Evaluation of a tissue homogenization technique that isolates nuclei for the in vivo single cell gel electrophoresis (comet) assay: a collaborative study by five laboratories.
We evaluated a tissue homogenization technique that isolates nuclei for use in the in vivo comet assay. Five laboratories independently tested the technique using the liver, kidney, lung, spleen, and bone marrow of untreated and mutagen-treated male CD-1 mice. The direct mutagen methylmethanesulfonate (MMS) or the promutagen diethylnitrosamine (DEN) were injected intraperitoneally at maximum tolerated doses. Three and twenty-four hours later, the organs were removed and, except for bone marrow, were minced and homogenized and a nuclear suspension was prepared. The nuclear suspensions and bone marrow cells were used in the comet assay. None of the nuclear suspensions from the non-treated mice induced a positive response. All nuclear suspensions derived from the MMS-treated mice and those of the liver, kidney, and lung from DEN-treated mice induced positive responses in all the laboratories similarly. Reproducibility was demonstrated by five replicate studies in one laboratory. Furthermore, the organ-specific responses to MMS and DEN reflected the characteristic genotoxicity of the chemicals. We concluded from these results that the homogenization technique is a valid one to be used for mouse organs in the in vivo comet assay.
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1-6, 2-chome, KashimaKasima, Yodogawa-ku, Osaka 532, Japan. youichi_miyamae@po.fujisawa.co.jp
Y
Y
Miyamae
M
M
Yamamoto
Y F
YF
Sasaki
H
H
Kobayashi
M
M
Igarashi-Soga
K
K
Shimoi
M
M
Hayashi
D000818:Animals; D001853:Bone Marrow / Q000187:drug effects / Q000648:ultrastructure; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004052:Diethylnitrosamine / Q000633:toxicity; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004996:Ethidium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007668:Kidney / Q000187:drug effects / Q000648:ultrastructure; D008099:Liver / Q000187:drug effects / Q000648:ultrastructure; D008168:Lung / Q000187:drug effects / Q000648:ultrastructure; D008297:Male; D008741:Methyl Methanesulfonate / Q000633:toxicity; D051379:Mice; D008856:Microscopy, Fluorescence; D009153:Mutagens / Q000633:toxicity; D015203:Reproducibility of Results; D013154:Spleen / Q000187:drug effects / Q000648:ultrastructure
D005456:Fluorescent Dyes; D009153:Mutagens; D004052:Diethylnitrosamine; D008741:Methyl Methanesulfonate; D004996:Ethidium