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Spermatozoa comprise a large and homogeneous population of cells that may serve as an alternative to resource-intensive assays of transmissible mutations based on progeny. To evaluate mutagenic responses in spermatozoa derived from germ cells exposed to a mutagen at different stages of spermatogenesis, we compared cII mutant frequencies (MFs) in spermatozoa collected from male lambda transgenic medaka exposed to ethylnitrosourea (ENU) as either post-meiotic or pre-meiotic germ cells. cII MFs in spermatozoa exposed to ENU as spermatogonial stem cells were induced significantly, 9-fold, compared to controls, whereas, cII MFs in spermatozoa exposed as spermatozoa/late spermatids were not elevated. To directly compare responses in spermatozoa with those in progeny, we analyzed cII MFs directly in spermatozoa and in the offspring produced from identical sperm samples of ENU-exposed males. cII MFs in isolated spermatozoa exposed to ENU as post-meiotic germ cells were not significantly elevated, whereas 11-30% of the progeny derived from the identically exposed germ cells exhibited significantly elevated cII MFs, approximately 2-fold to >130-fold, compared to controls. The contradictory responses between spermatozoa and progeny analyses can be attributed to induced pre-mutational lesions that remain intact in spermatozoa but were not detected as mutations. Progeny analyses, by contrast, revealed mutant individuals with elevated cII mutant frequencies because persistent DNA damage in the spermatozoa was fixed as mutations in cells of the early stage embryo. Spermatozoa exposed to a mutagen as spermatogonial stem cells can provide an efficient means to detect the portion of transmissible mutations that were fixed as mutations in spermatozoa. The caveat is that direct analyses of mutations in spermatozoa excludes the contribution of mutations that arise from post-fertilization processes in cells of early stage embryos, and therefore may underestimate the actual frequency of mutant offspring.

An accumulating body of research indicates there is an increased cancer risk associated with chronic infections. The genus Mycobacterium contains a number of species, including M. tuberculosis, which mount chronic infections and have been implicated in higher cancer risk. Several non-tuberculosis mycobacterial species, including M. marinum, are known to cause chronic infections in fish and like human tuberculosis, often go undetected. The elevated carcinogenic potential for fish colonies infected with Mycobacterium spp. could have far reaching implications because fish models are widely used to study human diseases. Japanese medaka (Oryzias latipes) is an established laboratory fish model for toxicology, mutagenesis, and carcinogenesis; and produces a chronic tuberculosis-like disease when infected by M. marinum. We examined the role that chronic mycobacterial infections play in cancer risk for medaka. Experimental M. marinum infections of medaka alone did not increase the mutational loads or proliferative lesion incidence in all tissues examined. However, we showed that chronic M. marinum infections increased hepatocellular proliferative lesions in fish also exposed to low doses of the mutagen benzo[a]pyrene. These results indicate that chronic mycobacterial infections of medaka are acting as tumor promoters and thereby suggest increased human risks for cancer promotion in human populations burdened with chronic tuberculosis infections.

Chromium is an increasing health concern for aquatic environments, however, the mechanism of chromium toxicity in aquatic species is yet unknown. We used a medaka (Oryzias latipes) fin cell line to investigate the cytotoxicity and genotoxicity of sodium chromate, a soluble form of hexavalent chromium. We used a clonogenic cytotoxicity assay to measure sodium chromate cytotoxicity, gamma-H2A.X immunofluoresence to measure DNA double-strand breaks, and chromosome damage to measure clastogenicity. We found that sodium chromate is cytotoxic to medaka fin cells, with toxicity increasing in a concentration-dependent manner. Treatments of 0.5, 1, 5, 10, 25, 50 and 100 microM sodium chromate caused 100, 103.5, 87.8, 77.5, 40.9, 15 and 2.7% survival, respectively, relative to the control. We visualized DNA double-strand breaks in medaka cells through the formation of gamma-H2A.X foci. Breaks could be detected at concentrations as low as 1 microM. We also found that sodium chromate induces chromosomal aberrations, causing chromatid lesions and exchanges that increase with concentration. Treatments of 0, 1, 5, 10 and 25 microM sodium chromate damaged 10.3, 17, 32.3, 43 and 51.6% of metaphases and induced 13, 23, 44, 69 and 118 total aberrations in 100 metaphases, respectively. These data show that hexavalent chromium is both cytotoxic and genotoxic to fish cells. Our results set the context for future work in the medaka cell culture model and provide important tools for investigating mechanisms of toxicity in aquatic organisms.

