不同传能线密度射线0~1Gy照射诱发染色体畸变的量-效关系研究

目的 建立0~1Gy范围内⁶⁰Coγ射线、 ²⁵²Cf 中子、单能中子及 ¹²C 重离子诱发染色体畸变剂量-效应曲线,用于评价职业受照或低剂量辐射损伤。方法 采用不同照射装置1Gy以内照射离体人外周血,于培养开始加秋水仙素,培养48h收获,制备染色体标本,Metafer中期细胞自动寻找系统,人机交互计数染色体畸变,最优拟合数据得到相应模型。结果 在低剂量范围内,以双着丝粒体+环(dic+r)为终点,⁶⁰Coγ射线为线性模型, ²⁵²Cf 中子、单能中子及 ¹²C 重离子为线性平方模型;以无着丝粒断片(ace)为终点,⁶⁰Coγ射线和 ¹²C 重离子为线性平方模型, ²⁵²Cf 中子和单能中子为线性模型;以总畸变为终点,4种射线均为线性平方模型。结论 高传能线密度(LET)射线损伤效应高于低LET射线,4种射线损伤效应依次为 ²⁵²Cf 混合中子 >单能中子> ¹²C 重离子> ⁶⁰Co γ射线。在低剂量范围内dic+r指标适用于高LET照射的剂量估算。     

Modelling the formation of polycentric chromosome aberrations.

Exchange-type chromosome aberrations produced by ionizing radiation or restriction enzymes are believed to result from pairwise interaction of DNA double-strand breaks (dsb). In addition to dicentrics, such aberrations may include higher-order polycentrics (tricentrics, tetracentrics, etc.). We have developed computer programs that calculate the probability of the various polycentrics for a given average number of pairwise interactions. Two models are used. Model I incorporates kinetic competition between restitution, complete exchanges (illegitimate recombination events), and incomplete exchanges. Model II allows unrestituted breaks even if there is no recombination. The models were applied to experimental observations of aberrations produced in G1 Chinese hamster ovary cells after electroporation with the restriction enzyme PvuII, which produces blunt-end dsb. We found, experimentally and theoretically, that there was a maximum in the number and multiplicity of polycentrics per cell: beyond a certain PvuII concentration no additional or higher-order polycentrics were produced. Computer-generated relationships, which were remarkably similar for both models and for all values of the adjustable parameters, were found between dicentrics per cell and higher-order polycentrics per cell. Excellent agreement was found between the experimental observations and the consensus theoretical curve relating tricentrics per cell to dicentrics per cell. The observed number of higher polycentrics per cell for a given number of dicentrics per cell was somewhat larger than the consensus theoretical prediction. The observed number of centric rings per cell was markedly larger than the consensus theoretical value, presumably owing to intrachromosomal localization ('proximity effects'). The computer models also provided estimates for the adjustable parameters; for example, in model I the fraction of incomplete exchanges was found to be about 35%.     

RBE of d(50)-Be neutrons for induction of chromosome aberrations in Allium cepa onion roots

RBE/absorbed dose relationship of d(50)-Be neutrons was determined for the induction of chromosome aberrations in Allium cepa onion roots. Neutrons are produced at the cyclotron "Cyclone" by bombarding a thick beryllium target with 50 MeV deuterons. Two biological criteria were selected: (1) mean number of aberrations (mainly breaks) per cell in anaphase and telophase, (2) fraction of intact cells in anaphase and telophase. For the two criteria, RBE increases continuously from about 7 to 12 as the neutron absorbed dose decreases from 0.4 to 0.1 Gy. RBE values for the first criterion are slightly higher than for the second one. This observation is interpreted in terms of the analysis of the distribution of the aberrations in the cells. In logarithmic coordinates, RBE/absorbed dose relationships for the two criteria are almost linear with a slope close to -1/2. RBE values observed for induction of chromosome aberrations in Allium cepa are higher than those generally observed for biological effects related to mammalian cell lethality.     

电离辐射诱发的联会复合体畸变研究

以雄性昆明种小鼠为实验材料，Ｘ射线一次全身均匀照射，照后不同时间取材，分别用电子显微镜观察精母细胞前期联会复合体畸变和光镜观察中期Ⅰ染色体畸变。结果表明，和中期Ⅰ染色体畸变相比，联会复合体的畸变类型更复杂，其发生频率也明显增高；回归分析表明，两者多价体发生率均随剂量增加而增加，剂量与多价体发生率之间的关系符合直线方程，Ｙｓｃ＝－０．８６＋１．６８Ｄ（ｒ＝０．９３）ＹＤ－ＭＩ＝－０．２０＋０．９０Ｄ（ｒ＝０．９９），说明联会复合体分析可以更敏感地检测畸变。因此，利用联会复合体检测技术来估价电离辐射对哺乳动物潜在的遗传危害优于常规染色体畸变分析。     

