Development and Distribution of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]‐pyridine (PhIP)‐induced Aberrant Crypt Foci in the Rat Large Intestine

Abstract

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

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

“…39 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 previously 40 and this did not change significantly during the experimental course (data not shown). No.…”

Hexosaminidase‐altered Aberrant Crypts, Carrying Decreased Hexosaminidase α and β Subunit mRNAs, in Colon of 1,2‐Dimethylhydrazine‐treated Rats

“…Crypt isolation The distal half of the colonic epithelium was isolated and treated with 30 mM ethylenediaminetetraacetic acid (EDTA) (Dojin, Kumamoto) in Hanks' balanced salt solution. The crypts were isolated as described elsewhere, [20][21][22] and fixed either in 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 for 2 h for hexosaminidase staining or in 70% ethanol for molecular analysis. Hexosaminidase staining The procedure for hexosaminidase staining was as described by Hayashi 23) with slight modifications.…”

“…However, ACF have been simply defined as having an aberrant appearance when viewed on the mucosal surface. Thus, to establish criteria for selection of aber-rant crypts upon crypt isolation, [20][21][22] hexosaminidase staining was utilized to distinguish aberrant crypts from normal ones. For comparison of selected isolated aberrant crypts with 'normal' counterparts, molecular analysis of the expression of hexosaminidase α-and β-subunit mRNAs was performed with β-actin mRNA as a reference, with the real-time relative quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) utilizing a LightCycler system to measure the decreased mRNA levels.…”

More Frequent β‐Catenin Gene Mutations in Adenomas than in Aberrant Crypt Foci or Adenocarcinomas in the Large Intestines of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)‐treated Rats

“…14) ACF were isolated and DNAs were extracted with InstaGene Purification matrix (BIO-RAD, Hercules, CA). Colon tumors appearing at weeks 50 and 75 were cut into two pieces.…”

“…We recently analyzed the morphology and localization of ACF, adenomas and adenocarcinomas sequentially in PhIP-treated F344 rats and obtained a sufficient number of samples for statistical analysis. 14) PhIP is the most abundant heterocyclic amine in the human environment, 15) being produced in broiled meat and fish during cooking, 16) and is considered one of the most important carcinogens taken in the daily diet. We here utilized the same samples (46 ACF, 7 adenomas and 12 adenocarcinomas) to evaluate the importance of β-catenin mutations during the progression of ACF through to adenocarcinomas.…”