Modification of cellular radiosensitivity by NO-synthase inhibitors

We recently reported that the treatment of V-79 and HeLa cells with nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly reduced the level of the radiation-induced unstable chromosome aberrations. The stereoisomer D-NAME had no effect.We suggest that the radioprotective effect of L-NAME resulted from the action on the generation reactive radicals due to the inhibition of the NOS-activity. We tested this suggestion on the NO-resistant (ECV-304) and NO-sensitive (HeLa) cells, which were treated with L-NAME or aminoguanidine or D-NAME or cysteamine before gamma-irradiation. There are no significantly differences in radiosensitivity between these cells estimated after exposure by gamma-rays with different doses. However, the radioprotective effect of the NOS-inhibitors manifested only for HeLa. D-NAME had no radioprotective effect neither HeLa nor ECV-304. In contrast NOS-inhibitors, cysteamine treatment EVC-304 reduced the radiation-induced level chromosome aberrations almost twofold. The different mechanisms of the modification of cellular radiosensitivity are discussed.     

Mechanism of the radioprotective effect of cysteamine

The radioprotective effect of cysteamine combined with the modification of the chromatin state by sodium butyrate has been studied using V-79 and CHEL lines of Chinese hamster cells and HeLa cells. Sodium butyrate enhances the chromatin sensitivity to nucleases and removes the radioprotective effect of cysteamine as measured by the yield of cells with chromosome aberrations. As is indicated by changes in the intensity of fluorescence of the DNA-ethidium bromide complex, measured by laser flow cytometry, the protective agent decreases the binding of the dye with both irradiated and nonirradiated DNA whereas ionizing radiation and sodium butyrate increase thereof. It is concluded that the radioprotective effect of cysteamine depends in its ability to reduce the susceptibility of DNA to nucleases.     

Effect of pulsed dose in simultaneous and sequential irradiation of V-79 cells by 14.8-MeV neutrons and 60Co photons

The effect of irradiating V-79 Chinese hamster cells with a mixture of 40% 14.8-MeV neutrons and 60% 60Co photons with simultaneous or sequential exposures is investigated. Sample doses are obtained by irradiating cells with alternating 3-min pulses of neutrons and photons (in the sequential case) or with mixed neutrons and photons followed by equal beam-off periods to ensure equal total exposure times for sequential and simultaneous irradiations. Differences between the survival results under each beam configuration that are consistent with previous observations with nonpulsed irradiations are observed.     

Cellular reactions induced by chronic exposure to 14C beta-rays

In V-79 cells the Effects of chronic exposure on induction of chromosome aberrations and abnormal metaphases as well as on efficiency of subsequent exposure to 2 Gy gamma-rays were investigated. It was found that chronic exposure increased the yield of chromosome aberrations as well as abnormal metaphases (spread-metaphases and apoptotic metaphases). In spite of the level of damages in cells, the chronic beta-exposure protected cells against the additional induction of chromosomal aberrations by subsequent exposure to higher acute dose of gamma-irradiation. Cytogenetic adaptive response was retained in the surviving progeny of irradiated cells which were cultured in free medium during 40, 70 days or one year after chronic exposure. At this time the level of residual chromosome aberrations, colony forming ability and distribution of the cells by the number of chromosomes were almost the same as in unirradiated cells. However, the high level of abnormal metaphases and half as much of cells in colony in the surviving progeny of irradiated cells in comparison with unirradiated cell, allow us to suggest that the chronic exposure induced the selection of adaptive forms in condition of the higher level of radiation.     

