Cytogenetic effects of low-dose radiation with different LET in human peripheral blood lymphocytes.

Chromosome damage and the spectrum of aberrations induced by low doses of gamma-irradiation, X-rays and accelerated carbon ions (195 MeV/u, LET 16.6 keV/microm) in peripheral blood lymphocytes of four donors were studied. G0-lymphocytes were exposed to 1-100 cGy, stimulated by PHA, and analyzed for chromosome aberrations at 48 h post-irradiation by the metaphase method. A complex nonlinear dose-effect dependence was observed over the range of 1 to 50 cGy. At 1-7 cGy, the cells showed the highest radiosensitivity per unit dose (hypersensitivity, HRS), which was mainly due to chromatid-type aberration. According to the classical theory of aberration formation, chromatid-type aberrations should not be induced by irradiation of unstimulated lymphocytes. With increasing dose, the frequency of aberrations decreased significantly, and in some cases it even reached the control level. At above 50 cGy the dose-effect curves became linear. In this dose range, the frequency of chromatid aberrations remained at a low constant level, while the chromosome-type aberrations increased linearly with dose. The high yield of chromatid-type aberrations observed in our experiments at low doses confirms the idea that the molecular mechanisms which underlie the HRS phenotype may differ from the classical mechanisms of radiation-induced aberration formation. The data presented, as well as recent literature data on bystander effects and genetic instability expressed as chromatid-type aberrations on a chromosomal level, are discussed with respect to possible common mechanisms underlying all low-dose phenomena.

[Cytogenetic effects of low dose radiation with different LET in human peripheral blood lymphocytes and possible mechanisms of their realization].

The induction of chromosome damage by the exposure to low doses of gamma-(60)Co and accelerated carbon ions 12C in peripheral blood lymphocytes of different donors was investigated. The complex nonlinear dose-effect dependence at the range from 1 to 50-70 cGy was observed. At the doses of 1-5 cGy the cells show the highest radiosensitivity (hypersensitivity), mainly due to the chromatid-type aberration, which is typical to those spontaneously generated in the cell and believed not to be induced by the irradiation of unstimulated lymphocytes according to the classical theory of aberration formation. With the increasing dose the frequency of the aberrations decreases significantly, in some cases up to the control level. At the doses over 50-70 cGy the dose-effect curve becomes linear. The possible role of the oxidative stress, caused by radiation-induced increase in mitochondrial reactive oxigen species (ROS) release in the phenomenon of hypersensitivity (HS) at low doses is discussed as well as cytoprotective mechanisms causing the increased radioresistance at higher doses.

[Induction of chromosome aberrations and micronuclei in human peripheral blood lymphocytes at low dose of radiation].

The chromosome damage induced by the doses of y-irradiation 6)Co in peripheral blood lymphocytes was studied using different cytogenetic assays. Isolated lymphocytes were exposed to 0.01-1.0 Gy, stimulated by PHA, and analysed for chromosome aberrations at 48 h postirradiation by metaphase method, at 49 h--by the anaphase method, at 58 h by micronucleus assay with cytochalasin B and, additionally, micronuclei were counted at 48 h on the slides prepared for the metaphase analysis without cytochalasin B. Despite of the quantitative differences in the amount of chromosome damage revealed by different methods all of them demonstrated complex nonlinear dose dependence of the frequency of aberrant cells and aberrations. At the dose range from 0.01 Gy to 0.05-0.07 Gy the cells had the highest radiosensitivity mainly due to chromatid-type aberration induction. With dose increasing the frequency of the aberrant cells and aberrations decreased significantly (in some cases to the control level). At the doses up to 0.5-0.7 Gy the dose-effect curves have become linear with the decreased slope compare to initial one (by factor of 5 to 10 for different criteria) reflecting the higher radioresistance of cells. These data confirm the idea that the direct linear extrapolation of high dose effect to low dose range--the procedure routinelly used to estimate genetic risk of low dose irradiation--cannot be effective and may lead to underestimation of chromosome damage produced by low radiation doses. Preferences and disadvantages of used cytogenetic assays and possible mechanisms of low ionising radiation doses action were discussed.

[Cytogenetic effects of low doses of radiation in mammalian cells: analysis of the hypersensitivity phenomenon and induced resistance]. / Tsitogeneticheskie éffekty malykh doz oblucheniia v kletkakh mlekopitaiushchikh: analiz fenomena giperchuvstvitel'nosti i indutsirovannoi rezistentnosti.

