Division of Pathology, National Institute of Health Sciences, 1181 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
1 To whom correspondence should be addressed. Fax: +81-337001425. E-mail: nishikaw{at}nihs.go.jp.
Received November 19, 2004; accepted January 2, 2005
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ABSTRACT |
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Key Words: MeIQx; heterocyclic amime; colon tumor; dextran sulfate sodium; mouse.
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INTRODUCTION |
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We have demonstrated that MeIQx is genotoxic in the colon as well as the liver in lacI transgenic C57BL/6N (Itoh et al., 2000) or gpt delta (Masumura et al., 2003
) C57BL/6J mice fed at a dose of 300 ppm. It was also documented that MeIQx induces aberrant crypt foci (ACFs), an intermediate biomarker for colon tumorigenicity, in rats (Kasahara et al., 1997
; Tanakamaru et al., 2001
) and mice (Okonogi et al., 1997
). Therefore, in the present study, in order to elucidate whether MeIQx can induce colon tumors in such transgenic mice, a 78-week feeding study of MeIQx was performed in gpt delta mice, and in order to neglect any possible involvement of the reporter genes to tumorigenicity, wild-type C57BL/6J mice were also fed MeIQx for 78 weeks. Then, the colon tumorigenicity of dietary MeIQx, IQ, or PhIP was investigated in C57BL/6J mice under a colon tumor-promotional condition due to dextran sulfate sodium (DSS), in order to confirm whether the positive genotoxicity of MeIQx found in lacI transgenic or gpt delta mice is sufficient to initiate colon tumors.
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MATERIALS AND METHODS |
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78-Week bioassay for tumorigenicity of dietary MeIQx in C57BL/6J mice.
Because MeIQx showed a genotoxicity in the colon in gpt delta C57BL/6J or lacI transgenic C57BL/6N mice at a dose of 300 ppm (Itoh et al., 2000; Masumura et al., 2003
), three groups of 5-week-old male C57BL/6J mice or gpt delta mice, each consisting of 20 animals, were fed 300 ppm or 30 ppm of MeIQx for 78 weeks. After 78 weeks, all survived animals were fully autopsied under anesthesia, and histological examination was performed for detecting neoplastic lesions.
Bioassay for tumorigenicity of MeIQx, IQ, or PhIP followed by DSS.
Based on the results of a preliminary study, the dose of DSS was determined to be 1.5% (data not shown). As shown in Fig. 1, one hundred and twenty 6-week-old male mice were randomized by weight into five groups. Group 1 mice, consisting of 15 animals, were fed MeIQx at a dose of 300 ppm for 12 weeks. Group 2 mice, consisting of 15 animals, received two cycles of 1-week 1.5% DSS treatment. Group 35 mice, each consisting of 30 animals, were fed MeIQx, IQ, and PhIP for 12 weeks, respectively, and then treated with two cycles of 1.5% DSS treatment, 2 weeks apart. In our previous studies, treatments with single gavage or short-term (4 weeks) feeding of MeIQx failed to induce genotoxicity in the colon as well as liver of reporter gene transgenic mice (Itoh et al., 2000; Nishikawa et al., 2001
), but the 12-week feedings significantly induced the genotoxicity (Itoh et al., 2000
; Masumura et al., 2003
), suggesting that it takes somewhat longer time for MeIQx to fix and accumulate the mutations. Therefore, in the present experiments MeIQx was fed for 12 weeks prior to DSS treatment. Twenty weeks after starting the experiment, all animals were sacrificed under anesthesia. The colons were cut longitudinally and examined for the presence of ACFs as reported previously (Tanaka et al., 2003
), and then all the macroscopical lesions were histopathologically determined. BrdU-labeling index on the nonlesional epithelia in Groups 1 and 4 was immunohistochemically determined according to the method described previously (Toyoda et al., 1994
).
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RESULTS |
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DISCUSSION |
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Recent epidemiologic studies have suggested that high red meat diets probably increase the risk of colorectal cancer (Butler et al., 2003; Norat and Riboli, 2001
; Sandhu et al., 2001
). Red meat, especially when cooked well done, may be a source of exposure to chemical carcinogens such as HCAs, polycyclic aromatic hydrocarbons, and other pyrolysis products (Butler et al., 2003
; Sugimura, 1985
). A number of HCAs have been identified in cooked meat and fish at levels that vary according to cooking methods, temperature and duration, and type of meat (Layton et al., 1995
; Skogg et al., 1998
), and PhIP and MeIQx are the most significant HCAs in terms of human exposure and carcinogenic potency (Layton et al., 1995
). A dose-dependent association was found between the HCA intake estimates and male rectal cancer, and this association was the strongest for MeIQx (Marchand et al., 2002
).
