Dose fractionation effects in plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts

A comparison of gamma-ray dose fractionation effects was made using plateau-phase cultures of C3H 10T1/2 cells and their transformed counterparts in an attempt to simulate basically similar populations of cells that differ primarily in their turnover rates. The status of cell populations with respect to their turnover rates may be an important factor influencing dose fractionation effects in early- and late-responding tissues. In this cell culture system, the rate of cell turnover was approximately three times higher for the plateau-phase transformed cultures. While the single acute dose survival curves for log-phase cells were indistinguishable, there were significant differences between the survival curves for plateau-phase cultures of the two cell types. These differences were qualitatively similar to the differences recently postulated for the survival of target cells governing early and late tissue responses. Both cell lines had a similar capacity for repair of sublethal damage, but untransformed cells had a much greater capacity to repair potentially lethal damage in plateau phase. Further, untransformed plateau-phase cultures were much more sensitive to a radiation-induced G1 (or G0 to G1) delay than transformed cultures. Multifraction survival curves were determined for both cell lines for doses per fraction ranging from 9.0 to 0.8 Gy, and from these isoeffect curves of log total dose versus dose per fraction were derived. The isoeffect curve for the slowly cycling, untransformed cells was found to be appreciably steeper than that for the more rapidly cycling transformed cells, a finding consistent with previously reported differences in dose fractionation isoeffect curves for early- and late-responding tissues in vivo.     

Multifraction radiation response of mouse lung

The response of mouse lung to repeated doses of 60Co gamma-rays of as low as 115 cGy per fraction was measured using death from pneumonitis between 80 and 120 days after irradiation as the endpoint. A fractionation interval of 3 h was maintained for most regimens but in the longer experiments some 12 h intervals were introduced for logistic reasons. The longest overall duration (for a 43 fraction regimen) was 8 days. The total doses required to produce 50 per cent mortality increased continuously as dose/fraction was decreased, even from 160 to 115 cGy per fraction. Of clinical relevance, the steepness of the isoeffect curve over the dose range 115-500 cGy indicates that the lung shows greater sparing from dose fractionation than is characteristic of more rapidly-responding normal tissues, resembling, in this respect, other more slowly-responding tissues such as spinal cord. The plot of the reciprocal of the LD50 values as a function of dose per fraction was non-linear, suggesting that a linear quadratic dose response model may not be appropriate or that repair of cellular injury in lung is not complete in 3 h, or both.     

Dose-rate effects in mammalian cells. IV. Repairable and nonrepairable damage in noncycling C3H 10T 1/2 cells

Repairable and nonrepairable components of gamma-ray damage leading to cell reproductive death were determined by measuring the range over which dose rate influenced the response of non-cycling C3H 10T 1/2 mouse cells. Cell proliferation and cell cycle redistribution were eliminated as factors influencing the dose-rate effect in the system by irradiating confluent monolayers of contact inhibited cells. The radiosensitivity of the cells did not change, and no selective loss of damaged cells occurred over the extended treatment times. A pronounced dose-rate effect was observed over the range between 55.6 and 0.29 Gy/hr, but a limit to the repair-dependent dose-rate effect was reached at 0.29 Gy/hr since no further reduction in effect per unit dose was observed when the dose rate was reduced to 0.17 or 0.06 Gy/hr. The survival curves, which were simple exponential functions of dose at dose rates of 0.29 Gy/hr and below, have a common Do of 7.32 Gy and represent an accurate measurement of the nonrepairable component of damage. Log-phase cultures showed remarkably different responses over the range of dose rates, due in large part to cell cycle redistribution and in some cases, cell proliferation during exposures. The results of these studies were compared with time-dose relationships used in clinical brachy-therapy and agree remarkably well with corrections in total dose suggested by R. Paterson [Br. J. Radiol. 25, 505-516 (1952)] and A.E.S. Green [cited in F. Ellis, Curr. Top. Radiat. Res. Q. 4, 357-397 (1968)] when the standard treatment time is changed. Comparison of our data with in vivo isoeffect curves of total dose vs dose per fraction for "early" and "late" tissue responses indicate that cell cycle redistribution should not be ignored as a factor influencing time-dose relationships in radiotherapy.     

