Claudio Tiribelli

Drinking habits as cofactors of risk for alcohol induced liver damage

Background—The Dionysos Study is a cohort study of the prevalence of chronic liver disease in the general population of two northern Italian communities. It included 6917 subjects, aged 12–65 (69% of the total population). Aims—The aim of this part of the study was to examine the relationship of daily alcohol intake, type of alcoholic beverage consumed, and drinking patterns to the presence of alcohol induced liver damage in an open population. Patients and methods—6534 subjects, free of virus related chronic liver disease and participating in the first cross-sectional part of the study, were fully examined. Each subject underwent: (a) medical history and physical examination, (b) evaluation of alcohol intake using an illustrated dietary questionnaire, and (c) routine blood tests. More invasive diagnostic procedures were performed when indicated. Results—Multivariate analysis showed that the risk threshold for developing either cirrhosis or non-cirrhotic liver damage (NCLD) was ingestion of more than 30 g alcohol per day in both sexes. Using this definition, 1349 individuals (21% of the population studied) were at risk. Of these, only 74 (5.5% of the individuals at risk) showed signs of liver damage. The prevalence of “pure” alcoholic cirrhosis was 0.43% (30 of 6917), representing 2.2% of the individuals at risk, with a ratio of men to women of 9:1, while 44 (3.3% of the individuals at risk) showed persistent signs of NCLD. After 50 years of age, the cumulative risk of developing both NCLD and cirrhosis was significantly higher (p<0.0001) for those individuals who regularly drank alcohol both with and without food than for those who drank only at mealtimes. Conclusions—Our data show that in an open population the risk threshold for developing cirrhosis and NCLD is 30 g ethanol/day, and this risk increases with increasing daily intake. Drinking alcohol outside mealtimes and drinking multiple different alcoholic beverages both increase the risk of developing alcohol induced liver damage.

Clinical course and risk factors of hepatitis C virus related liver disease in the general population: report from the Dionysos study

BACKGROUND The severity, clinical course, and risk of hepatitis C virus (HCV) related chronic liver disease are still rather poorly defined. AIMS To investigate the prevalence, risk factors, and severity of HCV related liver disease in the general population, and investigate whether infection with a specific genotype is associated with an increased risk of cirrhosis or hepatocellular carcinoma. METHODS HCV RNA determination by polymerase chain reaction (PCR) and HCV genotyping were performed in all anti-HCV positive subjects belonging to the Dionysos study (6917 subjects). Diagnosis of cirrhosis and hepatocellular carcinoma was established by liver biopsy in all cases. All the data were analysed by univariate and multivariate statistics in all the cohort. To investigate the natural history of HCV infection, anti-HCV positive subjects were followed up every six months for three years with liver function tests and ultrasonograms. RESULTS The overall prevalence of HCV RNA positivity was 2.3%. Positivity increased progressively with age, and was higher in women (ratio of men to women = 0.7). Genotypes 1b and 2a were the most frequent (42 and 24% of HCV RNA positive patients), with a prevalence of 1 and 0.6% respectively. Intravenous drug use, blood transfusions received before 1990, history of previous hepatitis among the cohabiting, and history of animal (mainly dogs) bites were significantly (p<0.05) associated with HCV infection, independently of age and sex. Multivariate analysis showed that, independently of age, sex, and alcohol intake, genotype 1b infection, with or without coinfection with other genotypes, is the major risk factor associated with the presence of cirrhosis and/or hepatocellular carcinoma. During the three years of follow up, 57 (35%) of the HCV RNA positive subjects had consistently normal alanine aminotransferase and γ-glutamyltransferase values. Two of the 22 HCV RNA positive cirrhotic patients, all drinking more than 90 g of alcohol a day, developed hepatocellular carcinoma (incidence rate = 3.0% per year). CONCLUSIONS In the general population of Northern Italy, HCV infection is widespread, but only less than 50% of the anti-HCV positive subjects, particularly those infected with genotype 1b, are associated with a more severe liver disease. Alcohol consumption greater that 30 g a day significantly aggravates the natural course of the disease.

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 μM H2O2. This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H2O2 treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H2O2 doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.

Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease

BackgroundIn vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH.MethodsHuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured.ResultsFFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species.ConclusionsThe exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.

The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1.

