Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells.
In this paper, we describe the evaluation of an automated in vitro micronucleus assay using CHO-K1 cells in 96-well plates. CHO-K1 cells were pre-loaded with a cell dye that stains the cytoplasm, after which the cells were treated with the test compounds for either 3h (for the +S9 condition) or 24h (for the -S9 condition). A total of 10 concentrations were tested, of which the top five concentrations were scored (limited by either cytotoxicity or solubility). At the end of the incubation period the cells were fixed and their DNA was stained with Hoechst. The visualization and scoring of the cells was done using an automated fluorescent microscope coupled with proprietary automated image analysis software provided by Cellomics (Pittsburg, PA). A total of 46 compounds were used in this evaluation, including 8 aneugens and 25 clastogens with varied mechanisms of action. Thirteen non-genotoxic compounds were also included. The automated scoring had a sensitivity of 88% and a specificity of 100%, with a predictive value positive of 100% and a predictive value negative of 76%, compared to data from the literature that was obtained with manual scoring. We also describe the incorporation of a metabolic activation system using rat liver S9 homogenates, and the use of cell number counts as a cytotoxicity index which is complementary to the CBPI- (cytokinesis-block proliferation index) based index. Finally, we also discuss the potential for artefactual findings due to fluorescent precipitate, which should be carefully monitored to prevent false positive results. In conclusion, the automated in vitro micronucleus scoring is a valid alternative to the manual scoring of slides, and it has the advantage of generating data in a rapid and consistent manner, and with low compound requirements, which makes it well suited as a screening assay in the early stages of compound development.
Diaz
Dolores
D
Cerep, Inc., 15318 NE 95th Street, Redmond, WA 98052, USA. d.diaz@cerep.com
Scott
Andrew
A
Carmichael
Paul
P
Shi
Wei
W
Costales
Chester
C
D000818:Animals; D001711:Biotransformation; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D007091:Image Processing, Computer-Assisted; D015162:Micronucleus Tests / Q000379:methods*; D008856:Microscopy, Fluorescence; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity