Mutation Research 616 (2007) 34–45

Effect of ATM heterozygosity on heritable DNA damagein mice following paternal F0 germline irradiation

Janet E. Baulch a,∗, Ming-Wen Li b, Otto G. Raabe b

a University of Maryland, Baltimore, Radiation Oncology Research Laboratory, BRB 7-002,655 West Baltimore Street, Baltimore, MD 21201, United States

b Center for Health and the Environment, University of California Davis, CA 95616, United States

Available online 11 December 2006

Abstract

The ataxia telangiectasia mutated (ATM) gene product maintains genome integrity and initiates cellular DNA repair pathwaysfollowing exposures to genotoxic agents. ATM also plays a significant role in meiotic recombination during spermatogenesis.Fertilization with sperm carrying damaged DNA could lead to adverse effects in offspring including developmental defects orincreased cancer susceptibility. Currently, there is little information regarding the effect of ATM heterozygosity on germline DNArepair and heritable effects of paternal germline-ionizing irradiation. We used neutral pH comet assays to evaluate spermatozoa 45days after acute whole-body irradiation of male mice (0.1 Gy, attenuated 137Cs � rays) to determine the effect of ATM heterozygosityon delayed DNA damage effects of Type A/B spermatogonial irradiation. Using the neutral pH sperm comet assay, significantirradiation-related differences were found in comet tail length, percent tail DNA and tail extent moment, but there were no observeddifferences in effect between wild-type and ATM +/− mice. However, evaluation of spermatozoa from third generation descendantsof irradiated male mice for heritable chromatin effects revealed significant differences in DNA electrophoretic mobility in the F3

descendants that were based upon the irradiated F0 sire’s genotype. In this study, radiation-induced chromatin alterations to TypeA/B spermatogonia, detected in mature sperm 45 days post-irradiation, led to chromatin effects in mature sperm three generations

later. The early cellular response to and repair of DNA damage is critical and appears to be affected by ATM zygosity. Our resultsindicate that there is potential for heritable genetic or epigenetic changes following Type A/B spermatogonial irradiation and thatATM heterozygosity increases this effect.© 2006 Elsevier B.V. All rights reserved.

Keywords: Radiation; Heritable effects; ATM; Sperm; Comet assay

1. Introduction

DNA damage evokes a range of acute cellular

responses that lead to delay of cell cycle progressionand initiation of DNA repair. DNA double strand breaks(DSBs) are generated by a variety of genotoxic agents

∗ Corresponding author. Tel.: +1 410 706 1572;fax: +1 410 706 6138.

E-mail address: jbaulch@som.umaryland.edu (J.E. Baulch).

0027-5107/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2006.11.020

including ionizing radiation. The gene product of ataxiatelangiectasia mutated (ATM) appears to play a crucialrole in surveillance of the genome and rapid detection ofDSBs [1]. Following the induction of DSB, ATM proteinkinase mediates the rapid induction of numerous cellu-lar responses that lead to damage repair, activation ofcell cycle checkpoints and other survival pathways [2,3].

Mis-sense or truncation mutations to the ATM gene inac-tivate ATM and result in the ataxia telangiectasia (AT)phenotype [4]. The AT phenotype manifests itself as asevere genomic instability syndrome in AT patients, con-

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erring cancer predisposition, radiation sensitivity andmpaired activation of pathways that mediate the cellularesponses to DNA DSBs [5]. ATM has been of additionalnterest to cancer epidemiologist and researchers as aesult of observations that clinically asymptomatic mem-ers of AT families, AT carriers, seem to have increasedadiation sensitivity and a higher incidence of cancer1,4,6]. Since the estimated frequency of AT carriersheterozygotes) in the general population is 0.4–2%,his increased risk has important implications for humanealth [1,4].

DSBs are usually repaired by non-homologous endoining (NHEJ) or homologous recombination and repairHRR) pathways. ATM is essential for HRR, activatingroteins including the RAD50/MRE11/NBS1 complexnd BRCA1 and BRCA2 [7]. In the testis, ATM HRRs especially important because endogenous DSBs arenduced during the first meiotic prophase [8]. In theestis of ATM −/− mice, spermatogenesis arrests at

eiotic prophase I due to the lack of functional ATMnd the subsequent inability to successfully completeeiotic recombination. Following meiotic arrest, germ

ell apoptosis occurs [7,9]. As a result of this germine apoptosis, ATM −/− male mice are sterile. Inild-type mice, however, an ATM-mediated DNA dam-

ge response is demonstrated in spermatogonial stageshrough the first meiotic prophase [10–12]. While sper-

atogenesis also seems to proceed normally in ATMeterozygotes, there is little or no information regard-ng the impact of ATM heterozygosity on the germ lineesponse to DNA damage, radiation sensitivity or trans-itted effects of paternal germ line irradiation observed

n the offspring of the irradiated male.Previous work in our laboratory using mice has

ocused on the heritable effects transmitted by sper-atozoa to offspring through the paternal germline

ollowing irradiation of premeiotic Type A/B spermato-onia [13–17]. Our studies and the studies of othersuggest that exposure of these spermatogonia to evenelatively low doses of DNA damaging agents increasehe frequency of genomic instability and other heritableffects observed in the offspring of the irradiated male18–21]. Epigenetics are thought to play a role in someypes of genomic instability, and we have hypothesizedhat the mechanism underlying the heritable effects thate observe may have an epigenetic component [15–17].

rrespective of the mechanism, these heritable effectsay predispose offspring to genetic diseases and cancer

22–25].In vitro and in vivo studies have demonstrated dose-

elated induction of DNA strand breaks in spermatozoasing comet assays and moderate to high doses of ion-

earch 616 (2007) 34–45 35

izing radiation [26–29]. The lowest radiation dose usedin these sperm comet assays was 0.25 Gy from X-raysand only sperm from the acutely irradiated mice wereevaluated [27–29]. We now report results of cometexperiments evaluating ATM heterozygous (+/−) andwild-type 129SvEv male mice for radiation-inducedeffects on spermatozoal DNA 45 days after Type A/Bspermatogonial irradiation using a 0.1 Gy dose of attenu-ated � radiation. Further, we report results of experimentsevaluating sperm from third generation (F3) descendantsof these irradiated F0 sires for heritable effects on chro-matin as measured by DNA electrophoretic mobility.

