Morphometric Analysis of Mast Cells in Tumor Predicts Recurrence of Hepatocellular Carcinoma After Liver Transplantation.

Tumor-infiltrating immune cells are relevant prognostic and immunotherapeutic targets in hepatocellular carcinoma (HCC). Mast cells play a key role in allergic response but may also be involved in anticancer immunity. Digital morphometric analysis of patient tissue sections has become increasingly available for clinical routine and provides unbiased quantitative data. Here, we apply morphometric analysis of mast cells to retrospectively evaluate their relevance for HCC recurrence in patients after orthotopic liver transplantation (OLT). A total of 173 patients underwent OLT for HCC at the Medical University of Vienna (21 women, 152 men; 55.2 ± 7.9 years; 74 beyond Milan criteria, 49 beyond up-to-7 criteria for liver transplantation). Tissue arrays from tumors and corresponding surrounding tissues were immunohistochemically stained for mast cell tryptase. Mast cells were quantified by digital tissue morphometric analysis and correlated with HCC recurrence. Mast cells were detected in 93% of HCC tumors and in all available surrounding liver tissues. Tumor tissues revealed lower mast cell density than corresponding surrounding tissues (P < 0.0001). Patients lacking intratumoral mast cells (iMCs) displayed larger tumors and higher tumor recurrence rates both in the whole cohort (hazard ratio [HR], 2.74; 95% confidence interval [CI], 1.09-6.93; P = 0.029) and in patients beyond transplant criteria (Milan HR, 2.81; 95% CI, 1.04-7.62; P = 0.01; up-to-7 HR, 3.58; 95% CI, 1.17-10.92; P = 0.02). Notably, high iMC identified additional patients at low risk classified outside the Milan and up-to-7 criteria, whereas low iMC identified additional patients at high risk classified within the alpha-fetoprotein French and Metroticket criteria. iMCs independently predicted tumor recurrence in a multivariate Cox regression analysis (Milan HR, 2.38; 95% CI, 1.16-4.91; P = 0.019; up-to-7 HR, 2.21; 95% CI, 1.05-4.62; P = 0.035). Conclusion: Hepatic mast cells might be implicated in antitumor immunity in HCC. Morphometric analysis of iMCs refines prognosis of HCC recurrence after liver transplantation.

Deviations of the immune cell landscape between healthy liver and hepatocellular carcinoma.

Tumor-infiltrating immune cells are highly relevant for prognosis and identification of immunotherapy targets in hepatocellular carcinoma (HCC). The recently developed CIBERSORT method allows immune cell profiling by deconvolution of gene expression microarray data. By applying CIBERSORT, we assessed the relative proportions of immune cells in 41 healthy human livers, 305 HCC samples and 82 HCC adjacent tissues. The obtained immune cell profiles provided enumeration and activation status of 22 immune cell subtypes. Mast cells were evaluated by immunohistochemistry in ten HCC patients. Activated mast cells, monocytes and plasma cells were decreased in HCC, while resting mast cells, total and naïve B cells, CD4+ memory resting and CD8+ T cells were increased when compared to healthy livers. Previously described S1, S2 and S3 molecular HCC subclasses demonstrated increased M1-polarized macrophages in the S3 subclass with good prognosis. Strong total immune cell infiltration into HCC correlated with total B cells, memory B cells, T follicular helper cells and M1 macrophages, whereas weak infiltration was linked to resting NK cells, neutrophils and resting mast cells. Immunohistochemical analysis of patient samples confirmed the reduced frequency of mast cells in human HCC tumor tissue as compared to tumor adjacent tissue. Our data demonstrate that deconvolution of gene expression data by CIBERSORT provides valuable information about immune cell composition of HCC patients.

Impact of glutathione peroxidase 4 on cell proliferation, angiogenesis and cytokine production in hepatocellular carcinoma.

