Non invasive in vivo investigation of hepatobiliary structure and function in STII medaka (Oryzias latipes): methodology and applications.

BACKGROUND: A novel transparent stock of medaka (Oryzias latipes; STII), recessive for all pigments found in chromatophores, permits transcutaneous imaging of internal organs and tissues in living individuals. Findings presented describe the development of methodologies for non invasive in vivo investigation in STII medaka, and the successful application of these methodologies to in vivo study of hepatobiliary structure, function, and xenobiotic response, in both 2 and 3 dimensions. RESULTS: Using brightfield, and widefield and confocal fluorescence microscopy, coupled with the in vivo application of fluorescent probes, structural and functional features of the hepatobiliary system, and xenobiotic induced toxicity, were imaged at the cellular level, with high resolution (< 1 microm), in living individuals. The findings presented demonstrate; (1) phenotypic response to xenobiotic exposure can be investigated/imaged in vivo with high resolution (< 1 microm), (2) hepatobiliary transport of solutes from blood to bile can be qualitatively and quantitatively studied/imaged in vivo, (3) hepatobiliary architecture in this lower vertebrate liver can be studied in 3 dimensions, and (4) non invasive in vivo imaging/description of hepatobiliary development in this model can be investigated. CONCLUSION: The non-invasive in vivo methodologies described are a unique means by which to investigate biological structure, function and xenobiotic response with high resolution in STII medaka. In vivo methodologies also provide the future opportunity to integrate molecular mechanisms (e.g., genomic, proteomic) of disease and toxicity with phenotypic changes at the cellular and system levels of biological organization. While our focus has been the hepatobiliary system, other organ systems are equally amenable to in vivo study, and we consider the potential for discovery, within the context of in vivo investigation in STII medaka, as significant.

Protective response of the Ah receptor to ANIT-induced biliary epithelial cell toxicity in see-through medaka.

The adaptive role of the aryl hydrocarbon receptor (Ah receptor or AHR) in protecting against disease-related conditions remains unclear in nonmammalian models, particularly teleosts. Therefore, this study focused on the potential role of AHR in response to biliary epithelial cell toxicity and hepatobiliary alteration in medaka. See-through medaka (STII strain) were exposed for 96 h using the biliary toxicant alpha-naphthylisothiocyanate (ANIT) as a reagent, and fish were evaluated daily using histological and ultrastructural analysis, and by imaging directly through the body wall of living fish. Brightfield and transmission electron microscopy showed that a single ANIT dose (40 mg/kg) specifically induced swelling and apoptosis of bile preductular epithelial cells (BPDECs) as early as 6 h after initial exposure. Following ANIT-induced BPDEC toxicity, in vivo imaging of STII medaka showed significant gallbladder discoloration from 48-72 h. Collectively, these pathologic data suggested that ANIT exposure resulted in acute hepatobiliary changes, lasting < 96 h following initial exposure. We then tested the potential role of AHR in response to ANIT-induced hepatobiliary alteration. Overall, we demonstrated that (1) transient AHR activation and cytochrome P450 1A (CYP1A) induction in livers occurred during ANIT-induced hepatobiliary impairment, (2) pretreatment with an AHR agonist partially protected against acute hepatobiliary alteration, and (3) using a luciferase-based reporter assay, the bile pigment bilirubin weakly activated mouse AHR and binding to medaka-specific CYP1A promoter, resulting in AHR element-driven transcription. Given that bile acids and pigments are present in mammalian and fish liver, these studies collectively suggest that bile-induced AHR activation may be conserved between teleosts and rodents.

An in vivo look at vertebrate liver architecture: three-dimensional reconstructions from medaka (Oryzias latipes).

Understanding three-dimensional (3D) hepatobiliary architecture is fundamental to elucidating structure/function relationships relevant to hepatobiliary metabolism, transport, and toxicity. To date, factual information on vertebrate liver architecture in 3 dimensions has remained limited. Applying noninvasive in vivo imaging to a living small fish animal model we elucidated, and present here, the 3D architecture of this lower vertebrate liver. Our investigations show that hepatobiliary architecture in medaka is based on a polyhedral (hexagonal) structural motif, that the intrahepatic biliary system is an interconnected network of canaliculi and bile-preductules, and that parenchymal architecture in this lower vertebrate is more related to that of the mammalian liver than previously believed. The in vivo findings presented advance our comparative 3D understanding of vertebrate liver structure/function, help clarify previous discrepancies among vertebrate liver conceptual models, and pose interesting questions regarding the "functional unit" of the vertebrate liver.

Resolving mechanisms of toxicity while pursuing ecotoxicological relevance?

In this age of modern biology, aquatic toxicological research has pursued mechanisms of action of toxicants. This has provided potential tools for ecotoxicologic investigations. However, problems of biocomplexity and issues at higher levels of biological organization remain a challenge. In the 1980s and 1990s and continuing to a lesser extent today, organisms residing in highly contaminated field sites or exposed in the laboratory to calibrated concentrations of individual compounds were carefully analyzed for their responses to priority pollutants. Correlation of biochemical and structural analyses in cultured cells and tissues, as well as the in vivo exposures led to the production and application of biomarkers of exposure and effect and to our awareness of genotoxicity and its chronic manifestations, such as neoplasms, in wild fishes. To gain acceptance of these findings in the greater environmental toxicology community, "validation of the model" versus other, better-established often rodent models, was necessary and became a major focus. Resultant biomarkers were applied to heavily contaminated and reference field sites as part of effects assessment and with investigations following large-scale disasters such as oil spills or industrial accidents. Over the past 15 years, in the laboratory, small aquarium fish models such as medaka (Oryzias latipes), zebrafish (Danio rerio), platyfish (Xiphophorus species), fathead minnow (Pimephales promelas), and sheepshead minnow (Cyprinodon variegatus) were increasingly used establishing mechanisms of toxicants. Today, the same organisms provide reliable information at higher levels of biological organization relevant to ecotoxicology. We review studies resolving mechanisms of toxicity and discuss ways to address biocomplexity, mixtures of contaminants, and the need to relate individual level responses to populations and communities.

Brevetoxin Degradation and By-Product Formation via Natural Sunlight.

We investigated the effects of solar radiation on brevetoxin (PbTx2). Our findings suggest that natural sunlight mediates brevetoxin (PbTx2) degradation and results in brevetoxin by-product formation via photochemical processes.