8-hydroxy-2--deoxyguanosine and Liver-Neoplasms

Oxidative stress may play pathogenic roles in the mechanisms underlying chronic hepatitis C (CHC). The impact of excessive oxidative stress and iron dysregulation on the development of hepatocellular carcinoma (HCC) after interferon therapy has not been established.. We investigated the impact of oxidative stress and iron deposition on HCC development after therapy with pegylated interferon (PegIFN)+ribavirin in CHC patients. Systemic and intracellular iron homeostasis was evaluated in liver tissues, peripheral blood mononuclear cells and sera.. Of 203 patients enrolled, 13 developed HCC during the 5.6-year follow-up. High hepatic 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were significantly associated with HCC development in multivariate analysis (p=0.0012) which was also significantly correlated with severity of hepatic iron deposition before therapy (p<0.0001). Systemic and intracellular iron regulators of hepcidin and F-box and leucine-rich repeat protein 5 (FBXL5) expression levels were significantly suppressed in CHC patients (p=0.0032 and p=0.016, respectively) despite their significantly higher levels of serum iron and ferritin compared with controls. However, intracellular iron regulators of FBXL5 and iron regulatory proteins were regulated in balance with hepatic iron deposition. Significant correlations were observed among IL-6, bone morphogenetic protein 6, hepcidin and ferroportin, as regards systemic iron regulation.. Measurement of hepatic oxidative stress before antiviral therapy is useful for the prediction of HCC development after interferon therapy. Low baseline levels of the intracellular iron regulators of FBXL5 in addition to a suppressed hepcidin level might be associated with severe hepatic iron deposition in CHC patients.. UMIN 000001031.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Antiviral Agents; Biomarkers; Carcinoma, Hepatocellular; Deoxyguanosine; Disease Progression; Drug Therapy, Combination; Female; Follow-Up Studies; Hepatitis C, Chronic; Humans; Interferon-alpha; Iron Metabolism Disorders; Leukocytes, Mononuclear; Liver; Liver Neoplasms; Logistic Models; Male; Middle Aged; Multivariate Analysis; Odds Ratio; Oxidative Stress; Polyethylene Glycols; Proportional Hazards Models; Recombinant Proteins; Ribavirin; Risk Factors; Severity of Illness Index; Time Factors; Treatment Outcome

Modulation of urinary excretion of green tea polyphenols (GTPs) and oxidative DNA damage biomarker, 8-hydroxydeoxyguanosine (8-OHdG), were assessed in urine samples collected from a randomized, double-blinded and placebo-controlled phase IIa chemoprevention trial with GTP in 124 individuals. These individuals were sero-positive for both HBsAg and aflatoxin-albumin adducts, and took GTP capsules daily at doses of 500 mg, 1000 mg or a placebo for 3 months. Twenty-four hour urine samples were collected before the intervention and at the first and third month of the study. Urinary excretion of 8-OHdG and GTP components was measured by HPLC-CoulArray electrochemical detection. The baseline levels of 8-OHdG and GTP components among the three groups showed homogeneity (P > 0.70), and a non-significant fluctuation was observed in the placebo group over the 3 months (P > 0.30). In GTP-treated groups, epigallocatechin (EGC) and epicatechin (EC) levels displayed significant and dose-dependent increases in both the 500 mg group and 1000 mg group (P < 0.05). The 8-OHdG levels did not differ between the three groups at the 1 month collection, with medians of 1.83, 2.08 and 1.86 ng/mg-creatinine for placebo, 500 and 1000 mg group, respectively (P = 0.999). At the end of the 3 months' intervention, 8-OHdG levels decreased significantly in both GTP-treated groups, with medians of 2.02, 1.03 and 1.15 ng/mg-creatinine for placebo, 500 mg and 1000 mg group, respectively (P = 0.007). These results suggest that urinary excretions of EGC and EC can serve as practical biomarkers for green tea consumption in human populations. The results also suggest that chemoprevention with GTP is effective in diminishing oxidative DNA damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers; Catechin; Chemoprevention; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Double-Blind Method; Female; Flavonoids; Humans; Liver Neoplasms; Male; Oxidative Stress; Phenols; Polyphenols; Risk Factors; Tea

Liver cancer is a critical clinical condition with augmented malignancy, rapid progression, and poor prognosis. Liver cancer often initiates as fibrosis, develops as cirrhosis, and results in cancer. For centuries, medicinal plants have been incorporated in various liver-associated complications, and recently, research has recognized that many bioactive compounds from medicinal plants may interact with targets related to liver disorders. Phyllanthin from the Phyllanthus species is one such compound extensively used by folklore practitioners for various health benefits. However, most practices continue to be unrecognized scientifically. Hence, in this work, we investigated the protective role of phyllanthin on diethylnitrosamine (DEN) induced liver carcinoma in Wistar Albino rats and the anti-tumor potential on human hepatocellular carcinoma (HCC) HepG2 cells. The DEN-challenged liver cancer in experimental rats caused increased liver weight, 8-OHD, hepatic tissue injury marker, lipid peroxidation, and tumor markers levels. Remarkably, phyllanthin counteracted the DEN effect by ameliorating all the liver function enzymes, oxidative DNA damage, and tumor-specific markers by enhanced anti-oxidant capacity and induced caspase-dependent apoptosis through the mTOR/ PI3K signaling pathway. MTT assay demonstrated that phyllanthin inhibited the HepG2 cell growth in a dose-dependent manner. Fascinatingly, phyllanthin did not demonstrate any substantial effect on the normal cell line, HL7702. In addition, HepG2 cells were found in the late apoptotic stage upon treatment with phyllanthin as depicted by acridine orange/ethidium bromide staining. Overall, this work offers scientific justification that phyllanthin can be claimed to be a safe candidate with potential chemotherapeutic activity against HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Biomarkers, Tumor; Body Weight; Cell Survival; Diethylnitrosamine; Disease Models, Animal; Hep G2 Cells; Humans; Lignans; Liver Neoplasms; Male; Oxidative Stress; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats, Wistar; TOR Serine-Threonine Kinases

Hepatocellular carcinoma (HCC) has a poor prognosis in the setting of chronic inflammation and fibrosis, both of which promote nuclear DNA oxidative damage. 8-hydroxy-deoxyguanosine (8-OHdG) DNA glycosylase (OGG1) enhances the repair of 8-OHdG, which is the primary oxidative stress-induced mutation that leads to malignant alterations. This study aims to clarify the relationships between oxidative stress-induced factors and HCC progression. The clinicopathological factors were compared with immunohistochemistry OGG1 and 8-OHdG expressions in 86 resected HCC specimens. High 8-OHdG expression was associated with high serum aspartate transaminase and total bilirubin levels, as well as a low platelet count, compared with low 8-OHdG expression. Histological liver cirrhosis and poor differentiation were more frequent in patients with high 8-OHdG expression than in those with low 8-OHdG expression. The 8-OHdG was negatively correlated with OGG1 expression in HCC patients. Therefore, we classified the patients into two groups, low OGG1/high 8-OHdG group and the other group. The patients with low OGG1/high 8-OHdG expressions had worse prognosis than those with the other expressions. Our results showed that low OGG1/high 8-OHdG expressions in nuclei influence HCC patient outcomes. Evaluating the patterns of OGG1 and 8-OHdG expressions might provide pivotal prognostic biomarkers in patients with HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Hepatocellular; Deoxyguanosine; Disease Progression; DNA Glycosylases; Female; Humans; Immunohistochemistry; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Oxidative Stress; Prognosis; Reactive Oxygen Species; Young Adult

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

Studies have shown that liraglutide, or human umbilical cord mesenchymal stem cell (hUC-MSCs) can improve non-alcoholic fatty liver disease (NAFLD). However there have been no studies on the combination of the two used to treat NAFLD. This study aimed to explore the therapeutic effects of combination of liraglutide and hUC-MSCs on liver injury in rats with type 2 diabetes mellitus (T2DM) and NAFLD, and further investigate their mechanisms. Sprague Dawley rats fed by a high fat and high sucrose diet were randomly divided into 5 groups, including NC group, T2DM/NAFLD group, liraglutide group (treated with liraglutide, 200 μg/kg, twice daily for 8 weeks), hUC-MSCs group (treated with hUC-MSCs at the first and fifth weeks), liraglutid＋hUC-MSCs group (treated with liraglutide and hUC-MSCs). Liver tissue was procured for histological examination, real-time qRT-PCR and Western blot analysis. After treatment, liraglutide and hUC-MSCs reduced serum ALT and AST levels, alleviate liver inflammation and improved liver histopathology. The expressions of inflammatory cytokines, TLR4 and NF-κB in serum and liver were significantly inhibited, particularly in the combination treatment group. Eight weeks after liraglutide or hUC-MSCs administration, FBG, HbA1c, HOMA-IR, ALT, AST, Liver wet eight and hepatic TLR4, NF-κB, IL-6, TNF-α, 8-OHdG mRNA and proteins were significantly decreased, and the levels of SOD expression were significantly increased in three treatment groups compared with T2DM/NAFLD group. This study suggests that liraglutide in combination with hUC-MSCs could significantly improve glycolipid metabolism, insulin resistance and liver injury in T2DM/NAFLD rats. Its mechanism may be related to the down-regulation of the TLR4/NF-κB inflammatory pathway and improvement in oxidative stress.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Combined Modality Therapy; Cytokines; Diabetes Mellitus, Type 2; Humans; Inflammation; Liraglutide; Liver; Liver Neoplasms; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; NF-kappa B; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Rats, Sprague-Dawley; Superoxide Dismutase; Toll-Like Receptor 4; Umbilical Cord

Molecular mechanisms underlying Hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) pathogenesis are still unclear. Therefore, we analyzed the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and other oxidative lesions at codon 176 of the p53 gene, as well as the generation of 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinoma, Hepatocellular; Cell Line, Tumor; Codon; Cytosine; DNA Adducts; Genes, p53; Hep G2 Cells; Hepacivirus; Hepatitis C; Humans; Lipid Peroxidation; Liver Neoplasms; Polymerase Chain Reaction; Reactive Oxygen Species

Troxerutin (TXER) a rutin derivative is known for its anticancer effect against hepatocellular carcinoma (HCC). As part of large study, recently we have shown TXER interact with genetic material and its anti-mutagenic property. In the present study we have explored its possible mode of action in HCC. Since TXER alone did not show significant anticancer effect on Huh-7 cells, in vitro biochemical assays were performed for determining anticancer efficacy of TXER + metal complex using transition metals such as Cu, Zn, and Fe. The anticancer efficacy of TXER + Cu on Huh-7 cells were evaluated using MTT assay, DCFDA, JC-1 staining, comet assay, cell cycle analysis, immunocytochemistry, and Western blotting. Non-toxic nature of TXER was analyzed on primary rat hepatocytes. The in vivo efficacy of TXER was tested in N-nitrosodiethylamine initiated and γ-benzene hexachloride and partial hepatectomy promoted rat liver cancer. Liver markers, transition metal levels, histopathological examination, and expression levels of GST-P, 8-OHdG and Ki-67 were studied to assess the in vivo anticancer effect of TXER. We observed that TXER + Cu induced extensive cellular death on Huh-7 cells through generating free radicals and did not possess any toxic effect on normal hepatocytes. The in vivo studies revealed that TXER possess significant anti-cancer effect as assessed through improved liver markers and suppressed GST-P, 8-OHdG, and Ki-67 expression. TXER treatment reduced the hepatic Cu level in cancer bearing animals. Current study brings the putative mechanism involved in anti-cancer effect of TXER, further it will help to formulate phytoconstituents coupled anti-cancer drug for effective treatment of HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Catalase; Cell Line, Tumor; Coordination Complexes; Copper; Deoxyguanosine; DNA Damage; Glutathione S-Transferase pi; Humans; Hydroxyethylrutoside; Ki-67 Antigen; Liver; Liver Neoplasms; Oxidative Stress; Rats; Rats, Wistar; Superoxide Dismutase; Superoxides

One mechanism by which alcoholic liver disease (ALD) progresses is oxidative stress and the generation of reactive oxygen species, among others due to the induction of cytochrome P-4502E1 (CYP2E1). Experimental data underline the key role of CYP2E1 because ALD could be partially prevented in rats by the administration of the specific CYP2E1 inhibitor chlormethiazole. As CYP2E1 is linked to the formation of carcinogenic etheno DNA adducts in ALD patients, a causal role of alcohol-induced CYP2E1 in hepatocarcinogenesis is implicated. The purpose of this study was to investigate CYP2E1 induction in ALD, and its correlation with oxidative DNA lesions and with hepatic histology.. Hepatic biopsies from 97 patients diagnosed with ALD were histologically scored for steatosis, inflammation, and fibrosis. CYP2E1 and the exocyclic etheno DNA adduct 1,N. A significant positive correlation was found between CYP2E1 and εdA (p < 0.0001) as well as between CYP2E1 and 8-OHdG (p = 0.039). Both CYP2E1 (p = 0.0094) and ɛdA (p < 0.0001) also correlated significantly with the stage of hepatic fibrosis. Furthermore, a significant correlation between the fibrosis stage and the grade of lobular inflammation (p < 0.0001) was observed. However, the amount of alcohol consumed did not correlate with any of the parameters determined.. These data suggest an important role of CYP2E1 in the generation of εdA, in the fibrotic progression of ALD, and thus in alcohol-mediated hepatocarcinogenesis. CYP2E1 may be a target in the treatment of ALD and a potential prognostic marker for disease progression.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinogenesis; Carcinoma, Hepatocellular; Cytochrome P-450 CYP2E1; Deoxyadenosines; Deoxyguanosine; Female; Fibrosis; Humans; Immunohistochemistry; Inflammation; Intra-Abdominal Fat; Liver; Liver Cirrhosis, Alcoholic; Liver Diseases, Alcoholic; Liver Neoplasms; Male; Middle Aged