Effect of nitric oxide donor (NaNO2) on the stability of non-transformed and malignant cells to ultraviolet and gamma-radiation

A decreased sensitivity of the Chinese hamster cells (line V-79) to gamma-radiation under the influence of nitric oxide induction was shown elsewhere. This effect is connected hypothetically with post-radiation reparation of DNA. The investigation of the nitric oxide donor effect on sensitivity of these to UV-radiation is of interest, because this radiation is an important ecological factor of the environment. The question of retention of nitric oxide positive effect on UV and gamma-radiation sensitivity in malignant HeLa cells is no less actual, because these cells significantly differ from normal cells of line V-79. We demonstrated that the donor of nitric oxide enhances stability of the Chinese hamster cells (line V-79) to UV-radiation, as well as to gamma-radiation independently of the time of cell incubation with sodium oxide donor before or after irradiation. The inefficiency of nitric oxide as a factor increasing UV-stability of cells was shown for malignant HeLa cells. A 1 h long incubation of these cells with NO-donor before gamma-irradiation decreased the number of chromosome aberrations, and conversely, the addition of this agent to the HeLa cell culture after gamma-irradiation did not change the radiostability. It may be inferred that distinctions in behaviour of nitric oxide in cultures of V-79 and HeLa cells using UV-radiation may be explained by transformation of the latter special features of their damage, and by the following reparation.     

Formation of chromosome aberrations at different stages of the mitotic cycle in a culture of human lymphocytes irradiated with neutrons of average energy 0.35 and 0.85 MeV

Incidence of chromosome aberrations at different mitotic cycle stages of a human lymphocyte culture has been studied after exposure to 60Co-gamma-quanta (2 Gy) and neutrons of 0.85 Mev (1 Gy) and 0.35 MeV (0.5 Gy). The yield of chromosome aberrations with both gamma- and neutron-radiation depends on a stage of the mitotic cycle. The S stage is the most radioresistant one with both types of ionizing radiation.     

Melanin decreases clastogenic effects of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response

Melanin’s influence on the chromosome aberration frequency induced by radiation in human lymphocytes and mouse bone marrow cells has been studied. We revealed earlier that melanin significantly decreases the frequencies of different radiation-induced mutations in animal germ cells. Melanin protection in somatic cells has been found to be less effective. The melanin effect in somatic cells depends on radiation dose: the lower the damage level, the better the melanin protection. In order to determine the influence of melanin at low radiation doses, the adaptive response was investigated in mouse bone marrow cells in vivo. The level of chromosome aberrations in these cells after fractionated irradiation of 0.2 Gy+1.5 Gy with a 4-h interval was about half that after a single dose of 1.7 Gy. If melanin was injected prior to irradiation, the aberration level decreased by a factor of about two in both cases. This observed result may be due to the potential radioprotective effect of melanin and to the absence of any adaptive response, whereas in the case of melanin application between the priming and challenge doses, the combined effect of the adaptive response as well as melanin protection resulted in a 4-fold decrease of chromosome aberrations. These results allow us to draw the following conclusions: adaptive response can be prevented by a radioprotector such as melanin, and melanin is capable of completely removing low-dose radiation effects. Received: 2 December 1998 / Accepted in revised form: 15 September 1999     

Effect of thyroxine in the G0 stage of the cell cycle on the yield of chromosome aberrations in rat hepatocytes and human lymphocytes after X-irradiation

A study was made of the mechanism of a modifying action of thyroxin (T4) on the chromosome integrity after the application thereof in vivo and in vitro following a single whole-body exposure to X-radiation with a dose of 2.19 Gy. It is concluded that T4 influences the quantity of cells with chromosome aberrations stimulating the recovery of the chromosome integrity: the stimulation can be brought about at the G0 stage of the cell cycle as a result of the direct effect of the hormone on a cell.     

The cytogenetic effects of 22-MeV neutrons

22-MeV neutrons affecting the culture of human peripheral blood lymphocytes have been studied for their cytogenetic peculiarities. Linear character of the dependence of chromosome aberrations yield within the dose range of 0.2-4.0 Gy is noted. Relative biological efficiency of 22-MeV neutrons reaches the highest values at low doses (18.8-2.4). Pair fragments and dicentrics prevail in spectrum of aberrations.     