2. Materials and methods

2.1. Mice

ATM mutant mice were originally generated using the129S6/SvEvTac strain (Taconic; Germantown, NY; 30), main-tained on this background and made commercially availablethrough Jackson Laboratory (Bar Harbor, ME). For thisreason, the 129S6/SvEv strain of mice is our wild-typecontrol. Single cohorts of 8–10-week-old ATM +/− andwild-type129S6/SvEv male mice were obtained from a sin-gle dedicated supply colony at Jackson Laboratory, rankedby body weight and randomly assigned to the irradiated (17mice/genotype) and the control (16 mice/genotype) groups.Since the dedicated supply colony was not sufficiently largeto provide the necessary numbers of female 129/S6SvEv micefor the study, all females were obtained from Taconic (13–14weeks old) and were also from single cohorts, ranked by bodyweight and randomly assigned to the irradiated and the controlgroups of F0 males.

Animal experimentation was carried out in accordance withthe principles of the American Association for Accreditationof Laboratory Animal Care (AAALAC) in fully accreditedfacilities.

2.2. Irradiation and dosimetry

The experimental males received an absorbed dose of0.1 Gy from acute whole-body attenuated 137Cs � irradiation.Mice were irradiated using a J.L. Shepherd & Associates(San Fernando, CA) Mark I Model 30 calibrated 137Cs � ray(0.662 MeV) irradiator at a dose rate of 0.017 Gy min−1 toobtain an absorbed dose of 0.1 Gy. Control mice were shamirradiated within the exposure chamber with the door closed butwithout � rays. The exposure for each group of mice was mea-sured using the average of three or more commercially suppliedthermoluminescent dosimeters (TLD-100 LiF powder, Engle-

hard Corp., Harshaw, OH), supplied and read by RadiationDetection Co. (Gilroy, CA). Air dose dosimeter measurementswere converted to tissue absorbed dose estimates using theratio of the energy mass absorption coefficients of � photonsfor tissue and air.

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2.3. Breeding and handling

Each male from the control and irradiated groups was matedto two female 129SvEv mice at 42–45 days post-irradiation(post-irradiation week 6) to obtain F1 offspring conceived ofsperm that were irradiated as Type A/B spermatogonia. At 45days post-irradiation, the acutely irradiated male mice were

sacrificed to obtain data on spermatogenic endpoints. The preg-nant females delivered their F1 litters and all offspring fromATM sires were genotyped. For each litter, the F1 male off-spring were weighed at 19 days of age and a mean maleoffspring body weight was obtained for the litter. For each

Fig. 1. Schematic representation of the breeding protocol for producing F3 offgous male mice received 0.1 Gy from attenuated 137Cs � rays and mated with nexperimental F1 animals with a Type A/B spermatogonial irradiation history.F1 male offspring were genotyped and one male of each genotype from each lgeneration offspring. The same genotyping and procedures were used to obtai12–16 weeks of age. All animals were tracked transgenerationally with respeobtained using the same protocol, but are not shown in the schematic. 129SvEthe same protocol, but without the necessity of genotyping. Male genotypes aobtained from the vendor for each generation’s breeding; their genotypes arelisted at the bottom of the breeding protocol and correspond to column headATM +/− F0 sire that have been +/+ for all three subsequent generations. ATat the F1 generation, but +/+ for the F2 and F3 generations. ATM-(−)+/+ denand F2 generations, but were +/+ at the F3 generation. ATM-(−)+/− denotes Fsubsequent generations. 129SvEv denotes F3 offspring from a 129SvEv F0 si

earch 616 (2007) 34–45

genotype, the F1 male closest in body weight to this meanbody weight was selected from each litter to sire the F2 gener-ation of offspring. Beginning at 15–19 weeks of age, these F1

males were mated to different pairs of females to obtain the F2

generation. The same breeding protocol was used to obtain theF3 generation. F3 generation males were sacrificed at 12–16weeks of age for sperm assays. The overall breeding protocol

is shown in Fig. 1.

New female mice were obtained from the vendor for eachgeneration’s matings in order to preclude inbreeding or closedcolony effects. As with the F0 dams, the F1 and F2 dams wereeach from a different, single cohort, ranked based on body

spring with a paternal F0 germline irradiation history. ATM heterozy-on-exposed 129SvEv females 42–45 days post-irradiation to produce

These F0 male mice were then sacrificed to obtain epididymides. Theitter was mated with a new, non-exposed 129SvEv female to obtain F2

n the F3 generation of offspring. F3 adult male mice were sacrificed atct to F0 sire and genotype. Sham-irradiated concurrent controls werev irradiation history and concurrent control mice were obtained usingre shown on the schematic. All female mice were 129SvEv (+/+), andnot shown on the schematic. F3 experimental group designations areings in other tables and figures. ATM+ denotes F3 offspring from anM-(+) denotes F3 offspring from an ATM +/− F0 sire that were +/−otes F3 offspring from an ATM +/− F0 sire that were +/− at the F1

3 offspring from an ATM +/− F0 sire that have been +/− for all threere.

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eight and randomly assigned to the irradiation history or con-urrent control experimental groups. Throughout the study F1,2, and F3 offspring were tracked with respect to the F0 sire.or a given generation, all experimental groups of mice wereoused together within the same room and maintained under a2 h light/12 h dark photoperiod.

.4. DNA purification and PCR genotyping

At 4 days of age ATM offspring were toe clipped for identi-cation purposes. DNA was purified from these mouse toe clipssing the QiaAmp Tissue Kit and protocol (Qiagen, Valencia,A).

