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Background: Alcoholic liver cirrhosis (ALC) endangering people's health. The association between aldehyde dehydrogenase 2 (ALDH2) gene polymorphisms and ALC is not clear. To analyze the relationship between ALDH2 and ALC among Hakka population in southern China., Methods: A total of 292 ALC patients and 278 controls were included in the study. The ALDH2 gene rs671 polymorphism was analyzed by polymerase chain reaction (PCR)-gene chip. Relevant information and medical records of these participants were collected., Results: The ALC patients had higher percentage of smoking, lower prevalence of hypertension, higher level of alanine aminotransferase (ALT), aspertate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), total bile acid (TBA), total bilirubin (Tbil), and direct bilirubin (Dbil), lower level of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) than controls. The proportions of the G/A genotype (p = 0.017), G/A plus A/A genotype (p = 0.023) and A allele (p = 0.031) were significantly higher in ALC patients than that of controls. ALC patients with G/A genotype had higher TC, HDL-C, and Apo-A1 than those with G/G genotype, while with A allele had higher HDL-C, and Apo-A1 than those with G allele. Logistic regression analysis indicated that ALDH2 SNP rs671 G/A plus A/A genotypes (A allele carriers) (OR 2.030, 95% CI 1.109-3.715, p = 0.022) in the dominant model was the risk factor for ALC., Conclusions: ALDH2 A allele (G/A + A/A genotypes) increased the risk of developing ALC among Hakka people in southern China. The results should enrich the relevant data and provide valuable information for the future related research., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Analyzing 12,361 all-cause cirrhosis cases and 790,095 controls from eight cohorts, we identify a common missense variant in the Mitochondrial Amidoxime Reducing Component 1 gene (MARC1 p.A165T) that associates with protection from all-cause cirrhosis (OR 0.91, p = 2.3*10-11). This same variant also associates with lower levels of hepatic fat on computed tomographic imaging and lower odds of physician-diagnosed fatty liver as well as lower blood levels of alanine transaminase (-0.025 SD, 3.7*10-43), alkaline phosphatase (-0.025 SD, 1.2*10-37), total cholesterol (-0.030 SD, p = 1.9*10-36) and LDL cholesterol (-0.027 SD, p = 5.1*10-30) levels. We identified a series of additional MARC1 alleles (low-frequency missense p.M187K and rare protein-truncating p.R200Ter) that also associated with lower cholesterol levels, liver enzyme levels and reduced risk of cirrhosis (0 cirrhosis cases for 238 R200Ter carriers versus 17,046 cases of cirrhosis among 759,027 non-carriers, p = 0.04) suggesting that deficiency of the MARC1 enzyme may lower blood cholesterol levels and protect against cirrhosis., Competing Interests: CAE reports consulting fees from Navitor Pharma, Novartis and Deerfield Management. AVK has received research grants from IBM Research and the Novartis Institute for Biomedical Research and has served as a consultant to or received honoraria from Color Genomics, Illumina, Novartis, Maze Therapeutics, and Navitor Pharmaceuticals; and has a patent related to a genetic risk predictor (20190017119). SK is an employee of Verve Therapeutics and has received a research grant from Bayer Healthcare; and consulting fees from Merck, Novartis, Sanofi, AstraZeneca, Alnylam Pharmaceuticals, Leerink Partners, Noble Insights, MedGenome, Aegerion Pharmaceuticals, Regeneron Pharmaceuticals, Quest Diagnostics, Color Genomics, Genomics PLC, and Eli Lilly and Company; and holds equity in San Therapeutics, Catabasis Pharmaceuticals, Verve Therapeutics and Maze Therapeutics. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Background: The multifactorial mechanisms driving negative health outcomes among risky drinkers with HIV may include immunosenescence. Immunosenescence, aging of the immune system, may be accentuated in HIV and leads to poor outcomes. The liver regulates innate immunity and adaptive immune tolerance. HIV-infected people have high prevalence of liver-related comorbidities. We hypothesize that advanced liver fibrosis/cirrhosis is associated with alterations in T-cell subsets consistent with immunosenescence., Methods: ART-naïve people with HIV with a recent history of heavy drinking were recruited into a clinical trial of zinc supplementation. Flow cytometry was used to characterize T-cell subsets. The two primary dependent variables were CD8+ and CD4+ T-cells expressing CD28-CD57+ (senescent cell phenotype). Secondary dependent variables were CD8+ and CD4+ T-cells expressing CD45RO + CD45RA- (memory phenotype), CD45RO-CD45RA+ (naïve phenotype), and the naïve phenotype to memory phenotype T-cell ratio (lower ratios associated with immunosenescence). Advanced liver fibrosis/cirrhosis was defined as FIB-4 > 3.25, APRI≥1.5, or Fibroscan measurement ≥10.5 kPa. Analyses were conducted using multiple linear regression adjusted for potential confounders., Results: Mean age was 34 years; 25% female; 88% hepatitis C. Those with advanced liver fibrosis/cirrhosis (N = 25) had higher HIV-1 RNA and more hepatitis C. Advanced liver fibrosis/cirrhosis was not significantly associated with primary or secondary outcomes in adjusted analyses., Conclusions: Advanced liver fibrosis/cirrhosis was not significantly associated with these senescent T-cell phenotypes in this exploratory study of recent drinkers with HIV. Future studies should assess whether liver fibrosis among those with HIV viral suppression and more advanced, longstanding liver disease is associated with changes in these and other potentially senescent T-cell subsets.

Background: Only a subset of patients with excessive alcohol use develop alcoholic liver disease (ALD), though the exact mechanism is not completely understood. Once ingested, alcohol is metabolized by 2 key oxidative enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). There are 2 major ALDH isoforms, cytosolic and mitochondrial, encoded by the aldehyde ALDH1 and ALDH2 genes, respectively. The ALDH2 gene was hypothesized to alter genetic susceptibility to alcohol dependence and alcohol-induced liver diseases. The aim of this study is to determine the association between aldehyde dehydrogenase 2 (rs671) glu504lys polymorphism and ALD., Methods: ALDH2 genotyping was performed in 535 healthy controls and 281 patients with ALD., Results: The prevalence of the common form of the single nucleotide polymorphism rs671, 504glu (glu/glu) was significantly higher in patients with ALD (95.4%) compared to that of controls (73.7%, P < 0.0001). Among controls, 23.7% had the heterozygous (glu/lys) genotype compared to 4.6% in those with ALD (odds ratio [OR] = 0.16, 95% CI: 0.09-0.28). The allele frequency for 504lys allele in patients with ALD was 2.3%, compared to 14.5% in healthy controls (OR = 0.13, 95% CI: 0.07-0.24)., Conclusions: Patients with ALDH2 504lys variant were less associated with ALD compared to those with ALDH2 504glu using both genotypic and allelic analyses., (Copyright © 2018 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Background/aims: Glutathione and glutathione S-transferases (GST) are involved in cell defence against reactive oxygen species, which induces oxidative stress and are associated with different chronic diseases. The aim of the present study was to determine the differences in reduced glutathione (GSH) and GST levels in patients with different liver diseases., Materials and Methods: Overall, 114 patients were enrolled in this study: 58 patients with colorectal cancer (18 without and 40 with liver metastases), 27 with liver steatosis, 29 with alcoholic cirrhosis and a group of 40 healthy volunteers. The levels of GSH and GST in blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's guidelines., Results: Significant differences in GSH and GST levels were observed in most of the groups compared to the healthy volunteers (GSH: 52.72 µg/mL, GST: 0.53 ng/mL): with hepatic steatosis (GSH: 17.04 µg/mL, p < 0.001; GST: 5.89 ng/mL, p < 0.001), alcoholic cirrhosis (GSH: 62.04 µg/mL, p < 0.003; GST: 0.94 ng/mL, p < 0.001) and liver metastases (GSH: 37.84 µg/mL, p < 0.001, GST: 1.25 ng/mL, p=0.747)., Conclusion: The different GSH and GST levels in patients with colorectal cancer liver metastases, liver steatosis and alcoholic cirrhosis indicate the differences in antioxidative system damage and its compensatory possibilities and could serve as potential biomarkers for its correction.

