CYTOGENETIC BIODOSIMETRY OF ACCIDENTAL EXPOSURES IN THE LONG TERMS AFTER IRRADIATION.

The group of radiation victims who had received radiation injures similar to those of Chernobyl accident victims was evaluated in terms of retrospective cytogenetic biodosimetry in the long term period of from 17 y up to 50 y after irradiation. Based on the existing results of the long-term cytogenetic examination of the victims injured after the Chernobyl accident, an original method was developed. This method of retrospective dose recovery was based on the use of a special computer program, the time elapsed after irradiation and the frequency of atypical chromosomes. Both patient groups were examined using conventional cytogenetic analysis. The new method of a retrospective biodosimetry was tested on the non-Chernobyl group. As a result the multiple regression equations which included frequency atypical chromosomes produced better results because the majority of the estimates of the retrospective doses fell into the 95%-prediction intervals for the reference group of the Chernobyl victims.

ACUTE MYELOID LEUKEMIA, PROSTATE AND SKIN CANCER IN ACUTE RADIATION SYNDROME SURVIVOR AFTER THE 1986 CHERNOBYL NUCLEAR ACCIDENT: CASE REPORT.

The development of hemoblastosis is often associated with the influence of various genotoxic unfavorable factors, in particular, with the effect of ionizing radiation. This article presents a case report of acute myeloid leukemia (AML) in a patient who was involved in the 1986 accident at the Chernobyl Nuclear Power Plant and suffered an acute radiation syndrome of degree II severity. Based on clinical and cytogenetic dosimetry, the average absorbed radiation dose to the whole body was estimated to be 4.3 Gy. During long-term clinical follow-up (27 years), moderate transient instability of hematological parameters was observed: lymphocytosis, leukopenia and thrombocytopenia, which was associated with chronic viral hepatitis C. Further cytogenetic investigations demonstrated a very high frequency of translocations, up to 50 times background values, that persisted over 3 decades. In 2014, the patient was diagnosed and operated on for prostate cancer and received a course of radiotherapy (total fractionated local dose of 35 Gy) in May 2015. From December 2015 through April 2016, the patient experienced general weakness and developed progressive cytopenia. A diagnosis of AML, resulting from a myelodysplastic syndrome, was confirmed by abnormal complex clones detected in 38% of metaphases by the mFISH-method, along with other chromosomal rearrangements. The patient underwent several chemotherapy treatments for AML but eventually died of bilateral pneumonia in March 2017.

Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25µM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-ß, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.

Genotoxicity of single-walled carbon nanotubes: in vitro study on human embryonic fibroblast cells.

The effects of single-walled carbon nanotubes on the levels of DNA aberrations, chromosome and genome disorders were studied on human embryonic fibroblasts, their karyotype was analyzed by the spectral karyotyping method. The level of DNA aberrations increased after 3-h exposure to the nanotubes. No appreciable increase in the incidence of aberrant metaphases, micronuclei, and chromosome 1, 6, 8, 11, X, and Y aneuploidy after 24- and 48-h incubation with the nanotubes were detected.

[Risk of genetic transformation of multipotent mesenchymal stromal cells in vitro].

Proof of the efficacy of cell therapy by numerous studies and clinical trials inevitably has raised the question of improving the regulatory framework that governs its use. Particular attention should be paid to the genetic safety of cell preparations. The immune, genetic, and pharmacological modification and expansion of cells in vitro can lead to an undesired effect, which not only has reduced the healing, recovery, and regulatory potential of cell therapy, but also increased the risk of accumulating genetically aberrant cells and the oncogenic transformation of cell preparations. The article has presented the estimation of the parameters of the genetic stability of cultured multipotent mesenchymal stromal cells (MSCs) derived from bone marrow and adipose tissue. The study was conducted using classic methods of genotoxicology, i.e., the individual cells gel-electrophoresis (DNA comets) and the micronucleus test. We described a basic level of DNA damage and the frequency of micronucleus, identified genetically instable cultures, and conducted the comparison of genetic variability of MSCs isolated from different tissues.

