Mutagenesis, Vol. 16, No. 1, 7-15,
January 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
p53 deficiency alters the yield and spectrum of radiation-induced lacZ mutants in the brain of transgenic mice
PN147, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA
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Abstract |
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Exposure to heavy particle radiation in the galacto-cosmic environment poses a significant risk in space exploration and the evaluation of radiation-induced genetic damage in tissues, especially in the central nervous system, is an important consideration in long-term manned space missions. We used a plasmid-based transgenic mouse model system, with the pUR288 lacZ transgene integrated in the genome of every cell of C57Bl/6(lacZ) mice, to evaluate the genetic damage induced by iron particle radiation. In order to examine the importance of genetic background on the radiation sensitivity of individuals, we cross-bred p53 wild-type lacZ transgenic mice with p53 nullizygous mice, producing lacZ transgenic mice that were either hemizygous or nullizygous for the p53 tumor suppressor gene. Animals were exposed to an acute dose of 1 Gy of iron particles and the lacZ mutation frequency (MF) in the brain was measured at time intervals from 1 to 16 weeks post-irradiation. Our results suggest that iron particles induced an increase in lacZ MF (2.4-fold increase in p53+/+ mice, 1.3-fold increase in p53+/– mice and 2.1-fold increase in p53–/– mice) and that this induction is both temporally regulated and p53 genotype dependent. Characterization of mutants based on their restriction patterns showed that the majority of the mutants arising spontaneously are derived from point mutations or small deletions in all three genotypes. Radiation induced alterations in the spectrum of deletion mutants and reorganization of the genome, as evidenced by the selection of mutants containing mouse genomic DNA. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported owing to technical limitations in most other mutation assays.
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Introduction |
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The space radiation environment encompasses a broad spectrum of radiation ranging from infra-red to galactic cosmic radiation (GCR). Highly energetic charged particle (HZE) radiation from, for example, the nucleus of iron particles accounts for the majority of absorbed cosmic radiation dose in the GCR (Badhwar, 1997
). The energy deposition pattern for charged particle radiation within a few nanometers around the ion track plays an important role in determining the biological effectiveness of the radiation (Ottolenghi et al., 1997; Durante et al., 1998). Biodosimetry, especially with the utilization of fluorescence in situ hybridization (FISH) for whole chromosomal painting to measure chromosomal aberrations in circulating T lymphocytes, has proved to be particularly useful in assessing radiation exposure (Edwards, 1997; Lucas, 1997; Tucker et al., 1997). Measurements of chromosomal aberrations in lymphocytes from blood samples obtained from Mir-18 crew members showed significant elevations in aberrations post-flight, compared with pre-flight measurements (Yang et al., 1997). However, because of the complex space radiation exposure environment, the reliability of these cytogenetic techniques is clouded by issues regarding the type of aberrations scored, radiation quality, non-uniformity of dose throughout the body and dose rate considerations (Straume and Bender, 1997; Testard and Sabatier, 1999). Chromosome aberrations also provide indirect information on the genotoxic effect of particle radiation in vivo and are not direct measurements of induced gene mutations. Published studies on charged particle radiation-induced mutation studies show dose-related increases in mutation frequencies (MF) in cells in culture (Kronenberg and Little, 1989; Tsuboi et al., 1992; Kronenberg et al., 1995; Belli et al., 1998). The MF at the hypoxanthine phosphoribosyltransferase (hprt) locus in T cells from five Russian cosmonauts measured 1–2 years after a space flight showed that the level of hprt MF remained 2.4- to 5-fold higher than that found in non-exposed individuals (Khaidakov et al., 1997). Despite the small sample sizes in this study, these results nevertheless demonstrate that very low doses of charged particle radiation received during space flight produced measurable and lasting genetic changes.
