Novel Transgenic Rat for In Vivo Genotoxicity AssaysUsing 6-Thioguanine and Spi- Selection

Hiroyuki Hayashi,1* Hiroshi Kondo,1 Ken-ichi Masumura,2

Yasuhiro Shindo,1 and Takehiko Nohmi21Pharmacology & Toxicology Research Laboratory,

Meiji Seika Kaisha, Yokohama, Japan2Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan

Transgenic rodents are valuable models for inves-tigating the genotoxicity of chemicals in vivo. Here,we report the establishment of a novel transgenicrat for genotoxicity analysis. In this model, about10 copies of �EG10 DNA carrying the gpt gene ofE. coli and the red/gam genes of � phage areintegrated per haploid genome of Sprague-Dawleyrats at position 4q24-q31. After recovery of�EG10 phage, point mutations in the gpt gene anddeletions in the red/gam genes are identified by6-thioguanine and Spi- selection, respectively. Toexamine the suitability of these rats for performingin vivo mutagenicity assays, rats were treated withsingle intraperitoneal injections of ethylnitrosourea(ENU; 100 mg/kg) or benzo[a]pyrene (B[a]P;62.5 and 125 mg/kg), and the mutant frequencies(MFs) in the liver were determined 7 days after the

treatment. ENU enhanced the gpt MF about 7-foldover the control while it did not significantly in-crease the Spi- MF. B[a]P increased both the gptand Spi- MFs several-fold in a dose-dependentmanner. To examine the kinetics of MF, ENU wasadministered (50 mg/kg/day for 5 successivedays) and gpt MFs in the liver were determined 7,21, 35, and 70 days after the last injection. TheMF increased to 8-fold and 13-fold over the controlat 7 and 35 days, respectively, after the last injec-tion and then slightly declined at 70 days. Thesekinetics are similar to those reported for ENU-treated lacZ transgenic mice. This novel transgenicrat could be useful for investigating species differ-ences between rats and mice in their response togenotoxic agents. Environ. Mol. Mutagen. 41:253–259, 2003. © 2003 Wiley-Liss, Inc.

Key words: gpt delta rat; 6-thioguanine selection; Spi- selection; deletion mutation

INTRODUCTION

Human risk associated with exposure to chemical com-pounds has been estimated from animal experiments,mostly using rats and mice. However, the extrapolation ofhuman risk is complicated by marked species differencesbetween rats and mice in their sensitivity to chemical car-cinogens [Tennant et al., 1986, 1987]. Indeed, some com-pounds are carcinogenic to rats but not to mice, and viceversa, and the target organs for cancer sometimes differbetween animal species. The International Conference forHarmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use (ICH) guidelines con-cerning testing for the carcinogenicity of pharmaceuticalsrecommends rats as a standard animal species, since rats aremore sensitive to chemical carcinogens than mice and pos-itive results in the liver of mice do not necessarily haverelevance to carcinogenic risk in humans [ICH SteeringCommittee, 1997]. Thus, it is important to develop geno-toxicity assays using rats for directly examining the rela-tionship between in vivo genotoxicity and carcinogenicity.

Previously, we developed the gpt delta transgenic mouseto detect mutations in vivo [Heddle et al., 2000; Nohmi etal., 1996, 1999]. A novel feature of this transgenic mutationassay is its ability to efficiently detect certain types of

deletions by Spi- (sensitive to P2 interference) selection, aswell as point mutations, i.e., base substitutions and frame-shifts, in the gpt gene by 6-thioguanine (6-TG) selection[Masumura et al., 1999; Nohmi et al., 1999]. Spi- selectiontakes advantage of the restricted growth of wildtype � phagein P2 lysogens [Ikeda et al., 1995; Shimizu et al., 1995].Only mutant � phages that are deficient in the functions ofboth the gam and redBA genes can grow well in P2 lysogensand display the Spi- phenotype. Simultaneous inactivationof both the gam and redBA genes is usually induced bydeletions. In the transgenic mice, about 80 copies of �EG10DNA, which carries the gam and redBA genes, are inte-

Grant sponsor: the Japan Health Science Foundation; Grant number:KH31029.