Background & Aims: Myeloperoxidase exocytosis and production of hydrogen peroxide via the neutrophil superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contribute to efficient elimination of bacteria. Cirrhosis impairs immune functions and increases susceptibility to bacterial infection. We recently showed that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a severe impairment of formylpeptide receptor (fPR)-mediated intracellular signaling and superoxide production. Here, we performed ex vivo studies with these patients' neutrophils to further investigate myeloperoxidase release, bactericidal capacity and signaling events following fPR stimulation by the formylpeptide formyl-met-leu-phe (fMLP)., Methods: Myeloperoxidase release was studied by measuring extracellular myeloperoxidase activity. Activation of signaling effectors was studied by Western blot and their respective contribution to myeloperoxidase release studied using pharmacological antagonists., Results: fMLP-induced myeloperoxidase release was strongly impaired in patients' neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced phosphorylation of major signaling effectors, AKT, ERK1/2 and p38-MAP-Kinases, was also strongly deficient despite a similar expression of signaling effectors or fPR. However, based on effector inhibition in healthy neutrophils, AKT and p38-MAPK but not ERK1/2 upregulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients' neutrophils exhibited a defective bactericidal capacity that was reversed ex vivo by the TLR7/8 agonist CL097, through potentiation of the fMLP-induced AKT/p38-MAPK signaling axis and myeloperoxidase release., Conclusions: We provide first evidence that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity, which can be reversed via TLR7/8 activation. These defects, together with the previously described severe deficient superoxide production, may increase cirrhotic patients' susceptibility to bacterial infections., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Alcoholic liver disease remains one of the most common causes of chronic liver disease worldwide. The aim of this study was to assess the usefulness of metalloproteinases (MMP1 and MMP13) as diagnostic markers of alcoholic liver disease and to determine the changes in free amino acid profile in the patients with alcoholic liver cirrhosis., Material and Methods: Sixty patients with alcoholic liver cirrhosis treated in various hospitals of the Lublin region were randomly enrolled. The control group consisted of 10 healthy individuals without liver disease, who did not drink alcohol. Additionally, a group of alcoholics (22 persons) without liver cirrhosis was included in the study. The activity of MMP-1 and MMP-13 in blood plasma of patients and controls was measured using the sandwich enzyme immunoassay technique with commercially available quantitative ELISA test kits. Amino acids were determined by automated ion-exchange chromatography., Results: No significant differences were observed in the activity of MMP-1 in alcoholics with or without liver cirrhosis or in controls. Increased serum MMP-13 was found in patients with liver cirrhosis (stage A, B, C) compared to the control group. Patients with alcoholic liver cirrhosis (stage A, B, C) demonstrated reduced concentrations of glutamic acid and glutamine compared to the control group. Plasma levels of valine, isoleucine, leucine, and tryptophan were significantly lower in patients with alcoholic liver cirrhosis (stage C) than in controls., Conclusions: MMP-13 can be useful to confirm the diagnosis of alcoholic liver cirrhosis, but levels of MMP-1 are not significantly increased in patients with liver cirrhosis compared to controls. The serum branched-chain amino acid (BCAA) is markedly reduced in patients with stage C alcoholic liver cirrhosis.

Background & Aims: Mitochondrial aldehyde dehydrogenase (ALDH2) plays a critical role in the detoxification of the ethanol metabolite acetaldehyde. This study was designed to examine the impact of global ALDH2 overexpression on alcohol-induced hepatic steatosis., Methods: Wild type Friend virus B (FVB) and ALDH2 transgenic mice were placed on a 4% alcohol or control diet for 12 weeks. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin and cholesterol, hepatic triglyceride, steatosis, fat metabolism-related proteins, pro-inflammatory cytokines, glutathione (GSH), oxidized glutathione (GSSG), autophagy and autophagy signalling were examined. The role of autophagy was evaluated in alcohol dehydrogenase 1 (ADH1)-transfected human hepatocellular liver carcinoma cells (VA-13) treated with or without the autophagy inducer rapamycin and lysosomal inhibitors., Results: Chronic alcohol intake led to elevated AST-, ALT-levels, bilirubin, AST/ALT ratio, cholesterol, hepatic triglycerides and hepatic fat deposition as evidenced by H&E and Oil Red O staining. Hepatic fat deposition was associated with disturbed levels of fat metabolism-related proteins (fatty acid synthase, SCD1), upregulated interleukin-6, TNF-α, cyclooxygenase, oxidative stress, and loss of autophagy, effects which were attenuated or ablated by the ALDH2 transgene. Moreover, ethanol (100 mM) and acetaldehyde (100 and 500 μM) increased levels of IL-6 and IFN-γ, and suppressed autophagy in VA-13 cells, effects which were markedly alleviated by rapamycin. In addition, lysosomal inhibitors mimicked ethanol-induced p62 accumulation with little additive effect with ethanol. Ethanol significantly suppressed LC3 conversion in the presence of lysosomal inhibitors., Conclusions: In summary, our results revealed that ALDH2 plays a beneficial role in ameliorating chronic alcohol intake-induced hepatic steatosis and inflammation through regulation of autophagy., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

With only 1.3-4.3% in total hepatic CYP content, human CYP2D6 can metabolize more than 160 drugs. It is a highly polymorphic enzyme and subject to marked inhibition by a number of drugs, causing a large interindividual variability in drug clearance and drug response and drug-drug interactions. The expression and activity of CYP2D6 are regulated by a number of physiological, pathological and environmental factors at transcriptional, post-transcriptional, translational and epigenetic levels. DNA hypermethylation and histone modifications can repress the expression of CYP2D6. Hepatocyte nuclear factor-4α binds to a directly repeated element in the promoter of CYP2D6 and thus regulates the expression of CYP2D6. Small heterodimer partner represses hepatocyte nuclear factor-4α-mediated transactivation of CYP2D6. GW4064, a farnesoid X receptor agonist, decreases hepatic CYP2D6 expression and activity while increasing small heterodimer partner expression and its recruitment to the CYP2D6 promoter. The genotypes are key determinants of interindividual variability in CYP2D6 expression and activity. Recent genome-wide association studies have identified a large number of genes that can regulate CYP2D6. Pregnancy induces CYP2D6 via unknown mechanisms. Renal or liver diseases, smoking and alcohol use have minor to moderate effects only on CYP2D6 activity. Unlike CYP1 and 3 and other CYP2 members, CYP2D6 is resistant to typical inducers such as rifampin, phenobarbital and dexamethasone. Post-translational modifications such as phosphorylation of CYP2D6 Ser135 have been observed, but the functional impact is unknown. Further functional and validation studies are needed to clarify the role of nuclear receptors, epigenetic factors and other factors in the regulation of CYP2D6.

Aim: To investigate the expression of P450 enzyme genes by using end-stage liver disease samples and trimmed normal Chinese donor livers., Methods: The end-stage liver disease samples [n = 93, including hepatocellular carcinoma (HCC), peri-HCC tissue, hepatitis B virus cirrhosis, alcoholic cirrhosis, and severe cirrhosis] and trimmed normal Chinese donor livers (n = 35) from The Institute of Organ Transplantation in Beijing, China. Total RNA was extracted, purified, and subjected to real-time RT-PCR analysis., Results: For cytochrome P450 enzymes 1 (CYP1) family, the expression of CYP1A2 was decreased 90% in HCC, 80% in alcoholic cirrhosis, and 65% in severe cirrhosis. For CYP2 family, the expression of CAR was decreased 50% in HCC, but increased 50% in peri-HCC tissues. Similar decreases (about 50%) of CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 were observed in HCC, as compared to peri-HCC tissues and normal livers. CYP2C19 were decreased in all end-stage liver diseases and CYP2E1 also decreased in alcoholic cirrhosis and severe cirrhosis. For CYP3 family, the expression of PXR was decreased 60% in HCC, together with decreases in CYP3A4, CYP3A5, and CYP3A7. In contrast, the expression of CYP3A7 was slightly increased in HBV cirrhosis. The expression of CYP4A11 was decreased 85% in HCC, 7% in alcoholic cirrhosis and severe liver cirrhosis, along with decreases in PPARα. The 93 end-stage livers had much higher inter-individual variations in gene expression than 35 normal livers., Conclusion: The expression of CYP enzyme genes and corresponding nuclear receptors was generally decreased in end-stage liver diseases, and significant differences in gene expression were evident between peri-HCC and HCC.

