Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

RBE: mechanisms inferred from cytogenetics.

Cyclotron-accelerated heavy ion beams provide a fine degree of control over the physical parameters of radiation. Cytogenetics affords a view into the irradiated cell at the resolution of chromosomes. Combined they form a powerful means to probe the mechanisms of RBE. Cytogenetic studies with high energy heavy ion beams reveal three LET-dependent trends for 1) level of initial damage, 2) distribution of damage among cells, and 3) lesion severity. The number of initial breaks per unit dose increases from a low-LET plateau to a peak at approximately 180 keV/micrometer and declines thereafter. Overdispersion of breaks is significant above approximately 100 keV/micrometer. Lesion severity, indicated by the level of chromosomal fragments that have not restituted even after long repair times, increases with LET. Similar studies with very low energy 238Pu alpha particles (120 keV/micrometer) reveal higher levels of initial breakage per unit dose, fewer residual fragments and a higher level of misrepair when compared to high energy heavy ions at the same LET. These observations would suggest that track structure is an important factor in genetic damage in addition to LET.     

Biological effects of heavy ions from the standpoint of target theory.

The biological effect of heavy ions is best described through the action cross section, as a function of the end-point of interest and the charge and speed of the ion. In track theory this is called the "ion-kill" cross section, for it is the effect produced by a single heavy ion and its delta rays. As with nuclear emulsions the biological track structure passes from the grain count regime to the track width regime to the thindown region with an increase in LET. With biological cells, as with any detector capable of storing sublethal damage, with low LET irradiation the action cross section (in the ion-kill mode) is increasingly obscured by the effect of "gamma-kill", by the influence of overlapping delta rays from neighboring heavy ions. Thus at low LET response is dominated by the gamma-kill mode, so that the RBE approaches 1. The theory requires 4 radiosensitivity parameters for biological detectors, extracted from survival curves at several high LET bombardments passing through the grain count regime, and at high doses. Once these are known the systematic response of biological detectors to all high LET bombardments can be unfolded separating ion kill from gamma kill, predicting the response to a mixed radiation environment, and predicting low dose response even at the level of a single heavy ion. Cell killing parameters are now available for a variety of cell lines. Newly added is a set of parameters for cell transformation.     

Microscopic track structure of equal-LET heavy ions.

The spatial distributions of ionization and energy deposition produced by high-velocity heavy ions are crucial to an understanding of their radiation quality as exhibited eg., in track segment experiments of cell survival and chromosome aberrations of mammalian cells. The stopping power (or LET) of a high velocity ion is proportional to the ratio z2/v2, apart from a slowly varying logarithmic factor. The maximum delta-ray energy that an ion can produce is proportional to v2 (non-relativistically). Therefore, two HZE ions having the same LET, but in general differing z and v will have different maximum delta-ray energies and consequently will produce different spatial patterns of energy deposition along their paths. To begin to explore the implications of this fact for the microscopic dosimetry of heavy ions, we have calculated radial distributions in energy imparted and ionization for iron and neon ions of approximately equal LET in order to make a direct comparison of their delta-ray track structure. Monte Carlo techniques are used for the charged particle radiation transport simulation.     

DNA damage and repair in oncogenic transformation by heavy ion radiation.

Energetic heavy ions are present in galactic cosmic rays and solar particle events. One of the most important late effects in risk assessment is carcinogenesis. We have studied the carcinogenic effects of heavy ions at the cellular and molecular levels and have obtained quantitative data on dose-response curves and on the repair of oncogenic lesions for heavy particles with various charges and energies. Studies with repair inhibitors and restriction endonucleases indicated that for oncogenic transformation DNA is the primary target. Results from heavy ion experiments showed that the cross section increased with LET and reached a maximum value of about 0.02 micrometer2 at about 500 keV/micrometer. This limited size of cross section suggests that only a fraction of cellular genomic DNA is important in radiogenic transformation. Free radical scavengers, such as DMSO, do not give any effect on induction of oncogenic transformation by 600 MeV/u iron particles, suggesting most oncogenic damage induced by high-LET heavy ions is through direct action. Repair studies with stationary phase cells showed that the amount of reparable oncogenic lesions decreased with an increase of LET and that heavy ions with LET greater than 200 keV/micrometer produced only irreparable oncogenic damage. An enhancement effect for oncogenic transformation was observed in cells irradiated by low-dose-rate argon ions (400 MeV/u; 120 keV/micrometer). Chromosomal aberrations, such as translocation and deletion, but not sister chromatid exchange, are essential for heavy-ion-induced oncogenic transformation. The basic mechanism(s) of misrepair of DNA damage, which form oncogenic lesions, is unknown.     