To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated λ 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 × 10−5) in the range of 10−4 to 10−3 were mosaic mutant offspring, whereas those with MFs approaching 1 × 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. Environ. Mol. Mutagen., 2008. © 2008 Wiley-Liss, Inc.

Perfluorooctanesulfonate (PFOS) has been widely detected in the environment, wildlife and humans, but few studies have ever examined its mutagenic effect in vivo. In the present study, we use a transgenic fish model, the λ transgenic medaka, to evaluate the potential mutagenicity of PFOS in vivo following a subchronic exposure of 30 days. The mutant frequency of cII target gene was 3.46 × 10−5 in liver tissue from control fish, which increased by 1.4-fold to 4.86 × 10−5 in fish exposed to 6.7 μg/L PFOS, 1.55-fold to 5.36 × 10−5 in fish exposed to 27.6 μg/L PFOS, and 2.02-fold to 6.99 × 10−5 in fish exposed to 87.6 μg/L PFOS. This dose-dependent increase of mutant frequency was also accompanied with mutational spectrum changes associated with PFOS exposure. In particular, PFOS-induced mutation was characterized by +1 frameshift mutations, which increased from 0% in control fish to 13.2% in fish exposed to 27.6 μg/L PFOS and 14.6% in fish exposed to 87.6 μg/L PFOS. Our findings provide the first evidence of PFOS’s mutagenicity in an aquatic model system. Given the fact that most conventional mutagenic assays were negative for PFOS, we propose that PFOS-induced mutation in liver tissue of λ transgenic medaka may be mediated through compromised liver function.

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 λ 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 × 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 × 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 λ transgenic medaka as a model for identifying and characterizing potential genetic health risks associated with chemical exposures in the environment.

Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment. Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others. Much still remains to be learned from these animals, and there is a growing need for new marine models. Models for expression of marine animal genes have been limited to heterologous expression systems. While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible. This has become particularly important with the development of functional genomics in marine models. These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology.

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.

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.

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 λ vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for λ transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by λ 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 λ 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 λ 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 λ 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.

Transgenic fish were developed using a variety of transgenes, methods, and species since the first transgenic fish were produced in China in 1985. Since that time, transgenic gene knockout and gene editing technologies have been adapted for fish experimentation. Early research emphasized refinement of transgenic methodology and development of novel strains of fish with commercially beneficial traits with emphasis on growth enhancement. The production of transgenic fish has since expanded to research focused on five general applications: to enhance traits of commercially important species, to develop fish as bioreactors to produce biomedically important proteins, to enhance the utility of fish as indicators of adverse health effects associated with exposure to toxicants in aquatic environments, to develop new nonmammalian animal models for comparative biomedical research and for functional genomics studies. Transgenic fish were produced that could benefit the aquaculture industry, but regulatory agencies have yet to approve them for commercial application.

This study describes the production of transgenic medaka ( Oryzias latipes ) and mummichog ( Fundulus heteroclitus ) containing multiple copies of the bacteriophage ΦX174 am3cs70 . This work is an initial approach for measuring mutations in aquatic species using the same gene target sequence in ish and laboratory mammals. The ΦX174 sequence is unique in that there is no detectable homology with chromosomal DNA of medaka, mummichog or mice. The authors have compared cytoplasmic injection of 1–2 cell embryos with linear single copy and catenated constructs of the phage DNA. The catenated construct results in greater efficiency of gene transfer for both species in terms of copies per cell. Analyses of DNA from founder transgenic fish with methylation sensitive (HpaII) and methylation insensitive (MspI) restriction enzyme isoschizmers indicates CpG methylation of the integrated ΦX174 sequence. This study also demonstrates the efficient rescue of live phage from the chromosomal DNA of founder fish in sufficient numbers to determine a spontaneous mutation frequency for reversion of am3 . A pooled sample of 20 μg DNA from four fish yielded 1.09 × 10 7 progeny phage with a spontaneous mutation frequency of 1.83 × 10 −7 . This spontaneous mutation frequency is similar to the spontaneous frequency for the same gene indictor recovered from transgenic mice. These results demonstrate that fish containing multiple copies of ΦX174 can be produced with no obvious detrimental effects and that the overall approach may be useful in basic and applied studies of environmental mutagenesis.

