Raymond Reif

Dexamethasone-dependent versus -independent markers of epithelial to mesenchymal transition in primary hepatocytes

Abstract Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4α, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.

Model-guided identification of a therapeutic strategy to reduce hyperammonemia in liver diseases

BACKGROUND & AIMS Recently, spatial-temporal/metabolic mathematical models have been established that allow the simulation of metabolic processes in tissues. We applied these models to decipher ammonia detoxification mechanisms in the liver. METHODS An integrated metabolic-spatial-temporal model was used to generate hypotheses of ammonia metabolism. Predicted mechanisms were validated using time-resolved analyses of nitrogen metabolism, activity analyses, immunostaining and gene expression after induction of liver damage in mice. Moreover, blood from the portal vein, liver vein and mixed venous blood was analyzed in a time dependent manner. RESULTS Modeling revealed an underestimation of ammonia consumption after liver damage when only the currently established mechanisms of ammonia detoxification were simulated. By iterative cycles of modeling and experiments, the reductive amidation of alpha-ketoglutarate (α-KG) via glutamate dehydrogenase (GDH) was identified as the lacking component. GDH is released from damaged hepatocytes into the blood where it consumes ammonia to generate glutamate, thereby providing systemic protection against hyperammonemia. This mechanism was exploited therapeutically in a mouse model of hyperammonemia by injecting GDH together with optimized doses of cofactors. Intravenous injection of GDH (720 U/kg), α-KG (280 mg/kg) and NADPH (180 mg/kg) reduced the elevated blood ammonia concentrations (>200 μM) to levels close to normal within only 15 min. CONCLUSION If successfully translated to patients the GDH-based therapy might provide a less aggressive therapeutic alternative for patients with severe hyperammonemia.

The ascending pathophysiology of cholestatic liver disease

In this review we develop the argument that cholestatic liver diseases, particularly primary biliary cholangitis and primary sclerosing cholangitis (PSC), evolve over time with anatomically an ascending course of the disease process. The first and early lesions are in “downstream” bile ducts. This eventually leads to cholestasis, and this causes bile salt (BS)–mediated toxic injury of the “upstream” liver parenchyma. BS are toxic in high concentration. These concentrations are present in the canalicular network, bile ducts, and gallbladder. Leakage of bile from this network and ducts could be an important driver of toxicity. The liver has a great capacity to adapt to cholestasis, and this may contribute to a variable symptom‐poor interval that is often observed. Current trials with drugs that target BS toxicity are effective in only about 50%‐60% of primary biliary cholangitis patients, with no effective therapy in PSC. This motivated us to develop and propose a new view on the pathophysiology of primary biliary cholangitis and PSC in the hope that these new drugs can be used more effectively. These views may lead to better stratification of these diseases and to recommendations on a more “tailored” use of the new therapeutic agents that are currently tested in clinical trials. Apical sodium‐dependent BS transporter inhibitors that reduce intestinal BS absorption lower the BS load and are best used in cholestatic patients. The effectiveness of BS synthesis–suppressing drugs, such as farnesoid X receptor agonists, is greatest when optimal adaptation is not yet established. By the time cytochrome P450 7A1 expression is reduced these drugs may be less effective. Anti‐inflammatory agents are probably most effective in early disease, while drugs that antagonize BS toxicity, such as ursodeoxycholic acid and nor‐ursodeoxycholic acid, may be effective at all disease stages. Endoscopic stenting in PSC should be reserved for situations of intercurrent cholestasis and cholangitis, not for cholestasis in end‐stage disease. These are arguments to consider a step‐wise pathophysiology for these diseases, with therapy adjusted to disease stage. An obstacle in such an approach is that disease stage–defining biomarkers are still lacking. This review is meant to serve as a call to prioritize the development of biomarkers that help to obtain a better stratification of these diseases. (Hepatology 2017;65:722‐738).

Adverse outcome pathways: opportunities, limitations and open questions

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

Bile canalicular dynamics in hepatocyte sandwich cultures

Many substances are hepatotoxic due to their ability to cause intrahepatic cholestasis. Therefore, there is a high demand for in vitro systems for the identification of cholestatic properties of new compounds. Primary hepatocytes cultivated in collagen sandwich cultures are known to establish bile canaliculi which enclose secreted biliary components. Cholestatic compounds are mainly known to inhibit bile excretion dynamics, but may also alter canalicular volume, or hepatocellular morphology. So far, techniques to assess time-resolved morphological changes of bile canaliculi in sandwich cultures are not available. In this study, we developed an automated system that quantifies dynamics of bile canaliculi recorded in conventional time-lapse image sequences. We validated the hepatocyte sandwich culture system as an appropriate model to study bile canaliculi in vitro by showing structural similarity measured as bile canaliculi length per hepatocyte to that observed in vivo. Moreover, bile canalicular excretion kinetics of CMFDA (5-chloromethylfluorescein diacetate) in sandwich cultures resembled closely the kinetics observed in vivo. The developed quantification technique enabled the quantification of dynamic changes in individual bile canaliculi. With this technique, we were able to clearly distinguish between sandwich cultures supplemented with dexamethasone and insulin from control cultures. In conclusion, the automated quantification system offers the possibility to systematically study the causal relationship between disturbed bile canalicular dynamics and cholestasis.

