The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice.

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.

Bile Microinfarcts in Cholestasis Are Initiated by Rupture of the Apical Hepatocyte Membrane and Cause Shunting of Bile to Sinusoidal Blood.

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

Pyrrolizidine alkaloid-induced alterations of prostanoid synthesis in human endothelial cells.

Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300 µM. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I2 as the main prostanoid of endothelial cells. All effects occurred only with in vitro-metabolically activated PA lasiocarpine and senecionine. By contrast, no effect was observed for the PA echimidine, heliotrine, lasiocarpine, senecionine, senkirkine and platyphylline in the absence of an external metabolizing system up to the highest tested concentration of 500 µM. Overall, our results confirm the metabolism-dependent toxification of PA and elucidate the involved pathways. These include induction of inflammatory cytokines and deregulation of the prostanoid synthesis pathway in endothelial cells, linking for the first time PA-dependent changes in prostanoid release to distinct alterations at the mRNA and protein levels of enzymes of prostanoid synthesis.

Adverse outcome pathways: opportunities, limitations and open questions.

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

The ascending pathophysiology of cholestatic liver disease.

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

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

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

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.

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.

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

Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear import of ErbB3 occurs via importin ß1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3 interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate that ErbB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1. Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating tissues.

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

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

Bile canalicular dynamics in hepatocyte sandwich cultures.

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

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.

Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis.

Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-µm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed 'liver architectural staining' for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled 'S-phase staining', where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified parameters in adult mice (C57Bl6/N) include the hepatocyte volume (5,128.3 ± 837.8 µm(3)) and the fraction of the hepatocyte surface in contact with the neighbouring hepatocytes (67.4 ± 6.7 %), sinusoids (22.1 ± 4.8 %) and bile canaliculi (9.9 ± 3.8 %). Parameters of the sinusoidal network that we also routinely quantify include the radius of the sinusoids (4.8 ± 2.25 µm), the branching angle (32.5 ± 11.2°), the length of intersection branches (23.93 ± 5.9 µm), the number of intersection nodes per mm(3) (120.3 × 103 ± 42.1 × 10(3)), the average length of sinusoidal vessel per mm(3) (5.4 × 10(3) ± 1.4 × 10(3)mm) and the percentage of vessel volume in relation to the whole liver volume (15.3 ± 3.9) (mean ± standard deviation). Moreover, the provided parameters of the bile canalicular network are: length of the first-order branches (7.5 ± 0.6 µm), length of the second-order branches (10.9 ± 1.8 µm), length of the dead-end branches (5.9 ± 0.7 µm), the number of intersection nodes per mm(3) (819.1 × 10(3) ± 180.7 × 10(3)), the number of dead-end branches per mm(3) (409.9 × 10(3) ± 95.6 × 10(3)), the length of the bile canalicular network per mm(3) (9.4 × 10(3) ± 0.7 × 10(3) mm) and the percentage of the bile canalicular volume with respect to the total liver volume (3.4 ± 0.005). A particular strength of our technique is that quantitative parameters of hepatocytes and bile canalicular as well as sinusoidal networks can be extracted from the same tissue block. Reconstructions and quantifications performed as described in the current protocols can be used for quantitative mathematical modelling of the underlying mechanisms. Furthermore, protocols are presented for both human and pig livers. The technique is also applicable for both vibratome blocks and conventional paraffin slices.

The transcription factor CHOP, a central component of the transcriptional regulatory network induced upon CCl4 intoxication in mouse liver, is not a critical mediator of hepatotoxicity.

Since xenobiotics enter the organism via the liver, hepatocytes must cope with numerous perturbations, including modifications of proteins leading to endoplasmic reticulum stress (ER-stress). This triggers a signaling pathway termed unfolded protein response (UPR) that aims to restore homeostasis or to eliminate disturbed hepatocytes by apoptosis. In the present study, we used the well-established CCl4 hepatotoxicity model in mice to address the questions whether CCl4 induces ER-stress and, if so, whether the well-known ER-stress effector CHOP is responsible for CCl4-induced apoptosis. For this purpose, we treated mice with a high dose of CCl4 injected i.p. and followed gene expression profile over time using Affymetrix gene array analysis. This time resolved gene expression analysis allowed the identification of gene clusters with overrepresented binding sites for the three most important ER-stress induced transcription factors, CHOP, XBP1 and ATF4. Such result was confirmed by the demonstration of CCl4-induced XBP1 splicing, upregulation of CHOP at mRNA and protein levels, and translocation of CHOP to the nucleus. Two observations indicated that CHOP may be responsible for CCl4-induced cell death: (1) Nuclear translocation of CHOP was exclusively observed in the pericentral fraction of hepatocytes that deteriorate in response to CCl4 and (2) CHOP-regulated genes with previously reported pro-apoptotic function such as GADD34, TRB3 and ERO1L were induced in the pericentral zone as well. Therefore, we compared CCl4 induced hepatotoxicity in CHOP knockout versus wild-type mice. Surprisingly, genetic depletion of CHOP did not afford protection against CCl4-induced damage as evidenced by serum GOT and GPT as well as quantification of dead tissue areas. The negative result was obtained at several time points (8, 24 and 72 h) and different CCl4 doses (1.6 and 0.132 g/kg). Overall, our results demonstrate that all branches of the UPR are activated in mouse liver upon CCl4 treatment. However, CHOP does not play a critical role in CCl4-induced cell death and cannot be considered as a biomarker strictly linked to hepatotoxicity. The role of alternative UPR effectors such as XBP1 remains to be investigated.

Interferon-inducible guanylate binding protein (GBP2) is associated with better prognosis in breast cancer and indicates an efficient T cell response.

BACKGROUND: Recently, interferon-inducible guanylate binding protein (GBP2) has been discussed as a possible control factor in tumor development, which is controlled by p53, and inhibits NF-Kappa B and Rac protein as well as expression of matrix metalloproteinase 9. However, the potential role that GBP2 plays in tumor development and prognosis has not yet been studied. METHODS: We analyzed whether GBP2 mRNA levels are associated with metastasis-free interval in 766 patients with node negative breast carcinomas who did not receive systemic chemotherapy. Furthermore, response to anthracycline-based chemotherapy was studied in 768 breast cancer patients. RESULTS: High expression of GBP2 in breast carcinomas was associated with better prognosis in the univariate (P < 0.001, hazard ratio 0.763, 95 % CI 0.650-0.896) as well as in the multivariate Cox analysis (P = 0.008, hazard ratio 0.731, 95 % CI 0.580-0.920) adjusted to the established clinical factors age, pT stage, grading, hormone and ERBB2 receptor status. The association was particularly strong in subgroups with high proliferation and positive estrogen receptor status but did not reach significance in carcinomas with low expression of proliferation associated genes. Besides its prognostic capacity, GBP2 also predicted pathologically complete response to anthracycline-based chemotherapy (P = 0.0037, odds ratio 1.39, 95 % CI 1.11-1.74). Interestingly, GBP2 correlated with a recently established T cell signature, indicating tumor infiltration with T cells (R = 0.607, P < 0.001). CONCLUSION: GBP2 is associated with better prognosis in fast proliferating tumors and probably represents a marker of an efficient T cell response.