Abstract
Purpose: We have studied the effect of ascorbic acid on radiation protection and our results have revealed that ascorbic acid cannot suppress the lethal effect induced by radiation, for example, chromosomal aberration, but can effectively suppress the non-lethal effect leading to mutation and cancer (Koyama et al. Mutat. Res. 1998). Now we focused on the induction of DNA double strand breaks, which is the main cause of the lethal effect, induced by radiation and examined whether ascorbic acid can suppress the induction of DNA double strand breaks.
Method: Human embryonic fibroblasts (HE17) cells were used. HE17 cells were cultured and the confluent cultures were treated with ascorbic acid (AsA, 5mM) or dimethyl sulfoxide (DMSO, 2%) for 2 hours and then were irradiated by X-ray. To visualize the site of DNA double strand breaks, immunofluorescence on p53-binding protein-1 (53BP1) foci, which are known to accumulate on this site, was performed. We counted the number of foci formed in nuclei at 15 minutes, 2 hours, and 24 hours after 1.8 Gy X-irradiation, and examined whether treatment with AsA or DMSO can decrease the number of foci. We counted the number of foci per 200 cells from each treatment group and evaluated using the average number of foci. Moreover, we examined the effect of AsA and DMSO on radiation protection by colony formation assay to confirm the effect of the same treatment as described above on survival fraction.
Results: In non-irradiated groups, there is no effect of AsA or DMSO treatment on the formation of 53BP1 foci at any time after X-irradiation. While in irradiated groups, we observed the number of 53BP1 foci at 15 minutes after X-irradiation as follows: non-treatment, 37.8; AsA treatment, 35.2; DMSO treatment, 27.0, and at 2 hours after X-irradiation as follows: non-treatment, 26.6; AsA, 24.8; DMSO, 17.0. Besides, we observed the number of 53BP1 foci at 24 hours after X-irradiation as follows: non-treatment, 4.0; AsA treatment, 3.6; DMSO, 2.2. These results indicate that treatment with AsA can little suppress the induction of 53BP1 foci formation by X-irradiation, while treatment with DMSO can. In other words, this suggests that AsA cannot suppress the induction of DNA double strand breaks, while DMSO can. In colony formation assay, we also observed that AsA cannot decrease lethal effects, while DMSO can. Taken together, these results suggest that AsA can little induce DNA double strand breaks and cannot decrease lethal damage.