The standard PCR protocol provided by Jackson laboratoryas used to genotype the ATM group mice. Primers 5′-GCT-CCATACTTGATCCATG-3′ and 5′-TCCGAATTTGCAG-AGTTG-3′ were used to obtain a 147 bp PCR prod-ct from ATM wild-type alleles. Primers 5′-CTTGGG-GGAGAGGCTAT-3′ and 5′-AGGTGAGATGACAGGAGA-′ were used to obtain a 280 bp PCR product from theeo insertion vector for animals carrying the ATM-targetedutation. PCR products were separated by agarose gel elec-

rophoresis on a 1.5% agarose gel.

.5. Sperm sample collection and assessment of motilitynd morphology

Adult male mice were sacrificed by cervical dislo-ation and weighed. Epididymides and testes were thenemoved and weighed. To obtain caudal epididymal spermor assessment of sperm motility, morphology and DNAntegrity, both caudal epididymides were placed in a Petriish containing 4 ml Dulbecco’s phosphate buffered salineDPBS) with 3 mg/ml bovine serum albumin (BSA) andently punched and pressed with a 30 G needle to releaseperm.

After adjusting sperm concentration to approximately05 sperm/ml, 6 �l of sperm suspension was mounted onto aicroscope slide and sperm motility was analyzed under phase-

ontrast microscope at 200× magnification. Sperm motilityas calculated as the percentage of motile sperm at room

emperature out of 200 sperm per animal.After motility analyses, 50 �l of sperm suspension from

ach animal was transferred into a microcentrifuge tube andtored on ice for the comet assay. Concentrated EDTA solu-ion was added to the sperm suspension (final concentration0 mM EDTA) to protect the DNA from DNase degradationntil comet slides were prepared.

About 200 air-dried sperm were scored under a phase-ontrast microscope at 400× magnification to obtain theercentage of sperm with normal head morphology. Sperm

mears were prepared for each animal by evenly spreading 5 �lf sperm suspension along the length of a microscope slide andir-drying. Sperm tail defects were not considered in this study.ormal sperm head morphology was judged as described byurruel et al. [30].

earch 616 (2007) 34–45 37

For each animal, the remaining sperm suspension with cau-dal epididymides tissue was frozen at −20 ◦C together with thecaput and corpus epididymides for later homogenization andepididymal sperm count.

2.6. Epididymal sperm counts

For each animal, both epididymides plus any reserved epi-didymal sperm suspension and tissues from the same animalthat had been used in other sperm assays were thawed andhomogenized together in DPBS containing 3 mg/ml BSA usinga manual glass homogenizer. The homogenate was mixed thor-oughly by vortexing and the sperm were counted using ahaemocytometer. An average of four independent counts wasused to calculate sperm numbers that were then expressed asnumber of sperm per milligram of epididymal weight.

2.7. Neutral pH sperm comet assay

The following procedure was performed under yellow lightto minimize light-induced damage to sperm DNA using thetechniques of Haines et al. [26], with some modifications.For each generation’s comet assays, one batch of all buffersand reagents were prepared and all slides for that given gen-eration were processed at the same time. Microscope slideswere coated with 1% normal melting-point agarose (NMA)in Ca2+,Mg2+-free PBS and then 85 �l of sperm suspensionin 0.7% low melting point agarose (LMA) in Ca2+,Mg2+-freePBS (9:1 v/v) was pipetted onto the slides and spread by cov-ering with a coverslip. The microgels were allowed to solidifyand then the coverslips were gently removed, and another85 �l layer of LMA was pipetted onto each slide and spreadwith a coverslip. The slides were then placed into coplin jarscontaining freshly prepared, ice-cold lysing solution (10 mMTris–HCl, pH 10.0 containing 2.5 M NaCl, 100 mM EDTA, 1%Triton X-100, and 10 mM dithiothreitol; 4 ◦C overnight). Thenext day, the lysing solution was drained off and replaced withfreshly made lysing solution containing 0.1 mg/ml DNase-freeProteinase K (Qiagen). After 4 h at 37 ◦C, the slides were placedin a 20 cm × 40 cm horizontal electrophoresis tank, and equi-librated in TAE buffer (100 mM Tris–HCl, pH 8.5, 100 mMsodium acetate, and 50 mM EDTA). Within each electrophore-sis run, balanced numbers of slides from each experimentalgroup and/or animal were represented until the entire genera-tion was complete. Slides were electrophoresed for 30 min at25 V and 35 mA. Following electrophoresis, the slides weredrained, washed, fixed, and air-dried at room temperature untilscored. The slides were coded randomly, stained with 2 �g/mlethidium bromide, and viewed using a Zeiss Photoscope flu-orescence microscope (200× magnification). Fifty sperm perslide were scored using Komet 5 software (Kinetic Imaging

Ltd., Liverpool, UK). Two slides were analyzed for a total of100 sperm per animal. Comet tail parameters evaluated includecomet tail length (the maximum distance [�m] the damagedDNA migrates from the center of the cell nucleus), the percentDNA in the tail (the percent of the total DNA that migrates

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38 J.E. Baulch et al. / Mutat

from the nucleus into the comet tail), and tail extent moment(TEM = [tail length × % tail DNA]/100), for an integrated mea-surement of overall DNA damage in the cell.

2.8. Statistical analyses

Comet data were log transformed to eliminate skewness andcontrasted by treatment group with respect to geometric mean(x̄g) and geometric standard error (S.E.g; [31]). Statistical eval-uations of comet data were performed using the non-parametricMann–Whitney test. Percent change of geometric mean valuesrelative to concurrent controls were contrasted by genotypeby two-tailed Student’s t-tests using the log transformed data.All other sperm measurements were evaluated by standardANOVA. For all tests, statistical significance was taken asα ≤ 0.05.

