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Fatty liver disease or the accumulation of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of a diet containing high fat and alcohol towards epigenetic aging, with respect to changes in transcriptional and epigenomic profiles. In this study, we took up a multi-omics approach and integrated gene expression, methylation signals, and chromatin signals to study the epigenomic effects of a high-fat and alcohol-containing diet on mouse hepatocytes. We identified four relevant gene network clusters that were associated with relevant pathways that promote steatosis. Using a machine learning approach, we predict specific transcription factors that might be responsible to modulate the functionally relevant clusters. Finally, we discover four additional CpG loci and validate aging-related differential CpG methylation. Differential CpG methylation linked to aging showed minimal overlap with altered methylation in steatosis., (© 2023. The Author(s).)

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (C max ) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC 10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients., Competing Interests: Declaration of interests K.N. is currently hired by AstraZeneca. P. Godoy is currently hired by Hoffman-La Roche, Ltd. A.S. is currently hired by Swiss-BioQuant AG. The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: In chronic liver diseases, inflammation induces oxidative stress and thus may contribute to the progression of liver injury, fibrosis, and carcinogenesis. The KEAP1/NRF2 axis is a major regulator of cellular redox balance. In the present study, we investigated whether the KEAP1/NRF2 system is involved in liver disease progression in humans and mice., Methods: The clinical relevance of oxidative stress was investigated by liver RNA sequencing in a well-characterized cohort of patients with non-alcoholic fatty liver disease (n = 63) and correlated with histological and clinical parameters. For functional analysis, hepatocyte-specific Nemo knockout (NEMO Δhepa ) mice were crossed with hepatocyte-specific Keap1 knockout (KEAP1 Δhepa ) mice., Results: Immunohistochemical analysis of human liver sections showed increased oxidative stress and high NRF2 expression in patients with chronic liver disease. RNA sequencing of liver samples in a human pediatric NAFLD cohort revealed a significant increase of NRF2 activation correlating with the grade of inflammation, but not with the grade of steatosis, which could be confirmed in a second adult NASH cohort. In mice, microarray analysis revealed that Keap1 deletion induces NRF2 target genes involved in glutathione metabolism and xenobiotic stress (e.g., Nqo1). Furthermore, deficiency of one of the most important antioxidants, glutathione (GSH), in NEMO Δhepa livers was rescued after deleting Keap1. As a consequence, NEMO Δhepa /KEAP1 Δhepa livers showed reduced apoptosis compared to NEMO Δhepa livers as well as a dramatic downregulation of genes involved in cell cycle regulation and DNA replication. Consequently, NEMO Δhepa /KEAP1 Δhepa compared to NEMO Δhepa livers displayed decreased fibrogenesis, lower tumor incidence, reduced tumor number, and decreased tumor size., Conclusions: NRF2 activation in patients with non-alcoholic steatohepatitis correlates with the grade of inflammation, but not steatosis. Functional analysis in mice demonstrated that NRF2 activation in chronic liver disease is protective by ameliorating fibrogenesis, initiation and progression of hepatocellular carcinogenesis., Lay Summary: The KEAP1 (Kelch-like ECH-associated protein-1)/NRF2 (erythroid 2-related factor 2) axis has a major role in regulating cellular redox balance. Herein, we show that NRF2 activity correlates with the grade of inflammation in patients with non-alcoholic steatohepatitis. Functional studies in mice actually show that NRF2 activation, resulting from KEAP1 deletion, protects against fibrosis and cancer., Competing Interests: Conflict of interest The authors have declared that no conflict of interest exists. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market. Therefore, there is an urgent need for reliable predictive in vitro tests, which unfailingly identify hepatotoxic drug candidates, reduce drug development time, expenses and the number of test animals. Currently, human hepatocytes represent the gold standard. However, the use of hepatocytes is challenging since the cells are not constantly available and lose their metabolic activity in culture. To solve these problems many different approaches have been developed in the past decades. The aim of this review is to present these approaches and to discuss the possibilities and limitations as well as future opportunities and directions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Drug-induced liver injury (DILI) represents one of the major causes why drugs have to be withdrawn from the market. In this study, we describe a new interaction between drug-exposed hepatocytes and natural killer (NK) cells. In a previous genome-wide expression analysis of primary human hepatocytes that had been exposed to clinically relevant concentrations of 148 drugs, we found that several activating ligands for NK cell receptors were regulated by various drugs (e.g., valproic acid, ketoconazole, promethazine, isoniazid). Especially expression of the activating NKG2D ligands (MICA, MICB and ULBPs) and the NKp30 ligand B7-H6 were upregulated in primary human hepatocytes upon exposure to many different drugs. Using the human hepatocyte cell lines Huh7 and HepG2, we confirmed that protein levels of activating NK cell ligands were elevated after drug exposure. Hepatocyte cell lines or primary human hepatocytes co-cultivated with NK cells caused enhanced NK cell activation after pretreatment with drugs at in vivo relevant concentrations compared to solvent controls. Enhanced NK cell activation was evident by increased cytotoxicity against hepatocytes and interferon (IFN)-γ production. NK cell activation could be blocked by specific antibodies against activating NK cell receptors. These data support the hypothesis that NK cells can modulate drug-induced liver injury by direct interaction with hepatocytes resulting in cytotoxicity and IFN-γ production.

