Medium-Term Bioassay Models for Environmental Carcinogens — Two-Step Liver and Multi-Organ Carcinogenesis Protocols

  • Nobuyuki Ito
  • Hiroyuki Tsuda
  • Ryohei Hasegawa
  • Masae Tatematsu
  • Katsumi Imaida
  • Makoto Asamoto


Epidemiological studies have made it increasingly clear that the variation in individual cancer rates in different regions of the world is a direct reflection of the presence or absence of exogenous, causal factors (Wynder and Gori, 1977; Doll, 1978). The investigation of possible nutritional and other environmentally derived influences in animal experiments has further established that a vast array of compounds are capable of playing a role in tumorigenesis, and it is now recognized that detection and appropriate regulation of these compounds are of prime importance for the management of neoplasia in man. Moreover, since it is now evident that neoplastic development is a multistep process, this question is complicated by the problem of different stages at which exogenous factors could interact (Foulds, 1969; Berenblum, 1974; Wynder, 1983). Until recently 2-year long-term in vivo testing using rats, mice or hamsters has been considered to be the most reliable method for the prediction of carcinogenic potential in man (NCI, 1976; IARC, 1980). However, to be internationally accepted these long-term tests must satisfy costly regulatory guidelines for appropriate facilities, long duration (2 years), maintenance of animals, sufficiently large numbers of rodents and careful histopathological examination. Therefore the vast number of compounds which have been introduced into our environment in recent years is far beyond our capacity to assess using such comprehensive carcinogenicity tests in each case.


Basal Diet Partial Hepatectomy Preneoplastic Lesion Environmental Carcinogen Hyperplastic Nodule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ames, B. N., McCann, J., and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutat. Res., 32: 347.Google Scholar
  2. Bannasch, P., Mayer, D., and Hacker, H. J., 1980, Hepatocellular glycogenesis and hepatocarcinogenesis, Biochim. Acta., 605: 217.Google Scholar
  3. Bannasch, P., Moore, M. A., Klimek, F., and Zerban, H., 1982, Biological markers of preneoplastic foci and neoplastic nodules in rodent liver, Toxicol. Pathol., 10: 19.CrossRefGoogle Scholar
  4. Bannasch, P., 1986, Preneoplastic lesions as end points in carcinogenicity testing. I. Hepatic neoplasia, Carcinogenesis, 7: 689.PubMedCrossRefGoogle Scholar
  5. Berenblum, I., 1974, “Carcinogenesis as a Biological Problem,” North-Holland Publ., Amsterdam.Google Scholar
  6. Butler, W. H., Hempsall, V., and Stewart, M. G., 1981, Histochemical studies on the early proliferating lesions induced in the rat liver by aflatoxin, J. Pathol., 133: 325.PubMedCrossRefGoogle Scholar
  7. Campbell, H. A., Pitot, H. C., Potter, V. R., and Laishes, B. A., 1982, Application of quantitative stereology to the evaluation of enzyme-altered foci in rat liver, Cancer Res., 42: 465.PubMedGoogle Scholar
  8. Cook, M. G., and McNamara, P., 1980, Effect of dietary vitamin E on dimethylhydrazine-induced colonic tumors in mice, Cancer Res., 40: 1329.PubMedGoogle Scholar
  9. Craddock, V. M., and Frei, J. V., 1974, Induction of liver cell adenoma in the rat by a single treatment with N-methyl-N-nitrosourea given at various times after partial hepatectomy, Br. J. Cancer, 30:503.Google Scholar
  10. Daikuhara, Y., Tamada, F., Takigawa, M., Takeda, Y., and Mori, Y., 1979, Changes in polyamine metabolism of rat liver after administration of D-galactosamine, Gastroloenterology, 77: 123.Google Scholar
  11. Decker, K., and Keppler, D., 1972, Galactosamine induced liver injury, in: “Progress in Liver Disease,” H. Popper, and S. Fenton, ed., Grune & Stratton, New York and London.Google Scholar
