Characterization of the adaptive response to ionizing radiation induced by low doses of X rays to human lymphocytes

In previous studies we have shown that low doses of radiation from incorporated tritiated thymidine can make human lymphocytes less susceptible to the genetic damage manifested as chromatid breakage induced by a subsequent high dose of X rays. We have also shown that this adaptive response to ionizing radiation can be induced by very low doses of X rays (0.01 Gy; i.e., 1 rad) delivered during S phase of the cell cycle. To see if a low dose of X rays could induce this response in cells at other phases of the cell cycle, human lymphocytes were irradiated with 0.01 or 0.05 Gy before stimulation by phytohemagglutinin (G0) or with 0.01 Gy at various times after stimulation (G1), followed by 1.5 Gy (150 rad) at G2 phase. Although G0 lymphocytes failed to exhibit an adaptive response, G1 cells irradiated as early as 4 h after stimulation did show the response. Experiments were also carried out to determine how long the adaptive response induced by 0.01 Gy could persist. A 0.01-Gy dose was delivered to lymphocytes in the first S phase, followed by 1.5 Gy in the same or subsequent cell cycles. Lymphocytes receiving a 1.5-Gy dose at 40, 48, or 66 h after stimulation exhibited an adaptive response, whereas those receiving a 1.5-Gy dose at 90 or 114 h did not. Duplicate cultures containing bromodeoxyuridine showed that at 40 h all the lymphocytes were in their first cell cycle after stimulation, at 48 h half of the lymphocytes were in their first cell cycle and half in their second, and at 66 h 80% of the lymphocytes were in their third cell cycle. Thus the adaptive response persists for at least three cell cycles after it is induced by 0.01 Gy of X rays. In other experiments, the time necessary for maximal expression of the adaptive response was determined by delivering 0.01 Gy at hourly intervals 1-6 h before the 1.5-Gy dose. While a 4-h interval was enough for expression of the adaptive response, shorter intervals were not.     

Induction of chromosome aberrations in G0 human lymphocytes by low doses of ionizing radiations of different quality

Human peripheral blood lymphocytes from two donors were exposed to low doses (0.05 to 2.0 Gy) of gamma rays, X rays, or fast neutrons of different energies. Chromosome aberrations were analyzed in metaphase of first-division cells after a culture time of 45-46 hr. At this time, less than 5% of the cells were found in second division. Different dose-response relationships were fitted to the data by using a maximum likelihood method; best fits for radiation-induced dicentric aberrations were obtained with the linear-quadratic law for all radiations. The linear component of this equation predominated, however, for neutrons in the range of doses studied, and the frequency of dicentrics induced by d(16)+Be neutrons up to 1.0 Gy could also be described by a linear relationship. The relative biological efficiency (RBE) of X rays and d(16)+Be, d(33)+Be, and d(50)+Be neutrons compared to 60Co gamma rays in the low dose range was calculated from the dose-effect relationships for the dicentrics produced. The RBE increased with decreasing neutron dose and with decreasing neutron energy from d(50)+Be to d(16)-+Be neutrons. The limiting RBE at low doses (RBEo) was calculated to be about 1.5 for X rays and 14.0, 6.2, and 4.7 for the d(16)+Be, d(33)+Be, and d(50)+Be neutrons, respectively.     

The enhanced radioresistance (adaptive response) in vivo of splenic colony-forming units (CFU-S) following the exposure of mice to 60Co gamma rays at low doses]

An increase of resistance to radiation damage of human lymphocytes previously exposed to low doses of ionizing radiation from incorporated tritiated thymidine was observed by G. Olivieri et al. in 1984. The phenomenon was named adaptive response and its occurrence was reported by others for many animal and plant cells. In this research we studied the adaptive response of spleen colony formation at different time after previous irradiation of mice with low doses of 60Co gamma rays. Our results suggest that the pretreatments protect spleen colony-formatting units (CFU-S) from the second damaging radiation dose of 1.5 Gy during long time (as many as one month).     

Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin

Human peripheral blood lymphocytes cultured in the presence of low concentrations of bleomycin (BLM), 0.01-0.1 microgram/ml, for 48 h and then treated with a high concentration (1.5 microgram/ml) of the same agent or with 1.5 Gy X-rays, became significantly less sensitive to the induction of chromosomal damage than those which did not receive the pre-treatment with BLM. They responded with lower frequencies of chromatid and isochromatid breaks. These results lend further support to the operation of an adaptive repair system in lymphocytes which offers resistance and cross-resistance to the induction of chromosomal damage by the same or similar DNA-damaging agents.     

Response of plateau-phase C3H 10T1/2 cells to radiation and concurrent administration of bleomycin

The mode and extent of interaction between bleomycin and radiation were assessed in contact-inhibited cultures of C3H 10T1/2 cells, which in confluent monolayers display a low turnover rate and behave more like late-responding normal tissues in vivo with respect to response to fractionated radiotherapy (i.e., having a low alpha/beta value). Plateau-phase C3H 10T1/2 cultures were exposed to gamma rays delivered in 1, 2, 5, or 10 fractions. The radiation doses administered ranged from 2 Gy in one exposure to 26 Gy in 10 fractions. Half of the cultures were also treated with 1 micrograms/ml of bleomycin for 5 days during which radiation was also given. It was found that 1 micrograms/ml of bleomycin sterilized approximately 40% of the C3H 10T1/2 cells in the cultures. The radiation dose-survival curves of various fractionation schedules (1, 2, 5, and 10 fractions) plus bleomycin were displaced downward (i.e., to lower survival levels) but not modified in shape. The alpha/beta ratios, parameters of the linear-quadratic model of cell survival, were 2.6 (2.2-3.1) and 2.4 (1.8-3.1) Gy for radiation only and radiation plus bleomycin, respectively. This observation indicates that the effect of combining irradiation and bleomycin on C3H 10T1/2 cells in monolayers was additive.     

低水平辐射诱导的细胞遗传学适应性反应

先用0.01GY x-射线(剂量率:0.01GY/分)体外照射人、兔外周血,经不同时间后再用1.5GY X-射线(0.44GY/分)照射,发现在G_0、G_1、S和G_2期受0.01GY X-射线照射后再给大剂量照射者,其染色体畸变率明显低于单纯受1.5GY X-射线照射组(P<0.01)。这一适应性反应能持续3个细胞周期,在接受小剂量照射后超过3个细胞周期再受大剂量照射者,染色体畸变率未见减少。若在第三细胞周期以后再次给予小剂量照射,可再次诱导适应性反应。用小鼠整体小剂量照射后骨髓细胞和生殖细胞亦出现这种适应性反应。另外也探讨了不同剂量和不同剂量率的预先照射对适应性反应的影响。     

Mathematical analysis of genetic effects of low doses of ionizing radiation

The comparative study of effects of low doses of radiation on peripheral blood lymphocytes of persons occupationally exposed to radiation and non-exposed ones was carried out. The main attention was paid to radio-adaptive response forming under consistent exposure to low (0.05 Gy) and damaging (2 Gy) doses of gamma-irradiation. Noticeable heterogeneity in capacity for adaptive response forming in occupational group was revealed. The mathematical model adequate to experimental material was constructed using Kohonen neuronets.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

Lack of adaptive response of gamma radiation for protection against neutron-induced teratogenesis