Repair but not potentiation observed in mouse lung irradiated with neon ions

The lungs of mice were irradiated with 1, 4, or 7 fractions of X rays or neon ions in a 4-cm spread Bragg peak. Lung function as a function of total radiation dose was tested at 7 and 12 months after irradiation by measuring the resting breathing rate in a whole-body plethysmograph. The isoeffect doses increased sequentially with X rays for 1 through 4 to 7 fractions, demonstrating repair of sublethal radiation injury as previously reported. There was also a significant increase of isoeffect dose with neon ions between 1 and 4 fractions but no further increase at 7 fractions. Thus repair instead of potentiation of radiation injury in lung clearly occurred after neon ion irradiation. The effectiveness of neon ions appeared to be closer to that of neutrons with a mean energy of 8 meV than those with a mean energy of 2.3 meV.     

Identification of Sensitive Serum microRNA Biomarkers for Radiation Biodosimetry

Exposure to ionizing radiation through environmental, occupational or a nuclear reactor accident such as the recent Fukushima Daiichi incident often results in major consequences to human health. The injury caused by radiation can manifest as acute radiation syndromes within weeks in organs with proliferating cells such as hematopoietic and gastrointestinal systems. Cancers, fibrosis and degenerative diseases are also reported in organs with differentiated cells, months or years later. Studies conducted on atom bomb survivors, nuclear reactor workers and animal models have shown a direct correlation of these effects with the absorbed dose. Physical dosimeters and the available radio-responsive biologics in body fluids, whose responses are rather indirect, have limitations to accurately evaluate the extent of post exposure damage. We have used an amplification-free, hybridization based quantitative assay utilizing the nCounter multiplex platform developed by nanoString Technologies to compare the levels of over 600 miRNAs in serum from mice irradiated at a range of 1 to 12 Gy at 24 and 48 hr time points. Development of a novel normalization strategy using multiple spike-in oligonucleotides allowed accurate measurement of radiation dose and time dependent changes in serum miRNAs. The response of several evolutionarily conserved miRNAs abundant in serum, were found to be robust and sensitive in the dose range relevant for medical triage and in patients who receive total body radiation as preparative regimen for bone marrow transplantation. Notably, miRNA-150, abundant in lymphocytes, exhibited a dose and time dependent decrease in serum, which we propose as a sensitive marker indicative of lymphocyte depletion and bone marrow damage. Our study has identified several markers useful for evaluation of an individual’s response by minimally invasive methods, relevant to triage in case of a radiation accident and evaluation of toxicity and response during and after therapeutic radiation.     

Effects of low level radiation upon the hematopoietic stem cell: Implications for leukemogenesis

Summary These studies have addressed firstly the effect of single small doses of x-rays upon murine hematopoietic stem cells to obtain a better estimate of theD _q . It is small, of the order of 20 rad.Secondly, a dose fractionation schedule that does not kill or perturb the kinetics of hemopoietic cell proliferation was sought in order to investigate the leukemogenic potential of low level radiation upon an unperturbed hemopoietic system. Doses used by others in past radiation leukemogenesis studies clearly perturb hemopoiesis and kill a detectable fraction of stem cells. The studies reported herein show that 1.25 rad every day decrease the CFU-S content of bone marrow by the time 80 rads are accumulated. Higher daily doses as used in published studies on radiation leukemogenesis produce greater effects.Studies on the effect of 0.5, 1.0, 2.0, and 3.0 rad 3 times per week are under way. Two rad 3 times per week produced a modest decrease in CFU-S content of bone marrow after an accumulation of 68 rad. With 3.0 rad 3 times per week an accumulation of 102 rad produced a significant decrease in CFU-S content of bone marrow. Dose fractionation at 0.5 and 1.0 rad 3 times per week has not produced a CFU-S depression after accumulation of 17 and 34 rad.Radiation leukemogenesis studies published to date have utilized single doses and chronic exposure schedules that probably have significantly perturbed the kinetics of hematopoietic stem cells. Whether radiation will produce leukemia in animal models with dose schedules that do not perturb kinetics of hematopoietic stem cells remains to be seen.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthdayResearch supported by the U.S. Department of Energy under contract DE-ACO2-7 6CH00016. Accordingly, the U.S. government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. government purpose     

