Radiation Quality and Mutagenesis in Human Lymphoblastoid Cells Author(s): Howard L. Liber, Rupa Idate, Christy Warner, and Susan M. Bailey Source: Radiation Research, 182(4):390-395. 2014. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR13817.1 URL: http://www.bioone.org/doi/full/10.1667/RR13817.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
RADIATION RESEARCH
182, 390–395 (2014)
0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13817.1
Radiation Quality and Mutagenesis in Human Lymphoblastoid Cells Howard L. Liber,1 Rupa Idate, Christy Warner and Susan M. Bailey Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80525
Furthermore, the nontargeted MFs appeared to reflect a mirror image of that for direct mutagenesis. Ó 2014 by Radiation
Liber, H. L., Idate, R., Warner, C. and Bailey, S. M. Radiation Quality and Mutagenesis in Human Lymphoblastoid Cells. Radiat. Res. 182, 390–395 (2014).
Research Society
An interesting problem associated with studying the effects of low doses of high atomic number and energy (HZE) particles, as found in space, is that not all cells will necessarily be similarly traversed during exposure, a scenario that greatly complicates the measurement of end points that require time to develop, gene-locus mutation being a perfect example. The standard protocol for measuring mutations at the heterozygous thymidine kinase locus in human lymphoblastoid cells involves waiting three days after treatment for newly induced mutants to fully express, at which time cells are then plated in the presence of the selective agent, and mutants are counted three weeks later. This approach is acceptable as long as all cells are uniformly affected, as is the case with low-linear energy transfer (LET) ionizing radiation. However, for HZE particles some fraction of cells may not be traversed or perhaps would receive fewer than the average number of ‘‘hits’’, and they would continue to grow at or closer to the normal rate, thus outpacing cells that received more damage. As a result, at three days posttreatment, more heavily damaged cells will have been ‘‘diluted’’ by the less damaged ones, and thus the measured mutant frequency (MF) will underestimate actual mutant frequency. We therefore developed a modified approach for measuring mutation that eliminates this problem and demonstrates that the mutagenicity of 1 GeV/n Fe ions are underestimated by a factor of two when using the standard MF protocol. Furthermore, we determined the mutagenic effects of a variety of heavy ions, all of which induced mutations in a linear fashion. We found that the maximal yield of mutations (i.e., highest relative biological efficiency) was about 7.5 times higher at an LET of 70 keV/l (400 MeV/n Si) than for gamma rays. Nontargeted mutagenicity after treatment with ionizing radiation was also investigated. For each particular ion/energy examined and in agreement with many previous studies, there was no clear evidence of a dose response for bystander mutagenesis, i.e., the MF plateaued. Interestingly, the magnitudes of the bystander MFs induced by different ion/energy combinations did vary, with bystander MFs ranging from 0.8 to 2.23 higher than the background.
INTRODUCTION
Current models used to estimate risk for radiation carcinogenesis are based primarily on data gathered from A-bomb survivors, a unique population exposed acutely to primarily low-linear energy transfer (LET) radiation. Assessment of radiation risks, including those associated with astronaut exposures, is based on extrapolation of the Japanese data and on assumptions about the relative biological efficiencies (RBE) of high atomic number and energy (HZE) and proton exposures. Inherent in these models is the assumption that, despite their differences in radiation quality, HZE particles have similar ability to induce cancers. These risk estimates are also based on linear-no-threshold (LNT) models, although our understanding of low-dose radiation effects raises the possibility of nonlinear responses. We are forced to make such assumptions in part because we still do not fully understand the molecular basis for radiation-induced cancers. The goal of the work described here was to address these critical concerns through a detailed analysis of the dose-response kinetics for mutagenesis at low to medium doses, as a function of radiation quality. Two major mutational dose-response studies covering a variety of HZE particles have been reported. Kronenberg and colleagues utilized a human lymphoblastoid cell system (1) similar to that used in the current study, and Chen and colleagues utilized normal human fibroblasts (2). Both groups used particles with a wide range of energies, and both developed detailed analyses of mutagenesis versus LET, however, some important questions have remained unaddressed. One particularly interesting issue emerges when considering that individual particles impart a fairly substantial dose when traversing a cell, and for the lower doses, the average number of particles traversing each cell would be small, but the damage would still be Poisson distributed [reviewed and discussed in refs. (3, 4)]. That is to say, if a heavy ion traversing a cell nucleus resulted in a
Editor’s note. The online version of this article (DOI: 10.1667/ RR13817.1) contains supplementary information that is available to all authorized users 1 Address for correspondence: Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523; e-mail: Howard.liber@ colostate.edu. 390
MUTAGENESIS OF HEAVY IONS IN HUMAN CELLS
dose of 30 cGy, then a 30 cGy dose to the population would produce an average of 1 ‘‘hit’’ per cell, meaning that 37% of the cells would not have been directly hit at all. In these earlier studies, the experimental approach for analyzing mutation required growth in nonselective medium after exposure, for newly induced mutants to become genotypically and phenotypically expressed. This generally requires 3–7 days, depending on the gene locus being studied. In that time period, less damaged cells (i.e., those with fewer than the average number of hits) would be expected to have a growth advantage and grow faster than the more severely damaged cells, which would as a result be ‘‘diluted’’. Since induced mutations would be more likely in the heavily damaged subpopulation, we hypothesized that these early experiments may have underestimated the mutant frequencies (MFs) induced by HZE particles. Similarly, our standard protocol for measuring mutation at the thymidine kinase (tk) locus in human lymphoblastoid cells involved waiting three days after exposure for the newly induced mutants to fully express. Cells were then plated on 96-well microtiter dishes in the presence of the selective agent trifluorothymidine (TFT) and the mutants counted three weeks later. Since the plating efficiency is also determined, the mutant fraction is a value that reflects induced mutants per surviving cell. This again is acceptable, as long as all cells are uniformly affected, as