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The modification effects of nimesulide, a cyclooxygenase (COX)-2 inhibitor, administration during the postinitiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Male Syrian hamsters were given 4 weekly s.c. injections of BOP at a dose of 10 mg/kg and thereafter administered 0, 100 or 400 ppm nimesulide in the diet for 36 weeks. Additional groups of hamsters were fed 400 ppm nimesulide without prior BOP initiation or nontreated. At week 40, all surviving animals were killed and development of neoplastic and preneoplastic lesions was assessed histopathologically. The incidence of pancreatic adenocarcinomas was significantly (p < 0.05) decreased in the BOP/400 ppm nimesulide group compared to the BOP alone group. The multiplicity of total lesions of pancreatic adenocarcinoma plus atypical hyperplasia was also significantly (p < 0.05) lowered. Immunohistochemically, COX-2 was clearly expressed in pancreatic and lung tumor cells, whereas expression was not remarkably affected by the 400 ppm nimesulide treatment. Proliferating cell nuclear antigen labeling indices of pancreatic ducts were significantly (p < 0.01) reduced by nimesulide. The incidence and multiplicity of neoplastic lesions in other organs did not significantly differ among the BOP-treated groups, though only the multiplicity of lung tumors showed a tendency to decrease. No neoplastic lesions were detected in animals receiving nimesulide alone. Our results clearly indicate that nimesulide protects against BOP-induced pancreatic tumors in hamsters., (Copyright 2003 Wiley-Liss, Inc.)

Background & Aims: Peroxisome proliferator-activated receptor gamma (PPARgamma) is known to regulate growth arrest and terminal differentiation of adipocytes and is used clinically as a new class of antidiabetic drugs. Recently, several studies have reported that treatment of cancer cells with PPARgamma ligands induces cell differentiation and apoptosis, suggesting a potential application as chemopreventive agents against carcinogenesis. However, contradictory results have been reported with regards to the biologic role of PPARgamma in carcinogenesis. Tanaka et al.(24) have recently reported the suppressive effect of a PPARgamma ligand, troglitazone (Tro), on the formation of aberrant crypt foci (ACF) in rats. In the present study, 3 different kinds of PPARgamma ligands were subjected to the experiments to confirm their suppressive effects on colon carcinogenesis., Methods: Three PPARgamma ligands, pioglitazone (Pio) (200 ppm), rosiglitazone (Rosi) (200 ppm), and Tro (1000 ppm) were investigated on the induction of ACF, a putative precancerous lesion of the colon, and colon tumor formation using an azoxymethane (AOM)-induced colon cancer model in BALB/c mice, and dose dependency of a PPARgamma ligand was also examined., Results: PPARgamma ligands reduced the ACF formation by AOM (10 mg/kg body weight) and induction of colon tumors were also markedly suppressed by a continuous feeding of Pio at 200 ppm., Conclusions: Our findings indicate that PPARgamma ligands are indeed potential chemopreventive agents for colon carcinogenesis.

Background: Recently, the acquisition by Helicobacter pylori of resistance to antibiotics has become a serious problem. Therefore, nonantibiotic substances are required to diminish H. pylori-induced gastric lesions. In the present study, the effects of Cladosiphon fucoidan were examined in terms of H. pylori attachment to porcine gastric mucin in vitro and Helicobacter pylori-induced gastritis in vivo., Methods: The inhibitory effect of Cladosiphon fucoidan and other polysaccharides on H. pylori attachment to porcine gastric mucin was assayed in vitro with mucin-coated microtiter plates. The effect of Cladosiphon fucoidan on H. pylori-induced gastritis was examined in vivo using Mongolian gerbils. H. pylori-inoculated gerbils were given fucoidan in drinking water. Six weeks after H. pylori-inoculation, gerbils were sacrificed for macroscopic and microscopic examination of gastric lesions and counting of viable H. pylori in the gastric mucosa., Results: Cladosiphon fucoidan inhibited the H. pylori attachment to porcine gastric mucin at pH 2.0 and 4.0. Two other sulfated polysaccharides, Fucus fucoidan and dextran sulfate sodium, also inhibited the attachment but only at pH 2.0. Inhibitory effects of these three sulfated polysaccharides were not observed at pH 7.2 and nonsulfated polysaccharides, such as mannan and dextran, exerted no influence at any pH. In the in vivo experiment, the H. pylori-induced gastritis and the prevalence of H. pylori infected animals were markedly reduced by fucoidan in a dose-dependent manner, at doses of 0.05 and 0.5% in the drinking water., Conclusion: Cladosiphon fucoidan may deserve particular attention as a safe agent that can prevent H. pylori infection and reduce the risk of associated gastric cancer.

The modifying effects of a cyclooxygenase (cox)-2 selective inhibitor nimesulide on tongue carcinogenesis were investigated in male F344 rats initiated with 4-nitroquinoline-1-oxide (4-NQO). The cell proliferation activity measured by proliferating cell nuclear antigen (PCNA)-positive index and apoptotic index, and the immunohistochemical expression of COX-2, and inducible nitric oxide synthase (iNOS) in the tongue mucosa or neoplasms were also examined for mechanistic analysis of modifying effects of nimesulide on tongue carcinogenesis. All animals except those treated with nimesulide alone and untreated rats were given 20 ppm 4-NQO in drinking water for 8 weeks to induce tongue neoplasms. Starting 1 week after the cessation of 4-NQO exposure, rats given 4-NQO were fed the experimental diets containing nimesulide (100 and 400 ppm) for 22 weeks. At week 32, the incidence of tongue squamous cell carcinoma was significantly reduced by feeding of the diet containing 400 ppm nimesulide. Feeding of nimesulide significantly decreased polyamine content and PCNA-labeling index in tongue carcinoma. Apoptotic index in tongue carcinoma was increased by feeding of nimesulide. In addition, nimesulide feeding reduced COX-2 and iNOS expression in the tongue dysplasia and neoplasms. These results suggest that 400 ppm nimesulide in diet, when given during the promotion phase, exerts chemopreventive ability against 4-NQO-induced tongue tumorigenesis through inhibition of cell proliferation activity in conjunction with modification of COX-2 and iNOS expression of the target lesions.

Helicobacter pylori infection is intimately involved in stomach cancer development and recent epidemiological studies have indicated that the consumption of allium vegetables reduces the risk of gastric neoplasia. Therefore, in the present study, we investigated the effect of a garlic extract on H. pylori-induced gastritis in Mongolian gerbils. Garlic extract was fed to animals at doses of 1, 2 and 4% in the diet from 4 h after H. pylori inoculation until the end of the experiment, at week 6. With the administration of garlic extract, H. pylori-induced gastritis in animals was decreased in a dose-dependent manner, and significantly so at 4%. The numbers of hemorrhagic spots in the glandular stomach and the microscopic score for gastritis were significantly reduced from 19.2+/-15.6 and 5.9+/-0.8 in control gerbils to 8.1+/-11.2 and 4.2+/-1.5, respectively, by 4% garlic extract treatment. The stomach wet weight (1.04+/-0.22 g) of control gerbils was also reduced by 4% garlic extract (0.86+/-0.18 g). However, the number of viable H. pylori was not changed by the garlic extract treatment. The above observations indicated that garlic extract might be useful as an agent for prevention of H. pylori-induced gastritis, leading to reduction in the risk of gastric cancer.

Cyclooxygenase (COX)-2, one enzyme isoform responsible for producing prostanoids from arachidonic acid, contributes to colon carcinogenesis. Recently, genetic disruption of COX-1, the other isoform, was shown to decrease the number of intestinal polyps and prostaglandin E(2) levels in intestinal mucosa, like the case with COX-2 gene disruption, in Min mice. We therefore investigated whether a COX-1 selective inhibitor, mofezolac, suppresses intestinal carcinogenesis in rodents. F344 male rats, receiving azoxymethane (AOM, 15 mg/kg body wt) s.c. injections at 5 and 6 weeks of age, were fed a diet containing 600 or 1200 p.p.m. mofezolac for 4 weeks. The number of aberrant crypt foci (ACFs) per rat and the bromodeoxyuridine labeling index of the crypt epithelium were dose-dependently decreased by administration of mofezolac, the value for the former at 1200 p.p.m. being 60% of control value. When Apc gene knockout mice (APC1309 mice) were given 600 or 1200 p.p.m. mofezolac in their diet for 8 weeks, the numbers of intestinal polyps were also dose-dependently decreased, with reduction to 59% of that in the control diet group at the higher dose. Nimesulide, a COX-2 selective inhibitor used as positive control, showed similar suppressive effects on the development of ACFs in AOM-treated rats and polyps in Apc gene knockout mice. The data indicate that both COX-1 and COX-2 can contribute to intestinal tumorigenesis.