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., (© 2018 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

The antihistaminic drug methapyrilene was withdrawn from the market in 1979 because of hepatocarcinogenicity in rats. Since then, the drug has been used as a model hepatotoxin especially for transcriptomic analyses using material from in vivo studies. Much less transcriptomics data are available from in vitro studies, and no studies have investigated proteomic effects of methapyrilene in vitro. Thus, the present study was aimed to characterize the proteomic response of primary rat hepatocytes to methapyrilene, to broaden our knowledge on the molecular mechanisms of methapyrilene toxicity, and to compare the results of collagen sandwich-cultured hepatocytes to in vivo data. In vitro methapyrilene concentrations (0.39 µM, 6.25 µM, and 100 µM) were chosen to cover an in vivo-relevant range. Based on published pharmacokinetic data they correspond to concentrations in portal vein blood for previously in vivo-tested doses of methapyrilene, up to a concentration showing slight cytotoxicity. Analysis of proteomic alterations by two-dimensional gel electrophoresis and mass-spectrometric protein identification demonstrated consistent and concentration-dependent effects of methapyrilene, in particular on mitochondrial proteins. Data suggest substantial deregulation of amino acid and ammonia metabolism and effects on mitochondrial energy supply pathways. The effects identified in vitro concur well with into previous in vivo observations. Several effects, for example, the influence of methapyrilene on S-adenosylmethionine metabolism, have not been described previously. The data suggest that already non-toxic concentrations of methapyrilene alter components of the intermediary metabolism, such as branched-chain amino acid metabolism, as well as urea and tricarboxylic cycle enzymes. In summary, data substantially add to our knowledge on molecular mechanisms of methapyrilene hepatotoxicity at the protein level.

Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC 50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC 50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC 50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC 50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity.

Transcriptomics is developing into an invaluable tool in toxicology. The aim of this study was, using a transcriptomics approach, to identify genes that respond similar to many different chemicals (including drugs and industrial compounds) in both rat liver in vivo and in cultivated hepatocytes. For this purpose, we analyzed Affymetrix microarray expression data from 162 compounds that were previously tested in a concentration-dependent manner in rat livers in vivo and in rat hepatocytes cultivated in sandwich culture. These data were obtained from the Japanese Toxicogenomics Project (TGP) and North Rhine-Westphalian (NRW) data sets, which represent 138 and 29 compounds, respectively, and have only 5 compounds in common between them. The in vitro gene expression data from the NRW data set were generated in the present study, while TGP is publicly available. For each of the data sets, the overlap between up- or down-regulated genes in vitro and in vivo was identified, and named in vitro-in vivo consensus genes. Interestingly, the in vivo-in vitro consensus genes overlapped to a remarkable extent between both data sets, and were 21-times (upregulated genes) or 12-times (down-regulated genes) enriched compared to random expectation. Finally, the genes in the TGP and NRW overlap were used to identify the upregulated genes with the highest compound coverage, resulting in a seven-gene set of Cyp1a1, Ugt2b1, Cdkn1a, Mdm2, Aldh1a1, Cyp4a3, and Ehhadh. This seven-gene set was then successfully tested with structural analogues of valproic acid that are not present in the TGP and NRW data sets. In conclusion, the seven-gene set identified in the present study responds similarly in vitro and in vivo to a wide range of different chemicals. Despite these promising results with the seven-gene set, transcriptomics with cultivated rat hepatocytes remains a challenge, because in general many genes are up- or downregulated by in vitro culture per se, respond differently to test compounds in vitro and in vivo, and/or show higher variability in the in vitro system compared to the corresponding in vivo data.

Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

The piperazine derivatives most frequently consumed for recreational purposes are 1-benzylpiperazine, 1-(3,4-methylenedioxybenzyl) piperazine, 1-(3-trifluoromethylphenyl) piperazine and 1-(4-methoxyphenyl) piperazine. Generally, they are consumed as capsules, tablets or pills but also in powder or liquid forms. Currently, the precise mechanism by which piperazine designer drugs induce hepatotoxicity and whether they act by a common pathway is unclear. To answer this question, we performed a gene array study with rat hepatocytes incubated with the four designer drugs. Non-cytotoxic concentrations were chosen that neither induce a decrease in reduced glutathione or ATP depletion. Analysis of the gene array data showed a large overlap of gene expression alterations induced by the four drugs. This 'piperazine designer drug consensus signature' included 101 up-regulated and 309 down-regulated probe sets (p < 0.05; FDR adjusted). In the up-regulated genes, GO groups of cholesterol biosynthesis represented a dominant overrepresented motif. Key enzymes of cholesterol biosynthesis up-regulated by all four piperazine drugs include sterol C4-methyloxidase, isopentyl-diphosphate-Δ-isomerase, Cyp51A1, squalene epoxidase and farnesyl diphosphate synthase. Additionally, glycoprotein transmembrane nmb, which participates in cell adhesion processes, and fatty acid desaturase 1, an enzyme that regulates unsaturation of fatty acids, were also up-regulated by the four piperazine designer drugs. Regarding the down-regulated probe sets, only one gene was common to all four piperazine derivatives, the betaine-homocysteine-S-methyltransferase 2. Analysis of transcription factor binding sites of the 'piperazine designer drug consensus signature' identified the sterol regulatory element binding protein (SREBP-1) as strongly overrepresented in the up-regulated genes. SREBP transcription factors are known to regulate multiple genes of cholesterol metabolism. In conclusion, the present study shows that piperazine designer drugs act by up-regulating key enzymes of cholesterol biosynthesis which is likely to increase the risk of phospholipidosis and steatosis.

Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The rodent liver eliminates toxic ammonia. In mammals, three enzymes (or enzyme systems) are involved in this process: glutaminase, glutamine synthetase and the urea cycle enzymes, represented by carbamoyl phosphate synthetase. The distribution of these enzymes for optimal ammonia detoxification was determined by numerical optimization. This in silico approach predicted that the enzymes have to be zonated in order to achieve maximal removal of toxic ammonia and minimal changes in glutamine concentration. Using 13 compartments, representing hepatocytes, the following predictions were generated: glutamine synthetase is active only within a narrow pericentral zone. Glutaminase and carbamoyl phosphate synthetase are located in the periportal zone in a non-homogeneous distribution. This correlates well with the paradoxical observation that in a first step glutamine-bound ammonia is released (by glutaminase) although one of the functions of the liver is detoxification by ammonia fixation. The in silico approach correctly predicted the in vivo enzyme distributions also for non-physiological conditions (e.g. starvation) and during regeneration after tetrachloromethane (CCl4) intoxication. Metabolite concentrations of glutamine, ammonia and urea in each compartment, representing individual hepatocytes, were predicted. Finally, a sensitivity analysis showed a striking robustness of the results. These bioinformatics predictions were validated experimentally by immunohistochemistry and are supported by the literature. In summary, optimization approaches like the one applied can provide valuable explanations and high-quality predictions for in vivo enzyme and metabolite distributions in tissues and can reveal unknown metabolic functions.

