This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Ittrich, C. Articles by Kopp-Schneider, A. Search for Related Content PubMed PubMed Citation Articles by Ittrich, C. Articles by Kopp-Schneider, A. Social Bookmarking                         What's this?

Prevalidation of a Rat Liver Foci Bioassay (RLFB) Based on Results from 1600 Rats: A Study Report

Central Unit Biostatistics German Cancer research Center (DKFZ), P.O. Box 101949, D-69009 Heidelberg, Germany

Erhard Deml

Department Technical Security GSF-National Research Center for Environment and Health, Ingolstädter Landstr.1, D-85764 Neuherberg, Germany

Doris Oesterle

Institute of Toxicology, GSF-National Research Center for Environment and Health, Ingolstädter Landstr.1, D-85764 Neuherberg, Germany

Karin Küttler

Product Safety-Regulations, Toxicology and Ecology, BASF AG, D-67056 Ludwigshafen/Rhein, Germany

Werner Mellert

Product Safety-Regulations, Toxicology and Ecology, BASF AG, D-67056 Ludwigshafen/Rhein, Germany

Susanne Brendler-Schwaab

Toxicology-Rodent Studies and Genetic Toxicology, Bayer AG, D-42096 Wuppertal, Germany

Harald Enzmann

Toxicology-Rodent Studies and Genetic Toxicology, Bayer AG, D-42096 Wuppertal, Germany

Ludwig Schladt

Toxicology-Rodent Studies and Genetic Toxicology, Bayer AG, D-42096 Wuppertal, Germany

Peter Bannasch

Department of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), P.O. Box 101949, D-69009 Heidelberg, Germany

Thomas Haertel

Department of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), P.O. Box 101949, D-69009 Heidelberg, Germany

Oliver Mönnikes

Department of Toxicology, Institute of Pharmacology and Toxicology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany

Michael Schwarz

Department of Toxicology, Institute of Pharmacology and Toxicology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany

Annette Kopp-Schneider

Central Unit Biostatistics German Cancer research Center (DKFZ), P.O. Box 101949, D-69009 Heidelberg, Germany, kopp{at}dkfz.de

A rat liver foci bioassay (RLFB) based on an initiation-promotion protocol employing preneoplastic foci of altered hepatocytes (FAH) as an endpoint, was prevalidated in 5 different laboratories. FAH were identified by immunohistochemical demonstration of glutathione-S-transferase (placental form, GSTP) and by staining with hematoxilin/eosin (H&E), and their area fraction was quantified morphometrically. The four model hepatocarcinogens N-nitrosomorpholine, 2-acetylaminofluoren, phenobarbital, and clofibrate were selected according to characteristic differences in their presumed mode of action, and tested in a total of 1,600 male and female rats at 2 different dose levels. The chemicals were found to differ characteristically in their potency and dose-response relationship to induce FAH when given alone or when administered following initiation with diethylnitrosamine. The interlaboratory variation was small for results obtained with the GSTP-stain and somewhat larger with respect to H&E. The assessment of the carcinogenic potential of the four chemicals by the different laboratories was in the same range and the nature of their dose-response relationships did not differ essentially between laboratories. Our results suggest that this RLFB is a sensitive bioassay, providing potentially valuable information for risk assessment including the classification of carcinogenic chemicals according to their mode of action.

Key Words: Hepatocarcinogenesis • foci of altered hepatocytes • rat liver foci bioassay • prevalidation study • phenobarbital • clofibrate • N-nitrosomorpholine • 2-acetylaminofluorene.