Insufficient supplementation with the micronutrient selenium and persistent hepatic inflammation predispose to hepatocellular carcinoma (HCC). Inflammation-associated reactive oxygen species attack membrane lipids and form lipid hydroperoxides able to propagate oxidative hepatic damage. Selenium-containing enzyme glutathione peroxidase 4 (GPx4) antagonizes this damage by reducing lipid hydroperoxides to respective hydroxides. However, the role of GPx4 in HCC remains elusive. We generated two human HCC cell lines with stable overexpression of GPx4, performed xenotransplantation into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) host mice and characterized the tumors formed. The experimental data were verified using gene expression data from two independent HCC patient cohorts. GPx4 overexpression protected from oxidative stress and reduced intracellular free radical level. GPx4-overexpressing cells displayed impaired tumor growth, reduced proliferation, altered angiogenesis and decreased expression of clinically relevant cytokine interleukin-8 and C-reactive protein. Moreover, GPx4 overexpression impaired migration of endothelial cells in vitro, and enhanced expression of thrombospondin 1, an endogenous inhibitor of angiogenesis. In patients, GPx4 expression in tumors positively correlated with survival and was linked to pathways which regulate cell proliferation, motility, tissue remodelling, immune response and M1 macrophage polarization. The patient data largely confirmed experimental findings especially in a subclass of poor prognosis tumors with high proliferation. GPx4 suppresses formation and progression of HCC by inhibition of angiogenesis and tumor cell proliferation as well as by immune-mediated mechanisms. Modification of GPx4 expression may represent a novel tool for HCC prevention or treatment.

Mesenchyme-derived factors enhance preneoplastic growth by non-genotoxic carcinogens in rat liver.

Many frequently prescribed drugs are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action and health risks for humans remain to be elucidated. Here, we investigated the impact of two model NGC, the anti-epileptic drug phenobarbital (PB) and the contraceptive cyproterone acetate (CPA), on intrahepatic epithelial-mesenchymal crosstalk and on growth of first stages of hepatocarcinogenesis. Unaltered hepatocytes (HC) and preneoplastic HC (HCPREN) were isolated from rat liver for primary culture. DNA replication of HC and HCPREN was increased by in vitro treatment with 10 µM CPA, but not 1 mM PB. Next, mesenchymal cells (MC) obtained from liver of rats treated with either PB (50 mg/kg bw/day) or CPA (100 mg/kg bw/day), were cultured. Supernatants from both types of MC raised DNA synthesis of HC and HCPREN. This indicates that PB induces replication of HC and HCPREN only indirectly, via growth factors secreted by MC. CPA, however, acts on HC and HCPREN directly as well as indirectly via mesenchymal factors. Transcriptomics and bio-informatics revealed that PB and CPA induce extensive changes in the expression profile of MC affecting many growth factors and pathways. MC from PB-treated rats produced and secreted enhanced levels of HBEGF and GDF15, factors found to suppress apoptosis and/or induce DNA synthesis in cultured HC and HCPREN. MC from CPA-treated animals showed enhanced expression and secretion of HGF, which strongly raised DNA replication of HC and HCPREN. In conclusion, our findings reveal profound effects of two prototypical NGC on the hepatic mesenchyme. The resulting release of factors, which suppress apoptosis and/or enhance cell replication preferentially in cancer prestages, appears to be crucial for tumor promotion by NGC in the liver.

The European Registered Toxicologist (ERT): Current status and prospects for advancement.

Following its inception in 1994, the certification of European Registered Toxicologists (ERT) by EUROTOX has been recognized as ensuring professional competence as well as scientific integrity and credibility. Criteria and procedures for registration are contained in the ERT "Guidelines for Registration 2012". The register of ERT currently has over 1900 members. In order to continue the harmonisation of requirements and processes between national registering bodies as a prerequisite for official recognition of the ERT title as a standard, and to take account of recent developments in toxicology, an update of the ERT Guidelines has been prepared in a series of workshops by the EUROTOX subcommittees for education and registration, in consultation with representatives of national toxicology societies and registers. The update includes details of topics and learning outcomes for theoretical training, and how these can be assessed. The importance of continuing professional development as the cornerstone of re-registration is emphasised. To help with the process of harmonisation, it is necessary to collate and share best practices of registration conditions and procedures across Europe. Importantly, this information can also be used to audit compliance with the EUROTOX standards. As recognition of professionals in toxicology, including specialist qualifications, is becoming more important than ever, we believe that this can best be achieved based on the steps for harmonisation outlined here together with the proposed new Guidelines.

Proinflammatory mesenchymal effects of the non-genotoxic hepatocarcinogen phenobarbital: a novel mechanism of antiapoptosis and tumor promotion.