Background & Aims: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories., Methods: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified., Results: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC., Conclusions: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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.

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 10(6) KC, 2.7 × 10(5) LEC and 4.7 × 10(5) HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4-5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor., (© 2014 by the Society for Experimental Biology and Medicine.)

A long-term goal of numerous research projects is to identify biomarkers for in vitro systems predicting toxicity in vivo. Often, transcriptomics data are used to identify candidates for further evaluation. However, a systematic directory summarizing key features of chemically influenced genes in human hepatocytes is not yet available. To bridge this gap, we used the Open TG-GATES database with Affymetrix files of cultivated human hepatocytes incubated with chemicals, further sets of gene array data with hepatocytes from human donors generated in this study, and publicly available genome-wide datasets of human liver tissue from patients with non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular cancer (HCC). After a curation procedure, expression data of 143 chemicals were included into a comprehensive biostatistical analysis. The results are summarized in the publicly available toxicotranscriptomics directory ( http://wiki.toxbank.net/toxicogenomics-map/ ) which provides information for all genes whether they are up- or downregulated by chemicals and, if yes, by which compounds. The directory also informs about the following key features of chemically influenced genes: (1) Stereotypical stress response. When chemicals induce strong expression alterations, this usually includes a complex but highly reproducible pattern named 'stereotypical response.' On the other hand, more specific expression responses exist that are induced only by individual compounds or small numbers of compounds. The directory differentiates if the gene is part of the stereotypical stress response or if it represents a more specific reaction. (2) Liver disease-associated genes. Approximately 20 % of the genes influenced by chemicals are up- or downregulated, also in liver disease. Liver disease genes deregulated in cirrhosis, HCC, and NASH that overlap with genes of the aforementioned stereotypical chemical stress response include CYP3A7, normally expressed in fetal liver; the phase II metabolizing enzyme SULT1C2; ALDH8A1, known to generate the ligand of RXR, one of the master regulators of gene expression in the liver; and several genes involved in normal liver functions: CPS1, PCK1, SLC2A2, CYP8B1, CYP4A11, ABCA8, and ADH4. (3) Unstable baseline genes. The process of isolating and the cultivation of hepatocytes was sufficient to induce some stress leading to alterations in the expression of genes, the so-called unstable baseline genes. (4) Biological function. Although more than 2,000 genes are transcriptionally influenced by chemicals, they can be assigned to a relatively small group of biological functions, including energy and lipid metabolism, inflammation and immune response, protein modification, endogenous and xenobiotic metabolism, cytoskeletal organization, stress response, and DNA repair. In conclusion, the introduced toxicotranscriptomics directory offers a basis for a rationale choice of candidate genes for biomarker evaluation studies and represents an easy to use source of background information on chemically influenced genes.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2-2,000 μM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC-ESI-MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 μM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 μM). In summary, the rate of AA-GSH formation was found to be about 1.5-3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 μM).

Cultivated hepatocytes represent a well-established in vitro system. However, the applicability of hepatocytes in toxicogenomics is still controversially discussed. Recently, an in vivo/in vitro discrepancy has been described, whereby the non-genotoxic rat liver carcinogen methapyrilene alters the expression of the metabolizing genes SULT1A1 and ABAT, as well as the DNA damage response gene GADD34 in vitro, but not in vivo. If the collagen sandwich cultures of hepatocytes really produce false-positive data, this would compromise its application in toxicogenomics. To revisit the putative in vivo/in vitro discrepancy, we first analyzed and modeled methapyrilene concentrations in the portal vein of rats. The relatively short half-life of 2.8 h implies a rapid decrease in orally administered methapyrilene in vivo below concentrations that can cause gene expression alterations. This corresponded to the time-dependent alteration levels of GADD34, ABAT and SULT1A1 RNA in the liver: RNA levels are altered 1, 6 and 12 h after methapyrilene administration, but return to control levels after 24 and 72 h. In contrast, methapyrilene concentrations in the culture medium supernatant of primary rat hepatocyte cultures decreased slowly. This explains why GADD34, ABAT and SULT1A1 were still deregulated after 24 h exposure in vitro, but not in vivo. It should also be considered that the earliest analyzed time point in the previous in vivo studies was 24 h after methapyrilene administration. In conclusion, previously observed in vitro/in vivo discrepancy can be explained by different pharmacokinetics present in vitro and in vivo. When the in vivo half-life is short, levels of some initially altered genes may have returned to control levels already 24 h after administration.