*Correspondence to: Hiroyuki Hayashi, Pharmacology & Toxicology Re-search Laboratory, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan. E-mail: Hiroyuki_Hayashi@meiji.co.jp

Received 12 September 2002; provisionally accepted 30 September 2002;and in final form 31 December 2002

DOI 10.1002/em.10152

Published online 16 April 2003 in Wiley InterScience (www.interscience.wiley.com).

Environmental and Molecular Mutagenesis 41:253–259 (2003)

© 2003 Wiley-Liss, Inc.

grated into each chromosome 17 of mice with a C57BL/6Jbackground [Masumura et al., 1999].

In the present report we describe a novel transgenic ratfor in vivo genotoxicity assay, a rat containing the �EG10transgene in a Sprague Dawley (SD) background. The trans-genic rats displayed sensitive responses to ethylnitrosourea(ENU) and benzo[a]pyrene (B[a]P) in the liver. The newlyestablished transgenic rats could be useful for addressingthe question of whether species differences in carcinogenicresponses are reflected in species differences in genotoxicresponses.

MATERIALS AND METHODS

Reagents and DNA

ENU (CAS no.759-73-9, purity 95%) was obtained from Nacalai Tesque(Tokyo, Japan) and B[a]P (CAS no. 50-32-8, purity 98%) from Wako PureChemical Industries (Tokyo, Japan). The �EG10 transgene was dissolvedin SM buffer at 12.5 mg/ml for microinjection. �EG10 DNA carries thered/gam genes in the stuffer region, and the gpt gene, the chloramphenicolacetyltransferase gene and the origin of replication for the pBR322 plasmidin a region flanked by two direct repeat loxP sequences [Nohmi et al.,1996].

Establishment of Transgenic Rat Lines

Fertilized zygotes for microinjection were prepared from SD or Wistarrats. Estral female rats (more than 6 weeks old) were selected using theImpedance Checker� (Muromachi Kikai, Tokyo, Japan). They showedelectric resistance of 5° or more in the vagina. Several pairs of estral femaleand male rats were placed together overnight and the fertilized zygoteswere taken from the oviducts of females with vaginal plugs. The fertilizedzygotes were cultured with M16 medium (Sigma, St. Louis, MO, USA) ina CO2 incubator until the pronucleus of the zygote became visible. The�EG10 solution was injected into the pronucleus of the fertilized zygote.Healthy microinjected zygotes were transplanted promptly into the ovi-ducts of female rats that had been mated with sterilized male rats. After21–25 days, the transplanted female rats yielded offspring. The blood fromweanling rats was examined for the presence of �EG10 DNA by PCRusing the primers described previously [Nohmi et al., 1996]. The founderswere maintained as transgenic lines by breeding with the original species.

Selection of Transgenic Rat Lines

�EG10 was recovered as � phage particles from the genomic DNA ofeach transgenic rat by in vitro packaging [Nohmi et al., 1996]. Thetransgenic lines suitable for genotoxicity assays were first selected by therescue efficiency of � phage. In the rescue assay, a freshly prepared cultureof E. coli C was used as the indicator and 8–10 �g DNA was used perpackaging reaction.

Southern Blot Analysis

Genomic DNAs (5 �g) from the tail and liver of rats were digested withDraI restriction endonuclease. Southern blot analysis was carried out asdescribed with 32P-labeled DraI-digested �EG10 DNA as the probe [No-hmi et al., 1996]. DraI-digested genomic DNAs (5 �g) from the tail andliver of gpt delta mice and �EG10 DNA were also analyzed as controls.

