* Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan; Department of Pathology, Sasaki Institute, 2-2 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan;
Department of Oncological Pathology, Cancer Center, Nara Medical University, 840 Sizyo-cho, Kashihara-shi, Nara 634-8521, Japan;
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; ¶ Department of Pathology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kida-gun, Kagawa 761-0793, Japan; || Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan; ||| Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa, Nagoya 464-8681, Japan; and |||| Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Received December 3, 2003; accepted February 5, 2004
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ABSTRACT |
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Key Words: PhIP; risk assessment; carcinogenicity threshold; PhIP carcinogenicity.
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INTRODUCTION |
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There are many genotoxic carcinogens occurring naturally in our environment, including the large group of heterocyclic amine mutagens (Sugimura et al., 1995, 2000
). The human daily intake of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of these food-derived agents, is estimated to be 0.113.8 µg/person (Wakabayashi et al., 1993
). PhIP can be detected in the urine of healthy volunteers after eating cooked meat (Donald et al., 1995
; Ushiyama et al., 1991
; Wakabayashi et al., 1993
) and in rats, it causes DNA adduct formation in the colon (Fretland et al., 2001
; Kaderlik et al., 1994
) and treatment at high doses induces carcinomas in the colon, breast, and prostate (Hasegawa et al., 1993
; Ito et al., 1991
; Shirai et al., 1997
).
Recently in vivo medium-term bioassays for carcinogens have been accepted as possible alternatives to long term carcinogenicity tests (Ito et al., 1988) and appropriate for assessment of low dose effects because of their high sensitivity. Aberrant crypt foci (ACF) are established preneoplastic markers in the colon of rats (Bird, 1987
; Tudek et al., 1989
) and their ready detectability underlies their acceptance as end-point lesions to assess carcinogenic responses in medium-term bioassays. PhIP has been shown to induce ACF in a dose-related fashion over a range of doses (Nakagawa et al., 2002
; Tudek et al., 1989
). In the present study, for clarification of human risk assessment of genotoxic carcinogens, we examined low dose carcinogenicity of PhIP in the rat colon in detail using a medium-term bioassay, with the primary aim of determining whether the response curve is indeed linear near zero.
DNA adduct formation is considered to be an important factor in carcinogenesis with heterocyclic amines and PhIP-DNA adducts are formed in rat colon (Ochiai et al., 1996). Generation of oxygen free radicals is also a key step in carcinogenesis, again induced by various heterocyclic amines (Maeda et al., 1995
). 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is the most abundant species of adduct associated with oxidative stress, resulting in DNA damage and specific types of mutation (Kasai et al., 1987
). Therefore, levels of PhIP-DNA adducts and 8-OHdG were also examined in the present study to cast further light on mechanistic aspects of PhIP carcinogenicity at low doses in the colon.
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MATERIALS AND METHODS |
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Experimental procedures. The experiment was started when the animals were six weeks of age. They received PhIP at doses of 0 (group 1, a control), 0.001 (group 2), 0.01 (group 3), 0.1 (group 4), 1 (group 5), 10 (group 6), 50 (group 7), 100 (group 8), and 400 ppm (group 9) in powdered basal diet (Oriental MF, Oriental Yeast Co., Tokyo, Japan) for 16 weeks, continuously. The lowest level, 0.001 ppm of PhIP was established as equivalent to the daily intake of this carcinogen in humans (Wakabayashi et al., 1993). Numbers of rats were 240 in group 1, 242 in group 2, 241 in group 3, 243 in group 4, 244 in group 5, 212 in group 6, 214 in group 7, 62 in group 8, and 61 in group 9. The rats were killed at the end of week 16 under ether anesthesia for examination of ACF (61 to 244 rats) in the colon. Additional rats in groups 1 to 9 were given diets containing PhIP and killed at week 4 for examination of PhIP-DNA adducts (three or four rats) and 8-OHdG (five rats each) in the colon. The animals were carefully observed during the course of the experiment, body weights, water intake, and food consumption were measured every week. Calculations to achieve precise mole-per-rat of total PhIP ingested in every initiated group was estimated.
ACF counts. Colons were quickly excised, flushed with saline, and inflated by intraluminal injection of 10% phosphate-buffered formalin solution, slit open along the longitudinal median axis from the cecum to anus, and fixed flat between two pieces of filter paper in 10% phosphate-buffered formalin. After fixation for at least 24 h at 4°C, the colons were all stained with 0.2% methylene blue (in H2O) for 35 min, and then examined for ACF by light microscopy at 40x and 100x magnification using the following criteria for identification: (1) increased size as compared to normal crypts, (2) enlarged pericryptal zone, (3) slight elevation above the surrounding mucosa, and (4) frequently more elongated shape of the luminal opening.