These HCAs have different organ specificity, although they have similar chemical structures and common metabolic pathways. MeIQx, alike other HCAs including MeIQ, IQ, and PhIP, is activated to genotoxic and carcinogenic intermediates through N-oxidations catalyzed by P450 1A2 in the liver (Rich et al., 1992; Shimada et al., 1989
; Turesky et al., 1998
) and further activated by N-acetyltransferases or sulfotransferases (Chou et al., 1995
; Minchin et al., 1992
; Turesky et al., 1991
). N2-(deoxyguanosin-8-yl)-MeIQx is a major DNA adduct detected in the liver and the other tissues of animals treated with MeIQx (Ochiai et al., 1993
; Schut and Snyderwine, 1999
; Snyderwine et al., 1993
). Although the level of MeIQx in cooked foods is the second highest following PhIP in this group of compounds, the level of human exposure is estimated to be 107- to 108-fold lower than the doses for the rodent bioassays (Kobayashi et al., 2002
; Layton et al., 1995
; Ushiyama et al., 1991
). It is important that the level of MeIQx-DNA adducts is greater in the human colon than in the colon of the rat or mouse strains, while the colon bioavailability of MeIQx and the types of DNA adducts appear similar between human and rodents (Mauthe et al., 1999
).
In comparison of three HCAs tested in the present study, the colon tumor-inducibility was the greatest with PhIP, followed by IQ and MeIQx, although the latter two were almost comparable. It has been reported that in vitro potencies of bacterial genotoxicity and micronucleus induction are in the order MeIQx > IQ > PhIP (Pfau et al., 1999). It has also been shown that in vivo potencies of genotoxicity in the colonic mucosa of mice by alkaline single cell gel electrophoresis (Comet) assay are almost comparable among the three HCAs (Sasaki et al., 1998
). Although the reasons for discrepancy between these previous data and the results in the present study remain unknown, one of the possibilities may be the differences in the tumor-promoting activity of the HCAs.
DSS is known to cause colitis in experimental animals, and DSS-induced colitis has been used as a model for ulcerative colitis in human (Okayasu et al., 1990). Although DSS itself is not genotoxic (Mori et al., 1984
), it has been shown that DSS enhances colon tumorigenesis and even induces colon tumors in rodents after long-term feeding, suggesting that DSS is a nongenotoxic colon carcinogen or a colon tumor-promoter (Hirono et al., 1981
; 1983
). The data for BrdU-labeling index in our preliminary study clearly indicate that DSS could be a strong colon tumor-promoter, although other factors such as inflammatory cytokines remain unknown. In the present study, under the condition that the DSS alone treatment did not induce any colon tumors, the HCAs MeIQx, IQ, and PhIP induced colon tumors. The fact that MeIQx induces colon tumors is very important, because cross associations between intake of MeIQx and risk of colon cancer have been reported in epidemiological studies (Marchand et al., 2002
; Sinha, 2002
). In this context, colon tumor-promotional conditions such as ulcerative colitis could be important to achieve the colon tumorigenicity of MeIQx.
Recently, we reported that MeIQx induces significant genotoxicity in the colon as well as the liver of gpt delta mice when given for 12 weeks (Masumura et al., 2003), but in the present study MeIQx failed to induce any tumors in both gpt delta and wild C57BL/6J mice even after feeding for 78 weeks. Although the transgenic reporter genes are known not to be transcribed (Ono et al., 1995
), wild mice were also tested for carcinogenicity in order to confirm that these genes would not influence genotoxicity or carcinogenicity. The results in the present study suggested that these animals carrying the transgene lambda EG10 should be biologically normal. In conclusion, our results clearly indicate that MeIQx, as well as IQ, can induce colon tumors in mice after sufficient tumor-promotion. This fact is in good agreement with epidemiological data and suggests that tumor-promoting factors are extremely important in colon carcinogenesis as well as tumor-initiating factors including HCAs. Further studies are needed to elucidate or neglect the possibilities that specific chemicals may promote spontaneously initiated cells. Not every tumor-promoter may promote all initiated cells the same way (Lee, 2000
), or transcriptional alterations may be involved in the targeted genes (Thilly, 2003
; Trosko, 1997
).
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ACKNOWLEDGMENTS |
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