The carcinogenic potential of phenobarbital (PB) was assessed in a mouse line carrying a mutant

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenoma; Animals; Apoptosis; Cell Proliferation; Deoxyguanosine; Disease Progression; DNA Glycosylases; Female; Immunohistochemistry; Liver Neoplasms; Male; Mice, Inbred C57BL; Mice, Mutant Strains; Models, Biological; NF-E2-Related Factor 2; Phenobarbital; Survival Analysis; Tandem Mass Spectrometry

Reactive oxygen species (ROS) is excessively generated in tumors creating an oxidative stress in tumor microenvironment. We investigated hepatic expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and 8-hydroxydeoxyguanosine (8-OHdG) in hepatocellular carcinoma (HCC) patients, and asked if ROS epigenetically upregulated Nrf2 and enhanced aggressiveness in HCC cells. Expression of Nrf2 (n = 100) and 8-OHdG (n = 53) was remarkably increased in HCC tissues compared with the noncancerous hepatic tissues. Elevated expression of 8-OHdG was associated with poor survival in HCC patients. H

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinoma, Hepatocellular; Deoxyguanosine; Disease Progression; Female; Hep G2 Cells; Humans; Hydrogen Peroxide; Liver Neoplasms; Male; Middle Aged; NF-E2-Related Factor 2; Oxidative Stress; Promoter Regions, Genetic; Reactive Oxygen Species

In viral hepatitis, inflammation is correlated with chronic oxidative stress, one of the biological events leading to DNA damage and hepatocellular carcinoma (HCC) development. Aim of this study was to investigate the complex molecular network linking oxidative damage to telomere length and telomerase activity and regulation in hepatitis C and B virus-related liver carcinogenesis. We investigated 142 patients: 21 with HCC (in both tumor and peritumor tissues) and 121 with chronic viral hepatitis in different stages. We evaluated 8-hydroxydeoxyguanosine (8-OHdG), marker of oxidative DNA damage, OGG1 gene polymorphism, telomere length, telomerase activity, TERT promoter methylation, and mitochondrial TERT localization. In hepatitis C-related damage, 8-OHdG levels increased since the early disease stages, whereas hepatitis B-related liver disease was characterized by a later and sharper 8-OHdG accumulation (P = 0.005). In C virus-infected patients, telomeres were shorter (P = 0.03), whereas telomerase activity was higher in tumors than that in the less advanced stages of disease in both groups (P = 0.0001, P = 0.05), with an earlier increase in hepatitis C. Similarly, TERT promoter methylation was higher in tumor and peritumor tissues in both groups (P = 0.02, P = 0.0001). Finally, TERT was localized in mitochondria in tumor and peritumor samples, with 8-OHdG levels significantly lower in mitochondrial than those in genomic DNA (P = 0.0003). These data describe a pathway in which oxidative DNA damage accumulates in correspondence with telomere shortening, telomerase activation, and TERT promoter methylation with a different time course in hepatitis B and C virus-related liver carcinogenesis. Finally, TERT localizes in mitochondria in HCC, where it lacks a canonical function.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinogenesis; Deoxyguanosine; DNA Damage; DNA Glycosylases; DNA, Mitochondrial; Gene Expression Regulation, Neoplastic; Hepacivirus; Hepatitis B; Hepatitis B virus; Hepatitis C; Humans; Liver Neoplasms; Oxidation-Reduction; Polymorphism, Genetic; Telomerase; Telomere Shortening

The aim of the present study was to investigate the role of interleukin (IL)-17A in the initiation and progression of hepatocellular carcinoma.. IL-17A deficient (KO) and wild-type (WT) mice were intraperitoneal injected with diethyl nitrosamine (DEN) to induce hepatocellular carcinoma, and the incidence of tumours was assessed 38 weeks later. In order to investigate the effects of DEN on hepatocytes in the acute phase of DEN administration, DEN-treated mice were sacrificed at designated time points. Serum and liver tissues were harvested for further analyses.. The tumor incidence was approximately 65 % in WT mice, but was significantly lower (by 20 %) in KO mice. The number of tumours was also less in KO mice. Serum ALT levels increased in WT mice 7 days after the administration of DEN, but were significantly lower in KO mice. Furthermore, the number of neutrophils and Kupffer cells, and the expression of TNF-α and IL-6 were reduced in KO mice. The intrahepatic expression of the oxidative DNA damage marker 8-OHdG and lipid oxidative marker 4-HNE was markedly increased in WT mice, but was significantly lower in KO mice. In addition, the increase of cell proliferation, as assessed by Ki-67 immunohistochemistry, in WT mice was significantly reduced in KO mice.. These results demonstrated that IL-17A plays a pivotal role in chemically induced hepatic carcinogenesis, which is most likely through inflammation-initiated oxidative DNA damage and cell proliferation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Carcinoma, Hepatocellular; Cell Proliferation; Deoxyguanosine; Diethylnitrosamine; DNA Damage; Gene Expression Regulation, Neoplastic; Interleukin-17; Interleukin-6; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Tumor Necrosis Factor-alpha

Non-alcoholic fatty liver disease (NAFLD) is an increasing cause of hepatocellular carcinoma (HCC). Previously, we reported that DNA oxidation induced epigenetic alteration of tumor suppressor genes (TSGs) and contributed to HCC emergence. Here, we examine the associations between clinicopathological characteristics of NAFLD and advanced oxidative DNA damage that is associated with TSG methylation in the NAFLD liver.. Liver biopsies from 65 NAFLD patients were analyzed for clinicopathological features and oxidative DNA damage using immunohistochemistry of 8-hydroxydeoxyguanosine (8-OHdG). Abnormal DNA methylation in the promoters of 6 TSGs, HIC1, GSTP1, SOCS1, RASSF1, CDKN2A, and APC, was examined using MethyLight. Associations between clinicopathological characteristics, methylation of TSGs, and accumulation of 8-OHdG were analyzed.. We found that aspartate aminotransferase/alanine aminotransferase ratio, the fibrosis-4 index, and serum α-fetoprotein (AFP) level were associated with degree of 8-OHdG, and AFP was an independent factor among them (P = 0.0271). Regarding pathological findings, hepatocellular ballooning and stage of fibrosis were also associated with oxidative DNA damage (P = 0.0021 and 0.0054); ballooning was an independent risk for detecting high degree of 8-OHdG in hepatocytes (odds ratio 7.38, 95% confidence interval 1.41-49.13, P = 0.0171). Accumulation of methylated TSGs was significantly associated with deposition of 8-OHdG (P = 0.0362).. Patients with high serum AFP and high degree of ballooning showed accumulation of oxidative DNA damage that could be a seed of DNA methylation responsible for hepatocarcinogenesis. These characteristics could be risk of HCC; such patients require urgent intervention such as lifestyle modification.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; alpha-Fetoproteins; Biopsy; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Deoxyguanosine; DNA Damage; DNA Methylation; Female; Gene Expression Regulation; Genes, Tumor Suppressor; Hepatocytes; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Non-alcoholic Fatty Liver Disease; Oxidation-Reduction; Promoter Regions, Genetic

The aim of this study was to examine the role of oxidative DNA damage in chronic liver inflammation in the evolution of hepatocellular carcinoma. The accumulated data demonstrated that oxidative DNA damage and chronic liver inflammation are involved in the transformation of normal hepatocytes and their evolution towards hepatocellular carcinoma. However, the levels of 8-oxy-2'-deoxy-guanosine (8-oxodG), a biomarker of oxidative DNA damage, were overestimated and underestimated in previous reports due to various technical limitations. The current techniques are not suitable to analyze the 8-oxodG levels in the non-malignant liver tissues and tumors of hepatocellular carcinoma patients unless they are modified. Therefore, in this study, the protocols for extraction and hydrolysis of DNA were optimized using 54 samples from hepatocellular carcinoma patients with various risk factors, and the 8-oxodG and 2'-deoxyguanosine (dG) levels were measured. The patients enrolled in the study include 23 from The Princess Alexandra Hospital and The Royal Brisbane and Women's Hospitals, Brisbane, Australia, and 31 from South Africa. This study revealed that the 8-oxodG/dG ratios tended to be higher in most non-malignant liver tissues compared to hepatocellular carcinoma tissue (p=0.2887). It also appeared that the ratio was higher in non-malignant liver tissue from Southern African patients (p=0.0479), but there was no difference in the 8-oxodG/dG ratios between non-malignant liver tissues and tumors of Australian hepatocellular carcinoma patients (p=0.7722). Additionally, this study also revealed a trend for a higher 8-oxodG/dG ratio in non-malignant liver tissues compared to tumoural tissues of patients with HBV. Significant differences were not observed in the 8-oxodG/dG ratios between non-cirrhotic and cirrhotic non-malignant liver tissues.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinogenesis; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Hepatocytes; Humans; Inflammation; Liver; Liver Neoplasms; Oxidation-Reduction; Oxidative Stress

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is present in high quantities in cigarette smoke and also in fried food, has been reported to be a probable human carcinogen. However, few studies have reported on the genotoxicity and oxidative stress induced by AαC. This study investigated the genotoxic effects of AαC in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells using the comet assay. Significant increases in DNA fragment migration indicated that AαC causes serious DNA damage in HepG2 and A549 cells. The role of oxidative stress in the mechanism of AαC-induced genotoxicity was clarified by measuring the level of intracellular reactive oxygen species (ROS), the GSH/GSSG ratio and the formation of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage. The results showed that the levels of ROS and 8-OHdG increased, whereas the GSH/GSSG ratio decreased. The concentration of 8-OHdG was positively related to DNA damage. Taken together, these results indicate that AαC can induce genotoxicity and oxidative stress and that AαC likely exerts genotoxicity in HepG2 and A549 cells through ROS-induced oxidative DNA damage. This is the first report to describe AαC-induced genotoxic and oxidative stress in HepG2 and A549 cells.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Biomarkers; Carbolines; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Comet Assay; Deoxyguanosine; DNA Damage; Glutathione; Hep G2 Cells; Humans; Liver Neoplasms; Mutagens; Osmolar Concentration; Oxidants; Oxidation-Reduction; Oxidative Stress; Pulmonary Alveoli; Reactive Oxygen Species; Respiratory Mucosa

Additional approaches to control malignancies are needed due to the emerging trends in the incidence of cancer of different organ sites. Due to the high frequency of hepatocellular carcinoma (HCC) and its poor prognosis, preventing HCC is an urgent priority. To explore the antioxidant and apoptotic pathways of grape seed extract (GSE) we induce HCC experimentally by diethylnitrosoamine (DEN) and treated the animals with low and high doses of GSE. The results indicate good therapeutic possibilities for GSE use in treatment of HCC., This was evidenced via regression of liver enzymes' function (ALT&AST), the HCC markers; α-fucosidase, α-fetoprotein and carcinoembrionic antigen (CEA) in HCC groups treated with the grape seed extract. Also, tumor necrosis factor (TNF-α) showed a significant decrease using GSE in HCC bearing animals. Regarding the apoptotic pathways of GSE, we found a significant down regulation of apoptosis enhancing nuclease (Aen), Bcl2-associated X protein (Bax), B-cell translocation gene 2(Btg2), Cyclin G1 (Ccng1) and Cyclin-dependent kinase inhibitor 1A (Cdkn1a) gene expression in HCC+GSE groups as compared to HCC bearing group. In the same trend, the necrotic/apoptotic rates were significantly higher in the HCC groups treated with GSE vs. the HCC bearing group. Finally, the 8-OHdG/2-dG generation decreased by 73.8% and 52.9% in HCC+GSE at low and high doses, respectively. Based on these encouraging observations, grape seed extract could be a promising natural remedy for attenuating hepatocellular carcinoma that has a great future in approaches directed towards control of HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; alpha-L-Fucosidase; Animals; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Deoxyguanosine; Down-Regulation; Gene Expression Regulation; Grape Seed Extract; Humans; Liver Neoplasms; Male; Necrosis; Oxygen; Prognosis; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Metabolic genotypes of 5,10-methylenetetrahydrofolate reductase (MTHFR) and folate status on oxidative DNA lesions in hepatocellular carcinoma (HCC) has not been elucidated. The aims of the study were to investigate the folate-polymorphic interactions on genetic oxidative damage in association with advanced HCC malignancy and prognosis.. The study included 232 HCC patients with folate nutrition, MTHFR C677T polymorphic, p53 genetic and tumour pathological data collected and analyzed for their survivals after a 7.8-years following up. By adjustment for oxidative risk factors of HCC, the compound CT and TT genotypes in relative to the CC wild-type were associated with 83% reduced lymphocytic p53 oxidative lesions of HCC patients with RBC folate lower than 688 ng/mL (OR: 0.17, 95%CI: 0.07-0.43). Such genetic protective effects by the CT/TT genotypes were 2-fold enhanced among those with high RBC folate (OR: 0.08, 95% CI: 0.03-0.21, P for interaction < 0.001). For those with non-folate-deficient status, the compound CT and TT vs. CC genotypes were associated with 80% reduced risks of advanced HCC stages (III&IV) (OR: 0.2, 95%CI: 0.08-0.56). Such protection was negated either by adjustment of lymphocytic p53 oxidative lesions or by 3-fold increased risks among those with high RBC status (OR: 0.6, 95%CI; 0.31-1.41, P for interaction = 0.009). Multivariate Cox proportional hazards analysis showed that the CT/TT genotypes vs. CC wild-type were the independent predictable factor for better survival outcome of HCC patients (HR: 0.48, CI = 0.30-0.79). For CC homozygote, the second vs. the bottom tertile levels of RBC status were associated with 2-fold increased mortality rate of HCC patients (HR: 2.05, CI = 1.0-4.1).. Our data demonstrated that reduced MTHFR activities associated with the MTHFR T allele may interact with RBC folate as the risk modifiers of lymphocytic p53 oxidative lesions of HCC patients. The CT/TT genotypes correlated with lower risks of late-stage HCC and a favorable survival of HCC patients, depending on p53 oxidative lesions or RBC folate status.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Erythrocytes; Female; Folic Acid; Gene Frequency; Genotype; Humans; Leukocytes, Mononuclear; Liver Neoplasms; Lymphocytes; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Multivariate Analysis; Oxidative Stress; Polymorphism, Genetic; Prognosis; Promoter Regions, Genetic; Proportional Hazards Models; Risk Factors; Tumor Suppressor Protein p53