The use of 'top-up' experiments to investigate the effect of very small doses per fraction in mouse skin

The partial tolerance type of 'top-up' experiment has been investigated to determine the resolution of this approach for studying the damage to mouse skin from very small doses of X-rays and neutrons. The effect of 20 fractions, each as small as 0.10 Gy of X-rays or of 0.05 Gy of neutrons, can be detected if 3 MeV neutrons are used as the 'top-up' reference radiation. This capability results from the almost linear underlying dose-response curve and highly reproducible dose-effect relationship for the low energy neutrons. The data fit the linear quadratic model of dose fractionation for X-rays down to fractional doses of 0.75 Gy, but at lower doses there is a trend towards an increase in the skin radiosensitivity. Modelling shows that this might be consistent with a sub-population of the cells showing an exceptional radiosensitivity, and a replenishment of this subpopulation occurring in the 8 h between small dose fractions. More experiments are needed at very low doses in order to confirm this hypothesis for skin and for other tissues.     

Multiple components of split-dose repair in plateau-phase mammalian cells: a new challenge for phenomenological modelers

Split-dose experiments using starved plateau-phase Chinese hamster ovary cells have been used to investigate the kinetics of repair, expressed in terms of enhancement of reproductive survival. The results show two distinct components of repair, one having a characteristic time of just over 1 h for the removal of a lesion, the other, about 18 h. The rate at which each component removes damage and the fraction of the total damage that each removes appear to be independent of the initial amount of damage produced, i.e., dose. This lack of dose dependence is not consistent with some simple models of ionizing radiation damage and repair, such as those which assume that saturation of a repair process, depletion of enzyme pools, or the interaction of pairs of sublesions is responsible for the curvature in the dose-response relationship. However, the relationship between the amounts of each type of damage and dose appears to be consistent with models that assume that only a portion of the initial damage is directly accessible to the repair systems or that the initial damage consists of a mixture of potentially lethal and sublethal lesions.     

Proliferation kinetics of NK/Ly tumor cells after single and fractionated irradiation with fast electrons]

With single irradiation, the inhibition of tumor cell division and DNA synthesis was more pronounced than with fractionation irradiation. In fractionated schedules, the dose increase per fraction, and the interval prolongation between fractions, with decrease of the number of fractions (within the same time of irradiation), enhanced the effect of radiation. Yield of pahologic mitoses and extent of morphologic injury of cells was less expressed with fractionated irradiation and did not depend on the schedule of fractionation of the total dose.     

Absence of correlation between the chromosomal radiosensitivity of peripheral blood lymphocytes and stem-cell spermatogonia in mammals

To evaluate the reliability of quantitative extrapolation of radiation-induced chromosomal damage from somatic cells to germ cells, data on the effects of several biological and physical factors on the chromosomal radiosensitivity of blood lymphocytes and stem-cell spermatogonia have been collected from the literature. The results show that most of the factors considered, such as chromosomal constitution, age, genetic constitution, species, sampling time and dose fractionation, had differential effects on the induction of chromosomal aberrations in both systems. These differential effects can easily be explained in terms of the biological differences between in-vitro-stimulated peripheral blood lymphocytes and stem-cell spermatogonia. It is concluded that only direct experiments on germ cells of higher primates and man can be used for a quantitative estimation of human genetic radiation risks arising from structural chromosomal aberrations.     