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: 1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me2SO), N, N- dimethylacetamide (DMA), N, N,-dimethyl formamide (DMF), and glycerol with concentrations of 5, 10, and 15% for 60 min of incubation at 4 °C; 2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and 3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5 and 10%) did not change the sperm motility after 30 min; Me2SO, DMA, and DMF (10 and 15%) and glycerol (5, 10 and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 °C/min to 25 °C/min) and were compared to Me2SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ≤ 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ≤ 10% which was significantly lower than that of methanol and ME. With Me2SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ≤ 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.

Recent changes in the risk assessment landscape underscore the need to be able to compare the results of toxicity and dose-response testing between a growing list of animal models and, quite possibly, an array of in vitro screening assays. How do we compare test results for a given compound between vastly different species? For example, what dose level in the ambient water of a small fish model would be equivalent to 10 ppm of a given compound in the rat's drinking water? Where do we begin? To initially address these questions, and in order to compare dose-response tests in a standard rodent model with a fish model, we used the concept of molecular dose. Assays that quantify types of DNA damage that are directly relevant to carcinogenesis integrate the factors such as chemical exposure, uptake, distribution, metabolism, etc. that tend to vary so widely between different phyletic levels. We performed parallel exposures in F344 rats and Japanese medaka (Oryzias latipes) to the alkylating hepatocarcinogen, dimethylnitrosamine (DMN). In both models, we measured the DNA adducts 8-hydroxyguanine, N(7)-methylguanine and O(6)-methylguanine in the liver; mutation frequency using lambda cII transgenic medaka and lambda cII transgenic (Big Blue(R)) rats; and early morphological changes in the livers of both models using histopathology and immunohistochemistry. Pulse dose levels in fish were 0, 10, 25, 50, or 100 ppm DMN in the ambient water for 14 days. Since rats are reported to be especially sensitive to DMN, they received 0, 0.1, 1, 5, 10, or 25 ppm DMN in the drinking water for the same time period. While liver DNA adduct concentrations were similar in magnitude, mutant frequencies in the DMN-exposed medaka were up to 20 times higher than in the Big Blue rats. Future work with other compounds will generate a more complete picture of comparative dose response between different phyletic levels and will help guide risk assessors using "alternative" models.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

The study of disease is, at its core, the study of abnormal structure and function spanning levels of organization from molecules to cells to tissues to organisms (and including populations and ecosystems in the disciplines of epidemiology and epizootiology). Fish and invertebrates from marine and freshwater environments have long provided valuable models for the study of basic biological processes. Some of the earliest studies of non-self recognition and phagocytosis were conducted using sea urchins (leading to the 1908 Nobel prize for Mechnikov) while the principles of signal propagation in axons were elucidated from studies of the giant axon of squid (leading a Nobel prize in 1963 for Hodgkin & Huxley). Aquatic invertebrates and fishes have proved to be valuable model organisms based on specific physical features, such as giant axons, as well as by providing an evolutionary perspective on structure and function in higher vertebrate systems. Less recognized, however, are the contributions that these organisms have made to the study of disease. Historically, such studies have tended to fall into two very broad categories, natural versus laboratory experiments. Natural experiments are those in which spontaneously occurring disease states are discovered in wild or laboratory animal populations as a result of injurious environmental factors, infectious agents and/or mutations. Outbreaks, or epizootics, of these diseases often serve as a first warning of serious environmental problems, such as when flatfish with liver tumors were found in high numbers in Eagle Harbor in the Puget Sound area of Washington State, USA (Myers et al, 2003). This observation led to the detection of high levels of polycyclic aromatic hydrocarbons in sediments at this site, resulting in the designation of the harbor as a “superfund site” and the eventual remediation of the toxic sediments. In laboratory experiments disease states are induced under controlled circumstances, often in an attempt to determine the causes and mechanisms of spontaneously occurring diseases or to evaluate the potential injurious effects of toxins or drugs. Disease research on aquatic animals involves a wide variety of disciplines such as physiology, pathology, toxicology, immunology, microbiology, embryology, molecular biology and genetics working and a wide phylogenetic range of organisms. To bring together investigators working in such varied fields of disease research, a series of meetings focusing on use of aquatic organisms as models of human disease was begun in 2000. This special issue of Comparative Biochemistry and Physiology contains 16 manuscripts from a conference entitled “Aquatic Animal Models of Human Disease” held from September 29 to October 2, 2005, at the University of Georgia, Athens, Georgia. This volume joins similar special issues developed from the two previous meetings in this series held in 2000 and 2003 (Walter 2001; Schmale, 2004). The studies presented in this volume demonstrate that fish models can be used to investigate a wide range of disease related questions and can provide unique models for human disease processes.

