David L. Stevens

M-FISH analysis shows that complex chromosome aberrations induced by α-particle tracks are cumulative products of localized rearrangements

Complex chromosome aberrations are characteristically induced after exposure to low doses of densely ionizing radiation, but little is understood about their formation. To address this issue, we irradiated human peripheral blood lymphocytes in vitro with 0.5 Gy densely ionizing α-particles (mean of 1 α-particle/cell) and analyzed the chromosome aberrations produced by using 24-color multiplex fluorescence in situ hybridization (M-FISH). Our data suggest that complex formation is a consequence of direct nuclear α-particle traversal and show that the likely product of illegitimate repair of damage from a single α-particle is a single complex exchange. From an assessment of the “cycle structure” of each complex exchange we predict α-particle-induced damage to be repaired at specific localized sites, and complexes to be formed as cumulative products of this repair.

Effect of Linear Energy Transfer (LET) on the Complexity of α-Particle-Induced Chromosome Aberrations in Human CD34+ Cells

Abstract Anderson, R. M, Stevens, D. L., Sumption, N. D., Townsend, K. M. S., Goodhead, D. T. and Hill, M. A. Effect of Linear Energy Transfer (LET) on the Complexity of α-Particle-Induced Chromosome Aberrations in Human CD34+ Cells. Radiat. Res. 167, 541–550 (2007). The aim of this study was to assess the relative influence of the linear energy transfer (LET) of α particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with α particles of various incident LETs (110–152 keV/μm, with mean LETs through the cell of 119–182 keV/μm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent “an area” of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single α particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the α particle increases, the likelihood of this aberration being classed as complex is greater.

The severity of alpha-particle-induced DNA damage is revealed by exposure to cell-free extracts

The rejoining of single-strand breaks induced by alpha-particle and gamma irradiation in plasmid DNA under two scavenging conditions has been compared. At the two scavenger capacities used of 1.5 x 10(7) and 3 x 10(6) s-1 using Tris-HCl as the scavenger, the ratio of single- to double-strand breaks for alpha particles is fivefold less than the corresponding ratios for gamma irradiation. The repair of such radiation-induced single-strand breaks has been examined using a cell-free system derived from human whole-cell extracts. We show that the rejoining of single-strand breaks for both alpha-particle- and gamma-irradiated plasmid is dependent upon the scavenging capacity and that the efficiency of rejoining of alpha-particle-induced single-strand breaks is significantly less than that observed for gamma-ray-induced breaks. In addition, for DNA that had been irradiated under conditions that mimic the cellular environment with respect to the radical scavenging capacity, 50% of alpha-particle-induced single-strand breaks are converted to double-strand breaks, in contrast with only approximately 12% conversion of gamma-ray-induced single-strand breaks, indicating that the initial damage caused by alpha particles is more severe. These studies provide experimental evidence for increased clustering of damage which may have important implications for the induction of cancer by low-level alpha-particle sources such as domestic radon.

Comments on the Recently Reported Low Biological Effectiveness of Ultrasoft X Rays

In a recent paper, C. K. Hill et al. (1) reported the results of experiments carried out at the University of Wisconsin in which Chinese hamster V79 and mouse C3H 10T1⁄2 cells were irradiated with synchrotronproduced 273 eV and 860 eV ultrasoft X rays. X rays at these energies are heavily attenuated through a single mammalian cell. Therefore, they chose two energies that would be equally attenuated, enabling a direct comparison between the biological effectiveness of the two energies. The two main results of the cell survival experiments carried out by the Wisconsin group were: 1. The biological effectiveness for these two energies, for both cell lines, was similar. 2. After the mean nuclear dose was calculated, taking into account attenuation through the cells, there were no significant differences in cell survival between these two ultrasoft X-ray energies and 250 kVp X rays. Based on this second result, the Wisconsin group (1) concluded that, in general, ultrasoft X rays are no more biologically effective per unit dose than are standard orthovoltage X rays and g rays, and hence they refuted the suggestion (2, 3) that the majority of critical damage that follows exposure to standard X rays and g rays results from low-energy, secondary electrons. Their second result is inconsistent with previously published experimental data from several other groups, and it therefore warrants close scrutiny to understand the origin of this difference, especially in view of the recent increase in the use of ultrasoft X rays for radiobiology experiments. The first result mentioned above is not inconsistent with other published experimental data or with the main interpretations that have been suggested for a high biological effectiveness of ultrasoft X rays. We first briefly discuss Result 1, and then we consider in more detail some potential reasons for the difference of Result 2.

