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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-α, UV-B irradiation, interleukin-1β and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways.

Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNF␣/ ActD)-induced apoptosis. Conclusion: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction. (HEPATOLOGY 2008;48:1942-1953

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor a (TNFa) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFa sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFa. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFa is clearly dependent on Bid. Moreover, both cJun N-terminal kinase (JNK) activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFa-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFa-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFa also sensitizes to agonistic anti-Fas-induced liver damage. Conclusion: Our data suggest that TNFa can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid. (HEPATOLOGY 2011;53:282-292) E nhanced apoptosis is critically involved in many acute and chronic liver diseases, and hepatocytes are the main cell type undergoing massive cell death during liver injury. This process is regulated by a complex network of soluble and cell-associated apoptotic and inflammatory signals.1 It is therefore increasingly important to obtain insight into the mechanistic interplay of these signals to define new therapeutic strategies. In Abbreviations: ActD, actinomycin D; AST, aspartate aminotransferase; Bak, B cell lymphoma 2 homologous antagonist/killer; Bax, B cell lymphoma 2-associated X protein; Bcl2, B cell lymphoma 2; BH3, B cell lymphoma 2 homology domain 3; c-FLIP, cellular Fas-associating protein with death domain-like interleukin-1 beta-converting enzyme (FLICE) inhibitory protein; cIAP, cellular inhibitor of apoptosis; Diablo, diablo homolog; DISC, death-inducing signaling complex; ELISA, enzyme-linked immunosorbent assay; FADD, Fas-associated death domain; FasL, Fas

BackgroundCytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization.ResultsThe present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately up-regulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1beta proposed an "IL-1beta-like effect" of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase activity of the up-regulated Zc3h12a have to be considered.ConclusionsOur microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signaling pathway. Gene expression profile suggests IL-17 alone and in concert with TNF-alpha a role in sustaining liver inflammatory processes. IL-17 might exceed this function by RNA stabilization.

We report on the structure elucidation of arnamial, a new Delta(2,4)-protoilludane everninate ester from the fungus Armillaria mellea, and on the apoptotic activity of arnamial as well as the cytotoxic activity of structurally related compounds on selected human cancer cells. Arnamial showed cytotoxicity against Jurkat T cells, MCF-7 breast adenocarcinoma, CCRF-CEM lymphoblastic leukemia, and HCT-116 colorectal carcinoma cells at IC50 = 3.9, 15.4, 8.9, and 10.7 microM, respectively, and the related aryl ester melledonal C showed cytotoxic activity against CCRF-CEM cells (IC50 = 14.75 microM). [1,2-13C2]Acetate feeding supports a polyketide origin of the orsellinic acid moiety of arnamial.

The proinflammatory cytokine TNFα fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Recently, we showed that TNFα is able to sensitize primary murine hepatocytes cultured on collagen to Fas ligand-induced apoptosis and presented a mathematical model of the sensitizing effect. Here, we analyze how TNFα induces apoptosis in combination with the transcriptional inhibitor actinomycin D (ActD). Accumulation of reactive oxygen species (ROS) in response to TNFR activation turns out to be critical for sustained activation of JNK which then triggers mitochondrial pathway-dependent apoptosis. In addition, the amount of JNK is strongly upregulated in a ROS-dependent way. In contrast to TNFα plus cycloheximide no cFLIP degradation is observed suggesting a different apoptosis pathway in which the Itch-mediated cFLIP degradation and predominantly caspase-8 activation is not involved. Time-resolved data of the respective pro- and antiapoptotic factors are obtained and subjected to mathematical modeling. On the basis of these data we developed a mathematical model which reproduces the complex interplay regulating the phosphorylation status of JNK and generation of ROS. This model was fully integrated with our model of TNFα/Fas ligand sensitizing as well as with a published NF-κB-model. The resulting comprehensive model delivers insight in the dynamical interplay between the TNFα and FasL pathways, NF-κB and ROS and gives an example for successful model integration.

Pancreatic cancer progresses aggressively and owing to chemoresistance responds poorly to chemotherapy. Thus, there is an urgent need to understand the mechanisms of cancer cell resistance to generate effective strategies to circumvent intrinsic chemoresistance in this tumour indication. In this study, three pancreatic cancer cell lines, MIA PaCa-2, MDAPanc-3 and AsPC-1, were treated with the proteasome inhibitor MG-132 together with camptothecin, doxorubicin or paclitaxel, and cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The combination of MG-132 and camptothecin at a ratio of 5 : 1 gave the most promising results and enhanced cytotoxicity compared with the single compounds in MIA PaCa-2 cells. The increase was shown to be due to enhanced caspase-3 activity resulting in apoptosis. Moreover, this combination upregulated the levels of the proapoptotic protein Noxa and reduced the levels of the antiapoptotic protein Mcl-1, as demonstrated by western blotting. In contrast, the combination of MG-132 with doxorubicin also induced increased cytotoxicity, but apoptosis was decreased. The lack of an enhanced apoptosis induction could be correlated with high levels of Mcl-1 in response to the combined treatment with MG-132 and doxorubicin. Thus, the results indicate that regulation of the antiapoptotic and proapoptotic Bcl-2 family members Noxa and Mcl-1 is predicative of the effectiveness of the combination of MG-132 with different anticancer agents on apoptosis induction in pancreatic cancer cells.