Search

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S-transferase placental form (GST-P)-positive foci (considered to be preneoplastic lesions) or 8-hydroxy-2'-deoxyguanosine in rat liver. Moreover a N-nitroso compound, N-nitrosodiethylamine, at low doses was also found not to induce GST-P-positive foci in rat liver. These results imply that there is a no-observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.

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%, P0.05, 1.2 versus 2.5, P0.05, 0.7 versus 1.4 cm(3), P0.01, respectively). Apoptosis was significantly increased in breast cancer cells by feeding of ONO-8711 at 800 p.p.m. of 158% (P0.05). 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.

9-(4′-Aminophenyl)-9 H -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.

We performed the in vitro micronucleus (MN) test on 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2 H -benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[ N -(2-cyanoethyl)-ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2 H -benzotriazole (PBTA-2), which are newly identified water pollutants from the Nishitakase river in Kyoto, Japan, and on their possible mother compounds (AZO DYE) and intermediates (non-ClPBTAs). We tested these compounds in the absence and presence of S9 mix in two Chinese hamster cell lines CHL and V79-MZ and scored MN, polynuclear and karyorrhectic (PN), and mitotic (M) cells. PBTA-2 in the absence of S9 mix induced the strongest responses in both cell lines. It was also a strong inducer of binucleate cells in PN cells in both cell lines, which suggested that it induced polyploidy. PBTA-1 showed clear positive results only in the absence of S9 mix and only in V79-MZ cells, inducing aneuploidy. In CHL cells AZO DYE-1 significantly induced MN cells in the presence of S9 mix, and AZO DYE-2 induced MN and PN cells, including binucleate cells and cells with a multilobed nucleus, in the absence of S9 mix. In V79-MZ cells, AZO DYE-1 and -2 induced primarily M cells in the presence of S9 mix. 9% of the M cells treated with 50 μg/ml AZO DYE-1 showed endoreduplication. AZO DYE-2 at 200 μg/ml condensed the chromatin in 100% of the cells. The non-ClPBTAs were a bit more cytotoxic than the other compounds and induced a slight increase in MN cells in both cell lines. Some of the chemicals tested induced a characteristic karyomorphology that might reflect abnormal cell division. Abnormalities of cell division could be detected in PN and M cells as well as in MN cells. Structure–activity relationships have also been discussed.

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.

Epidemiological and laboratory animal studies have suggested a positive association between Helicobacter pylori infection and gastric carcinogenesis. In Japan, gastric cancer is the most common and leading cause of cancer death. This study was designed to investigate the effects of fish meal on H. pyloriinduced gastritis in Mongolian gerbils in order to find dietary factors associated with gastric carcinogenesis. Male 7 week-old Mongolian gerbils were inoculated with H. pylori (ATCC43504) 0.5 ml (0.5×10^9CFUs) by gavage and a dietary regimen maintained as follows for 4 weeks : 0% fish meal (casein instead of fish meal in conventional diet); 10% fish meal (conventional diet); 20% fish meal (conventional diet plus 10% fish meal). All animals were sacrificed at 4 weeks after inoculation with H. pylori. In all gerbils inoculated with H. pylori, about 10^5 viable bacteria were counted, without any significant intergroup differences. In macroscopic findings, gastric edema and hemorrhage were observed in 10 and 5, 95 and 55, and 95 and 85% of animals in the 0, 10, and 20% fish meal diet groups, respectively. Histopathological examinations indicated that a fish meal diet significantly increased activity and inflammation indices of the stomach in a dose-dependent manner. These results suggest that some dietary factors in a fish meal diet enhance H. pylori-induced gastritis and may be dietary factors which enhance gastric carcinogenesis.

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AalphaC (2-amino-9H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AalphaC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.

微生物を用いた変異原性試験による環境汚染モニタリングの手法に関する基礎的資料を得ることを目的として, 変異原性試験用の共通試料として都市大気浮遊粒子の溶媒/超音波抽出物を調製し, 5機関3年間, 複数回にわたって, これを試料とする変異原性試験を実施した。-80℃で試料のDMSO溶液を保存したところ, 3年間保存での有意な変異原性の変化は認められなかった。試験菌株, 代謝活性化系など試験条件によると思われる機関内変動は1.5%～55%であった。これに対して機関間の変動は27%～69%であった。TA100及びTA98両菌株の結果を用いて統計的解析 (2レベル階層構造分散分析) を行ったところ, 機関間の変動は同一機関内での複数試験回の誤差と殆ど変わらなかった。

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 17β-estradiol (E2) at doses of 0, 30 or 50 μg/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.

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o -toluidine with S9 mix. When these β-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o -toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.