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.

The gene CTNNB1 encoding β-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation of β-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where β-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated β-catenin (human β-catenin(S33Y)) was established using the Cre/loxP system. Expression of several downstream targets of β-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the β-catenin(S33Y) transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated β-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated β-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of β-catenin in clusters of hepatocytes, suggesting a role of cell-cell communication in this process.

Background & Aims: In injured liver, TGF-β affects all hepatic cell types and participates in wound healing and fibrogenesis. TGF-β downstream signaling is highly complex and cell type dependent, involving Smad and non-Smad signaling cascades thus requiring tight regulation. Endocytosis has gained relevance as important mechanism to control signaling initiation and termination. In this study, we investigated endocytic mechanisms for TGF-β mediated Smad and non-Smad signaling in hepatocytes., Methods: Endocytosis in hepatocytes was elucidated using chemical inhibitors, RNAi, viral gene transfer and caveolin-1-/- mice. TGF-β signaling was monitored by Western blot, reporter assays and gene expression analysis., Results: In hepatocytes, Smad activation is to a large degree accomplished AP-2 complex dependent on the hepatocyte surface without the necessity of clathrin coated pit formation or an endocytic step. In contrast, non-Smad/AKT pathway activation required functional dynamin mediated endocytosis and the presence of caveolin-1, an essential protein for caveolae formation. Furthermore, these two TGF-β signaling initiation platforms discriminate distinct signaling routes that integrate at the transcriptional level as shown for TGF-β target genes, Id1, Smad7, and CTGF. Endocytosis inhibition increased canonical Smad signaling and culminated in a superinduction of Id1 and Smad7 expression, whereas caveolin-1 mediated AKT pathway activation was required for maximal CTGF induction., Conclusions: Endocytosis is critical for TGF-β signaling regulation in hepatocytes and determines gene expression signature and (patho)physiological outcome., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Unlabelled: The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte "plasticity" and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down-regulated, genes related to mitogen-activated protein kinase (MAPK) signaling and cell cycle were up-regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP-1 (Sp1 transcription factor) (P < 0.001), all depending on MAPK signaling. Time-dependent increase of ERK1/2 phosphorylation occurred during the first 48 hours (and beyond) in the absence of cytokines, accompanied by an enhanced bromodeoxyuridine labeling index of 20%. The MEK inhibitor PD98059 blunted these effects indicating MAPK signaling as major trigger for this cytokine-independent proliferative response. In line with these in vitro findings, liver tissue of mice challenged with CCl(4) displayed hepatocytes with intense p-ERK1/2 staining and nuclear SP-1 and E2F1 expression. Furthermore, differentially expressed genes in mice after partial hepatectomy contained overrepresented TFBS for ETF, E2F1, and SP-1 and displayed increased expression of E2F1., Conclusion: Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine-independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP-1 seem to play a pronounced role in mediating proliferation-dependent differential gene expression. Similar events, but on a shorter time-scale, occur very early after liver damage in vivo., (Copyright © 2010 American Association for the Study of Liver Diseases.)

Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.

Background/aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells., Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [(3)H]-thymidine assay., Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1)., Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling., (2010 Elsevier Inc. All rights reserved.)

Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured as monolayers on dried stiff collagen dedifferentiate, loosing specialized liver functions. In contrast, softer matrix systems like gelled collagen help to preserve these structural and functional features. We show that the de-differentiation process induced in conventional dry collagen is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix that results in apoptosis resistance. A dried stiff collagen activates Akt and ERK1/2 pathways that results in apoptosis resistance. In contrast to stiff collagen, a soft collagen gel does not activate these pathways keeping the hepatocytes in a state where they remain sensitive to TGF-beta-induced apoptosis. Finally, we show that matrix-induced apoptosis resistance is reversible by re-plating cells from dried stiff to soft gel collagen. Practical consequences of these observations are that differentiated functions of hepatocytes, such as metabolism, endocytosis, and apoptosis, should be studied in hepatocyte sandwiches. On the other hand, proliferation and regeneration associated signaling can better be studied in hepatocytes cultured on collagen monolayers. In this chapter we focus on mechanisms that influence apoptosis sensitivity in cultured mouse hepatocytes.

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, HNF4 alpha, 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.

Oxidative stress has become one of the most intensively studied topics in biomedical research and is an often observed mechanism of non-genotoxic carcinogens like carbon tetrachloride. To monitor the oxidative stress status in in vitro hepatocytes, we compared thermoluminescence (TL) measurements with biochemical standard methods for oxidative stress markers. In contrast to biochemical analysis, TL measurements can be performed without any time-consuming extraction procedures by using directly collected cell material. After incubation with CCl(4) (24 h), thermo-induced light emission increased with rising concentration of CCl(4) up to eightfold at 10 mM CCl(4). Simultaneously, we determined the content of different secondary oxidative stress products, like thiobarbituric acid reactive substances and malondialdehyde. The rise of all biochemical markers complied with the increasing concentration of CCl(4). Finally, we could show that the CCl(4)-induced increase of oxidative stress markers determined by time-consuming biochemical methods perfectly correlates with the increase of high temperature bands in rapid TL measurements.

Unlabelled: Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured on monolayers of dried stiff collagen dedifferentiate, losing specialized liver functions. In this study, we show that hepatocyte dedifferentiation is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix. A dried stiff collagen activates focal adhesion kinase (FAK) via Src, leading to activation of the Akt and extracellular signal-regulated kinase (ERK) 1/2 pathways. Akt causes resistance to transforming growth factor beta (TGF-beta)-induced apoptosis by antagonizing p38, whereas ERK1/2 signaling opens the route to epithelial-mesenchymal transition (EMT). Apoptosis resistance is reversible by inhibiting Akt or Src, and EMT can be abrogated by blocking the ERK1/2 pathway. In contrast to stiff collagen, a softer collagen gel does not activate FAK, keeping the hepatocytes in a state where they remain sensitive to TGF-beta-induced apoptosis and do not undergo EMT. In this culture system, inhibition of p38 as well as overexpression of constitutively active Akt causes apoptosis resistance, whereas constitutively active Ras induces EMT. Finally, we show that matrix-induced EMT is reversible by replating cells from dried stiff to soft gel collagen. Our results demonstrate that hepatocyte dedifferentiation in vitro is an active process driven by FAK-mediated Akt and ERK1/2 signaling. This leads to similar functional and morphological alterations as observed for regenerating hepatocytes in vivo and is reversible when Akt and/or ERK1/2 signaling pathways are antagonized., Conclusion: Hepatocytes can exist in a differentiated and a dedifferentiated state that are reversible and can be switched by manipulating the responsible key factors of the signaling network.

Carbon tetrachloride (CCl(4)) represents an excellent model to study oxidative injury of cells. It is widely accepted that hepatocellular injury is a consequence of the metabolic conversion of CCl(4) into highly reactive, free radical intermediates. Among the direct toxic effects of CCl(4), stimulation of lipid peroxidation and the binding of the electrophilic radicals to membrane lipids have been suggested to play important roles in the pathogenesis of irreversible cell damage. CCl(4)-induced liver damage was modeled in cultures of rat hepatocytes with the focus on alterations of phosphatidylcholine (PC). The PC acyl chain composition was analyzed by (31)P NMR spectroscopy and MALDI-TOF mass spectrometry. The content of the membrane arachidonoyl PC was decreased by almost 30% after incubation of the cells with CCl(4). This relative decrease was found to correlate with increased concentrations of the corresponding saturated lysophosphatidylcholine (LPC). It is concluded that LPC represents a useful biomarker of CCl(4)-mediated damaging of hepatocytes. It is also speculated that de novo biosynthesis of PC is influenced by CCl(4).