BACKGROUND: Although there are some reports on neutron teratology, there is little information on the adaptive response of gamma radiation for protection against neutron‐induced teratogenesis. This study examined whether or not a low dose of gamma radiation can induce an adaptive response in mouse fetuses exposed to a subsequent dose of neutrons in vivo. METHODS: Pregnant ICR mice were exposed to a priming dose of 0.3 Gy (0.9 Gy/min) of gamma rays on day 10.5 of gestation and challenged with 0.8 Gy (0.94 Gy/minute) of neutrons 24 hlater. The mice were sacrificed on day 18.5 of gestation. The fetuses were examined for mortality, growth retardation, and other morphologic abnormalities. RESULTS: The tail length in the 0.3 Gy of gamma rays + 0.8 Gy of neutrons group was significantly shorter than in the 0.8 Gy of neutrons group. Although there was no significant difference compared with the 0.8 Gy of neutrons group, the number of live fetuses in the 0.3 Gy of gamma rays +0.8 Gy of neutrons group was lower. There was no evidence of primed exposure‐related reductions in the malformed fetuses. Although there was no significant difference compared with the unprimed group, the number of malformed offspring in the primed group was higher. Furthermore, the incidence of kinked tail and adactyly was significantly higher in the primed mice than in the unprimed mice. CONCLUSIONS: Overall, this study shows that exposure to 0.3 Gy of gamma rays failed to induce an adaptive response of fetogenesis to a neutron challenge dose. Birth Defects Res (Part B) 83:502‐506, 2008. © 2008 Wiley‐Liss, Inc.     

A re-examination of the effects of ionizing radiation on lifespan and transformation of human diploid fibroblasts.

Human diploid fibroblasts, strain MRC-5, were sequentially irradiated with 60Co gamma rays at intervals during their in vitro lifespan. The results indicate that 3 or 6 doses of 1 Gy can increase lifespan, and the same was true for cells treated with 3 doses of 3 Gy. Higher doses (5 x 3 Gy) did reduce growth potential, suggesting either that mid-late passage cells become more sensitive to radiation, or that doses beyond a given threshold reduce population lifespan by multiple cellular hits. The life extension induced by gamma rays might be due to an induced hypermethylation of DNA. Alternatively, oxygen radicals produced by irradiation might trigger an adaptive stress response which would remove damaged macromolecules and thereby increase the cells' growth potential. Whichever explanation is correct, the results show that the human fibroblast system is not appropriate for the study of the well known effect of ionizing radiation in shortening the lifespan of experimental animals. Contrary to earlier published results, populations of cells treated with cumulative doses of 15 Gy or 18 Gy and held for nearly 3 months after they had reached senescence (Phase III), produced no foci of transformed cells.     

Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell

Chinese hamster ovary cells grown in vitro were treated with bleomycin or irradiated with high doses of 60Co gamma rays (200 and 400 Gy). DNA strand breaks in single cells were analysed by using our newly introduced microelectrophoretic technique. Bleomycin seems to act in a selective manner so that in some cells the DNA is heavily degraded while in others there is only moderate or no measurable damage. In contrast, a uniform response was found after gamma irradiation. To achieve the same magnitude of DNA fragmentation as in the most severely bleomycin-damaged cells, irradiation with more than 200 Gy is required. Some 8000 double-strand breaks per cell are produced by 200 Gy which will convert the molecular weight of the DNA to the range of 10(8)-10(9) dalton, and free migration of DNA fragments occurs during electrophoresis. We include also a detailed study of the DNA migration pattern following doses of 0-100 Gy gamma rays.     

The induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia: effects of low doses and low dose rates

The induction of reciprocal translocation in rhesus monkey spermatogonial stem cells was studied following exposure to low doses of acute X rays (0.25 Gy, 300 mGy/min) or to low-dose-rate X rays (1 Gy, 2 mGy/min) and gamma rays (1 Gy, 0.2 mGy/min). The results obtained at 0.25 Gy of X rays fitted exactly the linear extrapolation down from the 0.5 and 1.0 Gy points obtained earlier. Extension of X-ray exposure reduced the yield of translocations similar to that in the mouse by about 50%. The reduction to 40% of translocation rate after chronic gamma exposure was clearly less than the value of about 80% reported for the mouse over the same range of dose rates. Differential cell killing with ensuing differential elimination of aberration-carrying cells is the most likely explanation for the differences between mouse and monkey.     