would be the case with low-LET ionizing radiation exposure, however as we point out here, it may not be appropriate for damage that is Poisson distributed. To address such a possibility, we developed a protocol for measuring mutation in our lymphoblastoid cell model that eliminates this potential confounding factor. Our new approach does not allow cells to grow between irradiation and plating; instead genotypic and phenotypic expression is accomplished on the plates and the TFT selective agent is added three days after plating. Under these conditions, damaged cells that will become mutants can no longer be left behind by more rapidly growing cells, i.e., they are ‘‘locked’’ in place on the dish, and as long as they are viable and form a colony, they will contribute to the measured MF. Nontargeted effects, where unexposed cells are affected by nearby exposed cells, have a long and somewhat precarious history (5, 6). We have published a number of bystander studies utilizing medium transfer as the means to pass potential signals from irradiated to naı¨ve cells (7, 8). Pertinent to the current study, we found that bystander signals require 1 h to accumulate to a level that induces the maximal effect for mutagenesis. Furthermore, treatment times of 1–24 h with bystander signal all resulted in the same level of mutagenic response. Bystander effects have now been demonstrated in a number of systems, primarily for high-LET radiations. For example, targeted exposure of cells to alpha particles yielded increased levels of gene mutation, sister chromatid exchange, micronuclei, chromosome aberrations, cell killing and cell transformation in bystander cells [e.g., reviewed in refs. (5, 9)]. The
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potential for bystander effect involvement in high-LET radiation carcinogenesis is clear, especially for very lowdose exposures where not all of the cells experience a direct hit, and thus the transfer of a genotoxic signal to neighboring cells should effectively increase the target size for the radiation effect. However, questions remain, and a key one that was investigated here was whether and to what degree radiations of various LET elicit bystander effects. MATERIALS AND METHODS Cells WTK1 lymphoblastoid cells were maintained as suspension cultures in exponential growth at densities ranging from 3–12 3 105 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, at 378C in a humidified 5% CO2 atmosphere. WTK1 cells contain a mutation in codon 237 of p53 and are more mutable than cells with normal p53 (10). Prior to use in a mutation assay, WTK1 lymphoblasts were grown in CHAT medium (complete medium with 10 lM deoxycytidine, 200 lM hypoxanthine, 0.2 lM aminopterin and 17.5 lM thymidine) for one day to reduce the background level of spontaneous tk– mutants. This was followed by one day in CHT medium (CHAT without aminopterin). Cells were then resuspended in fresh medium (11). Radiation Exposures Gamma irradiations were done with a calibrated Mark1 6000 Ci Cs gamma-ray source (J.L. Shepherd and Associates, San Fernando, CA) at a dose rate of ;60 cGy per min, at Colorado State University (CSU). Heavy ion exposures were done at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). Samples of 20 million cells in T-25 flasks were irradiated with doses of 0, 10, 20, 30, 40, 50, 75 or 100 cGy. Dose rates ranged from 0.2– 0.8 Gy/min. The radiations used included c rays, 1,000 MeV protons, 250 MeV/n He, 250 MeV/n O, Si at 250, 400 or 1,000 MeV/n and Fe particles at 300, 600 or 1,000 MeV/n. 137
Mutagenesis Protocol Our historical and standard protocol for radiation mutagenesis has been described previously (11). Briefly, once a time interval for DNA damage repair is complete, cultures are grown in nonselective medium to allow phenotypic expression prior to determination of mutant fraction (MF). For WTK1, optimal expression time is three days for TFT resistance. A modification of the method described in ref. (12) is used to determine mutant fractions. After the expression period, cells are plated in 96-well microtiter dishes at a cell density of 2,000 cells/ well (2 dishes/point). The plating efficiency of each culture is also determined by plating at 1 cell/well in the absence of TFT. All plates are incubated for a total of 18 days prior to scoring colonies. Because TFT is labile, the mutation plates are fed with fresh selective agent between 9–12 days after the initial selection. Colonies are scored and the number of colony-forming units/well (cfu/w) are determined by the Poisson distribution. The MF equals the colony-forming units/well for mutation plates divided by the plating efficiency times the appropriate dilution factor. However, for the experiments described here, a variation of our standard protocol was developed. As above, plating efficiency dishes were plated at 1 cell/well, but cells on mutation plates were seeded at a lower cell density of 200 cells/well to allow room for growth (approximately 3 population doublings) during the first three days post exposure with no selective agent present. Cells were seeded in a
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TABLE 1 Mutagenicity of c Rays or 56Fe (1 GeV/n) to WTK1 Lymphoblasts Condition (Gy) 0 0.5 1.0 1.5 1.0
Radiation — Gamma Gamma Gamma Fe
MF using the standard protocol 91 470 753 1,272 1,206
volume of 150 ll (2 dishes/point) and 3 days later TFT was added to a final concentration of 2 lg/ml, in a volume of 50 ll. Feeding with fresh TFT was done 11 days later to restore the final concentration to 2 lg/ml. Colonies were scored four days after that. We can use this approach to predict the number of new spontaneous mutants that appear in the dishes between the plating and selection times. For each experiment, we calculated how many new spontaneous mutants appeared on the dishes between plating and selection. We did this by comparing the background MF in the 200 c/ w dishes with TFT applied three days later, with untreated cells plated in the standard fashion (2,000 c/w, immediate application of TFT). This indicated how many colonies needed to be subtracted from each of the experimental plates, to normalize the background MF. An additional adjustment was necessary because some of the treated cultures did not grow as well as the control the day after plating. We monitored the growth of the cultures in the flasks of the cells that had not been plated. So for example, if we subtracted 3 colonies from a control culture that doubled in the first day, we would subtract proportionately fewer for a treated culture that grew less well. No adjustment was necessary for radiation-induced mutants since they were induced at the time of irradiation. Experimental Approach T-25 flasks of WTK1 lymphoblastoid cells were shipped to BNL and grown for 3–5 days. Cells were then irradiated at NSRL with doses ranging from 0–100 cGy, and then incubated at 378C for 2 h to allow time for DNA repair and fixation of mutations. The cells were then cooled