RESUMO
Investigation of cell-free DNA (cf-DNA) in body fluids, as a potential biomarker for assessing the effect of ionizing radiation on the organism, is of considerable interest. We investigated changes in the contents of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in the urine of X-ray-exposed rats. Assays of cf-mtDNA and cf-nDNA were performed by a real-time PCR in rat urine collected before and after irradiation of animals with doses of 3 and 5 Gy. We also determined the presence of mutations in urine cf-mtDNA, as recognized by Surveyor nuclease. A sharp increase in cf-mtDNA and cf-nDNA in the urine of irradiated rats was observed within 24 h after exposure, followed by a decrease to normal levels. In all cases, the contents of cf-mtDNA fragment copies (estimated by gene tRNA) were significantly higher than those of cf-nDNA estimated by gene GAPDH. A certain portion of mutant cf-mtDNA fragments was detected in the urine of exposed rats, whereas they were absent in the urine of the same animals before irradiation. These preliminary data also suggest that the increased levels of urine cf-mtDNA and cf-nDNA may be a potential biomarker for noninvasive assessment of how the organism responds to ionizing radiation influence.
Assuntos
DNA/efeitos da radiação , DNA/urina , Animais , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Sistema Livre de Células , DNA/genética , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , DNA Mitocondrial/urina , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Ágar , Gliceraldeído-3-Fosfato Desidrogenases/genética , Masculino , Mutação , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Changes in the number of mitochondrial DNA (mtDNA) copies in the brain and spleen tissues of gamma-irradiated (3 Gy) mice were studied by comparative analysis of the long-extension PCR products of mtDNA (15.9 kb) and a fragment of the cluster nuclear beta-globin gene (8.7 kb) amplified simultaneously in one and the same test-tube within total DNA. The analysis showed that, compared to the nuclear beta-globin gene, an increase in mtDNA copy number (polyploidization) took place in the brain and spleen cells of mice exposed to gamma-radiation. This data led to the suggestion that the major mechanism for maintenance of the mitochondrial genome, which is constantly damaged by endogenous ROS and easily affected by ionizing radiation or other exogenous factors, is the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates because the repair systems in the mitochondria function at a low level of efficiency.
