Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response.

We have hypothesized that the adaptive response to low doses of ionizing radiation (IR) is mediated by oxidized cell-free DNA (cfDNA) fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production), induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs) with low doses of IR (10 cGy) leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.

[Cytogenetic damage and the degree of reparative synthesis in the lymphocytes of chemical industry workers]. / Tsitogeneticheskie povrezhdeniia i uroven' reparativnogo sinteza v limfotsitakh krovi rabochikh khimicheskogo proizvodstva.

A thiophosphamide-induced increase of repair DNA-synthesis in lymphocytes in shown. To include cytogenetic studies in a genetic- and epidemiologic programme of genetic monitoring to make a complex assessment of health status of chemical industry workers is recommended.

[The induction and elimination of cytogenetic disorders in monkey lymphocytes following thiophosphamide action]. / Induktsiia i éliminatsiia tsitogeneticheskikh narushenii v limfotsitakh obez'ian posle vozdeistviia tiofosfamidom.

The dynamics of sister chromatid exchanges and chromosome aberrations in lymphocyte of monkey has been investigated after a thiophosphamide exposure. The process of induction and elimination of cytogenetic damages was described by the mathematical model. Developing the model in detail will allow to make a cytogenetic prognosis of remote consequences of mutagenic exposure.

[Dynamics of sister chromatid exchange after in vivo exposure to thiophosphamide]. / Dinamika chastoti sestrinskikh khromatidnykh obmenov posle deistviia tiofosfamida in vivo.

Thiophosphamide (T) was i.v. administered into New Zealand rabbit (4 mg/kg). The rate of SCE in blood lymphocytes was increased up to 21st day after T administration. There are two phases in dynamics curve of SCE--a rapid phase and a slow one. The rapid decrease of SCE's rate depends on cell mortality. The slow decrease of SCE rate is connected with reparation of chromosome damages and selection of cells in lymphopoietic tissue. It was demonstrated that the special class of cells with intermediate number of SCE exists in blood after T administration.

[Dynamics of the frequencies of sister chromatid exchange and chromosome aberrations in vivo]. / Dinamika chastot sestrinskogo khromatidnogo obmena i aberratsii khromosom in vivo.

2.4 and 6 mg/kg thiophosphamide (T) was administered intravenously to New Zealand rabbits. A decrease in sister chromatid exchange (SCE) and chromosome aberration (CA) rate began immediately after the mutagenic action of T was over. The expected SCE rate was more than the investigated one. The difference between expected and investigated SCE rate increased with the dose of T. A calculation of SCE was based on the amount of the administered T, the rate of T removal and cell sensitivity to T. The death of cells with high number of SCE resulted in a fast decrease in SCE rate in the first 4 days. Reparative processes and cell proliferation in lymphocyte tissue resulted in a slow decrease in SCE rate after the 4th day. A number of nuclear cells in the blood was the smallest on the 4 th day, at the same time relative increase in CA rate was observed. The time of sampling and the dose of the substance tested should be taken into account for a more accurate estimation of mutagenic activity of some chemicals in in vivo cytogenetic tests.

[Quantitative comparison of the cytogenetic effect of thiophosphamide during in vivo and in vitro action on monkey lymphocytes]. / Kolichestvennoe sravnenie tsitogeneticheskogo éffekta tiofosfamida pri deistvii na limfotsity obez'ian in vivo i in vitro.

Mutagenic in vivo and in vitro effects were compared quantitatively by the investigation of sister chromatid exchange (SCE) rate and chromosomal aberrations caused by thiophosphamide in macaca rhesus lymphocytes. The integral of thiophosphamide concentration in the blood or culture fluid by a certain time period was used for the estimation of the dose of mutagenic exposure. It was shown that the dose-response relationships and corresponding regression coefficients were similar when the in vivo and in vitro results were compared. The data obtained indicate the possibility for quantitative extrapolation of the results obtained in vitro on the entire organism.

[Comparison of the cytogenetic effects of cyclophosphamide on monkey lymphocytes in vivo and in vitro]. / Sravnenie tsitogeneticheskikh éffektov pri deistvii tsiklofosfamida na limfotsity obez'ian in vivo i in vitro.

The comparative in vivo and in vitro study of chromosomal aberrations and SCE induced by cyclophosphamide (CP) in macaca rhesus lymphocytes was performed. The dose of mutagenic exposure for quantitative estimation of effects was determined as a product of concentration of alkylating CP metabolites on the exposure time. The mutagenic effect caused by the same doses of CP (CP metabolites) appeared similar in vivo and in vitro. This suggests that the results obtained in adequate in vitro mutagen-testing experiments may be quantitatively extrapolated for the in vivo conditions.

[Ratio of the frequency of chromosome aberrations and sister chromatid exchanges to the dose of the mutagenic exposure to cyclophosphamide in vivo and in vitro]. / Zavisimost' chastot khromosomnykh aberratsii i sestrinskikh khromatidnykh obmenov ot dozy mutagennogo vozdeistviia tsiklofosfamida in vivo i vitro.