In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

Transport and Metabolism at Blood–Brain Interfaces and in Neural Cells: Relevance to Bilirubin-Induced Encephalopathy

Bilirubin, the end-product of heme catabolism, circulates in non-pathological plasma mostly as a protein-bound species. When bilirubin concentration builds up, the free fraction of the molecule increases. Unbound bilirubin then diffuses across blood–brain interfaces (BBIs) into the brain, where it accumulates and exerts neurotoxic effects. In this classical view of bilirubin neurotoxicity, BBIs act merely as structural barriers impeding the penetration of the pigment-bound carrier protein, and neural cells are considered as passive targets of its toxicity. Yet, the role of BBIs in the occurrence of bilirubin encephalopathy appears more complex than being simple barriers to the diffusion of bilirubin, and neural cells such as astrocytes and neurons can play an active role in controlling the balance between the neuroprotective and neurotoxic effects of bilirubin. This article reviews the emerging in vivo and in vitro data showing that transport and metabolic detoxification mechanisms at the blood–brain and blood–cerebrospinal fluid barriers may modulate bilirubin flux across both cellular interfaces, and that these protective functions can be affected in chronic unconjugated hyperbilirubinemia. Then the in vivo and in vitro arguments in favor of the physiological antioxidant function of intracerebral bilirubin are presented, as well as the potential role of transporters such as ABCC1 and metabolizing enzymes such as cytochromes P-450 in setting the cerebral cell- and structure-specific toxicity of bilirubin following hyperbilirubinemia. The relevance of these data to the pathophysiology of bilirubin-induced neurological diseases is discussed.

Differential Proteomic Analysis of Subfractioned Human Hepatocellular Carcinoma Tissues

To discover new potential biomarkers of HCC, we used 2-DE gel separation and MALDI-TOF-MS analysis of partially enriched nuclear fractions from liver biopsies of 20 different patients. We obtained a proteomic map of subfractioned liver samples including about 200 common protein spots, among which identified components corresponded to expression products of 52 different genes. A differential analysis of proteins from tumoral and control tissues revealed a significant change in the expression level of 16 proteins associated to cytoskeletal, stress response and metabolic functions. These data may provide novel candidate biomarkers for HCC and useful insights for understanding the mechanisms of HCC pathogenesis and progression.

Modern strategies to identify new molecular targets for the treatment of liver diseases: The promising role of Proteomics and Redox Proteomics investigations.

Oxidative stress, due to an imbalance between the generation of ROS and the antioxidant defense capacity of the cell, is a major pathogenetic event occurring in several liver diseases, ranging from metabolic to proliferative. Main sources of ROS are represented by mitochondria and cytochrome P450 enzymes in the hepatocytes, Küppfer cells, and neutrophils. Oxidative stress affects major cellular components including lipids, DNA, and proteins. Through modulation of protein structure/function, ROS can influence gene expression profile by affecting intracellular signal transduction pathways. While several enzymatic and nonenzymatic markers of chronic oxidative stress are well known in liver, early protein targets of oxidative injury are yet poorly defined. Identification of these biomarkers will enable early detection of liver diseases and will allow monitoring the degree of liver damage, the response to pharmacological therapies, and the development of new therapeutic approaches. In the era of molecular medicine, new proteomic methodologies promise to establish a relationship between pathological hallmarks of the disease and protein structural/functional modifications, thus allowing a better understanding and a more rational therapy on liver disorders. Purpose of this review is to critically analyze the application of proteomic and redox proteomic approaches to the study of oxidative stress‐linked liver diseases.

X-ray fluorescence elemental mapping and microscopy to follow hepatic disposition of a Gd-based magnetic resonance imaging contrast agent.

1. Spatially resolved X‐ray fluorescence (XRF) spectroscopy with synchrotron radiation is a technique that allows imaging and quantification of chemical elements in biological specimens with high sensitivity. In the present study, we applied XRF techniques at a macro and micro level to carry out drug distribution studies on ex vivo models to confirm the hepatobiliary disposition of the Gd‐based magnetic resonance imaging contrast agent B22956/1.

The north-to-south gradient of hepatitis C virus infection.

