Title: Characterization of Dysplastic Aberrant Crypt Foci in the Rat Colon Induced by 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine
Abstract: 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 β-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 β-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. 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 β-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 β-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. Colon cancers develop after multistep accumulation of genetic and epigenetic alterations in both humans and experimental animals.1Kinzler KW Vogelstein B Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4328) Google Scholar, 2Nagao M Ushijima T Toyota M Inoue R Sugimura T Genetic changes induced by heterocyclic amines.Mutat Res. 1997; 376: 161-167Crossref PubMed Scopus (83) Google Scholar, 3Esteller M Corn PG Baylin SB Herman JG A gene hypermethylation profile of human cancer.Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar The adenomatous polyposis coli gene (APC) serves as a gatekeeper gene for the development of colon cancers and genetic alterations in APC and down-regulation of APC mRNA expression are frequently observed in human colon cancers.1Kinzler KW Vogelstein B Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4328) Google Scholar, 3Esteller M Corn PG Baylin SB Herman JG A gene hypermethylation profile of human cancer.Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar Mutations in several other genes involved in the WNT signaling pathway, such as CTNNB1, AXIN1, and AXIN2, are also found in some colon cancers.4Polakis P Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 5Liu W Dong X Mai M Seelan RS Taniguchi K Krishnadath KK Halling KC Cunningham JM Qian C Christensen E Roche PC Smith DI Thibodeau SN Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling.Nat Genet. 2000; 26: 146-147Crossref PubMed Scopus (447) Google Scholar, 6Webster MT Rozycka M Sara E Davis E Smalley M Young N Dale TC Wooster R Sequence variants of the axin gene in breast, colon, and other cancers: an analysis of mutations that interfere with GSK3 binding.Genes Chromosom Cancer. 2000; 28: 443-453Crossref PubMed Scopus (141) Google Scholar Aberrant crypt foci (ACF) were first described by Bird7Bird RP Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings.Cancer Lett. 1987; 37: 147-151Abstract Full Text PDF PubMed Scopus (1017) Google Scholar and claimed to be lesions composed of enlarged crypts, slightly elevated above the surrounding mucosa and more densely stained with methylene blue than normal crypts. ACF are considered as putative preneoplastic lesions of the colon based on the following observations in both humans and experimental animals. In the case of humans, large numbers of ACF are commonly observed in familial adenomatous polyposis patients, as well as in sporadic colorectal cancer patients.8Pretlow TP Barrow BJ Ashton WS O'Riordan MA Pretlow TG Jurcisek JA Stellato TA Aberrant crypts: putative preneoplastic foci in human colonic mucosa.Cancer Res. 1991; 51: 1564-1567PubMed Google Scholar, 9Roncucci L Stamp D Medline A Cullen JB Bruce WR Identification and quantification of aberrant crypt foci and microadenomas in the human colon.Hum Pathol. 1991; 22: 287-294Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 10Roncucci L Pedroni M Vaccina F Benatti P Marzona L De Pol A Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics.Cell Prolif. 2000; 33: 1-18Crossref PubMed Scopus (107) Google Scholar, 11Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, Apc, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar The number of foci decreases after treatment with nonsteroidal anti-inflammatory drugs,12Takayama T Katsuki S Takahashi Y Ohi M Nojiri S Sakamaki S Kato J Kogawa K Miyake H Niitsu Y Aberrant crypt foci of the colon as precursors of adenoma and cancer.N Engl J Med. 1998; 339: 1277-1284Crossref PubMed Scopus (536) Google Scholar which effectively reduce the numbers and sizes of polyps in familial adenomatous polyposis patients.13Jänne PA Mayer RJ Chemoprevention of colorectal cancer.N Engl J Med. 2000; 342: 1960-1968Crossref PubMed Scopus (449) Google Scholar In addition, increased proliferation activity10Roncucci L Pedroni M Vaccina F Benatti P Marzona L De Pol A Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics.Cell Prolif. 