The cabbage butterfly Pieris rapae contains a strong apoptosis-inducing substance, pierisin, against human cancer cell lines, which is thought to act via ADP-ribosylation. Here we report the purification and cloning of an apoptosis-inducing substance, designated as pierisin-2, from another cabbage butterfly, Pieris brassicae. Pierisin-2 was purified from pupae by sequential chromatography and its cytotoxic and apoptosis-inducing activities to various cancer cells were similar to those of pierisin, designated as pierisin-1, from P. rapae. cDNA cloning of pierisin-2 was performed on the basis of the partial amino-acid sequence. The nucleotide sequence indicated that the cDNA encodes an 850-amino-acid protein with a calculated molecular mass of 97 986. The deduced amino-acid sequence of pierisin-2 was 91% identical with that of pierisin-1. In vitro expressed protein in the reticulocyte lysate exhibited apoptosis-inducing activities against human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, similar to the purified native pierisin-2 from the pupae. Pierisin-2 shows regional sequence similarities with certain ADP-ribosylating toxins such as the A-subunit of cholera toxin. The results from site-directed mutagenesis at Glu165, a conserved residue among ADP-ribosylating enzymes necessary for NAD binding, and from experiments with ADP-ribosylating enzyme inhibitors suggested that pierisin-2 could be considered as an ADP-ribosylating toxin like pierisin-1.

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.

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.

Alteration of adenomatous polyposis coli (APC) is known to be an early event in neoplasia, causing activation of the beta-catenin / Tcf pathway. Although it is thought that alterations in APC and beta- catenin may complement one another, the contribution of beta-catenin mutations to colorectal carcinogenesis remains unclear. We therefore performed PCR-single strand conformation polymorphism analysis and direct sequencing of exon 3 of beta-catenin gene in adenomas, adenocarcinomas, and aberrant crypt foci (ACF), considered to be putative precursor lesions of colorectal neoplasias, in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) treated F344 rats. beta-Catenin mutations were identified in all of 7 adenomas (100%) and 6 of 12 (50%) adenocarcinomas. All of the mutations were found in codons 32 through 34, the serine encoded by codon 33 being an important phosphorylation site by glycogen synthase kinase-3beta. Regarding ACF, 14 of 46 (30.4%) were found to be mutated, eleven (78%) in codon 34, and the others in codon 45 (frequently altered in human colon cancer), and codons 47 and 56 (which have not been previously reported). The frequency of beta-catenin mutations in adenomas was significantly higher than in ACF (P < 0.001) and adenocarcinomas (P < 0.05). Thus, beta-catenin mutations may have more importance in the genesis of adenomas than ACF or adenocarcinomas in rat colon carcinogens by PhIP.

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.

The beta-catenin gene is frequently mutated at codons 33, 41 and 45 of the glycogen synthase kinase-3beta phosphorylation motif in human colon cancers in patients without APC mutations. Frequent mutations at codons 32 and 34, as well as 33 and 41, have been detected in rat colon tumors induced by azoxymethane (AOM), with the second G of CTGGA sequences being considered as a mutational hot-spot. In the present study, exon 3 of the beta-catenin gene in mouse colon tumors induced by AOM was amplified by PCR and mutations were detected by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. All 10 colon tumors tested were found to have beta-catenin mutations, four in codon 34, three in codon 33, two in codon 41 and one in codon 37, nine being G:C-->A:T transitions. However, no mutations were found in codon 32 of the mouse beta-catenin gene. On immmunostaining, beta-catenin was observed in the cytoplasm and nucleus of the tumor cells. The cytoplasmic staining was homogeneous, while both homogeneous and heterogeneous patterns were noted for the nuclei. Highly frequent mutations of the beta-catenin gene in AOM-induced mouse colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.

To clarify the mutagenic potential of nonagricultural surface soil in Japan, 110 soil samples were collected from five geographically different areas between November 1996 and March 1997, and organic extracts of the soil samples were examined by the Ames/Salmonella assay. Most of the soil extracts showed mutagenicity toward both strains TA98 and TA100 in the presence and/or absence of a mammalian metabolic activation system (S9 mix), suggesting that surface soil is largely contaminated with environmental mutagens. Soil samples collected at Hekinan, Kobe, and Osaka were highly mutagenic toward both strains, and their potencies toward TA98 without S9 mix were extremely high, inducing more than 12 000 revertants per gram of soil. On the other hand, soil samples from Muroran showed strong mutagenicity toward TA100 with S9 mix. Furthermore, 1, 3-dinitropyrene (DNP), 1,6-DNP, and 1,8-DNP in soil samples collected at 10 sampling sites in three metropolitan areas were quantified by fluorometric detection of the corresponding diaminopyrene isomers using high-performance liquid chromatography (HPLC). Three DNP isomers were detected in all soil samples, and the amounts of 1,3-, 1,6-, and 1,8-DNP isomers in the soil samples were 12-3270, 14-5587, and 13-6809 pg/g, respectively. The gross amount of three DNP isomers in surface soil collected at Hekinan was more than 10 ng per gram of soil. The highest contribution ratios of DNP isomers to the mutagenicity of soil extracts were observed for the samples collected at Osaka, and the total of the contribution ratios of three DNP isomers was about 50%. These results suggest that surface soil is largely contaminated with mutagenic compounds and that DNP isomers are one class of major mutagenic and carcinogenic compounds contaminating surface soil.