to room temperature, and the samples were returned to CSU and analyzed for MF using the variation of our standard protocol, described above. It is important to note that as soon as WTK1 lymphoblastoid cells fall below approximately 348C, they cease to grow, so cell concentrations did not change during shipment from BNL to CSU. Normal growth resumed as soon as the cells were returned to the incubator (data not shown). Two hours after a particular irradiation was completed, a sample of the media potentially containing bystander signals was removed from the various dose points and applied to unexposed, naı¨ve cells. In each case, media from 5 million irradiated cells was used to treat a naı¨ve sample also consisting of 5 million cells. The directly treated samples were centrifuged at high speed (2,000g) for 15 min. Only the upper 80% of the supernatant was removed so that no directly treated cells would be transferred to the naı¨ve sample (preliminary experiments had shown that this supernatant contained no viable cells, as evidenced by plating). After 24 h incubation with the bystander medium, cells were returned to CSU for processing. Because exposure to bystander signal is uniform, our standard protocol for determining mutant fractions was used, which generated a dose response for nontargeted mutagenesis. The times chosen for these experiments (i.e., harvest bystander signal-containing media 2 h after irradiation, and apply to naı¨ve cells overnight) were based on the experiments for c rays done previously (7, 8). They were chosen so that a maximal bystander effect could be induced. It is possible that there could be differences among the different radiations with respect to bystander kinetics, but that possibility was not investigated in the current work.
6 6 6 6 6
6
22 3 10 121 3 10 6 94 3 10 6 286 3 10 6 246 3 10 6
MF using revised plating protocol 77 426 846 1,432 2,460
6 6 6 6 6
15 3 10 6 68 3 10 6 131 3 10 6 211 3 10 6 393 3 10 6
RESULTS AND DISCUSSION
The major goals of this work were to produce detailed radiation dose-response curves for direct mutagenesis, and to determine whether radiations of different qualities induce nontargeted (bystander) mutagenesis. For the latter, we expected a yes or no response for each case, although it was also possible that we might see different magnitudes of bystander response. Standard Approaches for Measuring Mutagenesis Underestimate Mutant Fractions Induced by Heavy Ions
To demonstrate the feasibility of our new mutagenesis protocol, we first treated cells with 0–1.5 Gy of c rays and compared the two protocols for measuring MF (data shown in Table 1). There were no significant differences between the methods, as predicted for c rays, which uniformly damage all cells. However, when we compared the standard and alternative plating methods after treatment with 1 GeV/ n 56Fe, we observed that pre-expression plating yielded a significantly higher MF than did the standard protocol. It is important to note that HZE particles do induce mutation when they traverse lymphoblast cells. There has been some discussion as to whether the direct traversal of even a single HZE particle always kills the cell, and therefore the only effects observed among surviving cells are those associated with delta rays [e.g., see refs. (3, 4)]. Our treatments with 1 GeV/n Fe confirm that cells can indeed survive a direct traversal of an HZE particle, resulting in mutation. Theoretically, no more than one third of the total dose from 1 GeV/n 56Fe should be from delta rays [e.g., see refs. (13–15)], and delta rays should have a mutagenic efficiency approximately equal to that of c rays. We demonstrate here that for any particular dose of HZE, the observed MFs were always more than 3 times higher than what would normally have been seen from one third of that dose in c rays. This is evident from the data shown in Table 1, where the MF for 1 Gy of Fe ions is nearly 6 times higher than for 0.5 Gy c rays, confirming that direct traversals of HZE particles actually do induce mutation. Mutagenesis as a Function of Radiation Quality
Direct mutagenesis dose-response curves for WTK1 lymphoblastoid cells after exposures to different qualities of ionizing radiation are shown in Fig. 1. Each experiment
MUTAGENESIS OF HEAVY IONS IN HUMAN CELLS
393
FIG. 2. Mutant fractions induced by various ionizing radiations. Data points represent the slopes calculated from the dose-response curves shown in Fig. 1 and error bars are standard deviations. Nontargeted mutant fractions were subtracted. Statistical comparisons are shown in Table 2.
FIG. 1. Dose response for mutagenicity of various ionizing radiations. Error bars are standard deviations. Each data point is the average of four independently treated cultures in two separate experiments. Nontargeted (bystander) mutant fractions have been subtracted.
consisted of 7 doses (0, 10, 20, 30, 40, 50, 100 cGy), delivered at dose rates ranging from 10–80 cGy/min. These data points reflect calculated directly induced MFs, as they were derived by subtracting the induced bystander MFs (see Fig. 3) from the total observed MFs. However, because the
induced direct MFs were more than an order of magnitude greater than the bystander-induced MFs, the dose-response curves were not appreciably affected, i.e., the curves for total observed MF versus dose look very similar to those shown in the supplementary material (http://dx.doi.org/10. 1667/RR13817.1.S1). A plot of LET versus effectiveness (RBE) of inducing mutation is shown in Fig. 2. Since all of the dose-response curves were approximately linear, we plotted the slopes of the MF curves versus dose and thus the ordinates are in terms of MF/Gy. As can be seen, 400 MeV/n Si, with an intermediate LET of ;70 keV/l, was the most mutagenic ion/energy utilized, while gamma rays and 250 MeV/n helium were the least mutagenic. Statistical comparisons are shown in Table 2. Nontargeted mutagenesis studies were performed as described above, and there are two important findings. First, for each particular ion/energy examined, there was no clear evidence of a dose response for bystander mutagenesis, as shown in the example in Fig. 3, top panel, the MFs have clearly plateaued. This agrees with many published articles on bystander effects, although some articles have described dose-dependent responses [reviewed in ref. (9)]. Our other nontargeted dose-response curves can be found in the supplementary material (http://dx.doi.org/10.1667/ RR13817.1.S1). Statistical comparisons are shown in Table 2. The second finding, of particular interest, was that the magnitudes of the bystander MFs induced by different ion/ energy combinations did vary and bystander MFs ranged from ;0.8 to 2.23 higher than the background (Fig. 3 bottom panel). Furthermore, the data in Fig. 3 appear to resemble a mirror image of the data for direct mutagenesis shown in Fig. 2. It is also interesting that irradiation with
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LIBER ET AL.
