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...

We adapted transgenic rodent mutation assays based on fish carrying bacteriophage lambda and plasmid pUR288 vectors to address the needs for improved methods to assess health risks from exposure to environmental mutagens and also to establish new animal models to study in vivo mutagenesis. The approach entails separating the vectors from fish genomic DNA and then shuttling them into specialized strains of E. coli bacteria to analyze spontaneous and induced mutations in either lacI and cII or lacZ mutational targets. Fish exhibited low frequencies of spontaneous mutants comparable to the sensitivity of transgenic rodent models. Mutations detected after treating fish with chemical mutagens showed concentration-dependent, tissue-specific, and time-dependent relationships. Spontaneous and induced mutational spectra also were consistent with the specificity of known mutagens, further supporting the utility of transgenic fish for studies of in vivo mutagenesis.

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