3. Results

3.1. Breeding results

From the 17 irradiated ATM heterozygous and the17 129SvEv F0 male mice, 6 litters and 8 litters, respec-tively, were obtained that were bred to ultimately provideF3 offspring with a paternal irradiation history for thisstudy. From the 16 sham-irradiated concurrent controlATM heterozygous F0 male mice and the 16 sham-irradiated 129SvEv F0 male mice, 5 litters and 7 litters,respectively, were obtained that were bred to ultimately

provide concurrent control F3 offspring for this study.Each of the F3 adult male mice evaluated for a givenendpoint in each experimental group is a descendent ofa different one of these successful F0 mating events. The

Table 1Epididymal mass and sperm counts (x̄ ± S.E.) for F0 male mice acutely irrad

Genotype Exposure group

ATM +/− Sham-irradiated concurrent controls x̄

S.E.N 1

0.1 Gy acute irradiation x̄

S.E.N 1

129SvEv Sham-irradiated concurrent controls x̄

S.E.N 1

0.1 Gy acute irradiation x̄

S.E.N 1

Assays were performed at 45 days post-irradiation, corresponding to spermaindicate significant difference from concurrent controls. *P = 0.04; ANOVA.

earch 616 (2007) 34–45

only exception to this is the ATM+ irradiation historygroup where one lineage was lost because of a lack ofbreeding success during the second generation, result-ing in an N of 5. These breeding results demonstrate thatthere was no effect of acute irradiation using 0.1 Gy onin vivo fertility for either genotype of male mice.

3.2. Acutely irradiated F0 male mice

For the acutely irradiated F0 male mice there were noeffects of irradiation on body mass, testes mass, testessperm count, percent sperm motility, and percent nor-mal sperm morphology 45 days after acute irradiation.Although no effect on epididymides mass was observedin the male mice that had been exposed to a 0.1 Gydose of � rays, a decrease in epididymal sperm countswas observed for both the acutely irradiated ATM +/−and 129SvEv male mice relative to each group’s respec-tive concurrent controls (9.9% and 11.4%, respectively;P = 0.04; Table 1). The epididymal sperm correspondto spermatozoa that were irradiated 45 days earlieras premeiotic Type A/B spermatogonia. These resultsdemonstrate that ATM +/− and 129SvEv germlineshad similar sensitivities to radiation-induced spermato-gonial cell killing following acute � irradiation using0.1 Gy.

Neutral pH comet assays detected significantdecreases in TEM, tail length and percent tail DNA in

the sperm of the irradiated male mice relative to unir-radiated concurrent controls for both irradiated ATM+/− and 129SvEv male mice at 45 days post-irradiation(Table 2, Fig. 2). Student’s t-tests of the percent change

iated using a dose of 0.1 Gy from attenuated 137Cs � rays

Epididymal mass (g) Epididymal sperm count (×106 mg−1)

0.079 79.70.002 2.942 12

0.079 71.8*

0.001 2.181 11

0.076 84.00.002 2.720 10

0.078 74.4*

0.002 3.280 10

tozoa that were irradiated as Type A/B spermatogonia. Bold values

J.E. Baulch et al. / Mutation Research 616 (2007) 34–45 39

Table 2Effects of acute germline irradiation on DNA damage as measured by neutral pH comet analysis of spermatozoa 45 days after acute irradiation ofthe F0 male mouse

Genotype Exposure group Tail extent moment ([taillength × % tail DNA]/100)

Tail length (�m) Tail DNA (%)

ATM+/− Sham-irradiated concurrent controls x̄g 5.11 33.50 15.21S.E.g 1.03 1.02 1.02N 10 10 10

0.1 Gy irradiation history x̄g 3.89 28.58 13.61S.E.g 1.04 1.02 1.02N 8 8 8

P value <0.0001 <0.0001 <0.0001

129SvEv Sham-irradiated concurrent controls x̄g 3.73 27.73 13.43S.E.g 1.03 1.02 1.02N 10 10 10

0.1 Gy irradiation history x̄g 3.12 24.49 12.74S.E.g 1.03 1.01 1.02N 10 10 10

P value <0.0001 <0.0001 0.02

Data shown represent the geometric mean and geometric standard errors of tanimal, 50 cells were scored per slide from each of two slides for a total of 100 ctest. Bold P values indicate statistical significance.

Fig. 2. Effects of acute Type A/B spermatogonial irradiation (0.1 Gy,137Cs � rays) on DNA electrophoretic mobility as measured by neutralpH sperm comet analysis of the resulting F0 male spermatozoa 45 dayslater. Gray bars correspond to ATM +/− male mice; black bars corre-spond to 129SvEv male mice. Data shown represent percent change ofgeometric mean values with geometric S.E. for a given endpoint rela-tive to concurrent controls: tail extent moment (TEM = [tail length × %tail DNA]/100), tail length, percent tail DNA. Two-tailed Student’st-tests of the percent change of geometric mean values relative to con-current controls for the ATM +/− and 129SvEv mice demonstrateno significant effect of animal genotype on DNA damage endpoints45 days post-irradiation in sperm from acutely irradiated male mice.Original comet data and the results of non-parametric Mann–Whitneytests of these original data are given in Table 2.

he mean (x̄g ± S.E.g) and number of animals (N) evaluated. For eachells. P values were obtained using the Mann–Whitney non-parametric

in sperm DNA electrophoretic mobility relative to con-current controls demonstrated that irradiated ATM +/−and 129SvEv male mice are not significantly different intheir relative sensitivities to delayed radiation-inducedchromatin abnormalities 45 days after acute � irradiationusing 0.1 Gy.

3.3. Unirradiated F3 descendants of the γ

irradiated F0 sires

For all F3 generation experimental groups body mass,epididymal mass, testes mass, percent sperm motil-ity, and percent with normal sperm morphology weremeasured for heritable effects of paternal F0 germlineirradiation relative to concurrent controls (Table 3). Nosignificant effect was found on body, epididymal or testesmass. For the 129SvEv experimental group, an increasein percent with normal sperm morphology was observedin the irradiation history group relative to concurrentcontrols. While this increase was statistically signifi-cant, its biological relevance with respect to paternalF0 irradiation history is questionable since the effectwas not observed in any other experimental group andthe magnitude of the increase was just 5% (P = 0.03).