Chronic hepatitis C (CHC) triggers oxidative stress and contributes to the emergence of hepatocellular carcinoma (HCC). We previously reported that tumor suppressor gene (TSG) methylation is a critical factor during the early stages of hepatocarcinogenesis. In this study, we clarify the association between oxidative stress and epigenetic alterations during hepatocarcinogenesis. We examined DNA oxidation and methylation profiles in 128 liver biopsy samples from CHC patients. The DNA oxidation and methylated TSG numbers were quantified using immunohistochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) and quantitative PCR for 11 TSGs, respectively. The quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) assay in HepG2 and fetal liver Hc cells treated with H2O2 was used to quantify trimethyl-H3K4, acetylated-H4K16 (an active chromatin marker), trimethyl-H3K27 (a repressive chromatin marker) and 8-OHdG. We analyzed 30 promoters of 25 different TSGs by qPCR. The high levels of 8-OHdG was the only variable that was significantly associated with the increased number of methylated TSGs in CHC (p < 0.0001). The ChIP-qPCR revealed that after H2O2 treatment of the cell lines, the 8-OHdG-bound promoters showed a modification from an active chromatin (trimethyl-H3K4 and acetylated-H4K16 dominant) to a repressive chromatin (trimethyl-H3K27 dominant) status. We conclude that oxidative stress alters the chromatin status, which leads to abnormal methylation of TSGs, and contributes to hepatocarcinogenesis in CHC patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinogenesis; Carcinoma, Hepatocellular; Chromatin Immunoprecipitation; Chromosomal Instability; Deoxyguanosine; DNA Damage; DNA Methylation; Epigenesis, Genetic; Female; Genes, Tumor Suppressor; Histones; Humans; Hydrogen Peroxide; Immunohistochemistry; Liver; Liver Neoplasms; Male; Middle Aged; Protein Processing, Post-Translational; Reactive Oxygen Species

The rate of onset of hepatocellular carcinoma (HCC) in patients with nonalcoholic steatohepatitis (NASH) has been reported recently to be comparable to that of patients with chronic hepatitis C. However, the precise mechanism contributing to carcinogenesis in the former remains unclear. Although increased oxidative stress is presumed to play a role in carcinogenesis in patients with NASH, this relationship remains to be directly proven. In this study, we investigated the involvement of oxidative DNA damage in hepatocarcinogenesis in patients with NASH.. Patients with nonalcoholic fatty liver disease who were treated at our university hospital were eligible for enrolment in the study(n = 49). The study cohort included 30 patients with NASH without HCC (NASH without HCC), six HCC patients with NASH (NASH-HCC), and 13 patients with simple steatosis. Quantitative immunohistochemistry with a KS-400 image analyzing system was used for 8-hydroxy-2'-deoxyguanosine (8-OHdG) detection.. The 8-OHdG content in the liver tissue of NASH-HCC patients was significantly different from that in the other patients. The median immunostaining intensity was 8.605 in the NASH-HCC cases, which was significantly higher than that in the cases of NASH without HCC (4.845; P = 0.003). Multivariate analysis using hepatic 8-OHdG content as a factor in addition to age and fasting blood sugar revealed a significant difference in clinicopathological factors between NASH-HCC and NASH without HCC cases. Old age (P = 0.015) and high relative immunostaining intensity for intrahepatic 8-OHdG (P = 0.037) were identified as independent factors.. 8-OHdG content in liver tissue may serve a marker of oxidative stress and could be a particularly useful predictor of hepatocarcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Aged, 80 and over; Aldehydes; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Deoxyguanosine; DNA Damage; DNA, Neoplasm; Fatty Liver; Female; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Neoplasm Staging; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Young Adult

Hepatocyte apoptosis is a key feature of chronic liver disease including viral hepatitis and steatohepatitis. A previous study demonstrated that absence of the Bcl-2 family protein Mcl-1 led to increased hepatocyte apoptosis and development of liver tumors in mice. Since Mcl-1 not only inhibits the mitochondrial pathway of apoptosis but can also inhibit cell cycle progression and promote DNA repair, it remains to be proven whether the tumor suppressive effects of Mcl-1 are mediated by prevention of apoptosis.. We examined liver tumor development, fibrogenesis, and oxidative stress in livers of hepatocyte-specific knockout (KO) of Mcl-1 or Bcl-xL, another key antagonist of apoptosis in hepatocytes. We also examined the impact of additional KO of Bak, a downstream molecule of Mcl-1 towards apoptosis but not the cell cycle or DNA damage pathway, on tumor development, hepatocyte apoptosis, and inflammation.. Bcl-xL KO led to a high incidence of liver tumors in 1.5-year-old mice, similar to Mcl-1 KO. Bcl-xL- or Mcl-1-deficient livers showed higher levels of TNF-α production and oxidative stress than wild-type livers at as early as 6 weeks of age and oxidative DNA damage at 1.5 years. Deletion of Bak significantly inhibited hepatocyte apoptosis in Mcl-1 KO mice and reduced the incidence of liver cancer, coinciding with reduction of TNF-α production, oxidative stress, and oxidative DNA damage in non-cancerous livers.. Our findings strongly suggest that chronically increased apoptosis in hepatocytes is carcinogenic and offer genetic evidence that inhibition of apoptosis may suppress liver carcinogenesis in chronic liver disease.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aging; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Carcinoma, Hepatocellular; Deoxyguanosine; Gene Expression Regulation, Neoplastic; Genotype; Hepatitis; Hepatocytes; Humans; Liver Neoplasms; Mice; Mice, Knockout; Myeloid Cell Leukemia Sequence 1 Protein; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factor-alpha

MicroRNAs expression has been extensively studied in hepatocellular carcinoma but little is known regarding the relationship, if any, with inflammation, production of reactive oxygen species (ROS), host's repair mechanisms and cell immortalization. This study aimed at assessing the extent of oxidative DNA damage (8-hydroxydeoxyguanosine - 8-OHdG) in different phases of the carcinogenetic process, in relation to DNA repair gene polymorphism, telomeric dysfunction and to the expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death.. Tissue samples obtained either at surgery, [neoplastic (HCC) and adjacent non-cancerous cirrhotic tissues (NCCT)] at percutaneous or laparoscopic biopsy (patients with HCV or HBV-related hepatitis or patients undergoing cholecystectomy) were analysed for 8-OHdG (HPLC-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), telomerase activity, telomere length (T/S, by RT-PCR), Taqman microRNA assay and Bad/Bax mRNA (RT-PCR). Fifty-eight samples from 29 HCC patients (obtained in both neoplastic and peritumoral tissues), 22 from chronic hepatitis (CH) and 10 controls (cholecystectomy patients - CON) were examined.. Eight-OHdG levels were significantly higher in HCC and NCCT than in CH and CON (p=0.001). Telomerase activity was significantly higher in HCC than in the remaining subgroups (p=0.002); conversely T/S was significantly lower in HCC (p=0.05). MiR-199a-b, -195, -122, -92a and -145 were down-regulated in the majority of HCCs while miR-222 was up-regulated. A positive correlation was observed among 8-OHdG levels, disease stage, telomerase activity, OGG1 polymorphisms and ALT/GGT levels. In HCC, miR-92 expression correlated positively with telomerase activity, 8-OHdG levels and Bad/Bax mRNA.. The above findings confirm the accumulation, in the progression of chronic liver damage to HCC, of a ROS-mediated oxidative DNA damage, and suggest that this correlates with induction of telomerase activity and, as a novel finding, with over-expression of miR-92, a microRNA that plays a role in both the apoptotic process and in cellular proliferation pathways.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cluster Analysis; Deoxyguanosine; DNA Damage; DNA Glycosylases; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; MicroRNAs; Middle Aged; Polymorphism, Single Nucleotide; Telomerase; Telomere

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; DNA Repair; DNA-Binding Proteins; DNA, Mitochondrial; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Liver Neoplasms; Mitochondrial Proteins; Protein Binding; Protein Transport; Reactive Oxygen Species; RNA Interference; Transcription Factors; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

The cytoprotective mechanisms of ursodeoxycholic acid (UDCA) in primary biliary cirrhosis (PBC) have not been fully clarified. UDCA has some antioxidant properties. Nuclear factor-E2-related factor-2 (Nrf2) plays a critical role in protecting a variety of tissues against oxidative stress. Therefore, to investigate the potential antioxidant effects of UDCA in PBC, we determined the intracellular status of both oxidant stress and antioxidant defenses in paired pre- and posttreatment liver biopsies from 13 PBC patients by immunodetection of 8-hydroxydeoxyguanosine (8-OHdG), Nrf2-, and Nrf2-mediated antioxidant proteins. After UDCA treatment, the number of 8-OHdG-positive hepatocytes or bile duct cells decreased with improvement of hepatic injury. The hepatic levels of both total and phosphorylated Nrf2 protein were increased, along with upregulation of nuclear phosphorylated Nrf2 expression in bile duct cells. In addition, the levels of both thioredoxin (TRX) and thioredoxin reductase 1 (TrxR1) protein were increased in the liver after UDCA. The upregulation of hepatic TRX or TrxR1 protein expression positively correlated with that of total Nrf2 protein expression. In conclusion, UDCA treatment can enhance hepatic Nrf2 activation and upregulate hepatic TRX and TrxR1 protein expression. Hepatic Nrf2 activation may play a role in the therapeutic response to UDCA in PBC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Bile Acids and Salts; Biopsy; Deoxyguanosine; Female; Humans; Liver; Liver Cirrhosis, Biliary; Liver Neoplasms; Male; Middle Aged; NF-E2-Related Factor 2; Oxidative Stress; Thioredoxin Reductase 1; Ursodeoxycholic Acid

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acetylcysteine; Adenocarcinoma; Aldehydes; Animals; Antibiotics, Antineoplastic; Apoptosis; Caspase 3; Catheter Ablation; Chemotherapy, Adjuvant; Combined Modality Therapy; Deoxyguanosine; DNA Damage; Doxorubicin; Histones; HSP70 Heat-Shock Proteins; Humans; Liver Neoplasms; Mammary Neoplasms, Experimental; Oxidative Stress; Prospective Studies; Randomized Controlled Trials as Topic; Rats; Tyrosine

Amitrole (3-amino-1,2,4-triazole) is a widely used herbicide. Amitrole induces thyroid and liver tumors in rodents. However, the mechanism of carcinogenesis by amitrole remains to be clarified. To clarify the mechanism of carcinogenesis induced by amitrole, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic of oxidatively generated DNA damage, by an amitrole metabolite, 3-amino-5-mercapto-1,2,4-triazole (AMT), in the presence of Cu(II). The amount of 8-oxodG was increased by AMT in the presence of Cu(II). AMT-induced 8-oxodG formation was enhanced in deuterium oxide (D₂O), which prolongs the half life of singlet oxygen (¹O₂), more than that in H₂O. Sodium azide and 1,4-diazabicyclo[2,2,2]-octane (DABCO), potent and relatively specific scavengers of ¹O₂, inhibited AMT-mediated 8-oxodG formation. Bathocuproine, a Cu(I) chelator, also inhibited the 8-oxodG formation. On the other hand, typical OH scavengers did not inhibit the generation of 8-oxodG. AMT plus Cu(II) also induced piperidine-labile DNA lesions frequently at every guanine residue. These results suggest that ¹O₂ and Cu(I) play an important role in DNA damage induced by AMT. It is concluded that oxidatively generated DNA damage induced by AMT via the generation of ¹O₂ may contribute to carcinogenicity of amitrole.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amitrole; Carcinogens; Chelating Agents; Copper; Cyclin-Dependent Kinase Inhibitor p16; Deoxyguanosine; DNA Damage; Humans; Liver Neoplasms; Neoplasm Proteins; Oxygen; Phenanthrolines; ras Proteins; Sodium Azide; Thyroid Neoplasms; Triazoles; Tumor Suppressor Protein p53