In the present study we have established novel intermittent protocols featuring a high fat (HF) diet for efficient induction of large intestinal tumors with a relatively small amount of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In protocol 1, F344 male rats were first fed a diet containing 400 p.p.m. PhIP for 2 weeks, followed by continuous administration of a HF diet without PhIP for 108 weeks. In protocol 2, 2 week PhIP treatments were repeated three times with 4 week intervals on the HF diet alone, followed by continuous feeding of the HF diet for 42 weeks. At termination of the experiments, 16 (3 of 19) and 45% (9 of 20) of the rats had developed a total of three and 13 large intestinal tumors with protocols 1 and 2, respectively. The tumor incidence in protocol 2 was comparable with that observed with continuous feeding of 400 p.p.m. PhIP for 52 weeks, after exposure to only approximately 10% of the amount of carcinogen. Five of nine (55%) tumors harbored mutations in either the beta-catenin or Apc gene, while all demonstrated accumulation of beta-catenin protein in the cytoplasm and nucleus. This suggests that other unknown genetic alterations in the Wnt-Apc-beta-catenin signaling pathway could have been involved in the development of tumors. By further modifying this intermittent protocol with HF diet, one could expect more efficient induction of lesions with much smaller amounts of PhIP in a shorter period. In addition, this model could provide a means to elucidate genetic alterations in large intestinal tumors induced by relatively low levels of carcinogenic insult, mimicking the cases of human colon carcinogenesis induced by exposure to environmental carcinogens.

Accumulating evidence indicates that overproduction of prostanoids attributable to overexpression of cyclooxygenase-2 (COX-2) plays an important role in colon carcinogenesis. We have shown recently that the prostaglandin (PG) E receptor, EP(1), but not EP(3), is involved in mouse colon carcinogenesis. In line with our previous study, here we examined the role of prostanoid receptors in colon carcinogenesis using six additional lines of knockout mice deficient in prostanoid receptors EP(2), EP(4), DP, FP, IP, or TP. The animals were treated with the colon carcinogen, azoxymethane (AOM), and examined for the development of aberrant crypt foci (ACFs), putative preneoplastic lesions in the colon. Formation of ACFs was decreased only in the EP(4)-knockout mice, to 56% of the wild-type level. To confirm these results, we also examined the inhibitory effects of an EP(4)-selective antagonist, ONO-AE2-227, in the diet on the formation of AOM-induced colon ACFs in C57BL/6Cr mice and on the development of intestinal polyps in Min mice. ONO-AE2-227 at a dose of 400 ppm reduced the formation of ACFs to 67% of the control level, and intestinal polyp numbers in Min mice receiving 300 ppm were decreased to 69% of the control level. Plating efficiency assays showed that addition of 1.0 microM ONO-AE1-329, an EP(4)-selective agonist, resulted in a 1.8-fold increase in the colony number of the human colon cancer cell line, HCA-7, similar to the effect of PGE(2). Moreover, EP(4) mRNA expression was clearly observed in normal colon mucosa and colon tumors in mice. Our previous and present results indicate that PGE(2) contributes to colon carcinogenesis through its actions mediated through EP(1) and EP(4) receptors; therefore, antagonists for these two receptors may be good candidates as chemopreventive agents against colon cancer.

Accumulating evidence indicates that COX-2 inhibitors are involved in colon and breast cancer development. Our previous studies indicated that nimesulide and celecoxib, selective COX-2 inhibitors, show inhibitory effects of intestinal carcinogenesis in azoxymethane-treated rats and mice and in Min mice models. We recently found that nimesulide suppressed PhIP-induced breast cancer in female SD rats in which COX-2 protein was overexpressed. These results led us to investigate the effects of prostaglandin E2 (PGE2) in the target tissues. PGE2 showed its biological activity through binding to its membrane receptors, EP(1 to approximately 4). We also investigated the effects of EP receptors on colon carcinogenesis. We used receptor knockout mice and selective receptor antagonists. Our results indicated that the EP1 receptor plays a pivotal role in colon carcinogenesis. Selective EP1 receptor antagonists may be a new class of chemopreventive agents against colon cancer.

Levels of prostaglandin E(2) (PGE(2)) in human and rodent breast cancers are higher than surrounding normal tissues. PGE(2) exhibits biological activity through binding to membrane receptors, EP(1-4). The present study was designed to investigate the effects of ONO-8711, a newly synthesized selective PGE receptor EP(1) antagonist, on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced breast cancer development. Starting at 7 weeks of age, female Sprague-Dawley (SD) rats were given PhIP (85 mg/kg body weight) by gavage four times weekly for two weeks. Dietary administration of ONO-8711 at 400 or 800 p.p.m. delayed occurrence of breast tumors for 2 or 4 weeks, respectively. At 20 weeks after the last dosing of PhIP, all animals were killed and complete autopsy was made. All breast tumors were diagnosed as invasive ductal adenocarcinomas histopathologically. Administration of ONO-8711 at 800 p.p.m. significantly decreased PhIP-induced breast cancer incidence, multiplicity and volume compared with those of rats fed the control diet (56% versus 79%, P < 0.05, 1.2 versus 2.5, P < 0.05, 0.7 versus 1.4 cm(3), P < 0.01, respectively). Apoptosis was significantly increased in breast cancer cells by feeding of ONO-8711 at 800 p.p.m. of 158% (P < 0.05). EP(1) receptor was detected by reverse transcription-polymerase chain reaction (RT-PCR) in breast cancers, not in normal tissues. These results suggest that EP(1) receptor is associated with breast cancer development and selective PGE receptor EP(1) antagonists may possess chemopreventive effects through the induction of apoptosis without any side effects.

Background: Cyclooxygenases (COXs) and prostanoids play pivotal roles in colon carcinogenesis. This study was designed to determine the chemopreventive effects of ONO-8711, a selective prostaglandin E receptor EP1 antagonist, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats and to compare its potential with that of nimesulide, a well-documented selective COX-2 inhibitor., Materials and Methods: Five-week-old male F344 rats received s.c. injections of AOM (15 mg/kg body weight) or the saline vehicle once weekly for two weeks and were fed the control diet (AIN-76A) or the experimental diets containing 400 or 800 ppm of ONO-8711 or 400 ppm nimesulide for 5 weeks., Results: Administration of ONO-8711 at 800 ppm significantly reduced the total number of ACF/colon and 5-bromodeoxyuridine (BrdUrd) labeling index as compared to the control diet group (by 31% and 66%, respectively). As expected, dietary administration of nimesulide also suppressed the development of ACF and BrdUrd labeling index in the colon, by about 39% and 54%, respectively., Conclusion: Our finding that ONO-8711 significantly suppresses colonic ACF formation and cell proliferation strengthens the hypothesis that the selective prostaglandin E receptor EP1 antagonists possesses chemopreventive activity against colon cancer development.

9-(4'-Aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is a novel mutagenic heterocyclic amine, produced by the reaction of norharman with aniline in the presence of S9 mix. In the present study, the acute toxicity of this compound was investigated in male F344 rats. Ten-week-old animals were treated with a single intragastric injection of APNH at doses of 45 or 90 mg/kg body wt and euthanized 1, 3, or 6 days afterward. When APNH was administered at a dose of 90 mg/kg, vacuolation of Sertoli cells in the testis was seen at 1 day after treatment. The testicular damage had markedly progressed by day 6, with multinucleated giant cells and loss of round spermatids in the seminiferous tubules observed in groups 1 and 2 of the four histological categories of spermatogenesis. Numbers of spermatogonia were also decreased by APNH treatment. No toxic changes were observed in Leydig cells under these conditions and serum follicle-stimulating hormone and luteinizing hormone concentrations were also unchanged from control values. Such severe testicular damage was not observed at any time point at the 45 mg/kg dose level of APNH. Moreover, neither norharman nor aniline alone exerted acute testicular toxicity at doses equivalent to 90 mg/kg of APNH. In addition to the testicular lesions, erosive changes of urinary bladder, thymic atrophy, and panmyelophthisis were evident in rats given APNH at 90 mg/kg., (Copyright 2001 Academic Press.)