PhIP-DNA adducts and 8-OHdG formation. The colons were excised and flushed in saline, and the mucosa was scraped off to obtain samples, which were frozen in liquid nitrogen and stored at 80°C until the levels of PhIP-DNA adducts in the colon were measured by the 32p-postlabeling method as described previously (Uehara et al., 1996). Measurement of 8-OHdG levels in colon DNA was performed with the method of Nakae et al. (1997)
.
Statistical analyses. Statistical analysis of our data was performed using the StatView-J 5.0 program (Abacus Concepts, Inc., Berkeley, CA). Differences from the control values were evaluated for significance by the Dunnet-test.
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RESULTS |
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DISCUSSION |
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Recently we found that the hepatocarcinogens, MeIQx and DEN, do not induce the preneoplastic lesions, glutathione-S-tranceferase placented form (GST-P) positive foci, in rat liver at very low doses (Fukushima et al., 2002, 2003
). Morever, MeIQx-DNA adducts and particularly 8-OHdG levels demonstrated no-observed effect levels. Our findings thus indicated the existence of a threshold for carcinogenicity with genotoxic agents.
The present results for ACF, DNA-adducts, and 8-OHdG in the colons of rats treated with PhIP at various doses point to the same conclusion (Fig. 4). On the other hand, it is noteworthy to mention that the no-response level for adduct formation (below 0.01 ppm; about 21 adducts/cell), and for induction of ACF (below 50 ppm; about 60 adducts/cell), supports the notion that rather large threshold number of adducts must be exceeded in order to induce formation of ACF. i.e., here, the dose required to initiate ACF is approximately 5000 times higher than that for adduct formation. Maeda et al. (1995) reported that carcinogenic heterocyclic amines generate oxyradicals at differing levels, MeIQx giving the highest values as judged by electron-spin resonance (ESR) spin trapping and PhIP being much less active in this regard. Two different metabolic pathways have been indicated for activation of PhIP. One is the hepatic pathway, involving N-hydroxylation by CYP1A2 and O-acetylation by N-acetyltransferase-2 and the other is extrahepatic, rendering free-radical metabolites. On ESR examination, generation of free radicals was greater with 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) than PhIP although DNA analysis showed adduct formation to be similar with the two carcinogens (Moonen et al., 2002
). From this evidence, participation of 8-OHdG to colon carcinogenesis due to PhIP may not be of direct importance, although this parameter also demonstrated a threshold in the present study.
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In conclusion, a threshold may exist for the colon carcinogenic potential of PhIP, and by analogy, probably also for other colon-genotoxic carcinogens. Recently Waddell (2003) stressed the existence of a threshold for DEN carcinogenicity in the lever and esophagus of rodent in his review article. Williams et al. (2000)
postulated that mechanisms differ between low and high exposures, and reflect thresholds for hepatocellular initiating effects by low dose genotoxic carcinogens. Previously we provided evidence that genotoxic hepatocarcinogens may exhibit a threshold (Fukushima et al., 2002
). The present findings provide a new basis for extrapolation from animal carcinogenicity data to human risk assessment.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: +81(6)6646 3093. E-mail: fukuchan{at}med.osaka-cu.ac.jp.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Donald, S. D., Nigel, J. G., Stephen, M., Anthony, L., Rafael, D. T., Jorge, S., and Alan, R. B. (1995). Systemic exposure to dietary heterocyclic amines in man. In Heterocyclic Amines in Cooked Foods: Possible Human Carcinogens (R. H. Adamson, J.-Å. Gustafsson, N. Ito, M. Nagao, T. Sugimura, K. Wakabayashi, and Y. Yamazoe, Eds.), pp. 190196. Princeton Scientific Publishing, Princeton, NJ.
Fretland, A. J., Devanaboyina, U. S., Nangju, N. A., Xiao, G. H., Webb, S. J., Doll, M. A., and Hein, D. W. (2001). DNA adduct levels and absence of tumors in female rapid and slow acetylator congenic hamsters administered the rat mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. J. Biochem. Mol. Toxicol. 15, 2633.[CrossRef][ISI][Medline]
Fukushima, S., Wanibuchi, H., Morimura, K., Wei, M., Nakae, D., Konishi, Y., Tsuda, H., Uehara, N., Imaida, K., Shirai, T., Tatematsu, M., Tsukamoto, T., Hirose, M., Furukawa, F., Wakabayashi, K., and Totsuka, Y. (2002). Lack of a dose-response relationship for carcinogenicity in the rat liver with low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-nitrosodiethylamine. Jpn. J. Cancer Res. 93, 10761082.[ISI][Medline]
Fukushima, S., Wanibuchi, H., Morimura, K., Wei, M., Nakae, D., Konishi, Y., Tsuda, H., Takasuka, N., Imaida, K., Shirai, T., Tatemetsu, M., Tsukamoto, T., Hirose, M., and Furukawa, F. (2003). Lack of initiation activity in rat liver of low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Cancer Lett. 1911, 3540.