Cyclophosphamide (CP) and its metabolites were used to compare the rate of chromosomal aberrations (CA) and sister chromatid exchange (SCE) in the rabbit lymphocytes in vivo and in vitro. The dose-dependent increase of cytogenetic effects rate appeared to be of linear and exponential dependence for SCE and CA, respectively, both in vivo and in vitro. The regression equation coefficients coincided in in vivo and in vitro experiments.

Cytogenetic effects of cyclophosphamide on human lymphocytes in vivo and in vitro.

A comparative study of cytogenetic effects in human lymphocytes caused in vivo by cyclophosphamide (CP) after intravenous injection and in vitro by exposure of plasma of the same patients was carried out. It was found that the frequency of induced chromosome aberrations (CA) and sister-chromatid exchanges (SCE) increased linearly for SCE and exponentially for CA within the 'dose' of alkylating activity of CP metabolites. Parameters of 'cytogenetic effect-dose' in vivo and in vitro coincided. The intensity of cytogenetic effects varied between individuals.

[Induction of chromosome aberrations in human lymphocytes by cyclophosphamide action in vivo and in vitro]. / Induktsiia khromosomnykh aberratsii v limfotsitakh cheloveka pri deistvii tsiklofosfamida in vivo i in vitro.

The dose dependences of chromosome aberrations rate were investigated in lymphocytes of oncological patients after cyclophosphamide (CP) administration. It was shown that the rate of chromosome aberrations and the number of disruptions per cell in vivo and in vitro increases exponentially with the dose. At the same time, the parameters of regression equations coincide. This evidences that CP has the similar effect in vivo and in vitro.

Alterations in the baseline sister-chromatid exchange frequency in human lymphocyte culture following a number of cell divisions.

A significant decrease in the baseline of sister-chromatid exchanges (SCEs) was observed in cultured human lymphocytes, if 5-bromodeoxyuridine (BrdU) was added after 60 h of culture, and the cells were harvested at least 24-30 h after BrdU exposure. This decrease is supposed to occur if at least one cell division takes place before the addition of BrdU. For cytogenetic monitoring of mutagenic environmental factors, using human lymphocyte cultures, it is assumed that two time periods are sufficient for comparison.

[Proliferative activity of lymphocyte cultures due to the mutagenic action of thiophosphamide in vivo and in vitro]. / Proliferativnaia aktivnost' limfotsitov v kul'ture posle mutagennogo vozdeistviia tiofosfamida in vivo i in vitro.

Thiophosphamide is capable of inhibiting the cell cycle after the treatment of rabbit blood lymphocytes in vivo and in vitro. To assess the proliferative activity of the cells during cultivation, use was made of the index of the mean division number which was experienced by the cells after mutagenic exposure prior to fixation. As the mutagen dose was raised in vivo and in vitro, the mean division number decreased linearly, namely by approximately the same value with an equivalent dose variation. This indicates that thiophosphamide produces the same cytotoxic action both in vivo and in vitro.

[Changes in the occurrence of cytogenetic effects after mutagenic exposure in vivo]. / Dinamika izmeneniia chastot tsitogeneticheskikh éffektov posle mutagennogo vozdeistviia in vivo.

Cytogenetic effects after i. v. administration of thiophosphamide were investigated in rabbit lymphocytes. It was observed, that the rate of chromosome aberrations and SCE increased within a matter of first 6 hours.

[Comparison of the frequencies of sister chromatid exchanges induced by thiophosphamide in vivo and in vitro]. / Sravnenie chastot sestrinskikh khromatidnykh obmenov, indutsiro-vannykh tiofosfamidom in vivo i in vitro.

The effects of thiophosphamide on rabbit lymphocytes were studied and compared in vivo and in vitro. In the course of in vitro action the dose was calculated by multiplying the thiophosphamide concentration by the time of in vitro treatment. In vivo the dose was calculated as integral of concentration variation function of thiophosphamide from the time of injection till the time of blood collection. It was demonstrated that as the dose was raised, the rate of sister chromatid exchanges increased linearly in vivo and in vitro. The coefficients of linear regressions were found to coincide. The data point to equal efficacy of thiophosphamide in vivo and in vitro.

[Comparison of the frequency of chromosome aberrations induced by thiophosphamide in rabbit lymphocytes in vitro and in vivo]. / Sravnenie chastoty khromosomnykh aberratsii, indutsirovannykh tiofosfamidom v limfotsitakh krolika in vitro i in vivo.

Rabbit lymphocytes were treated with thiophosphamide in vivo and in vitro. In-vitro doses were calculated by multiplying thiophosphamide concentrations by the time of treatment (the doses ranged within 0-1500 mg/min/ml). In-vivo doses were calculated as integral of thiophosphamide concentration function from the time of administration till the time of blood sample collection (the doses ranged within 0-1900 mg/min/ml). It was shown that with the dose increase the rate of chromosome aberrations and the number of disruptions per cell rise exponentially in vivo and in vitro. At the same time the parameters of regression equations coincide. This evidences that thiophosphamide produces the same effect in vivo and in vitro.

[Analysis of certain mechanisms of antigen-specific suppression of the immune response]. / Analiz nekotorykh mekhanizmov antigenspetsificheskoi supressii immunnogo otveta.

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.