Bound PCNA in Nuclei of Primary Rat Tracheal Epithelial Cells after Exposure to Very Low Doses of Plutonium-238 α Particles

Abstract Hill, M. A., Ford, J. R., Clapham, P., Marsden, S. J., Stevens, D. L., Townsend, K. M. S. and Goodhead, D. T. Bound PCNA in Nuclei of Primary Rat Tracheal Epithelial Cells after Exposure to Very Low Doses of Plutonium-238 α Particles. Radiat. Res. 163, 36–44 (2005). Bystander effects from ionizing radiation have been detailed for a number of cell systems and a number of end points. We wished to use a cell culture/ex vivo rat model of respiratory tissue to determine whether a bystander effect detected in culture could also be shown in a tissue. Examination by immunofluorescence techniques of tracheal cell cultures after exposure to very low doses of α particles revealed a large proportion of cells with proliferating cell nuclear antigen (PCNA) bound in their nuclei. PCNA was selected as an end point because it is involved in both DNA repair and the changes in cell cycle that are typical of many reported bystander effects. Maximum response can be detected in up to 28% of the cells in sub-confluent cultures with a dose of only 2 mGy. At this dose less than 2% of the cell nuclei have experienced a particle traversal and less than 6% of the cells have experienced an α-particle traversal through either their nucleus or some part of their cytoplasm. The hypothesis that this bystander response in nontargeted cells is mediated through secreted factor(s) is presented, and supporting evidence was found using partial irradiation and co-culture experiments. Examination of the effect with excised pieces of trachea demonstrated a response similar to that seen in culture.

Relative sensitivities of repair-deficient mammalian cells for clonogenic survival after α-particle irradiation

Abstract Hill, M. A., Herdman, M. T., Stevens, D. L., Jones, N. J., Thacker, J. and Goodhead, D. T. Relative Sensitivities of Repair-Deficient Mammalian Cells for Clonogenic Survival after α-Particle Irradiation. Radiat. Res. 162, 667–676 (2004). The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after α-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single α-particle traversal, the average number of lethal lesions per track and per unit dose, along with the “intrinsic radiosensitivity” of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, α particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to α particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for α-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of α-particle-induced damage, although a large proportion of damage is not repaired by either pathway.

Is the increased relative biological effectiveness of high LET particles due to spatial or temporal effects? Characterization and OER in V79-4 cells.

The efficiency of producing biological damage varies with radiation quality. Conventional explanations rely on spatial differences in the radiation track structure; generally however there are also very large temporal differences in delivery of the radiation at the cellular level. High-LET radiation normally deposits substantial amounts of energy by individual heavily ionizing tracks on a timescale of the order of picoseconds. By contrast each low-LET radiation track deposits a small amount of energy. Many of these tracks, distributed over the whole cell, are required to deliver an equivalent dose to a high-LET track and they are usually delivered over much longer timescales (typically seconds) during which chemical, biochemical and biological processes are occurring. In this paper the design, characterization and initial application of a high-brightness, laser-plasma ultrasoft x-ray source is described. This has been used to investigate the importance of the temporal differences by irradiating mammalian cells with an energy deposition with spatial properties of low-LET radiation and temporal properties similar to high-LET radiation. The present system delivers a typical dose, to the incident surface of the cells, of 0.12 Gy per pulse delivered in <10 ps. The capabilities of the x-ray source were tested by determining the survival of V79-4 hamster cells irradiated with picosecond pulses of ultrasoft x-rays under aerobic and anaerobic conditions, which were found to be consistent with previously published non pulsed data with x-rays of similar energy. These results support the expectation that the disappearance of an oxygen effect for high-LET radiation particles is due to their spatial properties rather than the very short timescale of each particle traversal. For other effects, particularly non-targeted phenomena such as induced genomic instability, expectations may be less clear cut.

Ultrasoft 1.5 keV aluminum K X rays are efficient producers of complex chromosome exchange aberrations as revealed by fluorescence in situ hybridization.

