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Background: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized., Results: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake., Conclusions: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer., (© 2021. The Author(s).)

Monocyclic aromatic amines, o -toluidine ( o -Tol) and its structural analog o -anisidine ( o -Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl- N 4 -(2-methylphenyl) benzene-1,4-diamine (MMBD), a p -semidine-type homodimer of o -Tol, was detected and identified in an in vitro reaction of o -Tol with S9 mix and in vivo urinary samples of o -Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o -Ans is converted to active metabolites to induce DNA damage in a similar manner as o -Tol. In this study, we report that a novel o -Ans metabolite, 2-methoxy- N 4 -(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding ( p -semidine form), was for the first time identified in o -Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N -acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o -Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o -Ans was coincubated with o -Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o -Ans, o -Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

o -Toluidine ( o -Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o -Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o -Tol is metabolized by N -hydroxylation, N -acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8- o -Tol is thought to be a plausible DNA adduct of o -Tol exposure. However, direct detection of dG-C8- o -Tol in biological samples has not been reported yet. Here, we show that a novel o -Tol metabolite, 2-methyl- N 1 -(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o -Tol-exposed rat urine. MMBD was also detected in a reaction of o -Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N -acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o -Tol. Furthermore, a DNA adduct ( m / z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o -Tol. These results therefore suggested that o -Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

Aminomethylphenylnorharman (AMPNH) and aminophenylnorharman (APNH) are mutagenic norharman derivatives obtained from o-toluidine and aniline, respectively. APNH is carcinogenic to the urinary bladder of rats and present in urine samples of healthy volunteers, indicating that norharman derivatives may be associated with cancer development in the urinary bladder of humans. To evaluate the possible role of AMPNH and APNH in bladder carcinogenesis, we examined the formation of γ-H2AX, a DNA damage response marker, in the urinary bladder of rats. Seven-week-old male F344 rats were treated with 400 ppm AMPNH or 40 ppm APNH in the diet for 4 weeks. Animals were killed at the end of administration or after 2 weeks of recovery, and immunohistochemistry for γ-H2AX and Ki67, a cell proliferation marker, was performed. At week 4, γ-H2AX formation in bladder epithelial cells was significantly increased by APNH treatment as compared with that in controls. AMPNH also induced upregulation of γ-H2AX formation, although there was no statistical significance. After the recovery period, γ-H2AX-positive cells were reduced but remained significantly higher in AMPNH and APNH groups than in the control group. Ki67-positive cells were significantly increased by AMPNH and APNH at week 4 and reduced to the same level as the control after 2 weeks of recovery. Expression of KRT14, a bladder stem cell marker, was also increased in the basal layer by the two norharman derivatives. Thus, AMPNH and APNH showed in vivo genotoxicity in the bladder epithelium of rats, and APNH may be a potent causative agent of bladder carcinogenesis., (Copyright © 2017 John Wiley & Sons, Ltd.)

Tumor promotion potential of diacylglycerol (DAG)-rich edible oil was examined using a two-stage mouse skin carcinogenesis model initiated with 7,12-dimethylbenz[a]anthracene (DMBA). Topical treatment with 75 mg DAG oil once a day for 5 days/week for 35 weeks caused papillomas in 4 of 23 (17%) DMBA-treated female ICR mice, while DMBA initiation alone and DAG treatment without DMBA initiation did not induce any skin tumors. Doubling the daily treatment (twice a day x 5 days/week) at doses of 75 and 30 mg caused both papillomas and squamous cell carcinomas after DMBA initiation, the incidences of tumors being 48% (12/25) and 44% (11/25), respectively, significantly higher than the 4% (1/23) in the DMBA+ 85 mg triacylglycerol group and 0% (0/24) in the DMBA+ vehicle-treated group. The results indicate that DAG-rich oil has promoting potential for skin carcinogenesis, and thus, further investigations of its tumor-promoting potential in other organs are warranted.

Carcinogenic potential of heterocyclic amines (HCAs) was investigated using an in vivo 5-week initiation assay with quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver. Numbers of GST-P positive foci were significantly increased with individual administration of six different HCAs, indicating utility of the assay. It was therefore applied to investigate risk with multiple HCAs in combination. Unexpectedly, concomitant treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) did not result in any additive carcinogenicity. In the rats taking MeIQx prior to PhIP the value was almost equal to the sum total of individual data, indicating additive initiation activities. In contrast, simultaneous or prior administration of PhIP rather exerted inhibitory effects on the carcinogenic potential of MeIQx. Moreover, microarray and quantitative RT-PCR assessment revealed that PhIP induced cytochrome P450 1A1, responsible for both activation and detoxification of HCAs, more strongly than MeIQx. It is noteworthy that complex exposure to multiple HCAs is not necessarily associated with increased risk of carcinogenesis because they are simultaneously and continuously ingested under normal circumstances.

Several cooked meats such as beef (fried, coated-fried), pork (fried, coated-fried), and chicken (fried, griddled, coated-fried, roasted) were analyzed for the heterocyclic amine 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo[4,5- b]pyridine (4'-OH-PhIP) not commonly determined in food and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). The highest content of 4'-OH-PhIP was found in fried and griddled chicken breast, the concentration being 43.7 and 13.4 ng/g, respectively, whereas the corresponding PhIP concentrations were 19.2 and 5.8 ng/g. The estimated concentration of both pyridines in fried pork loin, in fried pork sausages, and in coated-fried chicken was below 2.5 ng/g. In the rest of the samples, 4'-OH-PhIP was not detected. The analyses were performed by solid-phase extraction and LC-MS/MS. The fragmentation of 4'-OH-PhIP in an ion trap mass analyzer was studied in order to provide information for the identification of 4'-OH-PhIP. Additionally, the effect of red wine marinades on the formation of 4'-OH-PhIP in fried chicken was examined, finding a notable reduction (69%) in the amine's occurrence.