Repairs of X-ray induced sublethal damage in class B oocytes of Drosophila melanogaster.

Repair of X-ray-induced sublethal damage (Elkind-type recovery) in class B oocytes of Drosophila melanogaster was studied. Newly hatched females of two different stocks were treated with either acute or fractionated exposures. For the fractionation experiments a constant time interval of one hour between the dose fractions was used. As genetic endpoints dominant lethality, chromosome aberrations (detachments) and non-disjunction were studied. The repair of X-ray-induced sublethal damage in class B oocytes is expressed as a reappearance on the initial shoulder in the fractionation curve. For dominant lethality it could be shown that less sublethal damage is repaired in oocytes of Berlin wild females than in those of attached-X females (on the average 76 per cent and 101 per cent respectively). Complete repair (about 100 per cent) was observed for detachments in occytes of attached-X females. Within the dose ranges used no radiation effects on non-disjunction could be observed. The results are interpreted to show that in class B oocytes (1) sublethal damage is due to chromosome breaks and/or lesions leading to breaks and (2) X-ray-induced dominant lethality is the consequence of chromosome damage (true dominant lethals).     

不同剂量的X-射线全身照射对小鼠健康状况及涎腺的影响

目的:通过直线加速器全身照射昆明小鼠建立辐射损伤模型,探索不同放射剂量对小鼠健康状况及涎腺功能和结构的影响。方法:选取八种不同剂量对昆明小鼠行体外全身照射,于照射后一个月内观察小鼠生长情况、体重变化;照射后一周、一个月检测各组小鼠血象的变化;测定放射半数致死剂量;照射后两个月,测定各组小鼠的唾液流量及唾液淀粉酶含量,并对下颌下腺组织切片行HE染色。结果:13Gy和15Gy照射组小鼠的体重逐渐下降,一周后死亡,其余组小鼠体重最终呈增加趋势。X-射线全身照射的半数致死量为10Gy。照射后一周,照射组小鼠的白细胞数目明显降低,与对照组比较有明显统计学差异(P0.01);在其他血象方面,除了7Gy组外,其他照射组与对照组比较也均有统计学差异(P0.05)。照射一个月后,各照射组小鼠的血象均恢复正常。照射后两个月,9Gy组和11Gy组小鼠的唾液流量及唾液淀粉酶含量均显著低于0Gy组,且11Gy组较9Gy组亦明显降低,差异均有统计学意义(P0.05)。随照射剂量的增加,小鼠的下颌下腺腺泡细胞数目逐步减少,结构排列紊乱,组织损伤逐渐加重。结论:X-射线全身照射引起小鼠健康状况受损,免疫功能减低,损伤程度与放射线强度呈剂量依赖性,小鼠半数致死量为10Gy,该剂量适合建立全身放射损伤模型。     

Radiation factors and their influence on induction of oxygen resistance

Shortly after gamma irradiation, flour beetles exhibited a decline in resistance to oxygen toxicity. Then, about 2 weeks after irradiation, the 50% lethal dose (LD50) exposure time in pure oxygen was much greater than that of nonirradiated beetles, and this enhanced resistance persisted for about 6 months. The magnitude of the enhancement was a function of dose, decreased with increasing age at irradiation, and was modified by radiation factors. Both dose protraction and dose fractionation reduced the development of oxygen resistance to approximately the same degree that it reduced acute radiation lethality. This suggests that both the initial sensitization and the later enhancement of resistance are correlated with the residual biological damage rather than with the physical dose or initial damage.     