For the ATM-(−)+/+ experimental group, a significantincrease in percent motile sperm was observed in theirradiation history group relative to concurrent controls(15% increase; P = 0.02). While the magnitude of this

40 J.E. Baulch et al. / Mutation Research 616 (2007) 34–45

Table 3Descriptive data from sperm assays for unirradiated F3 male descendants of the irradiated and sham-irradiated control F0 sires with mean values(x̄), standard errors of the mean (S.E.) and number of animals (N)

Experimentalgroup designation

Exposure group Bodymass (g)

Epididymalmass (g)

Testesmass (g)

Percent spermmotility

Percent normalsperm morphology

ATM+ Sham-irradiated concurrent controls x̄ 24.7 0.062 0.19 57.4 87.0S.E. 1.5 0.005 0.009 2.6 2.1N 5 5 5 5 5

0.1 Gy irradiation history x̄ 23.5 0.067 0.21 59.6 86.8S.E. 0.5 0.006 0.030 2.3 1.3N 5 5 5 5 5

ATM-(+) Sham-irradiated concurrent controls x̄ 23.9 0.069 0.21 64.4 89.8S.E. 0.8 0.005 0.008 1.2 1.4N 5 5 5 5 5

0.1 Gy irradiation history x̄ 23.6 0.067 0.22 61.7 89.3S.E. 0.5 0.005 0.013 2.2 1.2N 6 6 6 6 6

ATM-(−)+/+ Sham-irradiated concurrent controls x̄ 22.4 0.065 0.20 58.0 89.4S.E. 0.7 0.003 0.006 2.0 1.6N 5 5 5 5 5

0.1 Gy irradiation history x̄ 23.1 0.067 0.20 67.0* 91.5S.E. 0.7 0.002 0.005 2.4 1.1N 6 6 6 6 6

ATM-(−)+/− Sham-irradiated concurrent controls x̄ 22.7 0.070 0.20 62.4 88.0S.E. 1.4 0.005 0.008 2.3 2.1N 5 5 5 5 5

0.1 Gy irradiation history x̄ 23.3 0.067 0.21 62.3 89.2S.E. 0.5 0.001 0.004 2.8 1.5N 6 6 6 6 6

129SvEv Sham-irradiated concurrent controls x̄ 24.0 0.068 0.21 62.0 84.1S.E. 0.8 0.002 0.007 3.6 1.3N 6 7 7 7 7

0.1 Gy irradiation history x̄ 24.8 0.069 0.22 61.8 88.5**

S.E. 0.5 0.002 0.007 2.8 1.37

P = 0.0

N

Bold values indicate significant difference from concurrent controls. *

increase is more substantial, its biological relevance withrespect to paternal F0 irradiation history is also question-able since similar effects were not observed in any otherexperimental group.

3.4. Comet assays—unirradiated F3 descendants ofthe γ irradiated F0 sires

Using the neutral pH sperm comet assay, sperm DNA

was evaluated for unirradiated F3 generation descen-dants of the 129SvEv male mice and all ATM genotypes(Table 4, Fig. 3) [see Fig. 1 for experimental group desig-nations]. For all ATM experimental groups, significantly

8 8 8 8

2, **P = 0.03; ANOVA.

decreased TEMs were observed relative to each group’srespective concurrent controls. A significant increase inTEM relative to concurrent controls was observed forthe 129SvEv descendants, however. A decrease was alsoobserved for comet tail lengths for all ATM experimentalgroups, but no effect on comet tail length was detectedfor the 129SvEv mice. The ATM+ and ATM-(+) exper-imental groups also had significant decreases in percentDNA in the comet tail relative to concurrent controls,

but the ATM-(−)+/+ and ATM-(−)+/− groups were notaffected. The 129SvEv mice again demonstrated a signif-icant increase in percent tail DNA relative to concurrentcontrols.

J.E. Baulch et al. / Mutation Research 616 (2007) 34–45 41

Table 4Effects of genotype on comet measurements for sperm from unirradiated F3 male descendants of the irradiated and sham-irradiated F0 sires

Comet measurement Exposure group Experimental group designation

ATM+ ATM-(+) ATM-(−)+/+ ATM-(−)+/− 129SvEv

Tail extent moment Sham-irradiated concurrent controls x̄g 8.82 8.68 8.62 8.32 8.52S.E.g 1.02 1.02 1.02 1.02 1.02N 5 5 5 5 7

0.1 Gy irradiation history x̄g 6.51 6.73 8.12 7.73 9.03S.E.g 1.02 1.02 1.02 1.02 1.01N 5 6 6 6 8

P value <0.0001 <0.0001 0.03 0.007 0.01

Tail length (�m) Sham-irradiated concurrent controls x̄g 44.36 45.60 46.45 45.08 42.95S.E.g 1.01 1.01 1.01 1.01 1.01N 5 5 5 5 7

0.1 Gy irradiation history x̄g 37.84 40.27 43.45 42.07 42.85S.E.g 1.01 1.01 1.01 1.01 1.01N 5 6 6 6 8

P value <0.0001 <0.0001 <0.0001 <0.0001 0.4

Percent tail DNA (%) Sham-irradiated concurrent controls x̄g 19.68 19.05 18.58 18.41 19.86S.E.g 1.01 1.01 1.01 1.01 1.01N 5 5 5 5 7

0.1 Gy irradiation history x̄g 17.18 16.71 18.66 18.37 21.09S.E.g 1.01 1.01 1.01 1.01 1.01N 5 6 6 6 8

P value <0.0001 <0.0001 0.5 0.9 <0.0001

D valuatedt Whitne

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ata shown represent x̄g ± S.E.g. N indicates the number of animals eotal of 100 cells per animal. P values were obtained using the Mann–

.5. Impact of ATM heterozygosity on DNAbnormalities

For the acutely irradiated F0 male mice, the epi-idymal sperm counts demonstrated similar significantifferences in spermatogonial cell killing for bothenotypes (Table 1). Student’s t-tests of the percenthange in TEM, comet tail length, and percent DNAn the comet tail relative to concurrent control alsoemonstrated that ATM +/− and 129SvEv male miceere similarly different in their relative sensitivities

o delayed radiation-induced DNA effects 45 daysfter Type A/B spermatogonial irradiation using a dosef 0.1 Gy (Fig. 2). However, comparison of unirra-iated control ATM +/− and 129SvEv DNA usingann–Whitney tests, demonstrated a genotype-related

ncrease in comet measurements for the ATM +/− group

hat is not associated with irradiation history (Table 2,≤ 0.001).Unlike the acutely irradiated male mice, the unirradi-

ted F3 descendants of irradiated F0 sires demonstrated

. For each animal, 50 cells were scored from each of two slides for ay non-parametric test. Bold P values indicate statistical significance.

significant differences in sperm DNA abnormalitiesbased on the genotype of the irradiated F0 sire. Withrespect to each experimental group’s concurrent controlTEM, comet tail length, and percent DNA in the comettail were significantly different for all F3 ATM groupsas compared to the percent change relative to concurrentcontrols for the F3 129SvEv group (Fig. 3).