A novel heterocyclic amine, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), which is formed from nonmutagenic 9H-pyrido[3,4-b]indole (norharman) and aniline, is mutagenic to bacteria and mammalian cells and potently carcinogenic in rats. APNH is detected in human urine samples, suggesting that humans are continuously exposed to APNH. In the present study, (32)P-postlabelin analysis revealed that the levels of APNH-DNA adduct 24 hr after the treatment with APNH (1, 5 and 20 mg/kg body weight) in male ICR mice were increased in a dose-dependent manner in the colon and liver. Based on these findings, we determined the tumor-initiating potency of APNH in an inflammation-related and two-stage mouse colon carcinogenesis model. Male Crj: CD-1 (ICR) mice were given a single intragastric administration (1, 2, 5 or 10 mg/kg body weight) of APNH and subsequent 1-week oral exposure to dextran sodium sulfate (DSS, 2% in drinking water). Treatment with APNH and DSS resulted in numerous colon tumor development: the incidence and multiplicity of the tumors were the highest in the mice received 10 mg/kg body weight of APNH and followed by DSS. Development of colon tumors was dose-dependent of APNH. Seven of 9 (77.8%) colonic adenocarcinomas developed in mice treated with APNH (10 mg/kg body weight) and DSS had beta-catenin gene mutations at codons 32 and 37, being predominantly transversion. These findings indicate that APNH has an initiating activity in inflamed mouse colon and the APNH-DNA adduct formation correlates with its tumorigenic potential., ((c) 2007 Wiley-Liss, Inc.)

Epidemiologic data suggest that diabetes mellitus type II is a risk factor for several types of cancer, including pancreatic, liver, colon and thyroid cancers. In the present study, effects of diabetes/hyperlipidemia on N-nitrosobis(2-oxopropyl)amine (BOP)-induced cancer development were examined in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, model animals for non-insulin-dependent diabetes mellitus and Long-Evans Tokushima Otsuka (LETO) rats, appropriate controls. Males of both strains were given four subcutaneous injections of BOP (10 mg/kg body wt) or saline on alternative days, starting at 5 weeks of age. BOP induced tumors in a variety of tissues, including the thyroid gland, colon, kidney, liver and lung. The highest yields were noted for thyroid tumors, the incidence (P = 0.0182) and multiplicity (P < 0.001) of BOP-induced thyroid cancers with marked fibrosis being significantly higher in OLETF than in LETO rats. Interestingly, anaplastic thyroid carcinomas were observed limited to the BOP-treated OLETF rats. Additionally, a greater incidence and frequency of aberrant crypt foci, putative precursor lesions for colon tumors, was observed in the BOP-treated OLETF group. However, BOP was ineffective at inducing pancreatic ductal tumors. No thyroid, liver, lung or colon tumors were found in the OLETF and LETO rats receiving the vehicle. Significant increases in serum levels of insulin, glucose, phospholipids, triglycerides and total cholesterol were detected in the OLETF rats compared with the LETO rats, regardless of the treatment. Our results indicate that diabetic/hyperlipidemic state can enhance BOP-induced carcinogenesis of the thyroid gland and to a lesser extent the colon in OLETF rats.

Fat intake and obesity are positively correlated with pancreatic cancer in humans. N-nitrosobis(2-oxopropyl)amine (BOP) induces pancreatic ductal adenocarcinomas limited to Syrian golden hamsters, other rodents not being susceptible. In the present study, we found markedly high levels of serum triglycerides (TGs) and total cholesterol (TC) in Syrian golden hamsters, but not C57BL/6 mice, ICR mice, F344 rats and Wistar rats. Consistent with this, lipoprotein lipase (LPL) activities in the liver were lower in hamsters compared with mice and rats. To examine effects of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, on LPL expression, serum lipid levels and pancreatic cancer development, 6-week-old female Syrian golden hamsters were subcutaneously injected with BOP (10 mg/kg body wt) four times in a week and thereafter fed a diet containing 800 p.p.m. pioglitazone for 22 weeks. The treatment elevated LPL mRNA expression in the liver and significantly improved hyperlipidemia with serum levels of TG and TC being decreased to 62 and 71%, respectively, of the control values. Concurrently, the incidence and multiplicity of pancreatic ductal adenocarcinomas were significantly decreased by pioglitazone in comparison with the controls (38 versus 80%, P < 0.01 and 0.55 +/- 0.15 versus 1.37 +/- 0.22, P < 0.01, respectively). The suppression rates were greater in invasive adenocarcinomas than non-invasive ones. The incidence of cholangiocellular carcinomas was also reduced. Thus, suppression of pancreatic adenocarcinoma development by pioglitazone is possibly associated with improvement in the serum lipid profile, and hyperlipidemia could be an enhancing factor for development of pancreatic cancer in hamsters.

1,2-diacylglycerol (1,2-DAG) is involved in cell proliferation as an activator of protein kinase C (PKC) and has been shown to stimulate growth of cancer cells, raising the possibility of a role in tumor promotion. Ingested DAG oil, containing 70% 1,3-DAG and 30% 1,2-DAG, is digested and considered to be safe as edible oil. However, DAG may directly contact with oral cavity mucosa in undigested form. The present study was conducted to examine the effects of DAG oil on carcinogenesis in c-Ha-ras proto-oncogene transgenic (Tg) rats administered 4-nitroquinoline 1-oxide (4NQO, 10 ppm) in their drinking water for 10 weeks for initiation of mainly upper digestive organs. DAG oil added in basal diet at 5.5%, 2.75%, 1.38% and 0% with total fat made up to 5.5% with triacylglycerol (TAG) was administered during the initiation and post-initiation period. The study was terminated at week 12 (Tg females) and 20 (Tg males, wild females and males). The fatty acid composition of DAG oil was similar to TAG (linoleic acid 46.6% and oleic acid 38.9%). In Tg male rats, DAG oil administration was associated with significant increase (P<0.05) in the incidence of squamous cell carcinomas (SCC) of the tongue (5.5% DAG, 43.8%; 2.75% DAG, 20%; 1.38% DAG, 14.3%; 0%, 12.3%) with the Cochran-Armitage trend test and also number of tumors in coefficients for linear contrast trend tests. Tongue SCC induction of wild males and all females was not significant. The present results suggest that DAG oil may have enhancing and/or promotion potential for tongue carcinogenesis in male Tg featuring elevated ras expression.