TABLE 2 Statistical Comparisons by Student’s t Test of Data from Figs. 2 and 3 0 Gamma H-1,000 He-250 O-250 Si-250 Si-400 Si-1000 Fe-300 Fe-600 Fe-1,000
0
c
H-1,000
He-250
O-250
Si-250
Si-400
Si-1,000
Fe-300
Fe-600
Fe-1,000
– 0.008 0.001 ,0.001 0.005 0.042 0.463 0.126 0.028 0.047 0.040
,0.001 – 0.020 0.002 1.000 0.216 0.015 0.006 0.753 0.140 0.375
,0.001 0.067 – 0.031 0.010 0.007 0.001 0.001 0.083 0.005 0.012
,0.001 0.54 0.035 – ,0.001 ,0.001 ,0.001 ,0.001 0.009 ,0.001 0.002
,0.001 ,0.001 0.001 ,0.001 – 0.137 0.005 0.004 0.730 0.081 0.297
,0.001 ,0.001 ,0.001 ,0.001 0.005 – 0.072 0.014 0.250 0.732 0.756
,0.001 ,0.001 ,0.001 ,0.001 ,0.001 0.122 – 0.059 0.040 0.112 0.065
,0.001 0.003 0.009 0.003 0.420 0.005 0.001 – 0.012 0.018 0.014
,0.001 0.013 0.098 0.008 0.006 0.001 ,0.001 0.043 – 0.185 0.357
,0.001 0.019 0.171 0.012 0.005 0.001 ,0.001 0.032 0.700 – 0.542
,0.001 0.004 0.017 0.003 0.035 0.002 ,0.001 0.215 0.157 0.101 –
Notes. Direct mutagenesis P values are shown above and to the right of the diagonal, and bystander mutagenesis P values are below and to the left of the diagonal. Ion energies are in MeV/u. Significant differences are indicated in boldface.
400 MeV/n Si, the most directly mutagenic ion/energy, did not appear to generate detectable levels of bystander signals and 1,000 MeV/n Si also did not induce a response. These data in Fig. 3 are surprising, because we did not expect to see bystander responses plateauing at different magnitudes. At this time, we have no good explanation for this result. Questions arise regarding one possibility: Could there be multiple bystander signals produced, with different subsets of these released after different treatments? And could these different signals be additive? To summarize, we find that radiations of different qualities induce mutagenesis in an LET-dependent fashion. Further and irrespective of radiation quality, relatively low levels of mutation induced by bystander signals, compared to the much higher MFs observed in directly treated cells, indicate that for an irradiated population, even at very low doses, the great majority of mutants are induced directly. SUPPLEMENTARY INFORMATION
Figs. S1–S3. Total combined mutant frequencies (MF) observed in these experiments (slides 2–4). Figs. S4–S12. Dose-response curves for nontargeted mutagenesis (slides 5–13). ACKNOWLEDGMENT This work was funded by NASA grant NNX10AB36G. Received: June 2, 2014; accepted: July 14, 2014; published online: September 3, 2014
REFERENCES FIG. 3. Nontargeted effects of ionizing radiation on mutagenesis. The top panel shows the dose response at the TK locus for 300 MeV/n Fe ion, actually a plateau with an overall response of a 1.5-fold increase above background. The bottom panel shows nontargeted mutagenesis for all ions/energies. Values for all doses for a particular ion/energy were averaged and the background MFs were subtracted. Error bars are standard deviations. Statistical comparisons are shown in Table 2.
1. Kronenberg A. Mutation induction in human lymphoid cells by energetic heavy ions. Adv Space Res 1994; 14:339–46. 2. Chen DJ, Tsuboi K, Nguyen T, Yang TC. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts. Adv Space Res 1994; 14:347–54. 3. Peng Y, Borak TB, Bouffler SD, Ullrich RL, Weil MM, Bedford JS. Radiation leukemogenesis in mice: loss of PU.1 on chromosome 2 in CBA and C57BL/6 mice after irradiation with
MUTAGENESIS OF HEAVY IONS IN HUMAN CELLS
4.
5. 6. 7.
8.
9.
