Introduction. Aging is a complex process related with the gradual diminution in cellular and physiological functions. The geroprotective effect of 10 biologically active substances (BASs) – rutin, squalene, kaempferol, biochanin A, ursolic acid, chlorogenic acid, baicalin, mangiferin, quercetin and trans-cinnamic acid and 5 crude extracts (Ginkgo biloba L., Pulmonaria officinalis L., Scutellaria baicalensis Georgi, Hedysarum neglectum Ledeb. and Panax ginseng C.A. Mey) isolated from medicinal plants of Altai Region of Russia were evaluated for their influence on the accumulation of intestinal autofluorescence material (IAM) using Caenorhabditis elegans model.The aim of the study. IAM facilitates age-related decline and is a non-intrusive biomarker of senescence. This study assessed the impact of different bioactive substances in reducing the build-up of IAM using C. elegans model.Materials and methods. Gravid nematodes were synchronized, and then seeded in 96-well plates to develop to L4-stage. Each BAS in 200 µM, 100 µM, 50 µM and 10 µM concentrations and extracts with a tenth, hundredth and thousandth times-dilution were administered to each well in 6 replicates for each treatment group. On incubation days 1, 5, and 15, adult L4 nematodes underwent spectrofluorometric analysis to determine the effect of the BASs and extracts on IAM accumulation.Results. It was found that quercetin, kaempferol, baicalin, mangiferin, G. biloba and P. officinalis extracts exhibited the most profound inhibition of IAM accumulation compared to the control. It was noteworthy that the 10 µM concentration of mangiferin significantly inhibited IAM accumulation in a manner comparable to the 200 µM of baicalin and 100 µM of quercetin. In addition, the crude extracts of G. biloba and P. officinalis respectively exhibited 2.8- and 1.8-fold decrease in IAM accumulation.Discussion. The accretion of IAM is inversely proportional to longevity. Thus, the BASs identified in this study to modulate IAM accumulation could serve as important precursors or active ingredients for the pharmacosynthesis of geroprotective drugs in future research.

Aim: To study the patterns of changes in the number of foci of phosphorylated DNA double-strand break repair proteins H2AX (γH2AX) and ATM (pATM) in cultured human mesenchymal stem cells (MSCs) 1‒48 hours after exposure to X-ray radiation at doses of 40, 80, 160 and 250 mGy. Material and methods: We used the primary culture of human MSCs, obtained from the collection of LLC “BioloT” (Russia). Cells were irradiated using a RUB RUST-M1 X-ray biological unit (Diagnostika-M LLC, Moscow, Russia) equipped with two X-ray emitters at a dose rate of 40 mGy/min (voltage of 100 kV, an anode current of 8 mA, and a 1.5 mm Al filter) and 4 °C temperature. To quantify the yield of γH2AX and pATM foci immunocytochemical staining was carried out with the use of γH2AX and pATM antibody respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results: It was shown that the kinetics of changes in the number of γH2AX foci after irradiation at doses of 160 and 250 mGy and low (40‒80 mGy) doses are significantly different. In contrast to the significant (50‒60 %) decrease in the number of γH2AX foci observed 6 hours after irradiation at doses of 160 and 250 mGy, after irradiation at low doses, no significant decrease in γH2AX foci was observed at this time point. Analysis of the colocalization of γH2AX foci with pATM foci indicates that the mechanisms for maintaining a high number of γH2AX foci 24‒48 hours after low-dose irradiation are ATM independent. A hypothesis has been put forward to explain the phenomenon of maintaining the number of γH2AX foci 24‒48 hours after irradiation in low doses by replicative stress caused by stimulation of proliferation against the background of hyperproduction of free radicals, resulting in additional formation of DNA double-strand breaks and phosphorylation of H2AX by ATR kinase.