Mutagenic/carcinogenic 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman (APNH)] is formed from norharman and aniline in the presence of cytochrome P450 3A4/1A2. Because both precursors are widely distributed in the environment, human exposure is unavoidable. To clarify APNH formation in the human body, amounts of the compound in 24-h human urine collected from smokers and nonsmokers, eating a normal diet, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry. In addition, norharman and aniline were also analyzed by high-performance liquid chromatography and gas chromatography, respectively. APNH could be detected in all urine samples at levels 49 to 449 pg for smokers and 21 to 594 pg for nonsmokers per 24-h urine, respectively. The amounts of norharman and aniline were 46 to 185 ng and 0.70 to 8.10 microg for smokers and 52 to 447 ng and 0.49 to 5.72 microg for nonsmokers, respectively, per 24-h urine (none of the levels differing significantly between smokers and nonsmokers). To exclude exogenous exposure to norharman and aniline, we analyzed the levels of APNH, norharman, and aniline in urine samples collected from inpatients receiving parenteral alimentation. Similar to the healthy volunteers, all urine samples contained 12 to 338 pg of APNH, 6 to 75 ng of norharman, and 0.33 to 1.86 microg of aniline per 24-h urine. These results suggest that APNH should be considered as a novel endogenous mutagen/carcinogen; thus, it is very important to determine the biological significance of this carcinogen for human cancer development.

A method for direct palladium-catalyzed N-arylation reaction of nucleobases was developed for the convenient synthesis of DNA adducts with carcinogenic compounds. Using xantphos as the phosphine ligand and tetraethylammonium fluoride as the base in DMSO, several o-iodonitroarenes could be efficiently coupled with 2'-deoxyguanosine, 2'-deoxyadenosine, and 2'-deoxycytidine. The presence of a 3'-phosphate group in the deoxyribose moiety was found to be compatible with this N-arylation reaction; further, oligonucleotides could serve as substrates. The facile nitroreduction of the coupling compounds (12) yielded 2'-deoxyguanosin-N2-ylarylamine adducts, which are known to be biologically important. Compound 12 was easily converted to phosphoramidite derivatives, allowing the preparation of site-specific modified oligonucleotides with arylamine after the nitroreduction.

To clarify the in vivo genotoxic potential of kojic acid (KA), formation of DNA adducts and 8-hydroxy-deoxyguanosine (8-OHdG) in the thyroids of male rats subjected to dietary administration of 2% KA for 2 weeks were assessed by 32P-postlabeling analysis and with a high-performance liquid chromatography system coupled to an electrochemical detector (ECD), respectively. In addition, to investigate possible tumor initiation activity, male F344 rats were given diet containing 0, 0.02, 0.2 or 2% kojic acid for 8 weeks followed by administration of 0.1% sulfadimethoxine (SDM), a thyroid tumor promoter, in the drinking water for 23 weeks with a subsequent 13-week recovery period (two-stage thyroid tumorigenesis model). Rats given four times by s.c. injection of N-bis(2-hydroxypropyl)nitrosamine (DHPN; 700 mg/kg bw) during the initiation period followed by administration of 0.1% SDM and rats given diet containing 2% KA for the initial 8 weeks or for the entire 31 weeks of the experiment, or basal diet alone were provided as controls. DNA adducts were not formed, and the 8-OHdG level was not increased in the thyroids of rats given 2% KA for 2 weeks. In the two-stage thyroid tumorigenesis model, neither adenomas nor carcinomas were induced in the groups given 0, 0.02, 0.2 or 2% KA followed by 0.1% SDM administration, and incidences and multiplicities of focal follicular cell hyperplasias did not demonstrate any significant intergroup differences at the end of administration and recovery periods. In contrast, incidences and multiplicities of focal follicular cell hyperplasias, adenomas and carcinomas were all significantly increased in the DHPN + 0.1% SDM group. Although the incidences and multiplicities of focal follicular cell hyperplasias in the group given 2% KA for 31 weeks were greater than those in the 2% KA + 0.1% SDM group and an adenoma was observed in a rat at the end of the recovery period, no development of carcinomas was evident at either time point. No thyroid proliferative lesions were induced in the group given 2% KA for the initial 8 weeks only. The results of the present studies indicate that KA has neither in vivo genotoxic potential nor tumor initiation activity in the thyroid, and strongly suggest that the earlier observed thyroid tumorigenic activity of KA is attributable to a non-genotoxic mechanism.

The modifying effects of alpha-naphthyl isothiocyanate (ANIT) on 2-amino-3-methylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were investigated in female Sprague-Dawley (SD) rats, and the hepatic activities of the phase II detoxifying enzymes glutathione S-transferase (GST) and quinone reductase (QR) were also assayed. Ninety-eight rats were divided into 4 groups. Starting at 6 weeks of age, rats were fed the high-fat diet without ANIT (Groups 1 and 4) or the experimental diet (high-fat diet mixed with 400 ppm ANIT, Groups 2 and 3). At 7 weeks of age, Groups 1 and 2 were given PhIP in corn oil (85 mg/kg body weight, 8 times for 11 days) by intragastric intubation. One week after the last PhIP injection, 5 rats in each group were sacrificed to assay GST and QR activities, and the experimental diets for Groups 2 and 3 were switched to the high-fat diet without ANIT until termination of the experiment. Group 4 served as the vehicle control. All rats were sacrificed at 24 weeks after the start of the experiment. At termination of the experiment, mammary tumours were detected in Groups 1 (PhIP alone) and 2 (PhIP + ANIT) and were shown histologically to be adenocarcinomas; their incidences (multiplicities) were 56.3% (1.66 +/- 2.31/rat) in Group 1 and 6.7% (0.07 +/- 0.25/rat) in Group 2 (p < 0.001). Mean sizes of the tumours were 10.6 +/- 5.3 mm in Group 1 and 6.5 mm in Group 2. No mammary tumours were observed in rats of Groups 3 and 4. In addition, ANIT treatment significantly increased the activities of GST and QR in the livers of rats in Groups 2 and 3 as compared to Groups 1 and 4. These results imply that the isothiocyanate compound ANIT shows potent inhibitory effects on mammary carcinogenesis induced by PhIP in female SD rats when administered during the initiation stage., ((c) 2005 Wiley-Liss, Inc.)