Protective effect of ferulic acid on gamma-radiation-induced micronuclei, dicentric aberration and lipid peroxidation in human lymphocytes

In this study we examined radioprotective effect of ferulic acid (FA) on gamma radiation-induced dicentric aberration and lipid peroxidation with reference to alterations in cellular antioxidant status in cultured lymphocytes. To establish most effective protective support we used three different concentrations of FA (1, 5 and 10 microg/ml) and three different doses of gamma-radiation (1, 2 and 4 Gy). Treatment of lymphocytes with FA alone (at 10 microg/ml) gave no significant change in micronuclei (MN), dicentric aberration (DC), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPx) activities when compared with normal lymphocytes; irradiation at 1, 2 and 4 Gy increased the MN and DC frequencies in a dose-dependent manner. Treatment with FA for 30 min before radiation exposure resulted in a significant decline of MN and DC yields as FA concentration increased. Compared to 1 Gy exposure alone, the extent to which FA (1 microg/ml) reduced the MN and DC yields was 75% and 50%, respectively. With 4 Gy irradiation, FA (10 microg/ml) decreased 45% MN and 25% DC frequencies. FA-pretreated lymphocytes (1, 5 and 10 microg/ml) showed progressively decreased TBARS levels after irradiation. Irradiation (1, 2 and 4 Gy) significantly decreased GSH levels, SOD, CAT and GPx activities in a dose-dependent manner. Pretreatment with 10 microg/ml of FA significantly (p<0.05) prevented the decreases in the radiation-induced GSH, SOD, CAT and GPx activities. These findings suggest potential use and benefit of FA as a radioprotector.     

Kinetics of the Early Adaptive Response to Gamma Rays: Induction of a Cellular Radioprotective Mechanism in Murine Leukocytes <Emphasis Type="Italic">In vivo</Emphasis>

The aims of the study were to establish the kinetics of the early adaptive response and to determine the minimum adaptive dose of gamma rays capable of inducing this response. The minimum adaptive dose was determined by exposing groups of five BALB/c male mice to an adaptive dose of 0.005 or of 0.02 Gy γ rays from a ¹³⁷Cs source and challenge with 1.0 Gy 60 min later. The kinetics of adaptive response induction was established by exposing mice to an adaptive dose of 0.01 Gy, and subsequently to a challenge dose of 1.0 Gy at different times. Blood samples were collected from the tail immediately after exposure to the challenge dose, and the percentage of DNA-damaged cells and the extent of damaged were determined by single cell gel electrophoresis in 300 leukocytes per animal in five mice. The results confirms the capability of an in vivo induction of an early radioprotective process against the DNA-damage produced by gamma rays in murine leukocytes, and allows us to conclude that the minimum adaptive dose lies between 0.005 and 0.01Gy of gamma rays, and the early adaptive response is induced as early as 30 min after the exposure and persists for at least 18 hr.     

Induction and rejoining of large DNA fragments after ion irradiation.

Radiation-induced DNA double-strand breaks (DSBs) were analyzed by separating large DNA fragments by pulsed-field gel electrophoresis. Human U-343MG glioma and K562 erythroleukemia cells were irradiated with 60Co gamma rays or nitrogen ions with high linear energy transfer (125 keV/microm). By comparing the fraction of DNA released into the gel below different size thresholds, corresponding to megabase-pair-sized DNA fragments, the relative effectiveness of the nitrogen ions was found to be dependent on both dose and the threshold size used in the evaluation. This dose dependence was most evident for the smallest threshold (6 Mbp) and was due to a linear dose response for release of the fragments for the ions compared to the curvilinear response for the gamma rays. The two curves intersected, and the relative yield of fragments (nitrogen ions/gamma rays) decreased from more than 3 below 1.5 Gy to 0.8 at 30 Gy. For the larger sizes (6-10.5 Mbp), the relative yield was constant at around 0.7. Thus the ion-induced fragments were shifted to smaller sizes compared to the 60Co gamma rays, and the data for nitrogen ions could not be fitted to random fragment distributions at doses < or =20 Gy. From these results, we conclude that a substantial fraction of the DSBs induced by heavy ions were nonrandomly distributed, correlated with DSBs within a region of < or =2 Mbp. After a dose of 20 Gy, the rejoining curves for ion-induced DSBs were different for each fragment size, resulting in different levels of unrejoined breaks after 6 h.     