Many environmental pollutants and drugs, including steroid hormones, hypolipidemics and antiepileptics, are non-genotoxic carcinogens (NGC) in rodent liver. The mechanism of action and the risk for human health are still insufficiently known. Here, we study the effects of phenobarbital (PB), a widely used model NGC, on hepatic epithelial-mesenchymal crosstalk and the impact on hepatic apoptosis. Mesenchymal cells (MC) and hepatocytes (HC) were isolated from control and PB-treated rat livers. PB induced extensive changes in gene expression in MC and much less in HC as shown by transcriptomics with oligoarrays. In MC only, transcript levels of numerous proinflammatory cytokines were elevated. Correspondingly, ELISA on the supernatant of MC from PB-treated rats revealed enhanced release of various cytokines. In cultured HC, this supernatant caused (i) nuclear translocation and activation of nuclear factor-κB (shown by immunoblots of nuclear extracts and reporter gene assays), (ii) elevated expression of proinflammatory genes and (iii) protection from the proapoptotic action of transforming growth factor beta 1 (TGFß1). PB treatment in vivo or in vitro elevated the production and release of tumor necrosis factor alpha from MC, which was identified as mainly responsible for the inhibition of apoptosis in HC. In conclusion, our findings reveal profound proinflammatory effects of PB on hepatic mesenchyme and mesenchymal-epithelial interactions. The resulting release of cytokines acts antiapoptotic in HC, an effect crucial for tumor promotion and carcinogenesis by NGC.

Superoxide deficiency attenuates promotion of hepatocarcinogenesis by cytotoxicity in NADPH oxidase knockout mice.

Long-term exposure to carcinogens combined with chronic hepatitis contributes greatly to the worldwide high incidence of hepatocellular carcinoma (HCC). It is still unclear to which extent the release of pro-inflammatory reactive oxygen or nitrogen species contributes to the development of this malignancy. Here, we aim to elucidate the role of superoxide in a model of chemical hepatocarcinogenesis. p47(phox) knockout mice (KO), lacking superoxide formation by phagocytic NADPH oxidase (phox), and wild-type animals (WT) were subjected to two different initiation-promotion protocols: (1) single dose of diethylnitrosamine (DEN) at 6 weeks of age followed by phenobarbital (PB) via diet, or ethanol (EtOH) in drinking water; (2) DEN at neonatal age followed by three cytotoxic doses of DEN at intervals of 6-7 weeks. The appearance of tumors and prestages was quantified. There was no obvious difference in the capacity of DEN to initiate hepatocarcinogenesis in KO and WT mice. PB promoted tumor development in both genotypes without significant difference. EtOH induced steatosis significantly less in KO than in WT liver, but had no effect on tumor formation in either genotype. However, hepatocarcinogenesis by three cytotoxic DEN doses after neonatal initiation was attenuated significantly in KO. Macrophages/monocytes identified by the specific antigen F4/80 were more abundant in KO than in WT liver, possibly reflecting a compensatory response. We conclude that phox-derived superoxide is not essential but is supportive for the promotion of hepatocarcinogenesis by cytotoxic doses of DEN. The production of superoxide may therefore contribute to the promotion of hepatocarcinogenesis by cytotoxic/pro-inflammatory stimuli.

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma.

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Food-derived peroxidized fatty acids may trigger hepatic inflammation: a novel hypothesis to explain steatohepatitis.

BACKGROUND & AIMS: Obesity and hepatic steatosis are frequently associated with the development of a non-alcoholic steatohepatitis (NASH). The mechanisms driving progression of a non-inflamed steatosis to NASH are largely unknown. Here, we investigated whether ingestion of peroxidized lipids, as being present in Western style diet, triggers the development of hepatic inflammation. METHODS: Corn oil containing peroxidized fatty acids was administered to rats by gavage for 6 days. In a separate approach, hepatocytes (HC), endothelial (EC) and Kupffer cells (KC) were isolated from untreated livers, cultured, and incubated with peroxidized linoleic acid (LOOH; linoleic acid (LH) being the main fatty acid in corn oil). Samples obtained from in vivo and in vitro studies were mainly investigated by qRT-PCR and biochemical determinations of lipid peroxidation products. RESULTS: Rat treatment with peroxidized corn oil resulted in increased hepatic lipid peroxidation, upregulation of nitric oxide synthetase-2 (NOS-2), cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNFα), elevation of total nitric oxides, and increase in cd68-, cd163-, TNFα-, and/or COX-2 positive immune cells in the liver. When investigating liver cell types, LOOH elevated the secretion of TNFα, p38MAPK phosphorylation, and mRNA levels of NOS-2, COX-2, and TNFα, mainly in KC. The elevation of gene expression could be abrogated by inhibiting p38MAPK, which indicates that p38MAPK activation is involved in the pro-inflammatory effects of LOOH. CONCLUSIONS: These data show for the first time that ingestion of peroxidized fatty acids carries a considerable pro-inflammatory stimulus into the body which reaches the liver and may trigger the development of hepatic inflammation.