Both polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. They coexist widely in the environment at very low levels. Numerous studies indicated that aroclor1254 (one of PCBs mixture) is the inducer of cytochrome P450 1A enzyme acitivity. Benzo(a)pyrene (BaP) can cause a variety of toxicities in vitro, such as oxidative DNA damage and genotoxicity. In the present study, HepG2 cells were treated with either BaP (50 microM) or aroclor1254 at concentrations of 11.5 (low), 23.0 (medium), and 46.0 microM (high) alone, or pretreated the cells with aroclor1254 (11.5, 23.0, and 46.0 microM), followed by BaP (50 microM). It was found that 7-ethoxyresorufin-O-deetylase (EROD) activities of HepG2 cells exposed to either BaP or aroclor 1254 increased. DNA damage measured by DNA migration and the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) also increased in cells exposed to BaP, but not in cells exposed to aroclor1254. Under the Aroclor 1254 pretreatment condition, BaP-induced EROD activities was enhanced in cells exposed to the medium and high concentrations of aroclor1254 (P < 0.01 for both), whereas in all pretreatment groups aroclor1254 significantly increased BaP-induced DNA migration (P < 0.01 for all) and the 8-OHdG formation (P < 0.05 for all). In addition, there was positive correlation between the EROD induction activity and Olive tail moment (r(2) = 0.958, P < 0.01) or the levels of 8-OHdG (r(2) = 0.992, P < 0.01). The findings suggest that under the experimental conditions aroclor1254 may enhance BaP-induced DNA migration and oxidative DNA damage in HepG2, due to inducing CYP1A enzyme activity.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Benzo(a)pyrene; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorodiphenyl (54% Chlorine); Cytochrome P-450 CYP1A1; Deoxyguanosine; DNA; DNA Damage; Environmental Pollutants; Enzyme Induction; Humans; Liver Neoplasms; Toxicity Tests

Continuous oxidative stress (OS) plays an important role in the progression of chronic liver diseases and hepatocarcinogenesis through telomere shortening in hepatocytes. However, it has not been established how the OS influences the progression of human hepatocellular carcinomas (HCCs). We examined the correlations of OS with telomere length of cancer cells, telomerase activity and other clinicopathological factors in 68 HCCs.. The level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of OS was examined immunohistochemically and OS was scored in four grades (0-3). The telomere length of cancer cells was measured by quantitative fluorescence in situ hybridization. Telomerase activity was measured by (i) immunodetection of human telomerase reverse transcriptase (hTERT) and (ii) telomere repeat amplification protocol (TRAP) assay. Telomerase related proteins, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt, and other clinicopathological factors were also evaluated.. As the OS grade increased, the average telomere length became significantly shorter in HCCs, especially in the hTERT-negative group. In the state of high-grade OS, hTERT-positive HCC cells showed more proliferative and less apoptotic features compared with hTERT-negative HCC cells. Telomerase activity, as measured by the TRAP assay, was strongly correlated with OS grade in HCCs. Furthermore, a high OS grade was correlated with the downexpression of PTEN and the activation of Akt.. Oxidative stress enhanced the malignant potential of HCCs through the activation of telomerase, which raises the possibility of using OS as a marker for assessing the clinical state of HCCs.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Carcinoma, Hepatocellular; Deoxyguanosine; Disease Progression; Enzyme Activation; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; In Situ Nick-End Labeling; Liver Neoplasms; Male; Middle Aged; Oxidative Stress; PTEN Phosphohydrolase; Telomerase; Telomere

Several lines of evidence have suggested that oxidative stress plays an important role for the pathogenesis of nonalcoholic steatohepatitis (NASH). Therefore, by using immunohistochemical staining of liver biopsy samples, we measured hepatic 7,8-dihydro-8-oxo-2' deoxyguanosine (8-oxodG), a DNA base-modified product generated by hydroxyl radicals, of 38 NASH patients and compared with 24 simple steatosis and 10 healthy subjects. Relation of hepatic 8-oxodG with clinical, biochemical, and histologic variables and changes after iron reduction therapy (phlebotomy plus iron-restricted diet) were also examined. Hepatic 8-oxodG levels were significantly higher in NASH compared with simple steatosis (17.5 versus 2.0 8-oxodG-positive cells/10(5) microm(2); P < 0.0001). 8-oxodG was significantly related to iron overload condition, glucose-insulin metabolic abnormality, and severities of hepatic steatosis in NASH patients. Logistic regression analysis also showed that hepatic iron deposit and insulin resistance were independent variables associated with elevated hepatic 8-oxodG. After the iron reduction therapy, hepatic 8-oxodG levels were significantly decreased (from 20.7 to 13.8 positive cells/10(5) microm(2); P < 0.01) with concomitant reductions of serum transaminase levels in NASH patients. In conclusion, iron overload may play an important role in the pathogenesis of NASH by generating oxidative DNA damage and iron reduction therapy may reduce hepatocellular carcinoma incidence in patients with NASH.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Analysis of Variance; Carcinoma, Hepatocellular; Case-Control Studies; Deoxyguanosine; DNA Damage; Fatty Liver; Female; Humans; Insulin Resistance; Iron Overload; Liver Function Tests; Liver Neoplasms; Logistic Models; Male; Middle Aged; Oxidative Stress; Severity of Illness Index; Statistics, Nonparametric

To examine the effects of instant coffee consumption on cancer risk, we analyzed the oxidative DNA damage levels and the DNA repair and redox systems in the livers of coffee-fed mice. Three-week-old male ICR mice were fed with/without 0.1% (w/v) instant coffee solution. At 2, 4, and 8 mo, the levels of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, and the expression of mouse 8-OH-dG repair-associated genes and redox system-associated genes, the SOD activity, and the LPO level were analyzed. Simultaneously, half of the mice were fed a low vitamin (LV) diet (autoclaved diet) to disturb the defense system against oxidative stresses. As a result, the 8-OH-dG level was increased in the livers of LV diet (+ water)-fed mice for 8 mo, in comparison to those of the 0 M control mice and normal diet (+ water)-fed mice. However, no significant differences between water drinking and coffee drinking were observed, in terms of the 8-OH-dG level. In addition, the 8-OH-dG repair-associated gene expression, the SOD activity, and the LPO level also showed no significant differences between water drinking and coffee drinking in all mouse groups. On the other hand, among the redox system-associated genes, only the expression of GPx1 was changed. These results suggest that instant coffee consumption has little, if any, effect on the risk of liver cancer due to oxidative stresses.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Avitaminosis; Body Weight; Coffee; Deoxyguanosine; Diet; DNA Damage; DNA Repair; Food Handling; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Lipid Peroxidation; Liver; Liver Neoplasms; Male; Malondialdehyde; Mice; Mice, Inbred ICR; Oxidative Stress; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase; Time Factors

Increased production of reactive oxygen species, which cause oxidative DNA damage, is considered to be related to hepatocarcinogenesis. 8-Hydroxy-2'-deoxy-guanosine (8-OHdG) is a useful marker of DNA damage induced by oxidative stress. The aim of this study was to determine whether expression of 8-OHdG is a risk factor for the development of hepatocellular carcinoma (HCC) in patients with hepatitis C virus (HCV) infection.. The expression of 8-OHdG in liver biopsy specimens was assessed immunohistochemically. In total, 104 patients with chronic HCV infection who were diagnosed on liver biopsy between January 1987 and December 2002 were studied retrospectively. Univariate and multivariate analyses using age, gender, habitual drinking, tobacco exposure, diabetes mellitus, serum alanine aminotransferase level, HCV genotype, hepatic fibrosis, inflammation, steatosis, and 8-OHdG expression in liver biopsy specimens were conducted to identify factors related to the development of HCC.. On multivariate analysis, 8-OHdG and fibrosis were independent and significant risk factors for HCC development (relative risk, 2.48; P = 0.023; relative risk, 5.35; P = 0.001, respectively). Furthermore, the cumulative incidence rate of HCC in 39 patients with high 8-OHdG expression levels was significantly greater than that in 65 patients with low 8-OHdG expression levels (P = 0.043). In addition, liver 8-OHdG expression was correlated with hepatic inflammation.. 8-OHdG is a risk factor for the development of HCC in patients with chronic HCV infection. Patients with chronic HCV who express 8-OHdG should be monitored carefully for the development of HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers, Tumor; Biopsy; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Female; Hepatitis C, Chronic; Humans; Incidence; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Oxidative Stress; Retrospective Studies; Risk Assessment; Risk Factors; Severity of Illness Index; Time Factors; Up-Regulation

To study alone and combined effect of selenium and arsenic on oxidative stress, DNA oxidative damage and repair.. HepG2 cells were treated with selenium (2.5, 5.0 and 10.0 micromol/L sodium selenite) alone, arsenic (1.56, 3.13, 6.25, 12.5 and 25.0 micromol/L arsenious acid) alone and combined selenium plus arsenic. The quantitative analysis of malondialdehyde (MDA), 8-OHdG and hOGG1 was carried out by fluorometric method, HPLC-EC and Western Blot to represent oxidative stress, DNA oxidative damage and repair, respectively.. Under the condition of alone treatment, sodium selenite (5.0 and 10.0 micromol/L) as well as arsenious acid (6.25, 12.5 and 25.0 micromol/L) resulted in significant increased levels of MDA and 8-OHdG, and inhibition of hOGG1 expression in HepG2 cells compared with solvent control (P < 0.05, P < 0.01). Sodium selenite at the relative low dose (2.5 micromol/L) displayed certain anti-oxidative ability (P > 0.05). Combined treatment of sodium selenite (2.5 micromol/L) and arsenious acid (6.25 micromol/L) caused significant lower levels of MDA and 8-OHdG than those of correspondent arsenic alone treatment (P < 0.05). hOGG1 expression showed no difference between combined treatment (2.5 micromol/L of selenium selenite plus 6.25, 12.5 and 25.0 micromol/L of arsenious acid, respectively) and correspondent arsenic alone treatment (P > 0.05).. Sodium selenite at the concentrations of 5.0, 10.0 micromol/L and arsenious acid at the concentrations of 6.25, 12.5, 25.0 micromol/L induced enhanced oxidative stress and 8-OHdG production, and inhibition of hOGG1 expression, respectively. Selenium at certain concentration (2.5 micromol/L of selenium selenite) has ameliorative effects on oxidative stress and DNA oxidative damage induced by arsenic, but no effect on repair of DNA oxidative damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Arsenic; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Drug Synergism; Hep G2 Cells; Humans; Liver Neoplasms; Malondialdehyde; Oxidative Stress; Selenium

Although the oxidative stress frequently occurs in patients with chronic hepatitis C, its role in future hepatocellular carcinoma (HCC) development is unknown. Hepatic 8-hydroxydeoxyguanosine (8-OHdG) was quantified using liver biopsy samples from 118 naïve patients who underwent liver biopsy from 1995 to 2001. The predictability of 8-OHdG for future HCC development and its relations to epidemiologic, biochemical and histological baseline characteristics were evaluated. During the follow-up period (mean was 6.7+/-3.3 years), HCC was identified in 36 patients (30.5%). Univariate analysis revealed that 16 variables, including 8-OHdG counts (65.2+/-20.2 vs 40.0+/-23.5 cells per 10(5) microm2, P<0.0001), were significantly different between patients with and without HCC. Cox proportional hazard analysis showed that the hepatic 8-OHdG (P=0.0058) and fibrosis (P=0.0181) were independent predicting factors of HCC. Remarkably, 8-OHdG levels were positively correlated with body and hepatic iron storage markers (vs ferritin, P<0.0001 vs hepatic iron score, P<0.0001). This study showed that oxidative DNA damage is associated with increased risk for HCC and hepatic 8-OHdG levels are useful as markers to identify the extreme high-risk subgroup. The strong correlation between hepatic DNA damage and iron overload suggests that the iron content may be a strong mediator of oxidative stress and iron reduction may reduce HCC incidence in patients with chronic hepatitis C.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Female; Hepatitis C, Chronic; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Oxidative Stress; Reactive Oxygen Species

Trichloroethylene (TCE) is an environmental and industrial pollutant whose hepatotoxicity has been demonstrated in experimental animals. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of TCE and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. Exposure of the cells to TCE caused significant increase of DNA migration in comet assay and of micronuclei (MN) frequencies at all tested concentrations (0.5-4mM), respectively, which suggests that TCE caused DNA strand breaks and chromosome damage. The involvement of lipid peroxidation in the genotoxic properties of TCE was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). To elucidate the role of glutathione (GSH) in these effects, the intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by co-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to TCE-induced cytotoxicity and DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by TCE was almost completely prevented. These results indicate that TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Drug Evaluation, Preclinical; Glutathione; Humans; Lipid Peroxidation; Liver Neoplasms; Micronuclei, Chromosome-Defective; Mutagenicity Tests; Oxidative Stress; Solvents; Trichloroethylene; Tumor Cells, Cultured

