ABSTRACT
Retinopathy of animals is induced by many agents damaging DNA. 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 methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.
Subject(s)
DNA Repair , Epithelial Cells , Gamma Rays/adverse effects , Protons/adverse effects , Retina , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Differentiation , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , DNA Breaks, Single-Stranded/drug effects , DNA Breaks, Single-Stranded/radiation effects , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/radiation effects , Gene Expression , Methylnitrosourea/toxicity , Mice , Radiation Tolerance , Radiation, Ionizing , Retina/drug effects , Retina/metabolism , Retina/radiation effects , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Total repair capability is a widely used phenotypic marker of predisposition to cancer. Evaluation of this parameter implies using a challenge mutagen in an in vitro system to unmask latent genetic instability and repair insufficiency in the target cells. Traditionally, these investigations involve two tests, evaluation of mutagenic susceptibility (chromosomal aberrations) and genotoxic effect (DNA comet assay). The present study was focused on analysis of the effect of methylnitrosourea (MNU) on resting and PHA-stimulated lymphocytes from healthy donors and patients with gynecological cancer. Cytotoxic effect of MNU (apoptotic lymphocyte death) was estimated using two parameters, interaction of the cells with the annexin V-FITC complex, and morphological changes of the nuclei after their staining with the mixture of two DNA tropic dyes. The genotoxic effect of MNU, namely, secondary double-strand DNA breaks, was scored using the neutral comet assay, modified for the calculation of the comets produced exclusively by BrUdr-labeled proliferating lymphocytes. The proportion of these comets was represented as the proliferative cell index. It was shown that resting lymphocytes were resistant to genotoxic and cytotoxic effects of MNU. The response of proliferating cells to the action of MNU was expressed as the development of secondary DNA breaks (P <0.01), along with the increased frequency of apoptosis (P <0.05). The genotoxic effect of MNU on stimulated lymphocytes of gynecological cancer patients was fourfold lower compared to healthy donor lymphocytes. In response to the MNU action, patient lymphocytes did not change their proliferative index, while in healthy donor lymphocytes proliferative index was two times decreased in response to the MNU action. The data obtained pointed to the association between the cytotoxic response of the lymphocytes to the action of MNU and gynecological cancer. Since only proliferating lymphocytes response to the genotoxic effect of MNU, and the effect is revealed a day after the mutagen action, it is suggested that this phenomenon is associated with postreplicative repair, MMR, the substrate of which is O6-methylguanin. The MMR deficiency in patient lymphocytes determines their tolerance to the action of MNU. Genotoxic effect of lymphocytes to the action of MNU can serve as a marker of MMR, as well as of the MMR deficiency-associated gynecological cancer.
Subject(s)
DNA Mismatch Repair/drug effects , Genital Neoplasms, Female/genetics , Lymphocytes/drug effects , Methylnitrosourea/toxicity , Mutagens/toxicity , Apoptosis/drug effects , Chromosome Aberrations , Comet Assay , DNA, Neoplasm/metabolism , Female , Genital Neoplasms, Female/blood , Guanine/analogs & derivatives , Guanine/metabolism , HeLa Cells , Humans , In Vitro Techniques , Microsatellite Instability/drug effectsABSTRACT
Whole-body irradiation of mice with gamma-rays at 14 Gy causes DNA single and double strand breaks effectively repaired later. p53 is accumulated during the repair period. There is still some amount of DNA breaks 48-72 hours after the irradiation. Despite p53 accumulation and residual DNA lesions in the cells, mice retina demonstrated no morphological destructive changes or apoptosis signs. Retina resistance to apoptotic signals could derive from efficient repair of radiation-induced lesions in transcriptionally active regions of the genome of differentiated cells.
