bacterial activities of 7 alpha-dehydroxylase and cholesterol dehydrogenase were unaffected. The mode of cooking beef in experimental diets had no influence on the fecal bacterial enzymes and on the excretion of fecal bile acids and cholesterol metabolites.

The possible antimutagenic effects of butylated hydroxytoluene (BHT), ethoxyquin, disulfiram, indole-3-carbinol, indole-3-acetonitrile, sodium selenite and alpha-tocopherol on 3,2'-dimethyl-4-aminobiphenyl-induced mutagenicity were studied using the Ames Salmonella/mammalian microsome assay system with strains TA98 and TA100. All seven compounds were nonmutagenic in both bacterial tester strains. The addition of or 50-250 micrograms of sodium selenite, 5-50 mg of alpha-tocopherol or 50-250 microgram of BHT per plate inhibited DMAB-induced mutagenicity in TA98 and/or TA100. Ethoxyquin, disulfiram and indole-3-carbinol increased DMAB-induced mutagenicity in TA100, whereas these compounds had little or no effect in TA98-3-acetonitrile had very little effect in either strain.

1-Nitropyrene (100 mg/kg body wt.) was administered by gavage to conventional and germfree Fischer 344 rats. 1-Aminopyrene was detected in the feces of conventional rats (5–6% of the dose) but not in the feces of germfree rats. Conjugates of 1-aminopyrene were not detected in the feces of germfree rats and neither 1-aminopyrene nor its conjugates were found in the urine of either conventional or germfree rats. Since reduction of 1-nitropyrene to 1-aminopyrene is an activation process, the results indicate that intestinal microflora are important in the metabolic activation of 1-nitropyrene in vivo in the rat. Human intestinal microflora also reduced 1-nitropyrene to 1-aminopyrene.

Disaccharidase (maltase, invertase, trehalase, lactase, and cellobiase) activities have been studied in homogenates of small intestine of the germfree and conventional rats from birth through 120 days of age. The pH optimum values for germfree and conventional rats were comparable. In adult germfree rats, the pattern of distribution of disaccharidase activities in different sections of small intestine was similar to that of conventional rats. Lactase and cellobiase activities were substantial at birth, reached maximum at 7 days of age, and then decreased gradually to a plateau at 30 days of age in both groups. Maltase activity was low at birth, whereas no invertase and trehalase activities could be detected up to 7 days of age, and these activities developed rapidly during the third week of life in both groups. Differences in activity of these disaccharidases between germfree and conventional rats were not apparent until after weaning (21 days) when the germfree animal showed a higher activity than the conventional rat. When the germfree rats were conventionalized by the introduction of cecal contents from the conventional rats, the disaccharidase activities were reduced to normal conventional levels. These results indicate that intestinal disaccharidases are synthesized by the animal itself, and that the intestinal microorganisms do not contribute to any major extent to their production.