Oxidative DNA damage by reactive oxygen species is involved in the process of liver carcinogenesis. To test the hypothesis that a remedy containing Scutellaria baicalensis Georgi (Sb) and Bupleurum scorzonerifolfium Willd (Bs) (Sb/Bs remedy) modulates hepatic neoplastic growth, BOP (N-nitrosobis(2-oxopropyl)amine)-induced liver cancers in hamsters were established.. Parameters such as survival rate, tumour area, tumour foci, 8-hydroxydeoxyguanosine (8-OHdG), caspase-3, transforming growth factor (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) were measured after Sb/Bs remedy treatment during BOP-induced carcinogenesis.. The results showed that the Sb/Bs remedy and its constituents Sb and Bs suppressed the tumour area in BOP-induced liver tumours. Because selenium (Sel) is toxic at a high dose (10 mg/kg), with a low survival rate (0%), the combination of Sb/Bs remedy and low-dose Sel (1 mg/kg) was found to decrease the tumour area and the number of tumour foci while increasing serum TNF-alpha and TGF-beta1, but not IL-6 levels. Besides, the Sb/Bs remedy, when combined with low-dose Sel, not only decreased the expression of 8-OHdG and increased caspase-3 expression within the glutathione S-transferase placental form-positive tumour foci but also increased tumour apoptosis in BOP-induced hamsters.. We conclude that low-dose Sel has a chemoprevention effect on BOP-induced liver tumours and such an effect was more enhanced when combined with Sb/Bs treatment.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Anticarcinogenic Agents; Biomarkers, Tumor; Carcinoma, Hepatocellular; Caspase 3; Chemoprevention; Cricetinae; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Drugs, Chinese Herbal; Liver Neoplasms; Longevity; Male; Mesocricetus; Nitrosamines; Selenium; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

To estimate potential human risk of exposure to a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a 2-year carcinogenicity test was conducted using male F344 rats administered MeIQx-containing diet at doses of 0 (control), 0.001, 1, and 100 ppm. The lowest dose 0.001 ppm was established as equivalent to the daily intake of this carcinogen in humans (0.2 to 2.6 microg/man/day). Significant decreases of survival rate and body weight gain were observed in rats treated with 100 ppm MeIQx. Histopathological examination revealed significant induction of hepatocellular carcinomas, adenomas, and development of glutathione S-transferase placental form-positive foci with MeIQx at 100 ppm. Moreover, the incidences of Zymbal's glands carcinoma, mammary fibroadenoma, and subcutaneous fibroma were found significantly increased in a 100 ppm MeIQx group. However, no significant induction of altered preneoplastic hepatocellular foci was observed in 0.001 and 1 ppm groups as compared to the controls. 8-Hydroxy-2'-deoxyguanosine levels in the rat liver DNA of the 100 ppm-treated group were not elevated, but MeIQx-DNA adduct formation increased as compared with the 1 ppm case, albeit without significance. No significant induction of any other neoplastic lesions related to the carcinogen administration was found in MeIQx-administered groups except for 100 ppm. These results imply that 1 ppm may be a no-effect level for MeIQx carcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenoma, Liver Cell; Administration, Oral; Animal Feed; Animals; Carcinogens; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Adducts; Dose-Response Relationship, Drug; Glutathione Transferase; Liver; Liver Neoplasms; Longevity; Male; No-Observed-Adverse-Effect Level; Precancerous Conditions; Quinoxalines; Rats; Rats, Inbred F344

This study evaluated the relationship between inflammation, intra-hepatic oxidative stress, oxidative DNA damage and the progression of liver carcinogenesis in hepatitis C virus (HCV)-infected humans.. Non-cancerous liver tissues were collected from 30 patients with an HCV-associated solitary hepatocellular carcinoma (HCC) who received curative tumor removal. After surgery, the patients were followed at monthly intervals at the outpatient clinic. Distribution of the inflammatory cells (CD68+), the number of 8-hydroxydeoxyguanosine (8-OHdG) DNA adducts and 4-hydroxynonenal (HNE) protein adducts and the expression of apurinic/apyrimidinic endonuclease (APE) were determined by immunohistochemical analysis in serial liver sections from tumor-free parenchyma at the surgical margin around the tumor.. Significant positive correlations were observed between the number of CD68+ cells, the amount of HNE protein adducts, and the number of 8-OHdG adducts in liver tissue of patients with HCC and HCV. The cumulative disease-free survival was significantly shorter in patients with the highest percentage of 8-OHdG-positive hepatocytes. Using a Cox proportional hazard model, 8-OHdG, HNE and CD68 were determined to be good biomarkers for predicting disease-free survival in patients with HCC and HCV.. These results support the hypothesis that HCV-induced inflammation causes oxidative DNA damage and promotes hepatocarcinogenesis which directly affects the clinical outcome. Since patients with greater intra-hepatic oxidative stress had a higher incidence of HCC recurrence, we suggest that oxidative stress biomarkers could potentially be used as a useful clinical diagnostic tool to predict the duration of disease-free survival in patients with HCV-associated HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Alanine Transaminase; Aldehydes; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biomarkers, Tumor; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Adducts; DNA Damage; Female; Follow-Up Studies; Hepacivirus; Hepatectomy; Hepatitis C; Humans; Inflammation; Lipid Peroxidation; Liver Neoplasms; Male; Microfilament Proteins; Middle Aged; Neoplasm Recurrence, Local; Oxidative Stress; Prognosis; Reactive Oxygen Species; Risk Factors; Survival Rate; Vesicular Transport Proteins

Di(2-ethylhexyl)phthalate (DEHP), a commonly used industrial plasticizer, causes liver tumorigenesis presumably via activation of peroxisome proliferator-activated receptor alpha (PPARalpha). The mechanism of DEHP tumorigenesis has not been fully elucidated, and to clarify whether DEHP tumorigenesis is induced via PPARalpha, we compared DEHP-induced tumorigenesis in wild-type and Pparalpha-null mice. Mice of each genotype were divided into three groups, and treated for 22 months with diets containing 0, 0.01 or 0.05% DEHP. Surprisingly, the incidence of liver tumors was higher in Pparalpha-null mice exposed to 0.05% DEHP (25.8%) than in similarly exposed wild-type mice (10.0%). These results suggest the existence of pathways for DEHP-induced hepatic tumorigenesis that are independent of PPARalpha. The levels of 8-OHdG increased dose-dependently in mice of both genotypes, but the degree of increase was higher in Pparalpha-null than in wild-type mice. NFkappaB levels also significantly increased in a dose-dependent manner in Pparalpha-null mice. The protooncogene c-jun-mRNA was induced, and c-fos-mRNA tended to be induced only in Pparalpha-null mice fed a 0.05% DEHP-containing diet. These results suggest that increases in oxidative stress induced by DEHP exposure may lead to the induction of inflammation and/or the expression of protooncogenes, resulting in a high incidence of tumorigenesis in Pparalpha-null mice.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alanine Transaminase; Animals; Base Sequence; Deoxyguanosine; Inflammation; Japan; Liver Neoplasms; Male; Mice; NF-kappa B p50 Subunit; Oxidative Stress; Phthalic Acids; Polymerase Chain Reaction; PPAR alpha

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl dulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Bacterial Toxins; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Comet Assay; Cytochrome P-450 Enzyme System; Deoxyguanosine; Humans; Immunohistochemistry; L-Lactate Dehydrogenase; Liver Neoplasms; Marine Toxins; Microcystins; Polymerase Chain Reaction; Reactive Oxygen Species; RNA, Messenger

Although the importance of reactive oxygen species (ROS) in the pathogenesis of various diseases is stressed, clinical significance of the markers reflecting DNA oxidation such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) remains to be clarified.. To examine clinical usefulness of 8-OHdG in healthy individuals in comparison with liver disease patients, urinary excretion of 8-OHdG was measured in 336 healthy individuals and 110 patients with liver disease.. In healthy persons, the 8-OHdG excretion was increased in an age-dependent manner. It was positively correlated with cigarettes smoked a day and negatively correlated with body mass index (BMI) (P < 0.05, each). Age, smoking and BMI were independent predictors of urinary 8-OHdG excretion (P < 0.01, P < 0.01 and P < 0.05, respectively). In liver disease, the excretion of 8-OHdG was not changed, as compared with healthy individuals. However, the liver disease patients under the age of 40 had higher values of 8-OHdG than healthy persons. In addition, the urinary excretion of 8-OHdG was higher in patients with hepatitis C virus (HCV) infection than those with hepatitis B virus (HBV) infection.. The results of the present study suggest that measurement of urinary 8-OHdG excretion is useful in assessing DNA oxidation caused by aging, smoking, body composition and liver disease.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Age Factors; Aged; Aged, 80 and over; Body Mass Index; Deoxyguanosine; DNA; Female; Humans; Liver Neoplasms; Male; Middle Aged; Oxygen; Reactive Oxygen Species; Reference Values; Reproducibility of Results; Smoking

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was classified by the International Agency for Research on Cancer as a carcinogen in humans. It acts through an aryl hydrocarbon receptor-mediated mechanism, inducing the transcription of numerous genes, including various cytochrome P450s (CYPs - CYP1A1, 1A2, 1B1). Induction of CYPs may lead to genotoxicity by generating reactive oxygen species (ROS) which can damage DNA directly and/or via the generation of reactive metabolites. We determined ROS formation with the 2',7'-dihydrodichlorofluorescein diacetate fluorescence assay after incubation of HepG2 hepatoma cells or primary rat hepatocytes with TCDD. The amount of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA was measured using HPLC-MS/MS, the amount of CYP1A1 protein by Western blotting. The catalytic activity of CYP1A enzymes was determined as 7-ethoxyresorufin-O-deethylase (EROD) activity. Incubation of cells with TCDD for 48 h caused increased levels of ROS in primary rat hepatocytes as well as increased levels of 8-oxo-dG in DNA compared to untreated cells. In the HepG2 cell line no significant effects were observed for both ROS formation and 8-oxo-dG levels. Both effects were in good agreement with the extent of induction of CYP1A1 protein and EROD activity, suggesting that CYP1 induction is a major source of ROS formation in TCDD-treated hepatocytes.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Deoxyguanosine; Enzyme Induction; Fluoresceins; Humans; Liver; Liver Neoplasms; Polychlorinated Dibenzodioxins; Rats; Rats, Wistar; Reactive Oxygen Species

Difference of 8-hydroxy-deoxyguanosine (8-OH-dG) formation in liver DNA in C3H/HeN and in C57BL/6 mice--fed oxidized lard and dietary oils (soybean and sardine)--was investigated. The blank levels of 8-OH-dG were higher in C3H/HeN mice (highly sensitive to liver tumorigenesis) than in C57BL/6 mice (resistant strain). The level of 8-OH-dG increased much more in C3H/HeN mice than in the C57BL/6 mice fed by oxidized lard and dietary oil treatment. Feeding oxidized lard and dietary oils increased 8-oxo-guanine DNA glycosylase I (OGG1) and mRNA 8-oxo-dGTPase in C57BL/6 mice. On the other hand, no appreciable change of mRNA in the C3H/HeN mice was observed. The formation differences of 8-OH-dG from the two murine strains fed with oxidized lard and dietary oils may be associated with the different mRNA levels in the DNA repair enzymes because the mRNA levels in the DNA repair enzymes were much lower in C3H/HeN mice than in C57BL/6 mice.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Body Weight; Diet; Dietary Fats; DNA; DNA Damage; DNA Glycosylases; DNA Repair Enzymes; Fish Oils; Guanine; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Phosphoric Monoester Hydrolases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Soybean Oil

Hepatitis C virus (HCV) core protein is known to cause oxidative stress and alter apoptosis pathways. However, the apoptosis results are inconsistent, and the real significance of oxidative stress is not well known. The aim of this study was twofold. First, we wanted to confirm whether core-induced oxidative stress was really significant enough to cause DNA damage, and whether it induced cellular antioxidant responses. Second, we wanted to evaluate whether this core-induced oxidative stress and the antioxidant response to it was responsible for apoptosis changes.. HCV core protein was expressed under control of the Tet-Off promoter in Huh-7 cells and HeLa cells. We chose to use deoxycholic acid (DCA) as a model because it is known to produce both reactive oxygen species (ROS) and apoptosis.. Core expression uniformly increased ROS and 8-hydroxy-2'-deoxyguanosine (8-OHdG) under basal and DCA-stimulated conditions. Core protein expression also increased manganese superoxide dismutase levels. Core protein inhibited DCA-mediated mitochondrial membrane depolarization and DCA-mediated activation of caspase-9 and caspase-3, despite the increase in ROS by DCA. Core protein inhibited DCA-mediated apoptosis by increasing Bcl-x(L) protein and decreasing Bax protein, without affecting the proportion of Bax between mitochondria and cytosol, resulting in suppression of cytochrome c release from mitochondria into cytoplasm.. HCV core protein induces oxidative DNA damage, whereas it inhibits apoptosis that is accompanied by enhancement of ROS production. Thus, oxidative stress and apoptosis modulation by core protein are independent of each other.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochromes c; Cytosol; Dactinomycin; Deoxycholic Acid; Deoxyguanosine; Enzyme Activation; Humans; Intracellular Membranes; Liver Neoplasms; Mitochondria, Liver; Oxidative Stress; Protein Synthesis Inhibitors; Reactive Oxygen Species; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Viral Core Proteins

