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Escherichia coli containing polyketide synthase in the gut microbiota (pks + E coli) produce a polyketide‐peptide genotoxin, colibactin, and are suspected to play a role in the development of colorectal neoplasia. To clarify the role of pks + E coli in the early stage of tumorigenesis, we investigated whether the pks status of E coli was associated with the prevalence of colorectal neoplasia. This cross‐sectional analysis of data from a prospective cohort in Izu Oshima, Japan included asymptomatic residents aged 40‐79 years who underwent screening colonoscopy and provided a stool sample. We identified 543 participants with colorectal neoplasia (22 colorectal cancer and 521 adenoma) as cases and 425 participants with normal colon as controls. The pks status of E coli was assayed using stool DNA and specific primers that detected pks + E coli. The proportion of pks + E coli was 32.6% among cases and 30.8% among controls. Compared with those with pks − E coli, the odds ratio (OR) (95% confidence interval) for participants with pks + E coli was 1.04 (0.77‐1.41) after adjusting for potential confounders. No statistically significant associations were observed regardless of tumor site or number of colorectal adenoma lesions. However, stratified analyses revealed increased ORs among participants who consumed cereals over the median intake or vegetables under the median intake. Overall, we found no statistically significant association between pks + E coli and the prevalence of colorectal adenoma lesions among this Japanese cohort. However, positive associations were suggested under certain intake levels of cereals or vegetables., Overall, we found no statistically significant association between Escherichia coli containing polyketide synthase in the gut microbiota and the prevalence of colorectal adenoma lesions, based on a cross‐sectional analysis among this Japanese cohort aged 40‐79 years who underwent screening colonoscopy. On stratified analysis, however, positive associations were seen in participants with cereal intake above the median and in those with vegetable intake below the median.

Cyclooxygenase (COX) is a key enzyme in the formation of prostaglandins, and an inducible isoform of COX, COX-2, has been implicated in colorectal carcinogenesis. This study investigated the relation of COX-2 polymorphisms (–1195G>A, –765G>C and 8160A>G) to colorectal adenomas in a case–control study of male officials in the Self Defense Forces (SDF). The study subjects were 455 cases of colorectal adenoma and 1052 controls with no polyps who underwent total colonoscopy. Genotypes were determined using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method with genomic DNA extracted from the buffy coat. Statistical adjustment was made for age, hospital, rank in the SDF, body mass index (BMI), cigarette smoking, and alcohol intake. A statistically non-significant decrease in the risk of colorectal adenomas was observed for the AA versus GG genotype of –1195G>A polymorphism and for the GC versus GG genotype of –765G>C polymorphism. None had the –765CC genotype in either the case or control groups. No effect modification of overweight, smoking or alcohol use was observed for either –1195G>A or –765G>C polymorphism. The variant allele of the 8160A>G polymorphism was extremely rare. A haplotype of –1195G, –765G and 8160A alleles was associated with a modest increase in the risk (adjusted odds ratio [OR] 1.38, 95% confidence interval [CI] 0.99–1.91), and the increase was more evident for distal adenomas (adjusted OR 1.57, 95% CI 1.04–2.38). Another haplotype of –1195A, –765C and 8160A alleles showed an adjusted OR of 0.22 (95% CI 0.06–0.88). These findings add to evidence for the role of COX-2 in colorectal carcinogenesis and warrant further studies focusing on haplotypes. (Cancer Sci 2008; 99: 576–581)

Epidermal growth factor receptor (EGFR) gene alterations have been found in human lung cancers. However, there is no information on the factors inducing EGFR mutations. In rodents, K-ras mutations are frequently found in many lung carcinogenesis models, but hitherto, Egfr mutations have not been reported. Their presence was therefore investigated in representative lung carcinogenesis models with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosobis(2-hydroxypropyl)amine (BHP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and ethyl carbamate (urethane), as well as X-ray irradiation. With the chemical carcinogenesis models, no mutations were detected in Egfr, which is in clear contrast to the high rates observed in either codon 12 or 61 of K-ras (21/23 of the lung tumors induced with NNK, 4/5 with MeIQx, 1/4 with urethane and 7/18 with BHP). However, in the X-ray-induced lung tumors, Egfr mutations with amino acid substitution were observed in exons 18 and 21 (4/12, 33%), but no activating mutation of K-ras was detected. In addition, one and four silent mutations were identified in K-ras (exon 1) and Egfr (exons 18, 20 and 21), respectively. Most mutations in both Egfr and K-ras were G/CA/T transitions (7/8, 88% and 31/34, 91%, respectively). Although, the mutational patterns in equivalent human lesions were not completely coincident, this first report of Egfr mutations in an experimental lung tumor model suggests that X-rays or other factors producing oxygen radicals could cause EGFR mutations in some proportion of lung cancers in humans. (Cancer Sci 2008; 99: 241–245)

