To explore for practical thresholds for DNA-reactive carcinogens in rat liver carcinogenicity, we have conducted a series of exposure-response studies using 2 well-studied hepatocarcinogens, 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN). Findings with AAF, including as yet unpublished experiments, are reviewed here and related to DEN observations. In these studies, we have administered exact intragastric doses during an initiation segment (IS) of 12—16 weeks followed in some experiments by phenobarbital (PB) as a liver tumor promoter for 24 weeks to enhance manifestation of initiation. The cumulative doses (CD) of AAF at the end of ISs ranged from 0.094 to 282.2 mg/kg. Our findings for AAF in the IS can be summarized as follows: (1) the earliest parameter to be affected with administration of low doses was the appearance of DNA adducts (around 4 weeks), followed at higher doses by cell proliferation; (2) formation of DNA adducts was nonlinear, with a no-observed effect level (NOEL) at a CD of 0.094 mg/kg and a plateau at higher doses (94.1 mg/kg); (3) cytotoxicity (necrosis) showed a NOEL at a CD of 28.2 mg/kg; (4) compensatory hepatocellular proliferation showed a NOEL at a CD of 28.2 mg/kg and was supralinear at a high CD (282.2 mg/kg); (5) formation of preneoplastic hepatocellular altered foci (HAF) showed a NOEL at a CD of 28.2 mg/kg, and was supralinear at a high CD (282.2 mg/kg); (6) a NOEL (CD 28.2 mg/kg) was found for tumor development and the exposure—response was supralinear. We interpret these findings to reflect practical thresholds for hepatocellular initiating effects of AAF and exaggerated responses at high-exposures doses, as also found for DEN. Thus, mechanisms of carcinogenesis can differ between low and high doses.
Chen, B., Liu, L., Castonguay, A., Maronpot, R.R., Anderson, M.W., and You, M.L. (1993). Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone induced mouse lung tumors. Carcinogenesis14, 1603— 8.
2.
Cohen, S.M., and Ellwein, L.B. (1990). Cell proliferation in carcinogenesis. Science249, 1007—11.
3.
Counts, J.L., and Goodman, J.I. (1995). Principles underlying dose selection for, and extrapolation from, the carcinogen bioassay: dose influences mechanism. Reg Toxicol Pharmacol21, 418—21.
4.
Den Otter, W., Koten, J.W., Van Der Vegt, B.J.H., Beemer, F.A., Boxma, O.J., Derkinderen, D.J., De Graaf, P.W., Hiber, J., Lips, C.J.M., Roholl, P.M.L., Sluijter, F.J.H., Tan, K.E.W.P., Van Der Heyden, K.A., Van Der Ven, L., and Van Unnik, J.A.M. (1990). Oncogenesis by mutations in anti-oncogenes: a view. Anticancer Res10, 475—88.
5.
Dietrich, D. (1993). Toxicological and pathological applications of proliferating cell nuclear antigen (PCNA) a novel endogenous marker for cell proliferation. Crit Rev Toxicol23, 77—109.
6.
Enzmann, H., Zerban, H., Kopp-Schneider, A., Löser, A., and Bannasch, P. (1995). Effects of low doses of N-nitrosomorpholine on the development of early stages of hepatocarcinogenesis. Carcinogenesis16, 1513— 18.
7.
Farmer, J.H., Kodell, R.L., Greenman, D.L., and Shaw, G.W. (1980). Dose and time response models for the incidence of bladder and liver neoplasms in mice fed 2-acetylaminofluorene continuously . J Environ Pathol Toxicol3(3), 55—68.
8.
Fukushima, S. (1999). Low-dose carcinogenicity of a heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline: relevance to risk assessment . Cancer Lett143, 157—9.
9.
Gaylor, D.W. (1980). The ED01 study: summary and conclusions. J Environ Pathol Toxicol3(3), 179—83.
10.
Gebhardt, R., Tanaka, T., and Williams, G.M. (1989). Glutamine synthetase heterogenous expression as a marker for the cellular lineage of preneoplastic and neoplastic liver populations . Carcinogenesis10, 1917—23.
11.
Gebhardt, R., and Williams, G.M. (1995). Glutamine synthetase and hepatocarcinogenesis . Carcinogenesis16, 1673—81.
12.
Gupta, R.C. (1985). Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen-DNA adducts. Cancer Res45, 5656—662.
13.
Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell100, 57—70.
14.
Hengstler, J.G., Bogdanfly, M.S., Bolf, H.M., and Oesch, F. (2003). Challenging dogma: thresholds for genotoxic carcinogens? The case of vinyl acetate. Annu Rev Pharmacol Toxicol43, 485—520.
15.
Hino, O., and Kitagawa, T. (1981). Existence of a practical threshold dose for the hepatocarcinogen 3'-methyl-4-(dimethylamino)azobenzene in rat liver. Gann72, 637—8.
