Lack of large intestinal carcinogenicity of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine at low doses in rats initiated with azoxymethane

Abstract

2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), an abundant food-derived heterocyclic amine (HCA), has attracted particular attention as a human colon carcinogen. Humans are in fact exposed to continuous low doses of HCAs during lifetime. Therefore, we focused on rat large intestinal carcinogenicity of PhIP at levels that mimic practical human exposure. A total of 192 6-weekold male F344 rats were subcutaneously injected twice with 15 mg/ kg body weight azoxymethane (AOM), then continuously fed various doses (0, 0.001, 0.01, 0.1, 1, 10, 50 and 200 ppm) of PhIP in the diet. At week 16, aberrant crypt foci (ACF) were quantitatively analyzed. At week 36, tumor occurrence was pathologically analyzed. Then immunohistochemical examinations were performed. PhIP was found to enhance strongly AOM-initiated rat large intestinal tumorigenesis at high doses (50 and 200 ppm), while lower doses (0.001-10 ppm) had no apparent effects. High doses also caused variation in tumor histologic types and their distribution throughout the large intestinal segments. Frequencies of ACF/cm 2 did not meaningfully vary between the groups. Cellular proliferation activity in normal-appearing colonic mucosa was significantly increased at high doses. These novel findings may provide evidence of a low-dose potential for PhIP, with a no-observed effect level speculated to be 10 ppm in the present initiation-promotion experimental model. ' 2005 Wiley-Liss, Inc.Key words: rat; large intestine; carcinogenesis; PhIP; azoxymethane; no-observed effect level Carcinogenesis is widely accepted to be a multistep process caused by a series of genetic and/or subsequent epigenetic alterations that are indispensable to the different stages from initiation to promotion and progression of cancer development. 1,2 Humans are consistently exposed to very many naturally occurring or synthetic chemical carcinogens at quite low-dose levels in the environment, 3 although concomitantly they may consume mixtures of naturally occurring anticarcinogenic compounds in their daily diets. 4 One focus of attention has been food-derived heterocyclic amines (HCAs) generated in cooking processes that have proved to be carcinogenic in rodent species. 5,6 HCAs are highly mutagenic 6 and genotoxic carcinogens that are generally thought to have no threshold in exerting their carcinogenic potential, which was postulated to exhibit dose dependence in vivo. 3 However, in practice, low-dose administration has not necessarily led to cancer development in the rat liver. 7 Thereby, in our laboratory, the nonthreshold theory for hepatocarcinogenesis of one HCA, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), has been challenged, since in vivo dose-response curves do not show linearity at low-dose levels. 8,9 Moreover, thresholds for chemical carcinogenesis were concluded in a recent article. 10 In practice, the many factors active in vivo, from intake of carcinogens through to lesion development, mean that the processes are highly complicated, so that if the DNA-damaging pote...

Chemopreventive Action by Ethanol-extracted Brazilian Green Propolis on Post-initiation Phase of Inflammation-associated Rat Colon Tumorigenesis

“…The liver and kidneys were weighed and fixed in 10% phosphate-buffered formalin solution and 4-μm-thick sections were routinely prepared for hematoxylin and eosin (H&E) staining. The colons were prepared as previously described (33). In brief, normal saline (0.9%) was injected into the lumen of the colons and kept for a few minutes for trimming of external connective and adipose tissues.…”

Altered Gene Expression in Rat Colonic Adenocarcinomas Induced in an Azoxymethane plus 2-Amino-1-Methyl-6-Phenylimidazo[4,5-<i>b</i>]- Pyridine Initiation-Promotion Model

“…All tissue samples used were retrieved from our previous study samples with pathological confirmation of the histological types of lesions [18]. Briefly, 6-week-old, male F344 rats (Charles River Japan, Atsugi, Japan) were initially injected with AOM (Sigma Aldrich, St. Louis, Mo., USA) at a dose of 15 mg/kg body weight s.c., once a week for 2 weeks, and subsequently administered 0, 50 and 200 ppm of PhIP (Nard Institute, Osaka, Japan) in their MF basal diet (Oriental Yeast, Tokyo, Japan) for 34 weeks.…”

“…However, the yield of tumors is still low, and high-dose PhIP (50 and 200 ppm in diet) administered in the post-initiation phase after AOM initiation significantly increased colon tumor incidence (100%) and multiplicity (5.25/rat for 50 ppm and 13.79/rat for 200 ppm, respectively). Moreover, high doses significantly increased cell-proliferative activity, such as BrdU labeling or PCNA-positive indices in the rat colonic mucosae [18]. Based on these experimental findings, we hypothesized that high-dose PhIP administered in the post-initiation phase may affect preneoplastic cells and lesions mainly during late carcinogenic phases, because genotoxic/carcinogenic effects of PhIP alone are relatively limited even at high doses.…”

“…1 Description of PhIP-enhanced colon carcinogenesis initiated with AOM in rats. Our concept is based on our previous and other experimental data [8, 9, 17, 18]: when high-dose PhIP alone is chronically administered to rats ( a ), the yields of colon preneoplastic lesions (such as ACF) and neoplasms are relatively low; if AOM alone is injected to rats during the initial few weeks ( b ), much higher numbers of preneoplastic lesions (such as ACF) are noted, but tumor yields remain relatively low similar to the high-dose PhIP alone case; if high-dose PhIP is continuously administered to rats after initial AOM exposure ( c ), the preneoplastic lesion yield is almost equal to that in the AOM-alone group, but final tumor incidence and multiplicity can reach extremely high levels, and this may be linked to alterations in oncogenes and related mechanisms. …”

“…In our laboratory, different rat models of colon carcinogenesis have been employed, and PhIP-dose-dependent variation in the yield of aberrant crypt foci (ACF) and colon tumors has been well documented [17, 18]. ACF have been accepted as putative precursor lesions of colorectal neoplasms, both in humans and rodents, and have been frequently used in colon carcinogenesis models [19].…”