We demonstrated recently that mofezolac, a cyclooxygenase-1 (COX-1) selective inhibitor, suppresses the development of azoxymethane (AOM)-induced colonic aberrant crypt foci in F344 rats and intestinal polyps in APC1309 mice. In the present study, we therefore investigated the effects of mofezolac on colon cancer development. Male F344 rats were injected subcutaneously with 15 mg/kg body weight of AOM in the back twice at 7-day intervals from 5 weeks of age, and fed a diet containing 600 or 1200 ppm mofezolac for 32 weeks, starting 1 day before the first dosing of AOM. Treatment with 1200 ppm mofezolac significantly reduced the incidence, multiplicity and volume of colon carcinomas to 79%, 2.15 ± 1.65 and 7.5 ± 11.8 mm3, respectively, compared with 94%, 3.19 ± 1.87 and 23.7 ± 31.2 mm3 in the AOM treatment alone. Administration of 600 ppm mofezolac showed only a slight reduction. No side effects were observed in any of the groups. These results confirm that COX-1, as well as COX-2, contributes to colon carcinogenesis and that mofezolac may be a good chemopreventive agent for human colon cancer. (Cancer Sci 2006; 97: 1011–1014)

A new flavone derivative, chafuroside, has been isolated as a strong anti-inflammatory compound from oolong tea leaves, and its structure determined to be (2R,3S,4S,4aS,11bS)-3,4,11-trihydroxy-2-(hydroxymethyl)-8-(4-hydroxyphenyl)-3,4,4a,11b-tetrahydro-2H,10H-pyrano[2',3':4,5]furo[3,2-g]chromen-10-one. To assess its potential to inhibit intestinal carcinogenesis, 2.5, 5 and 10 p.p.m. chafuroside was given in the diet to Apc-deficient Min mice for 14 weeks from 6 weeks of age. Total numbers of polyps were reduced to 83, 73 and 56% of the control value, respectively. Moreover, dietary administration at 10 and 20 p.p.m. reduced azoxymethane (AOM)-induced colon aberrant crypt foci (ACF) development in rats to 69% of the AOM-treated control value with the higher dose. Chafuroside-associated toxicity was not observed at 2.5-10 p.p.m. in Min mice and 10-20 p.p.m. in AOM-treated rats. These results suggest that chafuroside might be a good chemopreventive agent for colon cancer.

Studies of colon carcinogenesis in animal models are very useful to elucidate mechanisms and provide pointers to potential prevention approaches in the human situation. In the rat colon carcinogenesis model induced by azoxymethane (AOM), we have documented frequent mutations of specific genes. K-ras mutations at codon 12 were found to be frequent in hyperplastic aberrant crypt foci (ACF) and large adenocarcinomas. In addition, mutations of the beta-catenin gene in its GSK-3beta phosphorylation consensus motif could also be identified in many adenomas and adenocarcinomas, and altered cellular localization of beta-catenin protein was observed in all of the dysplastic ACF, adenomas and adenocarcinomas examined, indicating that activation of Wnt signaling by accumulation of beta-catenin is a major mechanism in the AOM-induced colon carcinogenesis model. Frequent gene mutations of beta-catenin and altered cellular localization of the protein are also features of AOM-induced colon tumors in mice. Expression of enzymes associated with inflammation, such as inducible nitric oxide synthase (iNOS) and the inducible type of cyclooxygenase (COX), COX-2, is increased in AOM-induced rat colon carcinogenesis, and overproduction of nitric oxide (NO) and prostaglandins is considered to be involved in colon tumor development. We have demonstrated that increased expression of iNOS is an early and important event occurring in step with beta-catenin alteration in rat colon carcinogenesis. Activation of K-ras was also found to be involved in up-regulation of iNOS in the presence of inflammatory stimuli. In addition, expression levels of prostaglandin E(2) (PGE(2)) receptors may be altered in colon cancers. For example, the EP(1) and EP(2) subtypes have been shown to be up-regulated and EP(3) down-regulated in AOM-induced colon cancers in rats and mice. EP(1) and EP(4) appear to be involved in ACF formation, while alteration in EP(2) and EP(3) is considered to contribute to later steps in colon carcinogenesis. Increased expression of some other gene products, such as the targets of Wnt/beta-catenin signaling, have also been reported. The further accumulation of data with this chemically-induced animal colon carcinogenesis model should provide useful information for understanding colorectal neoplasia in man.

