Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice

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Fundamental and Molecular Mechanisms of Mutagenesis

Mutation Research 307 (1994) 479-487

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice Richard A. Winegar a , , Louise H. Lutze b Janice D. Hamer Kathleen G. O'Loughlin a, Jon C. Mirsalis a

a

a Toxicology Laboratory, SRI International, 333 RavenswoodAvenue, Menlo Park, CA 94025-3493, USA b Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143-0750, USA

(Received 29 June 1993; revision received 15 October 1993; accepted 19 October 1993)

Abstract

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis systems has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue T M lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei. C57B1/6 transgenic mice were irradiated with 137Cs y-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 y-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the alacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue TM l a d transgenic mouse system is sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene affects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57B1/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57B1/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage. Key words: LacI; Transgenic mice, lacI; Point mutations; Deletions; Micronuclei; Gene mutations; Ionizing radiation

* Corresponding author, Tel. 415 859 6457; Fax 415 859 2889. 0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0027-5107(93)E0232-F

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R.A. Winegar et al. / Mutation Research 307 (1994) 479-487

1. Introduction

2. Materials and methods

Transgenic mouse mutagenesis systems (Gossen et al., 1989; Kohler et al., 1991a) represent a major breakthrough in the field of genetic toxicology. Two of the major end points evaluated in traditional genetic toxicology studies are gene mutations and chromosome alterations. Although transgenic mice were designed specifically for the detection of gene mutations, ideally, they would be integrated into standard assays for cytogenetic damage such as the micronucleus assay. A number of factors could affect the sensitivity of a particular system to various types of genetic damage. The target gene itself may affect the sensitivity of the system by its size, chromosomal location, base sequence, and spectrum of sequence alterations that result in a detectable altered phenotype. We have been using the Big Blue T M lacI transgenic mouse to evaluate mutations and chromosome damage produced by ionizing radiation. This transgenic mouse contains a lambda shuttle vector (lambda LIZ) that carries a lacI target and an alacZ reporter gene integrated as a tandem repeat of 40 copies into mouse chromosome 4 (Kohler et al., 1991a). After treatment and expression, genomic DNA is isolated from mouse tissues and the shuttle vector is recovered by exposing the D N A to lambda phage in vitro packaging extracts. Mutations in the lacI target gene inactivate the repressor gene, allowing expression of the alacZ reporter gene. This gene complements the wlacZ in the bacterial host cell, resulting in blue mutant plaques when the cells are plated in the presence of the chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl-/3-Dgalactopyranoside). In this report we describe the response of the Big Blue T M transgenic mouse system to ionizing radiation. We analyzed the frequency and gross structure of mutations induced by y-rays and compared transgenic and parental mouse strains for spontaneous and induced micronucleus frequencies in the traditional mouse bone marrow micronucleus assay (Heddle et al., 1983; MacGregor et al., 1987).

Animals Mice were housed and cared for in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. The 1970 Animal Welfare Act and its amendments (P.L. 89-544 and P.L. 91-579) and the principles promulgated by the National Institutes of Health (NIH Publication No. 85-23) in its Guide for the Care and Use of Laboratory Animals were followed at all times. Male mice were obtained from Stratagene Cloning Systems (La Jolla, CA). The strains used were C57B1/6 (lambda LIZ: C57BI/6[LIZ]) or B6C3F1 (lambda LIZ: C57B1/6[LIZ] female X C3H male). Animals were approximately 6 weeks of age at the time of radiation exposure and were quarantined for at least 3 days before use. Mice were housed 5 per cage in polycarbonate, solidbottom, suspended drawer-type cages containing Sani-Chips hardwood bedding (P.J. Murphy Forest Products, Montville, N J), and were provided with deionized, UV-exposed water ad libitum. The light cycle was 12 h light : 12 h dark. Mice were euthanized by administration of 60 m g / k g sodium pentobarbital. For mutation analysis, spleens were removed, quick-frozen in liquid nitrogen, and stored at - 8 0 ° C until DNA isolation. Radiation exposures All irradiations were performed in a Mark I, Model 68-A self-shielded 137Cs irradiator (J.L. Shepherd, San Fernando, CA). The mice were placed unrestrained in a specially designed holder and centered in the field. Attenuators were placed in front of the source to attain the desired dose rate at the center of the field, as measured previously by thermoluminescent dosimeters taped onto the dorsal and ventral surfaces of mice. The mouse holder was on a platform that rotated at 6 rpm. For mutagenesis experiments, mice were divided into groups of two. Irradiations were performed at a dose rate of 11.5 c G y / m i n (0.1, 1 and 3 Gy total dose) or 395 c G y / m i n (7 and 14 Gy

R.A. Winegaret al. / Mutation Research 307 (1994) 479-487

total dose). Mice were sacrificed 2-14 days after irradiation. For micronucleus experiments mice were divided into two groups of five each. One group received 500 cGy of whole-body radiation. The second group served as an unirradiated control. 24 h later, all mice were sacrificed. The irradiation was at a dose rate of 227 cGy/min, and the irradiation field was uniform under these conditions. Isolation and packaging of DNA Genomic DNA was prepared from homogenized spleen tissue as described previously (Kohler et al., 1991). The lambda shuttle vector was recovered as viable phage by exposing the genomic DNA to lambda packaging extracts in vitro (Transpack; Stratagene). The phage particles were assayed for lacI mutations by infecting E. coli strain SCS-8 (Stratagene) (Kohler et al., 1991a), and screening for blue mutant plaques on 25-cm 2 NZY agar plates containing 70 mg X-gal per assay plate. The density of plaques per plate averaged 30 plaque forming units (PFU)/cm 2. Characterization of mutations 34 mutant plaques from irradiated tissues were selected for characterization. The plasmid containing the entire target gene region (including the lacI promoter and lacZ operator) was excised from the lambda phage as described by Short et al. (1988). Recovered plasmid DNA (designated pLIZ) was isolated by an alkaline lysis procedure and purified using MagicT M Miniprep columns (Promega). Gross structural changes were analyzed by digestion of mutant plasmid DNA with Rsa I and Hpa I. Products of the double digest were electrophoresed on a 1.2% agarose gel and visualized by ethidium bromide staining. The junction regions of mutants containing large rearrangements were sequenced by the dideoxy chain termination method (Sanger et al., 1977) with use of a Sequenase ® DNA sequencing kit (USB, Cleveland, OH) and primers specifically synthesized to anneal throughout the target region. Micronucleus studies Approximately 24 h after irradiation, mice were anesthetized with sodium pentobarbital (ap-

481

proximately 60 mg/kg), and sacrificed by cervical dislocation, and bone-marrow smears were prepared from one femur of each animal as described previously (Schmid, 1976). Briefly, cells were flushed from the femur into approximately 0.5 ml of fetal bovine serum in a 1.5-ml conical polycarbonate tube. Cells were concentrated by centrifugation, spread on ethanol-cleaned microscope slides, air-dried, and fixed for 5 min in absolute methanol. Before scoring, each slide was stained with acridine orange (Hayashi et al., 1983). Slides were evaluated by means of epifluorescence microscopy at a magnification of 630 × . The criteria for micronuclei were those described by Schmid (1976), with the additional requirement that they exhibit fluorescence characteristic of the stain used (i.e., bright yellow). The ratio of RNA-containing polychromatic erythrocytes (PCE) to total erythrocytes (RBC) was determined by scoring the number of RNA-positive cells among the first approximately 200 erythrocytes scored. The micronucleus frequency was determined by scoring approximately 2000 PCE per animal. Data were summarized with the use of an SRI-developed software package run on an IBM-PC. The micronucleus frequency data were analyzed by using the normal test for equality of binomial proportions (Kastenbaum and Bowman, 1970).

3. Results

Mutation studies Treatment of male B6C3F1 mice with 1 Gy or more of y-rays induced a significant increase in the frequency of lacI mutations (Table 1). At a dose of 1 Gy a longer expression period (14 days vs. 7 days) resulted in a doubling of the mutation frequency. In fact, the mutation frequency induced by 1 Gy with a 14-day expression period was higher than that induced by 3 or 14 Gy with shorter expression periods. Irradiation with 0.1 Gy resulted in approximately a doubling of the mutation frequency; however, this increase was not statistically significant.

