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Rapid revealing of mutations by endonuclease method in tissues and circulating mitochondrial dna after exposed to radiation


A.I. Gaziev, S.A. Abdullaev, V.N. Antipova, N.A. Gulyaeva, L.V. Malakhova, G.O. Shaikhaev, V.G. Bezlepkin

The screening DNA mutations at a level of the whole organism is the major task connected to studying of consequences of radiation expose, other physical and chemical agents, and an estimation genotoxic loаd. We determined mutations in tissues mtDNA of a brain and a spleen, and also in circulating mtDNA of blood plasmas of the mice subjected to radiation exposure. Revealing of mutations was carried out by a high-sensitivity method using CEL I endonuclease specifically cutting DNA on sites with the mismatches bases. The analyses was performed on 8, 14, 28 days after exposure to X-irradiation of mice at a dose of 5 Gy. The presence of mutations was judged from cleavage by CEL I nuclease of heteroduplexes obtained by hybridization of mtDNA PCR products (fragments of ND3 gene and two D-loop regions of different sizes), from tissues of exposed to radiation and control mice. As the control preparations were used heteroduplexes, obtained from PCR products of mtDNA of two unirradiated mice. The results show that heteroduplexes of PCR products of tissue mtDNA from one irradiated and one unirradiated mice were more prone to degradation by CEL I nuclease than those of mtDNA from two unirradiated (control) mice. These data indicates the presence of mutations in sites of mtDNA of the irradiated mice. A maximum degradation of heteroduplexes by CEL I nuclease was observed on day 8 after irradiation of mice. For heteroduplexes of mtDNA isolated on days 14 and 28 after irradiation, the cleavage by CEL I nuclease was significantly less. Results show that in tissues of a brain and a spleen the greatest level of mutant copies of mtDNA (on two sites D-loop and gene ND3) was observed on 8th day after irradiation of mice. Analyses of mutations in tissues mtDNA of mice, the next days after irradiation, have shown gradual decrease of their level by 28 day. Especially sharp decrease of the level of mutant copies of mtDNA to this term is registered in a spleen. The total mtDNA copy number for tissues of mice 8 to 28 days after their irradiation was reduced by 30-40% compared to the control. The share of mutant copies in circulating mtDNA (on the sites of D-loop and gene ND3) of blood plasma was two times higher, than those in mtDNA of tissues of the same mice for 8, 14 and 28 days of the post-radiatiation period. The highest percent of mutant copies circulating mtDNA of plasma and the maximal increase of its bulk contents was registered for 14 day after irradiation of mice. The results permit the suggestion that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the post-radiation period. The large sizes fragments of mutant and wild mtDNA are transfered in blood flow of irradiated animals.The appearance of circulating fragments of mutant copies mtDNA in blood plasma, at the further improvement endonuclease method of their determination, can serve as a sensitive marker for an estimation of radiation injury and others genotoxic influences on organism

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