Previous studies have shown that prostaglandin E(2) (PGE(2)) is involved in intestinal carcinogenesis through its binding to the PGE(2) receptor subtypes EP(1) and EP(4) and activation of downstream pathways. ONO-8711 and ONO-AE2-227, prostaglandin E receptor subtype EP(1)- and EP(4)-selective antagonists, respectively, are known to suppress formation of intestinal polyps in adenomatous polyposis coli gene-deficient mice. The present study was designed to investigate the combined effects of EP(1) and EP(4) antagonists on spontaneous polyp formation in APC1309 mice in order to determine the contribution of each receptor to intestinal tumorigenesis. APC1309 mice were treated with 400 ppm of ONO-8711 alone, 400 ppm of ONO-AE2-227 alone or both in combination in the diet for 6 weeks. The mean area of polyps found in the intestine, calculated as the longer diameter x the shorter diameter x pi, was reduced by 12%, 43% (P < 0.01) and 56% (P < 0.01) of the mean control value (8.8 mm(2)) in the ONO-8711 alone, ONO-AE2-227 alone and combination treatment groups, respectively, suggesting clear additive effects of the combination. The same additive tendency for suppression was also observed with respect to the numbers of polyps in the intestine. Polyp size reduction was more remarkable with the EP(4) antagonist, while the number reduction was more pronounced with the EP(1) antagonist. Our results indicate that EP(1) and EP(4) may have separate intrinsic roles and, to some extent, contribute to polyp formation independently. Thus, combination treatment has potential for the chemoprevention of colon carcinogenesis.

Recent epidemiological studies have shown a positive association of a high-fat diet with the risk of colon cancer. Indeed, increments in the serum levels of triglycerides (TG) and cholesterols are positively related with colon carcinogenesis. We previously reported that an age-dependent hyperlipidemic state is characteristic of Min mice, an animal model for human familial adenomatous polyposis (FAP). However, qualitative and quantitative changes of lipid metabolism are poorly understood in this state. Here, we provide detailed analysis of serum lipids in Min mice using reverse-phased liquid chromatography/electrospray ionization mass spectrometry (RPLC/ESI-MS). We also demonstrate local analysis of lipid droplets in the villi of the small intestine using laser capture microdissection and a sensitive chip-based nanoESI-MS system. As a result, oxidized phosphatidylcholines (PC) such as aldehyde and carboxylic acid types were increased, even at an early stage of intestinal polyp formation in serum. In addition, hydroperoxidizable TG precursors containing linoleic acid (18:2n-6) were deposited at the tip of the villi with aging, and these hydroperoxidized TG were also increased in serum. Meanwhile, increments of the oxidizable TG precursors in serum and small intestinal mucosa were suppressed by treatment with pitavastatin, a novel third generation lipophilic statin. These results suggest that quantitative and qualitative lipid changes such as hydroperoxidizable TG precursors are important in the course of intestinal polyp formation and oxidative stress might lead to the development of intestinal polyp formation in Min mice.