16.
Hussain, S.P., and Harris, C.C. (1998). Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Res58, 4023—37.
17.
Iatropoulos, M.J., Jeffrey, A.M., Enzmann, H.E., von Kentz, E., Schlueter, G., and Williams, G.M. (2001). Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of moxifloxacin, a quinolone antibiotic . Exp Toxicol Pathol53, 345—57.
18.
Keegan, T.E., Simmons, J.E., and Pegram, R.A. (1998). NOAEL and LOAEL determinations of acute hepatotoxicity for chloroform and bromodichloromethane delivered in an aqueous vehicle to F344 rats. J Toxicol Environ Health55, 65—75.
19.
Manam, S., Shinder, G.A., Joslyn, D.J., Kraynak, A.R., Hammermeister, C.L., Leander, K.R., Ledwith, D.J., Prahalada, S., van Zwieten, M.J., and Nichols, W.W. (1995). Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouselivertumors. Carcinogenesis16, 1113—9.
20.
Miller, J.A. (1994). Sulfonation in chemical carcinogenesis-history and present status. Chem-Biol Interact92, 329—41.
21.
National Research Council (NRC). (1977). Safe Drinking Water and Health. National Academy of Sciences , Washington, DC.
22.
Nestmann, E.R., Bryant, D.W., Carr, J., Fennell, T.T., Gallagher, J.E., Gorelick, N.J., Swenberg, J.A., and Williams, G.M. (1996). Toxicological significance of DNA adducts. Report of an expert panel. Reg Toxicol Pharmacol140, 254—63.
23.
Neumann, H.-G. (1986). The role of DNA damage in chemical carcinogenesis of aromatic amines. J Cancer Res Clin Oncol112, 100—6.
24.
Page, N.P., Singh, D.V., Farland, W., Goodman, J.I., Conolly, R.B., Anderson, M.E., Clewell, H.J., Frederick, C.B., Yamasaki, H., and Lucier, G. (1997). Implementation of EPA revised cancer assessment guidelines: incorporation of mechanistic and pharmacokinetic data. Fundam Appl Toxicol37, 16—36.
25.
Peto, R., Gray, R., Branton, P., and Grasso, P. (1991a). Effects on 4080 rats of chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine: a detailed dose-response study. Cancer Res51, 6415—51.
26.
Peto, R., Gray, R., Branton, P., and Grasso, P. (1991b). Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rats by chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine. Cancer Res51, 6452—69.
27.
Poirier, M.C., and Connor, R.J. (1982). Radioimmunoassay for 2-acetylaminofluorene-DNA adducts. Meth Enzymol84, 607—18.
28.
Randerath, K., and Randerath, E. (1994). 32P-postlabeling methods for DNA adduct detection: overview and critical evaluation. Drug Metab Rev26, 67—85.
29.
Reddy, M.V., and Randerath, K. (1986). Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis7, 1543—51.
30.
Ringer, D.R., and Norton, T.R. (1987). Further characterization of the ability of hepatocarcinogens to lower rat liver arylsulfotransferase activity. Carcinogenesis8, 1749—52.
31.
Root, M., Lange, T., and Campbell, T.C. (1997). Dissimilarity in aflatoxin dose-response relationships between DNA adduct formation and development of preneoplastic foci in rat liver. Chemico-Biolog Interact106, 213—27.
32.
Sato, K., Kitahara, A., Satoh, K., Ishikawa, T., Tatematsu, M., and Ito, N. (1984). The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis . GANN75, 199— 202.
33.
Swenberg, J.A., La, D.K., Scheller, N.A., and Wu, K.-Y. (1995). Dose-response relationships for carcinogens. Toxicol Lett82/83, 751—6.
34.
Tong, C., Fazio, M., and Williams, G.M. (1980). Cell cycle-specific mutagenesis at the hypoxanthine phosphoribosyl-transferase locus in adult rat live epithelial cells. Proc Natl Acad Sci USA77, 7377—9.
35.
Umemura, T., Tokumo, K., Sirma, H., Gebhardt, R., Poirier, M.C., and Williams, G.M. (1993). Dose response effects of 2-acetylaminofluorene on DNA damage, cytotoxicity, cell proliferation and neoplastic conversion in rat liver. Cancer Lett73, 1—10.
36.
Verna, L., Whysner, J., and Williams, G.M. (1996a). N-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation . Pharmacol Therap71, 57—82.
37.
Verna, L., Whysner, J., and Williams, G.M. (1996b). 2-acetylaminofluorene mechanistic data and risk assessment: DNA reactivity, enhanced cell proliferation and tumor initiation . Pharmacol Therap71, 83—106.
38.
Waddell, W.J. (2003a). Thresholds in chemical carcinogenesis: what are animal experiments telling us? Toxicol Pathol31, 260—2.
39.