1 GeV/nucleon 56Fe ions, X rays or gamma Rays. Part II. Theoretical considerations based on microdosimetry and the initial induction of chromosome aberrations. Radiat Res 2009; 171:484– 93. Peng Y, Brown N, Finnon R, Warner CL, Liu X, Genik PC, et al. Radiation leukemogenesis in mice: loss of PU.1 on chromosome 2 in CBA and C57BL/6 mice after irradiation with 1 GeV/nucleon 56Fe ions, X rays or gamma rays. Part I. Experimental observations. Radiat Res 2009; 171:474–83. Little JB. Genomic instability and bystander effects: a historical perspective. Oncogene 2003; 22:6978–87. Mothersill C, Seymour C. Radiation-induced bystander effects: past history and future directions. Radiat Res 2001; 155:759–67. Zhang Y, Zhou J, Held KD, Redmond RW, Prise KM, Liber HL. Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells. Radiat Res 2008; 169:197– 206. Zhang Y, Zhou J, Baldwin J, Held KD, Prise KM, Redmond RW, Liber HL. Ionizing radiation-induced bystander mutagenesis and adaptation: quantitative and temporal aspects. Mutat Res 2009; 671:20–5. Blyth BJ, Sykes PJ. Radiation-induced bystander effects: what are
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they, and how relevant are they to human radiation exposures? Radiat Res 2011; 176:139–57. Xia F, Wang X, Wang YH, Tsang NM, Yandell DW, Kelsey KT, Liber HL. Altered p53 status correlates with differences in sensitivity to radiation-induced mutation and apoptosis in two closely related human lymphoblast lines. Cancer Res 1995; 55:12– 5. Liber HL, Yandell DW, Little JB. A comparison of mutation induction at the tk and hprt loci in human lymphoblastoid cells; quantitative differences are due to an additional class of mutations at the autosomal tk locus. Mutat Res 1989; 216:9–17. Furth EE, Thilly WG, Penman BW, Liber HL, Rand WM. Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates. Anal Biochem 1981; 110:1–8. Chatterjee A, Schaefer HJ. Microdosimetric structure of heavy ion tracks in tissue. Radiat Environ Biophys 1976; 13:215–27. Cucinotta FA, Nikjoo H, Goodhead DT. The effects of delta rays on the number of particle-track traversals per cell in laboratory and space exposures. Radiat Res 1998; 150:115–9. Cucinotta FA, Nikjoo H, Goodhead DT. Model for radial dependence of frequency distributions for energy imparted in nanometer volumes from HZE particles. Radiat Res 2000; 153:459–68.
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
RADIATION RESEARCH
182, 390–395 (2014)
0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13817.1
Radiation Quality and Mutagenesis in Human Lymphoblastoid Cells Howard L. Liber,1 Rupa Idate, Christy Warner and Susan M. Bailey Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80525
Furthermore, the nontargeted MFs appeared to reflect a mirror image of that for direct mutagenesis. Ó 2014 by Radiation
Liber, H. L., Idate, R., Warner, C. and Bailey, S. M. Radiation Quality and Mutagenesis in Human Lymphoblastoid Cells. Radiat. Res. 182, 390–395 (2014).
Research Society
An interesting problem associated with studying the effects of low doses of high atomic number and energy (HZE) particles, as found in space, is that not all cells will necessarily be similarly traversed during exposure, a scenario that greatly complicates the measurement of end points that require time to develop, gene-locus mutation being a perfect example. The standard protocol for measuring mutations at the heterozygous thymidine kinase locus in human lymphoblastoid cells involves waiting three days after treatment for newly induced mutants to fully express, at which time cells are then plated in the presence of the selective agent, and mutants are counted three weeks later. This approach is acceptable as long as all cells are uniformly affected, as is the case with low-linear energy transfer (LET) ionizing radiation. However, for HZE particles some fraction of cells may not be traversed or perhaps would receive fewer than the average number of ‘‘hits’’, and they would continue to grow at or closer to the normal rate, thus outpacing cells that received more damage. As a result, at three days posttreatment, more heavily damaged cells will have been ‘‘diluted’’ by the less damaged ones, and thus the measured mutant frequency (MF) will underestimate actual mutant frequency. We therefore developed a modified approach for measuring mutation that eliminates this problem and demonstrates that the mutagenicity of 1 GeV/n Fe ions are underestimated by a factor of two when using the standard MF protocol. Furthermore, we determined the mutagenic effects of a variety of heavy ions, all of which induced mutations in a linear fashion. We found that the maximal yield of mutations (i.e., highest relative biological efficiency) was about 7.5 times higher at an LET of 70 keV/l (400 MeV/n Si) than for gamma rays. Nontargeted mutagenicity after treatment with ionizing radiation was also investigated. For each particular ion/energy examined and in agreement with many previous studies, there was no clear evidence of a dose response for bystander mutagenesis, i.e., the MF plateaued. Interestingly, the magnitudes of the bystander MFs induced by different ion/energy combinations did vary, with bystander MFs ranging from 0.8 to 2.23 higher than the background.