用染色体畸变研究AT细胞系辐射敏感性和适应性反应

目的探讨毛细血管扩张运动共济失调症(ataxiatelangiectasia,AT)细胞系的辐射敏感性和对低剂量辐射能否诱导细胞遗传学适应性反应。方法用2cGy60Coγ射线预先照射进入G1期的AT5B1VA(AT)细胞和GM0639(GM)细胞,培养6h后再照射1.0Gy或3.0Gy60Coγ射线,分析染色体畸变。结果G1期的GM细胞在预照射2cGy60Coγ射线的诱导剂量后能非常显著地降低随后照射1.0Gy或3.0Gy60Coγ射线诱发的染色体畸变率,染色体畸变的观察值非常显著地低于预期值(P<0.001),而对AT细胞则没有观察到这种现象,染色体畸变的观察值与预期值差异无统计学意义(P>0.05);另外,1.0Gy或3.0Gy60Coγ射线诱发AT细胞的各种畸变率(包括着丝粒畸变、无着丝粒畸变和染色单体型畸变)均显著地高于GM细胞(P<0.001)。结论低剂量电离辐射不能诱导AT细胞产生细胞遗传学适应性反应;AT细胞对电离辐射诱发染色体畸变高度敏感,可能是由于不能正确修复DNA双链断裂的结果。     

Induction of chromosome aberrations in unirradiated chromatin after partial irradiation of a cell nucleus

Purpose : It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. Materials and methods : Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X q domain) were labelled with 125 I-iododeoxyuridine (125 IdUrd) in a period of S-phase when the vast majority of the X q domain was not replicating. DNA damage from 125 I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125 I induced DSB in the immediate vicinity of the 125 I atom. Chromosome aberrations involving what is essentially the 125 I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125 IdUrd at a later period in S-phase when the X q domain replicates, yielding a labelled X q domain. Results : The 125 I-free X q domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125 I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125 I-free X q domain was approximately half of that observed in the 125 I-labelled X q domain. Conclusions : The involvement of the 125 I-free X q domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125 I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.     

Induction of chromosome aberrations in unirradiated chromatin after partial irradiation of a cell nucleus

PURPOSE: It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. MATERIALS AND METHODS: Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X(q) domain) were labelled with 125I-iododeoxyuridine (125IdUrd) in a period of S-phase when the vast majority of the X(q) domain was not replicating. DNA damage from 125I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125I induced DSB in the immediate vicinity of the 125I atom. Chromosome aberrations involving what is essentially the 125I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125IdUrd at a later period in S-phase when the X(q) domain replicates, yielding a labelled X(q) domain. RESULTS: The 125I-free X(q) domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125I-free X(q) domain was approximately half of that observed in the 125I-labelled X(q) domain. CONCLUSIONS: The involvement of the 125I-free X(q) domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.     

电离辐射诱发小鼠生殖细胞染色体畸变率的比较

利用小剂量(0～1.0Gy)的X线照射昆明种小白鼠,对雄性各类型生殖细胞,同一指标不同阶段照射以及雌、雄生殖细胞染色体畸变率进行了比较.结果表明,雄性生殖细胞中以次级精母细胞染色体畸变率最高,剂量效应曲线的斜率约是精原细胞的3.6倍;初级精母细胞的10倍.当以D-MI和MI期雄性生殖细胞为终点,观察不同阶段受照射时的染色体畸变,表现为初级精母细胞的终变期最敏感,从终变期至前期表现为染色体畸变率逐渐下降.照射后21天至120天的结果为分化型精原细胞和精原干细胞受照射所诱发的染色体畸变率,无明显差异.比较雌、雄生殖细胞MI染色体畸变率,以后者的畸变率高,平均畸变率为前者的4.4倍,曲线斜率约为6倍.     

Cytogenetic damage induced by radiotherapy. Evaluation of protection by amifostine and analysis of chromosome aberrations persistence

PURPOSE: To evaluate the cytogenetic damage induced by radiotherapy, the effect of concomitant amifostine and the persistence of translocations and dicentrics after the treatment. MATERIALS AND METHODS: Blood samples from 16 head and neck cancer patients were obtained at different times, just before treatment, at the 1st and 22nd sessions, at the end of radiotherapy, and one, four and 12 months later. Solid stain and fluorescent in situ hybridization (FISH) techniques were applied to analyse chromosome aberrations. RESULTS: In all the analysis the frequencies of dicentrics plus rings were slightly lower in the group of patients receiving concomitant amifostine, but in each sampling point the differences were not significant. The persistence of translocations and dicentrics one year after radiotherapy was very similar, with a decline of more than 50%. For all the chromosome aberrations considered, a negative correlation between their initial yield and the percentage of this yield remained 12 months after radiotherapy was observed (p < 0.05). CONCLUSION: No significant protection by amifostine against radiation-induced chromosome damage was observed in head and neck cancer patients treated only with radiotherapy. In these cases, the persistence of translocations and dicentrics during the first year after radiotherapy is similar and related to their initial yield.     

