In vivo imaging of hepatobiliary toxicity: canalicular attenuation/dilation and bile preductular lesions. (A) In vivo confocal image of untreated medaka liver (30 dpf), illustrating normal appearance of IHBPs, characterized by uniform diameter. (B – B1) In vivo confocal image, single optical section, of ANIT treated medaka liver (24 dpf) illustrating appearance of dilated and attenuated bile canaliculi (red arrowhead points to attenuation, white to dilation), 48 hrs post exposure to 2.5 μM ANIT. Canaliculi were elucidated with fluorescein isothiocyanate. Only the intrahepatic biliary passageways are fluorescent (green). Parenchyma is largely non fluorescent, aside from weak and diffuse fluorescence of hepatocellular cytosol. Canalicular dilation/attenuation appeared to be a canalicular constriction/dilation regulatory problem, as no clear alteration to hepatocyte morphology was observed in association with this change. (B1) Dilated canaliculi (gray arrowhead) were found to be up to approximately 3 times normal diameter (e.g., 3.9 μm diameter in dilated vs. 1.3 μm average diameter in normal canaliculi). Attenuated canaliculi were distinct, appearing as fine sinuous passageways measuring 0.4 μm to 0.8 μm in diameter. (C) Non invasive in vivo confocal image 10 days post exposure to 2.5 μM ANIT (chronic exposure) showing bile preductular lesions (red arrowhead), characterized by loss of preductule membrane integrity and loss of uniformity in preductule lumen diameter. Intrahepatic biliary passageways elucidated here with Bodipy C5 Ceramide. Black arrowhead illustrates normal appearance of bile preductule. (C1) Transmission electron micrograph illustrating changes to bile preductular epithelium (BPDEC) associated with preductular lesions, which showed increased cytosolic area and vacuolation (red arrowhead). In vivo observations helped lead to the hypothesis that ANIT induced BPDEC toxicity is responsible for bile preductular lesions observed, and that these cells are early targets of ANIT. (D) Example of a 3D reconstruction of damaged preductule that revealed the damaged bile passageway (green) was blind ending, not interconnected with other segments of the intrahepatic biliary network (atypical). Also shown are bile preductular epithelial cells (purple), illustrating the foci of alteration was a canaliculo-preductular junction. In (A), (B), and (B1), IHBPs elucidated with FITC, in (C) with Bodipy C5 ceramide.