Male F344 5-week-old rats were X-irradiated, and 16 weeks after the first dose, azoxymethane (AOM) was injected or 2-amino-1 -methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) was given by intragastric intubation. Tumors in the pylorus of the glandular stomach were observed in 4 out of the 29 animals receiving X-rays + AOM and in 4 out of the 25 animals receiving X-rays + PhIP, 12 months after administration. No such lesions were found in the chemical or X-ray alone groups. Intestinal metaplasia and some induced tumors were positive for CDX2. It was concluded that the presence of intestinal metaplasia may increase sensitivity to the induction of gastric tumors by colon carcinogens.

Heterocyclic amines are known to be important environmental carcinogens in several organs including the colon. The aim of this study was to induce colonic epithelial malignancies within a short-term period and analyze the expression of cycooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and beta-catenin, and mutations of beta-catenin gene in induced tumors. Male Crj: CD-1 mice were given a single i.g. administration (200 mg/kg body wt) of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) followed by 2% dextran sodium sulfate (DSS) in the drinking water for a week. The expression of beta-catenin, COX-2 and iNOS was immunohistochemically assessed in colonic epithelial lesions and the beta-catenin gene mutations in colonic adenocarcinomas induced were analyzed by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. At week 16, a high incidence of colonic neoplasms with dysplastic lesions developed in mice that received PhIP and DSS, but only a few developed in those given MeIQx and DSS. Immunohistochemically, the adenocarcinomas induced were all positive for three proteins. All seven adenocarcinomas induced by PhIP and DSS have mutations. The findings suggest that DSS exerts powerful tumor-promoting effects on PhIP-initiated colon carcinogenesis in mice and this mouse model is useful for investigating environment-related colon carcinogenesis within a short-term period.

Dibenzo[a,l]pyrene (DB[a,l]P), a notorious air pollutant, is the most powerful carcinogenic polycyclic aromatic hydrocarbon (PAH) ever tested. Although the carcinogenicity of PAH may be primarily mediated by the aryl hydrocarbon receptor (AhR), the in vivo role of AhR in skin carcinogenesis remains to be defined. In this context, we investigated the genotoxic and carcinogenic responses of the AhR-deficient mouse skin to DB[a,l]P. A single painting resulted in a striking epidermal hyperplasia in AhR+/+ mice but not in AhR-/- mice. Bromodeoxyuridine-labeling index and accumulation of p53 protein in epidermal cells of AhR+/+ mice were 8- and 33-fold higher than those of AhR-/- mice, respectively. 32P-Postlabeling assay for DB[a,l]P-DNA adducts displayed a 2-fold increase in the AhR+/+ mouse skin. After DB[a,l]P exposure, AhR-/- mice arranged a nearly 60% reduction in the induction of epidermal cytochrome P450 (CYP)1A1, but CYP1B1 was constitutively expressed in both genotypes of mice, irrespective of DB[a,l]P treatment. As compared with AhR+/+ mice, AhR-/- mice had both significantly lower incidence (100% vs. 33%) and multiplicity (2.7 vs. 0.46) of skin tumors by the complete carcinogenesis study. These observations indicate that a reduced tumor yield in AhR-/- mice may be secondary to reduction of inducible CYP1A1 activation and subsequent DNA adduction. It is evident from our continuous work that although AhR is likely to play a central role in epidermal proliferation and possibly neoplastic transformation, the relative importance of AhR for carcinogenesis may be different among PAH examined., (Copyright 2004 Wiley-Liss, Inc.)

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

Modulatory effects of beer consumption on azoxymethane (AOM)-induced rat colonic carcinogenesis in male Fischer 344 rats were investigated. Single cell gel electrophoresis assay indicated that DNA damage of colonocytes, induced by a single AOM injection (15 mg/kg body weight), was significantly reduced in rats fed beer or malt extract for 2 weeks. Examination of aberrant crypt foci (ACF) formation in colonic mucosa, induced by AOM (15 mg/kg body weight; twice weekly), revealed that feeding of beer during the whole experimental period of 5 weeks significantly reduced the number of ACF by 35%. In the post-initiation protocol, a reduction in ACF formation by 26% was not significant. The efficacy in inhibition of ACF formation varied with the brand of beer. ACF formation was significantly reduced in rats treated with freeze-dried beer (FD Beer), but not with ethanol, suggesting that nonvolatile components of beer are responsible for the reduction. Significant suppression of ACF formation was observed in groups treated with hot water extract of malt, especially with extracts of colored malts, although no reduction was observed by feeding with hops extract. A long-term experiment of 42 weeks indicated that intake of beer decreased tumor incidence by 22% and decreased the number of neoplastic lesions, including adenocarcinomas and adenomas, by 44%. These results suggest that components of beer have chemopreventive effects on colonic carcinogenesis induced by AOM and that intake of beer may contribute to a reduction in the risk of cancer susceptibility., (Copyright 2003 Wiley-Liss, Inc.)

