Enhanced activation of human NK cells by drug-exposed hepatocytes.

Drug-induced liver injury (DILI) represents one of the major causes why drugs have to be withdrawn from the market.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.

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Toxicogenomics directory of rat hepatotoxicants in vivo and in cultivated hepatocytes.

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.

Hepatotoxicity of piperazine designer drugs: up-regulation of key enzymes of cholesterol and lipid biosynthesis.

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.

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

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.

"Watching the Detectives" report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November 2015.

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.

Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells.

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.

A transcriptome-based classifier to identify developmental toxicants by stem cell testing: design, validation and optimization for histone deacetylase inhibitors.

Test systems to identify developmental toxicants are urgently needed. A combination of human stem cell technology and transcriptome analysis was to provide a proof of concept that toxicants with a related mode of action can be identified and grouped for read-across. We chose a test system of developmental toxicity, related to the generation of neuroectoderm from pluripotent stem cells (UKN1), and exposed cells for 6 days to the histone deacetylase inhibitors (HDACi) valproic acid, trichostatin A, vorinostat, belinostat, panobinostat and entinostat. To provide insight into their toxic action, we identified HDACi consensus genes, assigned them to superordinate biological processes and mapped them to a human transcription factor network constructed from hundreds of transcriptome data sets. We also tested a heterogeneous group of 'mercurials' (methylmercury, thimerosal, mercury(II)chloride, mercury(II)bromide, 4-chloromercuribenzoic acid, phenylmercuric acid). Microarray data were compared at the highest non-cytotoxic concentration for all 12 toxicants. A support vector machine (SVM)-based classifier predicted all HDACi correctly. For validation, the classifier was applied to legacy data sets of HDACi, and for each exposure situation, the SVM predictions correlated with the developmental toxicity. Finally, optimization of the classifier based on 100 probe sets showed that eight genes (F2RL2, TFAP2B, EDNRA, FOXD3, SIX3, MT1E, ETS1 and LHX2) are sufficient to separate HDACi from mercurials. Our data demonstrate how human stem cells and transcriptome analysis can be combined for mechanistic grouping and prediction of toxicants. Extension of this concept to mechanisms beyond HDACi would allow prediction of human developmental toxicity hazard of unknown compounds with the UKN1 test system.

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

Toxicogenomics directory of chemically exposed human hepatocytes.

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.

Pharmacokinetics explain in vivo/in vitro discrepancies of carcinogen-induced gene expression alterations in rat liver and cultivated hepatocytes.

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.

Transcriptomics of Hepatocytes Treated with Toxicants for Investigating Molecular Mechanisms Underlying Hepatotoxicity.

Transcriptomics is a powerful tool for high-throughput gene expression profiling. Transcriptome microarray experiments conducted with RNA isolated from hepatocytes after exposure to toxicants enable a deep insight into the molecular mechanisms of hepatotoxicity. This understanding, along with structure-activity relationships underlying hepatotoxicity, will provide a novel strategy to design cost-effective and safer therapeutics. Transcriptomics studies conducted with established hepatotoxic drugs in various in vitro and in vivo hepatotoxicity test systems have contributed to the elucidation of the mechanistic basis of liver insults, which were later on substantiated at the proteomics and metabolomics levels. The present chapter is focused on comprehensive transcriptomics of cultured primary hepatocytes treated with chemicals by applying Affymetrix microarray technology. It also describes the detailed protocol for culturing of hepatocytes, their exposure to toxicants as well as sample collection, including RNA isolation, RNA target preparation and finally the hybridization to gene chips for microarray expression analysis.