Waddell, W.J. (2003b). Thresholds of carcinogenicity in the EDO1 study . Toxicol Sci72, 158—63.
40.
Watanabe, L., and Williams, G.M. (1978). Enhancement of rat hepatocellular-altered foci by the liver tumor promoter phenobarbital: evidence that foci are precursors of neoplasms and that the promoter acts on carcinogen-induced lesions. J Natl Cancer Inst61, 1311—4.
41.
Whysner, J., Wang, C., Zang, E., Iatropoulos, M.J., and Williams, G.M. (1994). Dose response of promotion by butylated hydroxyanisole in chemically initiated tumours of the rat forestomach. Food Chem Toxicol32, 215—22.
42.
Williams, G.M. (1980). The pathogenesis of rat liver cancer caused by chemical carcinogens. Biochem Biophys Acta605, 167—89.
Williams, G.M. (1997). Chemicals with carcinogenic activity in the rodent liver; mechanistic evaluation of human risk. Cancer Lett118, 1—14.
45.
Williams, G.M. (1999a). Chemically induced rodent preneoplastic lesions as indicators of carcinogenic activity. In The Use of Short-and Medium-term Tests for Carcinogens and Data on Genetic Effects in Carcinogenic Hazard Evaluation (D. B. McGregor, J. M. Rice, and S. Venitt , eds.), pp. 185—202. IARC Scientific Publications No. 146. Lyon, France.
46.
Williams, G.M. (1999b). Mechanistic considerations in cancer risk assessment . Inhal Toxicol11, 549—54.
47.
Williams, G.M., and Iatropoulos, M.J. (2002). Alteration of liver cell function and proliferation: differentiation between adaptation and toxicity . Toxicol Pathol30, 41—53.
48.
Williams, G.M., Gebhardt, R., Sirma, H., and Stenbäck, F. (1993). Non-linearity of neoplastic conversion induced in rat liver by low exposures to diethylnitrosamine. Carcinogenesis14, 2149—56.
49.
Williams, G.M., Iatropoulos, M.J., and Jeffrey, A.M. (2000). Mechanistic basis for nonlinearities and thresholds in rat liver carcinogens by the DNA-reactive carcinogens 2-acetylaminofluorene and diethylnitrosamine. Toxicol Pathol28, 388—95.
50.
Williams, G.M., Iatropoulos, M.J., Jeffrey, A.M., Luo, F.-Q., and Wang, C.-X. (1998a). Diethylnitrosamine exposure-rsponses for DNA ethylation, hepatocellular proliferation and initiation of carcinogenesis in rat liver display non-linearities and thresholds. Arch Toxicol73, 394—402.
51.
Williams, G.M., Iatropoulos, M.J., Wang, C.X., Ali, N., Rivenson, A., Peterson, L.A., Schulz, C., and Gebhardt, R. (1996). Diethylnitrosamine exposure-responses for DNA damage, centrilobular cytotoxicity, cell proliferation and carcinogenesis in rat liver exhibit some non-linearities. Carcinogenesis17, 2253—8.
52.
Williams, G.M., Iatropoulos, M.J., Wang, C.-X., Jeffrey, A.M., Thompson, S., Pittman, B., Palasch, M., and Gebhardt, R. (1998b). Non-linearities in 2-acetylaminofluorene exposure-responses for genotoxic and epigenetic effects leading to initiation of carcinogenesis in rat liver. Toxicol Sci45, 152—61.
53.
Williams, G.M., Iatropoulos, M.J., and Whysner, J. (1999). Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives. Food Chem Toxicol37, 1027—38.
54.
Williams, G.M., Tanaka, T., Maruyama, H., Maeura, Y., Weisburger, J.H., and Zang, E. (1991). Modulation by butylated hydroxytoluene of liver and bladder carcinogenesis induced by chronic low level exposure to 2-acetylaminofluorene . Cancer Res51, 6224—30.
55.
Williams, G.M., and Watanabe, K. (1978). Quantitative kinetics of development of N-2-fluorenylacetamide-induced, altered (hyperplastic) hepatocellular foci resistant to iron accumulation and of their reversion of persistence following removal of carcinogen. J Natl Cancer Instit61, 113—121.
56.
Williams, G.M., and Whysner, J. (1995). Mechanistic considerations in risk assessment for epigenetic tumor-promoting carcinogens. In Growth Factors and Tumor Promotion: Implications for Risk Assessment (R. M. McClain, T. J. Slaga, R. LeBoeuf and H. Pitot, eds.), pp. 369—383. John Wiley and Sons, Inc.,New York.
57.
Williams, G.M., and Whysner, J. (1996). Epigenetic carcinogens: evaluation and risk assessment . Exp Toxicol Pathol48, 189—95.
58.
Wiltse, J., and Dellarco, V.L. (1996). U.S. Environmental Protection Agency guidelines for carcinogen risk assessment: Past and future. Mutat Res , 365, 3—15.
