Trangenic fish as models in environmental toxicology.

Historically, fish have played significant roles in assessing potential risks associated with exposure to chemical contamination in aquatic environments. Considering the contributions of transgenic rodent models to biomedicine, it is reasoned that the development of transgenic fish could enhance the role of fish in environmental toxicology. Application of transgenic fish in environmental studies remains at an early stage, but recent introduction of new models and methods demonstrates progress. Rapid advances are most evident in the area of in vivo mutagenesis using fish carrying transgenes that serve as recoverable mutational targets. These models highlight many advantages afforded by fish as models and illustrate important issues that apply broadly to transgenic fish in environmental toxicology. Development of fish models carrying identical transgenes to those found in rodents is beneficial and has revealed that numerous aspects of in vivo mutagenesis are similar between the two classes of vertebrates. Researchers have revealed that fish exhibit frequencies of spontaneous mutations similar to rodents and respond to mutagen exposure consistent with known mutagenic mechanisms. Results have demonstrated the feasibility of in vivo mutation analyses using transgenic fish and have illustrated their potential value as a comparative animal model. Challenges to development and application of transgenic fish relate to the needs for improved efficiencies in transgenic technology and in aspects of fish husbandry and use. By taking advantage of the valuable and unique attributes of fish as test organisms, it is anticipated that transgenic fish will make significant contributions to studies of environmentally induced diseases.

Investigation of the radioadaptive response in brain and liver of pUR288 lacZ transgenic mice.

The radioadaptive response, where a small priming dose of ionizing radiation can lessen the effects of subsequent exposure to a higher radiation challenge dose, was investigated in brain and liver within transgenic mice. Although it is well characterized in models in vitro, current radioadaptive response research has focused on particular cell types (i.e., lymphocytes) and does not provide comparative data for responses of multiple tissues within an organism. Transgenic animals are useful for such comparisons, because the transgene is integrated into all cells in the body. The pUR288 lacZ plasmid-based transgenic mouse model utilizes a plasmid vector allowing highly efficient recovery of mutational targets, including large size-change mutations that result from radiation exposure. Female C57BI/6 pUR288 lacZ mice were exposed to priming doses of 0.075- to 0.375-Gy x-rays over a 3-d period. After 3 wk, they received an acute challenge dose of 2.5-Gy x-rays. Spontaneous mutant frequencies in lacZ were significantly higher in liver than in brain (6.62 x 10(-5) vs. 3.51 x 10(-5)). In the absence of a priming dose, the 2.5-Gy challenge doubled the mutant frequency of both liver and brain (13.38 x 10(-5), and 7.63 x 10(-5) respectively). Priming doses of 0.15, 0.225, and 0.375 Gy significantly reduced (by 40%) the mutagenic effects of the 2.5-Gy challenge in brain. Restriction enzyme analysis of rescued mutant plasmids revealed a decrease in large size-change mutations at the three priming doses in brain. This study demonstrates the utility of this model for the investigation of radiological processes of large size-change mutations, as well as showing a radioadaptive response in brain, but not liver, of mice in vivo.

Bacteriophage lambda and plasmid pUR288 transgenic fish models for detecting in vivo mutations.

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.

Four resource centers for fishes: specifies, stocks, and services.

A conference on "Aquaria Fish Models of Human Disease" was held September 20-23, 2000, at Southwest Texas State University, San Marcos, Texas, USA. The meeting was sponsored by the National Cancer Institute (National Institutes of Health), the Roy and Joan Mitte Foundation, and Southwest Texas State University, home of the Xiphophorus Genetic Stock Center. In conjunction with the meeting, the conference organizers asked several participants to describe those components of their research programs that provide services and information to other researchers. This article summarizes their responses.

Aquaria fish models of human disease: reports and recommendations from the working groups.

On September 21-24, 2000, the National Cancer Institute, Southwest Texas State University, and the Roy and Joan Mitte Foundation sponsored an international conference entitled "Aquaria Fish Models of Human Disease" at Southwest Texas State University (SWT), San Marcos, Texas, USA. Over 100 scientists, representing various fish model systems, participated in four roundtable working groups. We considered the first step in promoting the exciting research with fish models was to unify the efforts within this scientific community towards accomplishing specific goals. With this objective in mind, the following four working groups were convened: (1) fish cancer models: sustenance and enhancement; (2) fish genomics and transgenics: resources and technology; (3) fish pathology: standards for tumor pathology classification; and, (4) resources underpinning aquaria fish research. Each working group was charged with preparing a report of their discussions with recommendations on how researchers and funding agencies might best direct and strengthen research support to ensure a healthy future for such work. Included are the final reports from these working groups, together with a brief summary of the discussions held during the sessions and the consensus recommendations from each group.

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

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 cII gene as a mutational target. We adapted a forward mutation assay, originally developed for lambda transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by lambda 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 lambda transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 x 10(-5) in liver, 2.9 x 10(-5) in whole fish, to 1.8 x 10(-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.