Severe combined immunodeficiency (Scid) mice have defects in V(D)J recombination and DNA double-strand breaks repair caused by an inherited genetic defect in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Scid mice are highly susceptible to development of T-cell lymphomas, and because of the nature of its association with DNA repair and recombination, DNA-PKcs is considered to belong to the caretaker class of tumor suppressor genes. In the present study, the susceptibility of Scid mice to colon carcinogenesis due to administration of azoxymethane (AOM) was investigated. Significantly higher susceptibility in terms of induction of both aberrant crypt foci (ACFs), putative pre-cancerous lesions of the colon and colon cancers was observed as compared with the isogenic strain, C.B-17 mice. The incidences of colon tumors, either adenomas or adenocarcinomas, in Scid and C.B-17 mice after administration of AOM (10 mg/kg body weight/week) for 6 weeks were 87% (26 of 30) and 50% (15 of 30), respectively, by experimental week 22 (P < 0.01). The multiplicity of colon tumors in Scid mice was also significantly higher than in C.B-17 mice, being 2.2 +/- 1.5 and 0.9 +/- 1.2, respectively (P < 0.001). The present study clearly demonstrated high susceptibility of Scid mice to colon carcinogenesis, which might be attributable to disruption of the caretaker role of DNA-PK in colonic epithelial cells.

Urease has been suggested to be essential for colonization and pathogenesis of Helicobacter pylori infection. In the present study, we evaluated the effects of urease inhibitors [acetohydroxamic acid (AHA) and flurofamide (FFA)] on H. pylori-induced gastritis in Mongolian gerbils. Animals were orally inoculated with H. pylori, and given urease inhibitors in their diet throughout the experimental period of six weeks or four weeks, starting from two weeks after H. pylori inoculation. With the administration of AHA at doses of 100, 500, and 2500 ppm throughout the experimental period, H. pylori-induced gastritis in animals was decreased in a dose-dependent manner, significantly so at 2500 ppm. Suppression of gastric lesions was also evident in animals administered 2500 ppm AHA after the H. pylori infection. Bacterial infection rates were reduced to 40-50% of the control value of 100%, by the highest dose of AHA. The potent urease inhibitor, FFA, also caused marked amelioration of H. pylori-associated gastritis on administration at 100 ppm throughout the six-week experimental period or for four weeks after H. pylori infection. Animals treated with FFA had few visible gastric lesions, and the proportion infected with H. pylori was reduced to less than 10%. Since antibiotic-resistant strains of H. pylori have become a serious problem, nonantibiotic urease inhibitors may be very useful to control H. pylori-associated gastroduodenal disease., (Copyright 2001 Academic Press.)

9-(4'-Aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), produced by the reaction of norharman with aniline in the presence of S9 mix, is a novel heterocyclic amine (HCA), with mutagenicity to Salmonella typhimurium TA 98 and YG 1024 comparable to that of other HCAs such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). This experiment was designed to investigate its potential to induce glutathione S-transferase placental form (GST-P) positive foci in the liver. Male F344 rats, 7 weeks old, were fed diet containing 0, 10, 20, or 50 ppm APNH for 4 weeks, killed by ether euthanasia and performed complete necropsy. Numbers of GST-P positive foci larger than 0.1 mm in diameter induced by APNH at the dose of 10, 20, and 50 ppm were increased in a dose dependent manner to 0.52, 1.3, and 21 foci/cm2, respectively, with areas of 0.006, 0.01, and 2.3 mm2/cm2. No such GST-P positive foci were observed in rats fed control diet. These findings suggest that APNH has hepatocarcinogenic potential in male F344 rats.

Heterocyclic amines (HCAs) present in cooked foods are suggested to be involved in human breast cancer development. Estrogen plays a pivotal role in mammary gland carcinogenesis. Therefore, we designed an in vivo experiment to investigate potential estrogenic effects of two HCAs, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induce mammary gland cancers in rodents, on the uterus of ovariectomized (OVX) Sprague-Dawley (SD) rats. Female SD rats ovariectomized at 35 days of age were given intraperitoneal injections of 17beta-estradiol (E2) at doses of 0, 30 or 50 microg/kg or one of the HCAs at a dose of 50 mg/kg b.w. once a day at 47, 48, and 49 days of age. E2 dramatically increased uterine weights, stromal thickness, epithelial cell height, and 5-bromo-2'-deoxyuridine (BrdU) positive cell counts in a dose dependent manner. Intraperitoneal administration of PhIP or IQ, in contrast, did not produce any estrogenic responses in this assay system. These results indicate that the carcinogenicities of these two HCAs in mammary glands are not associated with estrogenic potential.

Breast cancer is common in women all over the world, and exploration of chemopreventive approaches to this cancer is very important. Nimesulide, a selective inhibitor of cyclooxygenase-2 (COX-2), is a good candidate as a chemopreventive agent with low toxicity. We examined its effects on mammary tumor development in female Sprague-Dawley rats induced with the environmental carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rats at 7 weeks of age received intragastric intubations of PhIP (85 mg / kg body weight) 4 times weekly for 2 weeks and were maintained on control diet (high fat diet) or experimental diet (high fat diet supplemented with 400 ppm nimesulide) throughout the experiment. COX-2 protein was over-expressed in epithelial cancer cells and stromal cells of the PhIP-induced mammary carcinomas, but was weak or not apparent in normal mammary gland cells. The development of mammary carcinomas was clearly suppressed by administration of nimesulide. The carcinoma incidence was 51% as compared to 71% for the control diet group. The average multiplicity of carcinomas in the experimental diet group was 1.2 +/- 0.2 (P < 0.05), significantly smaller than the control diet group value (2.6 +/- 0. 5). The size of carcinomas was also clearly decreased; 1.1 +/- 0.4 cm(3) / rat in experimental diet group (P < 0.05), 4.1 +/- 1.3 cm(3) / rat in the control diet group. The results therefore provide evidence that the selective COX-2 inhibitor, nimesulide, possesses chemopreventive activity against PhIP-induced mammary carcinogenesis in rats.

We previously reported that prostaglandin E(2) contributes to colon carcinogenesis through its binding to the prostaglandin E receptor subtype EP(1) using a genetic approach in EP(1)-knockout mice and a pharmacological approach with the EP(1) selective antagonist, ONO-8711. In the present study, we examined the effects of another more selective EP(1) receptor antagonist, ONO-8713, on development of azoxymethane (AOM)-induced aberrant crypt foci (ACFs) in male C57BL/6J mice treated i.p. with 10mg/kg body weight AOM once a week for 3weeks. Administration of ONO-8713 at doses of 250, 500 and 1000ppm in diet during and post-AOM treatment for 5weeks resulted in a dose-dependent reduction of ACF formation, being 15, 30 and 36% inhibition of the control value, respectively. The level of inhibition was similar to that with ONO-8711. Moreover, ONO-8713 suppressed the development of ACF when administered at post-AOM, as in the case of ONO-8711. The data confirm EP(1) receptor involvement in colon carcinogenesis.

