Comet assay in HepG2 cells after compound exposure

ABSTRACT: To quantify oxidative DNA damage, we performed the comet assay on maturated HepG2 cells after 24 h of compound exposure. This was done using cells receiving repeated exposure, as well as cells receiving repeated exposure and recovery. The enzyme-modified alkaline comet assay was performed as described in [Collins et al. Nat Protoc. 2023;18(3):929–989]. This was done in the presence or absence of the enzyme DNA-formamidopyrimidine glycosylase (Fpg). At least 50 comets per gel were scored to obtain the median %DNA tail intensity (TI) and the mean of two replicate gels was calculated per sample. The results obtained after incubation without Fpg-enzyme only represent the DNA strand breaks and alkali-labile sites. The difference between the samples treated with Fpg-enzyme and those incubated without was calculated for each sample to derive the net Fpg-sensitive sites (%TI), which represent DNA oxidation. A positive assay control was included by using aliquots of frozen Hela cells that were exposed to 2mM KBrO3 for 1 hour (generating an oxidized DNA substrate) in the same batch analysis. The net Fpg results were normalized using this assay control to correct for inter-assay variation. This study was performed as part of the RISK-HUNT3R project and the PARC project.

ORGANISM(S): Homo sapiens (human)

SUBMITTER: Jeroen Pennings 

PROVIDER: S-BSST2297 | biostudies-other |

REPOSITORIES: biostudies-other