[Changes of mitochondrial DNA/nuclear DNA ratio in the blood serum following X-ray irradiation of mice at various doses].

Using quantitative real-time PCR, the copy number of mitochondrial and nuclear DNA fragments in mouse blood serum was estimated at different time points following X-ray irradiation at various doses (from 0.5 to 10 Gy). The changes in the correlation between mtDNA and nuclear DNA (mtDNA/nucDNA) in blood serum reflect the degree of radiation injury depending on the dose of irradiation. Exposure to radiation at 10 Gy and massive cell death caused by this lethal dose result in a sharp decrease by an order of magnitude of the mtDNA/nucDNA ratio in the mouse serum; the value of this parameter did not recover within the next 3 days. The opposite effect was revealed when mice were exposed to irradiation at the dose of I Gy, which is not followed by massive cell death, but leads to a higher level of the mtDNA damage as compared with the nuclear DNA protected by histones. Defective mtDNA molecules enter the bloodstream, which results in an increase of the mtDNA/nucDNA ratio in serum. Under irradiation of mice at the intermediate dose of 3 Gy the two processes described above are exhibited at once. During the first hours after irradiation an apoptotic death of radiosensitive cells and release of a large number of nuclear DNA fragments in the serum are initiated, which reduces the mtDNA/nucDNA ratio. However, at later times after irradiation, starting from 5 days, an increase of the mtDNA/nucDNA ratio is observed in the serum, presumably as a result of reparation and elimination of defective mtDNA. Thus, the mtDNA/nucDNA ratio in the serum of irradiated mice reflects the degree of the radiation damage to cells and may be considered as a biological marker of radiation injury in the future.

Mitochondrial DNA transcription in mouse liver, skeletal muscle, and brain following lethal x-ray irradiation.

Using quantitative real-time PCR, the levels of mitochondrial DNA transcripts in murine tissues (skeletal muscle, liver, and brain) were determined at different time points (1, 5, and 24 h) following X-ray irradiation at the dose of 10 Gy. One hour after irradiation the levels of mitochondrial transcripts ND2, ND4, CYTB, and ATP6 dramatically decreased by 85-95% and remained at the same minimum level for 24 h in all analyzed tissues. This decrease was not associated with depletion of mtDNA as a matrix for transcription, since mtDNA copy number increased after irradiation in all tissues. The decrease in mitochondrial transcription in liver, brain, and skeletal muscle did not generally result from the damage of cell transcription apparatus, because the transcription level of nuclear housekeeping gene BETA-ACTIN remained virtually unchanged after irradiation. The mitochondrial gene transcription decreased after irradiation in the same manner as that of the nuclear gene TFB2M encoding mitochondrial transcription factor, whose regulatory role under normal conditions is well understood.

[Mitochondrial genetic apparatus functioning in mice spleen cells under radiation-induced apoptosis].

An important role of mitochondria in the process of programmed cell death is widely accepted now. There is a set of nuclear-encoded mitochondrial proteins involved in this process. Apart from this, a mitochondrion contains its own genetic apparatus comprising mtDNA and replication, transcription, and translation systems. However, a mechanism of mitochondria genetic information realization under apoptosis-inducing conditions has been understood poorly. Here, using the real-time PCR technique the number of mitochondrial genes and their transcripts in mouse spleen cells after whole-body X-ray irradiation at the dosage of 10 Gy has been evaluated. During 5 h after the irradiation a nuclear DNA was subjected to fragmentation, whereas mtDNA remained intact. Moreover, in the course of time after irradiation the number of mtDNA copies increased threefold. A process of mtDNA transcription was more susceptible to the irradiation: in 1 h after exposure the number of ND2, ND4 and CYTB gene transcripts were sharply decreased. In 24 and 72 h after the irradiation the amount of ND2 and ND4 transcripts was restored to the control values, while the CYTB one remained low; the number of ATP6 transcripts was compared with the control within the whole period of observation. The difference in levels of mRNAs for the genes transcribed under the control of the same promoter for mice to be grown both under normal conditions and after x-ray irradiation allows us to propose the existing a posttranscriptional mechanism which regulates expression of mitochondrial genes and provides different recovery rates for different mitochondrial transcripts at the development of apoptosis.