  12. Doll, R., 1978, Strategy for detection of cancer hazards to man, Nature, 265: 589.CrossRefGoogle Scholar
  13. Enzmann, M., Edler, L., and Bannasch, P., 1987, Simple elementary method for the quantification of focal liver lesions induced by carcinogens, Carcinogenesis, 8: 231.PubMedCrossRefGoogle Scholar
  14. Farber, E., Parker, S., and Gruenstein, M., 1976, The resistance of putative premalignant liver cell populations, hyperplastic nodules, to the acute cytotoxic effects of some hepatocarcinogens, Cancer Res., 36: 3879.PubMedGoogle Scholar
  15. Farber, E., 1980, The sequential analysis of liver cancer induction, Biochim. Biophys. Acta, 605: 149.PubMedGoogle Scholar
  16. Foulds, L., 1969, “Neoplastic Development,” 1, Academic Press, New York.Google Scholar
  17. Foulds, L., 1975, “Neoplastic Development,” 2, Academic Press, New York.Google Scholar
  18. Fullman, R.L., 1953, Measurement of particle sizes in opaque bodies, Trans. AIME, 197: 447.Google Scholar
  19. Goldsworthy, T. L., Hanigan, M., and Pitot, H. C., 1986, Models of hepatocarcinogenesis in the rat - contrast and comparisons, CRC Crit Rev. Toxicol., 17: 61.CrossRefGoogle Scholar
  20. Hacker, H.-J., Moore, M. A., Mayer, D., and Bannasch, P., 1982, Correlative histochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver, Carcinogenesis, 3: 1265.PubMedCrossRefGoogle Scholar
  21. Hasegawa, R., Tatematsu, M., Tsuda, H., Shirai, T., Hagiwara, A., and Ito, N., 1982, Induction of hyperplastic liver nodules in hepatectomized rats treated with 3’-methyl-4dimethylaminoazobenzene, benzo(a)pyrene or phenobarbital before or after exposure to N-2-fluorenylacetamide, Gann, 73: 264.PubMedGoogle Scholar
  22. Hasegawa, R., Tsuda, H., Shirai, T., Kurata, Y., Masuda, A., and Ito, N., 1986, Effect of timing of partial hepatectomy on the induction of preneoplastic liver foci in rats given hepatocarcinogens, Cancer Lett., 32: 15.PubMedCrossRefGoogle Scholar
  23. Haworth, S., Lawlor, T., Morlelmans, K., Spesk, W., and Zeiger, E., 1983, Salmonella mutagenicity test results for 250 chemicals, Environ. Mutagen., Suppl. 1: 3.Google Scholar
  24. Hiasa, Y., Kitahori, Y., Konishi, N., Shimoyama, T., and Lin, J.-C., 1985, Sex differential and dose dependence of phenobarbital promoting activity in N-bis(2-hydroxypropyl)nitrosamine-initated thyroid tumorigenesis in rats, Cancer Res., 45: 4087.PubMedGoogle Scholar
  25. Hsu, S. M., Raine, L., and Fanger, H., 1981, Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29: 577.PubMedCrossRefGoogle Scholar
  26. IARC, 1974, IARC Monograph on the Evaluation of Carcinogenic Risk of Chemicals to Man, vol. 17, Lyon, France.Google Scholar
  27. IARC, 1980, Long-term and Short-term Screening Assays for Carcinogens. A Critical Appraisal, IARC Monographs, Suppl. 2 IARC Scientific Publications, Lyon.Google Scholar
  28. IARC, 1982, IARC Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 4, IARC Lyon.Google Scholar
  29. IARC, 1986, Long-term and Short-term Assays for Carcinogens: A Critical Appraisal, IARC Scientific Publications, No. 83, Lyon.Google Scholar
  30. ICPMC Publication No. 9, 1984, Report of ICPMC task group 5 on the differentiation between genotoxic and non-genotoxic carcinogens, Mutat. Res., 133: 1.Google Scholar
  31. Imaida, K., Fukushima, S., Shirai, T., Ohtani, M., Nakanishi, K., and Ito, N., 1983, Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of y-glutamyltranspeptidase-positive foci development in the liver of rats, Carcinogenesis, 4: 895.PubMedCrossRefGoogle Scholar
  32. Imaida, K., Fukushima, S., Shirai, T., Masui, T., Ogiso, T., and Ito, N., 1984, Promoting activities of butylated hydroxyanisole, butylated hydroxytoluene and sodium L-ascorbate on forestomach and urinary bladder carcinogenesis initiated with methylnitrosourea in F344 male rats,. Gann, 75: 769.PubMedGoogle Scholar