Group-A xeroderma pigmentosum (XPA) gene-deficient mice are defective in nucleotide-excision repair and highly susceptible to ultraviolet-B-, and 9,10-dimethyl-1,2-benz[ a ]anthracene (DMBA)-induced skin carcinogenesis [1] . In this study, changes of 2-amino-1-methyl-6-phenylimidazo[4,5– b ] pyridine (PhIP)-DNA adduct formations in the liver, colon and lung, as assessed by the 32 P-postlabeling method and immunohistochemical analysis, and carcinogenic and/or toxic susceptibility of both sexes of XPA-deficient mice (XPA −/− ) to PhIP, which is a carcinogenic heterocyclic amine, was examined. Levels of PhIP-DNA adduct formations in the liver, colon and lung, were almost twice as high in XPA −/− as in wild type mice (XPA +/+ ) mice, 7 days after a single i.g. administration of PhIP, and their delay in recovery was observed in XPA −/− mice. For the long-term experiment, XPA −/− and XPA +/+ type mice were treated with 80 ppm PhIP in the diet for the first 4 weeks followed by 40 ppm after a 2-week recovery period (long-term experiment I), or 40 ppm PhIP throughout the experiment (long-term experiment II). Severe toxicity, as evidenced by body weight retardation and poor survival, was observed in the PhIP treated XPA −/− mice of both sexes, but not in the XPA +/+ . At week 40 the experiments were terminated and histopathological examinations were performed after complete autopsy. Only lymphomas/leukemias were observed as neoplastic lesions, but no significant differences were observed between the groups. As non-neoplastic lesions, degenerating changes, for example in the pancreatic acinar cells, were observed with XPA −/− mice tending to be more sensitive than XPA +/+ mice. The present study demonstrated that PhIP-DNA adduct formations in the liver, colon and lung of XPA −/− mice were demonstrated and their recovery rate was more delayed than XPA +/+ mice, and furthermore, more severe toxicity to PhIP in XPA-deficient mice was observed, but they were not susceptible to PhIP carcinogenicity under the conditions of the experiment.

We have noticed the presence of cytotoxic activity in the body fluid of a butterfly, Ideopsis similis, in family Danaidae. The cytotoxic principles were purified and identified to be phenanthroindolizidine alkaloids. In addition, the cytotoxic activity was observed in the leaves of Tylophora tanakae, which is a host plant for caterpillars of I. similis. In the present study, cytotoxic principles in methanol extracts of the leaves against human gastric carcinoma TMK-1 cells were purified by acid-base partition, repeated HPLC and preparative TLC, and two cytotoxic compounds were isolated. From spectroscopic analysis of their structures, they were identified as phenanthroindolizidine alkaloids, trans-(+)-3, 14α dihydroxy-4, 6, 7-trimethoxyphenanthroindolizidine N-oxide and (-)-7-hydroxy-2, 3, 6-trimethoxyphenanthroindolizidine. The amounts of two compounds isolated from 200g of dried leaves were 3.5 and 4.0mg, respectively. These alkaloids are different from those in the fluid of I. similis. When TMK-1 cells were treated with these phenanthroindolizidine alkaloids, strong cytotoxic effects were observed, doses of 6ng/ml for trans-(+)-3, 14α-dihydroxy-4, 6, 7-trimethoxyphenanthroindolizidine N-oxide and 3ng/ml for (-)-7-hydroxy-2, 3, 6-trimethoxyphenanthroindolizidine inducing 50% cell growth inhibition (IC50).

The 2-phenylbenzotriazole (PBTA)-type water pollutant, 2-[2-(acetylamino)-4-[ N -(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2 H -benzotriazole (PBTA-2), has been recently identified in samples from the Nishitakase River in Kyoto, Japan, and shows potent mutagenic activities in Salmonella typhimurium in the presence of a microsomal metabolizing system (S9 mix). In the present study, we conducted the in vitro micronucleus (MN) test on PBTA-2 in the absence and presence of S9 mix in two Chinese hamster cell lines, CHL and V79-MZ. In the MN test, PBTA-2 was weakly positive in CHL cells and strongly positive in V79-MZ cells. Because the positive results were accompanied by a statistically significant increase in the number of polynuclear (PN) and/or mitotic (M) cells, we examined treated cells in metaphase to see if numerical chromosome aberrations were being induced. We found that PBTA-2 induces polyploidy in both CHL and V79-MZ cells. A detailed analysis of MN preparations showed that in CHL cells, PBTA-2 predominantly induces equal-sized binucleated cells. Rhodamine phalloidin staining revealed that PBTA-2 causes actin filament abnormalities in both cell lines similar to those caused by cytochalasin B. Cytochalasin B induced PN cells predominantly and dose dependently, and almost all the cells were equal-sized and binucleate. The results suggest that PBTA-2 has cytochalasin B-mimetic activity, although agents affecting actin filaments, such as cytochalasins, phallotoxins and chloropeptide, have been derived only from molds so far. This study also suggests that our MN test protocol may be used to identify chemicals that have cytochalasin B-mimetic activity as well as those that induce numerical aberrations.

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.