INTRODUCTION
Current models used to estimate risk for radiation carcinogenesis are based primarily on data gathered from A-bomb survivors, a unique population exposed acutely to primarily low-linear energy transfer (LET) radiation. Assessment of radiation risks, including those associated with astronaut exposures, is based on extrapolation of the Japanese data and on assumptions about the relative biological efficiencies (RBE) of high atomic number and energy (HZE) and proton exposures. Inherent in these models is the assumption that, despite their differences in radiation quality, HZE particles have similar ability to induce cancers. These risk estimates are also based on linear-no-threshold (LNT) models, although our understanding of low-dose radiation effects raises the possibility of nonlinear responses. We are forced to make such assumptions in part because we still do not fully understand the molecular basis for radiation-induced cancers. The goal of the work described here was to address these critical concerns through a detailed analysis of the dose-response kinetics for mutagenesis at low to medium doses, as a function of radiation quality. Two major mutational dose-response studies covering a variety of HZE particles have been reported. Kronenberg and colleagues utilized a human lymphoblastoid cell system (1) similar to that used in the current study, and Chen and colleagues utilized normal human fibroblasts (2). Both groups used particles with a wide range of energies, and both developed detailed analyses of mutagenesis versus LET, however, some important questions have remained unaddressed. One particularly interesting issue emerges when considering that individual particles impart a fairly substantial dose when traversing a cell, and for the lower doses, the average number of particles traversing each cell would be small, but the damage would still be Poisson distributed [reviewed and discussed in refs. (3, 4)]. That is to say, if a heavy ion traversing a cell nucleus resulted in a
Editor’s note. The online version of this article (DOI: 10.1667/ RR13817.1) contains supplementary information that is available to all authorized users 1 Address for correspondence: Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523; e-mail: Howard.liber@ colostate.edu. 390
MUTAGENESIS OF HEAVY IONS IN HUMAN CELLS
dose of 30 cGy, then a 30 cGy dose to the population would produce an average of 1 ‘‘hit’’ per cell, meaning that 37% of the cells would not have been directly hit at all. In these earlier studies, the experimental approach for analyzing mutation required growth in nonselective medium after exposure, for newly induced mutants to become genotypically and phenotypically expressed. This generally requires 3–7 days, depending on the gene locus being studied. In that time period, less damaged cells (i.e., those with fewer than the average number of hits) would be expected to have a growth advantage and grow faster than the more severely damaged cells, which would as a result be ‘‘diluted’’. Since induced mutations would be more likely in the heavily damaged subpopulation, we hypothesized that these early experiments may have underestimated the mutant frequencies (MFs) induced by HZE particles. Similarly, our standard protocol for measuring mutation at the thymidine kinase (tk) locus in human lymphoblastoid cells involved waiting three days after exposure for the newly induced mutants to fully express. Cells were then plated on 96-well microtiter dishes in the presence of the selective agent trifluorothymidine (TFT) and the mutants counted three weeks later. Since the plating efficiency is also determined, the mutant fraction is a value that reflects induced mutants per surviving cell. This again is acceptable, as long as all cells are uniformly affected, as would be the case with low-LET ionizing radiation exposure, however as we point out here, it may not be appropriate for damage that is Poisson distributed. To address such a possibility, we developed a protocol for measuring mutation in our lymphoblastoid cell model that eliminates this potential confounding factor. Our new approach does not allow cells to grow between irradiation and plating; instead genotypic and phenotypic expression is accomplished on the plates and the TFT selective agent is added three days after plating. Under these conditions, damaged cells that will become mutants can no longer be left behind by more rapidly growing cells, i.e., they are ‘‘locked’’ in place on the dish, and as long as they are viable and form a colony, they will contribute to the measured MF. Nontargeted effects, where unexposed cells are affected by nearby exposed cells, have a long and somewhat precarious history (5, 6). We have published a number of bystander studies utilizing medium transfer as the means to pass potential signals from irradiated to naı¨ve cells (7, 8). Pertinent to the current study, we found that bystander signals require 1 h to accumulate to a level that induces the maximal effect for mutagenesis. Furthermore, treatment times of 1–24 h with bystander signal all resulted in the same level of mutagenic response. Bystander effects have now been demonstrated in a number of systems, primarily for high-LET radiations. For example, targeted exposure of cells to alpha particles yielded increased levels of gene mutation, sister chromatid exchange, micronuclei, chromosome aberrations, cell killing and cell transformation in bystander cells [e.g., reviewed in refs. (5, 9)]. The
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potential for bystander effect involvement in high-LET radiation carcinogenesis is clear, especially for very lowdose exposures where not all of the cells experience a direct hit, and thus the transfer of a genotoxic signal to neighboring cells should effectively increase the target size for the radiation effect. However, questions remain, and a key one that was investigated here was whether and to what degree radiations of various LET elicit bystander effects. MATERIALS AND METHODS Cells WTK1 lymphoblastoid cells were maintained as suspension cultures in exponential growth at densities ranging from 3–12 3 105 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, at 378C in a humidified 5% CO2 atmosphere. WTK1 cells contain a mutation in codon 237 of p53 and are more mutable than cells with normal p53 (10). Prior to use in a mutation assay, WTK1 lymphoblasts were grown in CHAT medium (complete medium with 10 lM deoxycytidine, 200 lM hypoxanthine, 0.2 lM aminopterin and 17.5 lM thymidine) for one day to reduce the background level of spontaneous tk– mutants. This was followed by one day in CHT medium (CHAT without aminopterin). Cells were then resuspended in fresh medium (11). Radiation Exposures Gamma irradiations were done with a calibrated Mark1 6000 Ci Cs gamma-ray source (J.L. Shepherd and Associates, San Fernando, CA) at a dose rate of ;60 cGy per min, at Colorado State University (CSU). Heavy ion exposures were done at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). Samples of 20 million cells in T-25 flasks were irradiated with doses of 0, 10, 20, 30, 40, 50, 75 or 100 cGy. Dose rates ranged from 0.2– 0.8 Gy/min. The radiations used included c rays, 1,000 MeV protons, 250 MeV/n He, 250 MeV/n O, Si at 250, 400 or 1,000 MeV/n and Fe particles at 300, 600 or 1,000 MeV/n. 137