2000; 33: 1-18Crossref PubMed Scopus (107) Google Scholar and genetic alterations in K-RAS,11Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, Apc, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 14Pretlow TP Brasitus TA Fulton NC Cheyer C Kaplan EL K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (223) Google Scholar, 15Jen J Powell SM Papadopoulos N Smith KJ Hamilton SR Vogelstein B Kinzler KW Molecular determinants of dysplasia in colorectal lesions.Cancer Res. 1994; 54: 5523-5526PubMed Google Scholar, 16Smith AJ Stern HS Penner M Hay K Mitri A Bapat BV Gallinger S Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons.Cancer Res. 1994; 54: 5527-5530PubMed Google Scholar, 17Shivapurkar N Huang L Ruggeri B Swalsky PA Bakker A Finkelstein S Frost A Silverberg S K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients.Cancer Lett. 1997; 115: 39-46Abstract Full Text PDF PubMed Scopus (62) Google Scholar APC,15Jen J Powell SM Papadopoulos N Smith KJ Hamilton SR Vogelstein B Kinzler KW Molecular determinants of dysplasia in colorectal lesions.Cancer Res. 1994; 54: 5523-5526PubMed Google Scholar, 16Smith AJ Stern HS Penner M Hay K Mitri A Bapat BV Gallinger S Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons.Cancer Res. 1994; 54: 5527-5530PubMed Google Scholar and P53 genes,17Shivapurkar N Huang L Ruggeri B Swalsky PA Bakker A Finkelstein S Frost A Silverberg S K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients.Cancer Lett. 1997; 115: 39-46Abstract Full Text PDF PubMed Scopus (62) Google Scholar as well as microsatellite instability,18Augenlicht LH Richards C Corner G Pretlow TP Evidence for genomic instability in human colonic aberrant crypt foci.Oncogene. 1996; 12: 1767-1772PubMed Google Scholar, 19Heinen CD Shivapurkar N Tang Z Groden J Alabaster O Microsatellite instability in aberrant crypt foci from human colons.Cancer Res. 1996; 56: 5339-5341PubMed Google Scholar have been demonstrated in ACF of humans. In the rat model, ACF are induced by various types of colon carcinogens in a dose- and time-dependent manner.20McLellan EA Bird RP Aberrant crypts: potential preneoplastic lesions in the murine colon.Cancer Res. 1988; 48: 6187-6192PubMed Google Scholar, 21Tudek B Bird RP Bruce WR Foci of aberrant crypts in the colons of mice and rats exposed to carcinogens associated with foods.Cancer Res. 1989; 49: 1236-1240PubMed Google Scholar The formation of ACF is enhanced by dietary fats,20McLellan EA Bird RP Aberrant crypts: potential preneoplastic lesions in the murine colon.Cancer Res. 1988; 48: 6187-6192PubMed Google Scholar which have been demonstrated to have promoting effects on colon cancer development,22Reddy BS Maeura Y Tumor promotion by dietary fat in azoxymethane-induced colon carcinogenesis in female F344 rats: influence of amount and source of dietary fat.J Natl Cancer Inst. 1984; 72: 745-750PubMed Google Scholar and is suppressed by chemopreventive agents that have been demonstrated to suppress the development of colon cancers.23Pereira MA Barnes LH Rassman VL Kelloff GV Steele VE Use of azoxymethane-induced foci of aberrant crypts in rat colon to identify potential cancer chemopreventive agents.Carcinogenesis. 