Previous studies have identified two potent aromatic amine mutagens in the Nishitakase River, a tributary of the Yodo River, which serves as the main drinking water supply for the Osaka area in Japan. The two potent mutagens are 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). PBTA-1 and PBTA-2 are presumed to be formed from azo dyes discharged in a reduced form from dye factories to sewage treatment plants where they become chlorinated and are then discharged into the river. PBTA-1 and PBTA-2 account for 21% and 17% of the mutagenic activity of the Nishitakase River, respectively. Here we determined the mutation spectra induced by these two mutagens in TA98, TA100, and TA104 at 30-35, 8-10, and 2x, respectively, above the background. In TA98, the PBTA compounds produced identical mutation spectra, with 100% of the revertants containing the hotspot 2-base deletion of CG within the (CG)(4) sequence. In TA100, 73% of the revertants were GC-->TA transversions, with most of the remaining being GC-->AT transitions; the spectra produced by the two compounds in TA100 were not significantly different (p=0.8). In TA104, as in TA100, the majority (83%-87%) of the revertants were GC-->TA transversions, with most of the remaining revertants (11%-13%) being AT-->TA transversions. Thus, 83%-87% of the mutations induced by the PBTA compounds in TA104 were at G/C sites. The mutation spectra produced by the two compounds in TA104 were not significantly different (p0.08). PBTA-1 and PBTA-2 are structurally similar and have similar mutagenic potencies and mutation spectra in the respective strains. The mutation spectra produced by the PBTA compounds (100% hotspot deletion in TA98 and primarily GC-->TA transversions in TA100 and TA104) are similar to those produced by other potent aromatic amines, which is the class of compounds from which the PBTA mutagens derive.

Co-mutagenic beta-carbolines, such as norharman and harman, were quantified in mainstream and sidestream smoke condensates of six Japanese brands of cigarettes, and also in 13 kinds of cooked foods, using a combination of blue cotton treatment and HPLC. Norharman and harman were detected in all the cigarette smoke condensate samples. Their levels in the mainstream smoke case were 900-4240 ng per cigarette for norharman, and 360-2240 ng for harman, and in sidestream smoke, 4130-8990 ng for norharman and 2100-3000 ng for harman. These beta-carbolines were also found to be present in all the cooked food samples, at levels of 2.39-795 ng for norharman and 0.62-377 ng for harman per gram of cooked food. The observed concentrations are much higher than those found for mutagenic and carcinogenic heterocyclic amines (HCAs), suggesting that humans are exposed to norharman and harman in daily life to a larger extent than to HCAs.

The carcinogenic potential of 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) was evaluated in cynomolgus monkeys. The animals received MeIQx, beginning at the age of one year, at doses of 10 or 20 mg/kg body weight by gavage five times a week for 84 months and were autopsied 8 months thereafter. Although sporadic development of aberrant crypt foci in the colon and glutathione S‐transferase π‐positive foci in the liver as well as hyper plastic changes of the lymphatic tissue in the lung and gastro‐intestinal tract were observed in several monkeys, this was not treatment‐related. No neoplastic or preneoplastic lesions were found in other organs. Serum chemistry data and organ weights were also within the normal ranges. From these data, it is concluded that MeIQx is not carcinogenic in the cynomolgus monkey under the conditions examined. This lack of carcinogenicity is probably related to the poor activation of MeIQx due to the lack of constitutive expression of CYP1A2 as well as an inability of other cytochrome P450s to catalyze N‐ hydroxylation of MeIQx in the cynomolgus monkey.

In order to quantify 1,3-dinitropyrene (DNP), 1,6-DNP and 1,8-DNP in soil, we developed an efficient clean-up procedure and a sensitive determination method using fluorescence detection. DNP isomers were efficiently cleaned by three stages of fractionation, i.e., a silica gel open column chromatography using stepwise elution and two further purification steps by high-performance liquid chromatography (PPLC) using a monomeric-type octadecylsilyl (ODS) column and a polymeric-type ODS column. The recoveries of DNPs during the whole clean-up process were 94% or more. The fraction corresponding to DNPs was injected into an analytical polymeric-type ODS column for HPLC to separate DNP isomers. The effluent from the analytical ODS column was directly introduced to a catalyst column, which was packed with 5 microns alumina coated with platinum and rhodium (Pt-Rh), in order to reduce DNPs to diamino compounds, and then the fluorescence of diaminopyrenes was detected. The immediate detection of diaminopyrene isomers after on-line reduction afforded a sensitive detection of DNP isomers. The detection limits for DNPs were in the range of 0.7 to 4 pg. These developed methods were applied to four soil samples collected at parks in residential areas.

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‐aminobi‐phenyl (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.

Pierisin, a protein purified from pupae of the cabbage butterfly, Pieris rapae , exhibits cytotoxic effects against the human gastric cancer TMK-1 cell line, inducing apoptosis. The present study was performed to determine whether pierisin might exert a similar influence on nine other human cancer cell lines and human umbilical vein endothelial cells (HUVECs). Pierisin showed cytotoxic effects in all the human cells tested, with IC 50 values ranging from 0.043 ng/ml to 150 ng/ml. Among the target cells, the cervical carcinoma cell line, HeLa, was the most sensitive to pierisin, showing a 1000-fold less IC 50 value than that of HUVECs. While pierisin clearly induced apoptotic cell death in most cancer cell lines and HUVECs, the pathway appeared to be probably different from that involving anti-Fas, TNF-α and p53. Pierisin may thus be a promising new candidate for cancer therapy.

