Combined Effect of Low-Intensity Helium-Neon Laser and X-Ray Radiation on in Vivo Cellular Response of the Whole Blood and Lymphoid Organs in Mice.

We studied the effect of exposure to helium-neon laser (dose range 0.16-50 mJ/cm2) on activation of natural protection reserve in mice using the adaptive response test. DNA comets method revealed a protective response manifested in DNA damage level in whole blood leukocytes of mice and in lymphoid organs by the thymus and spleen weight index; preexposure to laser did not induce the adaptive response. ROS level in the whole blood was assessed by the level of zymosan-induced luminol chemiluminescence. In mice subjected to adaptive laser irradiation in doses of 0.16-5 mJ/cm2 followed by X-ray irradiation in a dose of 1.5 Gy, the activation index calculated as the ratio of induced to spontaneous area of luminescence was by 1.4 times lower than that in non-irradiated animals, which attested to reduced ROSgeneration reserve capacity of neutrophils.

[Induction of DNA damage in blood leucocytes and of cytogenetic injuries in bone marrow polychromatic erythrocytes in mice exposed to low-LET and high-LET radiation and in their progeny].

The present work was aimed at studying the molecular and cellular levels of the response of the hematopoietic system in mice and their progeny to the action of low-LET and high-LET radiation at different times after exposure. The damage to the genome at the molecular level was assessed by the comet assay in peripheral blood leucocytes, whereas at the cellular level it was estimated by means of the micronuclear test in the marrow cells, after exposure of mice to X-radiation of 1, 3 and 5 Gy and to a high-LET low-intensity radiation at thedoses of 0.14 and 0.35 Gy, as well as to a combined effect of these types of radiation. When accessing the level of the DNA damage to individual cells by the comet assay, we also used, apart from a commonly accepted parameter %TDNA, additional characteristics: the proportions of leucocytes with an intact and highly fragmented DNA. Using these parameters, we detected the changes characterizing the dynamics of the leukocyte population in mouse blood at different times after the action of X-ray and high-LET radiation. It was found that: (1) the DNA damage increases with the dose of high-LET radiation; (2) the level of damage in the progeny of the animals exposed to high-LET radiation does not differ from that in unirradiated animals both at the molecular and cytogenetic levels; and (3) a decrease in the radiosensitivity of the progeny of the mice exposed to high-LET radiation at a dose of 0.35 Gy makes itself evident only at the molecular level, which may point to the possible transgeneration transmission of genomic lesions.

A method of low-temperature storing of agarose slides with lysed cells.

A method has been developed for a long-term low-temperature storage (-10 to -15°C) of the agarose slides with nucleoids (lysed eukaryotic cells). After lysis of agarose-immobilized cells, the slides were incubated for 30 min in phosphate buffer with 50% glycerol and 100 mM EDTA, thereupon they were stored in a freezer at -10 to -15°C. After long-term storage, the slides were re-incubated for 30 min in lysing solution. The measurements of the baseline and in vitro induced DNA damage in nucleoids of the human and mouse leukocytes, which had been stored in agarose slides at low temperature, showed that DNA damage level determined after a 40-day storage did not significantly differ from that of the fresh slides. The advanced storage method is simple and reliable; it opens the way to avoid cryopreservation of the biological samples and to process little by little a great number of the identically prepared slides.

DNA-damaging effects of dental bleaching agents.

We studied DNA-damaging effects of dental bleaching systems containing hydrogen peroxide and/or carbamide peroxide by the "comet assay" (alkaline version). Dental bleaching systems in a hydrogen peroxide concentration range from 0.03 to 30 mM produced a genotoxic effect on isolated HeLa cells in vitro comparable with the effects of pharmacopoeial hydrogen peroxide or urea peroxide. Catalase protected the cells against products containing hydrogen peroxide and had no effect on the genotoxicity of samples containing carbamide peroxide.

Changes in the level of spontaneous DNA damage in whole blood leukocytes during storage.

