РАДИАЦИОННАЯ БИОЛОГИЯ. РАДИОЭКОЛОГИЯ, 2007, том 47, № 3, с. 297-301
PROCEEDINGS OF THE 4TH INTERNATIONAL WORKSHOP ON SPACE RADIATION RESEARCH = AND 17TH ANNUAL NASA SPACE RADIATION HEALTH INVESTIGATORS' WORKSHOP (Moscow-St.-Petersburg, June 5-9, 2006)
УДК 577.346:577.217:576.32/36
DEVELOPMENT OF GENETIC INSTABILITY IN MICE IN RESPONSE TO CHRONIC IRRADIATION SIMULATING HIGH-ALTITUDE FLIGHT CONDITIONS
© 2007 N. P. Sirota1*, E. A. Kuznetsova1, V. N. Peleshko2
1 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino 2 Institute of High Energy Physics, Russian Academy of Sciences, Protvino
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.
Comet-assay, whole blood, chronic irradiation, high-energy radiation field, genetic instability.
Genetic instability is often observed in human tumors and some individuals born with disorders that are associated with an increased level of spontaneous genomic instability are often cancer prone. In addition to persons with hereditary genetic instability syndromes, genetic instability can develop in individual cells within a healthy human organism when it is subjected to genotoxic influences. Ionizing radiation is a factor capable of inducing genetic instability. Since during high-altitude and long-term cosmic flights the man is subjected to a complex radiation [1], the study of risk of genetic instability development in pilots and spacemen is an important task. Therefore it is necessary to assess genetic instability risks in living organisms under conditions that can at least partially simulate flight conditions.
Radiation-induced genetic instability is defined as an increased rate of acquisition of alterations in the ge-
*Corresponding address: 142290 Pushchino, Moscow Region, ITEB RAS; tel.: (4967) 73-92-34; fax: (4967) 79-05-53; e-mail: sirota@iteb.ru.
nome. The occurrence of genetic instability has been detected by monitoring chromosome aberrations, delayed mutations, instability in DNA nucleotide repeats, cellular transformation as well as cell death [2, 3]. Some studies have monitored the development of clones with genetic instability. In some cases, a characteristic feature of such clones was an increased level of reactive oxygen species [4].
To estimate genetic instability, a number of assays can be used, including counting metaphase chromosome aberrations, micronuclei, or sister chromatid exchanges. These methods are labor-intensive and need highly qualified personnel for accurate analysis and interpretation of results (for instance, micronucleus assay and/or chromosome aberrations test). Recently, the "comet assay", which involves the electrophoresis of individual cells in agarose gels, has been widely used to study and evaluate DNA damage (single- and doublestrand breaks, and alkali labile sites in DNA chains), and DNA repair in order to determine the adverse effects of toxins on DNA [5].
The comet-assay possesses a number of definite advantages: it can be applied to cells of various tissue types, needs a small number of cells, and is suitable for detection of primary DNA lesions in individual cells [5]. The results are obtained several hours after sample collection, and the assay itself is inexpensive [6]. In addition, the comet assay is a simple, sensitive, and reliable laboratory method to assess in vitro genetic instability in molecular-epidemiological studies [7]. It was used successfully to detect genetic instability in 14 generations of Chinese hamster cells [8]. The comet assay was also applied to determine the level of genetic instability in mononuclear cells from the peripheral blood of breast cancer patients [9] and in blood samples from lung cancer patients [10].
In this work we compared the basal level of DNA damage in individual leukocytes of peripheral blood of un-irradiated mice with the level of blood cell DNA lesions in animals irradiated under conditions simulating the high-energy radiation field formed in the atmosphere at an altitude of 10 to 30 km.
MATERIALS AND METHODS
Irradiation. The external radiation field (behind the concrete shield) of the U-70 accelerator (Serpukhov, Russia) was used.
The neutron spectrum behind the concrete wall of the accelerator was measured with a Bonner spectrometer. The mean energy of neutrons was 40 MeV. The radiation field was monitored by an automatic system of radiation control of the accelerator.
The irradiation of animals was monitored by a neutron detector of the radiation control system (RM58), which measured the equivalent dose of neutrons with energies from thermal to 20 MeV, and by thermoluminescent dosimeters, which measured the absorbed dose of photons and protons. The dose of neutrons with energies up to 20 MeV at the point of measurement constituted half of the total dose of neutrons with energies from the thermal to 500 MeV.
Two-month old SHK mice were continuously irradiated: one group for 24 days (absorbed dose 21.5 cGy) and the other for 31 days (absorbed dose 31.5 cGy). Each group contained five animals.
Blood cells. Aliquots (5-15 ^l) of whole peripheral blood taken from animal tail were used. Blood was diluted 5 times with a physiological saline containing 1 mmol/l EDTA.
Comet-assay. The state of the genome of blood nucleated cells was assessed by the comet assay (alkaline version) 72 h after completion of chronic irradiation. Analyses were performed according to [11]. The slides obtained were analyzed under a fluorescent microscope "LUMAM-I3" ("LOMO", Sankt-Peterburg). The images were acquired by a digital camera "Nikon CoolPix 995" (Japan), transmitted to a computer and analyzed by specialized software [12]. Three slides were ana-
lyzed for each animal, and a minimum of 30 to 40 cells were scored on each slide. The damage level of each cell was expressed in %TDNA, the DNA amount found in the "comet tail", as a percentage of the total amount of DNA in the "comet". TDNA was calculated according to K. Konca et al [13].
Data collection, processing and statistical analysis. For each blood sample (control or ionizing radiationexposed), photometry was made on 3 slides. Routinely, images of 30-40 cells per slide were scored and processed. Then values of comet parameters were averaged for 30-40 cells. Mean values and standard errors of the mean (SEM) for each mice were calculated from three slides, mean values and SEM for each series were calculated using mean values for mice (independent experiments). Statistical analysis of data is based on the Student's t test (p < 0.05).
RESULTS AND DISCUSSION
The distributions of cells with respect to their individual levels of DNA damage are given in the Figure. As seen from the data presented, the fraction of cells without DNA damage (defined as a %TDNA of 0%) was 48% in the control (Figure, panel a) and in chronically irradiated animals it was 53.1 and 32.8% for 21.5 and 31.5 cGy, respectively (Figure, panels b and c). The fraction of cells with 0 to 20% TDNA in control animals was 50%, whereas in chronically irradiated animals, it was 44.0% for animals exposed to 21.5 cGy and 60.6% for animals exposed to 31.5 cGy. The irradiated animals showed a small but detectable fraction of cells with more than 20% TDNA, while few if any cells had this phenotype in the un-irradiated mice. Statistical treatment of the data revealed that in chronically-exposed animals, the mean %TDNA was equal to 6.00 ± 0.82% for animals chronically exposed to a dose of 31.5 cGy, which differed significantly from the control (mean %TDNA = 2.68 ± 0.56%, p = 0.011). For the dose 21.5 cGy, an increase in DNA damage (mean %TDNA = 3.88 ± 0.35%) was also observed but the difference from the control level was not statistically significant (p = 0.107).
The data indicate that the chronic irradiation that simulated high-altitude flight conditions resulted in an increase in the cells with elevated levels of DNA lesions. We have some suggestions concerning the origins of this effect. First, it might be due to an inadequate efficiency of DNA repair in some cells and, second, it may reflect the development of genetic instability as detected by an increased level of persistent DNA damage following exposure to chronic low-dose radiation from
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