In a search for dietary factors influencing Helicobacter pylori-induced gastritis, the effects of fish meal in the diet were examined in Mongolian gerbils. When a conventional diet containing 10% fish meal was given to Mongolian gerbils for 4 weeks after inoculation of H. pylori, edematous thickening with severe neutrophil and mononuclear cell infiltration in both the mucosa and submucosa was observed in the glandular stomach of 19 out of the 20 animals, and hemorrhagic spots were evident in 11 cases. These gastric lesions were enhanced by a 20% fish meal supplement, and edema and hemorrhage in the gastric mucosa were observed in 19 and 17 out of 20 animals, respectively. Although almost the same levels of viable bacteria were detected independent of the diet, edema and hemorrhage were seen in only 2 and 1 of 20 gerbils fed a diet containing 10% casein, instead of 10% fish meal, respectively. Neither edema nor hemorrhage was observed in 10% beef diet animals. These results suggest that fish meal contains factors which greatly enhance H. pylori-induced gastritis in Mongolian gerbils. Since the incidences of gastritis and gastric cancer are very high throughout the world, it is very important to identify these gastritis-enhancing factors.

Activation of the beta-catenin/T cell factor-mediated transcription pathway through mutations of the APC or beta-catenin gene is suggested to play an important role in colon carcinogenesis and there is great interest in the target genes. We have described the frequent mutation and an altered cellular localization of beta-catenin in rat colon adenocarcinomas induced by azoxymethane (AOM), along with up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. In the present study, the relation between beta-catenin alteration and expression of iNOS and COX-2 in AOM-induced rat colon carcinogenesis was examined in hyperplastic and dysplastic type aberrant crypt, adenoma and adenocarcinoma samples. K-ras gene mutations were also investigated. Mutation analysis by the PCR-single strand conformation polymorphism method and direct sequencing demonstrated the beta-catenin gene to be mutated in two of three dysplastic aberrant crypt foci (ACF), two of six adenomas and 20 of 26 adenocarcinomas, while K-ras was mutated in seven of 10 hyperplastic ACF and seven of 26 adenocarcinomas. Immunohistochemical staining showed an alteration in cellular localization of beta-catenin in all dysplastic ACF, adenomas and adenocarcinomas examined. iNOS expression was also observed in all but one of the lesions in which beta-catenin alterations were observed. Neither iNOS expression nor beta-catenin alterations were observed in any hyperplastic ACF. COX-2 expression in stromal elements was found even in normal colon mucosa and increased in adenomas and adenocarcinomas, while epithelial cells were only positive in large adenocarcinomas. These results show that beta-catenin alterations may be related to induction of iNOS expression, these being early events in AOM-induced colon tumorigenesis which may play important roles in causing dysplastic changes.

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.

Cyclooxygenase (COX)-2 has been suggested to play an important role in colon carcinogenesis. We found that the COX-2 selective inhibitor, nimesulide, reduces azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats and colon carcinogenesis in mice, as well as formation of intestinal polyps in Min mice. Thus, selective inhibitors of COX-2, which catalyzes the synthesis of prostanoids, could be good candidates as chemopreventive agents against colon cancer. Examination of the effect of prostanoid receptor deficiency and a selective antagonist of prostanoid receptor on the development of AOM-induced ACF in mice revealed the involvement of the EP1 receptor. Moreover, a selective EP1 antagonist reduced the number of intestinal polyps in Min mice. These results suggest that PGE2 contributes to colon carcinogenesis through binding to the EP1 receptor. Nitric oxide synthase (NOS) is known to be overexpressed in colon cancers of humans and rats, and a NOS inhibitor, L-NG-nitroarginine methyl ester, was found to inhibit the development of AOM-induced ACF in rats. Thus, NOS including iNOS could also be a good target for chemoprevention of colon cancer, as in the COX-2 case.

Nitric oxide synthase (NOS), an important bioregulator of a variety of biological processes, is overexpressed in colonic tumors of humans and rodents. In this study, effects of L-N(G)-nitroarginine methyl ester (L-NAME), a NOS inhibitor, on development of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in F344 male rats were investigated. Six-week-old male F344 rats were fed diets containing 0 or 100 ppm L-NAME, and given s.c. injections of AOM at 15 mg/kg body wt, once a week for 2 weeks. At 17 weeks of age, all animals were sacrificed and their colons were evaluated for numbers of ACF. Feeding of 100 ppm L-NAME inhibited the development of ACF in different sizes by 24-39%, those containing four or more crypts being most markedly affected. Assessment of silver-stained nucleolar organizer regions protein (AgNORs)/nucleus further revealed a 44% reduction by administration of L-NAME. These results suggest that the NOS inhibitor, L-NAME, may be an effective chemopreventive agent against colon carcinogenesis due to depression of cell proliferation.

Although the cyclooxygenase pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, the molecular species of prostanoids and receptors involved have not been fully elucidated yet. We examined the development of aberrant crypt foci (ACFs), putative preneoplastic lesions of the colon, in two lines of knockout mice, each deficient in prostaglandin E receptors, EP1 and EP3, by treatment with the colon carcinogen, azoxymethane. Formation of ACFs was decreased only in the EP1-knockout mice to approximately 60% of the level in wild-type mice. Administration of 250, 500, or 1000 ppm of a novel selective EP1 antagonist, ONO-8711, in the diet to azoxymethane-treated C57BL/6J mice also resulted in a dose-dependent reduction of ACF formation. Moreover, when Min mice, having a nonsense mutation in the adenomatous polyposis coli gene, were given 500 ppm ONO-8711 in the diet, the number of intestinal polyps was significantly reduced to 57% of that in the basal diet group. These results strongly suggest that prostaglandin E2 contributes to colon carcinogenesis to some extent through its action at the EP1 receptor. Thus, EP1 antagonists may be good candidates as chemopreventive agents for colon cancer.

S-methylmethane thiosulfonate (S-MMTS), isolated from cauliflower and having antiproliferative activity, and the non-steroidal anti-inflammatory drug sulindac have been shown to inhibit chemically induced colon carcinogenesis when they are administered during the initiation and/ or post-initiation stages. The present study was designed to investigate the chemopreventive efficacy of 80 p.p.m. S-MMTS administered during the initiation and post-initiation stages and of S-MMTS and sulindac administered together at low doses (40 and 160 p.p.m., respectively) during the promotion/progression phases (late in the premalignant stage) of colon carcinogenesis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0 (control diet) or 80 p.p.m. S-MMTS. At 7 and 8 weeks of age all rats except those in the vehicle-treated groups were given s.c. injections of 15 mg/kg body wt azoxymethane (AOM). Rats receiving the control diet and intended for the study of inhibition of colon carcinogenesis during the promotion/progression phases were continued on the control diet for 14 weeks after the second AOM treatment; they were then switched to experimental diets containing 80 p.p.m. S-MMTS, 160 p.p.m. sulindac or 40 p.p.m. S-MMTS plus 160 p. p.m. sulindac. The rats were maintained on their respective dietary regimens until 52 weeks after carcinogen treatment and were then killed. Colon tumors were evaluated histopathologically. Administration of 80 p.p.m. S-MMTS alone during the initiation and post-initiation stages and promotion/progression stages had no significant effect on colon tumor inhibition. In contrast, the administration of 160 p.p.m. sulindac during the promotion/progression stages did significantly inhibit total colon tumor multiplicity (P < 0.05). Moreover, co-administration of 40 p.p. m. S-MMTS with 160 p.p.m. sulindac during the promotion/progression stages suppressed the incidence and multiplicity of non-invasive adenocarcinomas (P < 0.05-0.01) and multiplicity of invasive and total adenocarcinomas of the colon to a significant degree (P < 0. 05-0.01). These findings have potential clinical implications.

Epidemiological and model studies with laboratory animals have provided evidence that nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer. Sulindac, a nonsteroidal anti-inflammatory drug, has been shown to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats when administered continuously before, during, and after carcinogen treatment (initiation and postinitiation periods) or when given continuously beginning 14 weeks after carcinogen administration (promotion/ progression stage). The present study was designed to investigate the chemopreventive efficacy of sulindac sulfone (exisulind), the sulfone metabolite of sulindac, when administered during the promotion/progression stage of colon carcinogenesis in comparison to the effect during the initiation and postinitiation periods. We have also studied the modulating effect of exisulind on colonic tumor apoptosis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0%, 0.06%, and 0.12% exisulind. At 7 weeks of age, groups of animals were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 weeks). Animals intended for the promotion/progression study and receiving 0% exisulind were switched to an experimental diet containing 0.12% exisulind at 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, 50 weeks after the second AOM injection. Colon tumors were evaluated histopathologically for tumor type. Administration of 0.06% and 0.12% exisulind during the initiation and postinitiation periods significantly inhibited the incidence and multiplicity of invasive and/or noninvasive adenocarcinomas of the colon. The inhibition of colon tumorigenesis by exisulind was associated with a significant retardation of body weight gain shortly after sulfone administration and increased apoptosis in the colon tumors. In contrast, administration of the higher dose (0.12%) of exisulind during the promotion/progression stage had only minimal effects on colon tumorigenesis and apoptosis in the colon tumors, suggesting that early administration, but not late administration, may be required for chemopreventive efficacy of this drug.