Assessment of radiation-induced damage to the CNS is important in determining long-term risks on extended space missions. Rats exposed to HZE radiation have been shown to demonstrate alterations in their ability to regulate their body temperature (Kandasamy et al., 1994). In addition, these animals have behavioral deficits reflecting accelerated brain aging (Joseph et al., 1992, 1993, 1994, 1999; Villalobos-Molina et al., 1994). Transgenic animals that carry selectable and recoverable target genes in every cell in the animal are the only systems currently available for quantitating and characterizing mutations in every tissue in response to genotoxic agents (Goldsworthy et al., 1994; Gossen et al., 1994; Mirsalis et al., 1994). Two of the most widely used models, the Big Blue mouse (Kohler et al., 1991) and the MutaMouse (Myhr, 1991), use a 
shuttle vector that carries a lacI or lacZ target gene. These shuttle vector transgenic systems are efficient models especially suited for detecting point mutations and small deletions and have been shown to provide mutation information comparable with that obtained from an endogenous Dlb-1 locus (Tao et al., 1993). However, mutations recoverable in the Big Blue and MutaMouse systems are restricted by the packaging machinery and large deletions arising from exposure to clastogenic agents [such as high linear energy transfer (LET) particle radiation] are difficult to detect in these systems. For our current studies of HZE-induced genetic damage we have chosen to use the plasmid-based transgenic mouse model system developed by Vijg and co-workers (Gossen et al., 1993; Boerrigter et al., 1995). Multiple copies of plasmid pUR288 containing the silent and inert lacZ reporter gene are integrated in tandem into every cell of the genome in C57Bl/6-TgN(LacZpl)60 mice. Recoverable deletions in this plasmid-based system are not subject to the fragment size constraints found in the -based systems. A study reporting the nature of background mutations in these transgenic mice showed that the majority of recovered mutants were mouse derived, with a constant but small subset of mutants (1.3x10–5) derived from the recovery process (Dolle et al., 1999). Spontaneous MF in eight tissues (liver, spleen, bone marrow, skin, stomach, kidney, lung and bladder) ranged between 2.5 and 7.0x10–5 in the lacI transgenic Big Blue mouse (de Boer et al., 1998), with G:C
A:T transitions being the predominant mutations for all the tissues examined. The background MF in the liver, lung and spleen of plasmid-based C57lacZ transgenic animals were higher, ranging between 4.2x10–5 and 9.8x10–5 (Gossen et al., 1995; Dolle et al., 1996). These differences are explained by the fact that large deletions can be recovered in the lacZ plasmid-based system but not in the lacI model (Gossen et al., 1989, 1993; Kohler et al., 1991; Douglas et al., 1994; Winegar et al., 1994).
Evaluation of MF in liver, spleen and lung tissue from lacZ mice exposed to fractionated X-irradiation shows tissue-specific differences in both the maximum mutant fraction and the subsequent recovery from X-ray exposure. These differences may be due to differences in the rate of cell turnover in stem cell compartments in the tissues examined (Gossen et al., 1995).
One of the factors that may influence an individual's response to ionizing radiation is genetic background. The tumor suppressor p53 gene is known to influence radiosensitivity through its function in regulating cell cycle and apoptosis in response to DNA damage. p53 protein has been found to accumulate in some tissues as a consequence of low dose X-irradiation (Wang et al., 1996a,b) and exposure to the space environment (Ohnishi et al., 1996). In apoptosis, p53 is known to play a major role in the complex signaling pathway leading to programmed cell death in several cell types (Lowe et al., 1993a,b). Wild-type p53 expression is induced by radiation and is associated with G1/S cell cycle arrest in cultured cells (Kuerbitz et al., 1992). p53 has also been linked to the radiation-induced G2/M transition, extending the role of p53 in radiation responsiveness in cells (Pellegata et al., 1996). Transgenic animals in which the p53 gene function is knocked out by insertion of a non-functional neo gene fragment (Donehower et al., 1992) have proved to be a useful model to study the effects of p53 genetic background on tumor progression (Donehower et al., 1995; Donehower, 1996) and responses to genotoxic agents. In adult mice, radiosensitivity to tumorigenesis is dependent on p53 status (Kemp et al., 1994). We are interested in the impact of variable p53 genetic background on individual susceptibility to heavy particle radiation-induced mutations. Cells with wild-type p53 are steered either toward repair of damage or, in the case of irreparable damage, towards apoptosis. However, p53-deficient cells may have a reduced repair capacity and do not undergo apoptosis after irreparable damage. Thus, in response to radiation-induced damage p53-deficient cells can be expected to be at increased risk of mutagenesis, resulting in adverse long-term consequences after exposure to radiation, although the risk may be dependant on the specific tissue examined.
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Materials and methods |
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Animal husbandry and handling
Mice were housed and cared for in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. All animal handling procedures were approved by the SRI International Institutional Animal Care and Use Committee, following the guidelines provided by the 1970 Animal Welfare Act and its amendments (P.L. 89-544 and P.L. 91-579) and the principles promulgated by the National Research Council in its Guide for the Care and Use of Laboratory Animals (1996). Mice were housed five per cage in polycarbonate, solid bottomed, suspended drawer-type cages containing Sani-Chip hardwood bedding (P.J. Murphy Forest Products, Montville, NJ). Purina Mills Diet 5001 and deionized, UV-exposed water were available to the mice ad libitum. The light cycle was 12 h light/12 h dark.