Gaylor, D. W. (1979). Summary and conclusions. J. Environ. Pathol. Toxicol. 3, 179183.[ISI]
Hasegawa, R., Sano, M., Tamano, S., Imaida, K., Shirai, T., Nagao, M., Sugimura, T., and Ito, N. (1993). Dose-dependence of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) carcinogenicity in rats. Carcinogenesis 14, 25532557.[Abstract]
Ito, N., Tsuda, H., Tatematsu, M., Inoue, T., Tagawa, Y., Aoki, T., Uwagawa, S., Kagawa, M., Ogiso, T., Masui, T., Imaida, K., Fukushima, S., and Asamoto, M. (1988). Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in ratan approach for a new medium-term bioassay system. Carcinogenesis 9, 387394.[Abstract]
Ito, N., Hasegawa, R., Sano, M., Tamano, S., Esumi, H., Takayama, S., and Sugimura, T. (1991). A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Carcinogenesis 12, 15031506.[Abstract]
Kaderlik, K. R., Minchin, R. F., Mulder, G. J., Ilrtt, K. F., Daugaard-Jenson, M., Teitel, C. H., and Kadlubar, F. F. (1994). Metabolic activation pathway for the formation of DNA adducts of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat extrahepatic tissues. Carcinogenesis 15, 17031709.[Abstract]
Kasai, H., Nishimura, S., Kurokawa, Y., and Hayashi, Y. (1987). Oral administration of a renal carcinogen, potassium bromate, specifically produces 8-hydroxydeoxyguanosine in rat target organ DNA. Carcinogenesis 8, 19591961.[Abstract]
Kitchin, K. T., and Brown, J. L. (1994). Dose-response relationship for rat liver DNA damage caused by 49 rodent carcinogens. Toxicology 88, 3149.[CrossRef][ISI][Medline]
Kleczkowska, H. E., and Althaus, F. R. (1996). Response of human keratinocytes to extremely low concertrations of N-methyl-N'-nitro-N-nitrosoguanidine. Mutat. Res. 367, 151159.[ISI][Medline]
Littlefield, N. A., Farmer, J. H., Gaylor, D. W., and Sheldon, W. G. (1979). Effects of dose and time in a long-term, low-dose carcinogenic study. J. Environ. Pathol. Toxicol. 3, 1734.[ISI]
Maeda, H., Sato, K., and Akaike, T. (1995). Superoxide radical generation from heterocyclic amines. In Heterocyclic Amines in Cooked Foods: Possible Human Carcinogens (R. H. Adamson, J.-Å Gustafsson, N. Ito, M. Nagao, T. Sugimura, K. Wakabayashi, and Y. Yamazoe, Eds.), pp. 103112. Princeton Scientific Publishing, Princeton, NJ.
Moonen, H. J., Briede, J. J., van Maanen, H. M., Kleinjans, J. C., and de Kok, T. M. (2002). Generation of free radicals and induction of DNA adducts by activation of heterocyclic aromatic amines via different metabolic pathways in vitro. Mol. Carcinog. 35, 196203.[CrossRef][ISI][Medline]
Nakae, D., Kobayashi, Y., Akai, H., Andoh, N., Satoh, H., Ohashi, K., Tsutsumi, M., and Konishi, Y. (1997). Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine. Cancer Res. 57, 12811287.[Abstract]
Nakagawa, H., Ochiai, M., Ubagai, T., Tajima, R., Fujiwara, K., Sugimura, T., and Nagao, M. (2002). A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP. Mutat Res. 506507, 137144.[ISI]
Ochiai, M., Watanabe, M., Kushida, H., Wakabayashi, K., Sugimura, T., and Nagao, M. (1996). DNA adduct formation, cell proliferation and aberrant crypt focus formation induced by PhIP in male and female rat colon with relevance to carcinogenesis. Carcinogenesis 17, 9598.[Abstract]
Olivieri, G., Bodycote, J., and Wolff, S. (1984). Adaptive response of human lymphocytes to low concentrations of radioactive thymidine. Science 223, 594597.[ISI][Medline]
Peto, R., Gray, R., Brantom, P., and Grasso, P. (1991). Effects on 4080 rats of chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine: A detailed dose-response study. Cancer Res. 51, 64156451.[Abstract]
Preussmann, R. (1980). The problem of thresholds in chemical carcinogenesissome views on theoretical and practical aspects. Cancer Res. Clin. Oncol. 97, 114.