Among the four ATM genotype experimental groups,there is also variation in the magnitude of the herita-ble effect of paternal irradiation on DNA electrophoreticmobility. The DNA abnormalities are consistently largerfor the ATM+ and ATM-(+) groups as compared to theATM-(−)+/+ and ATM-(−)+/− groups. The significantdecrease in TEM for the ATM+ and ATM-(+) groupsis driven by significant changes in both tail length andpercent tail DNA, while the significant decrease in TEMfor the ATM-(−)+/+ and ATM-(−)+/− groups is driven

solely by a significant decrease in comet tail length. Thesignificant increase in TEM observed for the 129SvEvgroup is driven by an increase in percent comet tail DNA.The mechanism governing these differences may involve

42 J.E. Baulch et al. / Mutation Res

Fig. 3. Heritable effects of paternal F0 germline irradiation on spermDNA electrophoretic mobility as measured by evaluation of cometendpoints (neutral pH) in unirradiated F3 male offspring. Data shownrepresent the percent change of the geometric mean values (with geo-metric S.E.) for a given endpoint in the unirradiated F3 offspring witha paternal F0 irradiation history relative to concurrent controls: (A) tailextent moment (TEM); (B) comet tail length; (C) percent DNA in tail.F3 experimental group designations correspond to those listed at thebottom of Fig. 1 breeding protocol. Two-tailed Student’s t-tests of thepercent change of geometric mean values relative to concurrent con-trols are used to demonstrate statistically significant between-genotypedifferences (P < 0.05); (a) ATM+; (b) ATM-(+); (c) ATM-(−)+/+; (d)ATM-(−)+/−; (e) 129SvEv. Original comet data and the results ofnon-parametric Mann–Whitney tests of these original data are givenin Table 4.

earch 616 (2007) 34–45

the types of chromatin modifications affected among thegroups based on ATM status.

As with the unirradiated F0 male mice, Mann–Whitney tests of control F3 offspring TEM and comettail length data suggested that there might be a genotype-related increase in basal levels of sperm DNA elec-trophoretic mobility for ATM lineage mice relative towild-type 129SvEv mice that is associated with genotyperather than a history of acute irradiation. This observa-tion is not is not supported by the percent comet tail DNAmeasurements, however.

4. Discussion

Evidence from mouse studies support the hypoth-esis that heritable genetic or epigenetic effects areinduced by low dose exposure of premeiotic Type A/Bspermatogonia to ionizing radiation. It is possible thatthese heritable effects may predispose subsequent gen-erations to increased radiation sensitivity and cancer.Alternatively, these heritable effects may represent aneutral or adaptive response. We have previously demon-strated that spermatogonial irradiation causes effectson embryonic cell proliferation rates and juvenile off-spring protein levels in as many as four generationsof offspring [13–17]. The current study using spermcomet assays supports our previous findings and clearlydemonstrates heritable effects of paternal F0 spermato-gonial irradiation history on chromatin. This studyalso clearly demonstrates that the ATM zygosity ofthe sire has a significant impact on these heritableeffects.

The literature is somewhat divided regarding thedetection of increased radiation sensitivity in ATMheterozygotes. In most studies AT carriers have beensuccessfully identified from wild-type or ATM null indi-viduals using screening methods including cytogenetics,comet assays and micronucleus assays [32–35]. Thereare however some studies that have not [36]. In our studyusing male mice heterozygous for ATM, we demon-strated that AT carriers and 129SvEv male mice arenot significantly different in their radiation sensitivity asmeasured by spermatogonial cell killing or measurablesperm DNA damage 45 days after receiving 0.1 Gy from� radiation. There is, however, a significant impact ofATM heterozygosity on the effect of acute germline irra-diation that is inherited by the offspring of the irradiatedmale mouse.

While we anticipated very few DNA DSBs as a resultof the acute irradiation, the overall decrease in DNAelectrophoretic mobility in sperm from the acutely irradi-ated males of both genotypes was unexpected. The most

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bvious explanations for the decreased electrophoreticobility of DNA in these comet assays involvesNA–protein crosslinking, DNA–DNA crosslinking orNA adduct formation [37]. These explanations seemnlikely, however, since the same decreased elec-rophoretic mobility phenomenon is not observed atigher doses of acute irradiation [16,27–29]. It alsoeems unlikely that DNA–protein crosslinking occurred,ince the use of relatively high concentration proteinase

treatments does not abrogate the observed effectsunpublished data).

It is also possible that the few DNA DSBs inducedy this 0.1 Gy dose of radiation are repaired and thatermline DNA repair is up regulated, resulting in a trueecrease in sperm DNA damage 45 days after irradia-ion in the F0 males. This argument seems somewhatnlikely, though, since decreased DNA damage effectsDSBs) were observed in sperm from F3 descendantsf ATM-irradiated male mice but not in sperm from F3escendants of 129SvEv-irradiated male mice. If DNASB repair were up regulated in the F0 germline, then weight expect heritably decreased DNA damage effects

n all groups of F3 offspring, rather than only the ATMroups.