  33. Institute of Laboratory Animal Resources, 1980, Histologic typing of liver tumors of the rat, J. Natl. Cancer Inst., 64: 179.Google Scholar
  34. Ishidate, M., Jr, Sofuni, T., and Yoshikawa, K., 1981, Chromosomal aberration test in vitro as a primary screening tool for environmental mutagens and/or carcinogens, in: “Mutation, Promotion and Transformation in vitro, Gann Monograph,” N. Inui, T. Kuroki, M. A. Yamada, and C. Heidelberger, eds., Japanese Scientific Society Press, Tokyo.Google Scholar
  35. Ito, N., Tatematsu, M., Nakanishi, K., Hasegawa, R., Takano, T., Imaida, K., and Ogiso, T., 1980, The effects of various chemicals on the development of a hyperplastic liver nodules in hepatectomized rats treated with N-nitrosodiethylamine or N-2-fluorenylacetamide, Gann, 71: 832.PubMedGoogle Scholar
  36. Ito, N., Tsuda, H., Hasegawa, R., and Imaida, K., 1982, Sequential observation of pathomorphologic alterations in preneoplastic lesions during the promoting stage for hepatocarcinogenesis and the development of short-term test system for hepatopromoters and hepatocarcinogens, Toxicol. Pathol., 10: 37.CrossRefGoogle Scholar
  37. Ito, N., Tsuda, H., Tatemastu, M., Inoue, T., Tagawa, Y., Aoki, T., Uwagawa, S., Ogiso, T., Masui, T., Imaida, K., Fukushima, S., and Asamoto, M., 1988, Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase-P form positive foci in rats - An approach for a new medium-term bioassay system, Carcinogenesis, 9: 387.PubMedCrossRefGoogle Scholar
  38. Ito, N., Imaida, K., de Camargo, J.L.V., Takahashi, S., Asamoto, M., and Tsuda, H., 1988, A new medium-term bioassay system for detection of environmental carcinogens using diethylnitrosamine-initiated rat liver followed by D-galactosamine treatment and partial hepatectomy, Jpn. J. Cancer Res. (Gann), 79: 573.Google Scholar
  39. Keppler, D., Lesch, R., Reutter, W., and Decker, K., 1968, Experimental hepatitis induced by D-galactosamine, Exp. Mol. Pathol., 9: 279.PubMedCrossRefGoogle Scholar
  40. Kier, L. W., Brusick, D. J., Auletta, A. E., Von Halle, E. S., Brown, M. M., Simmon, V. F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T. K., and Ray, V., 1986, The salmonella typhimurium/mammalian microsomal assay. A report on the U. S. Environmental Protection Agency Gene-Tox Program, Mutat. Res., 168:69..Google Scholar
  41. Kitagawa, T., 1971, Histochemical analysis of hyperplastic lesions and hepatomas of the liver of rats fed 2-fluorenylacetamide, Gann, 62: 207.PubMedGoogle Scholar
  42. Kluwe, W. M., Haseman, J. K., Douglas, J. F., and Huff, J. E., 1982, The carcinogenicity of dietary di(2-ethylhexyl)phthalate (DEHP) in Fischer 344 rats and B6C3Fi mice, J. Toxicol. Environ. Health., 10: 797.PubMedCrossRefGoogle Scholar
  43. Lamb, J. C., Huff, J. E., Haseman, J. K., Murth, A. S. K., and Lilga, H., 1986, Carcinogenesis studies of 4,4’-methylenedianiline dihydrochloride given in drinking water to F344/N rats and B6C3F1 mice, J. Toxicolg. Environ. Health, 18: 325.CrossRefGoogle Scholar
  44. Moore, M. A., Mayer, D., and Bannasch, P., 1982, Sequential appearance of putative preneoplastic populations induced in the rat liver by stop experiments with N-nitrosomorpholine, Carcinogenesis, 3: 1429.PubMedCrossRefGoogle Scholar
  45. Moore, M. A., Hacker, H.-J., Kunz, H. W., and Bannasch, P., 1983, Enhancement of NNM-induced carcinogenesis in the rat liver by phenobarbital: a combined morphological and enzyme histochemical approach, Carcinogenesis, 4: 473.PubMedCrossRefGoogle Scholar
  46. NCI, 1976, Guidelines for carcinogen bioassay in samll rodents, National Cancer Institute Carcinogenesis Technical Report Series, NCI, USA.Google Scholar