Mutagenesis Protocol Our historical and standard protocol for radiation mutagenesis has been described previously (11). Briefly, once a time interval for DNA damage repair is complete, cultures are grown in nonselective medium to allow phenotypic expression prior to determination of mutant fraction (MF). For WTK1, optimal expression time is three days for TFT resistance. A modification of the method described in ref. (12) is used to determine mutant fractions. After the expression period, cells are plated in 96-well microtiter dishes at a cell density of 2,000 cells/ well (2 dishes/point). The plating efficiency of each culture is also determined by plating at 1 cell/well in the absence of TFT. All plates are incubated for a total of 18 days prior to scoring colonies. Because TFT is labile, the mutation plates are fed with fresh selective agent between 9–12 days after the initial selection. Colonies are scored and the number of colony-forming units/well (cfu/w) are determined by the Poisson distribution. The MF equals the colony-forming units/well for mutation plates divided by the plating efficiency times the appropriate dilution factor. However, for the experiments described here, a variation of our standard protocol was developed. As above, plating efficiency dishes were plated at 1 cell/well, but cells on mutation plates were seeded at a lower cell density of 200 cells/well to allow room for growth (approximately 3 population doublings) during the first three days post exposure with no selective agent present. Cells were seeded in a
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TABLE 1 Mutagenicity of c Rays or 56Fe (1 GeV/n) to WTK1 Lymphoblasts Condition (Gy) 0 0.5 1.0 1.5 1.0
Radiation — Gamma Gamma Gamma Fe
MF using the standard protocol 91 470 753 1,272 1,206
volume of 150 ll (2 dishes/point) and 3 days later TFT was added to a final concentration of 2 lg/ml, in a volume of 50 ll. Feeding with fresh TFT was done 11 days later to restore the final concentration to 2 lg/ml. Colonies were scored four days after that. We can use this approach to predict the number of new spontaneous mutants that appear in the dishes between the plating and selection times. For each experiment, we calculated how many new spontaneous mutants appeared on the dishes between plating and selection. We did this by comparing the background MF in the 200 c/ w dishes with TFT applied three days later, with untreated cells plated in the standard fashion (2,000 c/w, immediate application of TFT). This indicated how many colonies needed to be subtracted from each of the experimental plates, to normalize the background MF. An additional adjustment was necessary because some of the treated cultures did not grow as well as the control the day after plating. We monitored the growth of the cultures in the flasks of the cells that had not been plated. So for example, if we subtracted 3 colonies from a control culture that doubled in the first day, we would subtract proportionately fewer for a treated culture that grew less well. No adjustment was necessary for radiation-induced mutants since they were induced at the time of irradiation. Experimental Approach T-25 flasks of WTK1 lymphoblastoid cells were shipped to BNL and grown for 3–5 days. Cells were then irradiated at NSRL with doses ranging from 0–100 cGy, and then incubated at 378C for 2 h to allow time for DNA repair and fixation of mutations. The cells were then cooled to room temperature, and the samples were returned to CSU and analyzed for MF using the variation of our standard protocol, described above. It is important to note that as soon as WTK1 lymphoblastoid cells fall below approximately 348C, they cease to grow, so cell concentrations did not change during shipment from BNL to CSU. Normal growth resumed as soon as the cells were returned to the incubator (data not shown). Two hours after a particular irradiation was completed, a sample of the media potentially containing bystander signals was removed from the various dose points and applied to unexposed, naı¨ve cells. In each case, media from 5 million irradiated cells was used to treat a naı¨ve sample also consisting of 5 million cells. The directly treated samples were centrifuged at high speed (2,000g) for 15 min. Only the upper 80% of the supernatant was removed so that no directly treated cells would be transferred to the naı¨ve sample (preliminary experiments had shown that this supernatant contained no viable cells, as evidenced by plating). After 24 h incubation with the bystander medium, cells were returned to CSU for processing. Because exposure to bystander signal is uniform, our standard protocol for determining mutant fractions was used, which generated a dose response for nontargeted mutagenesis. The times chosen for these experiments (i.e., harvest bystander signal-containing media 2 h after irradiation, and apply to naı¨ve cells overnight) were based on the experiments for c rays done previously (7, 8). They were chosen so that a maximal bystander effect could be induced. It is possible that there could be differences among the different radiations with respect to bystander kinetics, but that possibility was not investigated in the current work.
6 6 6 6 6
6
22 3 10 121 3 10 6 94 3 10 6 286 3 10 6 246 3 10 6
MF using revised plating protocol 77 426 846 1,432 2,460
6 6 6 6 6
15 3 10 6 68 3 10 6 131 3 10 6 211 3 10 6 393 3 10 6
RESULTS AND DISCUSSION
The major goals of this work were to produce detailed radiation dose-response curves for direct mutagenesis, and to determine whether radiations of different qualities induce nontargeted (bystander) mutagenesis. For the latter, we expected a yes or no response for each case, although it was also possible that we might see different magnitudes of bystander response. Standard Approaches for Measuring Mutagenesis Underestimate Mutant Fractions Induced by Heavy Ions
To demonstrate the feasibility of our new mutagenesis protocol, we first treated cells with 0–1.5 Gy of c rays and compared the two protocols for measuring MF (data shown in Table 1). There were no significant differences between the methods, as predicted for c rays, which uniformly damage all cells. However, when we compared the standard and alternative plating methods after treatment with 1 GeV/ n 56Fe, we observed that pre-expression plating yielded a significantly higher MF than did the standard protocol. It is important to note that HZE particles do induce mutation when they traverse lymphoblast cells. There has been some discussion as to whether the direct traversal of even a single HZE particle always kills the cell, and therefore the only effects observed among surviving cells are those associated with delta rays [e.g., see refs. (3, 4)]. Our treatments with 1 GeV/n Fe confirm that cells can indeed survive a direct traversal of an HZE particle, resulting in mutation. Theoretically, no more than one third of the total dose from 1 GeV/n 56Fe should be from delta rays [e.g., see refs. (13–15)], and delta rays should have a mutagenic efficiency approximately equal to that of c rays. We demonstrate here that for any particular dose of HZE, the observed MFs were always more than 3 times higher than what would normally have been seen from one third of that dose in c rays. This is evident from the data shown in Table 1, where the MF for 1 Gy of Fe ions is nearly 6 times higher than for 0.5 Gy c rays, confirming that direct traversals of HZE particles actually do induce mutation. Mutagenesis as a Function of Radiation Quality
Direct mutagenesis dose-response curves for WTK1 lymphoblastoid cells after exposures to different qualities of ionizing radiation are shown in Fig. 1. Each experiment
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FIG. 2. Mutant fractions induced by various ionizing radiations. Data points represent the slopes calculated from the dose-response curves shown in Fig. 1 and error bars are standard deviations. Nontargeted mutant fractions were subtracted. Statistical comparisons are shown in Table 2.