[Genetic safety of cellular therapy].

This paper presents the main results of the study on chromosome and genome variability of mesenchymal stem cell cultures from bone marrow and adipose tissue carried out in the Laboratory of Mutagenesis, Research Centre for Medical Genetics, over the last three years. Genome stability was assessed from DNA damage using the DNA comet assay, karyotyping and registration of aneuploidy by the FISH method. We found that DNA damage rate in MSC cultures from bone marrow was 3.9% and 3.8% at the early (2-5) passages and the late (10-15) passages respectively. The cultures were characterized by high dispersion of individual values. Karyotyping showed mosaicism in both types of MSC cultures at the early and late stages of cultivation. The fraction of abnormal cells in some cultures amounted to 80-90%. Evaluation of aneuploidy in interphase cells revealed 1.34% of aneuploid cells (on the average) per one "conventional" chromosome; their overall frequency in the genome amounted to 20-40%. The frequency of aneuploid cells was similar at the early and late passages. Cultures with clones of trisomic and monosomic cells were revealed. The probability of occurrence of abnormal cells may increase by virtue of de novo mutations in the culture and as a result of positive selection of the cells existing in the organism that exhibit a higher reproduction rate in culture. Based on the experimental data on mutational process, selection of mutant cells and clone formation, it is concluded that cytogenetic control of stem cells is necessary to ensure the safety of cellular therapy.

Assessment of DNA damage in human bone marrow cells and multipotent mesenchymal stromal cells.

We carried out a comparative analysis of DNA damage (percentage of DNA in comet tail) and frequencies of comets in apoptotic cells in BM samples and cultures of BM multipotent mesenchymal stromal cells at different terms of culturing (passages 3-11). The levels of DNA damage in mesenchymal stromal cells remained unchanged during culturing (3.5 ± 0.9 and 4.4 ± 1.2%) and did not differ from those in BM cells (3.6 ± 0.8%). In BM samples, 10-28% atypical cells with high level of DNA damage were detected. In mesenchymal stromal cells, 2.8 ± 0.9 and 3.6 ± 1.8% apoptotic cells were detected at early and late passages, respectively.

Study of genetic stability of human bone marrow multipotent mesenchymal stromal cells.

Immunophenotype, proliferation rate, and genetic stability parameters of bone marrow multipotent mesenchymal stromal cells were studied. Despite the reduction of proliferative activity by passages 11-12, the cells retained the characteristic immunophenotype. The incidence of spontaneous aneuploidy for autosomes 6, 8, 11 and sex chromosomes was evaluated. Two cultures of mesenchymal stromal cells carrying aneuploid cell clones were detected: with chromosome 8 trisomy and X chromosome monosomy. The results indicate the possibility of genetic transformation and selection of mesenchymal stromal cells with abnormal karyotype during in vitro culturing.

Spontaneous aneuploidy and clone formation in adipose tissue stem cells during different periods of culturing.

Cytogenetic analysis of 13 mesenchymal stem cell cultures isolated from normal human adipose tissue was carried out at different stages of culturing. The incidence of chromosomes 6, 8, 11, and X aneuploidy and polyploidy was studied by fluorescent in situ hybridization. During the early passages, monosomal cells were more often detected than trisomal ones. A clone with chromosome 6 monosomy was detected in three cultures during late passages.

Methodological guidelines for genetic safety testing of cell transplants.

A combination of karyotyping and aneuploidy analysis by interphase fluorescent in situ hybridization is a sensitive method for evaluation of genetic stability of stem cell cultures. The methodology and specific features of preparing and analyzing the cytogenetic preparations are described as exemplified by human multipotent mesenchymal stromal cells.

Aneuploidy of stem cells isolated from human adipose tissue.

The incidence of autosomes 6 and 8 aneuploidy in stem cell cultures derived from adipose tissue was evaluated at different stages of culturing. Monosomy was more incident than trisomy during the early passages. Distribution of cultures by the incidence of aneuploidy in different chromosomes was virtually the same. Clones with chromosome 6 monosomy were detected in two cultures during late passages.