Objective: The degree to which heterozygous forms of alpha-1 antitrypsin (A1AT), principally MZ, causes liver disease is uncertain. If heterozygosity is a relevant cofactor, over-representation in patients with end-stage liver disease would be predicted. We therefore assessed the prevalence and disease-related distribution of A1AT heterozygosity in the largest cohort to date for this purpose., Methods: We retrospectively analysed 1036 patients assessed for liver transplantation at our unit between 2003 and 2010. A1AT heterozygotes were identified on the basis of isoelectric focusing and/or histology, showing A1AT globule deposition consistent with an abnormal phenotype., Results: Z-allele frequency was the highest in patients with nonalcoholic steatohepatitis (NASH) cirrhosis (20.3%), followed by patients with 'other parenchymal' diseases (11.9%), alcohol-related liver disease (9.9%), autoimmune disease (8.6%), hepatitis C (6.1%), hepatitis B (3.0%) and biliary disease (1.9%). Compared with the heterozygote frequency in the general European population of 9.0%, the heterozygote frequency was significantly higher among patients with NASH cirrhosis (P≤0.0001) and lower in the biliary subgroup (P=0.004). The prevalence of MZ heterozygosity was significantly increased in cirrhosis because of both alcohol (9.9%) and NASH (17.3%) compared with the general European population (2.8%; P<0.0001)., Conclusion: Accumulation of misfolded A1AT aggregates appears to accelerate progression, in which the hepatocyte is the key injured cell. Heterozygous A1AT states worsen prognosis, particularly in NASH and alcohol-related cirrhosis, and should be identified at presentation. In cases in which genetic screening is not readily available, a low threshold for isoelectric focusing and routine specific histochemical staining of liver biopsy specimens are warranted to identify these patients.

Objective: To investigate the expression of the lysosomal enzyme acid sphingomyelinase (ASMase) in alcohol-induced hepatic fibrosis using a rat model., Methods: The model of liver fibrosis was induced by administration of alcohol and high fat diet using 20 rats. Six rats given no alcohol and normal diet served as the control group. Real-time PCR, western blotting, and immunohistochemistry were used to evaluate fibrosis-related changes in the mRNA and protein expressions of ASMase., Results: The fibrotic liver tissues of the model rats showed significantly higher expression levels of ASMase than the non-fibrotic liver tissues of the control rats (P less than 0.05)., Conclusion: Expression of ASMase is increased in the fibrotic liver tissue of an alcohol-induced hepatic fibrosis rat model, suggesting that this lysosomal enzyme may contribute to development of this disease condition.

The 148 Isoleucine to Methionine protein variant (I148M) of patatin-like phospholipase domain-containing 3 (PNPLA3), a protein is expressed in the liver and is involved in lipid metabolism, has recently been identified as a major determinant of liver fat content. Several studies confirmed that the I148M variant predisposes towards the full spectrum of liver damage associated with fatty liver: from simple steatosis to steatohepatitis and progressive fibrosis. Furthermore, the I148M variant represents a major determinant of progression of alcohol related steatohepatitis to cirrhosis, and to influence fibrogenesis and related clinical outcomes in chronic hepatitis C virus hepatitis, and possibly chronic hepatitis B virus hepatitis, hereditary hemochromatosis and primary sclerosing cholangitis. All in all, studies suggest that the I148M polymorphism may represent a general modifier of fibrogenesis in liver diseases. Remarkably, the effect of the I148M variant on fibrosis was independent of that on hepatic steatosis and inflammation, suggesting that it may affect both the quantity and quality of hepatic lipids and the biology of non-parenchymal liver cells besides hepatocytes, directly promoting fibrogenesis. Therefore, PNPLA3 is a key player in liver disease progression. Assessment of the I148M polymorphism will possibly inform clinical practice in the future, whereas the determination of the effect of the 148M variant will reveal mechanisms involved in hepatic fibrogenesis.

The hepatic growth factor hepatopoietin Cn (HPPCn) prevents liver injury induced by carbon tetrachloride in rats. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK). S1P and S1P receptors (S1PRs) are involved in liver fibrogenesis and oxidative injury. This work sought to understand the mechanism by which SphK/S1P/S1PRs are involved in the protective effects of HPPCn on ethanol-induced liver injury and fibrosis. Transgenic mice with liver-specific overexpression of HPPCn (HPPCn(liver) (+/+)) were generated. Two ethanol feeding protocols were used to assess the protective effect of HPPCn on acute and chronic liver injury in mice. Specific inhibitors of S1PR1, S1PR2 and S1PR3 and siRNA were used to examine the roles of S1PRs in hepatic stellate cell (HSC) activation and hepatocyte apoptosis. Increased HPPCn expression in transgenic mice attenuated fibrosis induced by ethanol and carbon tetrachloride (CCl4). Treatment with recombinant human HPPCn prevented human hepatocyte apoptosis and HSC activation. JTE-013 or S1PR2-siRNA attenuated the effect of HPPCn on HSC activation induced by tumour necrosis factor-α (TNF-α). Consistent with the effect of N,N-dimethylsphingosine (DMS), suramin or S1PR3-siRNA treatment blocked HPPCn-induced Erk1/2 phosphorylation in human hepatocytes. This study demonstrated that HPPCn attenuated oxidative injury and fibrosis induced by ethanol feeding and that the SphK1/S1P/S1PRs signalling pathway contributes to the protective effect of HPPCn on hepatocyte apoptosis and HSC activation., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: The development of ascites in cirrhotic patients generally heralds a deterioration in their clinical status. A differential gene expression profile between alcohol- and hepatitis C virus (HCV)-related cirrhosis has been described from liver biopsies, especially those associated with innate immune responses. The aim of this work was to identify functional differences in the inflammatory profile of monocyte-derived macrophages from ascites in cirrhotic patients of different etiologies in an attempt to extrapolate studies from liver biopsies to immune cells in ascites. To this end 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (n=15) or alcohol (n=30) were studied. Cytokines and the cell content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation in peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined., Results: A different pattern of leukocyte migration to the peritoneal cavity and differences in the primed status of macrophages in cirrhosis were observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral blood cell subpopulations could be observed, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in the HCV than the alcohol etiology. HCV-related cirrhosis etiology was associated with a decreased inflammatory profile in ascites compared with the alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were observed in ascitic fluid from the HCV group. Isolated peritoneal monocyte-derived macrophages maintained their primed status in vitro throughout the 24 h culture period. The level of ERK1/2 phosphorylation was higher in ALC peritoneal macrophages at baseline than in HCV patients, although the addition of LPS induced a greater increase in ERK1/2 phosphorylation in HCV than in ALC patients., Conclusions: The macrophage inflammatory status is higher in ascites of alcohol-related cirrhotic patients than in HCV-related patients, which could be related with differences in bacterial translocation episodes or regulatory T cell populations. These findings should contribute to identifying potential prognostic and/or therapeutic targets for chronic liver diseases of different etiology.

Background: Alcoholic cirrhosis constitutes a major public health problem in the world where ADH1B, ALDH2, and CYP2E1 polymorphisms could be playing an important role. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in healthy control individuals (C) and patients with alcoholic cirrhosis (AC) from western Mexico., Methods: Ninety C and 41 patients with AC were studied. Genotype and allele frequency were determined through polymerase chain reaction-restriction fragment length polymorphisms., Results: Polymorphic allele distribution in AC was 1.6%ADH1B*2, 0.0%ALDH2*2, and 19.5%CYP2E1*c2; in C: 6.1%ADH1B*2, 0%ALDH2*2, and 10.6%CYP2E1*c2. CYP2E1*c2 polymorphic allele and c1/c2 genotype frequency were significantly higher (p < 0.05 and p < 0.01, respectively) in patients with AC when compared to C. Patients with AC, carrying the CYP2E1*c2 allele, exhibited more decompensated liver functioning evaluated by total bilirubin and prothrombin time, than c1 allele carrying patients (p < 0.05). Cirrhosis severity, assessed by Child's Pugh score and mortality, was higher in patients carrying the c2 allele, although not statistically significant., Conclusions: In this study, CYP2E1*c2 allele was associated with susceptibility to AC; meanwhile, ADH1B*2 and ALDH2*2 alleles were not. CYP2E1*c2 allele was associated with AC severity, which could probably be attributed to the oxidative stress promoted by this polymorphic form. Further studies to clearly establish CYP2E1*c2 clinical relevance in the development of alcohol-induced liver damage and its usefulness as a probable prognostic marker, should be performed. Also, increasing the number of patients and including a control group conformed by alcoholic patients free of liver damage may render more conclusive results. These findings contribute to the understanding of the influence of gene variations in AC development among populations, alcohol metabolism, and pharmacogenetics., (Copyright © 2011 by the Research Society on Alcoholism.)

