Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells.

Abstract

The induction kinetics of genetic damage were measured in one clone of a mammalian cell line (CHO AS52) with three genotoxicity assays, the single cell gel electrophoresis (Comet) assay, laser beam flow cytometry and forward mutation. The first two assays allow for the rapid analysis of genotoxic damage in individual nuclei. The alkaline Comet assay detects DNA strand breaks, alkali-labile sites and incomplete excision repair sites. Flow cytometry measures chromosome damage that results in an unequal distribution of nuclear DNA in daughter cells. We calibrated these assays to compare acute DNA damage and longer term clastogenicity with forward mutation at the gpt locus using ethyl methanesulfonate (EMS). The EMS treatments were conducted in F12 medium for 2 h. AS52 cells carry a single functional gpt gene which provides for quantitation of gpt mutants by selecting for 6-thioguanine resistance. EMS induced a concentration-dependent response with median Comet tail moment values of 1.06 microns for the negative control and 64.6 microns with 20 mM. The coefficient of variation (CV) of the negative-control with flow cytometry was 2.33; the CV value increased to 4.87 in cells treated with 20 mM EMS, EMS (8 mM) induced a mutant frequency of 779.8 x 10(-6) at a relative survival of 64.4%. Genetic response factors were calculated and the data demonstrate that the induction kinetics of genetic damage as measured by the Comet assay (15.6) and flow cytometry (14.2) were more closely related than that determined for mutation induction (7.9). These three assays measure a wide spectrum of genetic events at the level of DNA, the gene and the chromosome and demonstrate the usefulness of the Comet assay and flow cytometry as two relatively rapid procedures to detect genotoxic damage in mammalian cells.