Aminophenylnorharman (APNH) is formed from non-mutagenic norharman and aniline, and is mutagenic to Salmonella typhimurium TA98 with S9 mix. Norharman and aniline are present in cigarette smoke and cooked foods and both compounds are detected in human urine samples, suggesting that APNH could be a mutagenic and carcinogenic human risk factor. The purpose of the present study was to determine the in vivo mutagenicity of APNH. Male gpt delta transgenic mice were fed a diet containing 10 or 20 p.p.m. APNH for 12 weeks. The gpt mutant frequency (MF) in the liver increased 10-fold in 20 p.p.m. APNH-treated mice, which was almost equivalent to the MF observed in the liver of the same transgenic mice treated with 300 p.p.m. 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline for 12 weeks. In the colon mucosa, the gpt MF increased approximately 5-fold in 20 p.p.m. APNH-treated mice. Our results suggest that APNH is a strong hepatic mutagen in mice. The APNH-induced gpt mutations in the liver were dominated by G:C to T:A transversions, followed by G:C to A:T transitions. They also included single G:C deletions in G:C run sequences and 2 bp deletions: GCGC to GC and CGCG to CG. The Spi- deletion MF in the liver was 13-fold higher in 20 p.p.m. APNH-treated mice, relative to the control, and were dominated by single base pair deletions, in particular, in G:C run sequences. Large deletions were rare. The mutational characteristics induced by APNH are compared with those induced by other heterocyclic amines, and the human risk of APNH is discussed.

Recent low-dose carcinogenesis studies, including major group projects are reviewed. The prevailing paradigm is that carcinogens, particularly genotoxic compounds, have no threshold in exerting their potential for cancer induction. However, the nonthreshold hypothesis can be challenged for cancer risk assessment in humans. A recent very large-scale cooperative effort in Japan furthermore showed that the genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, forms DNA adducts and 8-hydroxy-2'-deoxyguanosine at low doses, but does not induce glutathione S-transferase placental form (GST-P) positive foci as preneoplastic lesions in rat liver (< or = 10 ppm in diet). Moreover, very low doses of a N-nitroso compound. diethylnitrosamine (DEN), were also found not to significantly induce GST-P positive foci in rat liver (< or = 0.01 ppm in drinking water). Given the direct correlation between induction of the preneoplastic lesions in the short-term and carcinomas in the longer term with different carcinogens, the results imply a practical nonobserved effect level for hepatocarcinogenicity. Similar results were also observed with so-called nongenotoxic carcinogens such as phenobarbital (PB) and p,p-dichlorodiphenyltrichloroethane (DDT), which do not exert positive effects on lesion development at very low doses. Furthermore, experiments with application of PB and DDT after treatment with DEN indicate that at very low doses (< or = 2 ppm in diet), they may even inhibit the development of GST-P positive foci. The data reviewed provide evidence that preneoplastic foci in the liver can be employed as end-point lesions in place of tumors and that exposure to very low levels of carcinogens, typical of those found in the human environment, does not necessarily present as a risk factor.

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.

Norharman is not mutagenic to Salmonella strains, but becomes so to S. typhimurium TA98 and YG1024 with S9 mix in the presence of the non-mutagenic aromatic amine, aniline. The mutagenicity from norharman and aniline in the presence of S9 mix is reported to be due to formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH). In the present study, we examined which enzymes might be involved in in vitro formation of APNH from norharman and aniline. When norharman (5mg) and aniline (2.5mg) were incubated with S9 mix, the S9 fraction of which was prepared from the livers of rats treated with phenobarbital (PB) and beta-naphthoflavone (beta-NF), at 37 degrees C for 20 min, 496 ng of APNH was produced. Formation of APNH was also detected with the microsomal fraction, but not with the cytosol fraction. The addition of a p450 inhibitor, SKF-525A, to the reaction mixture at a dose of 5mM resulted in a decrease of APNH formation, to around 40% of the control level. These results suggest involvement of p450 enzyme(s) in the formation of APNH from norharman and aniline. Moreover, a microsomal fraction from human liver was demonstrated to have the capacity to produce APNH from norharman and aniline, similar to the case with the microsomal fraction from rat liver. When norharman (90 mg/kg) and aniline (90 mg/kg) were administered to rats by gavage, APNH could be detected in the urine, at a rate of 19.6 ng+/-16.9 ng per 24h. The level was increased by treatment of the urine samples with hydrochloric acid, suggesting some APNH was excreted into urine as conjugated forms. Thus, it is likely that APNH may be produced from norharman and aniline in the human body.

Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix. Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix. Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix. In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH. On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH. The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH. Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells. A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH. The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb). In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature. Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health.

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.

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.)

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.

Mechanisms underlying mammary carcinogenesis in female rat given nitrofurazone (NF) were examined. Experiment I: female Wistar rats were divided into three groups, and given diets containing 0, 500 or 1000 ppm NF for 5 weeks. At terminal sacrifice, body and uterus weights were the same in all groups, although ovary weights in NF-treated animals were significantly higher than in control animals, the increase being dose-dependent. Serum prolactin (PRL) concentrations in NF-treated groups at 17:00 h on the day of proestrus were also dose-dependently higher than that in control group. Experiment II: a two-stage rat mammary carcinogenesis protocol was performed. Rats were divided into four groups, Groups 2 and 4 being treated by 9,10-dimethyl-1,2-benzanthracene (DMBA) at 7-weeks-old. Groups 3 and 4 were given diets containing 1000 ppm of NF between 8 and 27 weeks of age, when all surviving rats were autopsied. DMBA-treated animals demonstrated mammary tumors at high incidences, 91.1 and 90.5%, respectively, in Groups 2 and 4, no tumor development being observed without the initial carcinogen exposure (Groups 1 and 3). The mean tumor weights and the mean numbers of tumors per tumor-bearing rats in Group 4 were increased as compared with Group 2, albeit not significantly. Serum PRL (proestrus day at 17:00 h) and progesterone (PG) (diestrus day at 10:00 h) concentrations in NF-treated animals (Groups 3 and 4) were significantly higher than those in untreated rats (Groups 1 and 2). These results suggest that increases of serum PRL and PG concentrations by NF may be the most important factors regarding its promotion of mammary tumor growth and/or enhancement of mammary carcinogenesis in female rats.

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.

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.

2-Amino-1-methyl-phenylimidazo[4,5b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine produced in cooked meat and fish, is known to be a carcinogen for rats and mice. This study provides the first evidence for hypertriglyceridemia in rats exposed to PhIP, suggesting its potential risk to induce not only carcinogenesis, but also atherosclerosis, and highlighting the potential importance of PhIP for humans.