Because of numerous limitations for liver biopsy, a noninvasive marker of liver cirrhosis is sought. Promising indicators seem to be matrix metalloproteinases (MMPs) that are responsible for degradation of extracellular matrix. The aim of the study was to evaluate the gelatinase activities (MMP-2 and MMP-9) in patients with different stages of alcoholic cirrhosis. Sixty-seven outpatients who presented various stages of alcoholic cirrhosis according to Child-Turcotte-Pugh criteria and 26 healthy control subjects were enrolled. Blood samples were collected for MMP-2 and MMP-9 activities. A significant decrease of serum MMP-2 activity was noted in stages B and C of cirrhosis in comparison with control. Serum MMP-9 activity did not depend on the stage of cirrhosis. The MMP-2 levels, but not those of MMP-9, may be of value in understanding the pathogenesis and progression of alcoholic cirrhosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Heme oxygenase 1 (HMOX1) is the rate limiting enzyme in heme degradation and a key regulator of inflammatory processes. In animal models the course of pancreatitis was ameliorated by up-regulation of HMOX1 expression. Additionally, carbon monoxide released during heme breakdown inhibited proliferation of pancreatic stellate cells and might thereby prevent the development of chronic pancreatitis (CP). Transcription of HMOX1 in humans is influenced by a GT-repeat located in the promoter. As such, HMOX1 variants might be of importance in the pathogenesis of pancreatitis., Methods: The GT-repeat and SNP rs2071746 were investigated with fluorescence labelled primers and by melting curve analysis in 285 patients with acute pancreatitis, 208 patients with alcoholic CP, 207 patients with idiopathic/hereditary CP, 147 patients with alcoholic liver cirrhosis, and in 289 controls, respectively. GT-repeat analysis was extended to a total of 446 alcoholic CP patients. In addition, we performed DNA sequencing in 145 patients with alcoholic CP, 138 patients with idiopathic/hereditary CP, 147 patients with alcoholic liver cirrhosis, and 151 controls. Exon 3 screening was extended to additional patients and controls., Results: S- and L-alleles of the GT-repeat, genotypes and alleles of SNP rs2071746 and non-synonymous variants detected by sequencing were found with similar frequencies in all groups., Conclusions: Although functional data implicate a potential influence of HMOX1 variants on the pathogenesis of pancreatitis, we did not find any association. As rare non-synonymous HMOX1 variants were found in patients and controls, it is rather unlikely that they will have functional consequences essential for pancreatitis development.

Background and Aim: The PNPLA3 rs738409 C>G polymorphism has been found to be strongly associated with non-alcoholic fatty liver disease and with alcoholic liver disease. Whether the PNPLA3 rs738409 polymorphism could be a risk factor for the development of hepatocellular carcinoma (HCC) in cirrhosis patients is unknown., Methods: This study included 483 (344 males) consecutive Italian patients of Caucasian ethnicity affected by cirrhosis, of whom 279 had undergone transplantation for end-stage liver disease while 204 had been referred to our liver and transplant unit for the diagnosis of cirrhosis. The aetiologies were hepatitis C virus=209, hepatitis B virus=76, alcohol=166, metabolic=32. Ile148Met rs738409 transversion was genotyped using an restriction fragment length polymorphism-based assay., Results: The genotype frequencies of the rs738409 polymorphism were distributed differently in patients with cirrhosis C/C=168, C/G=220, G/G=95 vs controls C/C=218, C/G=175, G/G=35 (P<0.0001). Among cirrhotics, the G allele was over-represented in alcoholic/metabolic (0.505) vs viral (0.368, P<0.001) liver disease. Patients with cirrhosis complicated by HCC were more likely to be G/G homozygotes (38/141) than the remaining patients (57/342, P<0.02). At multivariate analysis, the PNPLA3 rs738409 polymorphism was confirmed to be an independent predictor of HCC occurrence (odds ratio 1.76, 95% confidence interval 1.06-2.92, P<0.05). HCC rates increased from 13/116 (11.2%; female C/(*) carriers), to 97/295 (32.9%; male C/(*) carriers and female G/G homozygotes), to 31/72 (43.1%; male G/G homozygotes) (P<0.0001)., Conclusions: The PNPLA3 rs738409 C>G polymorphism is associated with cirrhosis. In synergy with gender, this polymorphism is a strong predictor of HCC occurrence among patients with cirrhosis., (© 2011 John Wiley & Sons A/S.)

Background: Polymorphism of N-acetyltransferase 2 gene was reported to be associated with the susceptibility of various cancers and liver diseases. However, its relationship to alcoholic liver disease is controversial and open to debate. The aim of this study was to evaluate the relationship of NAT2 genetic polymorphisms and the susceptibility to alcoholic liver cirrhosis (ALC) in Chinese, with special emphasis on the interaction of smoking., Methods: Peripheral white blood cell DNA from 148 patients with ALC, 104 patients with long-term alcoholic drinking but without cirrhosis (ANC) and 209 healthy controls were genotyped for NAT2 using a polymerase chain reaction-restriction fragment length polymorphism method. The possible confounding factors were included for analysis., Results: There was no statistical difference in the frequency of NAT2 genotype or NAT2 acetylator status among the 3 groups. However, among the chronic alcoholic drinkers, the rapid acetylators with smoking habits had higher percentage of ALC than those without smoking habit (18.9% vs. 9.5%, p=0.002). The adjusted odds ratio for rapid acetylator smoker to have ALC was 3.45 (95% CI=1.53 to 7.76, p=0.003)., Conclusions: The genetic factor, NAT2 polymorphism, may interact with environmental factor, smoking, to confer different susceptibilities to ALC. NAT2 rapid acetylators with smoking habit may increase the risk of ALC in Chinese., (Copyright © 2011 by the Research Society on Alcoholism.)

To validate the induction of blood lymphocyte cytochrome P450 2E1 (CYP2E1) expression in alcoholic liver cirrhosis and mRNA and protein expression of CYP2E1 in freshly prepared blood lymphocytes of alcoholic liver cirrhotic (ACP), nonalcoholic cirrhotic patients (NACP), alcoholic controls (ACs), and nonalcoholic controls (NACs) were investigated. Registered ACP and NACP patients at Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow, India along with NACs and ACs were included in the study. Real time polymerase chain reaction, enzyme-linked immunosorbent assay, and CYP2E1-dependent enzyme activity were determined in blood lymphocytes isolated from cases and controls. Significant increases in CYP2E1 mRNA and protein expression were observed in freshly prepared blood lymphocytes isolated from ACs and ACP patients as compared with respective NACs or NACP patients. A concomitant increase in N-nitrosodimethyamine demethylase activity was evident in the blood lymphocytes of ACs and ACP patients. Interestingly, the comparative increase observed in CYP2E1 expression was of greater magnitude in the blood lymphocytes isolated from ACP patients, although they abstained from alcohol drinking. Findings suggest that significant increase in the CYP2E1 mRNA and protein expression in the blood lymphocytes, isolated from early stage ACP patients, can be used to predict alcohol-induced toxicity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H₂O₂, endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H₂O₂, as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 μmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

The association of polymorphism of alcohol dehydrogenase (ADH) and its interaction with genes involved in the generation and detoxification of free radicals such as cytochrome P4502E1 (CYP2E1) and glutathione S-transferases M1 (GSTM1) were studied with alcoholic liver cirrhosis. The study included 175 alcoholic cirrhotic patients, 140 non-alcoholic cirrhotic patients, 255 non-alcoholic controls and 140 alcoholic controls. Our data revealed that the ADH1C*1/*1 genotype exhibited significant association with alcoholic liver cirrhosis while ADH1B genotypes did not show any significant association. A much higher risk to alcoholic liver cirrhosis was observed in patients carrying a combination of wild genotypes of ADH1C (ADH1C*1/*1) and variant genotype of ADH1B (ADH1B*2/*2) or CYP2E1 (CYP2E1*5B) or null genotype of GSTM1. Our data suggest a role for the interaction amongst the genes involved in metabolizing alcohol and in generating and detoxifying free radicals with susceptibility to alcoholic liver cirrhosis., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