References

• Alden CL, Smith PF, Piper CE, Brej L. ( 1996). A critical appraisal of the value of the mouse cancer bioassay in safety assessment. Toxicol Pathol 24: 722—725.[Abstract/Free Full Text]
• Altmann HW (1994). Hepatic neoformations. Pathol Res Pract 190: 513— 577.[Web of Science][Medline] [Order article via Infotrieve]
• Balls M., Blaauboer B., Brusick D., Frazier J., Lamb D., Pemberton M., Reinhardt C., Roberfroid M., Rosenkranz H., Schmid B., Spielmann H., Stammati A-L., Walum E. (1990). Report and recommendations of the CAAT/ERGATT workshop on the validation of toxicity test procedures. ATLA 18: 313—337.
• Balls M., Blaauboer BJ, Fentem JH, Bruner L., Combes RD, Ekwall B., Fielder RJ, Guillouzo A., Lewis RW, Lovell DP, Reinhardt CA, Repetto G., Sladowski D., Spielmann H., Zucco F. (1995). Practical aspects of the validation of toxicity test procedures. The report and recommendations of ECVAM Workshop 5. ATLA 23: 129—147.
• Balls M., Karcher W. (1995). The validation of alternative test methods. ATLA 23: 884—886.
• Bannasch P. (1968). The cytoplasm of hepatocytes during carcinogenesis. Electron and light microscopic investigations of the nitrosomorpholine-intoxicated rat liver. Recent Results Cancer Res 19: 1—100.
• Bannasch P. (1996). Pathogenesis of hepatocellular carcinoma: Sequential cellular, molecular, and metabolic changes. Prog Liver Dis 14: 161—197.[Medline] [Order article via Infotrieve]
• Bannasch P., Schröder C. (2002). Pathogenesis of primary liver tumours. In: Pathology of the Liver, Mac Sween RNM, Burt AD, Portman BC, Ishak KG, Scheuer PJ, Anthony PP (eds). Churchill Livingstone, New York, pp 777—825.
• Bannasch P., Zerban H. (1997). Experimental chemical hepatocarcinogenesis. In: Liver Cancer, Okuda K, Tabor E (eds). Churchill Livingstone New York, pp 213—253.
• Bannasch P., Zerban H., Hacker HJ (1997). Foci of altered hepatocytes, rat. In: Monographs on Pathology of Laboratory Animals, Digestive System, Jones TC, Popp JA, Mohr U (eds). 2nd edition. Springer-Verlag, Berlin, Heidelberg, New York, pp 3—37.
• Bitsch A., Hadjiolov N., Klohn PC, Bergmann O., Zwirner-Baier I., Neumann HG (2000). Dose response of early effects related to tumor promotion of 2-acetylaminofluorene. Toxicol Sci 55: 44—51.[Abstract/Free Full Text]
• Cattley RC, Kato M., Popp JA, Teets VJ, Voss KS ( 1994). Initiator-specific promotion of hepatocarcinogenesis by WY-14,643 and clofibrate. Carcinogenesis 15: 1763—1766.[Abstract/Free Full Text]
• Corton JC, Anderson SP, Stauber A. (2000). Central role of peroxisome proliferator-activated receptors in the action of peroxisome proliferators. Ann Rev Pharm Tox 40: 491—518.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Curren RD, Southee JA, Spielmann H., Liebsch M., Fentem JH, Balls M. (1995). The role of prevalidation in the development, validation and acceptance of alternative methods. ATLA 23: 211—217.
• Delesse MA (1847). Procede mecanique pour determiner la composition des roches. CR Acad Sci (Paris) 25: 544—545.
• Deml E., Oesterle D., Wolff T., Greim H. (1981). Age-, sex-, and strain-dependent differences in the induction of enzyme-altered islands in rat liver by diethylnitrosamine. J Cancer Res Clin Oncol 100: 125—134.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Emmelot P., Scherer E. (1980). The first relevant cell stage in rat liver carcinogenesis. A quantitative approach. Biochim Biophys Acta 605: 247—304.[Medline] [Order article via Infotrieve]
