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or aneugenic events during the early stages of experimental rat hepatocarcinogenesis, by fluorescence in situ hybridization
Abstracts/Mutation Research 360 (1996) 201-300
5-20
Identification of clastogenic and / or aneugenic events during the early stages of experimental rat hepatocarcinogenesis, by fluorescence in situ hybridization F. Van Goethem a, j. de Stoppelaar b, B. Hoebee b and M. Kirsch-Volders a; a Laboratory of Antropogenetics, Free University of Brussels, Pleinlaan 2, 1050 Brussels, Belgium, b Laboratory of Carcinogenesis and Mutagenesis, National Institute of Public Health and Environmental Protection (RIVM), Bilthoven, The Netherlands A growing body of evidence from human and animal cancer cytogenetics indicates that aneuploidy is an important chromosome change in carcinogenesis. Aneuploidy may be associated with a primary event in neoplastic transformation and to understand the role of this genetic phenomenon during the first steps of an experimental cancer model, molecular and cellular techniques were combined. A sequential cytogenetic study of a modified Solt-Farber liver cancer model in the rat was performed to identify the importance of chromosome versus genome mutations. Male Wistar rats were initiated by diethylnitrosamine (DENA), followed by a 2-acetylaminofluorene (2-AAF) exposure to select the resistant hepatocytes. Chronic phenobarbital (PB) treatment was used to induce promotion. Cell proliferation was induced by a necrogenic dose of CC14, administered during the selection period (Gerlans protocol) or 3 days before hepatocyte isolation (Experimental protocol). To identify clastogenic a n d / o r aneugenic events during the different stages (initiation, selection, promotion), non-radioactive in situ hybridization with a rat centromere satellite DNA probe was applied. Previous work showed that the expression of micronuclei (MN) can be considered as an adequate parameter to detect genetic alterations in isolated liver cells. Therefore micronucleated hepatocytes (MNH) were analysed for the presence of a centromere in the MN, in order to discriminate events causing chromosome breakage (clastogenic) from those that induce spindle malfunctioning (aneugenic).
259
Depending on the presence of a fluorescent signal in the MN following in situ hybridization with the centromere specific probe, MNH were scored as centromere positive (C + ) or centromere negative ( C - ) . Underestimation of aneuploidy might have occurred, since the used probe doesn't bind to the centromeric region of chromosome 1, 19, 20, X and Y. Although results showed that the majority of genetic damage, expressed as MN, was clastogenic, some aneugenic characteristics could be observed during PB treatment (promotion). 5-21
A flow-cytometer-based micronucleus assay for detection of chromosome aberrations in mice L. Abramsson-Zetterberg, L. Eriksson, J. Grawr, G. Zetterberg; Department of Genetics, Uppsala University, Sweden The micronucleus assay using erythrocytes from mice is the most frequently used in vivo short-term test in genetic toxicology. The scoring is simple and a great number of cells can be analysed which gives the test a high resolution also when the analyses are made with microscope. Using flow cytometry we have been able to automate the analyses of samples from both bone marrow and peripheral blood. Thiazole orange and Hoechst 33342 are used to stain RNA and DNA, respectively, allowing discrimination between young and mature erythrocytes as well as the identification of micronucleated cells and a contemporary analysis of their DNA content. The rate of analysis is about 1000 cells/s, which makes possible an increase of the number of analysed cells by two orders of magnitude compared to manual scoring. Results will be presented from experiments where we studied the effects of very low doses of ionizing radiation, as well as results from a comparison of the effects of clastogens and aneugens. In situ hybridization was used in outsorted micronucleated cells to discriminate between micronuclei containing centromere-less fragments and those containing intact chromosomes.
5-20
Identification of clastogenic and / or aneugenic events during the early stages of experimental rat hepatocarcinogenesis, by fluorescence in situ hybridization F. Van Goethem a, j. de Stoppelaar b, B. Hoebee b and M. Kirsch-Volders a; a Laboratory of Antropogenetics, Free University of Brussels, Pleinlaan 2, 1050 Brussels, Belgium, b Laboratory of Carcinogenesis and Mutagenesis, National Institute of Public Health and Environmental Protection (RIVM), Bilthoven, The Netherlands A growing body of evidence from human and animal cancer cytogenetics indicates that aneuploidy is an important chromosome change in carcinogenesis. Aneuploidy may be associated with a primary event in neoplastic transformation and to understand the role of this genetic phenomenon during the first steps of an experimental cancer model, molecular and cellular techniques were combined. A sequential cytogenetic study of a modified Solt-Farber liver cancer model in the rat was performed to identify the importance of chromosome versus genome mutations. Male Wistar rats were initiated by diethylnitrosamine (DENA), followed by a 2-acetylaminofluorene (2-AAF) exposure to select the resistant hepatocytes. Chronic phenobarbital (PB) treatment was used to induce promotion. Cell proliferation was induced by a necrogenic dose of CC14, administered during the selection period (Gerlans protocol) or 3 days before hepatocyte isolation (Experimental protocol). To identify clastogenic a n d / o r aneugenic events during the different stages (initiation, selection, promotion), non-radioactive in situ hybridization with a rat centromere satellite DNA probe was applied. Previous work showed that the expression of micronuclei (MN) can be considered as an adequate parameter to detect genetic alterations in isolated liver cells. Therefore micronucleated hepatocytes (MNH) were analysed for the presence of a centromere in the MN, in order to discriminate events causing chromosome breakage (clastogenic) from those that induce spindle malfunctioning (aneugenic).
259
Depending on the presence of a fluorescent signal in the MN following in situ hybridization with the centromere specific probe, MNH were scored as centromere positive (C + ) or centromere negative ( C - ) . Underestimation of aneuploidy might have occurred, since the used probe doesn't bind to the centromeric region of chromosome 1, 19, 20, X and Y. Although results showed that the majority of genetic damage, expressed as MN, was clastogenic, some aneugenic characteristics could be observed during PB treatment (promotion). 5-21
A flow-cytometer-based micronucleus assay for detection of chromosome aberrations in mice L. Abramsson-Zetterberg, L. Eriksson, J. Grawr, G. Zetterberg; Department of Genetics, Uppsala University, Sweden The micronucleus assay using erythrocytes from mice is the most frequently used in vivo short-term test in genetic toxicology. The scoring is simple and a great number of cells can be analysed which gives the test a high resolution also when the analyses are made with microscope. Using flow cytometry we have been able to automate the analyses of samples from both bone marrow and peripheral blood. Thiazole orange and Hoechst 33342 are used to stain RNA and DNA, respectively, allowing discrimination between young and mature erythrocytes as well as the identification of micronucleated cells and a contemporary analysis of their DNA content. The rate of analysis is about 1000 cells/s, which makes possible an increase of the number of analysed cells by two orders of magnitude compared to manual scoring. Results will be presented from experiments where we studied the effects of very low doses of ionizing radiation, as well as results from a comparison of the effects of clastogens and aneugens. In situ hybridization was used in outsorted micronucleated cells to discriminate between micronuclei containing centromere-less fragments and those containing intact chromosomes.