Purpose: To evaluate the frequency and spectrum of chromosome aberrations under X-Ray exposure at doses of 80, 250, and 1000 mGy in a human multipotent mesenchymal stromal cell (MMSC) cell line during long-term cultivation. Material and methods: MMSCs were isolated from human gingival mucosa by an enzymatic method and cultured in a serum-free medium. The presence of surface antigens was determined using the method of flow cytometry. The ability of the cell line to differentiate in the osteogenic, adipogenic, and chondrogenic directions was studied using induction media. Authentication was performed by genotyping of polymorphic STR loci, cytogenetic analysis was performed by multicolor fluorescent in situ hybridization (mFISH). Irradiation was carried out on an X-ray biological unit RUB RUST-M1 (Russia) at a dose rate of 40 mGy/min, a voltage of 100 kV, and a current of 0.8 mA. Results: At the first passage after irradiation, a statistically significant increase in the frequency of non-clonal CA compared with the control was recorded at a dose of 80, but not 250 and 1000 mGy. At the late stages of cultivation, the average frequency of breaks per chromosome in the group of non-irradiated cells did not differ from the values obtained after irradiation at doses of 80, 250, and 1000 mGy (p > 0.05). However, in MMSCs irradiated at a dose of 80 mGy, damage occurred more often in pairs of chromosomes 6 and 10, and at a dose of 1000 mGy, in a pair of chromosomes 9. A single irradiation of MMSCs in vitro did not affect the growth and progression of MMSCs characteristic of the studied primary cell line, of clonal cells with chromosome translocations and monosomy X, but led to an increase in the representation of a clone with tetrasomy 8. The total number of random clones with chromosome translocations that arose de novo increased after irradiation at a dose of 1000 mGy. Conclusion: Minor fluctuations in the proportion of cells with non-clonal CA, depending on the dose received in the early stages after irradiation (passage 1–4), disappeared at the later stages of cultivation (passage 8–14). There were no differences in mean frequencies between irradiated and non-irradiated MMSCs, but after irradiation, damage to some chromosomes could occur more frequently than others. A single X-ray irradiation of MMSCs can promote the growth and progression of primary pathological cytogenetic clones, regardless of the dose received, as well as an increase in the total number of de novo cell clones with chromosomal translocations that have arisen. A single X-ray irradiation of MMSCs can promote the growth and progression of primary pathological cytogenetic clones, regardless of the dose received, as well as an increase in the total number of de novo cell clones with chromosomal translocations that have arisen.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at damaged DNA sites, contributing to DNA damage tolerance. The contribution of PrimPol in response to the various types of DNA damage is not fully understood. We obtained the lung carcinoma cells A549 with PRIMPOL knockout and characterized its response to DNA damage caused by hydrogen peroxide, methylmethanesulfonate (MMS), cisplatin, bleomycin and ionizing radiation. Knockout of PRIMPOL reduced the number of proliferating cells and cells in G2 phase after treatment with MMS, caused a more pronounced delay of cisplatin-treated cells in S phase. A significant increase in the proportion of apoptotic cells was noted in PRIMPOL-/- cells in response to ionizing radiation at a dose of 10 Gy, while the proportion of cells prone to necroptosis increased significantly in both parental and knockout cells at any radiation dose. Under conditions of oxidative stress stimulated by hydrogen peroxide, PRIMPOL knockout increased cell viability, measured by the MTT method. The data obtained indicate the involvement of PRIMPOL in modulating stress-adaptive responses to various types of genotoxic stress.

Purpose: Comparative study of changes in the number of foci of DNA (DSB) marker proteins (γH2AX and 53BP1) in human mesenchymal stromal cells (MSCs) incubated with 3H-thymidine or HTO for 24, 48, and 72 h. Material and methods: We used the primary culture of human MSCs of passage 5–6, obtained from the collection of LLC “BioloT” (Russia). A sterile solution of 3H-thymidine or HTO with a specific activity of 100 to 400 MBq/l was added to the nutrient medium and incubated under standard conditions of a CO2 incubator for 24, 48, and 72 hours. To quantify γH2AX foci and the proportion of proliferating cells using antibodies to γH2AX, 53BP1 and Ki67 (a marker protein for cell proliferation), were used, respectively. Statistical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). To assess the significance of differences between samples, Student’s t-test was used. Results: Incubation of MSCs with 3H-thymidine with a specific radioactivity of 100-400 MBq/l in the first 24 hours leads to a dose-dependent increase in the number of γH2AX and 53BP1 foci. With a further increase in the incubation time to 48 h and 72 h, a saturation effect is observed ‒ the number of foci reaches a plateau. A statistically significant increase in the number of γH2AX and 53BP1 foci in MSCs incubated with HTO was observed only in actively proliferating cells during the first 24 h of incubation in a medium with specific radioactivity of 300 and 400 MBq/l, after which, with a decrease in proliferative activity, it decreased to control values. Calculations made on the basis of the results of a quantitative analysis of γH2AX and 53BP1 foci after 24 h of incubation of MSCs with tritium compounds obtained in the course of the work show, that under the influence of 3H-thymidine ~ 6 times more DNA double-strand breaks are induced than under the influence of HTO.