• Enzmann H., Ohlhauser D., Enzmann H., Dettler T., Benner U., Hacker HJ, Bannasch P. (1989). Unusual histochemical pattern in preneoplastic hepatic foci characterized by hyperactivity of several enzymes. Virchows Arch B (Cell Pathol.) 57: 99—108.[Web of Science][Medline] [Order article via Infotrieve]
• Enzmann H., Iatropoulos M., Brunnemann KD, Bomhard E., Ahr HJ, Schlueter G., Williams GM (1998). Short- and intermediate-term carcinogenicity testing—A review, part 2: Available experimental models. Food and Chem Toxicol 36: 997—1013.
• Friedrich-Freksa H., Papadopulu G., Gössner W. (1969). Histochemische Untersuchungen der Cancerogenese in der Rattenleber nach zeitlich begrenzter Verabfolgung von Diethylnitrosamin. Z Krebsforsch 72: 40—253.
• Goldberg AM, Frazier JM, Brusick D., Dickens MS, Flint O., Gettings SD, Hill RH, Lipnick RL, Renskers KJ, Bradlaw JA, Scala RA, Veronesi B., Green S., Wilcox NL, Curren RD (1993). Framework for validation and implementation of in vitro toxicity tests: Report of the validation and technology transfer committee of the John Hopkins Center for Alternatives to Animal Testing. J Am Coll Tox 12: 23—30.
• Goodman DG, Maronpot RR, Newberne PM, Popp JA, Squire RA (1994). Proliferative and selected lesions in the liver of rats. In: Guides for Toxicologic Pathologym, Street CS, Burek JD, Hardisty JF, Garner FM, Leininger JR, Pletscher JM, Moch RW (eds). Society of Toxicologic Pathologists/American Registry of Pathology/Armed Forces Institute of Patholgy Washington, DC, pp G1—5, 1—24.
• Grasl-Kraupp B., Luebeck G., Wagner A., Löw-Baselli A., de Gunst M., Waldhör T., Moolgavkar S., Schulte-Hermann R. (2000 ). Quantitative analysis of tumor initiation in rat liver: Role of cell replication and cell death (apoptosis). Carcinogenesis 21: 1411—1421.[Abstract/Free Full Text]
• Hasegawa R., Ito N. (1994). Hepatocarcinogenesis in the rat. In: Carcinogenesis, Waalkes MP, Ward JM (eds). Raven, New York, pp 39—65.
• Hollander M., Woolfe DA (1973). Nonparametric Statistical Methods. Wiley, New York.
• Holm S. (1979). A simple sequentially rejective multiple test procedure. Scand J Statist 6: 65—70.
• Honkakoski P., Zelko I., Sueyoshi T., Negishi M. (1998). The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol 18: 5652— 5658.[Abstract/Free Full Text]
• Hothorn LA (2002). Statistics of interlaboratory in vitro toxicological studies. ATLA (in press).
• Huff J., Cirvello J., Haseman J., Bucher J. (1991). Chemicals associated with site-specific neoplasia in 1394 long-term carcinogenesis experiments in laboratory rodents. Environ Health Perspect 93: 247—270.[Web of Science][Medline] [Order article via Infotrieve]
• Ihaka R., Gentleman R. (1996). R: A language for data analysis and graphics. J Comput Graph Stat 5: 299—314.[CrossRef]
• Imaida K., Tatematsu K., Kato T., Tsuda H., Ito N. (1989). Advantages and limitations of stereological estimation of placental glutathione S-transferase-positive rat liver cell foci by computerizedthree-dimensional reconstruction. Jpn J Cancer Res 80: 326—330.[CrossRef][Web of Science]
• Ito N., Shirai T., Hasegawa R. (1992). Medium-term bioassays for carcinogens. In: Mechanisms of Carcinogenesis in Risk Identification, Vainio H, Magee PN, McGregor DB, McMichael AJ (eds). International Agency for Research on Cancer, Lyon, pp 353—388.
• Kopp-Schneider A. (2002). Biostatistical evaluation of focal hepatic preneoplasia. Toxicol Pathol 31: this volume.