The major vault protein mediates resistance to epidermal growth factor receptor inhibition in human hepatoma cells.

To better understand the response of HCC to EGFR inhibition, we analyzed factors connected to the resistance of HCC cells against gefitinib. Sensitive HCC3 cells co-expressed EGFR and ErbB3 but lacked kinase-domain mutations in EGFR. Interestingly, expression of MVP was restricted to resistant cell lines, whereas ABCB1 and ABCC1 showed no association with gefitinib resistance. Moreover, ectopic MVP expression in HCC3 cells decreased gefitinib sensitivity, increased AKT phosphorylation and reduced the expression of inflammatory pathway-associated genes, whereas silencing of MVP in Hep3B and HepG2 cells increased sensitivity. These findings suggest MVP as a novel player in resistance against EGFR inhibition.

Antagonistic effects of selenium and lipid peroxides on growth control in early hepatocellular carcinoma.

UNLABELLED: Activation of the activator protein 1 (AP-1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium-lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC-1.2, HCC-3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL-8. LOOH up-regulated AP-1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock-down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL-8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP-1 component c-fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH-related antibodies (LOOH-Ab) were found, suggesting enhanced LOOH formation. LOOH-Ab correlated with serum VEGF and IL-8 and with AP-1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL-8, and HCC size (the latter only for tumors smaller than 3 cm). CONCLUSION: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP-1 activation and consequently to elevated expression of VEGF and IL-8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels.

Up-regulation of the fibroblast growth factor 8 subfamily in human hepatocellular carcinoma for cell survival and neoangiogenesis.

UNLABELLED: Fibroblast growth factors (FGFs) and their high-affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several non-liver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up-regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC-1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia-mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum-starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal-regulated kinase 1/2 and ribosomal protein S6. In contrast, down-modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells' potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC-derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. CONCLUSION: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy.

NORE1B is a putative tumor suppressor in hepatocarcinogenesis and may act via RASSF1A.

Recently, we found epigenetic silencing of the Ras effector genes NORE1B and/or RASSF1A in 97% of the hepatocellular carcinoma (HCC) investigated. This is strong evidence that the two genes are of major significance in hepatocarcinogenesis. Although RASSF1A serves as a tumor suppressor gene, the functions of NORE1B are largely unknown. Here, we studied the role of NORE1B for growth and transformation of cells. To understand the molecular mechanisms of action of the gene, we used the wild-type form and deletion mutants without the NH(2) terminus and CENTRAL domain, the Ras association (RA) domain, or the COOH-terminal SARAH-domain. Intact RA and SARAH-domains were found to be necessary for NORE1B (a) to increase the G(0)-G(1) fraction in hepatoma cells, (b) to suppress c-Myc/Ha-Ras-induced cell transformation, and (c) to interact closely with RASSF1A, as determined with fluorescence resonance energy transfer. In further studies, cell cycle delay by NORE1B was equally effective in hepatocyte cell lines with wild-type or mutant Ras suggesting that NORE1B does not interact with either Ras. In conclusion, NORE1B suppresses replication and transformation of cells as effectively as RASSF1A and thus is a putative tumor suppressor gene. NORE1B interacts physically with RASSF1A and functional loss of one of the interacting partners may lead to uncontrolled growth and transformation of hepatocytes. This may explain the frequent epigenetic silencing of NORE1B and/or RASSF1A in HCC.

Proteomics reveals acute pro-inflammatory and protective responses in rat Kupffer cells and hepatocytes after chemical initiation of liver cancer and after LPS and IL-6.