Phenotypic characteristics of human non-small cell lung cancer cells, A549 (p53 wild-type) and H1299 (p53-deficient) as well as their descendants surviving after multifraction X-ray irradiation at a cumulative dose of 60 Gy (sublines A549HR and H1299HR, respectively) were studied before and after additional 2 Gy single dose irradiation. In 24 h after the additional irradiation, we observed a significant increase in the proportion of cells with signs of entosis (by 5 times, p<0.05) and SA-β-gal+ cells (by 1.6 times, p<0.01) in the general population of A549HR cells. In contrast, a significant increase in the proportion of only SA-β-gal+ multinucleated giant cancer cells was revealed in the parental A549 cells. Additional single dose irradiation resulted in a significant (by 1.8 times, p<0.05) increase in the proportion of multinucleated giant cancer cells in H1299HR cells in comparison with their parental H1299 cells. These changes did not correlate with changes in the proportion of entotic cells, because their high basal content in the absence of functional p53 did not change in response to additional single dose irradiation. At the same time, both p53-deficient non-small cell lung cancer cell lines showed a significant (2.9-fold for H1299 and 5.5-fold for H1299HR cells, p<0.001) increase in the proportion of SA-β-gal+ cells in the general population, but not in the multinucleated giant cancer cells population.

Radioresistant sublines of non-small cell lung cancer cells differing in the p53 status were obtained: A549 (p53 wild type) and H1299 (p53 deficient). The exposure to ionizing radiation was carried out using a standard protocol developed on the basis of empirical clinical experience and consisting in exposure in a dose of 2 Gy once a day, 5 days a week up to total dose of 60 Gy. The cells survived after irradiation demonstrated reduced radiosensitivity, as well as changes in differential gene expression in comparison with parental cells. Some differences in the signaling pathways involved in DNA repair were revealed.

Abstract—Studies of the changes in the number of γH2AX foci (a DNA double-strand break protein-marker), and Rad51 foci (a key homologous recombination protein) were conducted on human fibroblast cultures during the 24 hours after exposure to low (80 mGy) and intermediate (250 and 1000 mGy) doses of X-ray irradiation. Based this data, exponential curves that approximated the experimental values were constructed, and the characteristic lifetimes of the γH2AX and Rad51 foci were evaluated using the method of least squares. The ratio of the areas under the curves of changes in the number of Rad51 and γH2AX foci, calculated by the trapezium method, divided by the ratio of the characteristic lifetimes of the Rad51 and H2AX foci was used to evaluate the contribution of homologous recombination in DNA double-strand break repair. It was shown that the contribution of homologous recombination in DNA double-strand break repair during the 24 hours after exposure to 80, 250 and 1000 mGy was approximately 16, 12 and 9% respectively. Thus, the relative contribution of homologous recombination in the DNA double-strand break repair after exposure to a low dose of X-ray irradiation was approximately 1.5 times higher than that after exposure to intermediate doses. Our results suggest that DNA double-strand break repair induced after exposure to 80 mGy of X-ray irradiation is more accurate than after exposure to 250 and 1000 mGy.

