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The 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 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, 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. The process of mtDNA transcription was more susceptible to irradiation: 1 h after exposure the number of ND2, ND4, and CYTB gene transcripts sharply decreased. At 24 and 72 h after irradiation, ND2 and ND4 transcripts recovered to the control values, while CYTB remained rather low; no significant decrease of ATP6 transcripts was observed within the whole period of observation. The difference in mRNA levels of 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 that there is a posttranscriptional mechanism which regulates expression of mitochondrial genes and provides different recovery rates for different mitochondrial transcripts during the development of apoptosis.

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.

Changes in the number of mitochondrial DNA (mtDNA) copies and alterations in its transcription were followed in the blood cells of mice after their exposure to X-rays; also, extracellular mtDNA fragments were registered in the blood serum of these mice. By a real time polymerase chain reaction (RT-PCR), a sharp increase in mtDNA copy number one hour after the exposure was detected in the blood cells of 1 Gy X-ray irradiated mice. This increase in mtDNA copies is considered as a result of a compensatory reaction developed in mice in response to the radiation damage in a part of mtDNA molecules. Exposure to X-ray radiation at a dose of 10 Gy resulted in an arrest of mtDNA replication in mouse blood cells within three hours after irradiation. At the same time, RT-PCR assays showed that at the same radiation dose, an increase in the number of mtRNA of the mitochondrial nd6 gene relative to mRNA of the nuclear gene gapdh occurs. This indicates that, although mtDNA is more damaged in comparison with nuclear DNA (nDNA), the process of transcription continues in the blood cell mitochondria of mice after their exposure to 10 Gy, whereas the transcription of damaged nDNA is arrested. After the irradiation of mice with 10 Gy, mtDNA fragments of 1841 bp were registered in the blood serum. Thus, in the blood cells of mice exposed to X-rays, the copy number of mtDNA and its transcription are sharply changed, and large mtDNA fragments are observed in the blood serum.

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.

The damage and the change in the number of mitochondrial DNA (mtDNA) copies in brain and spleen tissues of gamma-irradiated mice were studied. The changes in the number of mitochondrial DNA (mtDNA) copies were assayed by the comparative analysis of the density values of long-extension PCR products of the mtDNA fragments (16 kb) and the cluster nuclear gene of beta-globin (8.7 kb). PCRs of mtDNA fragments and the nuclear gene of beta-globin were carried out simultaneously in one test-tube within total DNA. Our results showed that in brain and in spleen cells of mice exposed to gamma-radiation an increase in copy number (polyploidization) of mtDNA with regard to the nuclear gene beta-globin took place. The induction of polyploidization of mtDNA observed in cells of gamma-irradiated animals is regarded as the development of a compensatory reaction because of the energy deficiency due to the increased ATP consumption and structural alteration of genes controlling OXPHOS. The data enabled the assumption that because of the low efficiency of repair systems in mitochondria the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates may be the major mechanism for the retention of the mitochondrial genome which is constantly damaged by the endogenous ROS and is affected by ionizing radiation and/or other exogenous factors.

Fragments of mitochondrial DNA are released from mitochondria upon opening of the mitochondrial permeability transition pore. Cyclosporin A, an inhibitor of pore opening, completely prevented the release of mitochondrial fragments. Induction of mitochondrial permeability transition and subsequent release of the fragments of mitochondrial DNA could be one cause of genomic instability in the cell.

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.

A study was made of the activity of adenylate cyclase and cAMP-phosphodiesterase in rat thymus and liver various time intervals following nonlethal fractionated gamma-irradiation (2 Gy three times at a week interval). There was a positive correlation between the activity of cAMP metabolism enzymes and the radiation modification of ornithine decarboxylase (ODC) observed before. It is suggested that cAMP system is involved in ODC activity regulation in the exposed tissue.

Basal and stimulated activity of adenylate cyclase (cyclizing ATP-pyrophosphate lyase, E.C. 4.6.1.1., AC) in plasma membranes of pulmonary tissue was being studied during a year after fractionated irradiation of rats (2 GyX3). Basal and hormone-stimulated activity of AC was shown to vary significantly from normal 6 and 12 months after irradiation. The exposed membranes responded differently to AC activation by isoproterenol and F-.

Possible reasons for the previously detected inhibitory effect of gamma-radiation (100-1000 Gy in vitro) on isoproterenol-stimulated activity of adenylate cyclase (AC) of the liver plasma membranes of a 20-day-old rat fetus were investigated. It was discovered that the number of beta-adrenergic receptors was reduced. Analysis of basal and fluorine-stimulated activities of AC attests to the radiation-induced damage to catalytic and N-regulatory proteins. The radiation-induced derangement of membrane lipids, where AC functions, is shown.

Ornithine decarboxylase activity in some rat tissues was shown to grow at remote times after gamma irradiation (60Co) with nonlethal doses. The authors suggest that ornithine decarboxylase activity should be used as a biochemical marker of remote effects of ionizing radiation.

The activity of adenylate cyclase (Ac), cAMP phosphodiesterase (PDE) and 5'-nucleotidase was studied in plasma membranes from the liver of rat embryo of the 20th day of development normally and after exposure to ionizing radiation. Gamma-irradiation of plasma membranes with doses ranging from 0.1 to 100 kR was shown to inhibit the activity of Ac, this effect being more pronounced during stimulation with higher doses of isoproterenol. The activity of 5'-nucleotidase and PDE remained unchanged up to the dose of 100 kR.

A change of enzymatic differentiation in the rat liver during the perinatal developmental period after gamma-irradiation on the 7-9th and 19th days of embryogenesis in doses 0.5, 2 and 6 Gr has been shown on the example of glucose-6-phosphatase (G-6-P-ase) and tyrosine aminotransferase (TAT). The protein-synthesizing machinery was not damaged at these doses. The radiation inhibition of G-6-P-ase synthesis was relieved by the injection of thyroxine. A dependence was shown between the radiation increase of TAT activity and changes in cAMP system (increase of cAMP level, decrease of phosphodiesterase activity, intensification of response of adenylate cyclase complex to biogenic amines). A suggestion is put forward that the radiation damage of the enzymes under study is mediated by a change in the number of hormonal inductors.