The C57Bl/6-TgN(LacZpl)60 (p53+/+) transgenic mice (Gossen et al., 1992, 1993; Boerrigter et al., 1995) were cross-bred with p53 knockouts (Jacks et al., 1994) to obtain the p53/lacZ hemizygote (p53+/–) and p53/lacZ nullizygote (p53–/–) double transgenics. The hybrids were bred to produce further generations of hybrid mice for experimental purposes. p53 genotype of each experimental animal was confirmed using PCR amplification of the p53 gene as previously reported (Chang et al., 2000). Six- to twelve-week-old mice were used for these studies. Animals were shipped from SRI International (SRI, Menlo Park, CA) to Brookhaven National Laboratory and were quarantined and acclimated for 1 week before irradiation. Three to four animals were used for each treatment condition. All animals, including the unirradiated controls, were transported via overnight delivery and returned to SRI within 24 h post-treatment.
Irradiation
Individual mice were placed in specialized holders and whole body irradiated with 1 Gy of 1 GeV/a.m.u. iron (56Fe) particle radiation at the Alternating Gradient Synchrotron at Brookhaven National Laboratory. A conventional dosimetry system consisting of three parallel plate ionization chambers upstream of the sample holder was used. Detailed dosimetry measurements and characterization of this beam have been published (Zeitlin et al., 1998). The uniformity of the 7 cm beam spot was confirmed by densitometric analysis of an exposed X-ray film. The average dose LET at the sample position was 146 keV/µm. Dose rates ranged from 0.7 to 1 Gy/min. The total time each animal was confined in the holders was <5 min.
lacZ rescue and mutation frequency measurements
Animals were necropsied at various times post-iron particle radiation. Selected tissues were harvested, snap frozen in liquid nitrogen and stored at –80°C. Frozen tissue samples were blind coded and sent to Leven Inc. (Bogart, GA) for processing. The mutant rescue procedure has been described in detail (Gossen et al., 1989, 1992, 1993; Gossen and Vijg, 1993; Boerrigter et al., 1995). Briefly, the lacZ transgene cassette was released from total mouse genomic DNA by HindIII (20 U/µl) digestion for 1 h at 37°C. The transgenes were preferentially selected from genomic DNA by binding to magnetic beads precoated with anti-ß-galactosidase antibody and the lacZ/lacI fusion protein. Bound lacZ DNA was eluted from beads with 5 µl of isopropyl-ß-D-thiogalactoside (IPTG). The rescued linear plasmids were circularized with 0.1 U T4 ligase and precipitated in sodium acetate and ethanol at –80°C, washed with 70% ethanol and resuspended in 5 µl of sterile water. Plasmid DNA was electroporated into 60 µl of electrocompetent Escherichia coli C and incubated in a shaker/incubator for 30 min at 37°C for the positive selection of mutants. Aliquots of 2 µl of the recovered cells were plated in rescue plating medium [25 µg/ml kanamycin, 75 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal), 75 µg/ml ampicillin and a small amount of 2,3,5-triphenyl-2H-tetrazolium chloride] and the rest of the 2 ml culture was plated in selective medium containing 0.3% phenyl-ß-D-galactoside (p-gal) and incubated overnight at 37°C. MF was calculated by dividing the number of red mutant colonies (on selective p-gal plates) per ml by the number of blue rescue colonies (on non-selective X-gal plates) per ml, corrected for the dilution factor (x1000).
