Abstract
Background: X-ray induced formation of micronuclei is generally thought to result from DNA double-strand breaks (DSBs). However, DNA DSBs inhibit the cell cycle progression that is required for micronucleus formation. In order to reconcile this apparent discrepancy, we investigated whether DNA DSBs induced during the G1 phase could lead to micronucleus formation using ascorbic acid (vitamin C).
Method: Human embryonic fibroblasts (HE17) cells were used. HE17 cells were cultured and their confluent cultures were maintained for a week to synchronize their cell cycle at G1 phase, and then were irradiated by X-ray. To visualize the site of DNA DSBs, cells were fixed by formalin at designated periods (between 15 minutes and 24 hours after X-irradiation), and immunofluorescence staining on p53-binding protein-1 (53BP1) foci, which are known to accumulate on this site, was performed. Also, in order to visualize micronucleus formation, cells were fixed by formalin at 24 hours after X-irradiation and DAPI staining was performed. Moreover, we investigated the contents of micronuclei using immunofluorescence staining and fluorescence in situ hybridization (FISH). In this study, we examined the relationship between DNA DSBs and micronucleus formation using two different radical scavengers from the point of view of radioprotection ability. Dimethyl sulfoxide (DMSO) was used as an inhibitor for DNA DSBs, because DMSO can capture irradiation-derived reactive radicals and inhibit cell death. On the other hand, vitamin C was used as an un-inhibitor for DNA DSBs, because vitamin C cannot inhibit lethal effect by X-irradiation.
Results: First, we examined the effect of radical scavengers on radioprotection using colony formation assay. The result showed that treatment with vitamin C (5 mM) before X-irradiation cannot cause radioprotection effect, but treatment with DMSO (2%, 256 mM) can, by comparison of survival fraction with non-treatment controls. Moreover, in evaluation of DNA DSBs using 53BP1 foci, we found that treatment with vitamin C cannot inhibit the number of 53BP1 foci after X-irradiation at all, but treatment with DMSO can significantly do. These results showed that DMSO can inhibit the generation of DNA DSBs via inhibition of reactive radicals during X-irradiation and this inhibition can lead to decrease cell lethal effect, while vitamin C cannot do. Next, we examined the inhibitory effect of radical scavengers on X-ray induced formation of micronuclei. The result showed that treatment with DMSO before X-irradiation (between 0.5 Gy and 2 Gy) cannot inhibit X-ray induced formation of micronuclei, but treatment with vitamin C can do. If X-ray induced formation of micronuclei results from DNA DSBs, treatment with DMSO should inhibit it. Actually, vitamin C can significantly inhibit X-ray induced formation of micronuclei. In addition, we found micronuclei with centromeric signals. Overall, we suggest that abnormalities in the cell division mechanism may have something to do with micronucleus formation after X-irradiation.
