Optimization of concentrations and exposure durations of commonly used positive controls in the in vitro alkaline comet assay

Abstract

Endogenous and exogenous factors cause DNA damage through chemical changes in the genomic DNA structure. The comet assay is a versatile, rapid, and sensitive method for evaluating DNA integrity at the individual cell level. It is used in human biomonitoring studies, the identification of DNA lesions, and the measurement of DNA repair capacity. Despite its widespread application, variations between studies remain problematic, often due to the lack of a common protocol and appropriate test controls. Using positive controls is essential to assess inter-experimental variability and ensure reliable results. Hydrogen peroxide (H₂O₂) is the most commonly used positive control, while potassium bromate (KBrO₃), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and etoposide are used less frequently. However, differences in concentrations and exposure durations prevent the confirmation of test method efficacy. This study investigates the dose-response relationship for H₂O₂, KBrO₃, MMS, EMS, ENU and etoposide in the comet assay for 30 and 60-minute exposure durations in 3T3 cell lines. Accordingly recommended concentrations and exposure durations were found to be 50 μM 30 minutes (H₂O₂); 500 μM 60 min. (MMS); 10 μM 30 min. (Etoposide); 0.2 mM 30 min. and 2 mM 60 min. (EMS); 2 mM 30 min. (ENU); 500 μM 30 min. and 50 μM 60 min. (KBrO₃). Our findings will contribute to reducing inter-laboratory variability by offering guidance on selecting doses and exposure durations for positive controls in the in vitro alkaline comet assay.

Keywords: Comet assay; Positive controls; Standardization; in vitro.

Fig. 1

The mean tail % intensity (n = 100) ± SEM in 3T3 cells following exposures to H₂O₂ for 30 and 60 min. ^*Statistically significant between control and exposed cells (p < 0.05). ^#Statistically significant between 30 and 60 min of exposure duration (p < 0.05).

Fig. 2

The mean tail % intensity (n = 100) ± SEM in 3T3 cells following exposures to MMS for 30 and 60 min. ^*Statistically significant between control and exposed cells (p < 0.05). ^#Statistically significant between 30 and 60 min of exposure duration (p < 0.05).

Fig. 3

The mean tail % intensity (n = 100) + SEM in 3T3 cells following exposures to etoposide for 30 and 60 min. ^*Statistically significant between control and exposed cells (p < 0.05). ^#Statistically significant between 30 and 60 min of exposure duration (p < 0.05).

Fig. 4

The mean tail % intensity (n = 100) ± SEM in 3T3 cells following exposures to EMS for 30 and 60 min. ^*Statistically significant between control and exposed cells (p < 0.05). ^#Statistically significant between 30 and 60 min of exposure duration (p < 0.05).

Fig. 5

The mean tail % intensity (n = 100) ± SEM in 3T3 cells following exposures to ENU for 30 and 60 min. ^*Statistically significant between control and exposed cells (p < 0.05). ^#Statistically significant between 30 and 60 min of exposure duration (p < 0.05).

Fig. 6

The mean tail % intensity (n=100) ± SEM In 3T3 cells following exposures to KBrO₃ for 30 and 60 min. ^*Statistically significant between control and exposed cells (p < 0.05). ^#Statistically significant between 30 and 60 min of exposure duration (p < 0.05).

Fig. 7

The cell viability for all positive controls at the concentrations and exposure duration given in Table 1 (Eto.: Etoposide).