We performed a comparative study of the formation of γН2АХ foci (a marker of DNA doublestrand breaks) in human bone marrow mesenchymal stem cells after 24-h incubation with 3 Н-thimidin and tritium oxide with low specific activities (50-800 MBq/liter). The dependence of the number of γH2AX foci on specific activity of 3H-thymidine was described by a linear equation y =2.21+43.45 x ( R 2 =0.96), where y is the number of γH2AX foci per nucleus and x is specific activity in 1000 MBq/liter. For tritium oxide, the relationship was described by a linear equation y =2.52+6.70 x ( R 2 =0.97). Thus, the yield of DNA double-strand breaks after exposure to 3 H-thymidine was 6.5-fold higher than after exposure to tritium oxide. Comparison of the effects of tritium oxide and X-ray radiation on the yield of DNA double-strand breaks showed that the relative biological efficiency of tritium oxide in a dose range of 3.78-60.26 mGy was 1.6-fold higher than that of X-ray radiation. Improvement of the methods of analysis of DNA double-strand breaks repair foci is highly promising in the context of creation of highly sensitive biodosimetry technologies for tritium compounds in humans.

Показано, что кинетика изменений количества фокусов H2AX (маркер двунитевых разрывов ДНК) в фибробластах кожи человека после воздействия рентгеновского излучения в малых дозах (20, 40 и 80 мГр) отличается от кинетики, наблюдаемой после облучения в умеренно низких дозах (160 и 250 мГр). После облучения в дозах 160 и 250 мГр наибольшее количество фокусов регистрировалось через 30 мин после воздействия радиации (первая экспериментальная точка), а далее наблюдалось снижение их уровня. При этом выделялась быстрая фаза (до 4 ч), за время которой происходит уменьшение количества фокусов на 50–60%, и медленная фаза репарации (от 4 до 24 ч). Через 24 ч оставалось всего 3 5 фокусов от количества, наблюдавшегося через 30 мин после облучения. После радиационного воздействия в малых дозах количество фокусов не снижалось в течение 2 ч, и даже через 24 ч после облучения их количество составляло 25 от наблюдавшегося в точках максимума (1 ч после облучения в дозах 40 и 80 мГр и 2 ч после облучения в дозе 20 мГр).

It was shown that the kinetics of changes of γH2AX foci number (marker of DNA double-strand breaks) in human skin fibroblasts after exposure to low doses of X-ray radiation (20, 40 and 80 mGy) differs from that observed after exposure to medium-low doses (160 and 240 mGy). After exposure to 160 and 240 mGy the highest number of γH2AX foci was detected at 30 min after exposure (first experimental point) and further their decrease was observed. At the same time we observed a fast phase of repair (upto 4 h), in which there was a decrease of the foci amount to ~50-60% and a slow phase of repair (from 4 h to 24 h). After 24 h only ~3-5% of the foci amount observed at 30 min after irradiation was left. After exposure to low doses, the foci number did not decrease during 2 h and even 24 h after exposure their amount was ~25% from that observed at maximum points (1 h after irradiation at 40 and 80 mGy and 2 h after irradiation at 20 mGy).

It is shown that exposure of 365 nm UV radiation at doses of 10, 20 and 50 kJ/m2 induces a dose-dependent increase in DNA single-strand breaks and alkali-labile sites (SSB and ALS) detected by comet and halo assays in human blood lymphocyte nucleoids. Adding 10% dimethyl sulfoxide (DMSO) reduces the SSB and ALS yields--in 3 times. A strong drop in the output of UV-A-induced SSB and ALS in lymphocyte nucleoids in the presence of DMSO shows the leading role of *OH radicals in this DNA damage formation under exposure to 365-nm UV-radiation.