1994; 15: 1049-1054Crossref PubMed Scopus (218) Google Scholar However, conflicting evidence has also been presented. For example, mutations in the K-RAS gene are relatively common in human ACF,11Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, Apc, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 14Pretlow TP Brasitus TA Fulton NC Cheyer C Kaplan EL K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (223) Google Scholar, 15Jen J Powell SM Papadopoulos N Smith KJ Hamilton SR Vogelstein B Kinzler KW Molecular determinants of dysplasia in colorectal lesions.Cancer Res. 1994; 54: 5523-5526PubMed Google Scholar, 16Smith AJ Stern HS Penner M Hay K Mitri A Bapat BV Gallinger S Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons.Cancer Res. 1994; 54: 5527-5530PubMed Google Scholar, 17Shivapurkar N Huang L Ruggeri B Swalsky PA Bakker A Finkelstein S Frost A Silverberg S K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients.Cancer Lett. 1997; 115: 39-46Abstract Full Text PDF PubMed Scopus (62) Google Scholar but are detected at a relatively late stage of colon cancer development.24Vogelstein B Fearon ER Hamilton SR Kern SE Preisinger AC Leppert M Nakamura Y White R Smits AM Bos JL Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (6128) Google Scholar In rat models, although hundreds of ACF are induced per animal by azoxymethane (AOM), and K-ras mutations are frequently observed in those ACF, only a few colon tumors are observed per animal.25Magnuson BA Carr I Bird RP Ability of aberrant crypt foci characteristics to predict colonic tumor incidence in rats fed cholic acid.Cancer Res. 1993; 53: 4499-4504PubMed Google Scholar, 26Takahashi M Fukutake M Isoi T Fukuda K Sato H Yazawa K Sugimura T Wakabayashi K Suppression of azoxymethane-induced rat colon carcinoma development by a fish oil component, docosahexaenoic acid (DHA).Carcinogenesis. 1997; 18: 1337-1342Crossref PubMed Scopus (79) Google Scholar, 27Takahashi M Mutoh M Kawamori T Sugimura T Wakabayashi K Altered expression of β-catenin, inducible nitric oxide synthase and cyclooxygenase-2 in azoxymethane-induced rat colon carcinogenesis.Carcinogenesis. 2000; 21: 1319-1327Crossref PubMed Google Scholar Moreover, some compounds that effectively suppress the induction of ACF by AOM in rats, eg, 2-(carboxyphenyl)retinamide or genistein, enhance the development of colon cancers.23Pereira MA Barnes LH Rassman VL Kelloff GV Steele VE Use of azoxymethane-induced foci of aberrant crypts in rat colon to identify potential cancer chemopreventive agents.Carcinogenesis. 1994; 15: 1049-1054Crossref PubMed Scopus (218) Google Scholar, 28Zheng Y Kramer PM Lubet RA Steele VE Kelloff GJ Pereira MA Effect of retinoids on AOM-induced colon cancer in rats: modulation of cell proliferation, apoptosis and aberrant crypt foci.Carcinogenesis. 1999; 20: 255-260Crossref PubMed Scopus (102) Google Scholar, 29Rao CV Wang CX Simi B Lubet R Kelloff G Steele V Reddy BS Enhancement of experimental colon cancer by genistein.Cancer Res. 1997; 57: 3717-3722PubMed Google Scholar This discrepancy could be caused by the heterogeneous nature of ACF and also by a wide range of biological consequences of ACF. (2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine PhIP), one of the most abundant heterocyclic amines produced during cooking of meat and fish,30Felton JS Jägerstad M Knize MG Skog K Wakabayashi K Contents in foods, beverages and tobacco.in: Nagao M Sugimura T Food Borne Carcinogens. John Wiley & Sons Ltd., Chichester2000: 31-71Google Scholar has been demonstrated to induce ACF in rats a few weeks after feeding a diet containing PhIP31Takahashi S Ogawa K Ohshima H Esumi H Ito N Sugimura T Induction of aberrant crypt foci in the large intestine of F344 rats by oral administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.Jpn J Cancer Res. 1991; 82: 135-137Crossref PubMed Scopus (60) Google Scholar and colon cancers 1 or 2 years later.32Ito N Hasegawa R Sano M Tamano S Esumi H Takayama S Sugimura T A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).Carcinogenesis. 1991; 12: 1503-1506Crossref PubMed Scopus (616) Google Scholar, 33Hasegawa R Sano M Tamano S Imaida K Shirai T Nagao M Sugimura T Ito N Dose-dependence of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenicity in rats.Carcinogenesis. 