Several epidemiological studies have suggested an inverse association between the risk of prostate cancer and intake of soybeans and their products. In vitro data pointing to possible anti-carcinogenic properties of the soybean isoflavone, genistein, led us to investigate the chemopreventive potential of soybean isoflavones in a rat carcinogenesis model induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB) and testosterone propionate (TP). Animals received DMAB s.c. injections at 2-week intervals for the first 20 weeks and implanted silicon tubes containing 40 mg of TP, replaced at 6-week intervals throughout the experiment. The soybean isoflavone mixture consisting of 74% genistein and 21% daidzein was mixed in basal diet (AIN-76A) at concentrations of 100 and 400 ppm and fed to F344 male rats throughout the experiment. Rats treated with carcinogens and administered isoflavone mixture at 100 and 400 ppm developed adenocarcinomas at incidences of 35% and 29%, respectively, in the prostate and seminal vesicles, whereas the figure was 60% for those maintained on control diet. Feeding of the isoflavone mixture at 100 and 400 ppm significantly inhibited the number of argyrophilic nucleolar organizer regions (AgNORs) in adenocarcinomas of the accessory sex glands as compared to those of rats fed control diet. No influence on the development of neoplastic lesions originating in other organs was noted. The results of this study provide evidence that soybean isoflavones may have potential as chemopreventive agents against carcinogenesis in the prostate.

Inducible nitric oxide synthase (iNOS) is overexpressed in colonic tumors of humans and also in rats treated with a colon carcinogen. iNOS appear to regulate cyclooxygenase-2 (COX-2) expression and production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to study the inhibitory effects of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT) a selective iNOS-specific inhibitor, measured against formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). Beginning at 5 weeks of age, male F344 rats were fed experimental diets containing 0 or 50 p.p.m. of PBIT, or 2000 p.p.m. of curcumin (non-specific iNOS inhibitor). One week later, rats were injected s.c. with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 17 weeks of age, all rats were killed, colons were evaluated for ACF formation and colonic mucosa was assayed for isoforms of COX and NOS activities. Both COX and iNOS activities in colonic mucosa of the AOM-treated rats were significantly induced. Importantly, 50 p.p.m. PBIT suppressed AOM-induced colonic ACF formation to 58% (P < 0.0001) and crypt multiplicity containing four or more crypts per focus to 78% (P < 0.0001); it also suppressed AOM-induced iNOS activity. Curcumin inhibited colonic ACF formation by 45% (P < 0.001). These observations suggest that iNOS may play a key regulatory role in colon carcinogenesis. Developing iNOS-specific inhibitors may provide a selective and safe chemopreventive strategy for colon cancer treatment.

Curcumin, derived from the rhizome of Curcuma longa L. and having both antioxidant and anti-inflammatory properties, inhibits chemically induced carcinogenesis in the skin, forestomach, and colon when it is administered during initiation and/or postinitiation stages. This study was designed to investigate the chemopreventive action of curcumin when it is administered (late in the premalignant stage) during the promotion/progression stage of colon carcinogenesis in male F344 rats. We also studied the modulating effect of this agent on apoptosis in the tumors. At 5 weeks of age, groups of male F344 rats were fed a control diet containing no curcumin and an experimental AIN-76A diet with 0.2% synthetically derived curcumin (purity, 99.9%). At 7 and 8 weeks of age, rats intended for carcinogen treatment were given s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight per week. Animals destined for the promotion/progression study received the AIN-76A control diet for 14 weeks after the second AOM treatment and were then switched to diets containing 0.2 and 0.6% curcumin. Premalignant lesions in the colon would have developed by week 14 following AOM treatment. They continued to receive their respective diets until 52 weeks after carcinogen treatment and were then sacrificed. The results confirmed our earlier study in that administration of 0.2% curcumin during both the initiation and postinitiation periods significantly inhibited colon tumorigenesis. In addition, administration of 0.2% and of 0.6% of the synthetic curcumin in the diet during the promotion/progression stage significantly suppressed the incidence and multiplicity of noninvasive adenocarcinomas and also strongly inhibited the multiplicity of invasive adenocarcinomas of the colon. The inhibition of adenocarcinomas of the colon was, in fact, dose dependent. Administration of curcumin to the rats during the initiation and postinitiation stages and throughout the promotion/progression stage increased apoptosis in the colon tumors as compared to colon tumors in the groups receiving AOM and the control diet. Thus, chemopreventive activity of curcumin is observed when it is administered prior to, during, and after carcinogen treatment as well as when it is given only during the promotion/progression phase (starting late in premalignant stage) of colon carcinogenesis.

Observational, clinical and experimental studies have suggested that dietary supplementation with selenium can inhibit the development of colon cancer. Since toxicity and chemopreventive efficacy of selenium compounds depend to a large extent, on the form of selenium the development of efficacious organoselenium compounds with low toxicity is being pursued in our laboratory. We have assessed the chemopreventive properties of a newly synthesized organoselenium compound, benzyl selenocyanate glutathione conjugate (BSeSG), and of benzyl selenocyanate (BSC), as a positive control, using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) as a measure of efficacy. Five-week-old male F344 rats were fed the control diet (modified AIN-76A) or experimental diets containing 10 or 20 ppm BSeSG (1.7 and 3.4 ppm as Se, respectively), or 10 ppm BSC (4.1 ppm as Se). One week later, all animals except those in vehicle (normal saline)-treated groups were s.c. injected with AOM (15 mg/kg of body weight, once weekly for 2 weeks). All animals were sacrificed 7 weeks after the last AOM injection, and the ACF, levels of prostaglandin E2 (PGE2), cyclooxygenase protein expression (COX-1 and -2), and glutathione S-transferase type mu (GST-mu) were determined in the colon. As expected, dietary administration of BSC suppressed ACF development by about 37%. In rats administered 10 or 20 ppm BSeSG, the frequencies of AOM-induced colonic ACF were significantly decreased compared to those of rats given AOM and control diet by about 41% (P<0.01) and 61% (P<0.001), respectively. Administration of BSeSG inhibited PGE2 production (81-88% inhibition) via COX-2 synthesis in the colonic mucosa (18-60% inhibition). Also, BSeSG increased GST-mu protein activity in colonic mucosa (30-32% increase). These data suggest that a newly synthesized organoselenium compound, BSeSG might be a promising chemopreventive agent against colon carcinogenesis.

Background: Although some cases of hepatocellular carcinoma (HCC) are known to possess a sarcomatous appearance, their cytologic characteristics in ascites have not been reported., Cases: Two Japanese males, aged 67 and 60 were admitted to the Gifu Municipal Hospital because of liver tumors. In the first case, although transcatheter arterial embolization (TAE) was performed, ascites with neoplastic cells continued to enlarge, and the patient died of liver failure. In the second case, although TAE, percutaneous ethanol injection therapy, chemotherapy and radiotherapy were performed, the patient died of tumor progression: expansion of the hepatic tumor, metastases to distal organs and increasing malignant ascites. Neoplastic cells in the ascites of both cases had nuclei that were shaped irregularly or were vesicular and contained conspicuous nucleoli. A number of multinucleated giant cells were also seen. Immunocytochemically, numerous neoplastic cells were positive for alpha-fetoprotein. These findings suggested sarcomatous HCC. Also, histologic findings of the liver tumors at autopsy showed the appearance of sarcomatous HCC., Conclusion: In both cases presented, HCC cells with sarcomatous change were observed in ascites. To our knowledge, this is the first report describing the cytologic diagnosis of sarcomatous HCC in ascites.