Effets de la radiothérapie sur la fonction testiculaire de l'adulte

Radiation therapy plays a major role in the curative management of numerous neoplasms, such as Hodgkin's disease or testicular cancer. However, the adverse effects of low-dose radiation scattered to radiosensitive normal tissues adjacent to the radiation fields, such as the testes, have been recognized. Experimental studies performed on healthy volunteers showed that no lesion was detectable on sperm counts or testicular biopsies after single doses of less than 10 cGy. Oligospermia has been reported after 15 cGy and 100 cGy result in a 90% incidence of azoospermia. In the radiotherapy of cancer, fractionated regimens are used to increase the differential effect between normal and tumoral tissues. For the same dose, a fractionated radiation regimen results in a higher incidence and a longer period of azoospermia than a single dose irradiation. Fractionated doses of >50 cGy result in a 100% incidence of azoospermia. For doses up to 200 cGy, recovery occurs but normal sperm production remains uncertain. Although the recovery time can be very long (more than 10 years), there is a risk of definitive azoospermia after doses of >200 cGy. Spermatogonia are the most radio-sensitive cell type and their depletion after small irradiation doses explain the effect of radiotherapy on fertility. Clinical hypogonadism is very unfrequent in usual practice, what seems to prove a relative radio-resistance of the Leydig cells. However, functionals studies show that there is a rise in serum LH with increasing dose to the testes. A decrease in testosterone levels has been reported after high testicular doses.     

Quantitative analysis of biological responses to ionizing radiation, including dose, irradiation time, and dose rate

Because biological responses to radiation are complex processes that depend on both irradiation time and total dose, consideration of both dose and dose rate is necessary to predict the risk from long-term irradiations at low dose rates. Here we mathematically and statistically analyzed the quantitative relationships between dose, dose rate and irradiation time using micronucleus formation and inhibition of proliferation of human osteosarcoma cells as indicators of biological response. While the dose-response curves did not change with exposure times of less than 20 h, at a given dose, both biological responses clearly were reduced as exposure time increased to more than 8 days. These responses became dependent on dose rate rather than on total dose when cells were irradiated for 20 to 27 days. Mathematical analysis demonstrates that the relationship between effective dose and dose rate is well described by an exponential function when the logarithm of effective dose is plotted as a function of the logarithm of dose rate. These results suggest that our model, the modified exponential (ME) model, can be applied to predict the risk from exposure to low-dose/low-dose-rate radiation.     

Quantitative aspects of repair of potentially lethal damage in mammalian cells.

Stationary cultures of Ehrlich ascites tumour cells have been irradiated with X-rays and then immediately or after a time interval trep plated to measure the survival. The increase in survival observed after delayed plating is interpreted as repair of potentially lethal damage. A cybernetic model is used to analyse these data. Three states of damage are assumed for the cells. In state A the cells can grow to macrocolonies, in state B the cells have suffered potentially lethal damage and can grow to macrocolonies only if they are allowed to repair the damage and in state C the cells are lethally damaged. A method of deriving the values of the parameters of the model from the experimental data is given. The dependence of the reaction rate constant of the repair of potentially lethal damage on the dose D is used to derive a possible mechanism for the production of the shoulder in the dose effect curve. Finally this model is compared with other models of radiation action on living cells.     

Molecular characterization of X-ray-induced mutations at the HPRT locus in plateau-phase Chinese hamster ovary cells

CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.     

全身放疗在造血干细胞移植中的研究进展

全身照射疗法(TBI)是一种姑息治疗,该方法已经成功地应用在慢性淋巴细胞白血病或滤泡性淋巴瘤等无干细胞支持的放射敏感的疾病中。目前,在血液系统恶性疾病中造血干细胞移植是较为有效的治疗手段之一,其中全身放射治疗与大剂量化疗是造血干细胞移植疗法的经典预处理方案。TBI方法主要应用在造血移植环境中,具有较强的周期非特异性抗肿瘤效应和免疫抑制效能。TBI给予干细胞移植病人超过正常骨髓的辐射耐受量,通过重建病人的造血和免疫来达到治疗目的。     

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668 Gy/min between 0 and 4°C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2–8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r_TM=0.999 and r_TL=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.