The association of polymorphism in cytochrome P450 2E1 (CYP2E1), the major microsomal ethanol metabolizing enzyme and its interaction with genes, involved in detoxification of reactive oxygen species, such as glutathione-S-transferases M1 (GSTM1) and alcohol intake, gamma-aminobutyric acid receptor gamma2 (GABRG2) was studied with the risk to alcoholic cirrhosis in a case-control study. A total of 160 alcoholic cirrhotic and 125 non-alcoholic cirrhotic cases, visiting the OPD facility of Gastroenterology Department of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGI), Lucknow, India and 250 non-alcoholic and 100 alcoholic controls having no evidence of liver disease were included in the study. PCR-based RFLP methodology was followed for genotyping studies. Our data revealed that the variant genotypes of CYP2E1 5B exhibited significant association with the alcoholic liver cirrhosis when compared to non-alcoholic controls (OR: 4.3; 95%CI: 1.5-12.4; p: 0.003) or non-alcoholic cirrhosis patients (OR: 5.4; 95%CI: 1.2-24.5; p: 0.01) or alcoholic controls (OR: 4.3; 95%CI: 0.95-19.62; p: 0.04). Haplotype approach revealed that haplotype T-A-T was found to be associated with more than 5-fold increase in risk for alcoholic cirrhosis. Likewise, combination of variant genotype of CYP2E1 5B with null genotype of GSTM1, a phase II detoxification enzyme, resulted in several fold increase in risk in alcoholic cirrhotic patients when compared with non-alcoholic controls or non-alcoholic cirrhotic patients. Further, the combination of variant genotype of CYP2E1 5B with GABRG2, significantly increased the risk upto 6.5-fold in alcoholic cirrhotic patients when compared with non-alcoholic controls thereby suggesting the role of gene-gene interaction in alcoholic cirrhosis.

In a case-control study, association of polymorphism in glutathione-S-transferases (GSTM1, GSTT1, GSTP1), involved in detoxification of reactive oxygen species (ROS), was studied with alcoholic liver cirrhosis. The study included 175 alcoholic cirrhotic patients (ACPs), 140 non-alcoholic cirrhotic patients (NACPs), visiting Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGI), Lucknow, India, 255 non-alcoholic controls and 140 alcoholic controls. The data showed an increase in risk to alcoholic cirrhosis in ACPs with GSTM1 (null) genotype when compared with non-alcoholic controls (OR: 1.7; 95% CI: 1.15-2.56) or alcoholic controls (OR: 1.7; 95% CI: 1.07-2.73). Significant increase in risk was also observed in ACPs with variant genotype of GSTP1 when compared with non-alcoholic controls (OR: 1.65; 95% CI: 1.12-2.43). A much higher risk to alcoholic liver cirrhosis was observed in patients carrying combination of null genotypes of GSTM1 and GSTT1 (OR: 2.8; 95% CI: 1.3-6.06) or variant genotype of GSTP1 and null genotype of GSTM1 (OR: 2.8; 95% CI: 1.58-4.90) or GSTT1 (OR: 2.16; 95% CI: 1.08-4.28). Likewise, greater risk for alcoholic cirrhosis was observed in patients carrying combination of GSTM1, GSTT1 (null) and variant genotype of GSTP1 (OR: 5.8; 95% CI: 2.17-15.80). Our data further showed that interaction of GSTs with variant genotype of manganese superoxide dismutase (MnSOD), which detoxifies free radicals, or cytochrome P450 2E1, which generates free radicals, resulted in several fold increase in risk to alcoholic liver cirrhosis in ACPs when compared with non-alcoholic controls thus demonstrating the role of gene-gene interactions in modulating the risk to alcoholic liver cirrhosis.

Objectives: The aim of the study was to determine the relationship between alcohol consumption and liver fibrosis as assessed by aspartate aminotransferase to platelet ratio index (APRI) in HIV-infected adults and to explore the relative contributions of alcohol and hepatitis C virus (HCV) to APRI among HIV/HCV-coinfected adults., Methods: We performed a cross-sectional analysis of data from an observational clinical cohort. Alcohol consumption was categorized according to National Institute on Alcohol Abuse and Alcoholism guidelines. We defined significant liver disease as APRI>1.5, and used multinomial logistic regression to identify correlates of increased APRI., Results: Among 1358 participants, 10.4% reported hazardous drinking. It was found that 11.6% had APRI>1.5, indicating liver fibrosis. Hazardous drinking was associated with increased APRI [adjusted relative risk ratio (RRR) 2.30; 95% confidence interval (CI) 1.26-4.17]. Other factors associated with increased APRI were male gender, viral hepatitis, and HIV transmission category of injecting drug use. Among coinfected individuals, 18.3% had APRI>1.5, and hazardous drinking was not associated with APRI. Among non-HCV-infected individuals, 5.3% had APRI>1.5 and hazardous drinking was associated with increased APRI (adjusted RRR 3.72; 95% CI 1.40-9.87)., Conclusions: Hazardous drinking is an important modifiable risk factor for liver fibrosis, particularly among non-HCV-infected patients. Clinicians and researchers must address alcohol use as the burden of liver disease increases among HIV-positive individuals.

Objective: We investigated patterns of use of alcohol and its clinical effects among cirrhotic subjects who participated in a randomized clinical trial comparing the efficacy of transjugular intravenous portosystemic shunt and distal splenorenal shunt., Methods: There were 132 cirrhotic subjects, 78 with alcoholic liver disease (ALD), who were followed for a median of 49 months (range 2-93 months). Alcohol use was assessed by patient questionnaire, with corroboration by family members., Results: Twenty-eight subjects (21%) were drinking at study entry and 60 subjects (45%) drank during follow-up. Heavy drinking (>4 drinks/day) was recorded in 25 ALD subjects, but in no non-ALD subjects (P < 0.0001). Drinking by ALD subjects was associated with a 153% increase in gamma-glutamyl transpeptidase (GGT) (P < 0.0001). The frequencies of death (46%vs 30%), ascites (33%vs 20%), encephalopathy (56%vs 42%), and variceal bleeding (11%vs 3%) were greater in the ALD group. In a Cox proportional hazards model only "ever heavy drinking" was associated with death (P= 0.0099), while recent heavy drinking increased the hazard of variceal hemorrhage dramatically (odds ratio 10.85)., Conclusions: Whereas most cirrhotic subjects, alcoholic or not, did not drink during 5 yr of observation, heavy alcohol use occurred exclusively in ALD patients. Alcohol use by ALD subjects was associated with elevations in GGT and was linked to death and with rebleeding from shunt dysfunction.

Unlabelled: The objective of this study was to assess the serum angiotensin converting enzyme (ACE) activity in patients with liver cirrhosis caused by chronic alcohol consumption, in order to get better insight into the function of the renin-angiotensin system., Patients and Methods: Serum level of ACE activity was measured by Neels spectrophotometry in 35 alcoholic liver cirrhosis patients classified according to Child-Pugh-Turcotte criteria and 35 dyspeptic patients with any liver disease excluded (control group)., Results: Serum values of ACE were statistically significantly higher (p < 0.00001) in the group of liver cirrhosis patients (x = 250.16 +/- 85.5 nmol) than in the control group (x = 115.88 +/- 58.19 nmol). The highest levels of ACE were measured in class B group of liver cirrhosis patients vs. class A and class B groups (p < 0.013)., Conclusion: It is concluded that liver cirrhosis patients have elevated ACE levels, which coud be useful in the diagnosis and follow up of these patients.

The liver is the main place of ethanol oxidation and is especially sensitive to the toxic effects of alcohol. Alcoholic liver disease is caused by chronic alcohol abuse. The article presents the negative effect of alcohol and its metabolites on the different biochemical processes in the liver.