Effects of eight heterocyclic amines (HCAs) on pancreatic duct carcinogenesis were investigated in a rapid production model in hamsters. N-Nitrosobis(2-oxopropyl)amine (BOP) was given to effect initiation, followed by augmentation pressure consisting of four daily i.p. injections of 500 mg/kg DL-ethionine, a choline-deficient (CD) diet, a single i.p. injection of 800 mg/kg L-methionine and a s.c. injection of 20 mg/kg BOP. After two cycles of augmentation pressure, the HCAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) or 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline [4,8-DiMeIQx) were administered in the diet for 50 or 70 days. The numbers of pancreatic ductal hyperplasias (H) and a total lesions including, atypical hyperplasia (AH), carcinomas in situ (CIS) and invasive carcinomas, were increased in hamsters given the diet containing 0.02% Trp-P-1 for 50 days. This result was confirmed and extended by the finding of increased numbers of invasive carcinomas in hamsters given 0.02% Trp-P-1 for 70 days. The number and incidence of invasive carcinomas were also elevated in hamsters given the diet containing 0.06% 4,8-DiMeIQx for 50 days. These results suggest a possible involvement of Trp-P-1 and 4,8-DiMeIQx in pancreatic duct carcinogenesis.

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 pi-positive foci in the liver as well as hyperplastic 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.

The purpose of this study was to determine the promotional role of estrogen and progestin in the development of radiation-induced mammary tumors. To eliminate the effects of endogenous ovarian hormones on tumor promotion, all rats were ovariectomized immediately after the initiation by irradiation with 2.6 Gy gamma-rays at day 21 of lactation, and were divided into 3 groups. For the control experiment, rats were implanted with a cholesterol pellet 1 month after the irradiation. Only one rat developed a fibroadenoma (4.3% mammary tumor incidence) during the 1 year period of the implantation. In the other two groups, chlormadinone acetate (CMA) to increase progestin level or diethylstilbestrol (DES) to increase estrogenic activity were administered, respectively, as tumor promoters for 1 year. Treatment with CMA did not significantly increase the incidence of mammary tumors as compared with the controls. However, administration of DES resulted in a significantly higher mammary tumor incidence (79.3%) than control treatment. Compared with cholesterol administration, DES treatment caused an increase in prolactin concentration in serum (5-fold), and reduction of estradiol-17beta concentration (22% of control). These results suggest that DES ia a potent effective promoter for tumorigenesis of radiation-initiated mammary cells, but CMA is not. DES may act directly on the irradiated mammary cells by binding to ER, and indirectly by stimulating prolactin secretion from the pituitary glands.

Docosahexaenoic acid (DHA), a major component of fish oil, suppresses the formation and growth of aberrant crypt foci induced by 1,2-dimethylhydrazine and azoxymethane. In the present study we examined the effects of intragastric gavage administration of DHA on the yield of rat colonic aberrant crypt foci due to treatment with a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induces colon cancer in male F344 rats and is considered to be a possible human colon carcinogen. Male F344 rats were given a standard diet (AIN-76A) and received 10 doses of PhIP (75 mg/kg body wt, by intragastric intubation, on days 1-5 and 8-12) with or without intragastric application of 1 ml DHA 4 h prior to each carcinogen treatment, followed by further DHA dosing. The numbers of PhIP-induced aberrant crypt foci per colon after 4 and 12 weeks DHA administration were significantly reduced to 47 and 38% respectively of the values obtained when PhIP alone was used. The mean number of aberrant crypts per focus was also decreased by DHA treatment. At week 4 the PhIP-DNA adduct levels in the colon of rats from the PhIP+DHA group were approximately two thirds of the PhIP group value. The results thus suggest that DHA exerts a preventive effect on PhIP-induced colon carcinogenesis.

The effects of intragastric gavage administration of docosahexaenoic acid (DNA) , a major component of fish oil, on azoxymethane (AOM)-induced colon carcinogenesis in rats was investigated. Male F344 rats were treated with 15 mg/kg body wt of AOM once a week, for two weeks. The animals were given either 1 ml of DHA or water intragastrically 5 times a week, starting the day before the first carcinogen treatment. The numbers of AOM-induced aberrant crypt foci in the rats given DHA were 76% and 62% of the control values, at 4 and 12 weeks, respectively. After 36 weeks of DHA treatment, colon tumors were counted and examined histologically. The blood plasma levels of prostaglandin E2 (PGE2) and polyunsaturated fatty acids were also quantified. The incidences of colon cancer did not differ, being 96% and 92% in the AOM and AOM+DHA groups, respectively. Colon cancer multiplicity was, however, significantly decreased by the DHA treatment; 3.65 +/- 2.18 in the AOM group and 2.41 +/- 1.58 in the AOM+DHA group (P <0.01). Notably, the numbers of moderately differentiated adenocarcinomas in the middle and distal colon in the DHA-treated group were lower than in the AOM group. The levels of PGE2 and arachidonic acid in the blood plasma of DHA-treated rats were also significantly lower than in the AOM group. These results suggest that DHA exerts its inhibitory effect on colon carcinogenesis by modulating lipid metabolism and inhibiting the arachidonic cascade.

Prostate tissues obtained from rats given a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), at a dose of 400 ppm in the diet for 52 weeks were histopathologically evaluated and found to contain prostate carcinomas limited to the ventral lobe in 18 of 27 cases. Atypical hyperplasias were also detected in the ventral and anterior prostate as well as the seminal vesicles. 32P-Postlabeling analysis of DNA demonstrated that PhIP-DNA adducts are produced in all lobes of the prostate of rats receiving PhIP. The findings indicate that PhIP is carcinogenic to rat prostate in addition to the previously demonstrated targeting of the colon and mammary glands, providing evidence of a possible role of PhIP in human prostate carcinogenesis and highlighting the potential importance of PhIP for man.