• Kopp-Schneider A., Portier C., Bannasch P. (1998). A model for hepatocarcinogenesis treating phenotypical changes in focal hepatocellular lesions as epigenetic events. Math Biosci 148: 181—204.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Kunz HW, Tennekes HA, Port RE, Schwarz M., Lorke D., Schaude G. (1983). Quantitative aspects of chemical carcinogenesis and tumor promotion in liver. Environm Health Perspect 50: 113—122.[CrossRef]
• Metzger C., Mayer D., Hoffmann H., Bocker T., Hobe G., Benner A., Bannasch P. (1995). Sequential appearance and ultrastructure of amphophilic cell foci, adenomas and carcinomas in the liver of male and female rats treated with dehydroepiandrosterone (DHEA). Toxicol Pathol 23: 591—605.[Abstract/Free Full Text]
• Montesano R, Bartsch H, Vainio H, Wilbourn J, Yamasaki H (eds) (1986). Long-term and short-term assays for carcinogens: A critical appraisal. International Agency for Research on Cancer, Lyon.
• Moolgavkar SH, Luebeck EG, de Gunst M., Port RE, Schwarz M. (1990). Quantitative analysis of enzyme-altered foci in rat hepatocarcinogenesis experiments—I. Single agent regimen. Carcinogenesis 11: 1271— 1278.[Abstract/Free Full Text]
• Moore MA, Mayer D., Bannasch P. (1982). The dose dependence and sequential appearance of putative preneoplastic populations induced in the rat liver by stop experiments with N-nitrosomorpholine. Carcinogenesis 3: 1429—1436.[Abstract/Free Full Text]
• Oecd (1996). Report of the OECD workshop on harmonisation of validation and acceptance criteria for alternative toxicological test methods. OECD Publications Office, Paris.
• Oesterle D., Deml E. (1983). Promoting effect of polychlorinated biphenyls on development of enzyme-altered islands in livers of weanling and adult rats. J Cancer Res Clin Oncol 105: 141—147.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Oesterle D., Gerbracht U., Deml E., Schlatterer B., Eigenbrodt E. (1989). Comparison of three rat liver foci bioassays—incidence of preneoplastic foci initiated by diethylnitrosamine. Carcinogenesis 10: 1891— 1895.[Abstract/Free Full Text]
• Peraino C., Fry RJ, Staffeldt E. (1971). Reduction and enhancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res 31: 1506—1512.[Abstract/Free Full Text]
• Pitot HC (1990). Altered hepatic foci: Their role in murine hepatocarcinogenesis. Ann Rev Pharmacol Toxicol 30: 465—500.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Pitot HC, Goldsworthy TL, Moran S., Kennan W., Glauert HP, Maronpot RR, Campbell HA (1987). A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci. Carcinogenesis 8: 1491— 1499.[Abstract/Free Full Text]
• Pitot HC, Sirica AE (1980). The stages of initiation and promotion in hepatocarcinogenesis. Biochim Biophys Acta 605: 191—215.[Medline] [Order article via Infotrieve]
• Pugh TD, King JH, Koen H., Nychka D., Chover J., Wahba G., He YH, Goldfarb S. (1983). Reliable stereological method for estimating the number of microscopic hepatocellular foci from their transections. Cancer Res 43: 1261—1268.[Abstract/Free Full Text]
• Rabes HM, Bücher T., Hartmann A., Linke I., Dünnwald M. (1982). Clonal growth of carcinogen-induced enzyme deficient preneoplastic cell population in mouse liver. Cancer Res 42: 3220—3227.[Abstract/Free Full Text]
• Russell WMS, Burch RL (1959). The Principles of Humane Experimental Technique. Methuen, London.
• Sato K. (1989). Glutathione transferases as markers of preneoplasia and neoplasia. Adv Cancer Res 52: 205—255.[Web of Science][Medline] [Order article via Infotrieve]