The induction of cytogenetic damage after irradiation of chinese hamster cells and human melanoma cells within a dose range 1-200 cGy was studied. The anaphase and metaphase analysis of chromosome damage and micronuclei test were applied. The hypersensitivity (HRS) at doses below 20 cGy and the increased radio-resistence at higher doses (IR) were shown with all cytogenetic critheria for both cell lines. The phenomenon of HRS/IR was reproduced in synchronic as well as in a synchronic population of chinese hamster cells. This fact shows that HRS was caused by high radiosensitivity of all cells and can not be explained by any differential sensitivity of cells in different phase of the cell cycle. So it was supposed that the increasing radio-resistence is determined by the inclusion of the inducible repair processes in all cells. This conclusion consents with the facts, that there was no evidence of HRS on dose-effect curves and that some parts of pre-existent damage was repaired after preliminary irradiation with low doses (1-20 cGy) which induce repair processes. It can be concluded that same inducible repair processes an analogous in mechanisms underlying in the base of HRS/IR phenomenon and adaptive response.

Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.

[The dose dependence of cytogenetic damages and the adaptive response of mammalian cells under the action of ionizing radiation at low doses]. / Dozovaia zavisimost' tsitogeneticheskikh povrezhdenii i adaptivnyi otvet kletok mlekopitaiushchikh pri deistvii ioniziruiushchego izlucheniia v malykh dozakh.

The dose-effect dependence of cytogenetic damage after single dose irradiation in the dose range of 0.1-2 Gy and the adaptive response after double-dose irradiation were studied on Chinese hamster and human melanoma cells in culture. The non-linear dose dependencies were found for the induction of chromosome aberrations with decrease in cell radiosensitivity in the definite dose range. This decrease started at 10 and 20 cGy for melanoma and Chinese hamster cells respectively. The maximal adaptive response was induced at 1 cGy for melanoma cells and at 20 cGy for Chinese hamster cells. It can be supposed that the same inducible repair processes are responsible for non-linearity of dose-effect curves and induction of the adaptive response. These processes are similar in mechanisms and different in quantitative proportion for different cell types.

[Effects of low radiation doses on Chinese hamster cells]. / Deistvie malykh doz oblucheniia na kletki kitaiskogo khomiachka.

The induction of cytogenetic damages after irradiation with single dose of gamma-rays (0.1-2 Gy) have been studied. It is shown non-linear curve for the induction of chromosome aberrations, detected by anaphase method. After irradiation in S-stage of the cell cycle at dose below 0.2 Gy the cells were more radiosensitive than after irradiation with doses 0.3-2 Gy. Between the phases of high radiosensitivity and radioresistance the reversal dose-effect relation was observed. This phenomenon was not marked for the cells after irradiation in G2-stage of the cell cycle. It is possible, this results could reflect an induced radioresistance at low dose of irradiation.

[The lethal and mutagenic actions of radiations with different LETs on mammalian cells]. / Letal'noe i mutagennoe deistvie izluchenii s raznoi LPE na kletki mlekopitaiushchikh.

Inactivation and induction of mutations in the HGPRT locus in Chinese hamster cells after irradiation with accelerated heavy ions in the LET range of 20 to 367 keV/micron were studied. In both cases, inactivation and induction of mutations, the LET dependence of RBE is described by a curve with a local maximum in the range of 80 to 100 keV/micron. The maximum RBE value for the mutagenic action is almost twice as high as that for inactivation. However, the RBE coefficients of the mutation induction criterion for a certain level of cell-survival is lower significantly and tend to decrease with an increase in inactivation. The obtained data show that the mutagenic effects, induction of chromosome aberrations, and deaths have their origins in the same kind of primary damages, i.e., double-strand breaks of DNA.

[The mutagenic action of gamma radiation on Chinese hamster cells. The cytogenetic characteristics of the mutants at the HPRT locus]. / Mutagennoe deistvie gamma-izlucheniia na kletki kitaiskogo khomiachka. Tsitogeneticheskaia kharakteristika mutantov po lokusu HPRT.

The peculiarities of mutation induction and cytogenetic characteristics of spontaneous and radiation-induced HPRT mutant clones have been studied. The linear-quadratic dependence of the mutation induction on radiation dose has been found. High heterogeneity of cytogenetic parameters (aneuploidy and chromosome aberration frequency) has been shown in the mutants.

[The effect of intracellular pH on radiation damage to mammalian cells]. / Vliianie vnutrikletochnogo pH na radiatsionnoe porazhenie kletok mlekopitaiushchikh.

In experiments with Chinese hamster cells and Ehrlich ascites tumor cells it has been shown that a decrease in intracellular pH from 7.0 to 6.0, produced by 0.1 M phosphate buffer, ameliorates the radiation injury to cells (DMF = 1.35) with respect to their clonogenic capacity and induction of chromosome aberrations. The rate of repair of DNA single-strand breaks, within the pH range under study, is invariable.

[Effects of a medium pH on the ability of Chinese hamster cells to repair radiation injuries]. / Vliianie pH sredy na sposobnost' kletok kitaiskogo khomiachka k vosstanovleniiu ot radiatsionnykh povrezhdenii.

In experiments with Chinese hamster cells at exponential and stationary growth phases, it has been shown that the postirradiation incubation of irradiated cells in a medium with low pH (up to 6.0) promotes the recovery of cells from potentially lethal damages; it has also been found that the recovery from sublethal radiation damages does not depend on the medium pH. The long-term incubation of nonirradiated cells with low pHc causes death of part of cells.