1993; 14: 2553-2557Crossref PubMed Scopus (140) Google Scholar A recent report by Sinha and colleagues34Sinha R Kulldorff M Chow WH Denobile J Rothman N Dietary intake of heterocyclic amines, meat-derived mutagenic activity, and risk of colorectal adenomas.Cancer Epidemiol Biomarkers Prev. 2001; 10: 559-562PubMed Google Scholar indicated that PhIP could be one of the causative agents for colon carcinogenesis in humans. To elucidate the biological significance of ACF in colon carcinogenesis, ACF induced in F344 male rats by PhIP were subjected to morphological and genetic analyses. Five-week-old F344 male rats were purchased from CLEA Japan (Tokyo, Japan) and housed in a ventilated, temperature-controlled room at 25°C with a 12-hour light/dark cycle. After a 1-week acclimatization to the housing environment on a basal diet (AIN-93G; Dyets, Bethlehem, PA), the rats were fed a basal diet (AIN-93G) containing 400 ppm of PhIP (Nard Institute, Osaka, Japan) for the first 2 weeks, followed by a high-fat diet (HF) containing hydrogenated oil PRIMEX (Dyets) for 4 weeks, as described previously, with minor modification.35Ubagai T Ochiai M Kawamori T Imai H Sugimura T Nagao M Nakagama H Efficient induction of rat large intestinal tumors with a new spectrum of mutations by intermittent administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in combination with a high fat diet.Carcinogenesis. 2002; 23: 197-200Crossref PubMed Scopus (34) Google Scholar Briefly, the 2-week administration of PhIP was repeated three times with 4-week intervals on the HF diet, and the HF diet without PhIP was given until the termination of the experiment at week 32 (Figure 1). Seven or 10 rats were killed at weeks 6, 18, 25, and 32 and subjected to histological and genetic analyses of ACF and colon tumors. The colons were resected and gently flushed with 10% neutralized formalin to remove residual bowel contents, cut open longitudinally, fixed flat between filter papers, and submerged in 10% neutralized formalin overnight at 4°C. Fixed colons were stained with 0.2% methylene blue, as described by Bird,7Bird RP Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings.Cancer Lett. 1987; 37: 147-151Abstract Full Text PDF PubMed Scopus (1017) Google Scholar and the numbers of ACF, and total numbers of aberrant crypts (ACs) comprising ACF were counted for each rat. ACF were identified as lesions composed of enlarged crypts, with an increased pericryptal area, slightly elevated appearance above the surrounding mucosa with an oval or slit-like orifice, and higher staining intensity with 0.2% methylene blue than normal crypts. When colon tumors were detected macroscopically, tissues were embedded in paraffin and histological evaluation was performed according to the intestinal tumor classification of the rat by Pozharisski36Pozharisski KM Tumors of the intestines.IARC Sci Pub (Lyon). 1990; 99: 159-198PubMed Google Scholar after hematoxylin and eosin (H&E) staining. ACF of different sizes were resected along with the surrounding normal colon tissues as transverse sections with less than 2-mm width, embedded in paraffin blocks, and subjected to microscopic observation. Serial paraffin sections were prepared for each lesion at 3.5-μm thickness, and one in every three sections was stained with H&E to evaluate the histological grade of ACF. According to their cytological and structural abnormalities, ACF were classified into two groups; nondysplastic ACF and dysplastic ACF. The former are characterized histologically as hyperplastic, and the latter as lesions with distortion of the crypt structure, decrease in goblet cells, nuclear stratification, and enlarged nuclei. Paraffin sections of ACF and colon tumors were stained with anti-β-catenin antibody (BD Transduction Laboratories, Lexington, KY) as reported previously.35Ubagai T Ochiai M Kawamori T Imai H Sugimura T Nagao M Nakagama H Efficient induction of rat large intestinal tumors with a new spectrum of mutations by intermittent administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in combination with a high fat diet.Carcinogenesis. 