Cell survival after DNA damage in the comet assay

Abstract

The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H₂O₂) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H₂O₂ or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.

Keywords: Cell death and comet assay; DNA damage; DNA repair.

Fig. 1

DNA strand breaks induced by 60 µM H₂O₂ over time in alkaline comet assay. *p ≤ 0.05 vs. Control at the same time point

Fig. 2

DNA strand breaks induced by 200 µM MMS over time in alkaline comet assay. *p ≤ 0.05 vs. Control at the same time point

Fig. 3

DNA strand breaks induced by 1 µM Etoposide over time in alkaline comet assay. *p ≤ 0.05 vs. Control at the same time point

Fig. 4

Concentration dependent increase in DNA damage after 0.5 h H₂O₂ treatment. *p ≤ 0.05 vs. 0 µM H₂O₂

Fig. 5

Concentration dependent increase in DNA damage after 4 h MMS treatment. *p ≤ 0.05 vs. 0 µM MMS

Fig. 6

Concentration dependent increase in DNA damage after 4 h etoposide treatment. *p ≤ 0.05 vs. 0 µM etoposide

Fig. 7

Correlation between the percentage of lost/apoptotic cells and the percentage of DNA in tail. (A) Correlation graph for H₂O₂ treatment. (B) Correlation graph for MMS treatment. (C) Correlation graph for etoposide treatment. (D) Correlation graphic for all three substances. Spearman correlation analysis was performed for each substance and the results showed a significant positive correlation between the percentage of lost/apoptotic cells and the percentage of DNA in tail

Fig. 8

DNA damage reduction after H₂O₂ treatment over time in the comet assay. Treatment duration was 0.5 h and DNA damage was measured after medium change. *p ≤ 0.05 vs. Control at the same time point and ∆ ≤ 0.05 vs. 60 µM H₂O₂ at 0 h

Fig. 9

DNA damage reduction after MMS treatment over time in the comet assay. Treatment duration was 4 h and DNA damage was measured after the medium change. *p ≤ 0.05 vs. Control at the same time point and ∆ ≤ 0.05 vs. 200 µM MMS at 0 h

Fig. 10

DNA damage reduction after etoposide treatment over time in the comet assay. Treatment duration was 4 h and DNA damage was measured after the medium change. *p ≤ 0.05 vs. Control at the same time point and ∆ ≤ 0.05 vs. 1 µM etoposide at 0 h