Mutagenesis vol. 18 no. 5 pp. 471-475,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Detection by dual color fluorescence in situ hybridization of in vivo chromosome damage in rat hepatocyte nuclei
Laboratory of Genetic Toxicology, The Institute of Environmental Toxicology, 4321 Uchimoriya-machi, Mitsukaido-shi, Ibaraki 303–0043, Japan
Our previous study using a multicolor fluorescence in situ hybridization (FISH) technique revealed that region-specific DNA probes for rat chromosome 1 enabled the detection of structural chromosome damage in rat interphase nuclei from peripheral blood and bone marrow cells. In the present study, this FISH technique was modified for application to a non-hematopoietic organ (liver) and the usefulness of the system was tested using diethylnitrosamine (DEN) as a model hepatocarcinogen. Male Sprague–Dawley rats were orally treated once with DEN at 200 mg/kg. Their livers were removed at 4, 7 or 14 days after treatment and homogenized with a tissue grinder to isolate hepatocyte nuclei. The nucleus suspension was fixed in methanol:acetic acid and air dried. Dual color FISH with two probes, one labeled with tetramethylrhodamine and one with digoxigenin, demonstrated that the maximum increase in the frequency of nuclei with spatially abnormal signals was observed 7 days after treatment. A dose–response relationship for induction of abnormal nuclei was observed. This improved dual color FISH system is potentially valuable for assessing in vivo clastogenicity in all rat organs.
1Tel: +81 297 27 4539; Fax: +81 297 27 4518; Email: matsumoto{at}iet.or.jp