X-ray computed tomography (CT) has been used for diagnoses of human pancreatic cancer. Although micro-CT is a useful approach to evaluate macromorphology of organs/tissue also in animal models, reports on pancreatic tumors are limited. In this study, the utility of micro-CT was assessed in characterizing chemically induced pancreatic tumors in Syrian hamsters. Hamsters treated with or without N-nitrosobis(2-oxopropyl)amine (BOP) were injected with the antispasmodic agent, scopolamine butylbromide, and contrast agents, 5 or 10 mL/kg body weight of iopamidol or Fenestra VC at 18-38 weeks, then examined by micro-CT scanning with a respiratory gating system. Both peristaltic and respiratory movements were substantially suppressed by the combination of scopolamine butylbromide treatment and the respiratory gating system, resulting in improvements of image qualities. Iopamidol clearly visualized the pancreatic parenchyma and contrasted the margins among the pancreas and other abdominal organs/tissue. Meanwhile Fenestra VC predominantly contrasted abdominal vascular systems, but the margins among pancreas and other organs/tissue remained obscure. Six pancreatic tumors of 4-13 mm in diameter were detected in four of 15 animals, but not the five tumors of 1-4 mm in diameter. The inner tumor images were heterogeneously or uniformly visualized by iopamidol and Fenestra VC. Overall, iopamidol could clearly contrast between pancreatic parenchyma and the tumors as compared with Fenestra VC. All tumors confirmed were histopathologically diagnosed as pancreatic ductal adenocarcinomas. Thus, micro-CT could be useful to evaluate the carcinogenic processes and preventive methods of pancreatic cancer in hamsters and to assess the novel contrast agents for detection of small pancreatic cancer in humans.

We have reported that a hyperlipidemic state is characteristic of Apc-deficient Min mice with multiple intestinal polyps. In our earlier case-control study, colorectal cancer risk showed positive relationships with erythrocyte membrane compositions of palmitic and oleic acids, but negative links with linoleic and arachidonic acids. To examine the roles of fatty acids in intestinal polyp formation, levels in plasma, erythrocytes, and intestinal polyps in Min mice were compared with those in wild-type mice. A diet free of eicosapentaenoic and docosahexaenoic acids with antineoplastic effects was fed to all mice from 6 to 15 weeks of age. Fatty acid levels were measured using accelerated solvent extraction and gas-liquid chromatography. Min mice with a hyperlipidemic state and multiple intestinal polyps had elevated values for palmitic and oleic acids in plasma and erythrocytes (at least P < 0.05), and higher plasma level of linoleic acid (P < 0.05). Arachidonic acid was 24.5% lower in erythrocytes (P < 0.0005), but did not differ in plasma. In Min mice, moreover, oleic and arachidonic acids were 1.78 and 1.43 times higher, respectively, in intestinal polyps than in paired normal mucosa (P < 0.05 and P < 0.01, respectively), but linoleic acid was 31.9% lower (P < 0.001). The present study suggests that palmitic, oleic, and arachidonic acids play key roles in intestinal polyp formation, and demonstrates reduced erythrocyte arachidonic acid values of Min mice, in line with our previous findings for patients with sporadic colorectal cancers.

X-ray microcomputed tomography (micro-CT) with a respiratory gating system is a useful non-invasive approach to evaluate lung tumor development in living animal models. Here micro-CT was applied for the detection of lung lesions induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice, at 2-week intervals from 10 to 30 weeks after carcinogen exposure. In micro-CT cross sections, lung tumor images were easily distinguished from surrounding non-tumorous tissues, the smallest detected tumor being approximately 0.5 mm in diameter. All of the urethane-treated mice (n = 15) developed lung tumors and the number of tumors developed in each mouse was 8.6 +/- 3.9. Six tumors, determined histopathologically to be adenocarcinomas, were detected, growing at different rates during the experimental period. The most aggressive carcinoma, increasing in diameter from 0.9 to 3.5 mm within 8 weeks, was a solid-type nodule with a clear tumor margin on the micro-CT imaging. Other tumors, histopathologically adenomas, grew slowly or moderately. The results provide evidence that micro-CT is a useful non-invasive imaging approach for evaluating the characteristics and growth of lung tumors in mice.