Epidemiological and laboratory studies suggest that nonsteroidal antiinflammatory drugs reduce the risk of colon cancer and that the inhibition of colon carcinogenesis is mediated through modulation of prostaglandin production by cyclooxygenase (COX) isozymes (COX-1 and -2). Overexpression of COX-2 has been observed in colon tumors; therefore, specific inhibitors of COX-2 activity could potentially serve as chemopreventive agents. Our recent study indicated that celecoxib (SC-58635), a specific COX-2 inhibitor, suppressed colonic aberrant crypt foci formation induced by azoxymethane in rats and led us to investigate more specifically the chemopreventive potential of this compound using colon tumors as end points. Five-week-old male F344 rats were fed the control diet (modified AIN-76A) or an experimental diet containing 1500 ppm celecoxib. Two weeks later, all animals except those in the saline-treated groups received s.c. injections of azoxymethane (15 mg/kg of body weight) once weekly for 2 weeks. All groups were kept on their regimen until the experiment was terminated, 50 weeks after carcinogen treatment. Colon tumors were evaluated histopathologically. Remarkably, dietary administration of celecoxib inhibited both incidence and multiplicity of colon tumors by about 93 and 97%, respectively. It also suppressed the overall colon tumor burden by more than 87%. The degree of tumor inhibition was more pronounced with celecoxib than it was with previously evaluated nonsteroidal anti-inflammatory drugs. The results of this study provide evidence, for the first time, that a specific COX-2 inhibitor, celecoxib, possesses strong chemopreventive activity against colon carcinogenesis.

The modifying effect of dietary administration of a xanthine oxidase inhibitor 1'-acetoxychavicol acetate (ACA) present in an edible plant Languas galanga in Thailand on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce colonic ACF. They were fed the diets containing 100 or 200 ppm ACA for 5 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (week 5), AOM induced 118 +/- 28 ACF/colon. Dietary administration of ACA caused significant reduction in the frequency of ACF (41% inhibition by 100 ppm ACA feeding and 37% inhibition by 200 ppm ACA feeding, P<0.01). Such inhibition might be associated with suppression of the proliferation biomarkers' expression such as ornithine decarboxylase activity in the colonic mucosa, number of silver-stained nucleolar organizer regions' protein in the colonic mucosal cell nuclei and blood polyamine content. These results indicate that ACA could inhibit the development of AOM-induced ACF through its suppression of cell proliferation in the colonic mucosa and ACA might be a possible chemopreventive agent against colon tumourigenesis.

The modifying effects of dietary exposure to two natural products, protocatechuic acid (PCA) and costunolide during the development of neoplasms in oral carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) were investigated in male Syrian golden hamsters. All hamsters except those in the test chemical alone and control groups received DMBA (0.5%) in mineral oil to the right buccal pouch 3 times per week for 4 or 6 weeks. At 13 weeks of age, the groups exposed to DMBA were fed diet containing PCA or costunolide at a dose of 0.2 g/kg diet (200 ppm) for 17 weeks. The other groups consisted of hamsters given mineral oil alone for 6 weeks, or given 200 ppm PCA or costunolide alone, or untreated. All animals were necropsied at the termination of the experiment (week 24). PCA or costunolide significantly decreased the tumor burden (P < 0.001-P < 0.05) and the extent of dysplastic areas (%) (P < 0.001-P < 0.05). PCA significantly decreased the mean number of AgNORs/nucleus (P < 0.05). The BrdUrd-labeling index was reduced by dietary administration of test compounds, though not significantly. These results suggest that PCA and costunolide inhibited hamster buccal pouch carcinogenesis and such inhibition may be related to suppression of cell proliferation in the buccal mucosa. It was also found that telomerase activity expressed in neoplastic and preneoplastic lesions of hamster buccal pouch epithelium after DMBA treatment correlated with the histopathological degree of malignancy.

Modifying effects of S-methyl methanethiosulfonate (MMTS) on diethylnitrosamine (DEN)-initiated and phenobarbital (PB)-promoted hepatocarcinogenesis were examined in rats. Five-week-old male F344 rats were divided into 8 groups. After a week, groups 1-5 were given DEN (100 mg/kg body weight, i.p.) once a week for 3 weeks, whereas groups 6-8 received vehicle treatment. Group 2 was given 100 ppm MMTS containing diet in the initiation phase. From 4 weeks after the start of experiment, groups 3 and 5 were fed MMTS, and groups 1-3 and 7 received drinking water containing 500 ppm PB. Group 6 was given MMTS diet alone throughout the experiment (24 weeks). The incidences of hepatocellular adenoma and total liver tumors were significantly smaller in group 3 than those of group 1. The average numbers of hepatocellular adenoma, carcinoma and total tumors in group 3 were significantly smaller than in group 1. Glutathione S-transferase placental form-positive foci were also significantly decreased by MMTS treatment in the promotion phase. MMTS treatment in the initiation or promotion phase reduced ornithine decarboxylase activity in the liver of rats given DEN. The antioxidant activity against lipid peroxidation of MMTS was confirmed in tests with rabbit erythrocyte membrane ghosts or rat hepatocytes. These results suggest that MMTS is a promising chemopreventive agent for liver neoplasia when concurrently administered with PB.

A number of naturally occurring compounds and several related synthetic agents were confirmed to exert chemopreventive properties against carcinogenesis in the digestive organs. Phenolic compounds, widely distributed as plant constituents, possess chemopreventive activities in tongue, liver, and large bowel of rodents. Of them, a simple phenolic protocatechuic acid seems to be a promising compound. Organosulfur compounds contained in the cruciferous vegetables and known to activate detoxifying enzymes are regarded as a candidate group for cancer preventive agents. We proved a strong protective effect of S-methylmethanethiosulfonate, a constituent in these vegetables, on azoxymethane (AOM)-induced large bowel carcinogenesis. Some oxygenated carotenoids (xanthophylls) are reported to have antitumor effects. Naturally occurring xanthophylls astaxanthin and canthaxanthin have considerable preventive activities on 4-nitroquinoline-1-oxide (4-NQO)-induced tongue carcinogenesis and AOM-induced large bowel carcinogenesis. A novel synthesized retinoidal butenolide, KYN-54, which suppresses large bowel as well as tongue carcinogenesis could be a useful agent for prevention of digestive organ cancers. Some trace elements are known to have anticarcinogenic effects. Magnesium hydroxide, a protective agent in colorectal carcinogenesis, inhibits c-myc expression and ornithine decarboxylase activity in the mucosal epithelium of the intestine. Our results show that many agents with preventive effects in tongue, liver, and large bowel control carcinogen-induced hyperproliferation of cells in these organs. Carcinogens used to induce large bowel cancers also induce apoptosis in the target sites. Telomerase activity is increased in the tissues of preneoplastic as well as neoplastic lesions in experimental models such as dimethylbenz[a]anthracene-induced oral carcinogenesis in hamsters. These could be useful biomarkers in studies for cancer chemoprevention.

Modifying effects of chlorophyllin (CHL) on the diethylnitrosamine (DEN)-phenobarbital (PB) hepatocarcinogenesis model were examined in rats. Five-week-old male F344 rats were divided into 8 groups. Groups 1 through 5 were given i.p. injections of DEN (100 mg/kg body weight) once a week for 3 weeks beginning one week after the start of the experiment, while groups 6 through 8 received vehicle treatment. Groups 1, 2, 3 and 7 received drinking water with 500 ppm PB from one week after the end of carcinogen or vehicle treatment. CHL-containing diet (2000 ppm) was given to group 2 during the initiation phase and to groups 3 and 5 during the promotion and the post-initiation phase, respectively. Group 6 was given the experimental diet alone throughout the experiment (24 weeks). Liver neoplasms were present in DEN-treated groups and PB treatment promoted liver tumorigenesis. The incidences of adenoma in groups 2 and 3 were significantly smaller than in group 1 (P<0.05 and P<0.02), although the reductions in the incidences of liver cell cancer were not significant. The average numbers of liver neoplasms/rat in group 2 were significantly smaller than in group 1 (P<0.05-P<0.005). Glutathione S-transferase placental form-positive foci were also significantly decreased by CHL treatment (P<0.05 and P<0.001). DEN and PB exposure increased liver ornithine decarboxylase activity and this increase was significantly inhibited by feeding of CHL during the initiation phase (P<0.001). These results suggest that CHL is a chemopreventive agent for liver neoplasia.