2002; 23: 197-200Crossref PubMed Scopus (34) Google Scholar Biotinylated goat anti-mouse IgG (Kirkegaard & Perry, Inc., Gaithersburg, MD) was used as the secondary antibody at a dilution of 1:200. Immunohistochemical staining of Ki-67 antigen, a cell-cycle-associated antigen that serves as an endogenous marker for cell proliferation, was performed using the Vectastain ABC system (Vector Laboratories, Burlingame, CA). Tissue sections were deparaffinized and the Ki-67 antigen was activated by boiling for 15 minutes in 10 mmol/L of citrate buffer (pH 6.0). A rabbit polyclonal antibody (Novocastra Laboratories, Newcastle on Tyne, UK), raised against human Ki-67 antigen, was used as the primary antibody at a dilution of 1:1000, and biotinylated goat anti-rabbit IgG (Vector Laboratories) as the secondary antibody at a dilution of 1:200. The labeling index for the Ki-67 antigen in ACF was calculated as the ratio of Ki-67-positive cells per total number of crypt cells using one representative tangential section for each ACF. DNA extraction from ACF and colon tumors was performed as follows. Lesions were microdissected from paraffin-embedded sections using the Pinpoint Slide DNA Isolation System (Zymo Research, Orange, CA) according to the manufacturer's protocol. Extracted DNA was then subjected to polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis.37Orita M Iwahana H Kanazawa H Hayashi K Sekiya T Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.Proc Natl Acad Sci USA. 1989; 86: 2766-2770Crossref PubMed Scopus (3480) Google Scholar PCR primers for the rat β-catenin gene were designed to amplify the 193-bp fragment from exon 2, corresponding to functionally important phosphorylation sites and mutation hot spots in β-catenin. For the Apc gene, four primer sets were designed to amplify the 5′-GGGA-3′ sites in exons 14 and 15, and the 5′-tagGGG-3′ site at the boundary of intron 10 and exon 11, where the Apc mutations are frequently found in PhIP-induced rat colon tumors.35Ubagai T Ochiai M Kawamori T Imai H Sugimura T Nagao M Nakagama H Efficient induction of rat large intestinal tumors with a new spectrum of mutations by intermittent administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in combination with a high fat diet.Carcinogenesis. 2002; 23: 197-200Crossref PubMed Scopus (34) Google Scholar, 38Kakiuchi H Watanabe M Ushijima T Toyota M Imai K Weisburger JH Sugimura T Nagao M Specific 5′-GGGA-3′ → 5′-GGA-3′ mutation of the Apc gene in rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.Proc Natl Acad Sci USA. 1995; 92: 910-914Crossref PubMed Scopus (162) Google Scholar Primer sequences, annealing temperatures, and expected sizes of PCR products are indicated in Table 1. PCR reactions were performed using AmpliTaq Gold DNA polymerase (Applied Biosystems Japan, Tokyo, Japan) and SSCP analysis was performed using the GenePhor DNA separation system (Amersham Pharmacia Biotech, Little Chalfont, UK), as described previously.35Ubagai T Ochiai M Kawamori T Imai H Sugimura T Nagao M Nakagama H Efficient induction of rat large intestinal tumors with a new spectrum of mutations by intermittent administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in combination with a high fat diet.Carcinogenesis. 2002; 23: 197-200Crossref PubMed Scopus (34) Google Scholar Nucleotide sequences of the bands with shifted mobilities were determined by PCR-based direct sequencing using the CEQ2000XL DNA analysis system (Beckman Coulter, Fullerton, CA). Mutational analyses for both ACF and colon tumors were repeated at least twice using serial paraffin sections to confirm the results.Table 1Oligonucleotide Primers Used for PCR-SSCP AnalysisGeneRegionNucleotides*Nucleotide numbers are assigned according to the rat β-catenin and Apc cDNA sequences.ForwardReverseAnnealling temperature (°C)Product size (bp)Ref.β-cateninExon 2172–3645′-TGACCTCATGGAGTTGGACA-3′5′-GCCTTGCTCCCACTCATAAA-3′6019335Ubagai T Ochiai M Kawamori T Imai H Sugimura T Nagao M Nakagama H Efficient induction of rat large intestinal tumors with a new spectrum of mutations by intermittent administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in combination with a high fat diet.Carcinogenesis. 