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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 [Lambda] vector that harbors the cll gene as a mutational target. We adapted a forward mutation assay, originally developed for [Lambda] transgenic rodents, to recover cll mutants efficiently from fish genomic DNA by [Lambda] in vitro packaging. After infecting and plating phage on a hfl- bacterial host, cll mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on [Lambda] transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 x [10.sup.-5] in liver, 2.9 x [10.sup.-5] in whole fish, to 1.8 x [10.sup.-5] in testes, were comparable to ranges in [Lambda] 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 [Lambda] 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

Mutagenicity assays with Salmonella have shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H ]-furanone (MX), a drinking-water disinfection by-product, is a potent mutagen, accounting for about one-third of the mutagenic potency/potential of chlorinated drinking water. The ability of MX to induce mutations was investigated in the liver of medaka ( Oryzias latipes ), a small fish model, utilizing the cII transgenic medaka strain that allows detection of in vivo mutations. Methylazoxymethanol acetate (MAMAc), a carcinogen in medaka, served as a positive control. Fish were exposed to MX at 0, 1, 10, or 30 mg/L for 96 h, whereas the MAMAc exposures were for 2 h at 0, 0.1, 1, or 10 mg/L. Both exposures were conducted under static water conditions and with fasted medaka. Following exposure, fish were returned to regular culture conditions to allow mutation expression for 15 or 40 d for MX or for 15 or 32 d for MAMAc. Mutations were not induced in medaka exposed to MX for 96 h. However, a concentration- and time-dependent increase in mutations was observed from the livers of fish exposed to 1 and 10 mg/L MAMAc. In conclusion, mutation induction was not observed in the livers of cII medaka exposed to MX for 96 h; however, studies are planned to examine mutation induction in the gills and skin to explore the possibility that MX-induced DNA damage occurs primarily in the tissues of initial contact. [ABSTRACT FROM AUTHOR]

Explores the detection of mutations in transgenic fish carrying a bacteriophage transgene target. Generation of a transgenic Medaka fish; Spontaneous and ethylnitrosourea-induced cll mutant frequencies; cll mutation spectra.

To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated lambda transgenic medaka, a small fish that carries the cII mutation target gene, as a new model for germ cell mutagenesis. Mutations in the cII gene in progeny derived from ethyl-nitrosourea (ENU)-exposed males were readily detected. Frequencies of mutant offspring, proportions of mosaic or whole body mutant offspring, and mutational spectra differed according to germ cell stage exposed to ENU. Postmeiotic germ cells (spermatozoa/late spermatids) generated a higher frequency of mutant offspring (11%) compared to premeiotic germ cells (3.5%). Individuals with cII mutant frequencies (MF) elevated more than threefold above the spontaneous MF (3 x 10(-5)) in the range of 10(-4) to 10(-3) were mosaic mutant offspring, whereas those with MFs approaching 1 x 10(-2) were whole body mutant offspring. Mosaic mutant offspring comprised the majority of mutant offspring derived from postmeiotic germ cells, and unexpectedly, from spermatogonial stem cells. Mutational spectra comprised of two different mutations, but at identical sites were unusual and characteristic of delayed mutations, in which fixation of a second mutation was delayed following fertilization. Delayed mutations and prevalence of mosaic mutant offspring add to growing evidence that implicates germ cells in mediating processes postfertilization that contribute to genomic instability in progeny. This model provides an efficient and sensitive approach to assess germ cell mutations, expands opportunities to increase understanding of fundamental mechanisms of mutagenesis, and provides a means for improved assessment of potential genetic health risks., (2007 Wiley-Liss, Inc)

1,1-Dichloropropene (1,1-DCP) is a contaminant present in both ground and surface waters used as sources for drinking water. Structural similarity to several compounds with known mutagenicity and carcinogenicity, and recent demonstration of mutagenicity in vitro, suggest this compound may be similarly mutagenic in vivo. A transgenic fish model, the lamda transgenic medaka, was used to evaluate the potential mutagenicity of this contaminant in vivo following sub-chronic exposure for 6 weeks. Mutant frequencies of the cII target gene (MF) increased six-fold in the livers of fish exposed to the lowest 1,1-DCP exposure concentration (0.44 mg/L, MF = 18.4 x 10(-5), and increased with each treatment, culminating in a 32-fold induction in fish from the highest 1,1-DCP treatment (16.60 mg/L, MF = 96.3 x 10(-5). Mutations recovered from treated fish showed a distinctive mutational spectrum comprised predominantly of +1 frameshift mutations, induced 166-fold above that of untreated animals. The majority of frameshifts were +1 insertions at thiamine and adenine. These results represent the first evidence of mutagenicity of 1,1-DCP in vivo, and of the highly characteristic spectrum of induced mutations dominated by +1 frameshift mutations. Based upon results from previous in vitro studies, the similar role of glutathione S-transferase (GSTT1-1) in the activation of 1,1-DCP to a mutagen in vivo is also suggested. This study further illustrates the utility of the lamda transgenic medaka as a model for identifying and characterizing potential genetic health risks associated with chemical exposures in the environment.

Mutagenicity assays with Salmonella have shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), a drinking-water disinfection by-product, is a potent mutagen, accounting for about one-third of the mutagenic potency/potential of chlorinated drinking water. The ability of MX to induce mutations was investigated in the liver of medaka (Oryzias latipes), a small fish model, utilizing the cII transgenic medaka strain that allows detection of in vivo mutations. Methylazoxymethanol acetate (MAMAc), a carcinogen in medaka, served as a positive control. Fish were exposed to MX at 0, 1, 10, or 30 mg/L for 96 h, whereas the MAMAc exposures were for 2 h at 0, 0.1, 1, or 10 mg/L. Both exposures were conducted under static water conditions and with fasted medaka. Following exposure, fish were returned to regular culture conditions to allow mutation expression for 15 or 40 d for MX or for 15 or 32 d for MAMAc. Mutations were not induced in medaka exposed to MX for 96 h. However, a concentration- and time-dependent increase in mutations was observed from the livers of fish exposed to 1 and 10 mg/L MAMAc. In conclusion, mutation induction was not observed in the livers of cII medaka exposed to MX for 96 h; however, studies are planned to examine mutation induction in the gills and skin to explore the possibility that MX-induced DNA damage occurs primarily in the tissues of initial contact.