Mutant characterization using restriction fragment length polymorphism (RFLP)
The spectrum of mutations of 58 lacZ mutants obtained from unirradiated control animals and 103 mutants harvested from irradiated animals were analyzed by RFLP. Harvested bacterial colonies containing mutant plasmids were grown in LB medium in the presence of ampicillin and kanamycin to maintain the selective pressure on the mutants. Total plasmid DNA was harvested using the Qiagen plasmid DNA extraction kit (Valencia, CA). The DNA was quantitated and 1 µg from each mutant was digested in two separate reactions: (i) 2 U of PstI and 1.5 U of SacI in 25 µl of reaction mixture; (ii) 1 U of RsaI in 25 µl of reaction mixture. PstI and SacI are both single cutters and concomitant digestion using both of these enzymes is expected to produce two restriction fragments, of 3.5 and 1.9 kb in plasmid pUR288 (Figure 1A). RsaI is a multi-cutter and is expected to produce seven fragments of 1.72, 0.85, 0.7/0.69, 0.59, 0.48 and 0.32 kb (Figure 1B). The restriction patterns obtained from both of these reactions were used to evaluate the alterations in size of plasmid pUR288 as a result of exposure to radiation. Parallel restriction digestions were done using DNA extracted from control plasmid pUR288 (a gift from Dr M.Boerrigter, Leven Inc.) containing the intact lacZ transgene, to confirm the sizes of the restriction fragments. Mutants were classified according to the size of deletions. Because of the limitations of resolving small (<50 bp) differences in DNA fragments, mutants with point mutations and small deletions were pooled into the same category and designated as `no change' when the PstI/SacI and RsaI restriction patterns appeared to be the same as those of plasmid pUR288. Mutant clones derived from the same animal that showed the same restriction patterns were considered clonally expanded colonies and were not counted as separate entities.
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Mouse genomic DNA
Mutant clones were hybridized to a mouse genomic DNA probe to detect the presence of flanking mouse sequences in the plasmid cassette. Total mouse genomic DNA (Clontech, Palo Alto, CA) was labeled with non-radioactive digoxigenin using the DIG-High prime DNA labeling kit (Roche Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. Briefly, 1 µg of mouse template DNA was denatured for 10 min at 95°C and incubated overnight at 37°C in the presence of DIG-High prime mix. The labeling reaction was terminated by inactivation at 65°C for 10 min. The yield of labeled probe was quantified by DIG quantification using control test strips provided by the manufacturer. Control plasmid pUR288 DNA was similarly DIG-labeled.
DNA from selected clones was dot blotted onto Hybond nylon filters (Amersham, Piscataway, NJ) and hybridized overnight in the presence of mouse probe (20 ng/ml of hybridization mix). These membranes were washed twice with 2x SSC, 0.1% SDS at room temperature, followed by two high stringency washes of 0.1x SSC, 0.1% SDS at 68°C. For chemiluminescence detection of hybridization signals the membranes were rinsed briefly with washing buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5), blocked for 30 min at room temperature with 1x blocking reagent and incubated for 30 min in 20 ml of anti-DIG–AP conjugate diluted to 75 mU/ml. Finally, the membranes were rinsed thoroughly with washing buffer and incubated for 30 min with CSPD for signal detection. Positive controls for mouse DNA and pUR288 DNA were included in each membrane. For membranes probed with mouse DNA, the mouse DNA spot showed a positive signal while the plasmid pUR288 DNA spot showed a negative signal. For membranes probed with the plasmid probe, the plasmid DNA spot showed a positive signal while the mouse genomic DNA spot showed a negative signal. The dot blot technique provided an efficient method to screen potential candidate mutants containing flanking mouse sequences. In order to confirm these results and to provide information on the size of the insertion, mouse positive clones were also Southern analyzed with the mouse probe and an example of a Southern blot is shown in Figure 2.
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Statistical analysis of data
A minimum of 300 000 transformants was counted for each sample. Standard deviations were calculated using the animal as the experimental unit.
2 comparisons were made between irradiated groups and their unirradiated controls for each genotype. The level of significance is guided by the degree of freedom in each data set and the critical value of the 2 distribution tables and are noted as footnotes in Tables I–III
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To test if the spectra of categories of mutations were dependent on differences in genotype or treatment conditions,
2 statistics was used to analyze the data in contingency tables containing pair-wise comparisons between the different experimental groups with their respective controls (Zar, 1974).
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Results |
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Animals with different p53 genetic status were exposed to an acute 1 Gy dose of iron particle radiation and brain tissues were harvested at various times ranging from 1 week up to 16 weeks post-irradiation. Tissues from unirradiated animals were harvested at the same time points to measure the spontaneous lacZ MF in the three different genotypes (Tables I–III
). The differences in spontaneous lacZ MF in the three strains were not significant with Z values (deviations from sample means of MF with pair-wise comparisons) of <1. In addition, the average spontaneous MF of 2.46 ± 1.34x10–5 in the C57lacZ (p53+/+) mice is consistent with the published lacZ MF in the brain of these transgenic mice (Dolle et al., 1999).