The electron pairs generated by ultrasoft 1.5 keV aluminum K X-ray photons deposit their energy in tracks of length < 70 nm and provide an ideal tool for analyzing the spatial distribution of breaks and misrepair processes. We have undertaken the analysis of changes in chromosome structure produced by aluminum K X rays in untransformed HF12 human fibroblasts in G1 phase using fluorescence in situ hybridization (FISH). Multicolored chromosome-specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe were used to examine in vitro radiation-induced chromosome-type exchange aberrations. After mean doses of 0.37-2.93 Gy the relative frequencies of complex exchanges, derived from three or more breaks in two or more chromosomes, ranged from 15-35%. For the classic break-age-and-rejoining theory to hold, very large interaction distances are needed to account for this high frequency of multibreak interactions, unless many sites pre-exist where several different chromosomes come very close together. Alternatively, damaged DNA may be able to interact with adjacent undamaged DNA, obviating the need for large rejoining distances.

The role of radiation quality in the stimulation of intercellular induction of apoptosis in transformed cells at very low doses.

An important stage in tumorigenesis is the ability of precancerous cells to escape natural anticancer signals. Apoptosis can be selectively induced in transformed cells by neighboring normal cells through cytokine and ROS/RNS signaling. The intercellular induction of apoptosis in transformed cells has previously been found to be enhanced after exposure of the normal cells to very low doses of both low- and high-LET ionizing radiation. Low-LET ultrasoft X rays with a range of irradiation masks were used to vary both the dose to the cells and the percentage of normal cells irradiated. The results obtained were compared with those after &agr;-particle irradiation. The intercellular induction of apoptosis in nonirradiated src-transformed 208Fsrc3 cells observed after exposure of normal 208F cells to ultrasoft X rays was similar to that observed for &ggr; rays. Intercellular induction of apoptosis was stimulated by irradiation of greater than 1% of the nontransformed 208F cells and increased with the fraction of cells irradiated. A maximal response was observed when ∼10–12% of the cells were irradiated, which gave a similar response to 100% irradiated cells. Between 1% and 10%, high-LET &agr; particles were more effective than low-LET ultrasoft X rays in stimulating intercellular induction of apoptosis for a given fraction of cells irradiated. Scavenger experiments show that the increase in intercellular induction of apoptosis results from NO• and peroxidase signaling mediated by TGF-&bgr;. In the absence of radiation, intercellular induction of apoptosis was also stimulated by TGF-&bgr; treatment of the nontransformed 208F cells prior to coculture; however, no additional increase in intercellular induction of apoptosis was observed if these cells were also irradiated. These data suggest that the TGF-&bgr;-mediated ROS/RNS production reaches a maximum at low doses or fluences of particles, leading to a plateau in radiation-stimulated intercellular induction of apoptosis at higher doses.

Induction of DNA-Protein Crosslinks in Chinese Hamster V79-4 Cells Exposed to High- and Low-Linear Energy Transfer Radiation

The induction of DNA-protein crosslinks (DPCs) in Chinese hamster V79-4 cells after irradiation under hypoxic and aerobic conditions at 277 K with 60Co gamma rays, 238Pu alpha particles and aluminum K (Al(K)) ultrasoft X rays has been determined using a nitrocellulose filter binding assay. The dose dependences for the induction of DPCs, which involves covalent linkage, are linear over the absorbed dose range used (0-400 Gy with alpha-particle and gamma radiation, 0-600 Gy with Al(K) X rays). The yield of DPCs induced under hypoxic conditions is 55, 51 and 25 DPCs per gray per cell for 60Co gamma rays, alpha particles and Al(K) X rays, respectively. The yield of DPCs is significantly reduced in the presence of oxygen by 20, 50 and 79% for 60Co gamma rays, alpha particles and Al(K) X rays, respectively. Since the mean size of the DNA attached to the protein is uniform for 60Co gamma rays and alpha particles, variations in the DNA size do not influence the yields of DPCs. Although a DPC may be considered as a complex lesion combining two macromolecules, the dependence of the yield of DPCs on LET does not reflect the ionizing density of the radiations used. Further, this dependence on LET and the effect of oxygen do not reflect the corresponding dependences determined for a variety of biological responses. From these findings and knowledge of the radiation tracks, it is proposed that DPCs induced particularly under aerobic conditions with 60Co gamma rays are formed mainly in the sparsely ionizing segments of the radiation track.