Antioxidative flavonoids, ubiquitously included in vegetables, fruits and teas, are expected to prevent degenerative diseases. It is unclear, however, whether flavonoids can enter the cellular nuclei and suppress the oxidative damage of DNA. Here, several flavonoids at the physiological concentration of 10 microM were dosed to 2.5x10(7) HepG2 cells. The nuclei were isolated and determined in the incorporated flavonoid levels, and simultaneously exposed to reactive oxygen generated from 25 mM of 2,2'-azobis(2-amidinopropane) dihydrochloride. Most of the tested flavonoids were incorporated into the cells in the range between 1000 and 1600 pmol/10(7) cells, and were in the nuclei at 250-450 pmol/10(7) cells at the maximum incorporation after 30min of cell incubation. In the cells, 23% of quercetin (3,5,7,3',4'-OH) and 8% of luteolin (5,7,3',4'-OH) were the original aglycone forms and the others were the methylated and gulucuronide/sulfate conjugates, while 72% of kaempferol (3,5,7,4'-OH) and 85% of apigenin (5,7,4'-OH) were aglycones and located in the nuclei at the similar ratio of metabolites. Quercetin and luteolin significantly suppressed the formation of 8-oxo-7,8-dihydrodeoxyguanosine by 25% and 15%, respectively, compared to those in 0-time incubated cells with the flavonoids. Under such conditions of low level and hydroxyl-masked in the nuclei, the limited flavonoids were bioavailable antioxidants to prevent genetic damage and they were B-ring catechols such as quercetin and luteolin.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Antioxidants; Biological Availability; Cell Line, Tumor; Deoxyguanosine; Dose-Response Relationship, Drug; Flavonoids; Hepatoblastoma; Humans; Liver Neoplasms

Microcystin-LR (MCYST-LR) and nodularin (NOD) are known as tumor promoters in experimental animals and so present potential health threats for humans. Although their hepatotoxic mechanisms have been very well documented, many other effects of these toxins are relatively undescribed, indeed controversial, notably those related to their genotoxicity. In the present investigation, we examined how these toxins could induce DNA damage using a combination of in vitro and in vivo approaches. We first used the (32)P-postlabeling assay to test hydrophobic adduct formation on DNA from primary cultured rat hepatocytes treated with noncytotoxic concentrations of MCYST-LR and NOD (2 and 10 ng/mL). Analysis of the autoradiograms of DNA digests isolated from the hepatocytes did not show any hydrophobic DNA adduct formation. However, these toxins significantly decreased the amount of hydrophobic endogenous adducts, termed I compounds. We next investigated oxidative DNA damage by using the (32)P-postlabeling assay to analyze 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content as a biomarker of possible DNA lesions. Both MCYST-LR and NOD significantly enhanced 8-oxo-dG in time- and dose-dependent manner in vitro in primary cultured hepatocytes and in vivo in rat liver cells. Thus, it appears that the depletion of endogenous DNA adducts (I compounds) and/or the increase of 8-oxo-dG levels by MCYST-LR and NOD could be involved in the formation of hepatic tumors during long-term exposure to these cyanobacterial hepatotoxins.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biomarkers; Cell Culture Techniques; Deoxyguanosine; DNA Damage; Enzyme Inhibitors; Hepatocytes; Liver; Liver Neoplasms; Male; Marine Toxins; Microcystins; Oxidative Stress; Peptides, Cyclic; Rats; Rats, Sprague-Dawley

The volatile extract from Allii Fistulosi Bulbus (VEAF) was isolated by steam distillation under reduced pressure, followed by continuous liquid-liquid extraction, and its effects on aflatoxin B1 (AFB1)-induced oxidative stress were investigated in human hepatoma cells (HepG2). The main constituents of the VEAF, identified by gas chromatography/mass spectrometry, were 2-octyl-5-methyl-3(2H)-furanone, 2-hexyl-5-methyl-3(2H)-furanone, 2,5-dimethylthiophene, 3,5-diethyl-1,2,4-trithiolane and 3,4-dimethyl-2,5-dihydro-thiophene-2-one. VEAF significantly inhibited the formation of intracellular reactive oxygen species caused by AFB1 in a dose-dependent manner, concomitant with a significant decrease in the AFB1-induced cytotoxicity. VEAF pretreatment significantly reduced the levels of thiobarbituric acid reactive substances, an indicator of lipid peroxidation, whereas increased the level of reduced glutathione. The level of 8-hydroxy-2'-deoxyguanosine, a DNA oxidative stress marker, was also decreased by 49-59% with pretreatment of VEAF. With respect to the activity of AFB1 metabolizing enzymes, VEAF significantly increased the activity of glutathione S-transferase, and significantly decreased the cytochrome (CYP) P450 3A4 activity, but had a little effect on the CYP1As. These results suggest that VEAF may be selectively effective in alleviating the AFB1-induced oxidative stress, and lead to cytoprotection against AFB1 exposure.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aflatoxin B1; Allium; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Deoxyguanosine; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Glutathione; Glutathione Transferase; Hepatocytes; Humans; Lipid Peroxidation; Liver Neoplasms; Oxidative Stress; Plant Extracts; Reactive Oxygen Species; Thiobarbituric Acid Reactive Substances; Volatilization

Nucleic acid damage by reactive nitrogen and oxygen species may contribute to inflammation-related carcinogenesis. To investigate the extent of nucleic acid damage in hepatitis C virus infection and its change after interferon treatment, we measured 8-nitroguanine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the liver of patients with chronic hepatitis C (CHC) before and after interferon therapy.. Hepatic accumulation of 8-nitroguanine and 8-OHdG was immunohistochemically evaluated in 20 CHC patients and 7 control patients with non-alcoholic fatty liver.. Immunoreactivities of 8-nitroguanine and 8-OHdG were strongly detected in the liver from patients with CHC, but not in control livers. 8-Nitroguanine accumulation was found not only in infiltrating inflammatory cells, but also hepatocytes particularly in the periportal area. The accumulation of 8-nitroguanine and 8-OHdG increased with inflammatory grade (8-nitroguanine; P = 0.0019, 8-OHdG; P = 0.0009). In the sustained virological responder group after interferon therapy, 8-nitroguanine and 8-OHdG accumulation were markedly decreased in the liver (8-nitroguanine; P = 0.018, 8-OHdG; P = 0.018).. In this study, we demonstrated for the first time that 8-nitroguanine accumulated in the liver of patients with CHC. 8-Nitroguanine is a useful biomarker to evaluate the severity of HCV-induced chronic inflammation in relation to hepatocellular carcinoma.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Biomarkers; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Female; Guanine; Hepatitis C, Chronic; Humans; Inflammation; Liver; Liver Neoplasms; Male; Middle Aged; Reactive Oxygen Species

Nitrative and oxidative DNA damage such as 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation has been implicated in initiation and/or promotion of inflammation-mediated carcinogenesis. The aim of this study is to clarify whether these DNA lesions participate in the progression of intrahepatic cholangiocarcinoma.. We investigated the relation of the formation of 8-nitroguanine and 8-oxodG and the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) with tumor invasion in 37 patients with intra-hepatic cholangiocarcinoma.. Immunohistochemical analyses revealed that 8-nitroguanine and 8-oxodG formation occurred to a much greater extent in cancerous tissues than in non-cancerous tissues. HIF-1alpha could be detected in cancerous tissues in all patients, suggesting low oxygen tension in the tumors. HIF-1alpha expression was correlated with inducible nitric oxide synthase (iNOS) expression (r = 0.369 and P = 0.025) and 8-oxodG formation (r = 0.398 and P = 0.015). Double immunofluorescence study revealed that iNOS and HIF-1alpha co-localized in cancerous tissues. Notably, the formation of 8-oxodG was correlated significantly with lymphatic invasion (r = 0.386 and P = 0.018). Moreover, 8-nitroguanine and 8-oxodG in non-cancerous tissues were associated significantly with neural invasion (P = 0.042 and P = 0.026, respectively). These results suggest that reciprocal activation between HIF-1alpha and iNOS mediates persistent DNA damage, which induces tumor invasiveness via mutations, resulting in poor prognosis.. The formation of 8-nitroguanine and 8-oxodG plays an important role in multiple steps of genetic changes leading to tumor progression, including invasiveness.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Case-Control Studies; Cholangiocarcinoma; Deoxyguanosine; DNA Damage; DNA-Binding Proteins; DNA, Neoplasm; Female; Guanine; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nuclear Proteins; Transcription Factors

To establish an optimized method to detect 8-OH-dG after DNA oxidation damage by capillary zone electrophoresis.. HepG2 cell was used as target cell and conditions for the separation and detection were obtained by studying the influence of pH of the running buffer, temperature, running voltage on the separation. 0 and 15 mmol/L H2O2 were added into two groups of HepG2 cells (5 x 10(7)) respectively for 24h. DNA was extracted by saturated salting out method to avoid the formation of additional 8-OH-dG by the method of phenol/chloroform extraction. DNA samples were digested to free nucleotides by incubation overnight at 37 degrees C with a mixture of DNase I, snake venom phosphoatase and alkaline phosphate. Proteins were removed and the supernatant was neutralized and then extracted with diethyl ether. The residue was evaporated to dryness and reconstituted and then analyzed under the optimized conditions by capillary zone electrophoresis.. The optimized conditions were: uncoated silica capillary (47 cm x 50 microm i.d.), 20 mmol/L borate buffer (pH 9.5), 25 degrees C, 25kV. The sample was injected by hydrostatic method for 20s. In either of H2O2-treated group and H2O2-untreated group, the peak of 8-OH-dG was detected. The 8-OH-dG content of H2O2-untreated DNA increased.. The method is convenient, rapid, sensitive and cheap and safe. It provides an experimental platform to the application of 8-OH-dG in the biological monitoring of population.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Electrophoresis, Capillary; Hep G2 Cells; Humans; Hydrogen Peroxide; Liver Neoplasms

Oxidative stress (OS) plays a major role in chronic hepatitis C. Various OS markers have been found to be elevated in hepatitis C virus (HCV)-related liver disease. This study detected the presence of OS in serum and liver biopsy specimens of HCV patients. Reactive oxygen molecules (ROM) in sera of 54 HCV patients were compared with 23 controls. OS markers 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal, malondialdehyde, and thioredoxin were measured in liver biopsy specimens of 18 HCV patients with fibrosis staging F1 (six); F2 (two), F3 (four), and F4 (six). The interferon (IFN) response and hepatocellular carcinoma (HCC) occurrence in the presence of OS markers were also evaluated. The level of ROM in HCV patients was 318 +/- 56.7 Carr compared with 248 +/- 40.8 Carr in controls (p=0.032). Multivariate analysis found age (p=0.0236) to be the only independent variable associated with increase in ROM in sera. In liver biopsy specimens, OS markers were found mainly around the area of piecemeal necrosis or the periportal area. The presence of OS markers seemed to increase with fibrosis staging, although not significantly. The OS DNA damage marker 8-OHdG was detected in the nucleus of hepatocytes. Thirteen patients received IFN therapy. During the 4-year follow-up period, HCC developed in four nonresponders to IFN and in one untreated patient. OS markers were stained in both HCC cells and non-HCC cells in HCC patients. OS markers were found in serum and liver specimens of HCV-associated liver disease and in HCC tissue. Detection of OS markers may be important for monitoring disease progression in HCV patients. Antioxidant therapy in combination with antiviral therapy may minimize liver damage and aid in the prevention and subsequent development of HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Age Factors; Aldehydes; Biomarkers; Carcinoma, Hepatocellular; Deoxyguanosine; Disease Progression; Female; Hepatitis C, Chronic; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Malondialdehyde; Oxidative Stress; Reactive Oxygen Species; Thioredoxins