R.A. Winegaret al. / Mutation Research 307 (1994) 479-487

482

Table 1 Induction of lad mutants by y-irradiation in spleen

approx. 1 kb I I

Dose Harvest Total lad Mutant Deletions/ (Gy) time plaques ~ mutants frequency mutants (days) (× 10-5) analyzed 0 0.1 1 3 14

7 14 7 14 7 2

297438 113391 203191 250852 218740 475665 335500

8 6 11 29 50 74 52

2.68 5.29 5.41 11.56 22.90 15.60 15.50

* * * *

N.D. b N.D. N.D. N.D. 1/9 0/10 3/15

"

R

R

R

H

H

I

I

I

I

I

I[ " 11

-_

14A-49 141]-1 t 41]-2

1A-14-1fi

a Plaque data are pooled from two animals. b Not determined. * Significantly greater than unirradiated control (p < 0.0001) by test of binomial proportions.

Characterization o f mutations A t o t a l o f 34 r a d i a t i o n - i n d u c e d m u t a n t s w e r e s e l e c t e d f r o m a m o n g 3 t r e a t m e n t g r o u p s for c h a r acterization. Digestion of wild-type pLIZ plasmid D N A w i t h R s a I a n d Hpa I p r o d u c e d 5 d i s t i n c t f r a g m e n t s r a n g i n g in size f r o m 359 to 1440 b p (Fig. 1). By s e p a r a t i n g t h e s e f r a g m e n t s o n a n a g a r o s e gel, it w a s p o s s i b l e to d e t e c t l a r g e - s c a l e a l t e r a t i o n s in p l a s m i d D N A e x c i s e d f r o m t h e mutant plaques. 4 of the mutants isolated from

Fig. 1. Linear map of pLIZ showing extent of four y-ray-induced deletions (heavy lines) as determined by restriction enzyme analysis. (R) Rsa I sites; (H) Hpa I sites.

spleens of irradiated animals had altered band p a t t e r n s ( T a b l e 1), w h i c h i n d i c a t e d t h e p r e s e n c e o f d e l e t i o n s r a n g i n g in size f r o m 50 to 500 b p (Fig. 1). S e q u e n c e analysis o f t h e r e a r r a n g e m e n t m u t a t i o n s is s h o w n in Fig. 2. T h e j u n c t i o n s for 3 o f t h e d e l e t i o n s ( 1 4 A - 4 9 , 1 4 B - l , 14B-2) h a d s h o r t regions of sequence homology. The fourth rearr a n g e m e n t ( 1 A - 1 4 - 1 5 ) h a d a d e l e t i o n o f 508 b p extending from within the alacZ region of the plasmid into the region immediately downstream o f alacZ. T h e j u n c t i o n h a d 43 b p o f s e q u e n c e homology.

Table 2 Induction of micronuclei by y-irradiation in transgenic or nontransgenic B6C3F1 or C57B1/6 mice Treatment

f'

Dose (Gy)

n

PCE/RBC (%) Mean _+S.E.

PCE with MN (%) Mean + S.E.

B6C3FI Non-transgenic Transgenic

0.0 0.0

5 5

56.71 + 6.08 48.88 _+3.24

0.26 + 0.01 0.20 +_ 0.05

Non-transgenic Transgenic

5.0 5.0

5 5

36.07 + 3.00 25.99 _+ 1.26

1.59 _+0.15 * 2.85 + 0.46 * +

C57Bl/6 Non-transgenic Hemizygous transgenic Homozygous transgenic

0.0 0.0 0.0

6 4 4

51.20 _+4.65 47.06 _+4.54 40.55 _+ 9.78

0.34 _+0.05 0.32 + 0.07 0.38 _+0.05

Non-transgenic Hemizygous transgenic Homozygous transgenic

5.0 5.0 5.0

4 5 5

35.25 _+5.30 34.31 _+ 2.80 34.04 + 3.62

2.45 + 0.50 * 1.25 ++0.05 * 1.65 _+0.20 *

* Significantly greater than unirradiated control (p < 0.01) by test for binomial proportions. + Significantly greater than irradiated nontransgenic (p < 0.01) by test for binomial proportions. PCE, polychromatic erythrocytes; RBC, total erythrocytes.