Fluorescent In Situ Hybridization (FISH) Analysis

Transgenic rat chromosome specimens were prepared from primarycultures prepared from fetuses that were obtained from 12-day pregnantrats mated with transgenic male rats. �EG10 DNA was labeled withdig11-dUTP using the Nick Translation kit� (Roche, Mannheim, Ger-many) and used as the detection probe for the transgene. The mixture ofdenatured detection probe and hybridization solution (50% of dextransulfate, 25 mg/ml bovine serum albumin and 10� sodium citrate-sodiumchloride (SSC)) was applied to chromosome spreads and the hybridizationreaction was carried out under humid conditions at 37°C overnight. Afterwashing, the specimens were stained with anti-digoxigenin-rhodamineantibody (Roche) and 4�, 6�-diamidino-2-phenylindole (DAPI; Roche).Properly stained metaphases were photographed with multiple exposuresfor identifying the signal of the transgene at 1,000� using an incident-lightfluorescence microscope. The chromosomal location of �EG10 in thegenome of SD-2 transgenic rats was determined by the position of theFISH signal on standard G-banded rat chromosomes [Satoh et al., 1989].

Validation Study for the gpt and Spi- Assays UsingTransgenic Rats

Male transgenic SD-2 rats (5 weeks old, two animals per group for ENU;9 or 10 weeks old, three animals per group for B[a]P) were treated withsingle intraperitoneal (i.p.) injections of mutagens. ENU was dissolved at25 mg/ml in phosphate-buffered saline (pH 6.0) for dosing at 100 mg/kgbody weight. B[a]P was dissolved at 6.25 and 12.5 mg/ml in olive oil fordosing at 62.5 and 125 mg/kg body weight, respectively. The animals weresacrificed 7 days after dosing and the livers were removed and stored at–80°C until use. The gpt and Spi- assays were performed as previouslydescribed [Nohmi et al., 2000]. At 48 hr after dosing, peripheral bloodspecimens were prepared from all the animals for concurrent evaluation ofmicronucleus (MN) formation as a blood exposure marker. The bloodsmears were stained with Acridine Orange as described by Hayashi et al.[1992]. The frequency of cells bearing MN was determined by observing2,000 polychromatic erythrocytes with an incident-light fluorescence mi-croscope.

Mutant Frequency Kinetics

ENU was intraperitoneally administered to SD-2 transgenic rats (10weeks old) once daily at 50 mg/kg/day for 5 consecutive days. Two malesand two females were euthanized 7, 21, 35, or 70 days after the finaladministration and the organs were immediately frozen in liquid nitrogenand stored at –80°C. Genomic DNA was extracted using the Recov-erEase™ Kit (Stratagene, La Jolla, CA, USA), and �EG10 was recoveredwith Transpack� Lambda Packaging Extract (Stratagene) to evaluate theoccurrence of gene mutation in the gpt assay.

RESULTS

Establishment of Transgenic Rat Lines

Wistar Rats

We injected �EG10 DNA into 870 fertilized zygotes andtransplanted 308 zygotes that were normal in appearanceinto recipient females. Of 47 offspring that were obtainedwe identified seven carrying the gpt gene. No �EG10 wasrescued, however, from five lines and the rescue efficienciesof the remaining two lines, i.e., Wistar-6 and Wistar-7, werebelow 105 pfu per packaging reaction. To improve the

254 Hayashi et al.

rescue efficiency, we made homozygous Wistar-6 trans-genic rats by sister–brother mating. However, the resultinghomozygous Wistar-6 line did not exhibit the desired rescueefficiency of 106 pfu per packaging reaction. In a secondtrial, we injected �EG10 DNA into 646 fertilized zygotesand transplanted 311 apparently normal zygotes into fe-males. In this trial, we injected more DNA than in theprevious experiments. Of 20 progeny obtained we identifiedthree carrying the gpt gene. However, one of these rats wassterile and neither of the remaining two produced progenythat carried the gpt gene. Thus, we examined the generationof transgenic SD rats, as described below.

SD Rats

We injected �EG10 DNA into 717 fertilized zygotes andtransplanted 270 apparently normal zygotes into females.Of 12 offspring, only one carried the gpt gene (SD-1).However, this founder resulted in a transgene recoverysimilar to that found for Wister-6 (data not shown). Toimprove the efficiency of generating transgenic lines, wetransplanted the fertilized SD zygotes into female Wistarrats. We have observed that Wistar rats are capable ofefficiently maintaining treated embryos, resulting in rela-tively high numbers of progeny (unpubl. results). We in-jected DNA into 192 fertilized SD zygotes and transplanted92 apparently normal zygotes into female Wister rats. Of 16progeny obtained, one was identified as carrying the gptgene. Since this line, i.e., SD-2, exhibited the best rescueefficiency (maximum 3 � 105 pfu per packaging reaction)and produced offspring carrying the gpt gene, we charac-terized this line thoroughly.

