Evaluation of EMS-induced DNA damage in the single cell gel electrophoresis (Comet) assay and with flow cytometric analysis of micronuclei.

Abstract

Toxic agents in the environment pose serious threats to ecosystems and to the public health. The single cell gel electrophoresis (SCGE) or Comet assay quantitatively measures genomic damage as DNA strand breaks. The micronucleus (MCN) test is an established assay that measures chromosomal damage. Micronuclei are formed from chromosome fragments or from whole chromosomes that have not undergone mitosis properly. This test is usually conducted microscopically. However, micronuclei can also be analyzed using flow cytometry. Chinese hamster ovary (CHO) cells were exposed to ethylmethanesulfonate (EMS), for 4 h in a total volume of 25 microl. These cells were immediately analyzed for genomic DNA damage by SCGE. In concurrent parallel experiments, CHO cells were treated with EMS in 6-well plates for 4 h, the cells were washed and fresh medium was added. The cells were allowed to grow for 45 to 48 h to express micronuclei. The data demonstrated that both DNA strand breaks and micronuclei were induced in a significant and concentration-dependent manner. There was a significant and high correlation (r = 0.91; P < or = 0.001) between the acute induction of DNA strand breaks and the subsequent generation of micronuclei. These data indicate that using molecular and computer technologies, the genotoxic impact of toxic and environmental agents can be rapidly and comprehensively evaluated in mammalian cell systems.