R.4. Winegar et aL / Mutation Research 307 (1994) 479-487

Micronucleus studies Treatment of male B6C3F1 or C57B1/6 mice with 5 Gy of y-radiation produced modest decreases in the P C E / R B C ratio, indicating that this dose of radiation produces mild suppression of erythropoiesis (Table 2). Radiation produced highly significant increases in the frequency of micronuclei in PCE. The increase in micronucleus frequency was significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; however, the converse was true in C57B1/6 mice: the increase in micronuclei was significantly higher in nontransgenic mice than in mice hemizygous or homozygous for lacI. There was no difference in the spontaneous (unirradiated) micronucleus frequencies between nontransgenic and transgenic mice of either strain. The C57B1/6 mice consistently had a higher spontaneous micronucleus frequency than B6C3F1 mice, regardless of the presence or absence of the lacI gene.

14 A - 4 9 :

17 177 C A A T T C A ......... 160bp ........ A C C G C G T

CAATTC A CCGCGT

14B-

1: 7O5

T C A A A T T C A .......... 4Sbp.......... T T C A A C A AA

TCAAATT

CA ACAAA

14 B - 2: 547

T G G A G i~;~

7~

.......... 249 bp........... C ~ , T

TGGAG

TAC C G

CA~TACCG

1A-14-15: 140g

1366

1874

c a c AC

1917 a ATc

c G cAC

AGArC

Fig. 2. D N A sequences at junction regions of deletions of four radiation-induced mutations. The shadowed bases are limited sequence homologies present at the junction. The positions of the junction regions in the parental sequences are shown above the sequences.

483

4. D i s c u s s i o n

We used the Big Blue T M lacI transgenic mouse mutagenesis assay (Kohler et al., 1991a) to evaluate the effects of radiation on chromosome damage and induction of mutations. For a mutation to be detected in this system, the sequence must be capable of (1) being packaged (it must contain two cos sites separated by 38-51 kb of DNA), (2) producing lytic phage particles (various lambda phage genes are required) and (3) producing blue plaques (it must have functional alacZ). Any mutation that does not meet any one of these requirements will not be detected. Thus, there are several constraints on the system's ability to detect deletions. Relatively small intragenic deletions (< 1 kb) are most amenable to detection. Larger intragenic (technically, intracopy) deletions are also detectable; however, the adjacent alacZ sequence must be functional for colorimetric detection. Very large intergenic deletions are detectable as long as one breakpoint is within the lacI and an intact alacZ gene is present (Fig. 3). With 40 copies of the ~ 45 kb lambda LIZ present as a tandem repeat this amounts to a potential target of approximately 1.8 megabases (Mb). Because of the relatively small size of the lacI gene and the constraints described above, it seemed likely that y-radiation-induced mutations, which are predominantly deletions, would not be readily detected in this system. We found, however, that treatment of mice with at least 1 Gy of y-irradiation produced a significant increase in mutations in the spleen. This effect increased dramatically with longer expression times. Similar time-dependent increases in mutation frequency have been observed after chemical mutagen treatments (Kohler et al., 1991b). Restriction enzyme analysis indicates that it is possible to detect relatively large rearrangements in the lacI gene. For Big BlueTM the spontaneous frequency of deletions > 50 bp has been approximately 1% (J. Short, personal communication); therefore, it is likely that the deletions reported in this study are actually radiation-induced. At the highest dose of exposure (14 Gy), 20% of recovered mutants contained deletions. With an in vitro shuttle vector system containing the en-