Number of Copies and Chromosome Location of�EG10 in SD-2 Rats

In order to determine the number of copies of �EG10 inthe genomic DNA of SD-2 rats, we prepared a Southern blotof Dra I-digested genomic DNAs from the tail and liver ofthe transgenic rats and hybridized with 32P-labeled �EG10DNA that had also been digested with Dra I (Fig. 1). Weestimated the genome of SD-2 rats contained about 10copies of �EG10 by comparing the density of hybridizationsignals among the samples and the positive controls. Asexpected, DNAs from gpt delta mice contained 100–300copies of �EG10 [Nohmi et al., 1996].

We then analyzed the chromosomal location of �EG10 inthe genome of SD-2 transgenic rats by FISH analysis (Fig.2). We concluded that �EG10 DNA was tandemly inte-grated in a single site of chromosome 4. The exact locationwas identified as 4q24-q31 by the position of the FISH

signal on a standard G-banded rat chromosome preparation[Satoh et al., 1989].

Sensitivity of the Novel Transgenic SD-2 Rat toStandard Mutagens

To examine whether the SD-2 rat exhibits reasonablesensitivity to chemical mutagens, we first treated two SD-2rats with ENU and examined the gpt and Spi- MFs in theliver (Table I). The gpt MF increased 5–13 times over thecontrol levels. In contrast, no increase in Spi- MF wasproduced by the treatment. The treatment also enhancedMN formation 6–10-fold over the control in specimens ofperipheral blood. MN was examined to confirm the ade-quate exposure of rats to mutagens.

To further validate the SD-2 line, we treated groups ofthree SD-2 rats with B[a]P at two different doses (Table II).B[a]P treatments enhanced the gpt MF in a dose-dependentmanner by 2- and 4-fold over the control levels. Similarly,the treatments increased the Spi- MF 2- and 9-fold over thecontrol levels. The treatments also increased MN formation4- and 9-fold over the controls.

Fig. 1. Estimation of the copy number of �EG10 in the SD-2 transgenicrat by Southern blot hybridization. Genomic DNAs (5 �g) from a gpt deltamouse (lanes 1–4) and rat (lanes 5 and 6) were digested with DraI andhybridized with 32P-labeled DraI-digested �EG10 DNA. Lane 1: tail DNAof a gpt delta female mouse; lane 2: liver DNA of a gpt delta female mouse;lane 3: tail DNA of a gpt delta male mouse; lane 4: liver DNA of a gpt deltamale mouse; lane 5: tail DNA of a gpt delta male SD-2 rat; lane 6: liverDNA of a gpt delta male SD-2 rat. Controls (lanes 7–12) contain definedamounts of DraI-digested �EG10 DNA. Lane 7: 0 copies; lane 8: 10copies; lane 9: 30 copies; lane 10: 100 copies; lane 11: 300 copies; lane 12:1,000 copies. The copy numbers for transgenic animals were estimated bycomparing the hybridization signals among the samples and controls.

Novel Transgenic Rats in In Vivo Genotoxicity 255

Kinetics of gpt MF in the Liver of SD-2 Rats TreatedWith ENU

To examine the kinetics of gpt MF in the liver, we treatedSD-2 rats with ENU and determined the gpt MF 7, 21, 35,and 70 days after the last injection. Four rats (two males and

two females) were used for each data point. The MF in-creased to 8-fold and 13-fold over the control at 7 and 35days, respectively, after the last injection and then slightlydeclined at 70 days (Fig. 3). The MFs of control rats did notchange significantly at 7 and 70 days after the injection ofPBS.

