Minako Nagao

A new approach to risk estimation of food-borne carcinogens : heterocyclic : amines : based on molecular information

Identification of causative agents for human cancers is the goal of our studies. We analyzed ordinary foods for mutagenicity, using the well-established Salmonella test. Heating fish and meat yielded mutagens that require metabolic activation for exhibition of mutagenicity. Structural determination revealed these mutagens to be heterocyclic amines (HCAs), their precursors in some cases being creatin(in)e, sugars and amino acids. Ten HCAs so far examined have all proved carcinogenic in mice and rats, inducing cancers in various organs such as in the mammary glands, prostate, lung, colon, skin, bladder and liver. Human exposure to HCAs is 0.1-12 microg/day, predominantly to 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). For these types of genotoxic carcinogens, DNA-adduct formation is crucially important and PhIP-DNA adducts have been detected in human tissues. However, the amounts of individual HCAs ingested by humans may not be sufficient to induce cancers by themselves and many environmental factors have also been implicated in neoplasia in man, with other considerable inter-individual variation in susceptibility, e.g., to colon carcinogenesis. This is in line with results obtained by feeding different strains of rats with HCA. Studies using lacI transgenic mice and rats have revealed that DNA adducts do not directly correlate with mutant frequencies at the organ level, or cancer incidence. However, sequencing of the Apc gene of rat colon tumors induced by PhIP revealed that it induces a signature mutation of G deletion from the GGGA sequence. This type of mutation is found in the p53 gene of 0.3% human cancers having p53-somatic mutations, and it has been calculated that 3%-10% of the p53 mutations detected in human cancers could be ascribable to PhIP. Although there remains the possibility that other carcinogens involved in human carcinogenesis cause the same signature mutation, the available data point to an important role for PhIP.

Characterization of Dysplastic Aberrant Crypt Foci in the Rat Colon Induced by 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine

The multistage model of colon carcinogenesis is well established in both humans and experimental animals, and aberrant crypt foci (ACF) are generally assumed to be putative preneoplastic lesions of the colon. However, morphological analyses of ACF have suggested that they are highly heterogeneous in nature and their role in tumorigenesis is still controversial. To better understand the biological significance of ACF in carcinogenesis, morphological and genetic analyses were performed using a rat colon cancer model induced by a food-borne colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). ACF of different sizes were collected at weeks 6, 18, 25, and 32 after three cycles of 2-week PhIP feeding (400 ppm in diet) with 4-week intervals on a high-fat diet, and a total of 110 ACF, representing approximately three-quarters of the total ACF, were subjected to histological evaluation. Thirty (27%) were diagnosed as dysplastic ACF, based on cytological and structural abnormalities of crypts. Dysplastic ACF were detected even at week 6 (0.4 per rat), and the numbers increased slightly at later time points, being 0.8, 1.4, and 0.8 per rat at weeks 18, 25, and 32, respectively. The sizes of these dysplastic ACF varied widely from 1 to 16 crypts and 50% (15 of 30) were composed of less than 4 crypts. Immunohistochemical analysis revealed that 83% (25 of 30) of dysplastic ACF demonstrated beta-catenin accumulation; 22 only in the cytoplasm and 3 in both the cytoplasm and nucleus, the latter manifesting a higher grade of dysplasia as compared with the former. Seven dysplastic ACF harbored beta-catenin mutations at codon 32, 34, or 36 in exon 2, and one had an Apc mutation at the boundary of intron 10 and exon 11. Mutations at these sites were also commonly found in colon tumors induced by PhIP. The results of our present study indicate that dysplastic ACF, which accounted for approximately one-fourth of the total ACF, are preneoplastic lesions of colon cancers induced by PhIP in rats.