Effect of ectopic superexperssion of the anti-apoptotic gene bcl-2 on the level and character of karyotypic instability in the CHLL V-79 RJK Chinese hamster cell line

Karyotypic destabilization in cells of Chinese hamster fibroblasts CHL V-79 RJK with ectopically overexpressed antiapoptotical human bcl-2 gene from pSFFV-bcl-2 vector has been analysed. Analysis of G-banded metaphase chromosomes from 4 clones with different levels of bcl-2 expression revealed an increased level of chromosomal instability in bcl-2-transfected cells. Besides, an increased percentage of aneu- and polyploid cells and high level of cells with different chromosomal aberrations was observed. The degree of karyotypic instability positively correlated with the level of bcl-2 expression in bcl-2-transfected cells. Cells of a clone with the highest bcl-2 expression at the 13th passage of cultivation displayed an almost 100% polyploidization and the presence of specific aberrations and a tricentric marker chromosome. Selection of cells with non-random specific chromosome changes was observed in pSFFV-bcl-2-transfected CHL V-79 RJK cells in the process of their long-term cultivation. By contrast, cells of the parental cell line, as well as the control pSFFV-neo transfectants, displayed a stable karyotype throughout the long period of cultivation. It is important that the presence of morphological markers of gene amplifications--DOO, DM, MH--was observed in bcl-2-transfected cells. These findings suggest that the overexpression of antiapoptotic human bcl-2 gene may result in destabilization of the karyotype structure in cells of Chinese hamster fibroblasts CHL V-79 RJK. The character and level of destabilization correlate with the level of ectopic overexpression of this gene.     

A simplified procedure for the observation in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in mammalian monolayer cell cultures

Chinese hamster V-79 cells are widely used in short term screening for potential physical or chemical mutagens of the environment. A simplified version of the standard Giemsa protocol of Moorhead and the Feulgen plus Giemsa protocol of Wolff and Perry is given which permits the observations in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in the Petri dishes for the culture of the V-79 cells.     

Effect of antioxidant-ambiol on the radiation adaptive response

For studying of the mechanism of adaptive response of plants the seeds of soft wheat Triticum aestivum cultivar Moscovskaya 39 were irradiated in doses 0.25, 50 and 0.25 + 50 Gy and the frequency of cells with aberrations and the mitotic activity in the meristem of seedlings were scored. The pre- and post-treatments of seeds with antioxidant--ambiol were also used. It was found that the exposure of seeds to 0.25 Gy reduce the effects of challenge dose of 50 Gy: the mitotic index increases and the frequency of cells with aberrations decreases--the adaptive response appear. It was also found that the pretreatment with ambiol reduce the effects of the irradiation in the dose of 50 Gy. Post-treatment was less efficiently. Both treatments raise the adaptive response. The correlation between the frequency of aberrant cells and the mitotic index was found and, regardless of the type of treatment all points of experiment fall on the common regression line with the regression coefficient -0.85 (p < 0.01). These facts serve as evidence (1) that the radioprotective effect by the pre- and post-treatment occurs by a common mechanism and (2) that the in the exhausted concentration antioxidant does not change the extent of genome damage inflicted by irradiation. The evidence is consistent with the hypothesis that a nonspecific inducible process of stimulated repopulation was a mechanism of adaptive response of plants.     

A Simplified Procedure for the Observation In Situ Of Chromosome Aberrations or Sister Chromatid Exchanges and the Estimation of the Mitotic Index in Mammalian Monolayer Cell Cultures

Chinese hamster V-79 cells are widely used in short term screening for potential physical or chemical mutagens of the environment. A simplified version of the standard Giemsa protocol of Moorhead and the Feulgen plus Giemsa protocol of Wolff and Perry is given which permits the observations in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in the Petri dishes used for the culture of the V-79 cells.     

Resveratrol reduces radiation-induced chromosome aberration frequencies in mouse bone marrow cells

Resveratrol, a polyphenol compound with reported antioxidant and anticarcinogenic effects, a wide range of molecular targets, and toxicity only at extreme doses, has received considerable attention. We evaluated the radioprotective effect of orally administered resveratrol on the frequencies of chromosome aberrations in irradiated mouse bone marrow cells. CBA/CaJ mice were divided into four groups: (1) no treatment, (2) resveratrol only, (3) radiation only, and (4) resveratrol and radiation. Resveratrol treatment (100 mg/kg daily) was initiated 2 days prior to irradiation. Bone marrow was then harvested at 1 and 30 days after a single dose of 3 Gy whole-body gamma radiation. A statistically significant (P < 0.05) reduction in the mean total chromosome aberration frequency per metaphase at both times postirradiation in the resveratrol and radiation group compared to the radiation-only group was observed. This study is the first to demonstrate that resveratrol has radioprotective effects in vivo. These results support the use of resveratrol as a radioprotector with the potential for widespread application.     