In recent intervention studies, beta-carotene failed to reduce or even increased the incidence of lung cancers in smokers. In the present investigation, the modifying effects of beta-carotene at various doses on the development of upper respiratory tract tumors were investigated in Syrian hamsters treated with diethylnitrosamine (DEN) and cigarette smoke. A total of 120 male 5-week-old hamsters were divided into 4 groups, each consisting of 30 animals. After a single subcutaneous (s.c.) injection of 100 mg/kg DEN, hamsters in groups 1-4 were respectively administered diets supplemented with beta-carotene at doses of 0.5%, 0.05%, 0.005% or 0% during experimental weeks 1 to 13, and simultaneously exposed to cigarette smoke. The duration of cigarette smoke exposure was 9 min twice a day, 5 days a week. Because of a marked reduction of body weight in group 1, the highest dose of beta-carotene was changed to 0.25% after 10 days. In all groups, epithelial hyperplasias and/or papillomas were induced in the larynx and trachea. However, the incidence and multiplicity of papillomas in group 1 were significantly (P < 0.05) lower than the group 4 values. Moreover, the beta-carotene treatments significantly (P < 0.05 or 0.01) reduced both the incidence and multiplicity of hyperplasias in a dose-dependent manner. The levels of retinol and beta-carotene in the serum, and the retinol level in the liver, were also elevated with dose dependence. Our results thus indicate that beta-carotene inhibits tumorigenesis, even at the high dose of 0.25%, under the present experimental conditions.

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue-stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, "single" with round bottoms and "bifurcating" displaying V-shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.

presented at the 6th International Conference on Carcinogenic and Mutagenic N-Substituted Aryl Compounds, p 89, November 4–8, 1995, Monterey, CA, USA 68 Lang, N.P., Butler, M.A., Massengill, J., Lawson, M., Stotts, R.C., Hauer-Jensen, M., and Kadlubar, F.F. (1994). Rapid metabolic phenotypes for acetyltransferase and cytochrome P4501A2 and putative exposure to food-borne heterocyclic amines increase the risk for colorectal cancer or polyps. Cancer Epidemiol. Biomarkers Prev. 3, 675–682 69 Roberts-Thomson, I.C., Ryan, P., Khoo, K.K., Hart, W.J., McMichael, A.J., and Butler, R.N. (1996). Diet, acetylator phenotype, and risk of colorectal neoplasia. Lancet 347, 1372–1374 70 Bjeldanes, L.F., Morris, M.M., Timourian, H., and Hatch, F.T. (1983). Effects of meat composition and cooking conditions on mutagenicity of fried ground beef. J. Agric Fd. Chem. 31, 18–21 71 Skog, K., Steineck, G., Augustsson, K., and Jagerstad, M. (1995). Effect of cooking temperature on the formation of heterocyclic amines in fried meat products and pan residues. Carcinogenesis 16

Two estrogen sulfates, pyridinium 3-methoxyestra-1,3, 5(10)-trien-6alpha-yl sulfate (3MeE-6alpha-S) and its 6beta-isomer (3MeE-6beta-S), synthesized as model compounds to demonstrate the carcinogenesis of estrogen, were found to react with calf thymus DNA to produce steroid-modified DNA adducts. Digestion of the DNA by nuclease P1 and phosphodiesterase I followed by alkaline phosphatase gave a deoxyribonucleoside fraction, of which N2-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyguanosine, N2-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyguanosine, N6-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyadenosine, and N6-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyadenosine (identified as a base adduct) were identified using HPLC by comparing them with authentic specimens prepared by reacting dG and dA with both sulfates. No steroid-dC adduct was detected in the digestion products of the DNA adduct, although dC reacted with the sulfates to form N4-[3-methoxyestra-1,3,5(10)-trien-6beta-yl]deoxycytidine. These results mean that estrogen 6-sulfate has an ability to modify DNA via the amino group of a guanine or adenine residue in DNA. The present studies imply that a sequential metabolism (hydroxylation and sulfation) at the C6-position of the estrogen molecule causes damage to DNA.

We previously isolated five mutagens, compounds I-V, in blue rayon-adsorbed materials from the Nishitakase River in Kyoto. The chemical structure of compound I, a major mutagen that accounted for 21% of the total mutagenicity, was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Compound II was also a major mutagen and accounted for 17% of the total mutagenicity. In this study, a large quantity (1.2 mg) of compound II was isolated from adsorbate to 27 kg of blue cotton, and its UV, mass, and 1H NMR spectra were analyzed. On the basis of the spectral data, compound II was deduced to be the PBTA-1 analogue 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). As with PBTA-1, PBTA-2 was synthesized from an azo dye by reduction and chlorination. Since all of the spectra of PBTA-2 coincided with those of compound II obtained from river water, compound II was concluded to be PBTA-2. PBTA-2 is a newly identified potent mutagen, which induces 93 000 and 3 200 000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. Like PBTA-1, PBTA-2 may also be produced from an azo dye during industrial processes in dyeing factories and treatment at sewage plants.

