Azhub I. Gaziev

Mitochondrial permeability transition triggers the release of mtDNA fragments

Abstract.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.

Dietary· supplements of antioxidants reduce hprt mutant frequency in splenocytes of aging mice

The level of spontaneous and gamma-radiation-induced mutations in the hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus as well as the decrease in frequency of these mutations in mice of various age pretreated with dietary supplements of an antioxidant mixture (vitamins C, E, beta-carotene, rutin, selenium, zinc) were studied in splenocytes of young (8-14-week-old) and aged (102-110-week-old) male C57BL/6 mice. The frequency of spontaneous mutations in splenocytes of 102-110-week-old mice was higher by 68-88% than that in mice aged 8-14 weeks. On gamma-irradiation (0.5-5.0 Gy) of mice, the frequency of radiation-induced mutations (Vf assay) in aged mice was 2.3 to 3.6 times (depending on dose) higher than in young ones. Daily supplements of an antioxidant mixture to the diet of mice prior to irradiation showed an antimutagenic effect. The values of mutant frequency reduction factor (MFRF) for 14-110-week-old mice fed with dietary antioxidants during 6 weeks prior to gamma-irradiation with doses of 2.0 and 5.0 Gy were 5.4 and 3.7, respectively. The frequency of radiation-induced mutations prevented or not prevented by antioxidants was much higher in aged mice than in young ones.

The prolongation of survival in mice by dietary antioxidants depends on their age by the start of feeding this diet

The effect of daily dietary supplements of an antioxidant mixture (AM) consisting of beta carotene, alpha tocopherol, ascorbic acid, rutin, selenium, and zinc on the survival of male C57BL/6 mice starting at 2, 9, 16, and 23 months of age was investigated. The survival of mice given AM starting at 2 and 9 months of age was found to increase significantly (from 86 to 108 days) compared to the control. The times, of 50, 90, and 100% mortality in mice given AM starting at 2 and 9 months of age increased by 16-9.5% compared to the control, whereas in mice given AM, starting at 16 and 23 months of age, no effect was observed.

The effect of dietary supplements on gene expression in mice tissues

Exposure of living organisms to various environmental stresses induces the synthesis of so-called shock/stress proteins; many of them can provide either immediate stress protection or participate in cellular repair processes. In the present study we focused our attention on the potential effect of dietary vitamins and microelements with antioxidant properties on stress protein gene expression. The analysis of gene expression in tissues of antioxidant-fed mice shows hsp-70 gene overexpression in liver and brain, but not in spleen and lung. Heat shock significantly induces gene expression that is less pronounced in antioxidant-fed animals in all analyzed tissues. Under conditions of oxidative stress, accumulation of lipid peroxidation products in liver homogenates is partially suppressed in mice subjected to heat shock, and significantly inhibited in antioxidant-fed mice and in antioxidant-fed mice subjected to heat shock. The glutathione content in liver homogenates of antioxidant-fed mice is higher than in the control group. Heat shock decreases the level of endogenous glutathione in both groups of animals, but it is still higher in the liver homogenate of antioxidant-fed mice. Thus, dietary supplements can modify gene expression induced by heat shock in vivo and protect rat tissues against oxidative stress by enhancing the level of endogenous antioxidants and inducing hsp-70 gene expression.

Mitochondrial function and mitochondrial DNA maintenance with advancing age

We review the impact of mitochondrial DNA (mtDNA) maintenance and mitochondrial function on the aging process. Mitochondrial function and mtDNA integrity are closely related. In order to create a protective barrier against reactive oxygen and nitrogen species (RONS) attacks and ensure mtDNA integrity, multiple cellular mtDNA copies are packaged together with various proteins in nucleoids. Regulation of antioxidant and RONS balance, DNA base excision repair, and selective degradation of damaged mtDNA copies preserves normal mtDNA quantities. Oxidative damage to mtDNA molecules does not substantially contribute to increased mtDNA mutation frequency; rather, mtDNA replication errors of DNA PolG are the main source of mtDNA mutations. Mitochondrial turnover is the major contributor to maintenance of mtDNA and functionally active mitochondria. Mitochondrial turnover involves mitochondrial biogenesis, mitochondrial dynamics, and selective autophagic removal of dysfunctional mitochondria (i.e., mitophagy). All of these processes exhibit decreased activity during aging and fall under greater nuclear genome control, possibly coincident with the emergence of nuclear genome instability. We suggest that the age-dependent accumulation of mutated mtDNA copies and dysfunctional mitochondria is associated primarily with decreased cellular autophagic and mitophagic activity.

PCR assay of DNA damage and repair at the gene level in brain and spleen of γ-irradiated young and old rats

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.

Modifying effect in vivo of interferon alpha on induction and repair of lesions of DNA of lymphoid cells of gamma-irradiated mice.

The induction of structural lesions and repair in DNA of lymphoid cells from the peripheral blood, spleen and thymus of mice treated with natural mouse interferon alpha (IFN-alpha) 24 and 48 h prior to gamma irradiation were studied using the comet assay and apurinic-apyrimidinic (AP) site radiolabeling. It was demonstrated that the radiation-induced damage assessed by the comet assay in the DNA of peripheral blood lymphocytes (PBLs), splenocytes and thymocytes of mice treated with IFN-alpha before irradiation was considerably less and was repaired more easily in the postirradiation period than that in untreated mice. The DNA of PBLs and splenocytes from interferon-treated mice showed a decrease in the spontaneously occurring and radiation-induced AP sites, as determined immediately and 90 min after irradiation, compared to the level of AP sites in the DNA of untreated mice. The results lead us to assume that IFN-alpha activates the DNA repair systems in the cells of lymphoid tissue.

Gamma Irradiation or Hydrocortisone Treatment of Rats Increases the Proteinase Activity Associated with Histones of Thymus Nuclei

An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after gamma irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiotreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis that several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats.

AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level γ-radiation

Abstract By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F 1 generation) of male mice chronically exposed to low-doses of γ-radiation was investigated. Male BALB / c mice exposed to 10–50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of “non-parental bands” in DNA-fingerprints from the progeny of males chronically exposed to γ-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F 1 generations.

Increased genomic instability in somatic cells of the progeny of female mice exposed to acute X-radiation in the preconceptional period

The level of genome instability (GI) was studied in the progeny of female mice exposed in the preconceptional period to radiation doses of 0.5, 1, and 2 Gy in comparison to that in the progeny of the same parent pairs born before irradiation of the females. To assess the level of genome instability, we analyzed polymorphism of DNA fragments from postmitotic (blood and brain) and proliferating (spleen and tail tip) tissues amplified by AP-PCR (PCR amplification with an arbitrary primer). It was found that polymorphism of the spectrum of AP-PCR products, which is a multilocus genetic marker (MGM), in the genome of somatic cells in the progeny of female mice exposed to 2 Gy was higher than in the progeny of male mice exposed to the same doses. In the progenies of female mice born before and after irradiation, tissue-specific variations in the level of DNA polymorphism were detected. The maximum value of this polymorphism (with respect to the frequency of “nonparental bands”) was determined for peripheral blood DNA in comparison with the other tissues. Estimations of the MGM polymorphism with the AP-PCR method demonstrate an increased level of genome instability in somatic cells of offsprings from female mice exposed to a single acute dose of X-rays (0.5, 1, and 2 Gy) in the preconceptional period. Radiation-induced transgenerational genome instability with an increase in the dose of preconceptional irradiation of female mice was more pronounced in DNA of the postmitotic tissues (blood and brain DNA) than in DNA of the proliferating tissues (spleen and tail tip epithelium).