Fig. 2. Chromosomal localization of �EG10. Chromosome preparationsfrom SD-2 transgenic rats were stained with anti-digoxigenin rhodamineand DAPI. �EG10 DNA was identified by the red signal of rhodamine.Properly stained metaphases were photographed with multiple exposures toidentify the transgene signal. In the four pairs of small photos, the left

shows the transgene signal and the right the G-band pattern. Judging fromthe G-band pattern [Satoh et al., 1989], we concluded that the transgene islocated in chromosome 4q24-31, as depicted in the pattern diagram ofchromosome 4.

TABLE I. Induction of gpt and Spi� mutations in ENU-treated SD-2 rats

Animal no.

gpt Spi�

%MN

pfu(�103) Mutants

MF(�10�6)

Mean MF(�10�6)

pfu(�103) Mutants

MF(�10�6)

Mean MF(�10�6)

Control 1 1,530 2 1.31 2.33 3,490 3 0.86 0.79 0.202 598 2 3.34 1,405 1 0.71 0.20

ENU 1 785 13 16.6 16.8 1,815 2 1.10 0.93 2.02 948 16 16.9 2,615 2 0.76 1.2

Male transgenic SD-2 rats (5-week-old) were treated with a single i.p. injection of ENU at a dose of 100 mg/kg body weight. The animals were sacrificed7 days after dosing and the gpt and Spi� MFs were determined in the liver. Control animals received PBS (pH 6.0). MN were assayed in blood takenfrom the tail vein 2 days after dosing [Hayashi et al., 1992].

256 Hayashi et al.

DISCUSSION

In this study we established a novel transgenic rat that candetect point mutations and deletions. Using some of thelimited number of these rats that are presently available, wealso demonstrated that these rats may be suitable for detect-ing the mutagenicity of potentially genotoxic agents. Pointmutations and deletions are separately identified by 6-TG(gpt gene) and Spi- (gam and redBA genes) selection, re-spectively. ENU, which predominantly induces base substi-tutions [Shibuya and Morimoto, 1993], enhanced gpt MFbut not Spi- MF. The positive response in the MN assay maybe related to the ability of ENU to induce DNA strandbreaks [Suzuki et al., 1997]. The damage responsible for

strands breaks and MN, however, apparently does not resultin a detectable increase in the intrachromosomal deletionsassayed by Spi- selection [Nohmi et al., 1999]. B[a]P en-hanced both gpt and Spi- MFs (Tables I, II). B[a]P inducesnot only point mutations but also deletions [Bishop et al.,2000; Hakura et al., 2000] and these activities are consistentwith the present results. However, there was a rat treatedwith 62.5 mg/kg B[a]P that was positive in the 6-TG assaybut negative for Spi- mutation and MN induction, andanother rat treated with 125 mg/kg that was negative in the6-TG assay but positive in the Spi- and MN assays (TableII). These apparent discrepancies may be due to variationsin the assays that might be improved by analysis of a greaternumber of transgenes.

The spontaneous gpt MF in the liver of the rats (1–3 �10-6) was less than half of that in the liver of mice (2–7 �10-6 [Masumura et al., 2002; and unpubl. results]. This mayreflect the lower rate of oxidative metabolism in rats com-pared with mice [Shigenaga et al., 1989]. A lower sponta-neous MF in rats than in mice was also reported for trans-genic rodents harboring the lacI reporter gene [Dycaico etal., 1994], which argues against the location of the transgenebeing responsible for the lower mutant frequency. Interest-ingly, the spontaneous Spi- MF in the liver of rats wassimilar to or only slightly lower than that found in the liverof gpt delta mice (Tables I, II). This raises the possibilitythat the rates of spontaneous double-strand breaks in DNAare similar between rats and mice. Since the transgenic ratsand mice harbor the same �EG10 DNA, the sensitivities tochemical mutagens can be directly compared between thesetwo species. On the basis of these present results, we believeit is important to carry out additional experiments with agreater number of animals in order to further clarify thefeatures of transgenic rats.