It is important to search for effective antioxidants to suppress formation of mutagenic and carcinogenic heterocyclic amines (HCAs), like 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), because these HCAs are considered to be probable human carcinogens. The effects of various food-derived antioxidants on MeIQx formation were examined by their addition (0.2 mmol each) to mixtures of creatine (0.4 mmol), glycine (0.4 mmol) and glucose (0.2 mmol), and heating at 128°C for 2 h. Glycine was replaced by l -phenylalanine in the case of PhIP formation. Among the 14 kinds of antioxidants tested, green tea catechins and the major component [(−)-epigallocatechin gallate], two flavonoids (luteolin and quercetin) and caffeic acid were found to clearly suppress the formation of both MeIQx and PhIP, being 3.2–75% of the level of the controls. These phenolic antioxidants also reduced the total mutagenicity of the heated mixtures. The results suggest that foodstuffs containing catechins, flavonoids and caffeic acid may suppress the formation of HCAs in cooked foods.

The carcinogenicity of 2-amino-3,4-dimethylimidazo[4,5- f ]quinoline (MeIQ) was examined in Big Blue® female mice with the genetic background of C57BL/6N. With the administration of 300 ppm of MeIQ in their diet for 92 weeks, the Big Blue® female mice developed intestinal tumors and hepatocellular carcinomas. The incidences of adenocarcinomas were 42% ( 8 19 ) in the colon and 68% ( 13 19 ) in the cecum. The incidence of hepatocellular carcinomas was 84% ( 16 19 ). No carcinomas of the intestine or the liver were induced in the control group. As we previously reported, administration of 300 ppm of MeIQ in a diet for 12 weeks induced lacI mutants at the highest frequency in colonocytes, and at only less than one-tenth of the colon in cells of the liver, forestomach and bone marrow, indicating no direct correlation between the lacI mutant frequency (MF) and cancer incidence (CI). The fate of cells with lacI mutation in each organ should be taken into consideration to validate MF as an indicator of carcinogenic potency of a chemical in different organs.

A substance strongly cytotoxic to human carcinoma cell line TMK-1 has been found in pupae, larvae and adults of the cabbage butterfly, Pieris rapae, and named pierisin. Pierisin was purified from the pupae of P. rapae by ammonium sulfate precipitation followed by DEAE-cellulose, Phenyl-Sepharose and hydroxyapatite column chromatographies. The molecular weight of the purified pierisin, which was homogenous on SDS-polyacrylamide gel, was analyzed by mass spectrometry and found to be 98 kDa. Pierisin showed a strong cytotoxic effect, with and IC50 of 0.75 ng/ml for human gastric carcinoma TMK-1 cells. The dying cells exhibited characteristic morphological features of apoptosis, such as cell shrinkage, chromatin condensation and nuclear fragmentation. Oligonucleosomal DNA fragmentation was also observed in DNA isolated from pierisin-treated cells. Moreover, similar characteristic changes showing apoptotic cell death were observed in TMK-1 cells treated with a crude extract of pupae of P. rapae. These results indicate that pierisin from the pupae of P. rapae induces apoptosis in human carcinoma cells.

Among five mutagenic compounds isolated from water samples, taken at sites below the sewage plants of the Nishitakase River in Kyoto, Japan, the structure of compound I has been determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Since this novel aromatic amine mutagen has characteristic substituents in its molecule, it is postulated that the azo dye, 2-[(2-bromo-4, 6-dinitrophenyl)azo]-4-methoxy-5-[bis(2-methoxyethyl)amino]acetoanili de (AZO DYE-1), used as an industrial material, is converted to the corresponding 2-phenylbenzotriazole derivative with a reducing reagent and subsequently to PBTA-1 by chlorination. In fact, AZO DYE-1 changed to the dechlorinated derivative of PBTA-1 (deClPBTA-1) on treatment with sodium hydrosulfite, and this reacted with sodium hypochlorite to produce PBTA-1. Moreover, the presence of deClPBTA-1 was confirmed in a river water sample, along with PBTA-1. PBTA-1 showed potent mutagenic activities in Salmonella typhimurium TA98 and YG1024, inducing 88 000 and 3 000 000 revertants, respectively, per microg, with S9 mix. deClPBTA-1 was also mutagenic, but less potent. From these observations, it is suggested that PBTA-1 is produced from AZO DYE-1 through deClPBTA-1, during industrial processes at dyeing factories and the treatment of wastewater at sewage plants.