Although attention has focused on the chemopreventive action of retinoic acid (RA) in hepatocarcinogenesis, the functional role of RA in the liver has yet to be clarified. To explore the role of RA in the liver, we developed transgenic mice expressing RA receptor (RAR) alpha- dominant negative form in hepatocytes using albumin promoter and enhancer. At 4 months of age, the RAR alpha- dominant negative form transgenic mice developed microvesicular steatosis and spotty focal necrosis. Mitochondrial beta-oxidation activity of fatty acids and expression of its related enzymes, including VLCAD, LCAD, and HCD, were down-regulated; on the other hand, peroxisomal beta-oxidation and its related enzymes, including AOX and BFE, were up-regulated. Expression of cytochrome p4504a10, cytochrome p4504a12, and cytochrome p4504a14 was increased, suggesting that omega-oxidation of fatty acids in microsomes was accelerated. In addition, formation of H2O2 and 8-hydroxy-2'-deoxyguanosine was increased. After 12 months of age, these mice developed hepatocellular carcinoma and adenoma of the liver. The incidence of tumor formation increased with age. Expression of beta-catenin and cyclin D1 was enhanced and the TCF-4/beta-catenin complex was increased, whereas the RAR alpha/ beta-catenin complex was decreased. Feeding on a high-RA diet reversed histological and biochemical abnormalities and inhibited the occurrence of liver tumors. These results suggest that hepatic loss of RA function leads to the development of steatohepatitis and liver tumors. In conclusion, RA plays an important role in preventing hepatocarcinogenesis in association with fatty acid metabolism and Wnt signaling.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; beta Catenin; Cytoskeletal Proteins; Deoxyguanosine; Diet; Dose-Response Relationship, Drug; Enzymes; Fatty Acids; Fatty Liver; Genes, Dominant; Hydrogen Peroxide; Liver; Liver Neoplasms; Male; Mice; Mice, Transgenic; Mitochondria, Liver; Oxidation-Reduction; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Trans-Activators; Tretinoin

Transgenic mice overexpressing hepatitis B x protein (HBx) show an increased susceptibility to mutations if exposed to mutagens. Also involved in HBx signalling, reactive oxygen intermediates (ROI) can induce DNA adducts such as 8-hydroxy-2'-deoxyguanosine that can in turn lead to G/T transversion mutations. Therefore, we investigated whether HBx expression increases the level of the mutational precursor 8-hydroxy-2'-deoxyguanosine in hepatocellular DNA.. 8-hydroxy-2'-deoxyguanosine concentrations of DNA hydrolysates of HBx protein expressing HepG2 cells and livers of HBx transgenic mouse lines were determined electrochemically after HPLC fractionation.. 8-hydroxy-2'-deoxyguanosine concentrations in genomic DNA of HBx protein expressing cell lines correlated with the factor of transactivation. The 8-hydroxy-2'-deoxyguanosine levels were reduced after incubation of HBx recombinant cell lines with 0.1 or 1 mM of the antioxidant N-acetylcysteine. Hepatic 8-hydroxy-2'-deoxyguanosine concentrations in DNA of old transgenic mice were significantly, i.e. twofold, (p < 0.01) increased as compared to those of old nontransgenic or young transgenic controls and of control mice expressing a second HBV transactivator (MHBs(t76)).. HBx expression results in elevated DNA adduct levels. This could reflect a direct inhibitory interaction of HBx with cellular repair mechanisms. Alternatively, this may be an effect of an increased generation of reactive oxygen intermediates through HBx.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acetylcysteine; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Deoxyguanosine; DNA Adducts; DNA, Recombinant; Humans; Liver; Liver Neoplasms; Mice; Mice, Transgenic; Trans-Activators; Viral Regulatory and Accessory Proteins

Reactive oxygen species may be involved in the progression of chronic liver disease and the occurrence of hepatocellular carcinoma (HCC). To clarify whether clinicopathological findings in liver diseases are related to oxidative DNA damage, hepatic expression of the 8-hydroxy-2'-deoxyguanosine (8-OHdG) was examined in 75 liver disease patients, which included 32 chronic hepatitis (CH), 13 liver cirrhosis (LC) and 30 HCC patients. The CH patients had higher 8-OHdG-positive hepatocytes than LC (P < 0.05). In CH and LC, the number of 8-OHdG-positive hepatocytes was correlated with alanine aminotransferase and asparate aminotransferase (P < 0.01 and P < 0.05, respectively). Of 30 HCC cases, 25 cases (83%) showed stronger immunoreactivity than non-cancerous counterparts. The patients with poorly differentiated HCC had a larger tumor size and higher levels of AFP, and exhibited higher labeling indices of PCNA-, TUNEL- and 8-OHdG-positive cells than those with well and moderately differentiated HCC. Our findings suggest that oxidative DNA damage is increased in association with necroinflammation in chronic liver disease and determination of 8-OHdG is useful in assessing high-grade malignancy in HCC.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers, Tumor; Carcinoma, Hepatocellular; Chronic Disease; Deoxyguanosine; DNA Damage; Female; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Liver Cirrhosis; Liver Neoplasms; Male; Middle Aged; Reactive Oxygen Species

We have recently shown that immunodeficient (SCID) mice, which lack functional T and B cells, are highly susceptible to low dose site specific induction of colon aberrant crypt foci (ACF), surrogates for colon tumors, by 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ). To test whether long-term exposure to a high dose in the diet might prove carcinogenic to the SCID mouse colon, in contrast to other mice strains tested to date, the compound was administered at 300 p.p.m. in the diet to female 6-7-week-old SCID mice for 32 weeks. IQ induced high numbers of ACF, hyperplastic polyps, dysplasia, and colon adenomas, as well as hepatocellular altered foci and liver adenomas. Induction of colon tumors did not correlate with the main sites where ACF developed, the proximal colon, however, being seen mainly in the mid and distal colon. Induction of colon tumors correlated significantly with the incidence of dysplasia, crypt height, the mitotic index, cell proliferation and numbers of 8-hydroxydeoxyguanosine (8-OHdG)-positive cells in the colon crypt, particularly in mid and distal colon. Administration of 20% omega-6 polyunsaturated fatty acids (corn oil), omega-3 polyunsaturated fatty acids (perilla oil), or monounsaturated fatty acids (olive oil) simultaneously with IQ in the diet resulted in: (i) inhibition of colon and liver tumor induction by corn and perilla oil, whereas olive oil showed no effects; (ii) no reduction in total numbers of ACF by corn oil or perilla oil but significant suppression in the olive oil treated group; (iii) inhibition of tumor development particularly by omega-3 polyunsaturated fatty acids in perilla oil, correlating significantly with decreased cell proliferation in both colon and liver and a marked decrease in crypt heights and mitotic indices. Selective reduction in the numbers of 8-OHdG-positive nuclei, mainly in the middle and distal colon crypts, was also found to correlate with tumor inhibition. Thus, the results indicate carcinogenicity of IQ in the colon of the SCID mouse and preventive effects of polyunsaturated fatty acids.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Apoptosis; Body Weight; Carcinogenicity Tests; Carcinogens; Cell Division; Colonic Neoplasms; Deoxyguanosine; Disease Models, Animal; Fatty Acids; Female; Incidence; Intestinal Mucosa; Kidney; Liver; Liver Neoplasms; Mice; Mice, SCID; Mitosis; Organ Size; Quinolines

Development of hepatocellular carcinomas in rats caused by a choline-deficient, L-amino acid-defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor-resistant C57BL/6J mice. Six-week-old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8-hydroxydeoxyguanosine (8-OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6 +/- 4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/24 (20.8%), and multiplicities of 1.42 +/- 1.32 and 0.29 +/- 0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8-OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenoma; Amino Acids; Animals; Carcinoma; Choline Deficiency; Deoxyguanosine; Female; Liver Cirrhosis, Experimental; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL

Expression of cyclooxygenase (COX)-2 protein during rat hepatocarcinogenesis associated with fatty change, fibrosis, cirrhosis and oxidative DNA damage, caused by a choline-deficient, L-amino acid-defined (CDAA) diet were investigated in F344 male rats, along with the chemopreventive efficacy of the specific COX-2 inhibitor, nimesulide (NIM). Nimesulide, which was administered in the diet at concentrations of 200, 400, 600 and 800 p.p.m. for 12 weeks, decreased the number and size of preneoplastic enzyme-altered liver foci, levels of oxidative DNA damage, and the grade and incidence of fibrosis in a dose-dependent manner. A preliminary long-term study of 65 weeks also revealed that 800 p.p.m. NIM decreased the multiplicity of neoplastic nodules and hepatocellular carcinomas and prevented the development of cirrhosis. Western blot analysis revealed that COX-2 protein was barely expressed in control livers and increased approximately 2.9-fold in the livers of rats fed on a CDAA diet for 12 weeks and approximately 4.5-5.4-fold in tumors, with a diameter larger than 5 mm, at 80 weeks. Immunohistochemically, COX-2 protein was positive in sinusoidal and stromal cells in fibrotic septa, which were identified by immunoelectron microscopy as Kupffer cells, macrophages, either activated Ito cells or fibroblasts, after exposure to the CDAA diet for 12 weeks, whereas it was only occasionally weakly positive in sinusoidal, probably Kupffer, cells in control livers. In neoplastic nodules in rats fed on a CDAA diet for 30 and 80 weeks, sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm were strongly positive for COX-2 protein, with the neoplastic hepatocytes in the minority of the nodules, but not the cancer cells, being moderately positive. These results clearly indicate that rat hepatocarcinogenesis, along with fatty change, fibrosis and cirrhosis, is associated with increased expression of COX-2 protein, and point to the chemopreventive efficacy of a selective COX-2 inhibitor against, at least, the early stages of hepatocarcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Amino Acids; Animal Nutritional Physiological Phenomena; Animals; Anticarcinogenic Agents; Blotting, Western; Cells, Cultured; Choline; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Fibrosis; Glutathione Transferase; Hepatocytes; Immunohistochemistry; Isoenzymes; Kupffer Cells; Liver; Liver Neoplasms; Male; Membrane Proteins; Microscopy, Immunoelectron; Organ Size; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred F344; Sulfonamides; Time Factors

Pentachlorophenol (PCP) is a widely used biocide that has been reported to be hepatocarcinogenic in mice. Its effects in rats are equivocal, but the liver clearly is not a target organ for carcinogenesis. The carcinogenic effects of PCP in mice may relate to reactive oxygen species generated during metabolism. PCP is known to increase the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine (ohdG), in the liver of exposed mice. To investigate whether the generation of oxidative DNA damage and direct DNA adducts may explain the species difference in carcinogenicity, we have analyzed ohdG in hepatic DNA from PCP-exposed rats. Rats were exposed acutely to PCP for 1 or 5 days. Tissues also were obtained from a 27 week interim sacrifice of the 2 year National Toxicology Program carcinogenesis bioassay. We used HPLC with electrochemical array detection for ohdG analysis. Single or 5 day exposure to PCP (up to 120 or 60 mg/kg/day, respectively) did not increase ohdG. Dietary exposure to 1000 p.p.m. PCP (equivalent to 60 mg/kg/day) for 27 weeks induced a 2-fold increase in ohdG (1.8 versus 0.91x10(-6) in controls). In parallel, formation of direct DNA adducts was analyzed by 32P-post-labeling following nuclease P1 adduct enrichment. We detected two major DNA adducts with relative adduct labeling of 0.78x10(7) adducts per total nucleotides. One of these adducts was found to co-migrate with the adduct induced by the metabolite, tetrachloro-1,4-benzoquinone. We observed differences in DNA adduct formation between acute and chronic studies, with acute studies not inducing any detectable amount of DNA adducts. These results indicated that chronic, but not acute exposure to PCP increased ohdG and direct adducts in hepatic DNA. As the same exposure conditions that enhanced ohdG did not produce liver cancer in rats, the generation of reactive oxygen species, oxidative DNA damage and direct DNA adducts is not sufficient for the induction of hepatocarcinogenesis by PCP in the rat.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biological Assay; Carcinogens; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Adducts; DNA Damage; Dose-Response Relationship, Drug; Environmental Pollutants; Liver Neoplasms; Mice; Models, Chemical; Oxygen; Pentachlorophenol; Rats; Rats, Inbred F344; Time Factors

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in acute intermittent porphyria (AIP) due to enzymatic deficiencies in the heme biosynthetic pathway Its accumulation has been associated with several symptoms, such as abdominal pain attacks, neuromuscular weaknesses, neuropsychiatric alterations and increased hepatocellular carcinoma (HCC) incidence. The use of exogenous ALA to elevate porphyrin levels in tumor photodynamic therapy, adds further significance to ALA toxicology. Under ferritin mediated and metal catalyzed oxidation, ALA produces reactive oxygen species that can damage plasmid and isolated DNA in vitro, and increases the steady-state level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in liver, spleen and kidney DNA and 5-hydroxy-2'-deoxycytidine in liver DNA of ALA-treated rats. The in vitro DNA damage could be partially inhibited by SOD, catalase, DTPA, mannitol and melatonin. ALA also promotes the formation of radical-induced base degradation products in isolated DNA. 4,5-Dioxovaleric acid, the final oxidation product of ALA, alkylates guanine moieties within both nucleoside and isolated DNA, producing two diastereoisomeric adducts. Dihydropyrazine derivatives of ALA generated by its dimerization, promote DNA strand-breaks and 8-oxodGuo formation in the presence of Cu2+. Together these results reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aminolevulinic Acid; Animals; Deoxyguanosine; DNA; DNA Damage; Liver Neoplasms; Metals; Plasmids; Porphyria, Acute Intermittent; Propionates; Pyrazines; Rats; Valerates

