A flow cytometry based in vitro micronucleus assay in TK6 cells--validation using early stage pharmaceutical development compounds.
The micronucleus test (MNT) is a well established test for detecting clastogenic and aneugenic compounds. Despite the assay's advantages, the MNT may produce false positive and false negative results in some conditions. This fact may be related to the underestimation of apoptosis or necrosis, the p53 status of the cell system or the cytotoxicity assay, and the top dose selection. The purpose of our studies was to contribute to the validation efforts of the flow cytometry based MNT. To identify the most reliable cytotoxicity assay for the top dose selection five parameters for relative survival were tested: relative cell count, relative population doubling, trypan blue supravital staining, relative ratio of scored nuclei to latex beads, and ethidium monoazide staining. For all compounds the least sensitive method was the relative cell count and the most reliable was the nuclei/beads ratio. The comparative evaluation of micronuclei induction in TK6 cells, analyzed with microscopy and flow cytometry, was performed with reference compounds and internal Novartis early development compounds with positive, weak positive, equivocal, and negative genotoxic effects. Our data document a good correlation between the MNT results obtained by flow cytometry and by microscopy. The results confirm that the method may be applied for routine testing in the pharmaceutical industry for the tested group of compounds, including compounds which require metabolic activation. However, further validation and miniaturization may be required.
Novartis Institutes for Biomedical Research, Preclinical Safety, Genetic Toxicology and Safety Pharmacology, Basel, Switzerland. magdalena.lukamowicz@novartis.com
Magdalena
M
Lukamowicz
Katherine
K
Woodward
Micheline
M
Kirsch-Volders
Willi
W
Suter
Azeddine
A
Elhajouji
D001564:Benzo(a)pyrene / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003520:Cyclophosphamide / Q000633:toxicity; D005434:Flow Cytometry; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D015162:Micronucleus Tests / Q000379:methods*; D016685:Mitomycin / Q000633:toxicity; D013395:Sucrose / Q000633:toxicity; D014747:Vinblastine / Q000633:toxicity
D001564:Benzo(a)pyrene; D016685:Mitomycin; D013395:Sucrose; D014747:Vinblastine; D003520:Cyclophosphamide; D008741:Methyl Methanesulfonate