Sandra Sagmeister

Potent protection of gallic acid against DNA oxidation: results of human and animal experiments.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation of oxidatively damaged DNA in humans.

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

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. (HEPATOLOGY 2012)

New cellular tools reveal complex epithelial–mesenchymal interactions in hepatocarcinogenesis

To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFβ-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.

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-1β (IL-1β), 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.

Lipid hydroperoxides from processed dietary oils enhance growth of hepatocarcinoma cells.

Linoleic acid, one of the major fatty acid in dietary oils, is an important source for hydroperoxides that may be formed in the presence of oxygen during food processing. Oxidized oils are absorbed in the intestine, transported as chylomicrones to the liver, and may affect unaltered hepatic cells as well as the process of hepatocarcinogenesis. We have studied the effects of linoleic acid hydroperoxides (LOOH) on growth and gene expression of cultured human hepatocellular carcinoma cells (HCC-1.2). The addition of LOOH to the medium of HCC-1.2 carcinoma cells caused dose-dependent cell loss and enhanced lactate dehydrogenase (LDH)-release. Under subtoxic conditions, LOOH induced intracellular hydrogen peroxide production, a decrease of glutathione content, elevated expression of the AP-1 components c-fos and c-jun as well as of the anti-apoptotic enzyme heme oxygenase 1 (HO-1). Furthermore, the cells were pushed by LOOH into the cell cycle as indicated by increased proportion of cells in the S- or G2/M-phase. The unoxidized linoleic acid was not active. Application of SnPPIX, a HO-1 inhibitor, decreased the viability of HCC-1.2 cells, indicating the protective role of HO-1 induction. This is the first evidence that lipid hydroperoxides of dietary origin may be an important driving force for carcinogenesis in the liver.

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.

Phenobarbital induces alterations in the proteome of hepatocytes and mesenchymal cells of rat livers.

Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

332 PEROXIDIZED FATTY ACIDS MAY INDUCE A PRO-INFLAMMATORY RESPONSE IN THE LIVER AND MAY TRIGGER THE OUTBREAK OF NON-ALCOHOLIC STEATOHEPATITIS (NASH)

Background: Retinal vessel diameter has been shown to be associated with CAD in the general population. However, it has not been studied in persons with NAFLD. Aim: Evaluate the relationship between Retinal vessel diameter and carotid arteries Intima-Media thickness (IMT) and coronary artery disease (CAD), in patients with NAFLD. Methods: 29 Patients with fatty liver (age 50±7) and low risk for CAD were included. Patients with Diabetes and Hypertension were excluded. 22 healthy individuals matched for age, gender and BMI served as controls. CAD was defined as a stenosis of >50% in at least 1 major coronary artery by Cardiac CT. Fatty Liver was defined as liver – spleen density (CT) <-10 HU. We measured (IMT) by Doppler US (IMT normal <0.7 women, <0.8 men), Retinal artery and vein diameter by retinal angiography, and biomarkers of insulin resistance, inflammation and oxidation. Results: NAFLD patients showed higher prevalence of CAD (38% vs. 14%, P < 0.001), coronary plaques (67% vs. 34%, P < 0.001), Higher IMT (0.98±0.3 Vs 0.83±0.1, P < 0.04), higher carotid plaques (60% Vs 40%, P < 0.001) higher HOMA (4.0±3.4 vs. 2.0±1.0, P < 0.005) and higher TG levels (200±80 vs. 150±60, P < 0.005) but Lower retinal artery-vein ratio (0.66±0.06 Vs 0.71±0.02. P < 0.01) and narrower retinal artery diameter (92.4±13 Vs 100.6±7, P < 0.04) than healthy controls. No significant difference in CRP, MDA, and Paraoxonase biomarkers levels was noted between the two groups. Multiple logistic regression showed IMT to be the strongest predictor of CAD (OR 2.3, CI 0.8–3.3, P < 0.001) in patients with NAFLD, followed by retinal A-V ratio (OR 1.5, CI 0.5–3, P < 0.01). Conclusion: Smaller retinal artery caliber and retinal A-V ratio may indicate an increased risk of CAD in patients with fatty liver even without diabetes and without hypertension.

New epithelial and mesenchymal cell lines from primary liver cancer to study cell interactions in hepatocarcinogenesis

To study cell interactions in tumor development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC), B-lymphoblastoid (BLC) and myofibroblastoid (MF) lines. In-depth characterization included cell kinetics, genotype, tumorigenicity, expression of cell-type specific markers and proteome patterns. Many functions of cells of origin were found to be preserved. Thus, HCC cells secrete albumin and α1-antitrypsin, BLC cells phagocytose and release TNF-β, other cytokines and reactive oxygen species upon stimulation, while MF cells express fibulin2, vimentin and hepatocyte growth factor (HGF). We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC and MF supernatants strongly increased DNA replication of premalignant hepatocytes. The stimulation by MF lines was mainly attributed to HGF secretion. In HCC cells, MF supernatant had only minor effects on cell growth but enhanced migration. MF lines also stimulated neoangiogenesis via vEGF release. BLC supernatant induced dramatically death of HCC cells, which could be largely abrogated by neutralizing the supernatant with TNF-β-antiserum. In conclusion, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumor cells. They offer new tools to unravel the role of the microenvironment during hepatocarcinogenesis. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Role of fibroblast growth factors in hepatocarcinogenesis of humans and rats

The fibroblast growth factors (FGF) FGF-1 and FGF-2 are often deregulated in hepatocellular carcinoma (HCC). Information is missing on the role of other FGF-family members for hepatocarcinogenesis. We therefore determined the function of FGF-8, FGF-17 and FGF-18 in the development and progression of liver cancer. Half of the rat liver tumors studied showed enhanced expression of FGF-18. In about 50% of human HCC, FGF-8, FGF-17 and FGF-18 and the corresponding FGF-receptors 3 and 4 were found to be upregulated. For functional studies we chose primary co-cultures of normal and premalignant (GSTp+) rat hepatocytes. DNA synthesis was significantly higher in GSTp+ than in normal hepatocytes indicating an inherent growth advantage of the premalignant cell population. FGF-8 and FGF-18 stimulated DNA preferentially in GSTp+ hepatocytes, while FGF-17 exhibited no effect. In HCC-derived cell lines FGF-8, FGF-17 and FGF-18 stimulated growth, which involved phosphorylation of ERK1/ 2 and S6. Conclusions: FGF-8 and FGF-18 induced preferential growth of (pre)malignant hepatocytes and are highly upregulated in liver tumors indicating autoand/or paracrine stimulation during formation and progression of the tumors. The results also show considerable similarities in the role of these FGFs in rat and human hepatocarcinogenesis implying that the molecular mechanisms underlying chemically induced rat hepatocarcinogenesis and human hepatocarcinogenesis are often identical. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007