8-hydroxy-2--deoxyguanosine and Hepatitis-B--Chronic

The hypomethylation of the Cyclin D1 (CCND1) promoter induced by excess oxidative stress likely promotes the development of hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). We aimed to evaluate methylation status of the CCND1 promoter as a new plasma marker for the detection of HBV-HCC.We consecutively recruited 191 participants, including 105 patients with HBV-HCC, 54 patients with chronic hepatitis B (CHB), and 32 healthy controls (HCs). Using methylation-specific polymerase chain reaction, we identified the methylation status of the CCND1 promoter in plasma samples. We analyzed the expression levels of the CCND1 mRNA in peripheral blood mononuclear cells by using quantitative real-time PCR. We assessed the plasma levels of superoxide dismutase, 8-hydroxydeoxyguanosine and malondialdehyde by using enzyme-linked immunosorbent assays.Patients with HBV-HCC (23.81%) presented a reduced methylation frequency compared with patients with CHB (64.81%) or HCs (78.13%) (P < .001). When receiver operating characteristic curves were plotted for patients with HBV-HCC versus CHB, the methylation status of the CCND1 promoter yielded diagnostic parameter values for the area under the curve of 0.705, sensitivity of 76.19%, and specificity of 64.81%, thus outperforming serum alpha-fetoprotein (AFP), which had an area under the curve of 0.531, sensitivity of 36.19%, and specificity of 90.74%. Methylation of the CCND1 promoter represents a prospective diagnostic marker for patients with AFP-negative HBV-HCC and AFP-positive CHB. The expression levels of CCND1 mRNA was increased in patients with HBV-HCC compared with patients with CHB (Z = -4.946, P < .001) and HCs (Z = -6.819, P < .001). Both the extent of oxidative injury and antioxidant capacity indicated by the superoxide dismutase, 8-hydroxydeoxyguanosine and malondialdehyde levels were increased in patients with HBV-HCC. Clinical follow up of patients with HBV-HCC revealed a worse overall survival (P = .012, log-rank test) and a decreased progression-free survival (HR = 0.109, 95%CI: 0.031-0.384) for the unmethylated CCND1 group than methylated CCND1 group.Our study confirms that oxidative stress appears to correlate with plasma levels of CCND1 promoter methylation, and the methylation status of the CCND1 promoter represents a prospective biomarker with better diagnostic performance than serum AFP levels.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cyclin D1; DNA Methylation; Early Detection of Cancer; Enzyme-Linked Immunosorbent Assay; Female; Hepatitis B, Chronic; Humans; Liver Neoplasms; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Promoter Regions, Genetic; Prospective Studies; Real-Time Polymerase Chain Reaction; ROC Curve; Sensitivity and Specificity; Superoxide Dismutase

Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes.. The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis.. In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2.. Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cell Line, Tumor; Deoxyguanosine; DNA Damage; DNA Methylation; DNA Repair Enzymes; Hepatitis B; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Humans; Hydrogen Peroxide; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphoric Monoester Hydrolases; Promoter Regions, Genetic; Pyrophosphatases; Reactive Oxygen Species; Trans-Activators; Viral Regulatory and Accessory Proteins

The significance of antibodies to cardiolipin (anti-CL) remains uncertain in patients with chronic hepatitis C (CH-C). The main purpose of this study was to elucidate the clinical characteristics of patients with CH-C seropositive for anti-CL. The prevalence of anti-CL and clinical parameters associated with anti-CL in those patients were examined. Six of the 45 (13%) patients with CH-C had anti-CL. However, none of these six CH-C patients fulfilled the criteria for antiphospholipid syndrome. Serum triglyceride and apolipoprotein B (ApoB) levels in CH-C patients with anti-CL were significantly higher than those in CH-C patients without anti-CL. Serum triglyceride levels positively correlated with serum ApoB levels. CH-C patients with anti-CL had significantly more progressive hepatic fibrosis than those without anti-CL. The degree of 8-hydroxy 2'-deoxyguanosine (8-OHdG) expression in the liver tissue was more severe in CH-C patients with anti-CL than in those without it. However, the emergence of anti-CL in CH-C patients was independent of insulin resistance, hepatic steatosis, and iron overload. These findings suggest that the emergence of anti-CL is associated with oxidative stress and that CH-C patients seropositive for anti-CL have clinical characteristics of hypertriglyceridemia, which derives from the facilitation of ApoB synthesis, and progressive hepatic fibrosis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Antibodies, Anticardiolipin; Apolipoproteins B; Deoxyguanosine; Fatty Liver; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; Histocytochemistry; Humans; Liver; Male; Middle Aged; Prevalence; Triglycerides