Modulation of gamma-ray-induced apoptosis in human peripheral blood leukocytes by famotidine and vitamin C

To study the radioprotective effects of vitamin C and famotidine against radiation-induced apoptosis in human peripheral blood leukocytes, peripheral blood was obtained from six healthy volunteers including three males and three females. Twelve microlitres of blood sample diluted in 1 ml complete RPMI-1640 medium was irradiated with various doses of gamma-rays (4, 8 and 12 Gy) in the presence or absence of various doses of vitamin C and famotidine. After 48 and 72 h incubation in a 37 degrees C CO(2) incubator, neutral comet assay was performed for all samples. At least 1000 cells were analyzed for each sample for presence of apoptosis. Data were statistically evaluated using Mann-Whitney non-parametric and ANOVA tests. Results show a significant increase in apoptosis induction following gamma-irradiation with a dose dependent manner compared to controls (p<0.001). Presence of famotidine at 200 microg/ml produced a significant protective effect against radiation-induced apoptosis for various doses of radiation. Similar effects were observed for vitamin C at much lower doses (10 microg/ml). Dose reduction factor (DRF) calculated for famotidine treatment was about 1.5, and above 2 for vitamin C treatment. These results suggest that both vitamin C and famotidine suppresses radiation-induced apoptosis when used with various doses of gamma-irradiation (4-12 Gy) probably via *OH radical scavenging and an intracellular antioxidation mechanism.     

Variability of the adaptive response to ionizing radiations in humans

Human lymphocytes exposed to low doses of ionizing radiations from incorporated tritiated thymidine ([3H]dThd) or from X-rays become less susceptible to the induction of chromatid aberrations by high doses of X-rays. This indicates that low doses of ionizing radiation can produce an effect similar to the adaptive response observed with alkylating agents in prokaryotes, animal and plant cells. To determine whether there is individual variability in the adaptive response to ionizing radiations we exposed human lymphocytes from 18 different healthy donors to 'adapting' doses of [3H]dThd (0.01 microCi/ml) or X-rays (0.01 Gy) and subsequently to a 'challenge' treatment of 0.75 Gy of X-rays delivered 2 h before fixation. Four of the 18 donors did not show an adaptive response; in some cases in these individuals a synergistic response of increased, rather than decreased, damage was found. Two of these 4 donors showed no adaptive response in 3 subsequent experiments separated by 4-month intervals. This suggests that the human population exhibits a heterogeneity in the adaptive response to ionizing radiations which might be, at least in part, genetically determined.     

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Protective effect of curcumin on gamma-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes

The present work is aimed at evaluating the radioprotective effect of curcumin, a naturally occurring phenolic compound on gamma-radiation induced toxicity. The cellular changes were estimated by using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analysed by using cytokinesis blocked micronucleus assay and dicentric aberration (DC). The gamma-radiation at different doses (1, 2 and 4Gy) were found to significantly increase micronuclei (MN), DC frequencies and TBARS level whereas the levels of GSH and antioxidant enzymes were significantly decreased. The maximum damage to lymphocytes was observed at 4Gy irradiation. Curcumin pretreatment (1, 5 and 10microg/ml) significantly decreased the frequency of MN and DC. The levels of TBARS decreased and activities of SOD, CAT and GPx significantly increased along with GSH levels. At 1Gy irradiation all the concentrations of curcumin (1, 5 and 10microg/ml) significantly protected the lymphocytes from radiation damage. At 2Gy irradiation, 5 and 10microg/ml of curcumin showed significant radioprotection. Since the highest damage was observed at 4Gy irradiation both 1 and 5microg/ml of curcumin pretreatment were not sufficient to protect the lymphocytes from radiation damage but 10microg/ml of curcumin significantly protected the cultured lymphocytes from radiation damage. Thus, pretreatment with curcumin gives protection to lymphocytes against gamma-radiation induced cellular damage.