FIG. 1. Dose response for mutagenicity of various ionizing radiations. Error bars are standard deviations. Each data point is the average of four independently treated cultures in two separate experiments. Nontargeted (bystander) mutant fractions have been subtracted.
consisted of 7 doses (0, 10, 20, 30, 40, 50, 100 cGy), delivered at dose rates ranging from 10–80 cGy/min. These data points reflect calculated directly induced MFs, as they were derived by subtracting the induced bystander MFs (see Fig. 3) from the total observed MFs. However, because the
induced direct MFs were more than an order of magnitude greater than the bystander-induced MFs, the dose-response curves were not appreciably affected, i.e., the curves for total observed MF versus dose look very similar to those shown in the supplementary material (http://dx.doi.org/10. 1667/RR13817.1.S1). A plot of LET versus effectiveness (RBE) of inducing mutation is shown in Fig. 2. Since all of the dose-response curves were approximately linear, we plotted the slopes of the MF curves versus dose and thus the ordinates are in terms of MF/Gy. As can be seen, 400 MeV/n Si, with an intermediate LET of ;70 keV/l, was the most mutagenic ion/energy utilized, while gamma rays and 250 MeV/n helium were the least mutagenic. Statistical comparisons are shown in Table 2. Nontargeted mutagenesis studies were performed as described above, and there are two important findings. First, for each particular ion/energy examined, there was no clear evidence of a dose response for bystander mutagenesis, as shown in the example in Fig. 3, top panel, the MFs have clearly plateaued. This agrees with many published articles on bystander effects, although some articles have described dose-dependent responses [reviewed in ref. (9)]. Our other nontargeted dose-response curves can be found in the supplementary material (http://dx.doi.org/10.1667/ RR13817.1.S1). Statistical comparisons are shown in Table 2. The second finding, of particular interest, was that the magnitudes of the bystander MFs induced by different ion/ energy combinations did vary and bystander MFs ranged from ;0.8 to 2.23 higher than the background (Fig. 3 bottom panel). Furthermore, the data in Fig. 3 appear to resemble a mirror image of the data for direct mutagenesis shown in Fig. 2. It is also interesting that irradiation with
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TABLE 2 Statistical Comparisons by Student’s t Test of Data from Figs. 2 and 3 0 Gamma H-1,000 He-250 O-250 Si-250 Si-400 Si-1000 Fe-300 Fe-600 Fe-1,000
0
c
H-1,000
He-250
O-250
Si-250
Si-400
Si-1,000
Fe-300
Fe-600
Fe-1,000
– 0.008 0.001 ,0.001 0.005 0.042 0.463 0.126 0.028 0.047 0.040
,0.001 – 0.020 0.002 1.000 0.216 0.015 0.006 0.753 0.140 0.375
,0.001 0.067 – 0.031 0.010 0.007 0.001 0.001 0.083 0.005 0.012
,0.001 0.54 0.035 – ,0.001 ,0.001 ,0.001 ,0.001 0.009 ,0.001 0.002
,0.001 ,0.001 0.001 ,0.001 – 0.137 0.005 0.004 0.730 0.081 0.297
,0.001 ,0.001 ,0.001 ,0.001 0.005 – 0.072 0.014 0.250 0.732 0.756
,0.001 ,0.001 ,0.001 ,0.001 ,0.001 0.122 – 0.059 0.040 0.112 0.065
,0.001 0.003 0.009 0.003 0.420 0.005 0.001 – 0.012 0.018 0.014
,0.001 0.013 0.098 0.008 0.006 0.001 ,0.001 0.043 – 0.185 0.357
,0.001 0.019 0.171 0.012 0.005 0.001 ,0.001 0.032 0.700 – 0.542
,0.001 0.004 0.017 0.003 0.035 0.002 ,0.001 0.215 0.157 0.101 –
Notes. Direct mutagenesis P values are shown above and to the right of the diagonal, and bystander mutagenesis P values are below and to the left of the diagonal. Ion energies are in MeV/u. Significant differences are indicated in boldface.
400 MeV/n Si, the most directly mutagenic ion/energy, did not appear to generate detectable levels of bystander signals and 1,000 MeV/n Si also did not induce a response. These data in Fig. 3 are surprising, because we did not expect to see bystander responses plateauing at different magnitudes. At this time, we have no good explanation for this result. Questions arise regarding one possibility: Could there be multiple bystander signals produced, with different subsets of these released after different treatments? And could these different signals be additive? To summarize, we find that radiations of different qualities induce mutagenesis in an LET-dependent fashion. Further and irrespective of radiation quality, relatively low levels of mutation induced by bystander signals, compared to the much higher MFs observed in directly treated cells, indicate that for an irradiated population, even at very low doses, the great majority of mutants are induced directly. SUPPLEMENTARY INFORMATION
Figs. S1–S3. Total combined mutant frequencies (MF) observed in these experiments (slides 2–4). Figs. S4–S12. Dose-response curves for nontargeted mutagenesis (slides 5–13). ACKNOWLEDGMENT This work was funded by NASA grant NNX10AB36G. Received: June 2, 2014; accepted: July 14, 2014; published online: September 3, 2014
REFERENCES FIG. 3. Nontargeted effects of ionizing radiation on mutagenesis. The top panel shows the dose response at the TK locus for 300 MeV/n Fe ion, actually a plateau with an overall response of a 1.5-fold increase above background. The bottom panel shows nontargeted mutagenesis for all ions/energies. Values for all doses for a particular ion/energy were averaged and the background MFs were subtracted. Error bars are standard deviations. Statistical comparisons are shown in Table 2.
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