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

Due to species differences, primary human hepatocytes are still the in vitro system of choice to analyse liver specific processes and functions. Human hepatocytes were cultured for several weeks in a serum-free two-dimensional culture system, which was used to study the effects of acetaminophen (APAP) on hepatocellular functions and vitality. Non-invasive determinations of albumin, urea and lactate dehydrogenase concentrations in cell culture supernatants allowed continuous monitoring for at least two weeks. APAP was applied every 4 days for 24 h. Each application reduced urea production by 25% and albumin synthesis by approximately 70% without any effects on cellular viability. After removal of the substance, hepatocellular functions returned to control levels within one (urea) to three (albumin) days. The repetitive analyses of APAP-mediated effects on cellular metabolism led to identical results for up to five cycles. The drug also caused reversible and repetitive ultrastructural modifications, in particular an almost complete replacement of rough endoplasmic reticulum by smooth endoplasmic reticulum and a massive degradation of glycogen stores. The data demonstrate the suitability of the culture system to serve as a model for repetitive testing of drug-mediated changes on hepatocellular functions, thereby reducing animal studies during drug development.

Aims: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo., Methods: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice., Results: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration., Conclusion: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

Polymorphic glutathione S-transferase (GST) enzymes are involved in the metabolism of xenobiotics. They are of particular interest when studying disease susceptibility and adverse drug responses. The present work deals with the genetic polymorphisms and expression of the five GST classes (alpha, mu, pi, theta and zeta) in human lung and hepatocyte cell lines. We have determined their bioavailability for in vitro approaches. Common genetic polymorphisms of GSTM1 (*0, null), GSTT1 (*0) and GSTP1 (*A/*B, I105V) are detectable. The frequencies of the polymorphisms are within the expected range for a Caucasian population with one exception. The GSTM1*0 allele is 1.5-fold more frequent in lung cell lines. GST mRNAs are frequently but not uniformly distributed among unstimulated in vitro conditions. Lung cell lines show an approximately six-fold higher total GST transcript expression than hepatocyte cell lines. Additional GST transcripts have been identified for GSTT1; they represent alternative new splicing variants that occur in cancerous cell lines and in healthy lung tissue and blood. GST enzyme activity is mainly influenced by GSTP1. The activity promoted by 1-chloro-2,4-dinitrobenzene is significantly correlated to the GSTP1 mRNA expression level (R2=0.77, P<0.001). Individual human cell lines thus express GST isoenzymes in a similar pattern to human tissue. The most common genetic polymorphisms are present among the cell lines and have to be considered for in vitro stimulation approaches in a combinatory pattern.

Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6beta, 16alpha, 2beta and 6alpha. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 microM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6beta-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16alpha- and 2beta-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells.

Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte does not yet exist there is a good chance that this aim may be achieved in the future.

Background & Aims: Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells., Methods: Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions., Results: In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed beta cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1., Conclusions: The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration.