Aim: to compare the repair process of DNA double-strand breaks in mammalian cells after acute versus prolonged exposure to X-ray irradiation with different dose rates. Material and methods. Studies were performed on primary human fibroblasts isolated from skin biopsies of healthy volunteers (women, 29 and 30 years). Cells were irradiated using an X-ray machine RUB RUST-M1 (JSC "Ruselectronics", Moscow, Russia) at 37°C temperature with a dose rate of 400 mGy/min (200 kV, 2*2.4 mA, a filter of 1.5mm AI) or 4 mGy/min (50 kV, 2*0.4 mA, a filter of 1.5 mm AI). Immuno-cytochemical protein staining was utilized for yH2AX and Rad51 foci analysis. Results. Phosphorylated histone H2AX (yH2AX) and the key protein of homologous recombination Rad51 foci formation and disappearance kinetics were investigated simultaneously in primary human dermal fibroblasts after acute and prolonged exposure to X-ray radiation at a same dose. It was shown that the relative yield of yH2AX foci per dose reduces with decrease in dose rate, while the relative yield of Rad51 foci conversely increases. Conclusion. Our findings suggest the fundamental differences in the ratio of non-homologous end joining and homologous recombination DNA repair in acute versus prolonged irradiated cells.

Показано, что воздействие 365 нм УФ-излучения в дозах 10, 20 и 50 кДж/м2 индуцирует дозозависимое увеличение однонитевых разрывов и щелочнолабильных сайтов (ОР и ЩС) ДНК, регистрируемых методами ДНК-комет и ДНК-гало, в нуклеоидах лимфоцитов периферической крови человека. Добавление 10% диметилсульфокида (ДМСО) снижает выход ОР и ЩС в 3 раза. Резкое снижение выхода УФ-А-индуцированных ОР и ЩС ДНК в нуклеоидах лимфоцитов в присутствии ДМСО свидетельствует о ведущей роли . радикала в образовании этих повреждений ДНК при воздействии 365 нм УФ-излучения.

Aim: to study the patterns of changes in the number of DNA double-strand breaks (DSB) in mammalian cells continuously exposed to low dose-rate y- radiation. Material and methods. Chinese hamster lung fibroblasts (V79) were used in this study. The y- irradiation of cells at a dose rate of 0.1 mGy/min was performed using the «Gamma-Panorama» unit (Cs-137). The fluorescence immunoassay of the phosphorylated H2AX-histone (y-H2AX) foci was used to investigate the DNA DSBs formation. Frequency of apoptotic cells was evaluated using «DNA halo» assay. 5 (6) — chloromethyl-2,7-dichlorodihydrofluorescein diacetate was used to estimate the reactive oxygen species (ROS) production. Results, it was showed that continuous low dose-rate irradiation of Chinese hamster V79 cells induces an increase of the y-H2AX foci number and ROS production rate at the early stages of exposure time (6-24 h, doses 3.6-14.4 cGy), while increasing exposition time and, therefore, the radiation dose (48-72 h, 28.8-43.2 cGy) caused a decrease in these endpoints to almost the control level. There was observed no significant changes in the frequency of apoptotic cells. Conclusion. It is assumed that the processes causing the DSB amount changes in mammalian cells continuously exposed to low dose-rate y-radiation are associated with the development of oxidative stress and subsequent activation of cellular antioxidant defense systems.

Using a modified DNA-halo method single-strand breaks and DNA alkaline-labile site induction were stud- ied in human peripheral blood lymphocytes after a short-term (up to 10 min) exposure in vitro to X-rays, hy- drogen peroxide and long-wave ultraviolet light (365 ± 10 nm). It was shown that the dose-effect dependence in thee X-ray dose range of 0.3-2 Gy approximates by a linear function of y = 0.25 + 0.42x (R2 = 0.98), where y is a DNA-halo index in standardized units, x--a radiation dose in Gy. The effect of "saturation" was ob- served in the range of 2-5 Gy. Under exposure to hydrogen peroxide up to a concentration of 25 μmol/L, the dose-effect is described by a linear function y = 0.23 + 0.033x (R2 = 0.96), where y is the DNA-halo index in standardized units, x--hydrogen peroxide concentration in μmol/L. UV exposure induced a linear in- crease of the DNA-halo index in the dose range of 2-10 kJ/m2 (y = 0.26 + 0.032x (R2 = 0.99), where y is theDNA-halo index in standardized units, x--a radiation dose in kJ/m2). In summary, the described modi- fication of the DNA-halo method provides a simple, sensitive, well reproducible and rapid assay for the anal- ysis of DNA single-strand breaks and alkaline-labile sites in living cells.