Aim: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha) expression in the liver., Methods: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-kappaB p65ìiNOS, eNOS and TNF-alpha protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-kappaB p65, iNOS and TNF-alpha protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB, and TNF-alpha mRNA expression., Conclusion: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB and TNF-alpha expression. eNOS activity is reduced, but its mRNA expression is not affected.

Manganese superoxide dismutase (MnSOD) converts the superoxide anion into H(2)O(2), which, unless it is detoxified by glutathione peroxidase 1 (GPx1), can increase hepatic iron and can react with iron to form genotoxic compounds. We investigated the role of Ala/Val-MnSOD and Pro/Leu-GPx1 polymorphisms on hepatic iron accumulation and hepatocellular carcinoma development in patients with alcoholic cirrhosis. Genotypes were determined in 162 alcoholic patients with cirrhosis but without hepatocellular carcinoma initially, who were prospectively followed up for hepatocellular carcinoma development. We found that patients with two Val-MnSOD alleles (slow H(2)O(2) production) and two Pro-GPx1 alleles (presumably quick H(2)O(2) detoxification) had a lower risk of hepatocellular carcinoma development than other patients (chi(2) trend test, P = 0.001; log-rank, P = 0.0009). Indeed, hepatocellular carcinoma percentage was 0% in subjects with this "2Val-MnSOD/2Pro-GPx1" genotype versus 16%, 27%, and 32% in "2Val-MnSOD/1or2Leu-GPx1," "1or2Ala-MnSOD/2Pro-GPx1," and "1or2Ala-MnSOD/1or2Leu-GPx1" patients, respectively. The percentage of patients with stainable hepatic iron increased progressively with these genotypic associations: 22%, 28%, 50%, and 53%, respectively (chi(2) trend test, P = 0.005). Stainable iron was a risk factor for hepatocellular carcinoma (log-rank, P = 0.0002; relative risk, 3.40). In conclusion, polymorphisms in antioxidant enzymes modulate hepatic iron accumulation and hepatocellular carcinoma development in French alcoholic patients with cirrhosis.

Alcohol dehydrogenase (ADH) was assessed in 81 patients admitted to hospital for treatment for alcohol dependence with or without liver cirrhosis, 20 patients with bipolar disorder treated with lithium carbonate and 41 patients with various mental disorders treated with psychopharmacologic agents. Testing the hypothesis of the arithmetic mean showed that in alcohol dependents the arithmetic mean of ADH activity (12.19 nkat/l+/-5.61) differs significantly from that in healthy subjects (4.45 nkat/l+/-2.31) and in the group with ethanol poisoning (6.24 nkat/l+/-3.65) there is none. In the group with bipolar disorder, treated with lithium (7.39 nkat/l+/-3.11) and, in the group of patients treated with psychiatric drugs because of various mental disorders (7.79 nkat/l+/-8.51), the differences are statistically significant. In our opinion, assessing ADH activity in the sera of alcohol dependents could be an additional marker advantageous to the diagnostics, course and monitoring of therapy in such patients. In the groups of patients with mental disorders treated with psychotropic drugs, the increased ADH activity was found to be a more sensitive marker for the detection of drug hepatotoxicity.

Background: Interstitial collagenase has been considered as an essential enzyme for collagenolysis in liver fibrosis, because type I and III collagens increase predominantly in liver fibrosis. The present study aimed to demonstrate the gene expression of matrix metalloproteinases-13 (MMP-13) in the progressive phases of ethanol induced experimental liver fibrosis in rats., Methods: Thirty-four Sprague-Dawley rats were randomly divided into two groups. The experimental group (24 rats) was given ethanol (44%, 7 g/kg) every day and the control group (10) was given normal saline. Liver samples were harvested from experimental rats at 4, 12 and 24 weeks respectively. The kinetics of MMP-13 mRNA expression was assayed by semi-quantity reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR (0.24+/-0.41). The gene expression of MMP-13 was increased in the liver of the rats treated with ethanol for 4 weeks (0.62+/-0.54), but it was not considered statistically significant (P>0.05). And the livers from 12-week-treated rats showed a marked mRNA expression (1.65+/-0.47, P<0.01). Once fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39+/-0.25)., Conclusion: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, and it was induced in a distinct time frame.

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, involved in the metabolism of purines, which generates superoxide and is thought to be involved in free radical-generated tissue injury. It is present at high concentrations in the liver, from where it may be released during liver injury into the circulation, binding to vascular endothelium and causing vascular dysfunction. The cellular localization of the enzyme, essential to understanding its function, is, however, still debated. The present study has used a highly specific mouse monoclonal antibody to define the cellular distribution of XOR in normal and cirrhotic human liver. As shown previously, XOR is present in hepatocytes. However, the novel finding of this study is that XOR is present in bile duct epithelial cells, where it is concentrated toward the luminal surface. Moreover, in liver disease, proliferating bile ducts are also strongly positive for XOR. These findings suggest that the enzyme is secreted into bile, and this was confirmed by analysis of human and rat bile. Xanthine oxidase activity was 10 to 20-fold higher in liver tissue obtained from patients with liver disease, than in healthy liver. We conclude that XOR is expressed primarily in hepatocytes, but is also present in bile duct epithelial cells and is secreted into bile. Its role in bile is unknown but it may be involved in innate immunity of the bowel muscosa.

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.

Excessive alcohol consumption may cause the development of pathologies in the liver and pancreas and various digestive tract cancers. The enzymes GSTM1, GSTT1, GSTP1, CYP1A1 and CYP2E1 are involved in the bioactivation and detoxification of a variety of xenobiotics present in food, organic solvents, tobacco smoke, drugs, pesticides, environmental pollutants and alcoholic drinks. Polymorphisms in the genes coding for these enzymes have been associated with susceptibility to different diseases, including ethanol-related diseases. To investigate whether these polymorphisms represent risk-modifying factors for ethanol-related diseases, a study was conducted involving 120 Brazilian alcoholics and 221 controls with similar ethnic backgrounds. The distribution of alcoholics groups was as follows: 65 with liver cirrhosis, 14 with chronic pancreatitis and 41 without cirrhosis or pancreatitis. The data revealed that carriers of the rare GSTP1 Val allele were at higher risk of liver cirrhosis and pancreatitis, since we found higher frequencies of the Val/Val genotype in alcoholics with liver cirrhosis (15.4%) and pancreatitis (28.6%) in comparison with alcoholics without disease (7.3%). No differences were found in the prevalences of the GSTM1 and GSTT1 null genotypes between alcoholics and the controls and no association was found between the rare CYP2E1 c2 allele and liver cirrhosis and pancreatitis. However, when the mutant CYP1A1 allele was compared between alcoholics and controls, the m2/m2 genotype was more prevalent in the liver cirrhosis alcoholics (7.7%) than in the controls (1.4%) and this difference was statistically significant (P = 0.03, OR = 5.33). In conclusion, our data indicate an association between occurrence of the Val/Val GSTP1 genotype and chronic pancreatitis and an association between the m2/m2 CYP1A1 genotype and alcoholic liver cirrhosis. This could indicate that persons with these genotypes are genetically more prone to the development of alcoholic pancreatitis and alcoholic cirrhosis, respectively.

Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. CYP2E1 is of interest because of its ability to metabolize and activate many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This Review Article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines developed to constitutively express the human CYP2E1 in assessing the actions of CYP2E1. Regulation of CYP2E1 is quite complex and will be briefly reviewed. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help to understand the actions of CYP2E1 and its role in alcoholic liver injury.

Chronic alcohol consumption is associated with increased incidence of variety of illnesses including cirrhosis. Studies have shown that ethanol consumption may result in increased oxidative stress with increased formation of lipid peroxides and free radicals. However, very few reports are available on their involvement in the toxicity of alcoholic cirrhosis. The present study was undertaken in 44 male subjects to evaluate the role of oxidative stress in liver injury with special reference to alcoholic or non alcoholic cirrhosis. It was observed that the parameters of liver function like total bilirubin, Alanine Transaminase (ALT), Aspartate Transaminase (AST), Alkaline Phosphatase (ALP), gamma Glutamyl transfarase (gammaGT) were increased in cirrhotic (alcoholic or non alcoholic) patients as compared to normal controls. However antioxidant enzymes like Superoxide dismutase (SOD) and Glutathine peroxidase (GPx) lipid peroxidation marker, Malondialdehyde (MDA) showed significant changes only in alcoholic cirrhosis and not in non alcoholic cirrhosis when compared with normal controls. The possibility of assessment of antioxidant enzymes to differentiate between alcoholic or non alcoholic or non alcoholic cirrhosis is postulated.