Inflammation is a key event in the development of liver cancer. We studied early inflammatory responses of Kupffer cells (KCs) and hepatocyte (HC) after cancer initiation. The chemical carcinogen N-nitrosomorpholine (NNM) was used in a rat model. We applied a comprehensive analytical strategy including metabolic labeling, 2-D PAGE, LC-MS/MS-based spot identification and shotgun proteomics and thus determined the rates of synthesis of individual proteins, compared whole tissue with isolated constituent cells and performed in vivo to in vitro comparisons of NNM effects. NNM increased synthesis of overall and 138 individual proteins identified in HC and/or KC, indicating reprogramming of metabolism favoring protection, repair and replacement of cell constituents in HC and KC. Secretome analysis by 2-D PAGE and shotgun proteomics of HC revealed the induction of acute phase proteins, in case of KC of proteases, cytokines and chemokines, indicating inflammatory effects. All responses were induced rapidly, independently of signals from other cells, and closely mimicked the pro-inflammatory and protective effects of inflammation modulators LPS in KC and IL-6 in HC. In conclusion, the carcinogen NNM exerts pro-inflammatory effects in the liver, partially by direct activation of KC. The acute inflammation and its protective component will enhance formation, survival and proliferation of initiated cells and may therefore act synergistically with the genotoxic action of the carcinogen.

Inhalative exposure to vanadium pentoxide causes DNA damage in workers: results of a multiple end point study.

BACKGROUND: Inhalative exposure to vanadium pentoxide (V(2)O(5)) causes lung cancer in rodents. OBJECTIVE: The aim of the study was to investigate the impact of V(2)O(5) on DNA stability in workers from a V(2)O(5) factory. METHODS: We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method. RESULTS: Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 microg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance. CONCLUSIONS: V(2)O(5) causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.

HB-EGF is a paracrine growth stimulator for early tumor prestages in inflammation-associated hepatocarcinogenesis.

BACKGROUND/AIMS: We studied the impact of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on inflammation-driven hepatocarcinogenesis. METHODS: HB-EGF expression was determined by qRT-PCR and immunodetection in hepatocellular adenoma and carcinoma and in mesenchymal (MC) and parenchymal liver cells obtained from different models of inflammation. The functions of HB-EGF in early hepatocarcinogenesis were assessed in co-cultures of unaltered and initiated/premalignant hepatocytes. RESULTS: In human and rat (pre)malignant liver lesions, HB-EGF levels were comparable to that of the surrounding tissue. In inflamed livers HB-EGF was expressed predominantly in MC and was further increased by pro-inflammatory lipopolysaccharide (LPS) or linoleic acid hydroperoxide (LOOH). In culture, DNA-replication occurred rather in initiated/premalignant than unaltered hepatocytes and was further elevated by LOOH- or LPS-stimulated MC-supernatants. The supernatant effects were abrogated by pre-incubation with HB-EGF-neutralizing antisera. HB-EGF itself induced DNA-replication and mitosis preferentially in the initiated/premalignant cells. When transducing hepatocytes with a dominant-negative ErbB1-construct, HB-EGF-induced DNA-replications were blocked completely in unaltered hepatocytes but incompletely in initiated/premalignant cells, which suggests elevated ErbB-mediated signal transduction in first stages of hepatocarcinogenesis. CONCLUSIONS: Pro-inflammatory stimuli induce the release of HB-EGF from MC, which stimulates DNA-replication in initiated/premalignant hepatocytes. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

Use of four new human-derived liver-cell lines for the detection of genotoxic compounds in the single-cell gel electrophoresis (SCGE) assay.

One of the main problems of in vitro genotoxicity assays is that the lack of adequate representation of drug-metabolising enzymes in indicator cell lines that are currently used in routine testing may lead to false results. In the present study, we investigated the ability of four new human-derived livercell lines to detect the DNA-damaging effects of representatives of different classes of genotoxic carcinogens that require metabolic activation, namely the nitrosamine N-nitrosodimethylamine (NDMA), the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), the polycyclic aromatic hydrocarbon benzo(a)pyrene (B(a)P) and the mycotoxin aflatoxin B1 (AFB1). Hydrogen peroxide (H2O2) was used in all experimental series as a positive control and parallel experiments were carried out with human HepG2 cells, which have been used in earlier studies. DNA damage was monitored in single-cell gel electrophoresis (SCGE) assays. Furthermore, RT-PCR experiments were carried out to study the expression of genes encoding for a panel of different phase-I and phase-II enzymes, which are involved in the activation/detoxification of genotoxic carcinogens. With one of the newly isolated hepatocellular lines, HCC1.2, positive results were obtained with all model compounds, two other new lines (HCC2 and HCC3), HepG2 and the virally immortalized line NKNT-3 were less sensitive and/or failed to detect some of the genotoxins. PCR analyses showed that all cell lines express genes coding for a variety of xenobiotic drug-metabolising enzymes. The highest levels were found in general in HCC1.2, while in NKNT-3 cells some genes were not transcribed. Overall, our results indicate that the line HCC1.2 may be useful for the development of improved in vitro genotoxicity test systems.