lacZ MF in the brain of the p53+/+ animals increased with increasing time (weeks) after whole body exposure to iron particle radiation, up to a maximum of 2.4-fold above background at 8 weeks post-irradiation (Figure 3A). We measured a small (~1.3-fold above background) increase in lacZ MF in the p53 hemizygotes at 1–8 weeks post-irradiation, but this slight elevation returned to the control level by 16 weeks post-irradiation (Figure 3B). Statistical analysis suggested that radiation-induced MF levels in these animals may not be significantly different from that of the controls. In the p53–/– animals lacZ MF increased to ~2.1-fold above control levels early (1 week) after radiation exposure but quickly returned to control levels at 4–8 weeks post-irradiation (Figure 3C). Only one p53 nullizygous animal survived at 16 weeks post-irradiation due to the propensity for spontaneous tumor development and subsequently a shortened lifespan in these animals (Donehower et al., 1992). Although no animal-to-animal comparisons can be made with this single measurement, nevertheless, the trend of the data suggests that the level of MF in the brain at 16 weeks post-irradiation remained at the control level.
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The spectrum of spontaneous mutants appeared to depend on the p53 status of the genome (Table IV
). Point or very small deletions (`no change' mutants) appeared to be the predominant lesion found in all three genotypes. The number of mutants in each category was small and comparisons using the more stringent pair-wise 2 analysis method showed that there were no significant differences between the three control spectra. These results suggest that p53 genotype did not influence the spectrum of lacZ mutants as distinguished by RFLP. It was also interesting that no spontaneous mutants in the p53+/+ and p53+/– animals showed mouse positive sequences, while one clone out of 18 tested in the p53–/– mutants showed the presence of mouse DNA.
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Analysis of the spectrum of mutants from irradiated animals showed that `no change' mutants continue to be the predominant lesion after radiation exposure. Pair-wise comparisons of control and irradiated samples for the p53+/+ and p53+/– animals showed no significant effect due to iron particle radiation. In contrast, radiation treatment altered the spectrum of mutants in the irradiated p53–/– animals at the
= 0.1 level. The percentage of `no change' mutants appeared to remain constant after irradiation in the p53–/– mutants, but more <1 kb mutants were harvested and mutants with >3 kb deletions were absent. Comparison of the mutant spectrum of irradiated p53+/+ with that of p53–/– animals also showed differences at the = 0.1 level, suggesting that processing of radiation damage in the brain may be dependent on the p53 status of the animal. Although no mouse positive clones were found in the spontaneous mutants, a few mouse positive clones were found in the samples from irradiated animals, showing that iron radiation resulted in rearrangements in the genome not otherwise present spontaneously. |
Discussion |
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Mutagenic changes that result from exposure to heavy particle radiation in outer space may affect the function of the brain and are an important consideration in the assessment of risk in long-term space flights. The plasmid-based lacZ transgenic mouse mutation model system provides a method to measure mutation in any tissues of the animal without requiring the preferential selection of clonogenic populations of cells. The lacZ transgene is neutral and alterations in MF suggest dynamic changes in the genome long after the initial dose of iron radiation. In the present plasmid-based lacZ model multiple 5.3 kb plasmid cassettes are integrated in tandem into the genome. Small deletions involving the origin of replication and ampicillin resistance sequences or large deletions involving multiple plasmid cassettes are not recovered. In addition, intercopy deletions involving several cassettes within the tandem arrays could not be distinguished from those resulting from intracopy deletions. During recovery recombination of residual cassettes may result in a single mutant plasmid showing small changes in RFLP patterns. The incidences of these events are expected to be very low. However, sequence analysis of mutants with small changes will reveal more information about the nature and characteristics of the mutants. Mutation analyses using endogenous selectable markers have shown that some initial HZE particle-induced deletions can range in size from 3 to 100 Mb. Although this system may not be sensitive enough to detect these initial megabase deletions after iron ion exposure, residual deletions and rearrangements long after the initial radiation insult are within the sensitivity range of this system. We propose that the gradual increase in MF in the p53+/+ transgenics from 1 to 8 weeks after radiation exposure reflects the temporal accumulation of residual recoverable mutants in the brain and measures late tissue responses.