We evaluated structural damage to DNA (%TDNA) in blood leukocytes from healthy donors of different age at different periods (0-6 days) of blood storage at 4-8°C. It was found that the basal level of DNA damage increased and intracellular antioxidant level decreased during storage. Mean %TDNA was 6.8±3.3% in the fresh blood and 19.2±8.1% after 5-day storage. The experiments with exposure to reactive oxygen species induced by irradiation suggest that depletion of low-molecular-weight endogenous antioxidants occurs as soon as after 5-h storage. Our results suggest that storage time should be taken into account when assessing the basal and induced levels of leukocyte DNA damage by the comet assay.

[Responses of thymocytes and splenocytes to low-intensity extremely high-frequency electromagnetic radiation in normal mice and in mice with systemic inflammation].

Changes in T cell subsets and expression of cytokine genes in thymocytes and splenocytes after exposure of BAL/c mice to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, exposure duration 20 min) under normal conditions and in systemic inflammation were studied using flow cytometry and the methods of reverse transcription and real-time polymerase chain reaction. It was found that the number of CD4+ and CD8+ T cells statistically significantly increased in the thymus and considerably decreased in the spleen of exposed animals. Apparently, the exposure of animals leads to an intensification of the host defense, by activating the T-cellular immunity. As for effector functions, the increased expression of IL-1beta and IFNgamma genes in thymocytes and essentially enhanced expression of IL-1beta, IL-10, and TNFalpha genes in splenocytes were observed in mice exposed against the background of a progressive inflammatory process. The experimental data obtained specify that the directed (anti-inflammatory) response of an organism to a specific combination of effective exposure parameters of electromagnetic radiation can be realized by the activation of particular immunocompetent cells and changes in the cytokine profile.

[The comet assay application in radiobiological investigations].

The analysis of the literature data on application of the gel electrophoresis of individual cells ("comet assay") in radiobiological investigations was carried out. The descriptions of various variants of the method are presented; its alkaline version is in more detail considered. The works concerning to induction and DNA damage repair of single stranded and double stranded DNA breaks, DNA alkali labile sites, crosslinks, DNA bases damage, cellular radiosensitivity and revealing of apoptotic cells were analyzed. The application of the method at biomonitoring of DNA damage level in cells of the person and the animals exposed to genotoxic agents, including ionizing radiation is described. The analysis of the literary data testifies to perceptivity of development and further uses of this method in radiobiological researches.

Spontaneous DNA damage in peripheral blood leukocytes from donors of different age.

Spontaneous DNA damage in peripheral blood cells was studied in healthy donors of different age (23-70 years). Alkaline comet assay was used to evaluate total DNA damage in individual cells. The individual variability in venous blood samples was higher than in capillary blood samples. The advantage of analysis of DNA damage in nucleated cells from the whole blood is more preferable compared to experiments with isolated lymphocytes because all cell populations in the sample are analyzed. Study of blood cells from healthy donors showed that the mean percent of DNA in the comet tail tended to decrease with age. However, correlation analysis revealed no relationship was found between donor age and degree of spontaneous DNA damage.

DNA damage in human mononuclear cells induced by bacterial endotoxin.

Damage to nuclear DNA in human peripheral blood mononuclear cells was studied after in vitro treatment with bacterial endotoxin by alkaline comet assay. It was found that LPS induced DNA damage as soon as over the first 30 min of incubation, while by the 4th hour of incubation DNA damage was found in more than 95% cells. Exogenous superoxide dismutase completely protected DNA, which suggests that superoxide radical is the primary extracellular damaging agent. Polyphenol antioxidant (water-soluble lignin) and specific NADPH oxidase inhibitor (diphenyleneiodonium chloride) also produced a protective effect. Our results show that LPS-activated mononuclear cells can be used ex vivo as a convenient and adequate experimental system for evaluation of the efficiency of various substances in protection of lymphocyte DNA from the damaging effect of reactive oxygen species of LPS-stimulated monocytes.

Development of genetic instability in mice in response to chronic irradiation simulating high-altitude flight conditions.

