Gunda Millonig

Extrahepatic cholestasis increases liver stiffness (FibroScan) irrespective of fibrosis

Transient elastography (FibroScan [FS]) is a novel non‐invasive tool to assess liver fibrosis/cirrhosis. However, it remains to be determined if other liver diseases such as extrahepatic cholestasis interfere with fibrosis assessment because liver stiffness is indirectly measured by the propagation velocity of an ultrasound wave within the liver. In this study, we measured liver stiffness immediately before endoscopic retrograde cholangiopancreatography and 3 to 12 days after successful biliary drainage in patients with extrahepatic cholestasis mostly due to neoplastic invasion of the biliary tree. Initially elevated liver stiffness decreased in 13 of 15 patients after intervention, in 10 of them markedly. In three patients, liver stiffness was elevated to a degree that suggested advanced liver cirrhosis (mean, 15.2 kPa). Successful drainage led to a drop of bilirubin by 2.8 to 9.8 mg/dL whereas liver stiffness almost normalized (mean, 7.1 kPa). In all patients with successful biliary drainage, the decrease of liver stiffness highly correlated with decreasing bilirubin (Spearmans ρ = 0.67, P < 0.05) with a mean decrease of liver stiffness of 1.2 ± 0.56 kPa per 1 g/dL bilirubin. Two patients, in whom liver stiffness did not decrease despite successful biliary drainage, had advanced liver cirrhosis and multiple liver metastases, respectively. The relationship between extrahepatic cholestasis and liver stiffness was reproduced in an animal model of bile duct ligation in landrace pigs where liver stiffness increased from 4.6 kPa to 8.8 kPa during 120 minutes of bile duct ligation and decreased to 6.1 kPa within 30 minutes after decompression. Conclusion: Extrahepatic cholestasis increases liver stiffness irrespective of fibrosis. Once extrahepatic cholestasis is excluded (e.g., by liver imaging and laboratory parameters) transient elastography is a valuable tool to assess liver fibrosis in chronic liver diseases. (HEPATOLOGY 2008.)

Liver stiffness is directly influenced by central venous pressure

BACKGROUND & AIMS Liver stiffness (LS) as measured by transient elastography [Fibroscan] offers a novel non-invasive approach to assess liver cirrhosis. Since Fibroscan seems to be unreliable in patients with congestive heart failure, it remains to be determined whether hemodynamic changes affect LS irrespective of fibrosis. METHODS & RESULTS Using landrace pigs, we studied the direct relationship between the central venous pressure and LS measured by Fibroscan. Clamping of the inferior caval vein increased LS from 3.1 to 27.8kPa while reopening reversed LS within 5min to almost normal values of 5.1kPa. We then studied LS as a function of venous pressure in the isolated pig liver by clamping the upper and lower caval, portal vein and hepatic artery. The stepwise increase of intravenous pressure to 36cm of water column (3.5kPa) linearly and reversibly increased LS to the upper detection limit of 75kPa. We finally measured LS in 10 patients with decompensated congestive heart failure before and after recompensation. Initial LS was elevated in all patients, in 8 of them to a degree that suggested liver cirrhosis (median 40.7kPa). Upon recompensation with a median weight loss of 3.0kg, LS decreased in all 10 patients down to a median LS of 17.8kPa. Inflammation could not account for increased LS since initial liver enzyme counts were only slightly elevated and did not change significantly. CONCLUSION LS is a direct function of central venous pressure which should be considered when assessing the degree of fibrosis.

Ethanol‐induced cytochrome P4502E1 causes carcinogenic etheno‐DNA lesions in alcoholic liver disease

Oxidative stress is thought to play a major role in the pathogenesis of hepatocellular cancer (HCC), a frequent complication of alcoholic liver disease (ALD). However, the underlying mechanisms are poorly understood. In hepatocytes of ALD patients, we recently detected by immunohistochemistry significantly increased levels of carcinogenic etheno‐DNA adducts that are formed by the reaction of the major lipid peroxidation product, 4‐hydroxynonenal (4‐HNE) with nucleobases. In the current study, we show that protein‐bound 4‐HNE and etheno‐DNA adducts both strongly correlate with cytochrome P450 2E1 (CYP2E1) expression in patients with ALD (r = 0.9, P < 0.01). Increased levels of etheno‐DNA adducts were also detected in the liver of alcohol‐fed lean (Fa/?) and obese (fa/fa) Zucker rats. The number of nuclei in hepatocytes stained positively for etheno‐DNA adducts correlated significantly with CYP2E1 expression (r = 0.6, P = 0.03). To further assess the role of CYP2E1 in the formation of etheno‐DNA adducts, HepG2 cells stably transfected with human CYP2E1 were exposed to ethanol with or without chlormethiazole (CMZ), a specific CYP2E1 inhibitor. Ethanol increased etheno‐DNA adducts in the nuclei of CYP2E1‐transfected HepG2 cells in a concentration‐dependent and time‐dependent manner, but not in vector mock‐transfected control cells. CMZ blocked the generation of etheno‐DNA adducts by 70%‐90% (P < 0.01). Conclusion: Our data support the assumption that ethanol‐mediated induction of hepatic CYP2E1 leading inter alia to highly miscoding lipid peroxidation–derived DNA lesions may play a central role in hepatocarcinogenesis in patients with ALD. (HEPATOLOGY 2009.)

Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.

Exposing cells to H2O2: A quantitative comparison between continuous low-dose and one-time high-dose treatments

Most studies investigating the influence of H2O2 on cells in culture apply nonphysiological concentrations over nonphysiological time periods (i.e., a one-time bolus that is metabolized in minutes). As an alternative, the glucose oxidase/catalase (GOX/CAT) system allows application of physiologically relevant H2O2 concentrations (300nM-10µM) over physiologically relevant time periods (up to 24h). Recent findings suggest that bolus and GOX/CAT treatments can lead to opposing cellular responses, thus warranting a quantitative comparison between the two approaches. First, we established a reaction-diffusion model that can predict the behavior of the GOX/CAT system with spatiotemporal resolution, thus aiding selection of optimal experimental conditions for its application. Measurements of H2O2 concentration in the cellular supernatant with the luminol/hypochlorite system were consistent with the predictions of the model. Second, we compared the impact of bolus and GOX/CAT treatments on cytosolic H2O2 levels over time. Intracellular H2O2 was monitored by the response of the thiol peroxidase Prx2 and the H2O2 sensor roGFP2-Orp1. We found that Prx2 rapidly and reversibly responds to submicromolar H2O2 levels and accurately reflects kinetic competition with cellular catalase. Our measurements reveal fundamental differences in the dynamic response of cellular H2O2 concentrations following either bolus or GOX/CAT treatments. Thus, different, or even opposing, biological outcomes from differing means of H2O2 delivery may be expected. Cellular responses induced by bolus treatment may not occur under GOX/CAT conditions, and vice versa.

Ethanol‐mediated carcinogenesis in the human esophagus implicates CYP2E1 induction and the generation of carcinogenic DNA‐lesions

Chronic alcohol consumption is a major risk factor for esophageal cancer. Various mechanisms may mediate carcinogenesis including the genotoxic effect of acetaldehyde and oxidative stress. Ethanol exerts its carcinogenic effect in the liver among others via the induction of cytochrome P450 2E1 (CYP2E1) and the generation of carcinogenic etheno‐DNA adducts. Here we investigated if such effects can also be observed in the human esophagus. We studied nontumorous esophageal biopsies of 37 patients with upper aerodigestive tract cancer and alcohol consumption of 102.3 ± 131.4 g/day (range: 15–600 g) as well as 16 controls without tumors (12 teetotalers and 4 subjects with a maximum of 25 g ethanol/day). CYP2E1, etheno‐DNA adducts and Ki67 as a marker for cell proliferation were determined immunohistologically. Chronic alcohol ingestion resulted in a significant induction of CYP2E1 (p = 0.015) which correlated with the amount of alcohol consumed (r = 0.6, p < 0.001). Furthermore, a significant correlation between CYP2E1 and the generation of the carcinogenic exocyclic etheno‐DNA adducts 1,N6‐ethenodeoxyadenosine (r = 0.93, p < 0.001) and 3,N4‐ethenodeoxycytidine (r = 0.92, p < 0.001) was observed. Etheno‐DNA adducts also correlated significantly with cell proliferation (p < 0.01), which was especially enhanced in patients who both drank and smoked (p < 0.001). Nonsmokers and nondrinkers had the lowest rate of cell proliferation, CYP2E1 expression and DNA lesions. Our data demonstrate for the first time an induction of CYP2E1 in the esophageal mucosa by ethanol in a dose dependent manner in man and may explain, at least in part, the generation of carcinogenic DNA lesions in this target organ.

The GOX/CAT system: A novel enzymatic method to independently control hydrogen peroxide and hypoxia in cell culture

The increasing demand in studying cellular functions in cultured cells under various levels of oxygen and hydrogen peroxide (H2O2) is only partly fulfilled by conventional approaches such as hypoxia chambers, bolus additions of H2O2 or redox-cycling drugs. This article describes the recently developed enzymatic GOX/CAT system consisting of glucose oxidase (GOX) and catalase (CAT) that allows the independent control and maintenance of both H2O2 and hypoxia in cell culture. In contrast to hypoxia chambers, the GOX/CAT system more rapidly induces hypoxia within minutes at a defined rate. The degree of hypoxia is dependent on the GOX activity and the diffusion distance of oxygen from the medium surface to the adherent cells. In contrast, H2O2 levels are solely controlled by the ratio of GOX and CAT activities. They can be adjusted at non-toxic or toxic dosages over 24 hours. Thus, the GOX/CAT system mimics a non-phosphorylating respiratory chain and allows to adjust H2O2 levels under hypoxic conditions truly simulating H2O2 release e.g. by inflammatory cells or intracellular sources. GOX/CAT can be employed to address many questions ranging from redox signaling to ischemia/reperfusion studies in transplantation medicine. Factors such as HIF1 alpha that respond both to hypoxia and H2O2 are an especially attractive target for the novel methodology. Several applications are discussed in detail to demonstrate the technical requirements and potentials. In addition, simplified protocols are presented for cell or molecular biology labs without dedicated biophysical equipment.