Many studies revealed that poly(ADP-ribose) polymerase (Pare) is involved in DNA-damage recovery, cell-death induction and maintenance of genomic stability. We generated mouse Parp+/- embryonic stem (ES) cell lines by disrupting one allele of Parp exon 1 with a neomycin-resistance gene-cassette and subsequently produced Parp-/- ES cells by disrupting the remaining allele with a puromycin-resistance genecassette. Parp activity was decreased to half in Parp+/- ES cell clones and lost in Parp-/- ES cell clones. Growth rates of Parp+/- and Parp-/- ES cell clones were similar to that of parental J1 ES cells, indicating that Parp is not required for ES cell proliferation. These Parp-/- ES cells will be useful to study biological role of poly(ADP-ribosyl)ation reaction.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is known to be a mutagen and carcinogen isolated from the charred parts of cooked foods. We found previously that Trp-P-1 enhanced UV-induced lethality and mutation frequency in Escherichia coli by inhibiting the repair of UV-induced DNA damage. In the present study, we investigated whether Trp-P-1 also potentiated UV-induced lethality by inhibiting the repair of UV-induced DNA damage in cultured mammalian cells. As a result, Trp-P-1 enhanced UV-induced lethality in a concentration-dependent manner in human and Chinese hamster cells. However, Trp-P-1 was unable to inhibit the repair of the two major photolesions (cyclobutane pyrimidine dimers and (6-4)photoproducts) from the genomic DNA, as determined using monoclonal antibodies specific for each type of lesion. On the other hand, Trp-P-1, with or without UV irradiation, efficiently suppressed DNA synthesis and arrested cells in S phase in concentration- and time-dependent manners, as measured by pulse-labelling with 3H-thymidine and flow cytometry. Thus, the present results suggest that Trp-P-1 potentiates UV-induced lethality in cultured mammalian cells by causing the S-phase arrest, not by inhibiting the repair of UV-induced DNA damage as observed in Escherichia coli.

Nonsteroidal anti-inflammatory drugs (NSAIDs) suppress colon carcinogenesis in man and experimental animals. However, conventional NSAIDs inhibit both cyclooxygenase (COX) isoforms, COX-1 and COX-2, and cause gastrointestinal side-effects. Nimesulide, a selective inhibitor of COX-2, is much less ulcerogenic. We, therefore, examined its influence on the development of intestinal polyps in Min mice. Female Min mice at 4 weeks old were given 400 ppm nimesulide in their diet for 11 weeks. This treatment resulted in a significant reduction of the numbers of both small and large intestinal polyps, the total being 52% of that in untreated control Min mice. The size of the polyps in the nimesulide-treated group was also significantly decreased. The results suggest that nimesulide is a good candidate as a chemopreventive agent for human colon cancer with low toxicity.

Modifying effects of diallyl disulfide (DAD), aspirin or DL-alpha-difluoromethylornithine (DFMO) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in SD rats were investigated. A total of 166 female rats, 6 weeks old, were divided into 8 groups. They were fed a high fat diet throughout the experiment. Starting at 7 weeks of age, groups 1-4 were given PhIP (85 mg/kg body weight in corn oil) by gavage 8 times in 10 days, and groups 5-8 were given corn oil alone. For the beginning 4 weeks, groups 2 and 5 were given DAD at 200 ppm in diet. Similarly groups 3 and 6, and groups 4 and 7 were given aspirin (400 ppm) and DFMO (400 ppm), respectively. Mammary carcinomas were only recognized in groups 1-4 at the termination (25 weeks after the start of experiment). Multiplicity (mean number/rat) of neoplasms in group 2 (PhIP+DAD, 0.90/rat) and group 3 (PhIP+aspirin, 1.37/rat) was significantly smaller than that in group 1 (PhIP alone, 2.45/ rat) (P < 0.005 and P < 0.05, respectively). These results indicate that dietary intake of DAD or aspirin during the time corresponding to initiation phase has chemopreventive potential on PhIP-induced mammary carcinogenesis in rats.

Although nitric oxide (NO) is an important biological mediator, its excessive production in inflammation is thought to be a causative factor for cellular injury and, over the long term, cancer. In the present study, the effects of several fatty acids on NO production in murine macrophage cell line RAW264 cells stimulated with lipopolysaccharide were examined. Suppression of NO production was observed with the omega 3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid, eicosapentaenoic acid and alpha-linolenic acid, in a dose-dependent fashion. In contrast, no inhibition was observed with omega 6 PUFA (linoleic acid), omega 9 PUFA (oleic acid) or a saturated fatty acid (stearic acid). Western and northern blot analyses suggested that suppression of the induction of inducible NO synthase gene expression is responsible for the inhibition of NO production by omega 3 PUFAs. The inhibitory effect of omega 3 PUFA on NO production in activated macrophages could contribute to their cancer chemopreventive influence.

The measurement of somatic cell mutation may assist in the assessment of human cancer risk. The glycophorin A (GPA) assay, which measures the frequency of variant erythrocytes in persons with blood type MN, was used to directly assess in vivo mutability in 30 patients with hepatocellular carcinoma (HCC). HCC patients showed significantly increased frequencies of both hemizygous (MO) and homozygous (MM) variants, due to somatic loss of expression of the N allele, when compared with 27 patients with chronic liver disease and 21 healthy controls. The mean elevations of the MO and MM variant frequencies (VF) in HCC patients were 2-3-fold greater than the comparable VF in the control groups. The mean MO and MM VF in the patients with chronic liver disease was slightly elevated compared to that in healthy controls, but the difference was not significant. In the 19 anti-hepatitis C virus (HCV)-positive patients with a history of blood transfusion, significant linear relations between VF and the duration of HCV infection were observed for MO and MM. These data indicate a high background frequency of somatic mutations in HCC patients. The GPA assay may prove to be a useful estimation of the individual's risk of development of HCC.