The purpose of this work was to study the chronic influence of the high-energy radiation field formed in the atmosphere at an altitude of 10 to 30 km on the level of DNA damage in leukocytes of peripheral blood in mice. The external radiation field (behind the concrete shield) of the U-70 accelerator (Serpukhov, Russia) was used for these studies. This radiation field simulates the components and spectral composition of the high-energy radiation field formed in the atmosphere at an altitude of 10 to 30 km. Two groups of SHK line mice were chronically irradiated with a total dose equivalent to 21.5 and 31.5 cGy. The state of the genome of nucleated blood cells was assessed by the Comet assay (alkaline version) 72 h after completion of chronic irradiation. The level of genome damage in individual peripheral blood leukocytes of irradiated animals was compared with the basal level of DNA lesions in peripheral blood leukocytes of unirradiated control mice. The damage was expressed in %TDNA (the amount of DNA found in the "comet tail" in percent of total DNA in the "comet"). It was found that in mice exposed to the radiation field of the accelerator, the mean value of DNA damage was: %TDNA = 3.88 +/- 0.35% for a dose of 21.5 cGy and % TDNA = 6.00 +/- 0.82% for a dose of 31.5 cGy. In mice irradiated at an X-ray therapeutic device with a dose of 150 cGy 24 h before the examination, %TDNA was 2.27 +/- 0.34% and this did not differ from %TDNA in unirradiated mice, 2.68 +/- 0.56%. We suggest that the increased level of DNA damage observed in mice irradiated with 31.5 cGy from the mixed radiation field at the Serpukhov accelerator points to the development of genetic instability in their leukocytes as a result of chronic exposure of animals to this particular radiation field.

Peculiarities of the effect of low-dose-rate radiation simulating high-altitude flight conditions on mice in vivo.

In the present work, the effect of a low-dose rate of high-LET radiation in polychromatic erythrocytes of mice bone marrow was investigated in vivo. The spectral and component composition of the radiation field used was similar to that present in the atmosphere at an altitude of about 10 km. The dose dependence, adaptive response, and genetic instability in the F1 generation born from males irradiated under these conditions were examined using the micronucleus test. Irradiation of the mice was performed for 24 h per day in the radiation field behind the concrete shield of the Serpukhov accelerator. Protons of 70 GeV were used over a period of 15-31 days, to accumulate doses of 11.5-31.5 cGy. The experiment demonstrated that irradiation of mice in vivo in this dose range leads to an increase in cytogenetic damage to bone marrow cells, but does not induce any adaptive response. In mice pre-irradiated with a dose of 11.5 cGy, an increase in sensitivity was observed after an additional irradiation with a dose of 1.5 Gy. The absence of an adaptive response suggests existence of genetic instability.

Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses.

Currently, the potential genotoxicity of high power microwave pulses (HPMP) is not clear. Using the alkaline single cell gel electrophoresis assay, also known as the alkaline comet assay, we studied the effects of HPMP (8.8 GHz, 180 ns pulse width, peak power 65 kW, pulse repetition frequency 50 Hz) on DNA of human whole-blood leukocytes and isolated lymphocytes. The cell suspensions were exposed to HPMP for 40 min in a rectangular waveguide. The average SAR calculated from the temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The steady-state temperature rise in the 50 microl samples exposed to HPMP was 3.5 +/- 0.1 degrees C. In independent experiments, we did not find any statistically significant DNA damage manifested immediately after in vitro HPMP exposure of human blood leukocytes or lymphocytes or after HPMP exposure of leukocytes subsequently incubated at 37 degrees C for 30 min. Our results indicate that HPMP under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites, and incomplete excision repair sites, which could be detected by the alkaline comet assay.

[Increase of mitochondrial DNA copies with low level of DNA repair in tissue cells of gamma-irradiated mice].

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.

[The individual differences in responses of cancer patient blood cells on radiation treatment during the chemotherapy].