  47. Ogawa, K., Solt, D. B., and Farber, E., 1980, Phenotypic diversity as an early property of putative preneoplastic hepatocyte populations in liver carcinogenesis, Cancer Res., 40: 725.PubMedGoogle Scholar
  48. Ogiso, T., Tatematsu, M., Tamano, S., Tsuda, H., and Ito, N., 1985, Comparative effects of carcinogens on the induction of placental glutathione S-transferase-positive liver nodules in a short-term assay and of hepatocellular carcinomas in a long-term assay, Toxicol. Pathol., 13:257..Google Scholar
  49. Peraino, C., Fry, R., Staffeldt, E., and Christopher, J. P., 1975, Comparative enhancing effect of phenobarbital, diphenylhydantoin, and dichlorodiphenyltrichloroethane on 2-acetylaminofluorene induced hepatic tumorigenesis in the rat, Cancer Res., 35: 2884.PubMedGoogle Scholar
  50. Pitot, H. C., Barsness, L., Goldsworthy, T., and Kitagawa, T., 1978, Biochemical characterization of stages of hepatocarcinogenesis after a single dose of diethylnitrosamine, Nature (Lond.), 271: 456.CrossRefGoogle Scholar
  51. Pitot, H. C., Goldsworthy, T., Campbell, H. A., and Poland, A., 1980, Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine, Cancer Res., 40: 3616.PubMedGoogle Scholar
  52. Pitot, H. C., and Sirica, A. E., 1980, The stages of initiation and promotion in hepatocarcinogenesis, Biochim. Biophys. Acta, 605: 191.PubMedGoogle Scholar
  53. Preat, V., de Gerlache, J., Lans, M., Taper, H., and Roberfroid, M., 1986, Comparative analysis of the effects of phenobarbital dichlorodiphenyltrichloroethane, butylated hydroxytoluene and nafenopin on rat hepatocarcinogenesis, Carcinogenesis, 7:1025.Google Scholar
  54. Pugh, T. D., and Goldfarb, S., 1978, Quantitative histochemical and autoradiographic studies of hepatocarcinogenesis in rats fed 2acetylaminofluorene followed by phenobarbital, Cancer Res., 38:4450.Google Scholar
  55. Rabes, H. M., Scholze, P. and Jantsch, B., 1972, Growth kinetics of diethylnitrosamine-induced enzyme-deficient “preneoplastic” liver cell populations in vivo and in vitro, Cancer Res., 32: 2577.PubMedGoogle Scholar
  56. Rappaport, A. M., 1979, Physioanatomical basis of toxic liver injury, in: “Toxic Injury of the Liver, Part A,” E. Farber, and M. M. Fischer, eds., Marcel Dekker, New York.Google Scholar
  57. Reddy, J. K., and Qureshi, S. A., 1979, Tumorigenicity of the hypolipidaemic peroxisome proliferator ethyl-a-pchlorophenoxyisobutyrate (clofibrate) in rats, Br. J. Cancer 40: 476.PubMedCrossRefGoogle Scholar
  58. Rinkus, S., and Legator, M. S., 1979, Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella typhimurium system, Cancer Res., 39:3289.Google Scholar
  59. Sato, K., Kitahara, A., Satoh, K., Ishikawa, T., Tatematsu, M., and Ito, N., 1984, The placental form of gluathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis, Gann, 75:199.Google Scholar
  60. Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hayama, I., and Sato, K, 1985, Purification, induction and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical carcinogenesis, Proc. Natl. Acad. Sci. USA, 82: 3964.PubMedCrossRefGoogle Scholar
  61. Scherer, E., 1984, Neoplastic progression in experimental hepatocarcinogenesis, Biochim. Biophys. Acta, 738: 219.PubMedGoogle Scholar
  62. Shirai, T., Fukushima, S., Ikawa, E., Tagawa, Y., and Ito, N., 1986, Induction of prostate carcinoma in situ at high incidence in F344 rats by a combination of 3,2’-dimethyl-4-aminobiphenyl and ethinyl estradiol, Cancer Res., 46: 6423.PubMedGoogle Scholar
  63. Shirai, T., Masuda, A., Imaida, K., Ogiso, T., and Ito, N., 1988, Effects of phenobarbital and carbazole on carcinogenesis of the lung, thyroid, kidney, and bladder of rats pretreated with N-bis(2hydroxypropyl)nitrosamine, Jpn. J. Cancer Res. (Gann), 79: 460.CrossRefGoogle Scholar
