Nitrosonium cations (NO+ ) are short-lived, so we hypothesized that they cause double-strand (DS) DNA breaks in the early stages of incubation of the NO+ donor with cells. By MTT-test and DNA-comet, methods on human breast cancer cells MCF-7 has been shown that the donor NO+ sodium nitroprusside leads to rapid cyto- and genotoxic effects before the start of reparative processes in the cells. The MTT-test showed the involvement of the NER repair mechanism in the occurrence of DS and a possible contribution to the genotoxic effect of NO+ .

The cytotoxic effect of dinitrosyl iron complex (DNIC) with mercaptosuccinate (MS) on MCF-7 human tumor cells is twice as great as the cytotoxic effect of the mixture of precursors of its synthesis (MS + Fe2+). It has been shown by the alkaline comet assay that the mixture (MS + Fe2+) induced single-strand breaks (SSBs) in DNA in cells. These lesions were completely repaired after 24 h. Although the DNIC–MS complex induced fewer SSBs in the cell DNA compared with the mixture, some of them remained unrepaired in 19

Systemic administration of N-methyl-N-nitrosourea in rats resulted in the death of retinal photoreceptors followed by differentiation of retinal Müller glial cells into photoreceptor-like cells [ 27 ]. However, mammalian Müller glial cells exhibit an extremely limited proliferative capacity, which correlates with the expression of histone γH2AX and p21 protein. These proteins are known to be components of the cellular response to DNA damage [ 26 ]. The restriction of proliferation of human Müller glial cells prevents retinal replacement therapy by cell transplantation. On the other hand, the mechanism that limits the proliferation of Müller glia in the mammalian retina remains to be elucidated. We examined the Müller glial proliferative response and the DNA damage response in Müller glia in the postreplicative stage, as well as the expression of the p53 protein in response to the influence of retinotoxic N-methyl-N-nitrosourea. It was shown that N‑methyl-N-nitrosourea induced retinal degeneration in mice via apoptosis of photoreceptors, whereas the other retinal layers retained intact morphology. Nevertheless, the formation of DNA breaks and alkali-labile sites was observed in all retinal cells 5 h after the N-methyl-N-nitrosourea injection; these formations completely disappeared 15 h after N-methyl-N-nitrosourea injection. By 72 h, a significant increase in the number of DNA breaks in Müller glial cells was observed. The absence of bromodeoxyuridine incorporation into the retinal cells later testifies to the absence of proliferation of Müller glial cells and DNA repair synthesis. At the same time, an increased expression of the p53 protein, a universal marker of DNA damage, was observed in the retina. Thus, our findings support the concept of the “DNA damage response” with respect to Müller glial cells, according to which the DNA damage in Müller glial cell is related to the restricted proliferation of these cells in mice. Postreplicative repair is considered as a probable mechanism of the formation of DNA breaks in postreplicative Müller glial cells.

Single intraperitoneal injection of methylnitrosourea (MNU) induces in mice the loss of retinal photoreceptors as a nonlinear dose response (Tronov et al. 2015). DNA repair was the putative mechanism for causing a threshold of DNA alkylation in retina cells. Photoreceptor degeneration can stimulate Müller glial cells to transdifferentiate into photoreceptor-like cells in adult mouse retina treated with MNU (Wan et al., 2008). In this paper, we evaluated Müller cell proliferative response to different doses of MNU and compared the response to DNA damage and repair in suspensions of retinal and Müller cells using a comet assay and BrdU (thymidine analogue) as a marker of proliferation. MNU administration in the dose ≤ 40 mg/kg did not result in the activation of Müller cell proliferation in 3 days after the treatment. By this time point, no DNA damage after this dose was observed. For MNU doses that exceed 50 mg/kg, TUNEL-detected death of retinal photoreceptors increased more than 10-fold in the proliferating pool of Müller cells. DNA breaks (single- and double-strand breaks and AP sites) were observed. The results are discussed within the framework of the concept of misbalance of excision repair that results in formation of cytotoxic intermediates in DNA initiated death of photoreceptors followed by activation of Müller retinal cells.

Exposure to a low level of stress, so-called preconditioning, has been shown to have a protective effect on the retina. Our previous studies demonstrated that there are nonlinear relationships between the doses of ionizing radiation and N-methyl-N-nitrosourea (MNU) and their cytotoxic effects on mouse retina; moreover, treatment with subcytotoxic doses of MNU increased the tolerance of the retina to the challenge-doses of the agent (Tronov et al., 2012). These results indicate the existence of a regeneration mechanism in the retina. In this work, we used an in vivo mouse model system for MNU-induced retinal degeneration to investigate the adaptive response of the retina to proton irradiation and a role of Müller glial cells in this process. We found that retina recovery following exposure to genotoxic stress that induced its degeneration was associated with an increased efficiency of DNA repair and a decrease in retinal photoreceptor death.

Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous studies have revealed a non-linear dose-response relationship for cytotoxic effect of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non-cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In the present study we used the mouse model for MNU-induced retinal degeneration to evaluate the adaptive response of the retina to proton irradiation and implication of glial Muller cells in this response. In this paper, we have shown that the recovery of the retina after exposure to genotoxic agents is associated with an increased efficiency of DNA damage repair and lowered death of retinal photoreceptors.

Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous study revealed non-linear dose-response relationship for cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In present study we used the mouse model for MNU-induced retinal degeneration to evaluate adaptive response of retina to proton irradiation and implication in it of glial Muller cells. The data showed that the recovery of retina after genotoxic agents has been associated with increased efficacy of DNA damage repair and lowered death of retinal photoreceptor cells.

The eye retina consists of terminally differentiated cells that have lost their ability to proliferate. The death of these cells leads tothe loss of sight. The mice retina is characterized by relatively high resistance to radiation, which is provided by its ability to repair damage caused by environmental factors. The aim of our work was to assess the damaging effect of ionizing radiation and methylnitrosourea (MNU) on the DNA structure in the mouse retina, the functional activity of the retina, and its ability to recover in vivo. The results confirm the ability of the mature retina to structural and functional recovery. Adapting influence of low dose chemical agent increases retina resistance to cytotoxic dose of genotoxicants and prevents degeneration of photoreceptor layer of the retina. The results show the possibility of neurohormesis effect in the mice retina after exposure to ionizing radiation and chemicals.

Сетчатка глаза состоит из терминально дифференцированных клеток, утративших способность к делению. Гибель этих клеток приводит к утрате зрения. Сетчатка мышей характеризуется сравнительно высокой устойчивостью к радиации. Эта устойчивость сетчатки обеспечивается ее способностью к восстановлению от повреждений, вызываемых факторами внешней среды. Цель работы заключалась в оценке повреждающего действия ионизирующего излучения и метилнитрозомочевины (МНМ) на структуру ДНК в сетчатке глаза мышей, функциональную активность сетчатки и в оценке ее способности к восстановлению in vivo. Результаты подтверждают способность зрелой сетчатки к структурному и функциональному восстановлению. Адаптирующее воздействие в малой дозе химического агента повышает устойчивость сетчатки к цитотоксической дозе этого генотоксиканта и препятствует дегенерации фоторецепторного слоя сетчатки. Полученные результаты указывают на возможность эффекта нейрогормезиса в сетчатке глаза мышей при действии ионизирующей радиации и химических веществ.

Retinopathy of animals is induced by many DNA-damaging agents. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation and single administration of methylnitrosourea (MNU) on mice retina. We assessed morphological changes, DNA damage and repair, as well as expression of proteins (p53, ATM, PARP, FasR, and caspase 3) participating in apoptosis in retina. 14 Gy was the equitoxic dose for induction of DNA single-strand breaks by both gamma- and proton irradiation. However, protons were twice as effective as γ rays in induction of DNA double-strand breaks. All breaks have been repaired for ≤10 h. Irradiation resulted in increased expression of p53 and ATM. Seven days after irradiation, no signs of cell death and retinal degeneration were observed. Proton irradiation with 25 Gy resulted in destructive changes in retina localized mainly in the photoreceptor layer. These changes were accompanied by enhanced expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, and Caspase 3 followed by destructive changes in retina with sings of apoptosis in photoreceptors. Similarly to irradiation, a halved MNU dose did not exhibit a cytotoxic effect on retina. A high level of spontaneous DNA damage at apurine and apyrimidine sites were observed in mouse retina. The results show that there is a genotoxic threshold in initiation of retinal cell death in vivo. It is suggested that topoisomerase 2 translates primary DNA damage into a cytotoxic effect in retina.

Melanoma is a highly aggressive tumor of melanocytes. The efficacy of different cytotoxic chemotherapy regimens does not exceed 20%. The search for markers for patient sensitivity to chemotherapy provides a rational basis for the development of cytotoxic chemotherapy. In this study, we evaluated six blood lymphocyte parameters (the efficacy of BER and MMR; the expression of MLH1, MSH2, FasR; and cell death) as prognostic markers for melanoma chemotherapy. We found that chemotherapy induced AP sites and ssDNA breaks in lymphocytes repaired through the BER pathway. However, ssDNA breaks were completely repaired after chemotherapy and, therefore, did not contribute to the toxic effect of chemotherapy. dsDNA breaks produced by a functioning MMR system appeared downstream of methylation adducts, which was confirmed by the linear correlation of these parameters in various patients. The number of dsDNA breaks, but not MMR, MLH1, and MSH2 expression, correlated to the efficacy of chemotherapy. A positive correlation was observed between lymphocyte death induced by chemotherapy and the patient’s clinical response, which may show that the nucleotide excision repair (NER) mechanism is implicated in the generation of double-strand breaks and the cytotoxic effect of chemotherapy.

