A comparison of the in vitro Comet assay with the in vitro chromosome aberration assay using whole human blood or Chinese hamster lung cells: validation study using a range of novel pharmaceuticals.
The in vitro Comet assay, a sensitive, quick and relatively cheap test, could become a valid alternative to the commonly used in vitro chromosomal aberration test, in the preliminary evaluation of new chemical entities early in the development of new pharmaceuticals. A validation of the Comet assay procedure using whole human blood or CHL cells was carried out in comparison with a cytogenetic test utilizing the same target cells with the following compounds which demonstrated positive results in standard chromosomal aberration tests: two well-documented clastogens, methyl methanesulphonate and cyclophosphamide, and eight novel drugs in early development. A 3 h exposure time, in both the absence and presence of metabolic activation, was used for the in vitro Comet assay. Agreement between the results of the Comet assay and the chromosomal aberration tests was found to be satisfactory on a qualitative basis, although positive results in the Comet assay were always at higher doses than in the cytogenetic test. This indicates a reduction in sensitivity using the former genotoxicity end-point. In order to try to explain this observation, a range of exposure times (0.25, 0.5, 1, 2 and 3 h) were investigated in two further experiments to determine the optimal time for detecting Comet induction in this assay procedure. Maximum levels of DNA damage (in terms of Comet induction) were recorded at earlier sampling times (0.25-1 h) in whole human blood using the same positive doses observed in HPLT. Further studies need to be performed to confirm these findings. It is possible that strand breaks are too short lived to allow detection after a 3 h treatment period (due to preferential repair), indicating the need for shorter exposure times in some cases to optimize their detection.
Giannotti
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E
Genetic Toxicology Laboratory, GlaxoSmithKline SpA, Via Alessandro Fleming 4, 37135 Verona, Italy. elg62175@gsk.co.uk
Vandin
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L
Repeto
P
P
Comelli
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R
D000818:Animals; D001769:Blood / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D002869:Chromosome Aberrations / Q000187:drug effects*; D002875:Chromosomes / Q000187:drug effects; D020552:Comet Assay; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D066298:In Vitro Techniques; D007769:Lactams / Q000378:metabolism / Q000494:pharmacology*; D008168:Lung / Q000737:chemistry*; D008214:Lymphocytes / Q000187:drug effects*; D008297:Male; D008677:Metaphase / Q000235:genetics*; D009152:Mutagenicity Tests; D051381:Rats; D017208:Rats, Wistar
D007769:Lactams