[Cellular therapy of hereditary diseases].

This analytical review of the literature on cellular therapy of hereditary diseases summarizes results of their treatment by stem cell transplantation. It describes the main sources of stem cells and considers prospects of and limitations on the use of cell therapy for the management of hereditary disorders. Cell technologies are considered to be promising for the treatment of a variety of inherited diseases including metabolic disturbances, hemopathies, hematopoietic disorders, pulmonary diseases in children, bone and nervous diseases. Special attention is given to the use of genetically modified stem cells. An international register of patients is deemed necessary for the evaluation of outcomes of cellular therapy.

[The protective action of vitamins in induced mutagenesis].

Peripheral blood leucocytes of healthy donors, both intact and mutagen-processed with cadmium chloride, dioxidine, and bleomycin in vitro, were studied with cytogenetic methods in G2 cell cycle period before, during, and after 14- and 30-day oral administration of vitamin and vitamin-mineral complexes. The study found that intake of complexes containing vitamins in total doses exceeding day requirements did not increase the rate of spontaneous mutations and decreased the sensitivity of human cells to the cytogenetic activity of chemical mutagens. At the same time, vitamin complexes of certain quantitative and qualitative composition were shown to be capable of giving rise to comutagenic modification of sensitive cells to the action of separate mutagens in certain concentrations in vitro. The above effects of anti- and comutagenic modification depend on the duration of intake, the quantitative and qualitative composition of the vitamin complex, the nature of mutagens, and the level of induced cytogenetic lesions.

Effect of vitamin therapy on sensitivity of peripheral blood lymphocytes to clastogenic effect of mutagens in vitro.

Spontaneous and in vitro induced (with 0.01 and 0.1 mg/ml dioxidine and 0.1 and 1.0 U/ml bleomycin) chromosome aberrations were counted in cultured peripheral blood cells from 11 donors before and after 2-week therapy with a vitamin complex. The complex contained the major vitamins in doses not surpassing the recommended daily doses. Vitamins had no effect on spontaneous mutations, but increased cell resistance to clastogenic effects of dioxidine in a concentration of 0.1, but not 0.01 mg/ml. Cell sensitivity to bleomycin notably increased after vitamin therapy in some donors and decreased in others, the mean parameters in the group remained virtually unchanged.

[Effects of biologically active food additives with different contents of vitamins on the vitamin status in humans]. / Vliianie biologicheski aktivnykh dobavok k pishche s razlichnym soderzhaniem vitaminov na vitaminnyi status cheloveka.

The comparative study of influence of two biologically active food additives with the different contents of vitamins is carried out: a drink "Zolotoi Shar", the dose of vitamins in which makes 50-90% from recommended daily consumption, and "Vitabalance 2000", the contents of vitamins in which at 2-17 of time exceeds need of organism. The use of both additives within 3 weeks resulted in increase of levels of vitamins C, A, E, B2, B6 and carotenoids in blood serum. However if in case of consumption of a drink an authentic level was reached only for vitamin C and beta-carotene, in a case "Vitabalance 2000" for all investigated vitamins except vitamin A. Thus, if the consumption of a drink has lowered frequency of deficiency of 3-4 vitamins, but has not allowed to liquidate it completely, in case of "Vitabalance 2000" consumption the simultaneous deficiency 3-4 vitamins. The received data allow to believe the biologically active food additives containing vitamins in amounts exceeding recommended consumption, are convenient for fast liquidation of hypovitaminoses, and the preparations containing vitamins in doses making 30-50% from need of organism, are acceptable for daily filling of insufficient consumption of vitamins with a usual diet for a long time.

Effect of regular intake of vitamin-mineral complex on spontaneous and on induced clustogenesis in humans.

Regular intake of a vitamin-mineral complex for 2 weeks had no effect on spontaneous clustogenesis in blood cells from healthy donors. Significant differences between the levels of chromosome aberrations induced by bleomycin and dioxidine in vitro before and after 2-week vitamin treatment indicate increased resistance of blood cells to clustogens.