Background: There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. In addition to mechanical devices supporting liver function, such as bioreactors, the transplantation of hepatocyte-like cells derived from (adult) stem cells offer great perspectives. We have generated hepatocyte-like (NeoHep) cells from terminally differentiated peripheral blood monocytes and, in this study, have evaluated these cells as a possible tool for autologous cell therapy., Methods: Peripheral blood monocytes were cultured under conditions that promote hepatocyte-like differentiation and were characterized for hepatocyte marker expression by reverse-transcriptase polymerase chain reaction, immunohistochemistry, and immunoblotting and for specific secretory and metabolic functions with the appropriate biochemical assays., Results: NeoHep cells resembled primary human hepatocytes with respect to morphology, expression of hepatocyte markers (albumin, cytochrome P450 isoenzymes, asialoglycoprotein receptor, coagulation factor VII), various secretory and metabolic functions (albumin secretion, urea production, lactate formation, and lactate dehydrogenase and aspartate transaminase release), and drug detoxification activities (phase I metabolization of ethoxycoumarin into 7OH-coumarin after stimulation with 3-methylcholanthren, induction of CYP3A4 activity, and phase II metabolization through UDP-glucuronidation of 4-methyl-umbelliferone)., Conclusions: These data convincingly show that NeoHep cells display a phenotype and specific in vitro metabolic functions that are quantitatively and qualitatively comparable in part with those of primary human hepatocytes. These cells could thus be clinically applied in an autologous setting for the treatment of end-stage liver diseases or for improving liver function in patients who have undergone critical liver-mass resection.

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is a psychoactive designer drug of abuse that is sold under the street names "Venus", "Bromo", "Erox", "XTC" or "Nexus". Concern has been raised because only little is known about its toxicity and metabolism in humans. In the present study we incubated 2C-B with human, monkey, dog, rabbit, rat and mouse hepatocytes to identify the metabolites formed and to determine possible toxic effects as evidenced by an ATP assay. Our data allow construction of the main metabolic pathways of 2C-B. Oxidative deamination results in the 2-(4-bromo-2,5-dimethoxyphenyl)-ethanol (BDMPE) and 4-bromo-2,5-dimethoxyphenylacetic acid (BDMPAA) metabolites. Additionally, 4-bromo-2,5-dimethoxybenzoic acid (BDMBA) can be produced also by oxidative deamination. Further metabolism of BDMPE and BDMPAA may occur by demethylation. Alternatively, the later metabolites can be generated by demethylation of 2C-B followed by oxidative deamination. Two remarkable interspecies differences in metabolism of 2C-B were observed (i) a hitherto unknown metabolite, 4-bromo-2,5-dimethoxy-phenol (BDMP), was identified after incubation only with mouse hepatocytes; (ii) 2-(4-bromo-2-hydroxy-5-methoxyphenyl)-ethanol (B-2-HMPE) was produced by hepatocytes from human, monkey and rabbit but not by dog, rat and mouse. Comparing the toxic effects of 2C-B between hepatocytes of the six examined species we observed only minor interspecies differences. However, large inter-individual differences in susceptibility of hepatocytes from three human donors were observed.

Several cases of death associated with 4-methylthioamphetamine (4-MTA) have raised public concern about the abuse of this designer drug that is usually sold as "Ecstasy" or "Flatliners". Since only very little is known about the metabolism of 4-MTA in humans we performed an in vitro study incubating racemic 4-MTA with primary hepatocytes isolated from three male human donors. Additionally, hepatocytes from male monkey (Cynomolgus), dog (Beagle), rabbit (Chinchilla), rat (Sprague-Dawley), and mouse (CD1) were examined for the metabolism of racemic 4-MTA. We observed that 4-MTA was not extensively metabolised by hepatocytes from all species examined. The main metabolite was identified as 4-methylthiobenzoic acid which, for the first time has been described as a human metabolite. In addition to metabolism we also examined 4-MTA-induced toxicity as evidenced by the ATP cellular content. Interestingly, one of the three human donors showed a dramatically increased sensitivity to the reduction in ATP content induced by 4-MTA. Comparing the species examined, the most extensive formation of 4-methylthiobenzoic acid was observed in the rabbit hepatocytes followed by human, monkey, dog and mouse hepatocytes, whereas no formation of 4-methylthiobenzoic acid was seen in the rat hepatocytes. Toxicity data suggest that rabbit hepatocytes are more resistant to 4-MTA than the other species, which may be due to the more extensive metabolism. In conclusion, we have shown that 4-methylthiobenzoic acid is the main metabolite formed from 4-MTA by human hepatocytes and also by the hepatocytes of the other tested species except the rat. Toxicity data suggest only moderate interspecies differences.

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.