Simple detection of chemical mutagens by the alkaline single-cell gel electrophoresis (Comet) assay in multiple mouse organs (liver, lung, spleen, kidney, and bone marrow).
Recently, we designed a fast and simple method to obtain nuclei for the alkaline SCG assay and we tested it with mouse liver, lung, kidney, spleen, and bone marrow. Instead of isolating organ cells by trypsinization, we homogenized tissue and isolated the nuclei. Each organ was minced, and the mince was suspended in chilled homogenizing buffer containing NaCl and Na2EDTA, homogenized gently using a Potter-type homogenizer set in ice, and then centrifuged. The nuclei from the precipitate were used for the assay. To evaluate the validity of this method, we tested the genotoxicity in mouse organs of 11 chemical mutagens with different modes of action. Mice were sacrificed 3 and 24 h after administration of each mutagen. Treatment with three alkylating agents (MMS, EMS, and MNNG), a DNA crosslinking agent (MMC), two aromatic amines (2-AAF and phenacetin), a polycyclic aromatic hydrocarbon (B[a]P), and two inorganic chemicals (KBrO3 and K2CrO4) increased migration of the DNA from mouse organs. 5-FU (a base analog) and colchicine (a spindle poison) treatment produced negative results in all organ studied. Considering that the alkaline SCG assay detects genotoxicity as DNA fragments derived from DNA single-strand breaks and alkali-labile damage, our results showed that the SCG assay using our homogenization technique detected chemical mutagens as a function of their modes of action.
Sasaki
Y F
YF
Laboratory of Genotoxicity, Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Aomori, Japan. yfsasaki-c@hachinohe-ct.ac.jp
Nishidate
E
E
Izumiyama
F
F
Matsusaka
N
N
Tsuda
S
S
D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D003078:Colchicine / Q000633:toxicity; D003432:Cross-Linking Reagents / Q000633:toxicity; D004586:Electrophoresis / Q000379:methods*; D005472:Fluorouracil / Q000633:toxicity; D007668:Kidney / Q000187:drug effects; D008099:Liver / Q000187:drug effects; D008168:Lung / Q000187:drug effects; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D016685:Mitomycin / Q000633:toxicity; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D010615:Phenacetin / Q000633:toxicity; D011084:Polycyclic Aromatic Hydrocarbons / Q000633:toxicity; D013154:Spleen / Q000187:drug effects
D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D009153:Mutagens; D011084:Polycyclic Aromatic Hydrocarbons; D016685:Mitomycin; D010615:Phenacetin; D003078:Colchicine; D005472:Fluorouracil