EVALUATION OF A TISSUE HOMOGENIZATION TECHNIQUE THAT ISOLATES NUCLEI FOR THE IN-VIVO SINGLE-CELL GEL-ELECTROPHORESIS (COMET) ASSAY - A COLLABORATIVE STUDY BY 5 LABORATORIES

We evaluated a tissue homogenization technique that isolates nuclei fo r use in the in vivo comet assay. Five laboratories independently test ed the technique using the liver, kidney, lung, spleen, and bone marro w of untreated and mutagen-treated male CD-I mice. The direct mutagen methylmethanesulfonate (MMS) or the promutagen diethylnitrosamine (DEN ) were injected intraperitoneally at maximum tolerated doses. Three an d twenty-four hours later, the organs were removed and, except for bon e marrow, were minced and homogenized and a nuclear suspension was pre pared. 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-tr eated mice induced positive responses in all the laboratories similarl y. Reproducibility was demonstrated by five replicate studies in one l aboratory. Furthermore, the organ-specific responses to MMS and DEN re flected the characteristic genotoxicity of the chemicals. We concluded from these results that the homogenization technique is a valid one t o be used for mouse organs in the in vivo comet assay. (C) 1998 Elsevi er Science B.V. All rights reserved.