Geographical difference in the prevalence and incidence of various diseases has long captured the interest of clinical epidemiologists and medical investigators. Hepatitis C is a major health problem, with some 150 million chronic HCV carriers throughout the world, an estimated 4 million living in the US and 5 million in Western Europe. Based on studies performed in representative cohorts, however, it is evident that the prevalence varies substantially from one geographical area to the other. In a serologic survey of a population-based sample consisting of more than 21,000 subjects participating in the Third National Health and Nutrition Survey (NHANES III) in the United States (conducted from 1988 to 1994) (1), a prevalence of anti-HCV of 1.8% was found. By contrast, household surveys of from 300 to more than 30,000 persons in both rural and urban areas of Africa indicate prevalence rates of more than 20% (2). Prevalence varies greatly and reaches a peak of 51% in an Egyptian population living along the Nile River delta (3). In Western Europe, overall prevalence is approximately 1%, increasing from North to South and is higher in Eastern than in Western Europe. Although evaluated only in blood donors, the prevalence of HCV infection in Eastern Europe ranges from 0.68% in the Czech Republic to 4.9% in Romania (4). This same regional difference is also evident in the case of Italy, where the prevalence of HCV infection was assessed in three regions moving from North to South. The Italian data reproduce what is observed in the rest of Europe, i.e. an increasing North-to-South gradient of prevalence ranging from 3.2% in the North (5, 6) to 8.4% in Central (7) and 12.6% in the South, with peaks greater than 20% (8). All these series concordantly indicated that HCV positivity was greater in females and increased with age, suggesting a ‘cohort effect’ (9). The finding that older subjects show a higher positivity for HCV indicates, in addition, that the infection occurred in the 1950s and 1960s, when circulation of the virus, associated with less stringent sanitary conditions, almost led to an epidemic. Direct proof of this hypothesis was recently obtained from a retrospective study on archival samples of liver cirrhosis (10). In an article in this issue of the journal (pp. 864–870), Dalgard et al. observed an overall prevalence of HCV positivity of 0.7% in a large community in the Oslo area. Although compliance was not optimal (less than 50%), their observation that the rate doubled (1.5%) in subjects 40–45 years old was interesting. This is in line with what is observed in Southern Europe, suggesting that most infections occurred in the past. What is intriguing, however, is the difference in the prevalence of HCV positivity moving from Northern to Southern Europe and then across the Mediterranean Sea. Assuming an overall prevalence of 1% in the North, this figure is 3-fold higher in the Northern regions of Italy and 12fold higher in the most southern parts of the country. In Egypt, a 50 times greater rate of infection is sometimes observed. So why does this occur? Epidemiologists have long been aware of the profound geographical difference in the spread of infectious diseases, and hepatotropic viruses make no difference. Transmission of the feco-oral viruses is related to hygienic conditions in the environment and to lack of proper sanitation procedures, particularly regarding water and food supplies and sewage treatment. In Italy, improved environmental conditions, along with a reduction in the size of families and socio-economic progress, have led to an impressive reduction in the rate of infection, particularly in the North, and have prevented the spread of the hepatitis E virus, despite the increasing number of migrant subjects (11). A similar approach, based on the epidemiologic characteristics (main routes of transmission, transmission rates, type and frequency of exposure), may help us understand the spatial-temporal difference in regard to the hepatotropic viruses with parenteral transmission. In addition, the infection distribution by cohorts indicates whether the determinants have disappeared or are still present. As observed for HCV, the geographical distribution of HBV also varies from areas of low to intermediate and high endemicity (9, 12). These areas do not fully overlap, and the exposures are frequently of different relevance. For instance, in hypoendemic areas, HCV transmission and HBV transmission are primarily linked to behavioural factors (i.e. injecting drug use) and to the residual iatrogenic risk (1). On the contrary, in hyperendemic areas the age at which HCV is first encountered is much younger (around 10 years) although older than is observed for HBV (3). This difference can be explained by the rate of perinatal transmission of HCV (around 5%) (13), which is much lower than that of HBV, particularly in areas where HBeAg is highly prevalent in pregnant women (12). In other words, HCV transmission in these areas is substantially horizontal, independently of certain major risk factors operating in developed countries—most of them unknown. This is not surprising, since it is well known that more than half of the HCV infections are not linked to any traceable exposure (cryptogenic infections). In general, it is assumed that the horizontal transmission is related to the hygienic level in the household and in other social settings, although rarely has a true risk EDITORIAL