Cell death and autophagy: cytokines, drugs, and nutritional factors.

Cells may use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, < or = 1 microM). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including GST-pi and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch, W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda, L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435-441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet, W., Nemes, Z., Bursch, W., Fésüs, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6), 1117-1128]. Autophagy also constitutes a cell's strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 microM), resulting in the lysis of almost all cells within 24h. However, a transient (1h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1-5 microM TAM, autophagy predominant; 7-9 microM, apoptosis predominant; 15 microM, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cell's major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF-beta1. The pro-apoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)environmental changes or cell death stimuli; a cell's response may shift gradually from the elimination of damaged proteins by autophagy and the recovery to autophagic or apoptotic pathways of cell death, the failure of which eventually may result in necrosis.

Review of the toxicology of chlorpyrifos with an emphasis on human exposure and neurodevelopment.

This review examines the large body of toxicological and epidemiological information on human exposures to chlorpyrifos, with an emphasis on the controversial potential for chlorpyrifos to induce neurodevelopmental effects at low doses. The results of this review demonstrate that the use of urinary 3,5,6-trichlorpyridinol (TCPy), a metabolite of chlorpyrifos as a biomarker of nonoccupational exposure is problematic and may overestimate nonoccupational exposures to chlorpyrifos by 10-to 20-fold because of the widespread presence of both TCPy and chlorpyrifos-methyl in the food supply. Current "background" (nonoccupational) levels of exposure to chlorpyrifos are several orders of magnitude lower than those required to inhibit plasma cholinesterase activity, which is a more sensitive target than nervous system cholinesterase. However, several in vitro studies have identified putative neurodevelopmental mechanisms that are altered at concentrations of chlorpyrifos below those that inhibit cholinesterases. Although one human cohort study reported an association between maternal and cord blood chlorpyrifos levels and several measures of neurodevelopment, two other cohort studies that utilized urinary TCPy as a surrogate for chlorpyrifos exposure did not demonstrate an association. Although the weight of the scientific evidence demonstrates that current levels of chlorpyrifos exposure will not have any adverse effects on neurodevelopment that might result from inhibition of nervous system cholinesterases, several recent studies propose alternative mechanisms. Thus, further in vivo investigation on neurodevelopment in an appropriate animal model is needed; additional epidemiological studies may be warranted if a suitable, chlorpyrifos-exposed cohort can be identified and more rigorous measures of exposure are utilized.

Activins and activin antagonists in hepatocellular carcinoma.

In many parts of the world hepatocellular carcinoma (HCC) is among the leading causes of cancer-related mortality but the underlying molecular pathology is still insufficiently understood. There is increasing evidence that activins, which are members of the transforming growth factor beta (TGFbeta) superfamily of growth and differentiation factors, could play important roles in liver carcinogenesis. Activins are disulphide-linked homo- or heterodimers formed from four different beta subunits termed betaA, betaB, betaC, and betaE, respectively. Activin A, the dimer of two betaA subunits, is critically involved in the regulation of cell growth, apoptosis, and tissue architecture in the liver, while the hepatic function of other activins is largely unexplored so far. Negative regulators of activin signals include antagonists in the extracellular space like the binding proteins follistatin and FLRG, and at the cell membrane antagonistic co-receptors like Cripto or BAMBI. Additionally, in the intracellular space inhibitory Smads can modulate and control activin activity. Accumulating data suggest that deregulation of activin signals contributes to pathologic conditions such as chronic inflammation, fibrosis and development of cancer. The current article reviews the alterations in components of the activin signaling pathway that have been observed in HCC and discusses their potential significance for liver tumorigenesis.