Background/aims: Genetic variations of ethanol-metabolizing enzymes can affect alcohol drinking behavior. The aims of this study were to investigate and compare the distributions of these genetic polymorphisms between a healthy control group and a heavy drinker group which included an alcoholic liver cirrhosis group., Methods: Genotypes of ADH2, ALDH2, CYP2E1, and catalase were identified by polymerase chain reaction and restriction fragment length polymorphism. Genomic DNA was extracted from peripheral leukocytes in 42 healthy controls, 12 heavy drinkers, and 30 alcoholic liver cirrhosis patients., Results: 1) The genotype frequencies of ALDH2 (1*1), ADH2 (1*1), CYP2E1 (c1c1), and catalase1 (TT) were 69%, 55%, 38%, and 12%, respectively in healthy Korean males. 2) There was a significant difference in the distribution of the genetic polymorphism of ALDH2 between the control group and heavy drinker group (12 heavy drinkers and 30 alcoholic liver cirrhosis patients). The genotype frequency of ALDH2 mutant, ALDH2 (1*2) and ALDH2 (2*2) in the heavy drinker group (12%) was significantly lower than that in the control group (30%). 3) We didn't find anyone with ALDH2 homozygote mutant (DD) in the heavy drinker group. 4) There was no significant difference in the distribution of genetic polymorphisms in ADH2, CYP2E1 and catalase1 between the two groups., Conclusions: These results suggest that the absence of ALDH2 mutant genotype is strongly related to heavy drinking behavior. We can not prove, however, any evidence that the polymorphisms of other ethanol-metabolizing enzymes are associated with the determination of alcohol-drinking behavior.

Autoimmune reactions are often associated with alcoholic liver disease; however, the mechanisms responsible are largely unknown. This study investigates the potential role of the immune response against hydroxyethyl free radical (HER)-derived antigens and of polymorphisms in immunoregulatory genes in the development of anti-cytochrome P4502E1 (CYP2E1) autoantibodies in alcohol abusers. Immunoglobulin G (IgG) recognizing human CYP2E1 and HER-derived epitopes were measured by microplate immunosorbent assay in the sera of 90 patients with alcoholic fibrosis/cirrhosis (ALD), 37 heavy drinkers without liver disease or steatosis only (HD), and 59 healthy subjects. Single nucleotide polymorphisms in the interleukin 10 (IL-10) promoter and in exon 1 of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. The titers and frequency of anti-CYP2E1 autoantibodies were significantly higher in ALD than in HD subjects or controls. ALD patients with anti-HER IgG had higher titers and a 4-fold increased risk (OR: 4.4 [1.8-10.9]) of developing anti-CYP2E1 autoantibodies than subjects without anti-HER antibodies. The mutant CTLA-4 G allele, but not the IL-10 polymorphism, was associated with an enhanced risk of developing anti-CYP2E1 IgG (OR: 3.8 [1.4-10.3]). CTLA-4 polymorphism did not influence antibody formation toward HER-antigens. ALD patients with concomitant anti-HER IgG and the CTLA-4 G allele had a 22-fold higher (OR: 22.9 [4.2-125.6]) risk of developing anti-CYP2E1 autoreactivity than subjects negative for these factors. In conclusion, antigenic stimulation by HER-modified CYP2E1 combined with an impaired control of T-cell proliferation by CTLA-4 mutation promotes the development of anti-CYP2E1 autoantibodies that might contribute to alcohol-induced liver injury.

Cerivastatine was administered as a reversible HMG-CoA reductase inhibitor (statine) to treat hypercholesterolemia until its withdrawal from the market following 52 reports of death due to drug-related rhabdomyolysis and acute renal failure. In most cases, cerivastatine was applied in combination with drugs which influenced the liver metabolism of cerivastatine via cytochromeoxidase P 450 isoenzymes. We report a well-documented case of acute rhabdomyolysis following cerivastatine monotherapy. The diagnosis was confirmed additionally by muscle biopsy.Finally,we give an overview of the current knowledge concerning therapy with HMG-CoA reductase inhibitors,1 year after the withdrawal of cerivastatine from the market.

The purpose of the study was to investigate the effect of mild liver impairment on the pharmacokinetics and metabolism of bosentan. Eight patients with mild liver impairment and 8 matching healthy subjects were treated with single and multiple oral 125-mg doses of bosentan. The pharmacokinetic parameters of bosentan and its metabolites were similar in both groups: geometric means for Cmax and AUC for bosentan were 2534 and 1980 ng/ml and 11,957 and 10,781 ng.h/ml after single doses and were 1831 and 1715 ng/ml and 7216 and 7838 ng.h/ml after multiple doses, respectively, in healthy subjects and patients. In both groups, the exposure to the metabolites was low when compared to that to bosentan. The decrease in exposure to bosentan after multiple dosing, indicative of autoinduction, tended to be less pronounced in patients as compared to healthy subjects. Bosentan was well tolerated in this study. In conclusion, the pharmacokinetics, metabolism, and tolerability of bosentan are similar in healthy subjects and patients with mild liver impairment.

Background: Ethanol may increase the production of reactive oxygen species (ROS) in the liver, which results in the development of alcoholic liver disease (ALD). Cytokine-activated blood leukocytes may also participate in this process. The aim of our study was to evaluate the oxidative stress level in the blood of patients with alcoholic liver cirrhosis., Material/methods: Blood neutrophils from 16 patients with compensated alcoholic liver cirrhosis and 28 patients with decompensated were evaluated for their ability to produce superoxide anion and hydrogen peroxide spontaneously and after PMA induction, in comparison to controls (16 healthy persons). Serum catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were also measured in both groups of patients and in the controls., Results: The neutrophils from patients with compensated liver cirrhosis spontaneously produced a normal level of O2*- and a high level of H2O2, but exhibited a defect in PMA-induced O2*- production. The neutrophils from patients with decompensated liver cirrhosis spontaneously produced more O2*- and H2O2 than the controls, but the PMA response was weak. There were no changes in the expression of GPx, but enhanced SOD activity was observed in the serum of patients with decompensated alcoholic liver cirrhosis. Increased CAT activity was detected only in serum from patients with compensated liver cirrhosis., Conclusions: These data point to oxidative stress in the blood of patients with decompensated alcoholic cirrhosis, since increased resting production of ROS in neutrophils was not accompanied by increased GPx and CAT activity in serum.

Alcohol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH) and cytochrome P-4502E1 (CYP2E1), and then to acetate by aldehyde dehydrogenase (ALDH). Polymorphisms of these ethanol-metabolizing enzymes may be associated with inter-individual difference in alcohol metabolism and susceptibility to alcoholic liver disease. We determined genotype and allele frequencies of ALDH2, CYP2E1, ADH2, and ADH3 in male Korean patients with alcoholic cirrhosis (n=56), alcoholics without evidence of liver disease (n=52), and nondrinkers (n=64) by using PCR or PCR-directed mutagenesis followed by restriction enzyme digestion. The prevalences of heterozygous ALDH2*1/*2 plus homozygous ALDH2*2/*2 in patients with alcoholic cirrhosis (7.1%) and alcoholics without evidence of liver disease (3.8%) were significantly lower than that in nondrinkers (45.3%). The c2 allele frequencies of the CYP2E1 in alcoholic cirrhosis, alcoholics without evidence of liver disease, and nondrinkers were 0.21, 0.20, and 0.20, respectively. Allele frequencies of ADH2*2 in the three groups were 0.78, 0.74, and 0.77 and those of ADH3*1 were 0.94, 0.98, and 0.95. Therefore, we confirmed the observation that the ALDH2*2 gene protects against the development of alcoholism. However, the development of cirrhosis in Korean alcoholic patients was not associated with polymorphisms of ethanol-metabolizing enzymes.