The number of copies of �EG10 DNA in the transgenicrats was estimated at 10 per haploid genome, which is about

Fig. 3. Kinetics of gpt MF in the liver of rats treated with ENU. Ten-week-old male and female rats (two of each per sampling time) weretreated with ENU (50 mg/kg for 5 consecutive days by i.p. injection) andthe gpt MF was determined 7, 21, 35, and 70 days after the last injection.Control rats were treated with PBS (pH 6.0) and the gpt MF was deter-mined 7 and 70 days after the last treatment. Bars represent the standarddeviations. There are significant differences between all ENU-induced MFsand the control MFs (P � 0.01). There are no significant differences amongthe four ENU-induced MFs (7, 21, 35, and 70 days) by Student’s t-test.

TABLE II. Induction of gpt and Spi� mutations by B[a]P treatment

Dose(mg/kg)

AnimalNo.

gpt Spi�

%MN

Pfu(�103) mutant

MF(�10�6)

Mean MF� SD

Pfu(�103) mutant

MF(�10�6)

Mean MF� SD

0 1 1.497 1 0.67 1,389 1 0.72 02 3,108 4 1.29 0.98a 2,714 2 0.74 0.49 � 0.42 0.053 ND 3,006 0 0 0.15

62.5 1 1,353 3 2.22 1,904 4 2.10 0.22 1,347 3 2.23 2.24* � 0.03 1,292 0 0 1.18 � 1.07 0.053 1,319 3 2.28 1,400 2 1.43 0.6

125 1 1,230 3 2.44 3,260 21 6.44 0.22 1,025 1 0.98 4.24 � 4.44 2,451 11 4.49 4.56* � 1.85 0.63 861 8 9.29 1,826 5 2.74 0.3

Male transgenic rats SD-2 (9 or 10 weeks old) were treated by single i.p. injection of B[a]P at doses of 62.5 or 125 mg/kg body weight. The animalswere sacrificed 7 days after dosing and the gpt and Spi� MFs were determined in the liver. Control animals received PBS (pH 6.0). Statistical analysiswas conducted by Student’s t-test for gpt and Spi� MFs (*P � 0.05). MN were assayed in blood taken from the tail vein 2 days after dosing [Hayashiet al., 1992]. MN induction in 62.5 mg/kg and 125 mg/kg was significant using Kastenbaum-Bowman tables (P � 0.01).aA mean value of 0.67 � 10�6 and 1.29 � 10�6. ND: The data were not determined due to contamination.

Novel Transgenic Rats in In Vivo Genotoxicity 257

one-tenth the number of copies per haploid genome in gptdelta mice (Fig. 1). In addition, the mice are homozygousfor the transgene so that the total number of copies in gptdelta mice is 160 [Nohmi et al., 1996]. Because of this lowcopy number, the rescue efficiency for the SD-2 rat was1–3 � 105 pfu per packaging reaction, which is one-tenth toone-third that of gpt delta mice. This low rescue efficiencyand the low spontaneous MF in SD-2 rats mean that severalpackaging reactions may be required for each rat to deter-mine reliable MFs. To improve the rescue efficiency, wemade a homozygous SD-2 rat line by sister–brother mating.However, most of the pups that were homozygous for thetransgene died before weaning, and some had deficiencies intheir teeth. Since �EG10 is integrated in chromosome 4q24-q31 (Fig. 2), we suspect that some important gene(s) forodontogenesis may be located in this area of the rat chro-mosome. At present, we are generating other transgeniclines in the Fischer 344 background, since this strain is mostoften used for cancer bioassay.

The period between the last treatment and sacrifice, var-iously referred to as the sampling time, expression time, ormutant manifestation time, is an important factor for theefficient detection of in vivo genotoxicity [Sun et al., 1999].The most appropriate sampling time may be different inrapidly proliferating tissues, such as bone marrow, andslowly dividing tissues, such as the liver. In fast-proliferat-ing tissues, MF often declines with long sampling times[Takeiri et al., 2003]. Douglas et al. [1996] carefully mon-itored lacZ MF in the livers of mice treated with ENU andfound that MF increased at least 10-fold over a period of 1month and thereafter slightly decreased. We were interestedin whether the kinetics of MF in mouse liver were similar tothose in the liver of rats, and determined the gpt MF in theliver 7, 21, 35, and 70 days after the last injection of ENU(Fig. 3). The MF increased more than 10-fold over thecontrol until 35 days after the last injection and then de-clined slightly. Although none of the MFs in treated ratswere significantly different from each other, the trend in thekinetics for rats was similar to that reported for ENU-treatedlacZ transgenic mice. Thus, the most appropriate samplingtime for transgenic mice might also be applicable to trans-genic rats.