Neoplastic cell transformation by energetic heavy ions and its modification with chemical agents.

For many years we have been interested in understanding the potential carcinogenic effects of cosmic rays. We have studied the oncogenic effects of cosmic rays with accelerator-produced heavy particle radiation and with a cultured mammalian cell system--C3H10T1/2 cells. Our quantitative data obtained with carbon, neon, silicon, and iron particles showed that RBE is both dose and LET dependent for neoplastic cell transformation. RBE is higher at lower dose, and RBE increases with LET up to about 200 keV/micrometer. In nonproliferation confluent cells, heavy-ion induced transformation damage may not be repairable, although a dose modifying factor of about 1.7 was observed for X-ray radiation. Our recent studies with super-heavy high-energy particles, e.g., 960 MeV/U U235 ions (LET = 1900 keV/micrometer), indicate that these ions with a high inactivation cross-section can cause neoplastic cell transformation. The induction of cell transformation by radiation can be modified with various chemicals. We have found that the presence of DMSO (either during or many days after irradiation) decreased the transformation frequency significantly. It is, therefore, potentially possible to reduce the oncogenic effect of cosmic rays in space through some chemical protection.     

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.     

Induction of chromosome aberrations in mammalian cells after heavy ion exposure.

The induction of chromosome aberrations by heavy charged particles was studied in V79 Chinese hamster cells over a wide range of energies (3-100 MeV/u) and LET (20-16000 keV/micrometer). For comparison, X-ray experiments were performed. Our data indicate quantitative and qualitative differences in the response of cells to particle and x-ray irradiation. For the same level of cell survival the amount of damaged cells which can be observed is smaller in heavy ion (11.4 MeV/u Ar) irradiated samples. The highest yield of damaged cells is found 8 to 12 hours after particle irradiation and 4 hours after x-irradiation. Differences in the amount of damaged cells are attributed to cell cycle perturbations which interfere with the expression of damage. After heavy ion exposure the amount of cells reaching mitosis (mitotic index) decreases drastically and not all damaged cells reach mitosis within 48 hours after exposure. A portion of cells die in interphase. Cell cycle delays induced by x-ray irradiation are less pronounced and all cells reach the first post-irradiation mitosis within 24 hours after irradiation. Additionally, the damage produced by charged particles seems to be more severe. The disintegration of chromosomes was only observed after high LET radiation: an indication of the high and local energy deposition in the particle track. Only cross sections for the induction of chromosome aberrations in mitotic cells were reported in this paper because of the problems arising from the drastic cell cycle perturbations. In this case, cells were irradiated in mitosis and assayed immediately.     

Quantitative interpretation of heavy ion effects: comparison of different systems and endpoints.

For a quantitative interpretation of biological heavy ion action the following parameters have to be taken into account: variations of energy depositions in microscopical sites, the dependence of primary lesion formation on local energy density and changes in reparability. They can be studied in objects of different size and with different sensitivities. Results on survival and mutation induction in yeast and in mammalian cells will be compared with theoretical predictions. It is shown that shouldered survival curves of diploid yeast can be adequately described if the final slope is adjusted according to the varying production of primary lesions. This is not the case for mammalian cells where the experiments show a rapid loss of the shoulder with LET, contrary to theoretical expectations. This behaviour is interpreted to mean that the reparability of heavy ion lesions is different in the two systems. Mutation induction is theoretically expected to decrease with higher LET. This is found in yeast but not in mammalian cells where it actually increases. These results suggest a higher rate of misrepair in mammalian cells.     

Cellular and subcellular effect of heavy ions: a comparison of the induction of strand breaks and chromosomal aberration with the incidence of inactivation and mutation.

Radiobiological effects of heavy charged particles are compared for a large variety of ions from Helium to Uranium and energies between 1 and 1000 MeV/u which correspond to LET values between 10 and 16000 keV/micrometers. The different cross section for the induction of strand breaks and chromosomal aberrations as well as for inactivation and mutation induction exhibit striking similarities when compared as function of the linear energy transfer (LET). At LET values below 100 keV/micrometers all data points of one specific effect form one single curve as a function of LET, independent of the atomic number of the ion. In this LET range, the biological effects are independ from the particle energy or track structure and depend only on the energy transfer. Therefore, LET is a good parameter in this regime. For LET values greater than 100 keV/micrometers, the curves for the different ions separate from the common curve in order of increasing atomic numbers. In this regime LET is no longer a good parameter and the physical parameters of the formation of particle tracks are important. The similarity of the sigma-LET curves for different endpoints indicates that the 'hook-structure' is produced by physical and chemical effects which occur before the biologically relevant lesions are formed. However, from the existing data of biological effects, it can be concluded that the efficiencies for cell killing are always smaller than those extrapolated from X-ray data on the basis of the energy deposition only. Therefore, cells which are directly hit by an HZE particle are not killed and undergo a finite risk of mutation and transformation.     

On the quality of mutations in mammalian cells induced by high LET radiations

The deleterious effects of accelerated heavy ions as component of the space radiation environment on living cells are of increasing importance for long duration human space flight activities. The most important aspect of such densely ionizing particle radiation is attributed to the type and quality of biological damage induced by them. This issue is addressed by investigating cell inactivation and mutation induction at the Hprt locus (coding for hypoxanthine-guanine-phosphoribosyl-transferase) of cultured V79 Chinese hamster cells exposed to densely ionizing radiation (accelerated heavy ions with different LETs from oxygen to gold, specific energies ranging from 1.9 to 69.7 MeV/u, corresponding LET values range from 62 to 13,223 keV/μm) and to sparsely ionizing radiation (200 kV X-rays). 30 spontaneous, 40 X-ray induced and 196 heavy ion induced 6-thioguanine resistant Hprt mutant colonies were characterized by Southern technique using the restriction enzymes EcoRI, PstI and BglII and a full length Hprt cDNA probe isolated from the plasmid pHPT12. Restriction patterns of the spontaneous Hprt mutants were indistinguishable from the wild type pattern, as these mutants probably contain only small deletions or even point mutations in the Hprt locus. In contrast, the overall spectrum of heavy ion induced mutations revealed a majority of partial or total deletions of the Hprt gene. With constant particle fluence (3 × 10⁶ particles/cm²) the quality of heavy ion induced mutations in the Hprt locus depends on physical parameters of the beam (atomic number, specific energy, LET). This finding suggests a relationship between the type of DNA damage and track structure. The fraction of mutants with severe deletions in the Hprt locus after exposure to oxygen ions increases from 65% at 60 keV/μm up to a maximum (100%) at 300 keV/μm and declines with higher LET values to 75% at 750 keV/μm. With heavier ions (Ca- and Au-ions) and even higher LET-values this mutant fraction decreases to 58% at 13,200 keV/μm. Heavy ion induced DNA break points in the Hprt locus are not randomly distributed.     

Estimation of risk based on multiple events in radiation carcinogenesis of rat skin.