The modulating effect of the combined dietary feeding of beta-carotene and perilla oil, which is rich in alpha-linolenic acid, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Rats received oral administration of beta-carotene (0, 50 or 200 mg/kg body weight/day) and fed a basal diet containing either 12% olive oil, 3% perilla oil plus 9% olive oil, or 12% perilla oil. A dose-dependent suppressive effect of perilla oil was found. The numbers of ACF were 42.0 and 18.4% of those of the 12% olive oil-fed controls in the rats fed 3% perilla oil plus 9% olive oil and 12% perilla oil, respectively. The development of ACF was also reduced significantly by the addition of dietary beta-carotene in each of the oil-fed groups (P < 0.05, respectively). The suppression by the combination of beta-carotene and perilla oil was synergistic, as the numbers of ACF were 12.9 and 8.9% of those of the 12% olive oil-fed controls in beta-carotene-treated rats fed 3% perilla oil plus 9% olive oil and 12% perilla oil, respectively. beta-carotene plus perilla oil also suppressed the numbers of silver-stained nucleolar organizer regions and the expression of ras mRNA in the colonic mucosa (cell proliferation biomarkers). Following administration of beta-carotene, a significant increase in the concentration of intact beta-carotene molecules was found in the colonic mucosa, livers, and sera. However, no accumulation of retinoids was observed in the colonic mucosa, suggesting that the inhibitory effect may not be related to the provitamin A activity. These results suggest that the combination of beta-carotene and perilla oil may be useful in the prevention of colon cancer.

We have investigated the modulatory effect of dietary perilla oil which is rich in the n-3 polyunsaturated fatty acid, alpha-linolenic acid, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACE. The rats were fed a basal diet containing either 12% olive oil, 12% safflower oil, 12% perilla oil, 6% perilla oil plus 6% olive oil, or 3% perilla oil plus 9% olive oil for 5 weeks, starting 1 week before the first dosing of AOM. All rats were sacrificed 2 weeks after the last AOM injection. The amount of food consumed and body weight gain were identical among every dietary group. The frequency of ACF was significantly lower in the rats fed 12% perilla oil than in those fed 12% olive oil or 12% safflower oil (P < 0.01 and P < 0.05, respectively). The suppressive effect of perilla oil was dose-dependent, as the number of ACF was 20.7, 40.7 and 47.4% of those of the 12% olive oil-fed controls in rats fed 12% perilla oil, 6% perilla oil plus 6% olive oil and 3% perilla oil plus 9% olive oil, respectively. Perilla oil significantly reduced ras expression as well as the AgNORs count (cell proliferation biomarkers) in the colonic mucosa, as compared with olive oil or safflower oil (P < 0.01, respectively). Marked increases in n-3 polyunsaturated fatty acids in membrane phospholipid fractions and decreased PGE2 levels were observed in colonic mucosa of perilla oil-fed rats. These results suggest that perilla oil, even in small amounts, suppresses the development of aberrant crypt foci, and is therefore a possible preventive agent in the early stage of colon carcinogenesis.

Chronic toxicity and tumorigenicity of purpurin, a natural hydroxyanthraquinone, was examined in two groups of male F344 rats. One group was given a basal diet mixed with purpurin at a concentration of 1% throughout the experiment (520 days). Another group was kept on the basal diet without purpurin during the experiment. Almost all animals given purpurin developed conspicuous changes of kidney resembling 'progressive chronic nephropathies', the severity and frequency of which were much stronger than in controls. In rats with purpurin treatment, marked hyperplasia of pelvic epithelium was frequently seen and several rats developed urinary bladder tumors (papilloma and carcinoma). Prominent crystallization in the renal pelvis and urinary bladder seems to be initially related to the toxic effects of this hydroxanthraquinone on the renal tubules and with the occurrence of epithelial hyperplasia and neoplasms.

The effect of a xanthine oxidase inhibitor, 1'-acetoxychavicol acetate (ACA), on 4-nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis was investigated in male F344 rats. All rats except those in the ACA-alone and untreated groups were given 4-NQO (20 ppm) In the drinking water for 8 weeks to induce oral cancer. Starting 1 week before the 4-NQO exposure, animals were fed diet containing 100 ppm or 500 ppm ACA for 10 weeks, followed by the basal diet without ACA for 22 weeks. Other groups were fed the diet containing ACA at 100 ppm or 500 ppm for 22 weeks, starting 1 week after the cessation of 4-NQO exposure. The remaining groups consisted of rats given 500 ppm ACA alone or untreated rats. At the termination of the experiment (32 weeks), the incidences of tongue neoplasms and preneoplastic lesions, polyamine levels in the tongue tissue, and cell proliferation activity estimated in terms of 5-bromodeoxyuridine (BrdU)-labeling index and by morphometric analysis of silver-stained nucleolar organizer regions' protein (AgNORs) were compared among the groups. Feeding of ACA at the two doses during initiation or postinitiation significantly decreased the development of tongue carcinoma (93-100% reduction, P < 0.001) and preneoplasia (43-50% reduction for hyperplasia and 34-48% reduction for dysplasia, P < 0.05). There were no such lesions in rats fed ACA alone or those in the untreated control group. The number of AgNORs per cell nucleus was significantly decreased by feeding of ACA at a high dose (500 ppm) (29% inhibition, P < 0.05). The BrdU-labeling index was also reduced by dietary administration of ACA (23-32% inhibition, P < 0.01). In addition, ACA feeding reduced tongue polyamine levels (35-40% inhibition, P < 0.05). These results indicate that ACA inhibited rat oral carcinogenesis, and such inhibition might be related to suppression of cell proliferation in the oral mucosa by the xanthine oxidase inhibitor.

The modifying effect of the monoterpenoid d-limonene in drinking water on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of d-limonene intake on ornithine decarboxylase (ODC) activity and on the silver stained nucleolar organizer region protein (AgNOR) count in the colonic mucosa were also estimated. Animals were given 3 weeks s.c. injections of AOM (15 mg/kg body wt) to induce ACF. These rats were treated with or without 0.5% d-limonene in the drinking water, starting 1 week before the first dosing with AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity and AgNOR count/nucleus in the colon. In rats given AOM and d-limonene the frequencies of ACF and aberrant crypts/colon, and aberrant crypts/focus were significantly decreased compared with those of rats given AOM alone (P < 0.01, P < 0.001 and P < 0.001 respectively). Number of AgNOR counts/nucleus of rats treated with AOM and d-limonene was significantly smaller than that of rats treated with AOM alone (P < 0.001). These results suggest that the monoterpenoid d-limonene might be a chemopreventive agent for colonic carcinogenesis in rats.

Modifying effects of taurine, a naturally occurring organosulfur compound, on diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis were examined in rats. Male F344 rats, 5 weeks old, were divided into 8 groups. Rats of groups 1 through 5 were given i.p. injections of DEN (100 mg/kg body weight) once a week for 3 weeks from one week after the start of the experiment. Of them, animals of group 2 received taurine mixed in a basal diet at a concentration of 2000 ppm for the initial 4 weeks, and those of groups 3 and 5 were given the agent starting 4 weeks after the beginning of the experiment until the end (24 weeks). Rats in groups 1, 4, 7 and 8 were kept on the basal diet throughout the experiment (24 weeks). Group 6 was given taurine throughout the experiment and group 8 was treated as a vehicle control. Animals of groups 1,2, 3 and 7 received PB in drinking water at a dose of 500 ppm from one week after the end of carcinogen or vehicle treatment. Liver neoplasms were recognized only in DEN-treated groups. The incidence and average number of liver neoplasms of group 3 were significantly lower than those of group 1. The number of glutathione S-transferase placental form (GST-P)-positive foci of group 2 or 3 was significantly smaller than that of group 1 (P < 0.01 or P < 0.005). The average and unit areas of GST-P-positive foci in groups 2 and 3 were also significantly smaller than those in group 1 (P < 0.005 and P < 0.0001 and P < 0.0001, respectively). In this study, the level of ornithine decarboxylase activity in non-neoplastic liver tissue was reduced by taurine treatment in both the initiation and postinitiation phases. These results suggest that taurine could be a chemopreventive agent for liver neoplasia.

