The aim of the investigation was to assess the availability of ozone to induce DNA damage in individual cells when analyzing them using the Comet assay.Materials and Methods. Experimental studies were performed on whole blood leukocytes of white non-linear intact male rats (n=16) weighing 250±25 g. Two series of experiments were made to induce DNA damage in leukocytes. During the first series the samples were exposed to gamma-radiation, and during the second series the slides were treated with ozonized phosphate buffer saline. Further the cells were exposed to cytolysis followed by DNA denaturation, electrophoresis, neutralization, DNA being stained with a SYBR GREEN I. Comet visualization (fluorescent microscopy) and scoring were performed.Results. The new version of the Comet assay was developed. Ozone concentration, 900 µg/L, in ozone-oxygen mixture, and the exposure time for 10 min on the cells on a microscope slides were found to be optimal for detection of DNA damage and its analysis. In addition, ozone application enables to minimize the drawbacks and limitations of gamma-radiation source.
Introduction. There is still an open question about the limits of medical use of low-intensity electromagnetic radiation of the optical range in tumor growth due to the risk of increased proliferation of tumor cells. The conditions under which the tumor process is stimulated, as well as the mechanisms of photobiomodulation in oncological pathology, remain unclear. The aim of the study - in vitro evaluation and comparison of the effect of low-intensity electromagnetic radiation with wavelengths of 400, 460 and 660 nm on the activity of free-radical processes in tumor tissue and blood in normal and growing experimental neoplasia. Materials and methods. The study was conducted on biological material obtained from white non-linear rats intact and with subcutaneously transplanted cholangiocellular cancer MS-1. The sources of low-intensity radiation with wavelengths of 400, 460 and 660 nm were led generators. The content of hemoglobin, the activity of superoxide dismutase and catalase, changes in the overall level of free radical processes and antioxidant activity by induced chemiluminescence, and DNA damage by the method of DNA comets were studied. Data analysis was performed using nonparametric statistics methods. Results. The multidirectional effect of radiation with wavelengths of 400, 460 and 660 nm on free-radical homeostasis indicators at the early and late stages of tumor growth, as well as the dependence of biological effects on the wavelength of radiation, was found. Conclusions. The results obtained allow making a number of assumptions about the mechanisms of action of the optical electromagnetic waves on tumor growth, modulating free radical processes in the tumor-bearing organism.
The aim of this research was (by using the DNA comet method) to study the level of DNA damage in leukocytes of the whole blood in tumor-bearing animals after photodynamic therapy (PDT) with the local administration of a photosensitizer.Materials and Methods. The experiments were performed on 30 non-linear male albino rats. As a model of neoplasia, the rat renal carcinoma strain was used. The animals were divided into three groups: intact (n=10), without exposure (n=10) and with PDT exposure (n=10); each group was then divided into two subgroups according to the initial tumor volume: A -less than 0.3 cm 3 and B -more than 0.5 cm 3 . In the PDT group, 0.3% Photosens (SRC "NIOPIK", Russia) was injected into the tumor. Then, 6-12 h after the injection, the tumor area was irradiated with a LED laser beam (λ=660±10 nm, P=100 mW/cm 2 ). The PDT sessions were conducted on the 15 th and 19 th day after the transplantation.The antitumor effect was evaluated by the absolute tumor growth rate. The DNA damage was assessed by the DNA comet assay adapted for this study. The %TDNA -the relative DNA content of the comet tail -was used for quantification.Results. A direct correlation between the DNA damage and the absolute tumor growth was found (Spearman rank correlation coefficient r s =0.85; p=0.006). Using the DNA comet method we observed an increased DNA damage in leukocytes of tumor-bearing animals exposed to PDT in the subgroup with initial tumor volumes <0.3 cm 3 ; whereas no such changes were found in the subgroup with tumors >0.5 cm 3 . When PDT was preceded by a Photosens injection, the tumors regressed in 50% of rats regardless of the initial tumor volume. In rats resistant to PDT, the tumor growth was stimulated in rats with the initial tumors <0.3 cm 3 . Conclusion. The level of DNA damage in blood leukocytes, determined with the alkaline version of the DNA comet method after author's modification, can be used to indirectly evaluate the growth rate of a malignant neoplasm and predict the tumor response to photodynamic therapy combined with a locally-administered photosensitizer.
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.
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