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R.A. Winegar et al. / Mutation Research 307 (1994) 479-487

tire lacZ gene, we have observed deletion frequencies of 17% (600 Gy X-rays) (Lutze and Winegar, 1990) and 67% (3 Gy radon) (Lutze et al., 1992). Thus, we have shown that it is possible to obtain similar results by using the lacI gene in vivo. The actual proportion of ionizing radiationinduced mutations that are deletions seems to depend on the radiation source, dose, target gene, and chromosomal location (see Hutchinson, 1993). The 4 rearrangement mutations were further characterized by DNA sequencing. Three of the rearrangements were deletions that ranged in size from 49 to 252 bp within the lacI gene. The fourth rearrangement (1A-14-15) had a 508 bp deletion that extended from within the alacZ gene into downstream sequences, with 43 bp of sequence homology at the breakpoint junction. Since the detection of mutations in this system requires the inactivation of the lacI gene and a functional odacZ gene, it would appear that there must be a point mutation in the lacI gene as well as the deletion in the alacZ. The downstream

J cos

region that the alacZ recombined with represents 43 bp of alacZ homology that are additional to the alacZ coding region and are presumably derived from a precursor plasmid used in constructing ,~LIZ (Kohler et al., 1991b). Apparently, this downstream region of alacZ homology recombined in frame with the 127 bp of alacZ remaining in the mutant to give 170 bp of alacZ sequence that were sufficient to provide a-complementation. It has been reported (Riither, 1980) that just 59 amino acids (corresponding to 177 bp) of /3-galactosidase are sufficient to provide a-complementation. The deletion size range that we detected in this limited sample is smaller than that which we have observed with the use of a shuttle vector containing the entire lacZ gene (Lutze and Winegar, 1990). This may be due to the constraints imposed by the size of the gene, the close proximity of the alacZ reporter gene to the lacI target gene, and the requirements for packaging. It is possible, however, that some deletions could actually be intergenic deletions. Sequence analysis

1

[

2

pr~

cos

pLr7

.... co5

40 plI~

I cos

a

b C

d e

Fig. 3. Map of lambda LIZ, which is present at approximately 40 head-to-tail concatemers ( ~ 45 kb each) integrated into chromosome 4 (map not drawn to scale). Each copy is bound at each end by a cos site, which allows excision and packaging into phage heads, pLIZ is excisable as a phagemid and contains the lacI target gene (light shading) and the alacZ target gene (dark shading). Shown is a classification of possible deletions (heavy lines). (a) intragenic deletions (approximately 0-8 kb). (b) Nondetectable intragenic deletions ( a l a c Z reporter gene deleted). (c) Nondetectable intergenic deletions. (d) Intergenic deletions that appear to be intragenic deletions; the size deletions will be 38-51 kb plus multiples of 45 kb. (e) Intragenic deletions that can be distinguished. Breakpoint in the upstream copy is 3' to the breakpoint in the downstream copy.

R.A. Bqnegar et al. / Mutation Research 307 (1994) 479-487

can identify only those intergenic deletions in which the breakpoint in the upstream copy of lacI occurs 5' relative to the breakpoint in the downstream copy of lacI (Fig. 3). This should occur in approximately 50% of intergenic deletions. None of the 4 deletions could be classified unambiguously as intergenic. Although most of the radiation-induced mutations were classified by restriction enzyme analysis as point mutations, many of them may actually be deletions smaller than that which can be resolved by gel electrophoresis (Grosovsky et al., 1988). For example, the point mutation 14A-5 actually contains a 7-bp deletion. A striking characteristic of the large deletions observed in these studies is the presence of short regions of homology at the breakpoint junctions. In previous studies we have reported that short regions of homology play a role in deletion formation by radon (Lutze et al., 1992) and 3'-rays (Winegar et al., 1993) in vitro. The present results indicate that short regions of homology play a role in the rejoining of DNA breaks in vivo as well. It is possible that DNA double-strand breaks are subject to exonucleolytic activity that exposes regions of various lengths. Pairing of exposed single strands could then occur at regions of limited homology, which would facilitate rejoining (Lutze et al., 1993). As well as inducing gene mutations, ionizing radiation induces cytogenetic damage. The cytogenetic consequence of integrating 1.8 Mb of repetitive foreign DNA is unclear. Previous reports have indicated that tandem repeats can produce chromosome instability, fragile sites and other effects. Studies in Chinese hamster cells indicate that chromosomes containing amplified sequences undergo frequent rearrangement (Ottaggio et al., 1988) and these sequences are preferentially lost spontaneously (Miele et al., 1989). Spontaneous and induced micronucleus frequencies have been found to be higher in cells containing amplified CAD (carbamylphosphate synthetase, aspartate transcarbamoylase, dihydroorotase) sequences than in parental cell lines. This increase is found because the marker chromosome is a target for both clastogenesis and aneugenesis (Ottaggio et al., 1993). Amplification