Effect of preliminary chronic irradiation and alpha-tocopherol on the frequency of chromosome aberrations in mouse bone marrow cells, induced by exposure to acute gamma-irradiation

The incidence of chromosome aberrations in bone marrow cells of femur did not exceed the spontaneous one in CBA mice exposed, during 70 days, to gamma-radiation at dose--rates of 33.7-35.8 nA/kg and cumulative dose of 2.75 Gy. A single acute exposure of intact animals to a dose of 2.98 Gy increased significantly the mutation level. Preirradiation with small doses increased the resistance of hereditary structures to sublethal radiation doses. Exogenous alpha-tocopherol (0.06 mg/20 g mass) protected the genetic apparatus of cells from total-body irradiation and was an additional factor decreasing the mutation level after acute exposure of mice at the background of long-term irradiation with small doses.     

Rapid dicentric assay of human blood lymphocytes after exposure to low doses of ionizing radiation

The probability of losses of different chromosome aberrations during the dicentric chromosome assay of metaphase cells with incomplete sets of chromosome centromeres was estimated using a mathematical model for low doses of ionizing radiation. A dicentric assay of human blood lymphocytes without determination of the total amount of chromosome centromeres in cells without chromosome aberrations (rapid dicentric assay) has been proposed. The rapid dicentric analysis allows to register chromosome aberrations in full compliance with the conventional classification. The experimental data have shown no statistically significant difference between the frequencies of dicentric chromosomes detected by rapid and classical dicentric chromosome assays of human lymphocytes exposed to 0.5 Gy of 60Co gamma-rays. The rate of the rapid dicentric assay was almost twice as high as that of the classical dicentric assay.     

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.     

Action of mixed gamma-neutron radiation with various dose rates on the number of cells in thymus and chromosomes of mice bone marrow and human lymphocytes

The irradiation with mixed gamma-neutron radiation was carried out at the pulse nuclear reactor on fast neutrons BARS-6 in a regimen of one pulse (100 micros) and in a regimen of continuous irradiation during 60 minutes. Was shown, that the irradiation of mice with pulse radiation was 1.3-1.8 times more effective in the induction of the chromosome aberrations in bone marrow cells in comparison with the continuous regimen of irradiation. At the same time, other biological tests (yield of chromosome aberrations in human lymphocytes, decreasing the number of cells in thymus) demonstrated that pulsed and continuous regimens have almost equal biological effectiveness.     

Study of external low irradiation dose effects on induction of chromosome aberrations in Pisum sativum root tip meristem

The effects of low doses of ionizing radiation have been a matter of important debate over the last few years. The point of discussion concerns the validity of the linear dose-response extrapolation for low doses, used by international organizations, to establish radio-protection norms. Here, we contributed to this discussion by investigating the induction of chromosome aberrations by low to moderate doses ranging from 0 to 10 Gy in root meristem cells of 6-day-old Pisum plantlets. After acute irradiation of plantlets by a (60)Co source, the percentage of root tip meristem cells displaying chromosome aberrations was estimated immediately after irradiation and after 20 h recovery time. The dose-effect curves show non-linear responses, especially in the low dose range (0- 1 Gy), which is of particular interest. After 20 h of recovery, a steep increase of aberrations was observed for cells exposed to 0.4 Gy, followed by a plateau for doses until 1 Gy. There was an irradiation effect on plant growth during the first and second generations, showing the persistence of cell division anomalies as a long term effect of acute irradiation. This result suggests the induction of a genomic instability.Our results, in agreement with some obtained in animals, show rather non-linear dose-effect responses, with notably higher biological effects of low doses than expected.     

The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.