In vivo response of mouse liver to gamma-radiation assessed by the comet assay.

摘要

The alkaline comet assay was used to measure DNA damage induced in liver cells of mice irradiated with gamma-radiation, as well as the repair competency of these cells. A simplified procedure for the isolation of nuclei from cells in solid tissues was developed. This simplified method allows nuclei to be processed into lysis only 5 min after briefly chilling the tissue to depress any enzymatic activity. The nuclei were spontaneously released by a sharp cut of the tissue and exposure of the cut to a drop of 50 mM sodium-phosphate buffer at pH 7.2, immediately before adding the low melting agarose. Thus, the procedure minimizes time-dependent modification of the endogenous level of damage by reducing additional strand breaks or repair produced during processing. The induction of DNA damage by gamma-radiation behaved as a one-hit event in the liver cells, as there was a positive linear correlation between the radiation dose and the fraction of DNA migrated into the comet tails. The level of DNA damage produced by gamma-radiation was highly significant at doses of 0.5 and 1 Gy. Based on the mean extent of DNA migration, the level of damage was not reduced following only one hour of repair time however, after two hours, there was a significant reduction in DNA migration. To increase the resolution of the statistical analysis, the nuclei of each sample were distributed in five types of comets, according to the percentage of DNA in the tail. To compare the frequency distributions of these types of comets between different experimental situations, a Pearson chi-square statistical analysis was applied. It was found, by this analysis, that the DNA repair which occurred 1 h after 1 Gy of gamma-irradiation is significant and that, after 2 h, more DNA repair occurs, but a significant residual damage still persists when comparing this sample with the control.