Shirai, T., Sano, M., Tamano, S., Takahashi, S., Hirose, M., Futakuchi, M., Hasegawa, R., Imaida, K., Matsumoto, K., Wakabayashi, K., Sugimura, T., and Ito, N. (1997). The prostate: A target for carcinogenicity of 2-amino-1-methy-6-phenylimidazo[4,5-b]pyridine (PhIP)derived from cooked foods. Cancer Res. 57, 195198.[Abstract]
Sugimura, T. (1995). History, present and future, of heterocyclic amines, cooked food mutagens. In Heterocyclic Amines in Cooked Foods: Possible Human Carcinogens (R.H. Adamson, J.-Å. Gustafsson, N. Ito, M. Nagao, T. Sugimura, K. Wakabayashi, and Y. Yamazoe, Eds.), pp. 214231. Princeton Scientific Publishing, Princeton, NJ.
Sugimura, T. (2000). Nutrition and dietary carcinogens. Carcinogenesis 21, 387395.
Tomatis, L., Huff, J., Hertz-Picciotto, I., Sandler, D. P., Bucher, J., Boffetta, P., Axelson, O., Blair, A., Taylor, J., Stayner, L., and Barrett, J. C. (1997). Avoided and avoidable risk of cancer. Carcinogenesis 18, 97105.[Abstract]
Tudek, B., Bird, R. P., and Bruce, W. R. (1989). Foci of aberrant crypts in the colons of mice and rats exposed to carcinogens associated with foods. Cancer Res. 49, 12361240.[Abstract]
Uehara, N., Iwahori, Y., Asamoto, M., Baba-Toriyama, H., Iigo, M., Ochiai, M., Nagao, M., Nakayama, M., Degawa, M., Matsumoto, K., Hirono, I., Beppu, H., Fujita, K. and Tsuda H. (1996). Decreased levels of 2-amino-3 methylimidazo[4,5-f]quinoline-DNA adducts in rats treated with ß-carotene, -tocopherol and freeze-dried aloe. Jpn. J. Cancer Res. 87, 342348.[ISI][Medline]
Ushiyama, H., Wakabayashi, K., Hirose, M., Itoh, H., Sugimura, T., and Nagao, M. (1991). Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal diet, but not in patients receiving parenteral alimentation. Carcinogenesis 12, 14171422.[Abstract]
Waddell, W. J. (2003). Thresholds in chemical carcinogenesis: What are animal experiments telling us? Toxicol. Pathol. 31, 260262.[CrossRef][ISI][Medline]
Wakabayashi, K., Ushizima, H., Takahashi, M., Nukaya, H., Kim, S. B., Hirose, M., Ochiai, M., Sugimura, T., and Nagao, M. (1993). Exposure to heterocyclic amines. Environ. Health Perspect. 99, 129134.[ISI][Medline]
Williams, G. M., Iatropoulos, M. J., Wang, C. X., Jeffrey, A. M., Thompson, X., Pittman, B., Palasch, M., and Gebhardt, R. (1998). Nonlinearities in 2-acetylaminofluorene exposure responses for genotoxic and epigenetic effects leading to initiation of carcinogenesis in rat liver. Toxicol. Sci. 45, 152161.[Abstract]
Williams, G. M., Iatropoulos, M. J., and Jeffrey A. M. (2000). Mechanistic basis for nonlinearities and thresholds in rat liver carcinogenesis by the DNA-reactive carcinogens 2-acetylaminofluorene and diethylnitrosamine. Toxicol. Pathol. 28(3), 388395.[ISI][Medline]
Wollff, S. (1998). The adaptive response in radiobiology: Evolving insights and implications. Environ. Health Perspect. 106(Suppl. 1), 277283.[ISI][Medline]
Yoshino, H., Ichihara, T., Tamano, S., Tsutsumi, T., Wanibuchi, H., Morimura, K. and Hagiwara, A. (2002). Estimation of a practical threshold level for 2-acetylaminofluene, a genotoxic liver carcinogen, in a 16-week feeding study with F344 rats. Proceedings of 61st Annual Meeting of the Japanese Cancer Association 204, 2658.