A recently agreed consensus for conducting transgenicrodent genotoxicity assays proposes treatment on 28 con-secutive days and sacrifice 3 days after the last treatment[Thybaud et al., 2003]. This proposal is partially based onthe theory that transgenes are genetically neutral and freefrom selection bias in vivo so that the mutations in thetransgenes can accumulate during the treatment time [Co-sentino and Heddle, 2000]. In fact, multiple treatmentsproduce a larger response in the transgene than single treat-ments [Zhang et al., 1996]. Since 28 days of treatment is theprotocol commonly used for subchronic toxicity assays inrats, i.e., 28-day repeated dose toxicity study, the consensusrecommendation for transgenic assays raises the possibility

of unifying the protocols for subchronic toxicity and in vivogenotoxicity assays using transgenic rats. This arrangementcould potentially examine the relationship between geno-toxicity and other toxicities induced by chemicals using asingle set of animals. In future studies, we will test thevalidity of the 28 plus 3 day protocol using our transgenicrat model.

In summary, a novel transgenic rat for detecting in vivogenotoxicity has been established. The availability of trans-genic rats greatly increases the potential of the Spi- and6-TG selection systems. Comparison of genotoxic re-sponses in rats and mice could help elucidate the mecha-nism(s) of species differences to chemical carcinogens.

ACKNOWLEDGMENT

We thank Dr. Hitoshi Satoh, Institute of Medical Science,University of Tokyo, for help with FISH analysis.

REFERENCES

Bishop AJ, Kosaras B, Sidman RL, Schiestl RH. 2000. Benzo(a)pyrene andX-rays induce reversions of the pink-eyed unstable mutation in theretinal pigment epithelium of mice. Mutat Res 457:31–40.

Cosentino L, Heddle JA. 2000. Differential mutation of transgenic andendogenous loci in vivo. Mutat Res 454:1–10.

Douglas GR, Jiao J, Gingerich JD, Soper LM, Gossen JA. 1996. Temporaland molecular characteristics of lacZ mutations in somatic tissuesof transgenic mice. Environ Mol Mutagen 28:317–324.

Dycaico MJ, Provost GS, Kretz PL, Ransom SL, Moores JC, Short JM.1994. The use of shuttle vectors for mutation analysis in transgenicmice and rats. Mutat Res 307:461–478.

Hakura A, Tsutsui Y, Sonoda J, Tsukidate K, Mikami T, Sagami F. 2000.Comparison of the mutational spectra of the lacZ transgene in fourorgans of the MutaMouse treated with benzo[a]pyrene: target organspecificity. Mutat Res 447:239–247.

Hayashi M, Kodama Y, Awogi T, Suzuki T, Asita AO, Sofuni T. 1992. Themicronucleus assay using peripheral blood reticulocytes from mit-omycin C- and cyclophosphamide-treated rats. Mutat Res 278:209–213.

Heddle JA, Dean S, Nohmi T, Boerrigter M, Casciano D, Douglas GR,Glickman BW, Gorelick NJ, Mirsalis JC, Martus HJ, Skopek TR,Thybaud V, Tindall KR, Yajima N. 2000. In vivo transgenicmutation assays. Environ Mol Mutagen 35:253–259.

ICH Steering Committee. 1997. Testing for carcinogenicity of pharmaceu-ticals. ICH Harmonized Tripartite Guideline. p 1–6.

Ikeda H, Shimizu H, Ukita T, Kumagai M. 1995. A novel assay forillegitimate recombination in Escherichia coli: stimulation oflambda bio transducing phage formation by ultra-violet light and itsindependence from RecA function. Adv Biophys 31:197–208.

