Recent Work
Our focus is to gain a deeper understanding of bile transport, and the mechanisms that govern transport, in piscine species. This is important and highly relevant to both fish and humans for two primary reasons; (1) aquatic species are seeing increasing application as biomedical surrogate models for human health and disease, (2) the majority of our understanding of vertebrate hepatobiliary structure/function relationships, and in turn vertebrate biliary disease and toxicity, has been derived from mammalian liver studies. We know comparatively less about the piscine biliary system, though are we gaining greater insight into piscine hepatobiliary structure/function relationships. Because our understanding of the piscine biliary system has lagged, particularly in a comparative sense, our ability to interpret and communicate biliary disease and toxicity in piscine species has been limited. By example, cholestasis (impaired/inhibited bile transport) has never been described in fish, a fact more representative of our lack of understanding (investigation), as opposed to the lack of occurrence of this response in piscine systems. This a critical need: bile synthesis and transport, performed by the hepatobiliary system, are essential life functions; fundamental to the elimination of endogenous and exogenous metabolic byproducts, and vital to the assimilation of lipid soluble nutrients (e.g. vitamins A, K, E, triacylglycerols). Impairment or inhibition of bile synthesis & transport (cholestasis), a common response of the mammalian hepatobiliary system to xenobiotic insult, results in morbidity and mortality; the result of systemic accumulation of endogenous & exogenous compounds and their metabolites, which have deliterious effects on biological functions.
To study vertebrate liver sturcture, function and xenobitoic response we employ a novel transparent small fish animal model, the "see-through" medaka (STII: Oryzias latipes). This unique animal model permits in vivo study in living individual, and for biological questions such as cholestasis which are best studies in vivo, the STII medaka are idealy suited. For decades various color mutant strains of medaka (Oryzias latipes), acquired from natural and commercially available populations, have been maintained in the Laboratory of Freshwater Fish Stocks at Nagoya University, Japan. Cross breeding from these stocks was used to produce a stable “transparent” strain of medaka in the 1990's. See-through (STII) medaka are homozygous recessive for all four pigments (iridophores, leucophores, xanthophores, melanophores), and, exhibiting no expression of leucophores and melanophores, and minimal expression of xanthophores and iridiophores, are essentially transparent throughout their life cycle (Wakamatsu et al. 2001). We have developed and appleid non invasive in vivo imaging methodlogies that permit high resolution (< 1µm) non invasive in vivo imaging of internal organs and tissues at the subcellular level.
Our recent in vivo investigations in STII medaka elucidated structure/function relationships in both 2 and 3 dimensional contexts, revealing medaka livers to be replete with bile preductular epithelial cells (BPDECs), and the transitional biliary passageways (bile preductules, BPDs) associated with them. These investigations showed the intrahepatic biliary system in medaka to largely be an interconnected network of equidiameter (1-2 µm) canaliculi and bile preductules, organized through a polyhedral (hexagonal) structural motif, that occupies the majority of the liver corpus (~95%) uniformly. Larger bile ductules and ducts were predominantly found in the hilar and peri-hilar region of the liver, and it follows, an arborizing biliary tree (as described in mammals) was largely absent, seen only in the rudimentary branching of intrahepatic ducts from the hilar hepatic duct). From prior investigations we recognized injury to BPDECs may serve to distort bile preductular lumina and result in transient or longer alterations to intrahepatic bile flow, and that attention to BPDECs/BPDs, and their relationship to the interconnected intrahepatic biliary network, is essential to understanding the spectrum of responses of the piscine hepatobiliary system to xenobiotics that target this organ system.
With a better comparative understanding of the medaka hepatobiliary system established in our prior studies, and normalcy characterized, we have been able to investigate response of the hepatobiliary system to xenobiotics in vivo. By example: we used α-naphthylisothiocyanate (ANIT), a well described hepatotoxicant that induces hallmark responses in the mammalian biliary system, namely: cytotoxicity in biliary epithelium of bile ductules and ducts (e.g. impaired mitochondrial function, necrosis), cholestasis, and biliary tree arborization (biliary epithelial cell hyperplasia). Because biliary toxicity and cholestasis are poorly understood in piscine species, and because we now understand the medaka hepatobiliary system to be more similar to mammalian liver architecture than previously considered, we considered ANIT good toxicant for investigation of comparative hepatology. The goals of these investigations were to determine the cellular targets of ANIT in medaka and to characterize toxic response, relative to what is known regarding ANIT induced hepatotoxicity in mammalian liver.
These findings revealed the intrahepatic biliary system of medaka to be targeted by the reference hepatotoxicant, ANIT. The cytological changes (e.g. BEC hyperplasia, cytomegaly, hydropic vacuolation, attenuated/dilated canaliculi), and putative changes in bile volume and transport in ANIT exposed medaka, were consistent with those observed in rodent ANIT studies. Biliary tree “arborization”, or rather, the interpretation of this response, differs, since the vast majority of the liver corpus of medaka is comprised of a canaliculo-bile preductular network, while BECs, and their associated bile ductules and ducts, are largely localized to the liver hilus.
These findings, which describe similarities and differences between mammalian and medaka hepatobiliary systems in response to a reference hepatotoxicant (ANIT), in conjunction with our prior in vivo work characterizing normalcy, illustrate the importance of our comparative understanding of the vertebrate liver, and the significance of this understanding on the interpretation and communication of xenobiotic induced injury in piscine livers. From these and previous findings it is apparent that appreciating the spectrum of responses of the piscine liver to xenobiotics that target the this organ system, particularly in a comparative sense, requires more attention to bile preductular epithelial cells, bile preductules, and their relationship to the interconnected intrahepatic biliary network. This is becoming increasingly important given that toxicity screening in embryos and eleutheroembryos is a key factor in the regulatory evaluation of chemicals of environmental concern (e.g. REACh protocol; regulatory framework for Registration, Evaluation, Authorization and Restriction of Chemicals) (ECHA 2007), and that the liver is a key target organ of toxicity.
Our in vivo xenobiotic response work also showed for the first time in vivo evaluation of toxicity in the STII medaka, and demonstrate the ability to study and image, with high resolution, normalcy and toxicity in living individuals; a valuable diagnostic and investigatory tool. Given the described coupling of in vivo and ex vivo investigations, this suggests the future ability to integrate molecular mechanisms of disease and toxicity to system level phenotypes, a current research aim in this laboratory. (End)