A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines contained in cooked meat and fish, and induces aberrant crypt foci (ACF), putative preneoplastic lesions of the colon, and colon cancers in male rats when administered orally. As has been reported previously, F344 rats are susceptible to induction of ACF by PhIP, while ACI rats being relatively resistant. Approximately one-fourth of ACF induced by PhIP in F344 rats are dysplastic; exhibiting lesions with structural distortion of the crypt, decrease of goblet cells, nuclear stratification and enlargement of nuclei. Dysplastic ACF demonstrate beta-catenin accumulation, mainly in the cytoplasm, and increased cell proliferation in crypts. These dysplastic ACF are, therefore, strongly considered to be putative preneoplastic lesions of the colon.A genetic trait affecting the susceptibility to colon carcinogenesis in F344 rats was mapped to chromosome 16, between D16Rat17 and D16Wox3, using the number of ACF as a surrogate biomarker for colon carcinogenesis. Since the number of dysplastic lesions is well correlated with the total number of ACF, being approximately one-fourth of the total ACF as described above in F344 rats and will be described elsewhere in ACI rats, the gene involved in the susceptibility to ACF induction may possibly be partly responsible for the susceptibility to colon carcinogenesis by PhIP. We, thus, tentatively referred the name of the candidate susceptibility gene on rat chromosome 16 as susceptibility to colon tumor (Sct). In the present study, the colonic lesions induced by PhIP were well refined histologically and genetically, and the multi-step profiles of colon cancer development by PhIP were well characterized and revealed to be similar to the multi-step model of colon carcinogenesis in humans. The PhIP-induced colon cancer model in rats, thus contributes as a relevant tool to elucidate genetic factors responsible for susceptibility to colon carcinogenesis in human. Other unknown genetic or epigenetic alterations, which are essential for the development of early lesions of colon carcinogenesis, could also be clarified using this system.

LacI transgenic animal study: relationships among DNA-adduct levels, mutant frequencies and cancer incidences

In the processes of carcinogenesis caused by genotoxic carcinogens, DNA-adduct formation and resultant genetic changes are crucially important. In this report, the relationship between DNA-adduct levels and mutant frequencies (MFs), DNA-adduct levels and cancer incidences, and MFs and cancer incidences induced by heterocyclic amines (HCAs), to which humans are exposed on daily basis were investigated. There was no direct correlation between adduct levels and MFs detected after feeding Big Blue mice with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), in a comparison among various organs. Further, there was no direct correlation between DNA-adduct levels and cancer incidences, in a comparison among various organs of F344 rats. Since DNA-adducts are fixed as mutations after cell proliferation, and mutations in cancer-related genes result in cancer development, it is expected that MFs directly correlate with cancer incidences. However, there was no direct correlation between MFs and cancer incidences. Possible mechanisms involved in the discordance between DNA damage markers and cancer incidences are discussed.

Effective pretreatment of human serum samples for dioxin analysis by solid phase extraction and blue-chitin column cleanup.

For the analysis of dioxins, polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/Fs) and coplanar polychlorinated biphenyls (Co-PCBs), devising simple and economical methods is important, especially for mass screening of human exposure. Pretreatment of samples, namely the extraction and cleanup methods that are widely used at present, needs to be improved for savings in time, manpower, and solvent consumption. In the present study, we applied solid phase extraction (SPE) using octadecyl (C18) and a blue-chitin column in place of liquid-liquid extraction (LE) and an active-carbon column with serum samples, frequently used for assessment of human exposure. Efficacy of the new pretreatment methods was demonstrated by successful high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) of the major 17 PCDDs/Fs and 12 Co-PCBs that are on the list of WHO/IPCS (1997) hazardous dioxins with toxic equivalent factor (TEF) values. SPE is timesaving and requires less manpower and organic solvent as compared with the LE that is presently widely used. Concerning cleanup with blue-chitin, the amount of toluene applied as eluent could be reduced to 1/3, as compared with the active-carbon case. The combination of SPE and blue-chitin for pretreatment of serum saves time and manpower, is accurate and uses less organic solvent than LE with active carbon cleanup.

Frequent and Multiple Mutations at Minisatellite Loci in Sporadic Human Colorectal and Gastric Cancers—Possible Mechanistic Differences from Microsatellite Instability in Cancer Cells

Minisatellites (MNs), composed of 5 to 100 nucleotide repeat units, range from 0.5 to 30 kb in length, and have been reported to be mutated in various human malignancies. In this study, frequencies of MN mutations in sporadic human colorectal (34 cases) and gastric cancers (24 cases) at various clinicopathological stages were assessed by multilocus DNA fingerprint analysis with three MN probes, Pc‐1, 33.6 and 33.15. MN mutations were observed in both colorectal and gastric cancers, but at a significantly higher frequency in the former (56%) than in the latter (25%). Multiplicities of MN mutations were 1.50±1.81 and 0.46±1.10 in colorectal and gastric cancers, respectively, and the difference was also significant. Neither the presence nor multiplicity of MN mutations in either colorectal or gastric cancer cases had any correlation with the pathological stage, histological grading or the presence of microsatellite instability (MSI). Although the biological relevance of MN mutations still remains to be clarified, a subset of colorectal and gastric cancers could feature a new type of genomic instability, distinct from MSI.