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Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F+ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.

Primary sclerosing cholangitis (PSC) is an idiopathic chronic immune-mediated cholestatic liver disease characterized by fibro-inflammatory bile duct strictures, progressive hepatobiliary fibrosis, and gut-liver axis disruption. The pathophysiology of PSC remains insufficiently characterized, which hampers the development of effective therapies. Hepatic macrophages (MFs) such as Kupffer cells (KCs) are implicated in PSC pathogenesis, but their exact role is unclear. Using the latest markers to discriminate resident KCs (ResKCs) from their monocyte-derived counterparts (MoKCs), and two models of intrahepatic and extrahepatic cholestasis, respectively, this study showed that CLEC4F+TIM4+ ResKCs were depleted after chronic cholestatic liver injury. The infiltrating CLEC4F+TIM4- MoKCs were already enriched during the acute phase of PSC. Transcriptional profiling of hepatic MF subsets during early cholestatic injury indicated that ResKCs were indeed activated and that MoKCs expressed higher levels of pro-inflammatory and proliferative markers compared with those of ResKCs. As indicated in experiments with Clec4fDTR transgenic mice, conditional depletion of KCs, before and during early cholestasis induction, had no effect on the composition of the hepatic myeloid cell pool following injury progression and did not affect disease outcomes. Taken together, these results provide new insights into the heterogeneity of the MF pool during experimental PSC and evidence that depletion of resident and activated KCs during sclerosing cholangitis does not affect disease outcome in mice.

Cholestatic liver disease denotes any situation associated with impaired bile flow concomitant with a noxious bile acid accumulation in the liver and/or systemic circulation. Cholestatic liver disease can be subdivided into different types according to its clinical phenotype, such as biliary atresia, drug‐induced cholestasis, gallstone liver disease, intrahepatic cholestasis of pregnancy, primary biliary cholangitis and primary sclerosing cholangitis. Considerable effort has been devoted to elucidating underlying mechanisms of cholestatic liver injuries and explore novel therapeutic and diagnostic strategies using animal models. Animal models employed according to their appropriate applicability domain herein play a crucial role. This review provides an overview of currently available in vivo animal models, fit‐for‐purpose in modelling different types of cholestatic liver diseases. Moreover, a practical guide and workflow is provided which can be used for translational research purposes, including all advantages and disadvantages of currently available in vivo animal models.

Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information.

Adverse outcome pathways (AOPs) are tools to capture and visualize mechanisms driving toxicological effects. They share a common structure consisting of a molecular initiating event, a series of key events connected by key event relationships and an adverse outcome. Development and evaluation of AOPs ideally comply with guidelines issued by the Organization for Economic Cooperation and Development. AOPs have been introduced for major types of hepatotoxicity, which is not a surprise, as the liver is a frequent target for systemic adversity. Various applications for AOPs have been proposed in the areas of toxicology and chemical risk assessment, in particular in relation to the establishment of quantitative structure–activity relationships, the elaboration of prioritization strategies, and the development of novel in vitro toxicity screening tests and testing strategies.

Adverse outcome pathways (AOPs) are tools to capture and visualize mechanisms driving toxicological effects. They share a common structure consisting of a molecular initiating event, a series of key events connected by key event relationships and an adverse outcome. Development and evaluation of AOPs ideally comply with guidelines issued by the Organization for Economic Cooperation and Development. AOPs have been introduced for major types of hepatotoxicity, which is not a surprise, as the liver is a frequent target for systemic adversity. Various applications for AOPs have been proposed in the areas of toxicology and chemical risk assessment, in particular in relation to the establishment of quantitative structure-activity relationships, the elaboration of prioritization strategies, and the development of novel in vitro toxicity screening tests and testing strategies.

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

The provided dataset describes the differential gene expression profile of human hepatoma HepaRG cells cultured in monolayer configuration upon treatment with chemical compounds with cholestatic potential, including food additives sunset yellow and tartrazine and cosmetic ingredient triclosan, while being exposed to a highly concentrated bile acid mixture. Whole genome microarray Affymetrix Human U133 plus 2.0 was used to obtain the raw data followed by normalization, summarization and background adjustments by means of Robust Multichip Average Express software. Raw data of the different conditions were included as .CEL files in the Gene Expression Omnibus with accession number GSE169072. These data may serve as the basis for further refinement studies to establish an adequate transcriptomic signature of chemical-induced cholestasis fit-for-purpose in screening the cholestatic liability of different types of chemical compounds. (C) 2021 The Authors. Published by Elsevier Inc.