Pregnant Wistar-MS strain rats were irradiated with 2.6 Gy of gamma-rays at day 20 of pregnancy. Rats in the control group (n = 48) were then implanted with a diethylstilbestrol (DES) pellet at 35 days after weaning, while being fed a control (MB-1) diet. The incidence of mammary tumors was 89.6% within 1 year. In the experimental group (n = 22), a bezafibrate (0.15%) diet was initiated immediately after weaning, and 35 days after weaning a DES pellet was implanted. Administration of dietary bezafibrate together with DES-implantation continued for a period of 1 year, at which time the experiment was terminated. The incidence (27.3%) of the mammary tumors in the bezafibrate-fed rats was less than one-third of that in the control rats. Compared with the control group, the number of mammary tumors per tumor-bearing rat in the bezafibrate-treated group was reduced. For clarification of the mechanism of the chemopreventive effects of bezafibrate, lipid and hormone concentrations in serum were measured. Bezafibrate-fed rats showed a significant decrease in serum prolactin (56%) and triglyceride (63%) concentrations, and a significant increase in serum estradiol-17beta (3.8-fold), cholesterol ester (2.0-fold) and TSH (2.0-fold) concentrations in comparison with the control rats. The bezafibrate diet inhibited the formation of DES-induced pituitary tumors. However, the development of mammary glands in the bezafibrate-fed rats was stimulated more than that in the control rats treated with DES alone. The present results demonstrate that bezafibrate is effective in preventing mammary tumors induced by radiation together with DES, possibly by reducing prolactin and triglyceride concentrations.

One of the mutagenic and carcinogenic heterocyclic amines (HCAs), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), is present in cooked foods and we are chronically exposed to this compound in our daily life. To study the role of HCAs in human carcinogenesis, we analyzed MeIQx-DNA adducts in 38 DNA samples obtained from surgical and autopsy specimens by the 32P-postlabeling method under adduct-intensification conditions with the modification of additional digestion with nuclease P1 and phosphodiesterase I after 32P-labeling at 5'-hydroxyl termini. This modified 32P-postlabeling method can detect N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo- [4,5-f]quinoxaline 5'-monophosphate (5'-pdG-C8-MeIQx) at levels down to 1/10(10) nucleotides. The DNA samples from colon and rectum surgical specimens and a kidney taken at autopsy were found to contain an adduct spot corresponding to that of standard 5'-pdG-C8-MeIQx on TLC at levels of 14,18 and 1.8 per 10(10) nucleotides, respectively. Each adduct spot was extracted from TLC and identified to be 5'-pdG-C8-MeIQx by HPLC. Thus, MeIQx-DNA adducts actually exist in human tissues and this adduct formation may be involved in human cancer development.

Effects of pregnancy and lactation on a food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were investigated in female Sprague-Dawley rats. When rats were administrated PhIP in the diet (100 ppm) for 15 weeks, palpable subcutaneous tumors were first detected at week 24 in three nulliparous rats, while tumor latency was apparently increased in animals undergoing delivery and weaning during the PhIP administration period. However, since the incidence of palpable tumors was not consistently increased in the nulliparous animals, possibly due to the short PhIP-treatment, and the fact that spontaneous tumors developed in parous animals after week 44, the final incidences and multiplicities of histopathologically confirmed adenocarcinomas at week 48 were not significantly different between the two groups. These findings art partly supported by the epidemiological evidence that breast cancer is more prevalent in nulliparous than in parous women.

Male Wistar-MS (W/MS), Fisher-344 (F-344) and Sprague-Dawley (SD) rats were divided into four groups including a control group implanted with a cholesterol pellet. Rats in the three experimental groups were treated with gamma-ray irradiation (260 cGyt) alone, diethylstilbestrol (DES) pellet implantation alone or both irradiation and DES, and all rats were observed for detection of mammary tumors for 1 year. Morphologically, well-developed mammary glands were observed in the SD rats at ages corresponding to the time of irradiation. But, the mammary glands in the W/MS and F-344 rats showed a lower degree of differentiation than those in the SD rats. No mammary tumor developed spontaneously in the W/MS and F-344 strains of rats during the experimental period. The rats administered both DES and irradiation showed significantly increased incidence of mammary tumors compared with the control, the incidence being 80.9% in the SD rats, 35.0% in the W/MS rats, and 9.4% in the F-344 rats, respectively. The incidence of tumor in the SD rats treated with irradiation alone and with DES alone was 9.5% and 14.3%, respectively, but no tumor development was observed in the F-344 rats treated with either irradiation alone or DES alone or in the W/MS rats treated with DES alone. The magnitude of the decrease of testicular weight in the SD rats implanted with DES after irradiation (to 70% of the control weight) was slightly less marked than that in either the W/MS (35%) or F-344 (16%) rats. The testicular atrophy showed a correlation with the accessory sex organ weight at the end of the experiment, serum testosterone concentration, and incidence of mammary tumors. Following administration of DES pellets after the irradiation, the activity of delta 5-3 beta- and of 17 beta-hydroxysteroid dehydrogenase in the testes showed the order F-344 < W/MS = SD and F-344 = W/MS < SD, respectively. Compared with the control, the irradiated F-344 rats implanted with DES pellets showed hypertrophied pituitary glands (10.7-fold, P < 0.01) as well as increased serum prolactin concentration (21.4-fold, P < 0.01). Of the three strains treated with both irradiation and DES, the F-344 rats showed the highest concentration of serum prolactin but the lowest incidence of mammary tumors. Our results suggest that W/MS, F-344 and SD male rats have differing susceptibilities for the induction of mammary tumor following irradiation. We discuss the relationship between testicular and pituitary functions and male mammary tumorigenesis.