  64. Solt, D., and Farber. E., 1976, New principle for the analysis of chemical carcinogenesis, Nature (London), 262: 701.CrossRefGoogle Scholar
  65. Tamano, S., Tsuda, H., Fukushima, S., Masui, T., Hosoda, K., and Ito, N., 1983, Dose and sex dependent effects of 2-acetylaminofluorene, 3’methyl-4-dimethylaminoazobenzene and DL-ethionine in induction of yglutamyltranspeptidase-positive liver cell foci in rats, Cancer Lett., 20: 313.PubMedCrossRefGoogle Scholar
  66. Tatematsu, M., Hasegawa, R., Imaida, K., Tsuda, H., and Ito, N., 1983, Survey of various chemicals for initiating and promoting activities in a short-term in vivo system based on generation of hyperplastic liver nodules in rats, Carcinogenesis, 4: 381.PubMedCrossRefGoogle Scholar
  67. Tatematsu, M., Mera, Y., Ito, N., Satoh, K., and Sato, K., 1985, Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of y-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats, Carcinogenesis, 6: 1621.PubMedCrossRefGoogle Scholar
  68. Thamavit, W., Tatematsu, M., Ogiso, T., Mera, Y., Tsuda, H., and Ito, N., 1985, Dose-dependent effects of butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquin in induction of foci of rat liver cells containing the placental form of glutathione S-transferase, Cancer Lett., 27: 295.PubMedCrossRefGoogle Scholar
  69. Tsuda, H., Lee, G., and Farber, E., 1980, Induction of resistant hepatocytes as a new principle for a possible short-term in vivo test for carcinogens, Cancer Res., 40: 1157.PubMedGoogle Scholar
  70. Tsuda, H., Sakata, T., Tamano, S., Okumura, M., and Ito, N., 1983, Sequential observations on the appearance of neoplastic lesions in the liver and kidney after treatment with N-ethyl-Nhydroxyethylnitrosamine followed by partial hepatectomy and unilateral nephrectomy, Carcinogenesis, 4: 523.PubMedCrossRefGoogle Scholar
  71. Tsuda, H., Sakata, T., Shirai, T., Kurata, Y., Tamano, S., and Ito, N., 1984, Modification of N-methyl-N-nitrosurea initiated carcinogenesis in the rat by subsequent treatment with antioxidants, phenobarbital and ethinyl estradiol, Cancer Lett., 24: 19.PubMedCrossRefGoogle Scholar
  72. Tsuda, H., Masui, T., Ikawa, E., Imaida, K., and Ito, N., 1987, Compared promoting potential of D-galactosamine, carbon tetrachloride and partial hepatectomy in rapid induction of preneoplastic liver lesions in the rat, Cancer Lett., 37: 163.PubMedCrossRefGoogle Scholar
  73. Vesselinovitch, S. D., and Mihailovich, M., 1983, Kinetics of diethylnitrosamine hepatocarcinogenesis in the infant mouse, Cancer Res., 43: 4253.PubMedGoogle Scholar
  74. Williams, G. M., 1980, The pathogenesis of rat liver cancer caused by chemical carcinogens, Biochim. Biophys. Acta, 605: 167.PubMedGoogle Scholar
  75. Williams, G. M., 1982, Phenotypic properties of preneoplastic rat liver lesions and applications to detection of carcinogens and tumor promoters, Toxicol. Pathol., 10: 3.CrossRefGoogle Scholar
  76. Wynder, E. L., and Gori, G. B., 1977, Contribution of the environment to cancer incidence: An epidemiologic exercise, J. Natl. Cancer Inst., 58: 825.PubMedGoogle Scholar
  77. Wynder, E. L., 1983, Tumor enhancers: underestimated factors in the epidemiology of life style-associated cancers, Environ. Health Perspect., 50: 15.PubMedCrossRefGoogle Scholar
  78. Zeiger, E., 1987, Carcinogenesis of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity, Cancer Res., 47: 1287.PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • Nobuyuki Ito
    • 1
  • Hiroyuki Tsuda
    • 1
  • Ryohei Hasegawa
    • 1
  • Masae Tatematsu
    • 1
  • Katsumi Imaida
    • 1
  • Makoto Asamoto
    • 1
  1. 1.First Department of PathologyNagoya City University Medical SchoolMizuho-cho, Mizuho-ku, Nagoya 467Japan

Personalised recommendations