The endogenous DNA adducts O(6)-carboxymethyl-deoxyguanosine (O(6)-CM-dG) and 3-ethanesulfonic acid-deoxycytidine (3-ESA-dC) are produced from N-nitroso bile acid conjugates, such as N-nitrosoglycocholic acid (NO-GCA) and N-nitrosotaurocholic acid (NO-TCA), respectively. Formation of these DNA adducts in vivo was here analyzed by 32P-postlabeling in the glandular stomach of rats subjected to duodenal content reflux surgery. In this model, all duodenal contents, including bile acid conjugates, flow back from the jejunum into the gastric corpus. The levels of O(6)-CM-dG found at 4 and 8 weeks after surgery were 40.9 +/- 9.4 and 56.3 +/- 3.2 per 10(8) nucleotides, respectively, whereas the sham operation groups had values of 5.8 +/- 2.3 and 5.9 +/- 0.5 per 10(8) nucleotides. Moreover, adduct spots corresponding to 3-ESA-dC were detected in both duodenal reflux and sham operation groups and levels in the duodenal reflux groups were around four-fold elevated at 11.2 +/- 1.0 and 8.9 +/- 1.0 per 10(8) nucleotides after 4 and 8 weeks, respectively. When the duodenal reflux animals were treated with a nitrite trapping agent, thiazolidine- 4-carboxylic acid (thioproline, TPRO), the levels of O(6)-CM-dG and 3-ESA-dC were reduced to the same levels as in the sham operation animals. These observations suggest that NO-TCA and NO-GCA are formed by nitrosation of glycocholic acid and taurocholic acid, respectively, and these nitroso compounds produce DNA adducts in the glandular stomach of rats subjected to duodenal content reflux surgery.

A new flavone derivative, chafuroside, has been isolated as a strong anti-inflammatory compound from oolong tea leaves, and its structure determined to be (2R,3S,4S,4aS,11bS)-3,4,11-trihydroxy-2-(hydroxymethyl)-8-(4-hydroxyphenyl)-3,4,4a,11b-tetrahydro-2H,10H-pyrano[2',3':4,5]furo[3,2-g]chromen-10-one. To assess its potential to inhibit intestinal carcinogenesis, 2.5, 5 and 10 p.p.m. chafuroside was given in the diet to Apc-deficient Min mice for 14 weeks from 6 weeks of age. Total numbers of polyps were reduced to 83, 73 and 56% of the control value, respectively. Moreover, dietary administration at 10 and 20 p.p.m. reduced azoxymethane (AOM)-induced colon aberrant crypt foci (ACF) development in rats to 69% of the AOM-treated control value with the higher dose. Chafuroside-associated toxicity was not observed at 2.5-10 p.p.m. in Min mice and 10-20 p.p.m. in AOM-treated rats. These results suggest that chafuroside might be a good chemopreventive agent for colon cancer.

Prostaglandin E2 is involved in colon carcinogenesis through its binding to the PGE2 receptor subtypes EP1, EP2, EP3 and EP4. We have demonstrated that administration of ONO-8711, an EP1-selective antagonist, suppresses development of AOM-induced ACF in C57BL/6 mice and F344 rats. ONO-8711 also reduced the numbers of intestinal polyps in Min mice. In the present study, we investigated the long-term effects of ONO-8711 on colon cancer development in rats treated with AOM. Male F344 rats were injected subcutaneously with AOM (15 mg/kg body weight) once a week for the first 2 weeks to develop colon cancer. Administration of 400 or 800 p.p.m. ONO-8711 in their diets for 32 weeks reduced the incidence, multiplicity and volume of colon carcinomas. The incidence of colon adenocarcinomas in AOM-treated rats was 97, 83 and 76% (P