In the multistage theory of carcinogenesis, cells progress to cancer through a series of discrete, irreversible, heritable genetic alterations or mutations. However data on radiation-induced cancer incidence in rat skin suggests that some part of an intermediate repairable alteration may occur. Data are presented on cancer induction in rat skin exposed to the following radiations: 1. an electron beam (LET=0.34 keV/um, 2. a neon ion beam (LET=25 keV/um and 3. an argon ion beam (LET=125 keV/um. The latter 2 beams were generated by the Bevalac at the Lawrence Berkeley Laboratory, Berkeley, CA. About 6.0 cm2 of skin was irradiated per rat. The rats were observed every 6 weeks for at least 78 weeks and tumors were scored at first occurrence. Several histological types of cancer, including squamous and basal cell carcinomas, were induced. The cancer yield versus radiation dose was fitted by the quadratic equation (Y(D)=CLD+BD2), and the parameters C and B were estimated for each type of radiation. Analysis of the DNA from the electron-induced carcinomas indicated that K-ras and/or c-myc oncogenes were activated in all tumors tested, although only a small proportion of neon-induced tumors showed similar activation. In situ hybridization indicated that the cancers contain subpopulations of cells with differing amounts of c-myc and H-ras amplification. The results are consistent with the idea that ionizing radiation produces carcinogenically relevant lesions via 2 repairable events at low LET and via a non-repairable, linked event pathway at high LET; either pathway may advance the cell by 1 stage in the multistage model. The model, if validated, permits the direct calculation of cancer risk in rat skin in a way that can be subjected to experimental testing.     

The role of repair in the survival of mammalian cells from heavy ion irradiation: approximation to the ideal case of target theory.

Theories of cellular radiation sensitivity that preclude a significant role for cellular repair processes in the final biological expression of cellular damage induced by ionizing radiation are unsound. Experiments are discussed here in which the cell-cycle dependency of the repair deficiency of the S/S variant, of the L5178Y murine leukemic lymphoblast was examined by treatment with the heavy ions, 20Ne, 28Si, 40Ar, 56Fe and 93Nb. Evidence from those studies, which will be described in detail elsewhere, provide support for the notion that as the linear energy transfer (LET infinity) of the incident radiation increases the ability of the S/S cell to repair radiation damage decreases until effectively it is eliminated around 500 keV/micrometer. In the region of the latter LET infinity value, the behavior of the S/S cell approximates the ideal case of target theory where post-irradiation metabolism (repair) does not influence cell survival. The expression of this phenomenon among different cell types and tissues will depend upon the actual repair systems involved and other considerations.     

Radiation effects on late cytopathological parameters in the murine lens relative to particle fluence.

Lenses of mice irradiated with 250 MeV protons, 670 MeV/amu 20Ne, 600 MeV/amu 56Fe, 600 MeV/amu 93Nb and 593 MeV/amu 139La ions were evaluated by analyzing cytopathological indicators which have been implicated in the cataractogenic process. The LETs ranged from 0.40 keV/micrometer to 953 keV/micrometer and fluences from 1.31 10(3)/mm2 to 4.99 x 10(7)/mm2. 60Co gamma-rays were used as the reference radiation. The doses ranged from 10 to 40 cGy. The lenses were assessed 64 weeks post irradiation in order to observe the late effects of LET and dose on the target cell population of the lens epithelium. Our study shows that growth dependent pathological changes occur at the cellular level as a function of dose and LET. The shapes of the RBE-LET and RBE-dose curves are consistent with previous work on eye and other biological systems done in both our laboratory and others. The RBEmax's were estimated, for the most radiation cataract related cytological changes, MN frequency and MR disorganization, by calculating the ratio of the initial slopes of dose effect curve for various heavy ions to that of 60Co gamma-ray. For each ion studied, the RBEmax derived from micronucleus (MN) frequency is similar to that derived from meridional row (MR) disorganization, suggesting that heavy ions are equally efficient at producing each type of damage. Furthermore, on a per particle basis (particle/cell nucleus), both MN frequency and MR disorganization are LET dependent indicating that these classic precataractogenic indicators are multi-gene effects. Poisson probability analysis of the particle number traversing cell nuclei (average area = 24 micrometers2) suggested that single nuclear traversals determine these changes. By virtue of their precataractogenic nature the data on these endpoints intimate that radiation cataract may also be the consequence of single hits. In any case, these observations are consistent with the current theory of the mechanism of radiation cataractogenesis, which proposes that genomic damage to the epithelial cells surviving the exposure is responsible for opacification.     