Our results suggest that a single dose of iron particles produced a measurable change in the lacZ MF in the brain tissue of p53+/+ animals. Ono et al. (1999) reported that the MutaMouse exposed to a high 200 Gy dose of X-rays showed a 4.8-fold increase in MF in the brain, compared with the unirradiated controls, with deletions being the predominant type of mutations in the irradiated tissue. We observed a much lower induction in lacZ MF after iron particle radiation. One possible interpretation is that an acute exposure of 1 Gy iron particle radiation is not optimal for maximal induction of lacZ mutation. Animals exposed to different doses of iron particle radiation will provide additional information on the dose dependence of lacZ MF. Another possibility is that iron particles are not very mutagenic, in that most cells hit are so severely damaged that they do not survive long enough for mutation measurement at 1 week post-irradiation. Additional dose–effect studies with beams of different LET, such as X-rays and protons, will allow us to compare the relative mutagenic effectiveness of the iron beam in this transgenic mouse mutation system.
Of the mutants harvested from brain tissue of control and irradiated animals, an insignificant number were derived from clonal expansion of damaged cells. This substantiates our expectation that the cellular turnover in the brain is low. A predominant percentage of mutations in the brain appear to be `no change' mutants in both the control and irradiated animals, suggesting that very small deletions arise spontaneously in these animals. p53 genetic background does not appear to affect the spectrum of spontaneous mutants. Among the mutants analyzed we noted an increase in `no-change' mutants harvested from irradiated p53+/+ animals and an increase in the percentage of the larger deletions mutants (sum of categories <1, 1–3 and >3 kb) from the irradiated p53+/– and p53–/– animals. However, statistical analysis showed that the radiation-induced changes in mutation spectrum may only be significant in the case of the p53–/– animals. This could be due to the small number of samples in each category. Mouse genomic DNA probes were used to test for the presence of mouse genomic DNA in the plasmid mutants. Since no mutants harvested in the p53+/+ and p53 +/– genotypes showed mouse DNA, the presence of several mutants in the treated samples suggests that radiation contributed to an increase in the incidences of recombination/rearrangements in the genomes of these animals.
The number of functional copies of p53 appeared to affect both the MF and the characteristics of the spectrum of mutants. In the wild-type p53 animals iron radiation induced a temporal increase in lacZ MF but not a significant change in the spectrum of mutants. In the p53+/– lacZ animals radiation induced only a slight increase in MF. The lack of one functional copy of p53 did not appear to significantly alter the spectrum of recovered mutants. Brain tissue from the p53–/– animals, with no functional copies of p53, showed an initial transient but significant increase in MF after irradiation and a shift in the spectrum of mutants, from mutants with 1 to >3 kb deletions to those with deletions <3 kb. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported due to technical limitations in most other mutation assays.
In the p53+/+ animals, brain cells that are heavily damaged by iron particle radiation may be lost through p53-dependent apoptotic mechanisms. Although the observed increases in MF were small compared with those reported in published X-ray studies of other tissues in these transgenic animals (Gossen et al., 1995), molecular analysis of the recovered mutants in our study revealed changes in the proportion and size of deletions, with some alterations extending beyond the transgene locus and into the flanking mouse sequence, in the irradiated compared with unirradiated animals. These genetic alterations are important consequences to consider in assessment of late tissue damage. Although p53 signal transduction has been shown to be important for a myriad of functions vital for damage surveillance in normal cells, several p53-independent mechanisms have been proposed to protect the genome in the absence of p53 in neural tissues (Wood and Youle, 1995; Haas-Kogan et al., 1996; Johnson et al., 1998; Frenkel et al., 1999). In particular, mechanisms involving ceramide-induced Fas signaling (Yount et al., 1999) have been suggested. Our results suggest that these mechanisms may also play an important role in the accumulation as well as the determination of characteristics of lacZ mutations in response to HZE radiation exposure.
In vivo systems are far more complex than in vitro assays in that many cell types are involved and our current measurements using total genomic DNA from whole brain do not provide information on contributions from individual tissue types. For some of the samples in this study we measured the MF in the cortical and non-cortical sections of the brain and found no significant difference between the sections. However, analysis of samples derived from further microdissection of specific regions in the brain may provide important information relating tissue damage to known functional roles in the brain.
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Acknowledgments |
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We would like to extend our thanks to the AGS crew and especially Drs John Gately and Marcello Vasques for their assistance with radiation exposures, to Ms Maryann Kershaw for assistance in the animal facilities at BNL and the Mammalian Toxicology group at SRI for technical assistance with the experiments. We would also like to acknowledge Dr Michael Boerrigter for his assistance in the selection of lacZ mutants. This work was supported by NASA grant, NAG.5-6177.
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Notes |
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1 To whom correspondence should be addressed. Email: pchang{at}sri.com
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References |
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Received on March 2, 2000; accepted on July 17, 2000.
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