Sperm comet studies from other laboratories haveemonstrated significant increases in sperm DNA dam-ge 45 days after acute irradiation of male mice using.5 Gy doses of X-rays [28,29], but, no significantffect was observed using 0.25 Gy [28]. Similarly,n a recent study from our laboratory we observedncreases in spermatozoal DNA damage in 129SvEv

ice 45 days after irradiation using 0.5 Gy of � radi-tion. However, at a dose of 0.1 Gy, decreased DNAamage was observed [16]. Our results at 0.1 Gy mayndicate real differences in germline DNA repair fol-owing lower dose irradiation as compared to higherose irradiation. Alternatively, the results may indi-ate differences in mouse strains or between-laboratoryifferences in experimental methods. At this time, weypothesize that DNA damage was repaired follow-ng spermatogonial irradiation, but that this exposureaused subtle heritable radiation-induced chromatinlterations that are not repaired by conventional cellulareans.Based on the findings of the current study, it seems

hat ATM plays a critical role in the spermatogonialadiation response and that ATM heterozygotes have aompromised or altered radiation response. This role was

ot revealed to us by evaluating the acutely irradiatedale mouse, but by evaluating the offspring descended

rom that irradiated male mouse. Our results suggesthat the initiating mechanism underlying the heritable

earch 616 (2007) 34–45 43

response to paternal spermatogonial irradiation involvesATM and is affected by ATM heterozygosity. Whilethere is little apparent difference in the quality of theDNA abnormalities in the mature sperm 45 days afteracute spermatogonial irradiation when the ATM +/− and129SvEv mice are compared, there are marked differ-ences in the quality of the DNA abnormalities in thedescendants of the irradiated male mice based on pater-nal F0 genotype.

In this study, it is clear that ATM heterozygosity of theF0 sire affects DNA damage endpoints in the descendentsof the irradiated male. As these animals were bred trans-generationally, the mutant ATM allele was replaced witha normal allele at a different generation for each of thefour ATM F3 experimental groups. It is not clear whetherthis transgenerational change in genotype affects thisheritable DNA effect. The magnitude of decrease in elec-trophoretic mobility of sperm DNA in comet assays forthe ATM+ and ATM-(+) groups relative to the ATM-(−)+/+ and ATM-(−)+/− groups indicates that ATMheterozygosity affects the electrophoretic mobility ofgermline DNA of offspring of irradiated male mice. Thisoutcome suggests that ATM heterozygosity affects theinitiating event in the germline of the irradiated malemouse, but that ATM heterozygosity may not affect thepropagation of these heritable DNA modifications acrossgenerations.

In this study, we did not detect increases in DNA dam-age in sperm from acutely irradiated male mice followingthe 0.1 Gy dose of radiation. It is apparent, however,that some chromatin alteration occurred in the irradi-ated Type A/B spermatogonia that resulted in effectsin mature sperm 45 days later and to chromatin effectsthree generations later. The early cellular response toand repair of DNA damage is critical and is most likelyaffected by ATM zygosity. Our results suggest that thereis a potential for heritable genetic or epigenetic changesassociated inheritance of a paternal F0 spermatogonialirradiation history. Further, our results suggest that thereis an increase in this potential that is associated withATM heterozygosity, most likely as a result of a com-promised early cellular radiation response in the acutelyirradiated spermatogonia of the heterozygous malemouse.

While a link to the human from these animal stud-ies has yet to be found [16–19], there should be concernfor the children of men receiving spermatogonial irra-diation. Fertilization with sperm carrying compromised

or altered genetic material may lead to effects includingdevelopmental defects, increased sensitivity to chemicalor radiation exposures and cancer susceptibility in theoffspring.

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Acknowledgements

The authors thank David Hayes for his expert techni-cal assistance. This work was conducted at the Center forHealth and the Environment, University of California,Davis and supported by Biological and EnvironmentalResearch Program (BER), United States Department ofEnergy Grant No. DE-FG03-01ER63225 and NIH RO1ES06516 to Dr. J.E. Baulch.

References

[1] Y. Shiloh, ATM and related protein kinases: safeguarding genomeintegrity, Nat. Rev. Cancer 3 (2003) 155–168.

[2] G. Rotman, Y. Shiloh, ATM: a mediator of multiple responses togenotoxic stress, Oncogene 18 (1999) 6135–6144.

[3] Y. Shiloh, ATM and ATR: networking cellular responses to DNAdamage, Curr. Opin. Genet. Dev. 11 (2001) 71–77.

[4] M. Fernet, N. Moullan, A. Lauge, D. Stoppa-Lyonnet, J. Hall,Cellular responses to ionising radiation of AT heterozygotes: dif-ferences between missense and truncation mutation carriers, Brit.J. Cancer 90 (2004) 866–873.

[5] M.F. Lavin, Radiosensitivity and oxidative signalling in ataxiatelangiectasia: an update, Radiother. Oncol. 47 (1998) 113–123.

[6] K.K. Khanna, Cancer risk and the ATM gene: a continuing debate,J. Natl. Cancer Inst. 92 (2000) 795–802.

[7] G. Hamer, H.B. Kal, C.H. Westphal, T. Ashley, D. de Rooij, Ataxiatelangiectasia mutated expression and activation in the testis, Biol.Reprod. 70 (2004) 1206–1212.

[8] S.K. Mahadevaiah, J.M. Turner, F. Baudat, E.P. Rogakou, P. deBoer, J. Blanco-Rodrigues, M. Jasin, S. Keeney, W.M. Bonner,P.S. Burgoyne, Recombinational DNA double-strand breaks inmice precede synapsis, Nat. Genet. 27 (2001) 271–276.

[9] C. Barlow, M. Liyange, P.B. Moens, M. Tarsounas, K. Nagashima,K. Brown, S. Rottinghaus, S.P. Jackson, D. Tagle, T. Ried, A.Wynshaw-Boris, Atm deficiency results in severe meiotic disrup-tion as early as leptonema of prophase I, Development 125 (1998)4007–4017.

10] A.W. Plug, A.H. Peters, Y. Xu, K.S. Keegan, M.F. Hoekstra,D. Baltimore, P. de Boer, T. Ashley, ATM and RPA in meioticchromosome synapsis and recombination, Nat. Genet. 17 (1997)457–461.