Chronic hepatitis B (CHB) appears to progress more rapidly in males than in females, and CHB-related hepatic cirrhosis and hepatocellular carcinoma are predominately diseases that tend to occur in men and postmenopausal women. To obtain more insight into the underlying mechanisms of gender disparity of CHB progress, two-dimensional difference gel electrophoresis was employed to compare liver proteome of C57BL/6 and HBV transgenic (HBV-Tg) mice both in male and female groups. We identified 8 differently expressed proteins in male HBV-Tg mice and 12 in female HBV-Tg mice. Apolipoprotein A-I (Apo A-I) was found to be down-regulated in male and female HBV-Tg mouse liver. It is more interesting that the pattern of liver Apo A-I isoforms was altered in male HBV-Tg mice but not in female HBV-Tg mice. Our further results indicated that the basic Apo A-I isoform, based on pI positions from serum 2-dimensional Western blotting, increased in male CHB patient sera but not in female CHB patient sera. Finally, we identified that the oxidative modification Apo A-I mainly reside in basic isoform. This pattern of selectively modified Apo A-I isoforms may be considered as a pathological hallmark that may extend our knowledge of the molecular pathogenesis of CHB progression.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Apolipoprotein A-I; Blotting, Western; Deoxyguanosine; Disease Progression; DNA Damage; Electrophoresis, Gel, Two-Dimensional; Female; Hepatitis B, Chronic; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxidation-Reduction; Protein Isoforms; Sex Factors; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

8-Hydroxydeoxyguanosine (8-OHdG) is a promutagenic DNA lesion produced by hydroxyl radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress. The aim of this study was to clarify the clinical significance of hepatic 8-OHdG levels in patients with chronic viral hepatitis. Hepatic 8-OHdG accumulation was investigated in patients with chronic hepatitis C (CH-C) (n = 77) and chronic hepatitis B (CH-B) (n = 34) by immunohistochemical staining of liver biopsy samples. 8-OHdG positive hepatocytes were significantly higher in patients with CH-C compared to CH-B (median 55.0 vs 18.8 cells/10(5) mum(2), P < 0.0001). The number of positive hepatocytes significantly increased with the elevation of serum aminotransferase levels, especially in CH-C patients (8-OHdG vs alanine aminotransferase (ALT)/aspartate aminotrasferase (AST) were r = 0.738/0.720 in CH-C and 0.506/0.515 in CH-B). 8-OHdG reactivity was strongly correlated with body and hepatic iron storage markers in CH-C (vs serum ferritin, r = 0.615; vs hepatic total iron score, r = 0.520; vs hepatic hepcidin mRNA levels, r = 0.571), although it was related to serum HBV-DNA titers (r = 0.540) and age of patients (r = -0.559) in CH-B. These results indicate that hepatic oxidative DNA damage is common in chronic viral hepatitis, in particular chronic HCV-infected patients, suggesting a possible link between chronic hepatic inflammation and hepatocarcinogenesis. The strong positive correlation between hepatic DNA damage and iron overload suggests that iron content is one of the most likely mediators of hepatic oxidative stress and iron reduction may be beneficial to reduce the incidence of hepatic cancer in CH-C patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alanine Transaminase; Aspartate Aminotransferases; Deoxyguanosine; DNA Damage; Female; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Iron Overload; Liver; Male; Middle Aged; Oxidative Stress

8-Hydroxydeoxyguanosine (8-OHdG) is a promutagenic DNA lesion produced by oxygen radicals and is recognized as a useful marker in estimating DNA damage induced by oxidative stress.. Hepatic expression of 8-OHdG was immunohistochemically investigated in control and diseased human livers.. While no positive immunolabeling for 8-OHdG was observed in control livers, 8-OHdG was widely evident in diseased livers. Nuclear expression of 8-OHdG in the hepatocytes and bile duct cells were found in various forms of chronic hepatitis. 8-OHdG-positive hepatocytes were especially abundant in the periportal area with piecemeal necrosis and prominent cell infiltration. The number of positive hepatocytes significantly increased with the progression of severity of chronic hepatitis activity (r(s)=0.68, P<0.05). In alcoholic liver disease, nuclear expression of 8-OHdG was detected in the hepatocytes in the area of alcoholic hepatitis. Regarding primary biliary cirrhosis, 8-OHdG was preferentially detected in the nuclei of injured bile ducts (11 of 12 cases, 91.7%) and occasionally (2 of 12 cases, 16.7%) in the nuclei of hepatocytes around the bile duct lesions.. These results indicate that oxidative DNA damage is common in various forms of chronic liver disease suggesting a possible link between chronic inflammation and hepatocarcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Bile Duct Diseases; Biomarkers; Deoxyguanosine; DNA Damage; Hepatitis; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Immunohistochemistry; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Liver Diseases, Alcoholic; Middle Aged; Mitotic Index; Necrosis; Oxidation-Reduction