Mongolian gerbils are considered to be a good animal model for understanding the development of Helicobacter pylori-associated diseases. However, limitations regarding the genetic information available for this animal species hamper the elucidation of underlying mechanisms. Thus, we have focused on identifying the nucleotide sequences of cDNAs encoding Mongolian gerbil inflammatory proteins, such as interleukin-1 (IL-1beta), tumor necrosis factor alpha (TNF-alpha), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Furthermore, we examined the mRNA expression of these genes in the glandular stomach by RT-PCR at 1-8 weeks after H. pylori infection. The deduced amino acid homologies to mouse, rat and human proteins were 86.2%, 83.6% and 67.8% for IL-1beta, 87.2%, 85.1% and 78.4% for TNF-alpha , 91.9%, 90.2% and 84.8% for COX-2 and 90.8%, 89.1% and 80.1% for iNOS, respectively. The average stomach weight of Mongolian gerbils inoculated with H. pylori was increased in a time-dependent manner at 1, 2, 4 and 8 weeks after inoculation. In the pyloric region, mRNA expression levels of IL-1beta, TNF-alpha and iNOS were increased in H. pylori-infected animals at the 2 weeks time point, while in the fundic region, expression levels of IL-1beta, TNF-alpha and iNOS were elevated at 4 and 8 weeks. The COX-2 expression level in the fundic region was clearly elevated in infected animals compared with control animals at 4 and 8 weeks, but in the pyloric region, expression levels were similar in both infected and control animals. Thus, our results indicate that oxidative stress occurs from an early stage of H. pylori infection in the glandular stomach of Mongolian gerbils.

Research leading to the discovery of a series of mutagenic and carcinogenic heterocyclic amines (HCAs) was inspired by the idea that smoke produced during cooking of food, especially meat or fish, might be carcinogenic. More than ten kinds of HCAs, actually produced by cooking or heating of meat or fish, have now been isolated and their structures determined, most being previously unregistered compounds. They are highly mutagenic towards Salmonella typhimurium in the presence of S9 mix and are also mutagenic in vitro and in vivo toward mammalian cells. HCAs have now been chemically synthesized in quantity and subjected to long-term animal testing. When HCAs were fed in the diet, rodents developed cancers in many organs, including the colon, breast and prostate, and one HCA produced hepatomas in monkeys. The lesions exhibited alteration in genes including Apc, beta-catenin and Ha-ras, and these changes provide clues to the induction mechanisms. The HCAs are oxidized to hydroxyamino derivatives by cytochrome P450s, and further converted to ester forms by acetyltransferase and sulfotransferase. Eventually, they produce DNA adducts through the formation of N-C bonds at guanine bases. There are HCA-sensitive and resistant strains of rodents and a search for the responsible genes is now under way. While the content of HCAs in dishes consumed in ordinary life is low and not sufficient in itself to explain human cancer, the coexistence of many other mutagens/carcinogens of either autobiotic or xenobiotic type and the possibility that HCAs induce genomic instability and heightened sensitivity to tumor promoters suggest that avoidance of exposure to HCAs or reduction of HCAs' biological effects as far as possible are to be highly recommended. Usage of microwave ovens for cooking and supplementation of the diet, for example with soy-isoflavones, which have been found to suppress the occurrence of HCA-induced breast cancers, should be encouraged. Advice to the general public about how to reduce the carcinogenic load imposed by HCAs would be an important contribution to cancer prevention.

In our previous study, a peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist, pioglitazone, suppressed both hyperlipidemia and intestinal polyp formation in Apc(1309) mice at doses of 100 and 200 ppm in the diet. In contrast, it has been reported that doses of 1500 or 2000 ppm of another PPAR gamma agonist, troglitazone, enhanced colon polyp development in Min mice. In the present study, we therefore investigated the effects of a wide range of pioglitazone doses on both hyperlipidemia and intestinal polyp formation in Min mice. Serum triglycerides and very low density lipoprotein (VLDL) cholesterol in the basal diet group were elevated to levels 13-15 times higher than those in the wild-type counterparts at 20 weeks of age. They were reduced dose-dependently by treatment with 100, 200, 400 and 1600 ppm pioglitazone from 6-20 weeks of age with suppression to almost the wild-type level at the highest dose. Moreover, up-regulation of the liver mRNA levels for lipoprotein lipase (LPL) was evident in the pioglitazone-treated animals. Dose-dependent reduction of intestinal polyps was observed in Min mice given 100-1600 ppm for 14 weeks, total numbers being decreased to 63-9% of the control value. A suppressive effect of pioglitazone on colon polyp formation was also found. The PPAR gamma agonist, pioglitazone, may thus be a promising candidate chemopreventive agent for colon cancer.