130nm体硅反相器链的单粒子瞬态脉宽特性研究

针对130 nm体硅反相器链,利用脉冲激光和重离子实验研究了目标电路单粒子瞬态（SET）的脉宽特性,并分析了电路被辐射诱发的SET脉宽特性受激光能量、重离子线性能量传递（LET）值、PMOS管栅长尺寸等因素的影响机制。重离子和脉冲激光实验结果类似,均表现为随激光能量、LET值的增加,电路被辐射诱发的SET脉宽逐步增大,且表现出明显的双（多）峰分布趋势,但辐射诱发的SET脉冲个数呈先增加再减少规律。此外,实验结果表明,在不同激光能量、LET值下,PMOS管栅长尺寸影响反相器链SET脉冲的特征不同。当激光能量、LET值较低时,PMOS管栅长尺寸大的电路产生的SET脉宽较大,而当激光能量、LET值较大时,PMOS管栅长尺寸小的电路反而产生更宽的SET脉冲。分析表明,较高激光能量、LET辐照时,寄生双极放大效应被触发可能是导致PMOS管栅长尺寸影响电路SET特征差异的主要原因。      

Biochemical mechanisms and clusters of damage for high-LET radiation.

Using mechanisms of indirect and direct radiation, a generalized theory has been developed to account for strand break yields by high-LET particles. The major assumptions of this theory are: (i) damage at deoxyribose sites results primarily in strand break formation and (2) damage to bases leads to a variety of base alterations. Results of the present theory compare well with cellular data without enzymatic repair. As an extension of this theory, we show that damage clusters are formed near each double strand break for high-LET radiation only. For 10 MeV/n (LET = 450 keV/micrometer) neon ions, the results show that on average there are approximately 3 additional breaks and approximately 3 damaged bases formed near each double strand break. For 100 MeV/n helium ions (LET = 3 keV/micrometer), less than 1% of the strand breaks have additional damage within 10 base pairs.     

Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.     

Cell inactivation, repair and mutation induction in bacteria after heavy ion exposure: results from experiments at accelerators and in space.

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.     

Analysis of secondary electron emission spectra of equal-LET protons and alpha particles for purposes of radiation quality and spaceflight hazard assessment.

Amongst the great variety of heavy particles present in the galactic and solar cosmic ray spectra, hydrogen and helium nuclei are significantly more abundant than all other heavier ions and, as such, represent a major radiation hazard to humans in space. Experimental data have suggested that differences in relative biological effectiveness (RBE) exist between the two species at the same value of linear energy transfer (LET). This has consequences for heavily ionising radiation protection procedures, which currently still assume a simple dependence of radiation quality on LET. By analysing the secondary electron (delta-ray) emission spectra of protons and alpha particles, in terms of the spatial characteristics of energy deposition in cellular targets and the likelihood of complex lesion formation, a numerical quantity representing biological effectiveness is generated. When expressed relative to a reference radiation, this quantity is found to differ for protons and a particles of the same LET, demonstrating not only the ion-specific nature of RBE but also the inadequacy of specifying radiation quality as a function of LET only. Such a method for numerically assessing radiation quality may have implications for procedures for heavy ion protection in space at low doses and for understanding the initial mechanisms of radiation action.     

HZE effects on mammalian cells.

In track segment experiments cell survival and chromosome aberrations of mammalian cells have been measured for various heavy ion beams between helium and uranium in the energy range between 0.5 and 960 MeV/u, corresponding to a velocity range of 0.03 to 0.87 C, and an LET spectrum from 10 to 15 000 keV/micrometers. At low LET, the cross section (sigma) for cell killing increases with increasing LET and shows a common curve for all ions regardless of the atomic number. This indicates that in this region the track structure of the different ions is of only a minor influence, and it is rather the total energy transfer, which is important for cell killing. At higher LET values, deviations from a common sigma-LET curve can be observed which indicate a saturation effect. The saturation of the lighter ions occurs at lower LET values than for the heavier ions. These findings are also confirmed by the chromosome data, where the efficiency for the induction of chromosomal aberrations for high LET particles depends on the track structure and is nearly independent of LET. In the heavier beams (Z > or = 10) individual particles cause multiple chromosome breaks in mitotic cells.