11] Y. Xu, T. Ashley, E.E. Brainerd, R.T. Bronson, M.S. Meyn, D. Bal-timore, Targeted disruption of ATM leads to growth retardation,chromosomal fragmentation during meiosis, immune defects, andthymic lymphoma, Genes Dev. 10 (1996) 2411–2422.

12] T.K. Pandita, C.H. Westphal, M. Anger, S.G. Sawant, C.R. Geard,R.K. Pandita, H. Scherthan, Atm inactivation results in aberranttelomere clustering during meiotic prophase, Mol. Cell Biol. 19(1999) 5096–5105.

13] L.M. Wiley, J.E. Baulch, O.G. Raabe, T. Straume, Impaired cellproliferation in mice that persists across at least two generationsafter paternal irradiation, Radiat. Res. 148 (1997) 1–7.

14] J.E. Baulch, O.G. Raabe, L.M. Wiley, Heritable effects of paternal

irradiation in mice on signaling protein kinase activities in F3

offspring, Mutagenesis 16 (2001) 17–23.15] M.M. Vance, J.E. Baulch, O.G. Raabe, L.M. Wiley, J.W. Over-

street, Cellular reprogramming in the F3 mouse with paternal F0

radiation history, Int. J. Radiat. Biol. 78 (2002) 513–526.

[

earch 616 (2007) 34–45

16] M.W. Li, J.E. Baulch, Heritable effects on DNA damage followingpaternal F0 germline irradiation, Male Mediat. Dev. Toxic. (2007),in press.

17] J.E. Baulch, O.G. Raabe, Gamma irradiation of Type B spermato-gonia leads to heritable genomic instability in four generations ofmice, Mutagenesis 20 (2005) 337–343.

18] Y.E. Dubrova, Radiation-induced transgenerational instability,Oncogene 22 (2003) 7087–7093.

19] Y.E. Dubrova, Long-term genetic effects of radiation exposure,Mutat. Res. 544 (2003) 433–439.

20] W.F. Morgan, Non-targeted and delayed effects of exposure toionizing radiation. II. Radiation-induced genomic instability andbystander effects in vivo, clastogenic factors and transgenera-tional effects, Radiat. Res. 159 (2003) 581–596.

21] C.L. Yauk, Advances in the application of germline tandemrepeat instability for in situ monitoring, Mutat. Res. 566 (2004)169–182.

22] W.L. Russell, J.W. Bangham, L.B. Russell, Differentialresponse of mouse male germ-cell stages to radiation-inducedspecific-locus and dominant mutations, Genetics 148 (1998)1567–1578.

23] B.I. Lord, Transgenerational susceptibility to leukaemia inductionresulting form preconception paternal irradiation, Biol. Reprod.67 (1999) 854–861.

24] M.H. Brinkworth, Paternal transmission of genetic damage: find-ings in animals and humans, Int. J. Androl. 23 (2000) 123–135.

25] K.P. Hoyes, B.I. Lord, C. McCann, J.H. Hendry, I.D. Morris,Transgenerational effects of preconception paternal contamina-tion with (55)Fe, Radiat. Res. 156 (2001) 488–494.

26] G. Haines, B. Marples, P. Daniel, I. Morris, DNA damages inhuman and mouse spermatozoa after in vitro-irradiation assessedby the comet assay, Adv. Exp. Med. Biol. 444 (1998) 79–93.

27] G.A. Haines, J.H. Hendry, C.P. Daniel, I.D. Morris, Increasedlevels of comet-detected spermatozoal DNA damage followingin vivo isotopic- or x-irradiation of spermatogonia, Mutat. Res.495 (2001) 21–32.

28] G.A. Haines, J.H. Hendry, C.P. Daniel, I.D. Morris, Germ celland dose-depended DNA damage measured by the comet assayin murine spermatozoa after testicular x-irradiation, Biol. Reprod.67 (2002) 854–861.

29] E. Cordelli, A.M. Fresegna, G. Leter, E.M. Spano, P. Villani,Evaluation of DNA damage in different stages of mouse sper-matogenesis after testicular x irradiation, Radiat. Res. 160 (2003)443–451.

30] V.R. Burruel, R. Yanagimachi, W.K. Witten, Normal mice developfrom oocytes injected with spermatozoa with grossly misshapenheads, Biol. Reprod. 55 (1996) 709–714.

31] S.J. Wiklund, E. Agurell, Aspects of design and statistical analysisin the comet assay, Mutagenesis 18 (2003) 167–175.

32] M.R. Chowdhury, G. Singh, A. Shulka, I.C. Verma, Cytogeneticstudies in ataxia telangiectasia and their use in prenatal diagnosis,Indian J. Med. Res. 103 (1996) 155–158.

33] D. Scott, Q. Hu, S.A. Roberts, Dose-rate sparing for micronucleusinduction in lymphocytes of controls and ataxia-telangiectasiaheterozygotes exposed to Co60 gamma-irradiation in vitro, Int. J.Radiat. Biol. 70 (1996) 521–527.

34] U. Nachtrab, U. Oppitz, M. Flentje, H. Stopper, Radiation-

induced micronucleus formation in human skin fibroblasts ofpatients showing severe and normal tissue damage after radio-therapy, Int. J. Radiat. Biol. 73 (1998) 279–287.

35] C.S. Djuzenova, D. Schindler, H. Stopper, H. Hoehn, M.Flentje, U. Oppitz, Identification of ataxia telangiectasia het-

ion Res

[

J.E. Baulch et al. / Mutat

erozygotes, a cancer-prone population, using the single-cellgel electrophoresis (comet) assay, Lab. Invest. 79 (1999) 699–705.

36] A.J. Bishop, C. Barlow, A.J. Wynshaw-Boris, R.H. Schiestl,Atm deficiency causes an increased frequency of intrachromoso-

[

earch 616 (2007) 34–45 45

mal homologous recombination in mice, Cancer Res. 60 (2000)395–399.

37] M.S. Marty, N.P. Singh, M.P. Holsapple, B.B. Gollapudi, Influ-ence of p53 zygosity on select sperm parameters of the mouse,Mutat. Res. 427 (1999) 39–45.