In vivo imaging of systemic transport and elimination of xenobiotics and endogenous molecules in mice.

We describe a two-photon microscopy-based method to evaluate the in vivo systemic transport of compounds. This method comprises imaging of the intact liver, kidney and intestine, the main organs responsible for uptake and elimination of xenobiotics and endogenous molecules. The image quality of the acquired movies was sufficient to distinguish subcellular structures like organelles and vesicles. Quantification of the movement of fluorescent dextran and fluorescent cholic acid derivatives in different organs and their sub-compartments over time revealed significant dynamic differences. Calculated half-lives were similar in the capillaries of all investigated organs but differed in the specific sub-compartments, such as parenchymal cells and bile canaliculi of the liver, glomeruli, proximal and distal tubules of the kidney and lymph vessels (lacteals) of the small intestine. Moreover, tools to image immune cells, which can influence transport processes in inflamed tissues, are described. This powerful approach provides new possibilities for the analysis of compound transport in multiple organs and can support physiologically based pharmacokinetic modeling, in order to obtain more precise predictions at the whole body scale.

Activated ErbB3 Translocates to the Nucleus via Clathrin-independent Endocytosis, Which Is Associated with Proliferating Cells.

Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear import of ErbB3 occurs via importin β1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3 interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate that ErbB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1. Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating tissues.

In Vitro Systems for Hepatotoxicity Testing

Hepatotoxicity is the most frequent reason of drug withdrawal from the market. Therefore, hepatocyte in vitro systems that predict human hepatotoxicity are of high importance. Although some progress has been achieved in predicting toxicity formation of major metabolites and enzyme induction ( Hewitt et al., Drug Metab Rev 39:159–234, 2007; Hengstler et al., Chem Biol Interact 125:51–73, 2000) it is still diffi cult to reliably predict idiosyncratic drug-induced liver injury (iDILI), a particularly worrying form of hepatotoxicity that can arise from many commonly prescribed drugs (Godoy et al., Arch Toxicol 87:1315–1530, 2013; Amacher, Expert Opin Drug Metab Toxicol 8:335–347, 2012). This chapter describes currently available hepatocyte in vitro systems and their possibilities as well as limitations in studying hepatotoxicity and ADME.

Bile micro-infarcts in cholestasis are initiated by rupture of the apical hepatocyte membrane and cause shunting of bile to sinusoidal blood

Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so‐called bile infarcts that correspond to Charcot‐Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon–based imaging of BDL mice was performed with fluorescent bile salts (BS) and non‐BS organic anion analogues. Key findings were followed up by matrix‐assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1‐3 days after BDL, BS concentrations in bile increased and single‐cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a “domino effect” of further death events of neighboring hepatocytes. Bile infarcts provided a trans‐epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS‐overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.

Toxicology of magnetic nanoparticles: disturbed body iron homeostasis?

Nanotoxicology is one of the cutting-edge topics of ourjournal (Gehrke et al. 2011; Xie et al. 2010; Jeong et al.2010; Marano et al. 2011). As the use of nanoparticles incommercially available products is rapidly growing, toxi-cological research has to address the risk of unintendednanomaterial exposure (Foldbjerg et al. 2011; van Berloet al. 2010; Kim et al. 2011a, b; Truong et al. 2011;Leppa¨nen et al. 2011). On the other hand, the use ofnanomaterials in diagnostics and cancer therapy is alsogrowing (Weiss and Diabate´ 2011; Gibson et al. 2011;Hoshino et al. 2011). Here, nanotoxicology has to identifypossible toxic mechanisms to avoid side effects ofpharmaceutical nanoproducts.One of the widely used pharmaceutical nanomaterials ismagnetic nanoparticle. Therefore, the editors are pleasedthat Dr. Cho from Seoul National University has acceptedour invitation and contributed a comprehensive state of theart review about application and possible toxicity of mag-neticnanoparticles(Kimetal.2011a,b;thisissue).Magneticnanoparticles are used in magnetic resonance imaging.Moreover, they allow binding of biomolecules, such asantibodies, proteins, ligand or therapeutic gens, and canthereforeimprovedeliveryofbiomoleculesanddrugs.Whenweconsiderpossibletoxiceffectsofmagneticnanoparticles,iron toxicity is one of the first possible mechanisms thatcomes to ones mind. However, small amounts of ironreleased from magnetic nanoparticles do not necessarilyhave toinduceadverse health effects, since iron isefficientlymetabolized by the reticuloendothelial system, used forbiosynthesis of hemoglobin or excreted via the kidney. Inrats even relatively high doses of 100 mg/kg of iron did notinduce toxic effects (discussed in Kim et al. 2011a, b).Therefore, the question is relevant whether magnetic nano-particles can cause a systemic or local iron overload that ishigh enough to induce toxic effects. The current article ofCho gives a comprehensive summary of animal studiesaddressing organ toxicity, particularly placenta and testis,the nervous, cardiovascular, respiratory and immune systemas well as liver and kidney. The review is highly recom-mended to anyone interested in application and possibletoxicity of magnetic nanoparticles.References