Cholestasis is a major pathological manifestation, often resulting in detrimental liver conditions, which occurs in a variety of indications collectively termed cholestatic liver diseases. The frequent asymptomatic character and complexity of cholestasis, together with the lack of a straightforward biomarker, hampers early detection and treatment of the condition. The 'omics' era, however, has resulted in a plethora of cholestatic indicators, yet a single clinically applicable biomarker for a given cholestatic disease remains missing. The criteria to fulfil as an ideal biomarker as well as the challenging molecular pathways in cholestatic liver diseases advocate for a scenario in which multiple biomarkers, originating from different domains, will be assessed concomitantly. This review gives an overview of classical clinical and novel molecular biomarkers in cholestasis, focusing on their benefits and drawbacks. ispartof: BIOMARKERS IN MEDICINE vol:15 issue:6 pages:437-454 ispartof: location:England status: published

The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:21 issue:18 ispartof: location:Switzerland status: published

Made available in DSpace on 2018-12-11T17:21:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 FP7 Ideas: European Research Council Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis. Department of In Vitro Toxicology and Dermato-Cosmetology Vrije Universiteit Brussel Department of Pathology School of Veterinary Medicine and Animal Science University of São Paulo Bascom Palmer Eye Institute Department of Ophthalmology University of Miami Miller School of Medicine Department of Cell Biology and Anatomy University of Miami Miller School of Medicine Department of Food and Experimental Nutrition Faculty of Pharmaceutical Sciences University of São Paulo Department of Pathology Botucatu Medical School UNESP-São Paulo State University Department of Pathology Botucatu Medical School UNESP-São Paulo State University FP7 Ideas: European Research Council: 335476

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury. ispartof: ARCHIVES OF TOXICOLOGY vol:92 issue:6 pages:1939-1952 ispartof: location:Germany status: published

Drug-induced cholestasis (DIC) poses a major challenge to the pharmaceutical industry and regulatory agencies. It causes both drug attrition and post-approval withdrawal of drugs. DIC represents itself as an impaired secretion and flow of bile, leading to the pathological hepatic and/or systemic accumulation of bile acids (BAs) and their conjugate bile salts. Due to the high number of mechanisms underlying DIC, predicting a compound's cholestatic potential during early stages of drug development remains elusive. A profound understanding of the different molecular mechanisms of DIC is, therefore, of utmost importance. Although many knowledge gaps and caveats still exist, it is generally accepted that alterations of certain hepatobiliary membrane transporters and changes in hepatocellular morphology may cause DIC. Consequently, liver models, which represent most of these mechanisms, are valuable tools to predict human DIC. Some of these models, such as membrane-based in vitro models, are exceptionally well-suited to investigate specific mechanisms (i.e. transporter inhibition) of DIC, while others, such as liver slices, encompass all relevant biological processes and, therefore, offer a better representation of the in vivo situation. In the current review, we highlight the principal molecular mechanisms associated with DIC and offer an overview and critical appraisal of the different liver models that are currently being used to predict the cholestatic potential of drugs.

Drug-induced liver injury is a major reason for discontinuation of drug development and withdrawal of drugs from the market. Intensive efforts in the last decades have focused on the establishment and finetuning of liver-based in vitro models for reliable prediction of hepatotoxicity triggered by drug candidates. Of those, primary hepatocytes and their cultures still are considered the gold standard, as they provide an acceptable reflection of the hepatic in vivo situation. Nevertheless, these in vitro systems cope with gradual deterioration of the differentiated morphological and functional phenotype. The present paper gives an overview of traditional and more recently introduced strategies to counteract this dedifferentiation process in an attempt to set up culture models that can be used for long-term testing purposes. The relevance and applicability of such optimized cultures of primary hepatocytes for the testing of drug-induced cholestatic liver injury is demonstrated.

Drug-induced liver injury is a major reason for discontinuation of drug development and withdrawal of drugs from the market. Intensive efforts in the last decades have focused on the establishment and finetuning of liver-based in vitro models for reliable prediction of hepatotoxicity triggered by drug candidates. Of those, primary hepatocytes and their cultures still are considered the gold standard, as they provide an acceptable reflection of the hepatic in vivo situation. Nevertheless, these in vitro systems cope with gradual deterioration of the differentiated morphological and functional phenotype. The present paper gives an overview of traditional and more recently introduced strategies to counteract this dedifferentiation process in an attempt to set up culture models that can be used for long-term testing purposes. The relevance and applicability of such optimized cultures of primary hepatocytes for the testing of drug-induced cholestatic liver injury is demonstrated.

A frequent side effect of many drugs includes the occurrence of cholestatic liver toxicity. Over the past couple of decades, drug-induced cholestasis has gained considerable attention, resulting in a plethora of data regarding its prevalence and mechanistic basis. Likewise, several food additives and dietary supplements have been reported to cause cholestatic liver insults in the past few years. The induction of cholestatic hepatotoxicity by other types of chemicals, in particular synthetic compounds, such as industrial chemicals, biocides, and cosmetic ingredients, has been much less documented. Such information can be found in occasional clinical case reports of accidental intake or suicide attempts as well as in basic and translational study reports on mechanisms or testing of new therapeutics in cholestatic animal models. This paper focuses on such nonpharmaceutical and nondietary synthetic chemical inducers of cholestatic liver injury, in particular alpha-naphthylisocyanate, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, methylenedianiline, paraquat, tartrazine, triclosan, 2-octynoic acid, and 2-nonynoic acid. Most of these cholestatic compounds act by similar mechanisms. This could open perspectives for the prediction of cholestatic potential of chemicals.

Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided.

Cholestasis can be defined as any situation of impaired bile secretion with concomitant accumulation of bile acids in the liver or in the systemic circulation. A variety of factors may evoke cholestasis, including genetic disorders, metabolic pathologies, infectious diseases, immunogenic stimuli, and drugs. Drug-induced cholestasis is a mechanistically complex process. At least three triggering factors of drug-induced cholestasis have been described, including effects on drug transporters, various hepatocellular changes, and altered bile canaliculi dynamics. These stimuli induce two cellular responses, each typified by a number of key events, namely a deteriorative response activated by bile acid accumulation and an adaptive response aimed at decreasing the uptake and increasing the export of bile acids into and from the liver, respectively. The mechanistic scenario of drug-induced cholestasis is described in this chapter.

Primary hepatocytes and their adherent cultures are still considered as the golden standard in the field of liver-based in vitro modeling. However, they cope with progressive deterioration of their in vivo-like morphological and functional phenotype. Among the various strategies that are used to counteract this dedifferentiation process is the seeding and cultivation of freshly isolated or cryopreserved hepatocytes between two layers of extracellular matrix scaffolds. This so-called sandwich culture system allows to restore cell-extracellular matrix interactions and thereby to delay dedifferentiation. The practical setup of the sandwich culture system of primary hepatocytes is described in this chapter.

Cholestasis can be defined as any situation of impaired bile secretion with concomitant accumulation of bile acids in the liver or in the systemic circulation. A variety of factors may evoke cholestasis, including genetic disorders, metabolic pathologies, infectious diseases, immunogenic stimuli, and drugs. Drug-induced cholestasis is a mechanistically complex process. At least three triggering factors of drug-induced cholestasis have been described, including effects on drug transporters, various hepatocellular changes, and altered bile canaliculi dynamics. These stimuli induce two cellular responses, each typified by a number of key events, namely a deteriorative response activated by bile acid accumulation and an adaptive response aimed at decreasing the uptake and increasing the export of bile acids into and from the liver, respectively. The mechanistic scenario of drug-induced cholestasis is described in this chapter.

Primary hepatocytes and their adherent cultures are still considered as the golden standard in the field of liver-based in vitro modeling. However, they cope with progressive deterioration of their in vivo-like morphological and functional phenotype. Among the various strategies that are used to counteract this dedifferentiation process is the seeding and cultivation of freshly isolated or cryopreserved hepatocytes between two layers of extracellular matrix scaffolds. This so-called sandwich culture system allows to restore cell-extracellular matrix interactions and thereby to delay dedifferentiation. The practical setup of the sandwich culture system of primary hepatocytes is described in this chapter.

Bile acid accumulation and subsequent liver damage is a frequent adverse effect induced by drugs. Considerable efforts have therefore been focused on the introduction and characterization of tools that allow reliable prediction of this type of drug-induced liver injury. Among those are the cholestatic index and transcriptomic profiling, which are typically assessed in in vitro settings. The present study was set up to test the applicability of both tools to non-pharmaceutical compounds with cholestatic potential, including the industrial compound bis(2-ethylhexyl)phthalate, the cosmetic ingredients triclosan and octynoic acid, the herbicides paraquat and quizalofop-para-ethyl, and the food additives sunset yellow and tartrazine, in a human hepatoma cell culture model of cholestatic liver injury. The cholestatic index method showed cholestatic liability of sunset yellow, tartrazine and triclosan. Of those, tartrazine induced transcriptional changes reminiscent of the transcriptional profile of cholestatic drugs. Furthermore, a number of genes were found to be uniquely modulated by tartrazine, in accordance with the cholestatic drugs atazanavir, cyclosporin A and nefazodone, which may have potential as novel transcriptomic biomarkers of chemical-induced cholestatic liver injury. In conclusion, unambiguous identification of the non-pharmaceutical compounds tested in this study as inducers of cholestasis could not be achieved.

The transcriptomic dataset (whole genome microarray Affymetrix Human U133 plus 2.0 and Affymetrix Mouse Genome 430 2.0) presented in this paper describes the differential gene expression profile of a human in vitro model of drug-induced cholestasis and a well-known mouse in vivo model of cholestasis. The in vitro model consists of human hepatoma HepaRG cells in monolayer configuration exposed to 3 different cholestatic drugs with or without bile acids. For in vivo modelling of cholestasis, mice were subjected to bile duct ligation surgery. Consecutive normalization, summarization and background adjustments have been made by means of Robust Multichip Average Express software. (C) 2020 The Author(s). Published by Elsevier Inc.

Chemical-induced liver injury can be manifested in a number of ways, such as cholestasis, steatosis, fibrosis, and cancer. The mechanisms driving these toxicological processes have been well characterized and have been embedded in adverse outcome pathway frameworks in recent years. This article provides a concise overview of these constructs.