A food-borne heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), induces hepatocellular carcinomas (HCCs) in F344 male rats at an incidence of 95%, when fed in the diet at 400 ppm for 61 weeks. In this study, the effect of a low dose of MeIQx was examined in Long-Evans with cinnamon-like coat color (LEC) rats, which have a mutation in Atp7b and suffer from hereditary hepatitis and HCCs, with high levels of copper accumulation in the liver. Rats of the LEC and Long-Evans with agouti coat color (LEA) sibling lines were given a diet containing 40 ppm MeIQx from the age of 23 weeks to 63 weeks, for a total administration period of 40 weeks. In LEC rats, HCCs were observed in 8/8 animals administered MeIQx, and 2/8 rats receiving a normal diet. The number of HCCs per rat (mean +/- SD) was 2.8 +/- 2.0 and 0.3 +/- 0.5, respectively. In the LEA rats, however, no tumors were induced by administration of MeIQx. These results indicate that damaged liver associated with compensatory cell proliferation is much more susceptible to chemical hepatocarcinogens, including MeIQx, than the normal liver.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces colon tumors in male, but not female, F344 rats. We investigated the mechanisms leading to this difference by measuring the level of PhIP-DNA adducts, the enhancement of cell proliferation and aberrant crypt focus (ACF) formation in colon mucosa. PhIP was administered in the diet at a level of 0.04% to both male and female F344 rats for 1-8 weeks. The level of DNA adducts in the colon mucosa was measured using the 32P-postlabeling method. Four major PhIP-DNA adducts were detected in fairly constant proportions in all the animals examined. The level of PhIP-DNA adducts in male and female rats was the same, indicating no direct correlation between adduct levels and carcinogenesis. Labeling indices (LIs) were determined by measuring BrdU incorporation in rats after feeding with a PhIP diet for 4, 8 and 12 weeks. After 8 weeks administration the LI had increased 1.5-fold in the colon of the male rats, but no increase was observed in the female rats. ACF formation was examined after feeding with a PhIP diet for 14 weeks. The number of aberrant crypt foci was 6.6 +/- 1.5 per rat in males and 1.9 +/- 0.5 per rat in females. Thus differences in colon tumor development in male and female rats takes place at an early stage(s). Our results suggest that, in addition to DNA adduct formation, enhanced proliferation contributes to the formation of ACFs, which are premalignant lesions of the colon.

The effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins (GTC), alpha-tocopherol, beta-carotene, chlorophyllin, phenylethylisothiocyanate (PEITC), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethyl ascorbic acid (DAsA), n-tritriacontane-16,18-dione (TTAD) and d-limonene on 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)- or dimethylnitrosamine (DMN)-induced hepatocarcinogenesis, and the dose dependence of HTHQ inhibition of Glu-P-1- or 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx)-influence on lesion development were examined in a rat medium-term liver bioassay system featuring diethylnitrosamine initiation and partial hepatectomy. At the end of week 8, the number and total area of glutathione S-transferase placental form (GST-P) positive liver foci in rats treated with 0.03% Glu-P-1 alone were increased significantly (46.8 +/- 11.0 and 12.0 +/- 5.6 respectively) as compared to the control values (3.8 +/- 1.6 and 0.4 +/- 0.2). Combined treatment with 1% HTHQ remarkably reduced both of these parameters (8.1 +/- 2.1 and 0.6 +/- 0.2). GTC (1%), PEITC (0.1%), beta-carotene (0.1%) and DAsA (1%) also demonstrated inhibition but less than HTHQ. On the other hand, these antioxidants did not influence development of foci initiated by 0.002% DMN. In the dose-response study, up to 0.125% HTHQ significantly reduced the effects of 0.02% Glu-P-1 or 0.03% MeIQx on the number and area of foci. These results indicate that several antioxidants exert chemopreventive effects against heterocyclic amine (HCA)-induced hepatocarcinogenesis, and particularly HTHQ which thus deserves further attention as a chemopreventor in the contest of the environmentally important HCA group of carcinogens.

Effects of transplacental and trans-breast milk exposure to a food-derived mammary and colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were investigated in rats. Female Sprague-Dawley rats were administered PhIP in the diet (100 ppm) for 4 weeks before mating with nontreated males and also during gestation and lactation. As controls, additional females were maintained on the basal diet without PhIP and mated as with the treated animals. The offspring of both groups were subdivided for each sex at weaning into three dietary groups receiving 100, 25, and 0 ppm and were killed at 47 weeks of age. Effects of the transplacental and neonatal exposure to PhIP on mammary carcinogenesis were most evident in females administered 25 ppm PhIP after weaning; the incidence and multiplicity of adenocarcinomas in offspring from the PhIP fed dams (42.9%, 0.62/rat) was significantly higher than the value for offspring from nontreated dams (4.8%, 0.05/rat). Furthermore, in the basal diet groups, the incidence of adenocarcinomas in females was higher, albeit not significantly, in offspring of the PhIP-treated than the nontreated dams (16.7%, 0.22/rat as compared with 3.3%, 0.07/rat). Although the highest incidence of mammary adenocarcinomas was found in the female progeny given 100 ppm PhIP from PhIP-treated dams (70.0%, 1.55/rat), this was only slightly higher than the 61.9% and 0.90/rat of the same dose group from the nontreated dams. In males, no apparent effects of transplacental and neonatal exposures were evident. In a separate experiment, excretion of PhIP into breast milk and transfer of PhIP to fetuses and neonates with resultant hepatic PhIP-DNA adduct formation were demonstrated. Thus, maternal exposure to this food-derived carcinogen may be a critical risk factor for generation of mammary carcinomas.

Effects of chronic administration of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) at 0.4, 8 and 400 ppm in the diet on DNA adduct formation and removal in the rat liver were examined by the 32P-postlabeling method. The 0.4 and 8 ppm doses for 40 weeks resulted in time-dependent increases in MeIQx-DNA adduct levels until 16 and 8 weeks, respectively, with constant values being maintained thereafter. In the case of a carcinogenic dose (400 ppm) of MeIQx, the adduct levels reached a maximum at week 12, and then gradually decreased. Alteration of metabolism of MeIQx during liver carcinogensis might be related to this decrease in DNA adduct levels. When MeIQx administration was stopped at week 20, 60-90% of the MeIQx-DNA adducts formed with the three doses (0.4, 8 and 400 ppm) of MeIQx were removed in a biphasic manner after return to a basal diet, with initial rapid removal followed by a slow change. No difference in the pattern of MeIQx-DNA adducts was detected on thin layer chromatography at any dose at any time point. Thus, it is suggested that there may be at least two types of damaged DNA, susceptible and resistant to removal of MeIQx-DNA adducts, after chronic administration of MeIQx.