Reaction kinetics for the development of radiation-induced chromosome aberrations.

The formation of chromosome aberrations from DNA double-strand breaks (dsb) following ionizing irradiation of cells is analysed using a stochastic, continuous-time Markov chain formalism. A restitution/complete exchange model is proposed which incorporates kinetic competition between dsb restitution and chromosome exchange; it applies primarily to those dsb whose broken ends are held in close proximity by proteins. Some additional pathways for damage evolution are also considered. The calculations are compared in detail to the experiments on dicentric yield and variance in human lymphocytes following acute low-LET irradiation summarized by Lloyd and Edwards (1983) and Lloyd et al. (1987). It was found that dicentric formation by pairwise dsb interactions can lead to a dicentric yield/dose curve with a significant linear component even if one-track action is neglected. In other words a dicentric formation rate quadratic in the dsb number does not automatically imply a dicentric yield quadratic in the dose. However, the data indicate that at least in some experiments there could have been a significant one-track contribution to the dicentric yield in addition to the intertrack contributions analysed in the present paper. From the data the order of magnitude of the dsb interaction rate constant is estimated to be at most approximately 3 x 10(-3) exchanges per dsb pair per hour. It was found that for the reaction pathways considered, a initial Poisson distribution of dsb leads to an underdispersed distribution of dicentrics; the amount of underdispersion is strongly model-dependent and the variance data are consistent with the restitution/complete exchange model. Finally, a general mathematical theorem on Markov models is presented. It implies that assays performed after repair and exchange are completed cannot determine absolute repair or exchange rates, only their ratio (which may depend on lesion number).     

Induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells by carbon ions

PURPOSE: To study the induction of reproductive cell death and chromosome aberrations in radioresistant tumour cells exposed to carbon ions in vitro. MATERIALS AND METHODS: X-ray-resistant colon carcinoma cells (WiDr) were used. Confluent G0/G1 cells were irradiated in vitro with graded doses of 100/200/400 MeV u(-1) carbon ions and carbon ions from the middle of a 1 cm extended Bragg peak, and 200 kV X-rays for comparison. Cells were harvested in their first post-irradiation division and aberrations were analysed either by the Giemsa/Hoechst 33258-staining technique or by the fluorescent in-situ hybridization technique involving whole chromosome hybridization and 4',6-diaminido-2-phenylidole (DAPI)-staining. Whole chromosome probes were used for chromosomes 2, 4 and 5, and the chromosome painting patterns were classified according published protocols. Reproductive cell survival was determined by a standard clonogenic assay. RESULTS: With respect to the induction of reproductive cell death and chromosome aberrations, carbon ions of different energies were more effective than 200 kV X-rays. As expected, irradiation in the extended Bragg peak was the most efficient mode. For cell killing, relative biological effectiveness increased with linear energy transfer up to 2.9. The frequencies of total dicentrics and excess acentric fragments as determined in Giemsa-stained cells were higher in cells irradiated with carbon ions than in cells with X-rays. For 100 MeV u(-1) ions, the dose dependence of apparently simple dicentrics as determined for chromosomes 2, 4 and 5 by single-colour fluorescent in-situ hybridization was linear up to 4 Gy, and linear-quadratic for excess acentric fragments and apparently simple translocations. After irradiation with D=4 Gy carbon ions with energy of 100 MeV u(-1) and from the extended Bragg peak, 12 and 54% of cells displayed complex exchanges, respectively. In contrast, after irradiation with D=4 Gy X-rays, only 1% of cells displayed complex aberrations. Hence, the number of cells with complex exchange aberrations increased strongly after irradiation with carbon ions. CONCLUSION: An increased biological efficiency of carbon ions could be confirmed in radioresistant tumour cells with respect to the induction of reproductive cell death and of unstable as well as stable chromosome aberrations. Relative biological effectiveness reached 2.9 for cell killing by carbon ions from the extended Bragg peak. The yields of apparently simple dicentrics as well as of total dicentrics, i.e. simple dicentrics plus dicentrics belonging to complex exchanges, evaluated in Giemsa-stained metaphases as observed in first post-irradiation mitoses were rather low. In contrast, apparently simple translocations displayed yields systematically higher than simple dicentrics in WiDr cells irradiated with either X-rays or 100 MeV u(-1) or Bragg peak carbon ions. Frequencies o f cells containing complex aberrations increased dramatically after carbon ion irradiation, reaching a maximum for ions from the extended Bragg peak.     

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.     

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations

Purpose : To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. Methods : Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. Results : When mediated by a DSB-repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose- responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear-quadratic model. Conclusions : Using a minimal number of assumptions, the kinetics and dose-response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose-response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.