Masumura K, Matsui M, Katoh M, Horiya N, Ueda O, Tanabe H, YamadaM, Suzuki H, Sofuni T, Nohmi T. 1999. Spectra of gpt mutationsin ethylnitrosourea-treated and untreated transgenic mice. EnvironMol Mutagen 34:1–8.

Masumura K, Kuniya K, Kurobe T, Fukuoka M, Yatagai F, Nohmi T.2002. Heavy-ion-induced mutations in the gpt delta transgenicmouse: comparison of mutation spectra induced by heavy-ion,X-ray and �-ray radiation. Environ Mol Mutagen 40: 207–215.

258 Hayashi et al.

Nohmi T, Katoh M, Suzuki H, Matsui M, Yamada M, Watanabe M, SuzukiM, Horiya N, Ueda O, Shibuya T, Ikeda H, Sofuni T. 1996. A newtransgenic mouse mutagenesis test system using Spi- and 6-thio-guanine selections. Environ Mol Mutagen 28:465–470.

Nohmi T, Suzuki M, Masumura K, Yamada M, Matsui K, Ueda O, SuzukiH, Katoh M, Ikeda H, Sofuni T. 1999. Spi- selection: an efficientmethod to detect gamma-ray-induced deletions in transgenic mice.Environ Mol Mutagen 34:9–15.

Nohmi T, Suzuki T, Masumura K. 2000. Recent advances in the protocolsof transgenic mouse mutation assays. Mutat Res 455:191–215.

Satoh H, Yoshida MC, Sasaki M. 1989. High resolution chromosomebanding in the Norway rat, Rattus norvegicus. Cytogenet CellGenet 50:151–154.

Shibuya T, Morimoto K. 1993. A review of the genotoxicity of 1-ethyl-1-nitrosourea. Mutat Res 297:3–38.

Shigenaga MK, Gimeno CJ, Ames BN. 1989. Urinary 8-hydroxy-2�-deoxyguanosine as a biological marker of in vivo oxidative DNAdamage. Proc Natl Acad Sci USA 86:9697–9701.

Shimizu H, Yamaguchi H, Ikeda H. 1995. Molecular analysis of lambdabio transducing phage produced by oxolinic acid-induced illegiti-mate recombination in vivo. Genetics 140:889–896.

Sun B, Shima N, Heddle JA. 1999. Somatic mutation in the mammarygland: influence of time and estrus. Mutat Res 427:11–19.

Suzuki T, Hayashi M, Wang X, Yamamoto K, Ono T, Myhr BC, Sofuni T.

1997. A comparison of the genotoxicity of ethylnitrosourea andethyl methanesulfonate in lacZ transgenic mice (Muta Mouse).Mutat Res 395:75–82.

Takeiri A, Mishima M, Tanaka K, Shioda A, Ueda O, Suzuki H, Inoue M,Masumura K, Nohmi T. Molecular characterization of MitomycinC-induced large deletions and tandem-base substitutions in thebone marrow of gpt delta transgenic mice. Chem Res Toxicol16:171–179.

Thybaud V, Dean S, Nohmi T, Boer J, Douglas GR, Glickman BW,Gorelick NJ, Heddle JA, Heflich RH, Lambert I, Martus H, MirsalisJC, Suzuki T, Nobuhiro Y. 2002. In vivo transgenic mutationassays. Mutat Res (in press).

Tennant RW, Stasiewicz S, Spalding JW. 1986. Comparison of multipleparameters of rodent carcinogenicity and in vitro genetic toxicity.Environ Mutagen 8:205–227.

Tennant RW, Margolin BH, Shelby MD, Zeiger E, Haseman JK, SpaldingJ, Caspary W, Resnick M, Stasiewicz S, Anderson B. 1987. Pre-diction of chemical carcinogenicity in rodents from in vitro genetictoxicity assays. Science 236:933–941.

Zhang XB, Felton JS, Tucker JD, Urlando C, Heddle JA. 1996. Intestinalmutagenicity of two carcinogenic food mutagens in transgenicmice: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and ami-no(alpha)carboline. Carcinogenesis 17:2259–2265.

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