Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.

To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by in vitro packaging. After infecting and plating phage on a hfl؊ bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 ؋ 10 ؊5 in liver, 2.9 ؋ 10 ؊5 in whole fish, to 1.8 ؋ 10 ؊5 in testes, were comparable to ranges in transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7-and 6.7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models. medaka ͉ ethylnitrosourea A major challenge to the detection of spontaneous and induced mutations is the difficulty with which mutant genes can be efficiently recovered and accurately identified in vivo. Considering that mutations must be detected at low frequencies (e.g., Ϸ1 spontaneous mutation͞10 5 -10 7 loci), and that sufficient DNA sequence information must be available to distinguish mutant from nonmutant genes, the problem of efficiently detecting and quantifying mutations in whole animals can be formidable. Transgenic animals that carry specific genes for quantitation of spontaneous and induced mutations have been developed to assist in improving in vivo mutation analyses (1). In this approach, a transgenic animal carries a prokaryotic vector that harbors a gene that serves as a mutational target. After mutagen exposure, the vector is separated from the animal's genomic DNA and shuttled into indicator bacteria where mutant and nonmutant genes are readily quantified (2, 3). Transgenic mutation assays offer numerous benefits for in vivo mutation detection not available by using other approaches. Benefits include the ability to screen rapidly statistically meaningful numbers of genetically neutral mutational targets in a variety of tissues and the ability to characterize mutations to aid in disclosing possible mechanisms of mutagen action. † A significant additional attribute is the pot...

Section: ϫ5supporting

confidence: 77%

Detection of mutations in transgenic fish carrying a bacteriophage λ cII transgene target

Winn

Norris²,

Brayer³

et al. 2000

“…In addition, the ENU-induced adducts 04-ethylthymine and 02-ethylthymine induce ART --GC transitions (34) and ART -* T-A transversions (35), respectively. Slow repair of 02-and 04-ethylthymine adducts has been suggested to contribute to increases in observed mutations at A-T base pairs over time (36,37) and, therefore, should be detected at 90 days, as seen in our studies ( Table 2). This result suggests that the observed spectra result from adduct-specific repair rates and the mutagenic potential of the ENU-induced adducts.…”

Section: Discussionsupporting

confidence: 57%

Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice.

Provost¹,

Short²

1994

Transgenic B6C3F1 mice carrying a lacl target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2-and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lacl genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-flve percent (11/13) of the spontaneous mutatious resulted in G-C -* AT transitions, all ofwhich occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G-C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at APT base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in viva spectra seen in ENU studies by using transmissibiliy assays and the hpilgene. These findings represent significant progress toward dening the in vivo spectra of Ethylnitrosourea (ENU) induces a broad spectrum of promutagenic lesions in vivo (6, 7) and has been studied by using a variety of transmissibility assays (8)(9)(10)(11)(12)(13)(14)(15). Although ENU has been well characterized as a mouse germ cell mutagen, the spectrum of ENU-induced mutations in mouse germ cells remains largely undefined. To date, only six germ-line mutations identified in ENU-treated mice offspring have been characterized at the nucleotide level and reported, all of which occur at either adenine or thymine residues (10-13, 16). The experiments described here characterize the mutagenic response oftransgenic mouse testicular germ cells to acute-and multiple-dose ENU exposure and outline the spectra of mutants recovered after these exposures. These findings represent progress toward defining the in vivo spectrum of ENU mutagenesis in germ cells and explore the relationship to the in vivo somatic cell spectrum in the endogenous hprt gene. MATERIALS AND METHODSTransgenic Mice. Transgenic B6C3F1 hybrid mice were generated from natural matings (performed at Taconic Farms) of inbred female transgenic hemizygous A/lacd C57BL/6 mice (2) and male nontransgenic C3H mice. DNA was prepared from tail tips of the resulting offspring and screened for the transgene by dot-blot DNA hybridization, as described (2). Animals were maintained on a 12-hr light schedule in cages containing heat-sterilized wood chips and were provided with F4 rodent block food (Harlan Bioproducts, Haslett, MI) and tap water ad libitum.Mutagen Treatment. ENU (Sigma, Lot no. 20H0369) was dissolved in Dulbecco's phosphate-buffered saline (pH 6.0) (GIBCO) and administered within 30 min of solution preparation. Four 8-to 10-week-old male transgenic B6C3F1 mice (Big Blue mouse, Stratagene) were given three i.p. injections of 100 mg (± 2%) of ENU per kg of body weight at 7-day intervals for a cumulative multiple dos...

“…They occur predominantly in A-T base pairs (70%), indicating that this lacZ surrogate mutation model detects a high proportion of ENU-induced mutations similar to, but at a somewhat lower frequency than, those observed in some endogenous mammalian genes-for example, offspring in the specific locus test [86% (28)] and in the somatic hprt mutations in the mouse Genetics: Douglas et al [90% (29)] and monkey [94% (30)], although any differences may simply be the result of different experimental conditions including dose (31). The proportion of mutations in AT vs. G-C base pairs in the present study is nearly identical to that reported for the lacI transgenic model [i.e., 68% (5)].…”

Section: Discussionmentioning

confidence: 99%

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

Douglas

Jiao

Gingerich

et al. 1995

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by -35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than -35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial mutations. The most prominent feature of the ENU-induced basepair mutations in testicular germ cells sampled 55 days after treatment is that 70%o are induced in AT base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.in bacteria, A-T base pairs are the predominate source of mutations in mammalian species in vivo (13). This change is related to the reduced capacity of DNA alkyltransferase to remove ethyl adducts other than 06-ethylguanine from mammalian cells (13). ENU induces both spermatogonial stem cell and postspermatogonial mutations in mice (14). The former are characterized by base-substitution mutations in A-T base pairs (15-19); the latter have been shown to result in relatively more multilocus lesions (20) and, if adducts are induced late in development, mosaic mutations in the progeny result (21).In this study we have used a lacZ transgenic mouse (Muta mouse) model to determine the timing of ENU-induced mutation induction in a mixed population of germ cells isolated from seminiferous tubules and in spermatozoa isolated from the vas deferens. The mutation spectrum for mutations from mixed germ cells was also determined. It is anticipated that such information will be influential in determining the acceptability of transgenic mouse models for the study of germ-cell mutagenicity, in suggesting appropriate experimental protocols for future studies, and in alleviating the need to conduct costly experiments on the offspring of exposed animals when it is not necessary. It is envisaged that such a transgenic mouse germ-cell assay, once validated, may become a companion to the dominant lethal test in terms of its role in hazard identification.The establishment of transgenic mouse models for the quantitative detection of gene mutations (1, 2) has provided unprecedented access to the study of mutagenesis ...