2002; 23: 197-200Crossref PubMed Scopus (34) Google ScholarApcIntron 10/ exon 11Exon/intron junction5′-CGATTATGGATTATTGTATG-3′5′-AAGTGAGCACCTTAGGAAGT-3′5230038Kakiuchi H Watanabe M Ushijima T Toyota M Imai K Weisburger JH Sugimura T Nagao M Specific 5′-GGGA-3′ → 5′-GGA-3′ mutation of the Apc gene in rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.Proc Natl Acad Sci USA. 1995; 92: 910-914Crossref PubMed Scopus (162) Google ScholarExon 141791–20105′-GAATCAACTCTCAAAAGCGT-3′5′-TATACCTGTGGTCTTCGTTG-3′52220Exon 152492–27245′-CACTGGAAACATGACTGTTC-3′5′-CTTCCATCACTTTGGCTATC-3′54233Exon 154081–43515′-CCGCCAGGCATAAAGCTGTT-3′5′-CTTCTGCTTGGTGGCATGGT-3′58271* Nucleotide numbers are assigned according to the rat β-catenin and Apc cDNA sequences. Open table in a new tab All statistical analyses were performed by the Kruskal-Wallis test using SPSS for Macintosh (SPSS Inc., Tokyo, Japan). Differences were considered significant when the P value was less than 0.05. The average numbers of ACF, total ACs, and large ACF (≥4 ACs) per animal observed at weeks 6, 18, 25, and 32 are summarized in Table 2. These values at week 6 after one 2-week administration of PhIP in this study were essentially equivalent to those in our previous report using the same protocol.39Ochiai M Nakagama H Watanabe M Ishiguro Y Sugimura T Nagao M Efficient method for rapid induction of aberrant crypt foci in rats with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.Jpn J Cancer Res. 1996; 87: 1029-1033Crossref PubMed Scopus (26) Google Scholar A slight increase of these values was observed at weeks 18 and 25 after three cycles of PhIP administration, and the values at week 32 were similar to those at week 25. Regarding the site distribution, ACF were most frequent in the mid-colon as reported previously40Tsukamoto T Kozaki K Nishikawa Y Yamamoto M Fukami H Inoue M Wakabayashi K Tatematsu M Development and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci in the rat large intestine.Jpn J Cancer Res. 1999; 90: 720-725Crossref PubMed Scopus (23) Google Scholar and this did not change significantly during the experimental course (data not shown).Table 2Number of Aberrant Crypt Foci Induced by PhIPExp. periods (weeks)PhIP-exposure (weeks)No. of rats analyzedNo. of ACF*All the values for ACF presented in this table are expressed as means ± SD.No. of ACNo. of large ACF (≥4 ACs)Histological characterization of ACF†Of the total 144 ACF, 19 of 27, 27 of 37, 26 of 33, and 38 of 47 ACF at weeks 6, 18, 25, and 32, respectively, were subjected to the histological evaluation. The values listed in this column were based on the observation of these analyzed ACF, representing 70 to 80% of total ACF at respective time points, and therefore, simple summation of the numbers of dysplastic ACF and nondysplastic ones does not match the total number of ACF per rat presented in the fourth column from the left.Dysplastic ACFNondysplastic ACFNo.No. of ACF with β-catenin accumulationNo.No. of ACF with β-catenin accumulation62102.7 ± 1.56.3 ± 3.10.6 ± 0.70.4 ± 0.50.4 ± 0.51.5 ± 1.00186103.7 ± 2.211.7 ± 8.01.0 ± 0.90.8 ± 1.00.6 ± 0.71.9 ± 1.4025674.7 ± 2.014.6 ± 9.41.3 ± 1.41.4 ± 1.21.0 ± 0.82.3 ± 2.00.1 ± 0.4326104.7 ± 2.713.5 ± 11.41.3 ± 2.00.8 ± 1.00.7 ± 0.83.0 ± 1.40.1 ± 0.3* All the values for ACF presented in this table are expressed as means ± SD.† Of the total 144 ACF, 19 of 27, 27 of 37, 26 of 33, and 38 of 47 ACF at weeks 6, 18, 25, and 32, respectively, were subjected to the histological evaluation. The values listed in this column were based on the observation of these analyzed ACF, representing 70 to 80% of total ACF at respective time points, and therefore, simple summation of the numbers of dysplastic ACF and nondysplastic ones does not match the total number of ACF per rat presented in the fourth column from the left. Open table in a new tab Of 144 ACF induced in 37 rats, 110 ACF of different sizes were subjected to histological evaluation; namely 19, 27, 26, and 38 ACF at weeks 6, 18, 25, and 32, respectively (Table 2). Thirty ACF were diagnosed as dysplastic (Figure 2, B and C), and the remaining 80 as nondysplastic (Figure 2A). Dysplastic ACF were detected as early as 6 weeks and their numbers per rat increased slightly at later time points, those being 0.4, 0.8, 1.4, and 0.8 at weeks 6, 18