We recently demonstrated that a novel heterocyclic amine, 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ), is produced from glucose and l-tryptophan by the Maillard reaction at physiological temperature and pH, and that ABAQ was strongly mutagenic for Salmonella strains in the presence of S9 mix. Here, we present the results of three in vivo genotoxicity assays of ABAQ. The comet assay revealed that DNA damage was significantly increased in the livers, kidneys, lungs, and bone marrows of ICR mice, 3h after i.p. injection of ABAQ (50mg/kg body weight (bw)). To evaluate clastogenicity, the peripheral blood micronucleus test was performed, also in ICR mice. ABAQ induced micronucleated reticulocytes (MNRETs) in a dose-dependent manner; the frequency of MNRETs was significantly elevated at all i.p. doses (12.5, 25, and 50mg/kg bw) after 48h. To investigate the mutagenicity of ABAQ in vivo, gpt delta transgenic mice were treated with five consecutive administrations of ABAQ by gavage at doses of 25 or 50mg/kg per week for 3 weeks. The frequencies of gpt mutations (MF) in the liver of mice increased significantly compared with controls, in a dose-dependent manner. No significant increase of gpt MF was detected in the kidneys. Base substitutions predominated; both G:C→A:T and A:T→C:G mutations were significantly increased by ABAQ. The Spi(-) MF was also significantly increased in the liver after ABAQ treatment. If formed in vivo, ABAQ may give rise to adverse genotoxic effects.

Helicobacter pylori (Hp) infection causes gastritis and is considered a gastric cancer risk factor. We have previously reported that codfish meal markedly enhanced Hp-induced gastritis in Mongolian gerbils. In the present study, we sought the responsible components in codfish meal.Codfish were divided into three parts (meat, viscera and 'other parts', including bone), and administered to Hp-infected gerbils. Subsequently, cod bone, sardine bone and prawn shell were tested, along with major calcium components, hydroxyapatite and calcium carbonate, in bone and shell, respectively.'Other parts' and cod bone enhanced Hp-induced gastritis, as was observed for whole codfish. Similarly, sardine bone and prawn shell, as well as 0.22-0.88% hydroxyapatite and calcium carbonate, enhanced gastritis. In contrast, administration of a higher dose of the calcium compounds exerted protective effects.Intake of calcium compounds may contribute to enhancement of Hp-induced gastritis.

Cytotoxic activity in extracts of pupae and adults of various kinds of butterflies and moths was tested in vitro against the human gastric carcinoma cell line, TMK-1, which was chosen as an example of human carcinoma cells. Among the species examined, cytotoxicity was limited to Pieris rapae, Pieris napi and Pieris brassicae. Activity was found down to a dilution of 1/10(4), while with the other butterflies and moths no activity was observed, even at 1/10(2). When the cytotoxicity of the three developmental stages, larvae, pupae and adults, of Pieris rapae was compared, the pupae showed the strongest activity, the IC50 against TMK-1 cells being at the 1/10(6) dilution. For larvae and adults, the respective IC50 values were at the 1/10(5) and 5/10(5) dilutions. The active principle in the pupae of Pieris rapae was found to be heat-labile and not extractable with organic solvents, but precipitated with ammonium sulfate and digested by proteases, suggesting that it is a protein. This cytotoxic factor was named pierisin.

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), which is a heterocyclic amine found in food and the potent mutagen in S. typhimurium TA98, was examined for its genotoxic potential using lacI transgenic mice (Big Blue, C57BL/6N lineage). Female mice, at 7 weeks of age, were given a diet containing 0.03% MeIQ for 1, 4 and 12 weeks, and mutant frequencies (MF) were analyzed in the bone marrow, liver, forestomach, colon and heart. The MF increased in a feeding period-dependent manner. Relative to untreated mice, the MF after a 12-week-feeding of MeIQ was 38 times higher in the colon, 5.8 times higher in the bone marrow, 4.6 times higher in the liver, and 2.6 times higher in the forestomach. No increase in MF was detected in the heart, where no tumors develop.

Non-steroidal anti-inflammatory drugs, such as piroxicam and sulindac, are known to inhibit development of aberrant crypt foci (ACF) and cancer in the colon. However, these agents cause gastrointestinal side-effects. Nimesulide is a selective inhibitor of cyclooxygenase 2 and has been shown to have a more potent anti-inflammatory action than piroxicam, but be less ulcerogenic and, therefore, a potentially more useful chemopreventive agent. To assess this possibility the inhibitory effects of nimesulide on the formation of ACF induced by azoxymethane in rat colon were investigated, and compared with those of piroxicam and sulindac. Male F344 rats were treated s.c. with 15 mg/kg body weight azoxymethane once a week for 2 weeks and given 50, 100 or 200 ppm nimesulide, 200 ppm piroxicam, or 200 ppm sulindac in their diet from the day before the first carcinogen treatment until the end of the experiment at week 4. At this time, nimesulide at doses of 50, 100 and 200 ppm had reduced the numbers of azoxymethane-induced ACF to 75%, 71% and 65% respectively compared to the control. The number of azoxymethane-induced ACF per colon in the group given 200 ppm nimesulide was almost the same as in those given 200 ppm piroxicam, and lower than that in the group given 200 ppm sulindac. These results suggest that nimesulide, a selective cyclooxygenase 2 inhibitor, warrants attention as a candidate for chemopreventive agent with low toxicity, active against colon carcinogenesis.