485

of the CAD gene is due to selective pressure provided by a drug. Since the 40 integrated lambda LIZ sequences are unlikely to provide any selective advantage to a cell, there is no pressure for amplification to occur. The mere presence of repetitive copies of sequence, does not appear to increase instability. Heartlein et al. (1988) found that amplified DHFR (dihydrofolate reductase) DNA with or without a poly(dG-T) insert caused no increase in instability. In contrast, ceils with copies of amplified DHFR containing 0.34 kb human alphoid DNA showed marked chromosomal instability. Thus any chromosomal instability may be sequence dependent. The micronucleus assay permits the assessment of spontaneous and induced cytogenetic aberrations in mice with or without the integrated lambda LIZ. Suzuki et al. (1993) have reported that spontaneous micronucleus frequencies in the MutaXMMouse (lacZ) are slightly elevated compared with those in historical controls and that mitomycin C-induced micronucleus frequencies are similar to those in historical controls. In the present study we found that the presence of 40 or 80 copies of the lacI transgene, in hemizygous and homozygous mice, respectively, did not affect the spontaneous incidence of micronuclei (Table 2). The induction of micronuclei by 3' irradiation does appear to be affected by the presence of the transgene. Male B6C3F1 mice that were hemizygous for 40 copies of lacI had nearly twice the incidence of micronuclei as did nontransgenic litter mates (2.85% vs. 1.59%) when exposed to 5 Gy of radiation. This effect was consistent in all irradiated mice, and the difference was highly significant. This would suggest that the presence of the tandem repeats of lacI may produce an unstable region of chromosome 4 that is more susceptible to radiation-induced damage. This result is contradicted, however, by results in C57B1/6 mice that demonstrated higher micronucleus frequencies in nontransgenic mice than in either hemizygous or homozygous litter mates. It is difficult to interpret these results. The contradictory findings between the two strains of mice suggest that the lacI transgene itself does not cause the increased radiosensitivity. One possibility is that there are differences in the rate of

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R.A. Winegar et al. / Mutation Research 307 (1994) 479-487

cell turnover, which could affect when the peak y-ray-induced micronucleus frequency is detected (Jagetia, 1990). This possibility could be examined by performing time-course studies. In addition, studies using in situ hybridization should be performed to determine if the transgene occurs in micronuclei at a frequency different from that expected by chance. In summary, y-irradiation produced significant chromosomal damage in bone marrow of lacl mice and a significant increase in mutations in spleen. These mutations were predominantly point mutations; however, approximately 14% were large-scale rearrangements. Given the 50% probability of detecting intergenic deletions, it seems possible that additional deletion mutations were produced that were not detected in this system. These results indicate that this system is amenable to studying radiation-induced mutations and chromosomal damage. The data from the micronucleus experiments suggest that the l a d transgene does not cause chromosome instability as measured by spontaneous micronucleus frequencies. However, the response of transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

Acknowledgement The authors express their appreciation to Dr. Jay Short for many helpful suggestions. Irradiation was performed by Dr. Virginia Langmuir and Ms. Holly Mendonca. This work was supported in part by Stratagene Cloning Systems (La Jolla, CA), National Institutes of Health grant R29 GM46563-01 (RAW), and contract DE-AC03-76SF01012 from the Office of Health and Environmental Research, U.S. Department of Energy (LHL).

References Gossen, J.A., W.J.F. de Leeuw, C.H.T. Tan, E.C. Zwarthoff, P.H.M. Lohman, F. Berends, D.L. Knook and J. Vijg

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