A comparative comet-assay study of X-ray influence on DNA of leukocytes of peripheral blood from both cancer patients in the course of chemotherapy and on healthy donors was carried out. The amount of DNA registered in comet tails of blood samples from 18 healthy donors was between 0.8-3.6%. The mean value was 2.9 +/- 0.5%. In the preparations of cancer patients, an increase in comet tail DNA was observed for each chemotherapy course and in each subsequent course compared to the previous one. The individual variations were found in the level of DNA damage in the response to the administration of cyclophosphane, of methotrexate, of 5-fluorourocil (CMF protocol). The X-ray radiation (4 Gy) challenge test of blood cells showed an increase in comet tail DNA, the dynamics of radiation-induced lesions varying between individuals. The combined use of X-ray radiation and of the comet-assay in evaluating the capacity of the defence systems of the whole blood cells during chemotherapy let us to hold the monitoring of the state of genome of leukocytes without their isolation. This approach enables additional information on leukocyte genome to be rapidly obtained.

[The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific]. / Tkanespetsificheskii kharakter povysheniia variabel'nosti mikrosatellit-assotsiirovannykh povtorov v genome potomstva gamma-obluchennykh samtsov myshei.

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.

[Effects of low-intensity extremely high frequency electromagnetic radiation on chromatin structure of lymphoid cells in vivo and in vitro]. / Vliianie nizkointensivnogo krainevysokochastotnogo élektromagnitnogo izlucheniia na strukturu khromatina limfoidnykh kletok in vivo i in vitro.

Using a comet assay technique, it was shown for the first time that low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) in vivo causes oppositely directed effects on spatial organization of chromatin in cells of lymphoid organs. In 3 hrs after single whole-body exposure of NMRI mice for 20 min at 42.0 GHz and 0.15 mW/cm2, an increase by 16% (p < 0.03 as compared with control) and a decrease by 16% (p < 0.001) in fluorescence intensity of nucleoids stained with ethidium bromide were found in thymocytes and splenocytes, respectively. The fluorescence intensity of stained nucleoids in peripheral blood leukocytes was not changed after the exposure. The exposure of cells of Raji hunan lymphoid line and peripheral blood leukocytes to the EHF EMR in vitro induced a decrease in fluorescence intensity by 23% (p < 0.001) and 18% (p < 0.05), respectively. These effects can be determined by changes in a number of physiological alkali-labile sites in DNA of exposed cells. We suggested that the effects of low-intensity EHF EMR on the immune system cells are realized with the participation of neuroendocrine and central nervous systems.

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

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of gamma-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 gamma-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 F1 generations.

[Computer analysis of band variability in DNA fingerprints]. / Komp'iuternyi analiz variabel'nosti polos na DNK-fingerprintakh.

Through the example of the distribution of PCR products DNA matrices of mouse tail tissue, a method of comparative analysis of DNA fingerprints is described. The PCR products were obtained using a 20-mer random primer flanking the Atp1b2 locus on mouse chromosome 11. A software program was designed that permits the simplification of comparison of DNA fragments variability or polymorphism detected on electrophoregrams from different individuals.

[Study of genome instability using DNA fingerprinting of the offspring of male mice subjected to chronic low dose gamma irradiation]. / Issledovanie nestabil'nosti genoma metodom analiza fingerprintov DNK potomstva samtsov myshei, podvergnutykh khronicheskomu gamma-oblucheniiu v malykh dozakh.

By a polymerase chain reaction with an arbitrary primer (AP-PCR), the possibility of transmission of genome instability to somatic cells of the offspring (F1 generation) from male parents of mice exposed to chronic low-level gamma-radiation was studied. Male BALB/c mice 15 days after exposure to 10-50 cGy were mated with unirradiated females. Biopsies were taken from tale tips of two month-old offspring mice and DNA was isolated. The primer in the AP-PCR was a 20-mer oligonucleotide flanking the microsatellite locus Atp1b2 on chromosome 11 of the mouse. A comparative analysis of individual fingerprints of AP-PCR products on DNA-templates from the offspring of irradiated and unirradiated male mice revealed an increased variability of microsatellite-associated sequences in the genome of the offspring of the males exposed to 25 and 50 cGy. The DNA-fingerprints of the offspring of male mice exposed to chronic irradiation with the doses 10 and 25 cGy 15 days before fertilization (at the post-meiotic stage of spermatogenesis) showed an increased frequency of "non-parent bands". The results of the study point to the possibility of transmission to the offspring somatic cells of changes increasing genome instability from male parents exposed to chronic low-level radiation prior to fertilization.