The modulating effects of dietary feeding of two xanthophylls, astaxanthin (AX) and canthaxanthin (CX) during the postinitiation phase on colon carcinogenesis initiated with azoxymethane (AOM) were investigated in male F344 rats. Animals were initiated with AOM by weekly s.c. injections of 15 mg/kg body wt for 3 weeks and then they were fed the diets containing AX or CX at concentrations of 100 and 500 p.p.m. for 34 weeks. The others contained the groups of rats treated with AX or CX alone and untreated. At the end of the study (week 37), the incidence and multiplicity of neoplasms (adenoma and adenocarcinoma) in the large intestine of rats initiated with AOM and followed by AX or CX containing diet at a high dose (500 p.p.m.) were significantly smaller than those of rats given AOM alone (P < 0.001). In addition, AX or CX feeding significantly inhibited the development of aberrant crypt foci induced by AOM. Dietary exposure to AX or CX also decreased cell proliferation activity as revealed by measuring 5'-bromodeoxyuridine-labeling index as crypt cells, colonic mucosal ornithine decarboxylase activity and blood polyamine levels. These results indicate that AX and CX are possible chemopreventers for carcinogenesis of colon in addition to urinary bladder and oral cavity and such effects may be partly due to suppression of cell proliferation.

The modifying effect of dietary administration of protocatechuic acid (PCA) during the initiation and postinitiation phases on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder carcinogenesis was investigated in male F344 rats. Animals were divided into nine groups and groups 1-7 were given 0.05% BBN in drinking water for 6 weeks to induce bladder neoplasms. Rats in groups 2, 3 and 4 were fed diets containing 500, 1000 and 2000 p.p.m. PCA respectively for 8 weeks, starting 1 week before BBN exposure. Groups 5, 6 and 7 were fed the PCA-containing diets at three dose levels for 33 weeks. Group 8 was fed the diet containing 2000 p.p.m. PCA alone throughout the study. Group 9 was given tap water without BBN and the basal diet without PCA and served as an untreated control. At 41 weeks after the start, all animals were killed. The incidence of bladder tumors and preneoplastic lesions, and cell proliferation activity estimated by the numbers of silver-stained nucleolar organizer regions proteins (AgNORs) and proliferating cell nuclear antigen (PCNA)-immunoreactive cells were compared among the groups. PCA administration at 1000 and 2000 p.p.m. during the initiation and postinitiation phases significantly decreased the carcinoma incidence in a dose-dependent manner. Also, PCA at all doses given during either initiation or postinitiation phases reduced the development of the preneoplastic lesions. PCA feeding significantly reduced the numbers of AgNORs and PCNA-positive cells in the non-lesional transitional epithelium, preneoplasms, and neoplasms in the urinary bladder of rats treated with BBN. These results indicate that dietary administration of PCA is quite effective in preventing BBN-induced bladder carcinogenesis.

Modifying effects of dietary exposure of S-methyl methane thiosulfonate (MMTS) isolated from cauliflower Brassica oleracea L. var. botrytis on rat colon carcinogenesis induced by azoxymethane (AOM) and on the expression of cell proliferation biomarkers were investigated in two experiments. In experiment 1, male F344 rats were given three s.c. injections of AOM (15 mg/kg body weight) and fed 100 ppm MMTS for 5 weeks, starting 1 week before the first dose of AOM. The frequency of colonic aberrant crypt foci was determined at 5 weeks after the start. Feeding of 100 ppm MMTS for 5 weeks significantly decreased the number of aberrant crypt foci/colon. Colonic mucosal ornithine decarboxylase activity and the number of silver-stained nucleolar organizer regions per nucleus in colonic epithelium were significantly decreased by MMTS treatment compared with those of AOM alone. In experiment 2, effects of dietary feeding of MMTS at two doses (20 and 100 ppm) during the postinitiation phase on intestinal tumorigenesis initiated with AOM were investigated by using a long-term experiments in male F344 rats. Incidence of intestinal neoplasms of rats fed MMTS-containing diets after AOM exposure were reduced in a dose-dependent manner. Feeding of MMTS during the postinitiation phase decreased the number of aberrant crypt foci/colon, colonic ornithine decarboxylase activity, 5-bromodeoxyuridine-labeling index in colonic epithelium, and polyamine level in blood compared with those of AOM alone. These results suggest that MMTS might be a possible chemopreventive agent for intestinal neoplasia.

The modifying effect of dietary exposure to a novel synthesized retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone (KYN-5) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given weekly s.c. injections of AOM (15 mg/kg body wt.) for 3 weeks to induce ACF. These rats were fed diet containing 100 or 200 ppm KYN-54 for 5 weeks, starting 1 week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ornithine decarboxylase (ODC) activity, mucosal polyamine level, and silver-stained nucleolar organizer regions protein (AgNORs) count per nucleus in the colon. In rats given AOM and KYN-54, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone. ODC activity and polyamine levels, and the mean AgNORs number in the colon of rats given AOM and KYN-54 at both doses were also significantly lower than that of rats treated with AOM alone. These results provide further evidence that KYN-54 could be a chemopreventive agent against rat colon carcinogenesis.

The present study was designed to investigate the modifying effects of dietary 5-hydroxy-4-(2-phenyl-(E)-ethenyl)-2(5H)-furanone (KYN-54), a new synthetic retinoidal butenolide, during the post-initiation phase on azoxymethane (AOM)-induced rat intestinal carcinogenesis. The number of aberrant crypt foci (ACF) in rat colon, colonic ornithine decarboxylase (ODC) activity and bromodeoxy-uridine (BrdUrd) labeling index in rat colonic epithelium were also assessed. At 7 weeks of age, male F344 rats (except the KYN-54 alone and control groups) were given weekly s.c. injections of AOM at 15 mg/kg body wt for 3 weeks. Starting 1 week after the last injection of AOM, rats (except the control group) were fed a diet containing KYN-54 at concentrations of 100 or 200 p.p.m. throughout the experiment. All animals were necropsied at 32 weeks after the start of the experiment. Compared with the AOM alone group, KYN-54 at both doses reduced the incidence and multiplicity of tumors in entire intestine (small and large intestines). In the 200 p.p.m. KYN-54 fed group especially, tumor incidence and multiplicity in the entire intestine were lower compared with the AOM alone group (P < 0.005 and P < 0.05 respectively). Also, the number of ACF/cm2 colon in the groups of rats treated with AOM and KYN-54 at both doses were significantly lower than that of rats treated with AOM alone (P < 0.05). Colonic ODC activity and BrdUrd labeling index in the groups of rats treated with AOM and KYN-54 at both doses were slightly lower than those treated with AOM alone. KYN-54 at 200 p.p.m. significantly lowered BrdUrd labeling index induced by AOM (P < 0.005). These results suggest that KYN-54 might be a promising chemopreventive agent for intestinal neoplasia.

1-Hydroxyanthraquinone (1-HA), which is present in some herbs, and methylazoxymethanol (MAM) acetate, a metabolite of azoxymethane, show synergistic carcinogenicity in rat colon, and 1-HA induces ulcerative changes with simultaneous severe inflammation of the entire colon. In this study, mutations in Ki-ras (exons 1 and 2) and p53 (exons 4-7) were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. Of 18 adenomas and 38 adenocarcinomas induced in male F344 rats (52 tumors induced by 1-HA plus MAM acetate, three by 1-HA alone, and one by MAM acetate alone), no mutations in Ki-ras or p53 were detected under two conditions of PCR-SSCP analysis. Because human colon carcinomas from patients with ulcerative colitis have a very low incidence of Ki-ras mutation, this experimental system would be a good animal model of human colon carcinomas with ulcerative colitis and of human colon carcinomas without Ki-ras or p53 mutations.