Background and Aim: Acute alcohol withdrawal causes changes in hepatic blood flow and metabolism that may result in liver damage. This study aims to assess liver function tests and markers of hepatic fibrogenesis following alcohol withdrawal in alcoholics with clinically compensated liver disease., Methods: Serial liver function tests and clinical assessments were performed on 22 male alcoholics during alcohol withdrawal. Plasma tissue inhibitor of metalloproteinase 1 (TIMP1), an inhibitor of collagen degradation, and plasma amino-terminal procollagen III peptide (PIIINP), a collagen precursor molecule, were measured in these alcoholics and in 11 control subjects., Results: Transaminase levels did not change significantly over 7 days when all subjects were analyzed together. However, 32% of subjects showed a marked transaminase rise. These subjects did not differ from the others in baseline characteristics or short-term outcome, but had a greater benzodiazepine requirement. Only one subject consumed paracetamol (acetaminophen; 1-2 g/day). He had the largest transaminase rise. By comparing PIIINP assays, intact PIIINP concentration appears to increase following alcohol withdrawal. The TIMP1 levels were elevated in alcoholic subjects, but did not change following withdrawal., Conclusions: Increasing PIIINP suggests that hepatic fibrogenesis increases, or hepatic clearance falls, during acute alcohol withdrawal. The TIMP1 elevation in these alcoholics suggests that the inhibition of collagen degradation occurs while liver disease is still compensated. The period following alcohol withdrawal may be a time of marked increased susceptibility to paracetamol. The biochemical changes we observed were not associated with adverse short-term outcome, but the cumulative effect after repeated episodes of abrupt withdrawal may be of concern.

Alcoholism is related to malnutrition and low levels of several vitamins that take part in the metabolism of homocysteine. The objective of the study was to analyze the prevalence of hyperhomocysteinemia in patients with heavy alcohol intake and the factors on which it depends. Included in the study were 103 hospitalized heavy drinkers (i.e., patients with an intake of alcohol greater than 80 g per day). Serum homocysteine, folate, and vitamin B(12) levels, plasma vitamin B(6) levels, and CT677 polymorphisms of methylenetetrahydrofolate reductase (MTHFR) were determined. We also recorded the intensity of alcoholism, the status of nutrition, and the existence of liver cirrhosis. Determination of biochemical data was repeated after 15 days of withdrawal. Serum homocysteine levels were found to be significantly elevated, whereas serum folate and plasma B(6) levels were significantly decreased. Serum homocysteine levels were significantly higher in those heavy drinkers who showed the TT polymorphism of MTHFR, with a prevalence of hyperhomocysteinemia of 84.2% in the homozygote TT, 54.3% in the heterozygote CT, and 31.6% in the normal CC genotype. Serum homocysteine inversely correlated with serum folate, serum B(12), and plasma B(6) levels. We did not find any relation between serum homocysteine and intensity of alcoholism, nutritional status, or liver cirrhosis. Serum folate levels were significantly decreased in heavy drinkers, mainly depending on irregular feeding and malnutrition. After 15 days of withdrawal, serum homocysteine levels significantly decreased, whereas folate, B(12), and B(6) levels significantly increased. The conclusion is that heavy drinkers show a high prevalence of hyperhomocysteinemia related to low levels of folate, B(6), and B(12) and to the TT polymorphism of MTHFR., (Copyright 2001 Elsevier Science Inc.)

Arylsulfatase (ASA) enzyme deficiency is associated with metachromatic leukodystrophy (MLD), which is a hereditary myelin metabolic disease. It has been proposed that in alcoholic subjects with abnormal ASA, the accumulation of sulfatides may lead to demyelinization and generalized cerebral atrophy. ASA may be diminished in subjects with alcoholic cirrhosis having encephalopathic manifestations. This idea has not been previously proposed. Leukocyte arylsulfatase A (ASA) activity was measured in 30 healthy male volunteers and 28 patients with alcohol-related cirrhosis. The patients were divided into two groups: patients with alcohol-related cirrhosis with hepatic encephalopathy history and patients with alcoholic cirrhosis without history of hepatic encephalopathy. Alcoholic cirrhotic patients with history of encephalopathy showed 58.21% (40.95 nmol/mg protein/h) less enzymatic activity than a control group (98.00 nmol/mg protein/h), whereas the group without history of encephalopathy showed an ASA value which was 38.2% (60.55 nmol/mg protein/h) less than the control group. The results suggest that the low ASA activity is a factor associated to the appearance of encephalopathy in patients with alcohol-related cirrhosis.

Nitric oxide (NO) has been proposed to have an important role in the immune response. Plasma nitrate levels increase during acute rejection and decrease after treatment with corticosteroids, but little is known about its potential cellular source. We studied inducible NO synthase (iNOS) expression in liver biopsy specimens of 12 patients with acute rejection compared with biopsy specimens from the same patients after treatment with high doses of intravenous corticosteroids. We also compared iNOS expression during acute rejection with a control group (9 patients without histological rejection). iNOS expression was assessed by immunohistochemistry. Intrahepatic iNOS expression was only observed in the cytoplasm of hepatocytes, which were diffusely distributed throughout hepatic lobules. iNOS expression could not be shown in portal tracts, inflammatory cells, or endothelial and sinusoidal lining cells. In patients with acute rejection, iNOS expression was significantly stronger than in the control group (2 +/- 0.7 v 0.6 +/- 0.7; P <.05). After treatment with corticosteroids, iNOS expression decreased significantly (2 +/- 0.7 v 1.3 +/- 0.9; P <.05). In conclusion, the findings of the present study show that during acute liver rejection, hepatocytes are the main cellular source for NO production and treatment with corticosteroids induces significant downregulation of intrahepatic iNOS expression.

Aim/background: In various hepatobiliary diseases mast cells have been found to be associated with fibrogenesis. However, mast cell subpopulations have not been investigated in the human liver in normal subjects or in disease. Human mast cells are categorized into mast cells positive for tryptase (MC(T)) only and mast cells positive for both tryptase and chymase (MC(TC))., Methods: In this study we investigated mast cell subpopulations (MC(T) and MC(TC)) by double immunostaining for mast cell tryptase and chymase as well as by a computer-aided quantitative morphometry in 13 normal livers and in 193 liver tissue specimens comprising of primary biliary cirrhosis (n=43), autoimmune hepatitis (n=11), chronic hepatitis B (n=37), chronic hepatitis C (n=41), alcoholic liver disease (n=40) and hepatolithiasis (n=21)., Results: The densities of MC(T) and MC(TC) per 1 mm2 stroma were low in normal livers but high in chronic liver diseases, and correlated positively with the degree of fibrosis. The percentages of MC(T) and MC(TC) subpopulations were 25% and 75%, respectively. The percentage was almost the same in normal livers and various hepatobliliary diseases, as well as between less fibrotic cases and more fibrotic cases in liver diseases., Conclusions: These results suggest that MC(T) and MC(TC) subpopulations in healthy and diseased livers do not change during liver fibrosis of any etiology.

Fibroblast activation protein (FAP) is a cell surface-bound protease of the prolyl oligopeptidase gene family expressed at sites of tissue remodelling. This study aimed to delineate the expression of FAP in cirrhotic human liver and examine its biochemical activities. Seventeen cirrhotic and 8 normal liver samples were examined by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Hepatic stellate cells (HSC) were isolated and immunostained. Recombinant FAP and immunopurified, natural FAP were analyzed for protease activities and similarities to dipeptidyl peptidase IV (DPPIV), a structurally related enzyme. FAP-specific messenger RNA and immunoreactivity were detected in cirrhotic, but not normal, livers. FAP immunoreactivity was most intense on perisinusoidal cells of the periseptal regions within regenerative nodules (15 of 15 cases); this pattern coincides with the tissue remodelling interface. In addition, human FAP was expressed by cells within the fibrous septa (10 of 15 cases). Cell morphology, location, and colocalization with glial fibrillary acidic protein (GFAP) indicated that FAP is present on HSC in vivo. Similarly, isolated HSC expressed FAP in vitro. Both natural FAP from cirrhotic liver and recombinant FAP were shown to have gelatinase and dipeptidyl peptidase activities. FAP is a cell-bound, dual-specificity dipeptidyl peptidase and gelatinase expressed by activated HSC at the tissue remodelling interface in human cirrhosis. FAP may contribute to the HSC-induced extracellular matrix (ECM) changes of cirrhosis.