[Effects of induced hyperglycemia on metastasis of Lewis pulmonary carcinoma]. / Vliianie iskusstvennoi giperglikemii na metastazirovanie kartsinomy legkikh Liuis.

The effect of induced hyperglycemia on the intensity of metastatic spreading of Lewis lung carcinoma was investigated in experiments on mice. Neither long-, nor short-term hyperglycemia, induced at different phases of dissemination, influenced the intensity of metastatic spreading, primary tumor growth, the time of appearance of metastases, and the average life duration of tumor-bearing animals.

[Effects of At-211 alpha irradiation on Ehrlich ascites tumor cells]. / Deistvie alfa-izlucheniia 211-At na kletki astsitnogo raka Erlikha.

The biological effect of 211At alpha-particles has been investigated using the Chinese hamster fibroblasts and Ehrlich carcinoma cells growth in vitro. The mean energy of 211At alpha-particles is 6.8 MeV, LET in tissue is 70-160 keV/microns; the half-life period of decomposition of 211At is 7.2 h. The end-points used were a decrease in the mitotic activity, an elevation of the number of degenerating cells, cell with chromosome aberrations and the cell survival. The RBE of alpha-particles in comparison with 60Co gamma-rays is close to 3.

[Patterns in the combined action of irradiation and glucose loading on tumor cells]. / Zakonomernosti kombinirovannogo deistviia oblucheniia i gliukoznoi nagruzki na opukholevye kletki.

It was shown that incubation of Ehrlich ascites tumor cells with glucose or in buffer solutions of low pH decreases their viability. The cell survival rate depends on pH values irrespective of the protoxidation method and oxygenation conditions used. At the same time, radiosensitivity of Ehrlich ascites tumor cells is practically invariable with pH being decreased from 7.0 to 5.0. The effects of glucose and radiation are additive in conditions simulating the effect of hyperglycaemia in vivo.

[Lethal effect of glucose load on tumor cells in hypoxia]. / Letal'noe deistvie gliukoznoi nagruzki na opukholevye kletki v usloviiakh gipoksii.

The in vitro treatment of the Ehrlich ascites tumour (EAT) cells with glucose for 15 and 60 min under hypoxic conditions leads to a decrease in their survival by a factor of 10(2) and 10(4). Glucose load is ineffective under normal oxygenation. The mass destruction of EAT cells under the influence of glucose takes place within the first 24 hours in the interphase. The lethal effect of different pH values on EAT cells is independent of the way by which the given pH value was reached (glucose load or phosphate buffer). The same values of pH lead to the same effect on EAT cells. The lethal effect markedly increased when the value of pH was lower than 5.6. It is concluded that the lethal effect of the glucose load is due to self acidification of EAT cells.

Mathematical model of Ehrlich ascites tumor growth from in vitro treated cells.

A mathematical model is proposed describing the dependence of lethality and average life-span in mice on the number of Ehrlich ascites tumor (EAT) cells inoculated both intact or treated in vitro with various agents. Particularly, the effect of ionizing radiation is discussed, and the effect of combined action of two agents is also considered.

Lethal effect of glucose load on malignant cells.

Ehrlich ascites tumor (EAT) cells were treated by glucose load under anoxic conditions (for 15 or 60 min) and/or by gamma-radiation (20 Gy). The efficiency of the treatment was judged from the tumorigenic activity of EAT cell inocula. The markedly increased efficiency of the combined treatment of EAT cells by glucose load in anoxia and by gamma-radiation is due to the additive action of both agents. The glucose load in anoxia leads to extensive desintegration of tumor cells. Further, the lethal effect of various pH values on EAT cells was investigated. Different pH values were obtained by means of both glucose load and phosphate buffers. The effect was investigated by determining the tumorigenic activity of EAT cells tested in vivo in mice and by determining the radiosensitivity of treated EAT cells. The results allowed us to conclude that the same values of pH lead to the same effect on EAT cells independently of the way by which the given pH value was reached.

[The role of hypoxia in the effect of glucose load on irradiated tumor cells]. / Rol' gipoksii v deistvii gliukoznoi nagruzki na obluchennye opukholevye kletki.

In experiments on Ehrlich ascites tumor cells it was shown that hypoxia, which reduces the lethal effect of gamma-rays, can considerably enhance the injury of cells by glucose. Treatment of tumor cells with glucose in hypoxic conditions followed by exposure to ionizing radiation under both hypoxia and normal aeration causes a 6-7-fold increase in cell injury as compared to irradiation alone. Moreover, the glucose treatment in hypoxic conditions (without concomitant irradiation) may cause approximately 99% death of tumor cells. The data obtained permit to consider the glucose treatment as an effective means by breaking the tumor radioresistance conditioned by a pool of hypoxic cells.