• Sato K., Kitahara A., Satoh K., Ishikawa T., Tatematsu M., Ito N. (1984). The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical carcinogenesis. Gann 75: 199—202.[Web of Science][Medline] [Order article via Infotrieve]
• Schauer A., Kunze E. (1968). Enzymhistochemische und autoradiographische Untersuchungen während der Kanzerisierung der Rattenleber durch Diäthylnitrosamin. Z Krebsforsch 70: 252—266.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Scherer E., Hoffmann M., Emmelot P., Friedrich-Freksa H. (1972). Quantitative study on foci of altered liver cells induced in the rat by a single dose of diethylnitrosamine and partial hepatectomy. J Natl Cancer Inst 49: 93— 106.[Web of Science][Medline] [Order article via Infotrieve]
• Schwarz M., Buchmann A., Robertson LW, Kunz W. ( 1990). Cell proliferation and hepatocarcinogenesis. In: Scientific Issues in Quantitative Cancer Risk Assessment, Moolgavkar SH (ed). Birkhäuser Press, Boston, pp 96— 115.
• Solt D., Farber E. (1976). New principle for the analysis of chemical carcinogenesis. Nature 236: 702—703.
• Spielmann H., Liebsch M., Reinhardt C. (1998). ERGATT/ECVAM workshop on acceptance of validated alternative methods: Amden III. ALTEX 15: 18—22.[Medline] [Order article via Infotrieve]
• Squire RA, Levitt MH (1975). Report of a workshop on classification of specific hepatocellular lesions in rats. Cancer Ress 35: 3214— 3223.
• Stewart HL, Williams G., Keysser CH, Lombard LS, Montali RJ (1980). Histologic typing of liver tumors of the rat. J Natl Cancer Inst 65: 179—206.
• Su Q., Benner A., Hofmann WJ, Otto G., Pichlmayr R., Bannasch P. (1997). Human hepatic preneoplasia: Phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia. Virchows Arch 431: 391—406.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Tsuda H., Hasegawa R., Imaida K., Masui T., Moore MA, Ito N. (1984). Modifying potential of thirty-one chemicals on short-term development of -glutamyl transpeptidase-positive foci in diethylnitrosamine-initiated rat liver. Gann 74: 876—883.
• US Food and Drug Administration (1998). International Conference on Harmonisation; Guidance on testing for carcinogenicity of pharmaceuticals. Fed Reg 63: 8983—8986.
• US National Institute of Environmental Health Sciences (1989). Significance of foci of cellular alteration. A symposium. Toxicol Pathol 17: 557— 735.[Medline] [Order article via Infotrieve]
• Vainio H, Magee P, McGregor D, McMichael AJ (eds) (1994). Mechansims of carcinogenesis in risk identification, International Agency for Research on Cancer, Lyon.
• Wei P., Zhang J., Egan-Hafley M., Liang S., Moore DD (2000). The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407: 920—923.[CrossRef][Medline] [Order article via Infotrieve]
• Weinberg WC, Berkwits L., Innaccone PM (1987). The clonal nature of carcinogen-induced altered foci of gamma-glutamyl transpeptidase expression in rat liver. Carcinogenesis 8: 565—570.[Abstract/Free Full Text]
• Williams GM, Enzmann H. (1999). The rat liver hepatocellular-altered focus-limited bioassay for chemicals with carcinogenic activity. In: Carcinogenicity: Testing, Predicting and Interpreting Chemical Effects, Kitchin KT (ed). Marcel Dekker, New York, pp 361—394.

Toxicologic Pathology, Vol. 31, No. 1, 60-79 (2003)
DOI: 10.1080/01926230390173888


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Ittrich, C. Articles by Kopp-Schneider, A. Search for Related Content PubMed PubMed Citation Articles by Ittrich, C. Articles by Kopp-Schneider, A. Social Bookmarking                         What's this?