Patients with advanced malignant melanoma have poor prognosis as conventional chemotherapy induces complete response in a very small fraction (not more than 20%). One of research strategies aimed at raising its efficiency is the search for markers predicting individual response to chemotherapy. Our study was concerned with evaluation of the potential of DNA damage, repair (BER, MMR), expression of proteins MLH1, MSH2 and FasR as prognosticators of chemotherapy. These parameters were assessed in lymphocytes sampled from the blood of patients with metastatic cutaneous melanoma before and after one cycle of chemotherapy with lomustine, dacarbazine, cisplatin and interferon-gamma (LDCI). Clinical response was evaluated after a full course of therapy. We established that the major DNA damage induced by chemotherapy occurred on the levels of AP sites and single strand (SS) breaks. Despite the individual variations in BER efficacy, complete repair of SS breaks was reported in lymphocytes of all patients 30 days after the first cycle of chemotherapy. As a consequence, this type of damage and relevant BER efficacy did not correlate with clinical response. Conversely, the number of DNA double strand breaks detected in lymphocytes after the first cycle of chemotherapy was in good correlation with positive clinical response (p < 0.001). This parameter does not fully represent MMR function and, if coupled with cytotoxic effect of chemotherapy on lymphocytes, may be used as a predictive marker for clinical response to LDCI chemotherapy regimens for melanoma.

Melanoma is a highly aggressive neoplastic disease attributed to transformed melanocytes. The efficacy of regimens of cytotoxic chemotherapy for advanced stage patients does not exceed 20%. Search for lymphocyte markers of patients' sensitivity to chemotherapy provides a rational basis for development of cytotoxic chemotherapy. Using blood lymphocytes we evaluated efficacy of BER and MMR, expression of MLH1, MSH2 and FasR, and cell death in melanoma patients relative to clinical response to chemotherapy. We found that LDCI-chemotherapy (lomustine, dacarbazine, cisplatin and interferon gamma), induced AP sites and DNA ss-breaks which repaired trough BER pathway. However, neither initial DNA damage nor the rate of their repair correlated with clinical response. This result prompts us to think that this type of damage is not crucial in cytotoxic effect of LDCI-regimen of chemotherapy. DNA ds-breakes appeared downstream ss-breakes were attributed to repair of 06-methylguanine by MMR mechanism in PHA-stimulated lymphocytes. The number of ds-breakes appeared by 48 correlated with positive clinical response of patients to chemotherapy. The same link was observed between clinical response and the number of dead lymphocytes. However, there was no correlation between clinical response and expression of MLHI + MSH2 and FasR. These results imply possible contribution of crosslink repair through NER pathway to formation of DNA ds-breaks as well as to cytotoxicity of LDCl-therapy. The observed link between high level of secondary ds-breaks and positive response to chemotherapy indicates the potential of these instruments to serve as prognostic end point in clinical trials.

Melanoma is among the most aggressive malignancies. Tumors with a thickness of 4 mm can produce metastases, and the mean survival of the patients is 9 months. The review presents modern classification of the melanoma types based on cytological and morphological indices (Clark model). Alterations of genes in melanomas are discussed in detail. These genes include tumor suppressors, proliferative response genes (oncogenes), and transcription factors. Alterations in the Wnt signaling, MAPK cascade, and Fas signaling pathways are considered. Changes in the mismatch repair (MMR) genes are also analyzed. From practical perspective, understanding the genetic alterations provides identification of potential targets for therapeutic exposure and enables prognosis of the tumor response to chemotherapy.

The regularities of the induction of DNA double strand breaks (DSB) in human lymphocytes after irradiation by different doses of accelerated lithium and carbon ions (33 and 480 MeV/nucleon, LET = 20 and 10.6 keV/microm, respectively) and gamma-rays 60Co by using of comet assay were investigated. It was shown that the dependence of DSB formation increases linearly with growing of the dose of lithium and carbon ions and gamma-rays. The biological effectiveness of carbon ions with high energy was similar with gamma-rays, lithium ions possess greater biological effectiveness in comparison with gamma-rays and value of RBE of lithium ions amount 1.6 +/- 0.1. The kinetic of DNA repair from DSB in human lymphocytes after irradiation by lithium and carbon ions and gamma-rays was studied. It is revealed that the reparation proceeds effectively with heavy ion and gamma-ray irradiation by exponential kinetics.