The goal of this study was to provide data supporting the use of lambda transgenic medaka (Oryzias latipes) embryos to evaluate mutagens in sediments. Embryos incubated directly on sediments dosed with the reference mutagen, benzo[alpha]pyrene (BaP), were examined for BaP uptake and metabolism. Mutant frequency and mutational spectrum were assessed in the cII transgene recovered from adult medaka livers exposed as embryos. Embryos rapidly accumulated 14C-BaP and metabolized BaP to polar metabolites, indicating sediment-sorbed BaP is available for bioaccumulation and medaka embryos are capable of bioactivating this mutagen. Exposure of embryos to BaP dosed sediments significantly induced cII transgene mutant frequencies with mutations predominantly being in G:C base pairs, consistent with known mechanisms of BaP mutagenesis in transgenic mice and fish.

We examined the ability of a mixture of the predominant green tea polyphenolic compounds (GTP) to reduce benzo[a]pyrene (B[a]P)-induced mutations in the cII gene of the λ transgenic medaka. Fish were treated with 50 ppb B[a]P for 24 hr, followed by exposure to 2 ppm or 10 ppm GTP for 28 days. cII mutations in livers of fish exposed to B[a]P were increased significantly, 2.6-fold above controls. In contrast, the addition of GTP significantly reduced the frequency of cII mutants by 84%, comparable to that of controls. The frequencies of mutations at G:C basepairs, mutations that are highly characteristic of B[a]P exposure, were elevated significantly in treated fish. By comparison, B[a]P-exposed fish also treated with GTP showed reductions in these mutations, demonstrating a protective effect of GTP against B[a]P-induced mutagenesis. The antioxidant mechanism of GTP possibly played an important role in the reduction of B[a]P mutagenicity. These results corroborate findings from rodent models, showing that the protective effects of green tea extend to different species, and suggesting that similar mechanisms of B[a]P mutagenesis and GTP antimutagenesis are shared among the models. These studies illustrate the utility of λ transgenic medaka for in vivo mutation analyses and suggest that this fish may be a valuable model in chemoprevention studies. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc.

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.

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.

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.

Transgenic medaka and mummichog were developed that carry multiple copies of either lambda bacteriophage, or plasmid vectors that harbor target genes (lacZ, lacI, or cII) for mutation detection. The common approach used in the three mutation assays entails exposing fish to a potential mutagen, followed by recovering the vector from the genomic DNA, and quantifying mutations in the target genes via indicator bacteria. A number of features of the fish mutation assays were evaluated related to their potential utility in genotoxicology. The vectors were recovered from transgenic fish genomic DNA with efficiencies that surpassed those of transgenic mice that carry identical vectors. The spontaneous mutation frequencies of the three target genes obtained from the fish were comparable to one another, as well as, to transgenic mice. The induction of mutations following chemical mutagen exposure indicated predictable and sensitive responses. Individual mutations were examined to provide spontaneous and induced mutational spectra. These analyses demonstrate that the fundamental requirements for mutation detection using transgenic fish have been met.