Evaluation of DNA damage induced by proton and lithium beams in murine melanoma cells

IBAñEZ IL; BRACALENTE C; EDREIRA M; PALMIERI MA; MOLINARI B; POLICASTRO LL; KREINER A; BURLóN A; VALDA A; GARCíA MORILLO MF; DAVIDSON J; DAVIDSON M; VáZQUEZ M; OZAFRáN M; DURáN H

Buenos Aires

Congreso; 12th International Congress of the International Radiation Protection Association; 2008

The aim of the present study was to evaluate the response of melanoma cells to low and high linear energy transfer (LET) radiation, i.e. gamma rays and proton and lithium beams. For this purpose, DNA damage and survival were determined as a function of dose in the mouse melanoma cell line B16-F0. Irradiations were performed with monoenergetic proton (14 MeV, 3.4 keV/µm and 3 MeV, 14 keV/µm) and lithium (7 MeV, 250 keV/µm) beams generated by the Tandem accelerator TANDAR (Buenos Aires, Argentina) and with a 137Cs g source. Cells were irradiated (0-8 Gy) and survival curves were obtained by clonogenic assay and fitted to the linear quadratic model. Radiation-induced DNA damage was evaluated as follows. As a measure of DNA single strand breaks (SSBs) the alkaline single cell gel electrophoresis assay (comet assay) was performed and data of head and tail fluorescence intensity and the tail moment were obtained from digital images of nuclei. DNA double strand breaks (DSBs) were assessed by the detection of phosphorylated histone H2AX (ãH2AX) foci by immunofluorescence. The phosphorylation of H2AX on serine 139 occurs at sites flanking DSBs and is a measure of the number of DSBs per cell. Results showed an increase in the á parameter and a decrease in the â parameter of the survival curves as a function of LET with the disappearance of the shoulder of the curve for high LET radiation. Results of the comet assay showed an increase in DNA SSBs as a function of dose for all the radiations evaluated. The tail moment was the most sensitive parameter. However, no significant differences were found in the induction of SSBs by proton beams of both energies tested as compared to g rays. Results of ãH2AX labeling demonstrated that the number of foci increases as a function of dose. When ãH2AX foci were determined 6 h post-irradiation, a decrease in the number of foci was found for 1 and 2 Gy of g rays and 14 MeV protons, revealing the repair capacity of DSBs for low LET radiation. The comparison of ãH2AX labeling results for low and high LET radiations demonstrated an increase in the number of foci as a function of LET. Moreover, there was an increase in the size of ãH2AX foci in cells irradiated with lithium beams, which could be attributed to the clusters of DSBs induced by high LET radiation. Concluding, our results showed a good correlation between the evaluation of ãH2AX and survival, which is consistent with the fact that DSBs are difficult to repair and may be correlated with cell death.