Sustained submicromolar H2O2 levels induce hepcidin via Signal Transducer and Activator of Transcription 3 (STAT3)

Background: Hepcidin, the systemic iron regulator, is induced during inflammation and leads to low circulating and increased intracellular iron levels. Results: (Patho)physiologically relevant H2O2 levels up-regulate hepcidin via STAT3 in cultured liver cells. Conclusion: Intracellular and extracellular H2O2 acts similarly to IL-6 on hepcidin up-regulation and requires a functional STAT3-binding site. Significance: H2O2 is an important link between inflammation and iron metabolism. The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H2O2 (0.3–6 μm), concentrations that are comparable with levels of H2O2 released by inflammatory cells. In contrast, bolus treatment of H2O2 has no effect at low concentrations and even suppresses hepcidin at concentrations of >50 μm. H2O2 treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H2O2-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H2O2 responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H2O2 released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H2O2. Thus, similar to cytokines, H2O2 provides an important regulatory link between inflammation and iron metabolism.

Assessment of renal allograft fibrosis by transient elastography

Transient elastography (TE, Fibroscan) has been established as a noninvasive assessment tool of liver fibrosis. We evaluated potentials and limitations of TE for identifying renal allograft fibrosis. The technical possibility of kidney examination by TE was assessed in two 10‐week‐old German landrace pigs and kidney stiffness (KS) was evaluated in 164 renal transplant patients. KS could be determined in all animals at the pole and pars media (29 ± 10 kPa vs. 31 ± 17 kPa). In human renal allografts KS was successfully performed in 94.5% of the test series with reliable results in 72% of the measurements. Mean KS at the pole or pars media were comparable (35.0 ± 19.9 kPa vs. 33.2 ± 18.6 kPa). Significantly higher KS was detected in renal allografts with histologically confirmed advanced fibrosis. Body‐mass‐index, skin‐allograft distance, and peri or intrarenal fluid accumulation were important confounders of successful KS measurements (BMI: r = −0.31; P < 0.001; distance: r = −0.50; P < 0.001). Notably, KS did not correlate with renal function. TE represents a noninvasive approach in selected transplant recipients to identify allografts with severe fibrosis. The heterogeneous kidney morphology and several other confounding factors negatively affect measurability of KS by TE. Further technical modifications are required to improve applicability of TE for kidney assessment.

Alcohol and Colorectal Cancer: The Role of Alcohol Dehydrogenase 1C Polymorphism

BACKGROUND Chronic alcohol consumption is a risk factor for colorectal cancer. Animal experiments as well as genetic linkage studies in Japanese individuals with inactive acetaldehyde dehydrogenase leading to elevated acetaldehyde concentrations following ethanol ingestion support the hypothesis that acetaldehyde may be responsible for this carcinogenic effect of alcohol. In Caucasians, a polymorphism of alcohol dehydrogenase 1C (ADH1C) exists resulting in different acetaldehyde concentrations following ethanol oxidation. METHODS To evaluate whether the association between alcohol consumption and colorectal tumor development is modified by ADH1C polymorphism, we recruited 173 individuals with colorectal tumors diagnosed by colonoscopy and 788 control individuals without colorectal tumors. Genotyping was performed using genomic DNA extracted from whole blood followed by polymerase chain reaction. RESULTS Genotype ADH1C*1/1 was more frequent in patients with alcohol-associated colorectal neoplasia compared to patients without cancers in the multivariate model controlling for age, gender, and alcohol intake (odds ratio = 1.674, 95% confidence interval = 1.110-2.524, 2-sided p from Wald test = 0.0139). In addition, the joint test of the genetic effect and interaction between ADH1C genotype and alcohol intake (2-sided p = 0.0007) indicated that the difference in ADH1C*1 polymorphisms between controls and colorectal neoplasia is strongly influenced by the alcohol consumption and that only individuals drinking more than 30 g ethanol per day with the genotype ADH1C*1/1 had an increased risk for colorectal tumors. CONCLUSIONS These data identify ADH1C homozygosity as a genetic risk marker for colorectal tumors in individuals consuming more than 30 g alcohol per day and emphasize the role of acetaldehyde as a carcinogenic agent in alcohol-related colorectal carcinogenesis.