Oxidative stress plays an important role in hepatocarcinogenesis. Although Sho-saiko-to (TJ-9), a Japanese herbal medicine which has been recently administered to patients with chronic liver disease in Japan, prevents hepatocarcinogenesis, the mechanism by which TJ-9 protects against cancer development is not fully understood. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a DNA adduct by reactive oxygen species, is known as a parameter of genetic risk for hepatocarcinogenesis. To clarify whether the preventive effect on hepatocarcinogenesis by TJ-9 is dependent on 8-OHdG, the effect on 8-OHdG levels by TJ-9 was examined by using high-performance liquid chromatography-mass spectrometry (LC-MS) in a diethylnitrosamine (DEN)-induced hepatocarcinogenesis model of male Fisher rats. TJ-9 reduced the number of preneoplastic cells, detected as the glutathione S transferase P (GST-P)-positive hepatocytes, and inhibited the development of liver tumors. TJ-9 also significantly decreased the formation of 8-OHdG, as indicated by LC-MS and immunohistochemical analysis. In addition, ornithine decarboxylase (ODC) activity and the number of proliferating cell nuclear antigen (PCNA)-positive cells were not altered. An electron paramagnetic resonance spin-trapping technique showed that TJ-9 scavenges hydroxyl radicals in a dose-dependent manner. In conclusion, the results of the present study suggest that TJ-9 prevents hepatocarcinogenesis in association with inhibition of 8-OHdG formation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Anticarcinogenic Agents; Biomarkers, Tumor; Cell Count; Deoxyguanosine; Drugs, Chinese Herbal; Glutathione Transferase; Hydroxyl Radical; Liver Neoplasms; Male; Ornithine Decarboxylase; Oxidative Stress; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344

Since oxidative DNA damage plays a role in experimental carcinogen-induced cancers, the purpose of the present study was to determine if hepatic oxidative DNA damage was increased in patients with HCC compared to patients with benign hepatic tumors or hepatic metastases (non-HCC) or to patients with end-stage alcoholic liver disease undergoing liver transplantation. Oxidative DNA damage was assessed by 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Results showed that peritumoral 8-OH-dG was markedly increased in HCC (N= 51) (180 +/- 74 vs 32 +/- 58-OH-dG/10(6)dG for tumor, P < 0.005) in contrast to patients with non-HCC (N = 17), in whom the peritumoral 8-OH-dG did not differ from that in tumor (39 +/- 7 vs. 31 +/- 108-OH-dG/10(6)dG). Oxidative DNA damage can be both mutagenic and carcinogenic; our data suggested it will be important in future studies to determine the chronology of this type of liver injury relative to hepatocarcinogenesis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Carcinoma, Hepatocellular; Deoxyguanosine; DNA Damage; Humans; Liver; Liver Diseases, Alcoholic; Liver Neoplasms

The authors have revealed a significant association between hepatoblastoma and low birth weight. This study was done to explore the evidence that liver cells were oxidatively damaged, based on the hypothesis that oxidative damage to DNA is involved in the development of hepatoblastoma in children of low birth weight.. Oxidative DNA damage in the liver was examined by immunohistochemically detecting the presence of a DNA repair product, 8-hydroxy-2'-deoxyguanosine (8-OHdG), in five patients with hepatoblastoma and 14 children with non-neoplastic disease.. Positive staining for 8-OHdG was observed in all five patients with hepatoblastoma. Distribution of 8-OHdG positivity was diffuse in the intralobular area in one patient and was restricted to the periportal area of the lobules in four patients. There was no apparent correlation between birth weight of the patients, histological findings in the liver, and the distribution of 8-OHdG positivity. In children with non-neoplastic disease, 8-OHdG was detected in nine of 14 patients, and 8-OHdG was positive in the intralobular area of the liver parenchyma except in one patient.. These results suggest that the cause of oxidative DNA damage in patients with hepatoblastoma may be different from the cause, extensive parenchymal damage to the liver, in children with non-neoplastic disease, but the 8-OHdG formation is not specific to hepatoblastoma patients of low birth weight. Further studies to elucidate the true reason for the high incidence of hepatoblastoma in children of low birth weight are necessary.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Case-Control Studies; Child, Preschool; Deoxyguanosine; DNA Damage; Female; Hepatoblastoma; Humans; Infant; Infant, Low Birth Weight; Infant, Newborn; Liver; Liver Neoplasms; Male

Hydroxychavicol (HC) is the major safrole urinary metabolite in rats and humans. The cytotoxic potential of HC in metabolically competent cells has yet to be studied. HC alone was slightly toxic to HepG2 cells. However, the cytotoxicity increased significantly (P<0.05) when HepG2 cells were pretreated with buthionine sulfoximine (BSO), suggesting that endogenous glutathione participates in HC-induced cytotoxicity. Addition of catalase or N-acetylcysteine prevented the BSO plus HC-mediated cytotoxicity. HC also increased 8-hydroxy-2'-deoxyguanosine formation and apoptosis in BSO-pretreated HepG2 cells and this increase could also be suppressed by catalase. These data suggest that BSO pretreatment enhanced HC-induced cytotoxic effects in HepG2 cells, which are related to oxidative DNA damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acetylcysteine; Antimetabolites, Antineoplastic; Apoptosis; Buthionine Sulfoximine; Catalase; Cell Survival; Deoxyguanosine; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Electrons; Eugenol; Free Radical Scavengers; Glutathione; Humans; Liver Neoplasms; Mutagens; Oxidative Stress; Reactive Oxygen Species; Time Factors; Tumor Cells, Cultured

Aflatoxin B(1) (AFB(1)), a potent hepatocarcinogen, enhances ROS formation and causes oxidative DNA damage, which may play a role in its carcinogenicity. We have demonstrated recently that ebselen, an organic selenium compound, protects against the cytotoxicity of AFB(1) through its antioxidant capability. The present study was designed to investigate the effect of ebselen on AFB(1)-induced hepatocarcinogenesis in an animal model. Fischer 344 rats were first treated with either deionized water or ebselen (5 mg/kg, 5 days/week) via gavage for 4 weeks, then given AFB(1) (0.4 mg/kg, gavage, once a week) or AFB(1) plus ebselen (5 mg/kg, 5 days/week) for another 24 weeks. The results showed that the hepatocarcinogenicity of AFB(1) in rats was significantly reduced by ebselen treatment as indicated by a decrease in: (i) serum gamma-glutamyl transpeptidase activity; (ii) expression of mRNAs of liver alpha-fetoprotein and the placental form of glutathione S-transferase (GST-P); and (iii) the area and mean density of staining of liver GST-P foci. Ebselen treatment significantly reduced the formation of hepatic AFB(1)-DNA adducts and 8-hydroxydeoxyguanosine caused by AFB(1) exposure. These findings suggest that ebselen can inhibit the carcinogenicity of AFB(1). In addition to the reduction of AFB(1)-DNA adduct formation, the protective effect of ebselen against AFB(1)-induced oxidative DNA damage may also, at least in part, contribute to its anticarcinogenic property.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aflatoxin B1; alpha-Fetoproteins; Animals; Anticarcinogenic Agents; Antioxidants; Azoles; Deoxyguanosine; DNA Adducts; gamma-Glutamyltransferase; Glutathione Transferase; Isoenzymes; Isoindoles; Liver; Liver Neoplasms; Male; Organoselenium Compounds; Rats; Rats, Inbred F344; Reactive Oxygen Species; RNA, Messenger; Transcription, Genetic

Susceptibility to spontaneous or chemically-induced liver tumors in mice has been demonstrated to be strain dependent, with the tumor development or promotion phase contributing most to variability. Since reactive oxygen species are thought to play a role in carcinogenesis, especially tumor promotion, we investigated steady-state levels of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in hepatic DNA from age matched (7 months), untreated mice of three inbred strains, that differ significantly in their susceptibility to liver carcinogenesis. Male mice of strain C3H, highly sensitive to liver tumorigenesis, had significantly higher levels of hepatic 8-oxo-dG (3.3 SE 0.2 8-oxo-dG/10(5) dG) than resistant strains C57BL (2.1 SE 0.3/10(5) dG; p < 0.01) and A/JCr (2.5 SE 0.1/10(5) dG; p < 0.015). In contrast, levels of 8-oxo-dG in livers of female A/JCr mice (3.1 SE 0.3/10(5) dG) were higher than in those of C3H (2.2 SE 0.1/10(5) dG; p < 0.025) and C57BL females (2.5 SE 0.3/10(5) dG; p = NS). Male C3H livers presented significantly more 8-oxo-dG than those of C3H females (p < 0.002). These results suggest that steady-state levels of 8-oxo-dG may contribute to the inherent differences in susceptibility to hepatocarcinogenesis in inbred strains of mice, especially the high sensitivity of C3H males.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Carcinoma, Hepatocellular; Deoxyguanosine; DNA; DNA Damage; Female; Genetic Predisposition to Disease; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred Strains; Oxidative Stress; Sex Factors; Species Specificity

It has been reported that flumequine (FLU) induces hepatic tumors in mice when given orally for 18 months. We investigated possible underlying mechanisms using a two-stage mouse hepatocarcinogenesis model. After initiation with a single intraperitoneal injection of 100 mg/kg body weight diethylnitrosamine (DEN) or saline, male CD-1 mice were given 4000 ppm FLU in the diet or 500 ppm phenobarbital (PB) in drinking water for 9, 19, 24 or 30 weeks. Toxicity, evidenced by centrilobular swollen and polar hepatocytes with fatty droplets, infiltration of inflammatory cells and increased numbers of mitosis in hepatocytes, was apparent in the livers of mice treated with FLU at all time points, but its severity declined towards the termination. FLU did not induce cytochrome P-450 enzymes such as 1A1, 2B1 and 3A2 as assessed immunohistochemically, while positive expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was increased in hepatocytes of both DEN + FLU and FLU groups compared with the relevant controls. In animals given PB, eosinophilic swelling of hepatocytes was prominent, and the hepatocytes showed strongly positive reactions for CYP 1A1 and 3A2. Altered cell foci were induced in the livers of FLU-treated animals both with and without DEN initiation, especially the former, and their development paralleled the degree of hepatic toxicity. These results suggest that FLU hepatocarcinogenicity in mice is dependent on hepatotoxic damage and consequently increased cell proliferation. Oxidative damage to DNA may also be a crucial factor.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenoma; Animals; Anti-Infective Agents; Carcinogenicity Tests; Cell Division; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP2B1; Cytochrome P-450 Enzyme System; Deoxyguanosine; Diethylnitrosamine; Fluoroquinolones; Immunohistochemistry; Liver; Liver Neoplasms; Male; Mice; Phenobarbital; Proliferating Cell Nuclear Antigen; Quinolizines; Steroid Hydroxylases

Phosphine (PH3), from hydrolysis of metal phosphides, is an important insecticide (aluminum phosphide) and rodenticide (zinc phosphide) and is considered genotoxic and cytotoxic in mammals. This study tests the hypothesis that PH3-induced genotoxicity and cytotoxicity are associated with oxidative stress by examining liver (Hepa 1c1c7) cells for possible relationships among cell death, increases in reactive oxygen species (ROS) and lipid peroxidation, and elevated 8-hydroxyguanine (8-OH-Gua) in DNA. PH3 was generated from 0.5 mM magnesium phosphide (Mg3P2) to give 1 mM PH3 as the nominal and maximal concentration. This level causes 31% cell death at 6 h, measured by lactate dehydrogenase leakage, with appropriate dependence on concentration and time. The intracellular ROS level is elevated within 0.5 h following exposure to PH3, peaking at 235% of the control by about 1 h. Lipid peroxidation (measured as malondialdehyde plus 4-hydroxyalkenals) is increased up to 504% by PH3 at 6 h in a time-dependent manner. The level of 8-OH-Gua in DNA, a biomarker of mutagenic oxidative DNA damage analyzed by GC/MS, increases to 259% at 6 h after PH3 treatment. Antioxidants significantly attenuate the PH3-induced ROS formation, lipid peroxidation, 8-OH-Gua formation in DNA, and cell death, with the general order for effectiveness of GSH (5 mM) and D-mannitol (10 mM) (hydroxyl radical scavengers), then Tempol (2.5 mM) and sodium azide (3 mM) (superoxide anion and singlet oxygen scavengers, respectively). These studies support the hypothesis that PH3-induced mutagenic and cytotoxic effects are due to increased ROS levels, probably hydroxyl radicals, initiating oxidative damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cell Death; Coloring Agents; Deoxyguanosine; DNA; Gas Chromatography-Mass Spectrometry; Hydrogen Peroxide; Insecticides; L-Lactate Dehydrogenase; Lipid Peroxidation; Liver; Liver Neoplasms; Mice; Mutagenicity Tests; Oxidants; Oxidative Stress; Phosphines; Reactive Oxygen Species; Trypan Blue; Tumor Cells, Cultured

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cell Division; Deoxyguanosine; DNA Damage; Hepatitis B virus; Hepatitis, Chronic; Liver Neoplasms; Liver Neoplasms, Experimental; Mice; Mice, Transgenic; Oxidation-Reduction; Precancerous Conditions; Reactive Oxygen Species; Superoxides; Viral Proteins

Acute hepatitis spontaneously develops in the Long-Evans Cinnamon rat at the age of 4 mo, and eventually hepatocellular carcinoma develops after the chronic hepatitis that persists for over a year. Previously, abnormal copper accumulation was found in the livers of Long-Evans Cinnamon rats from birth, and it was reported that short-term administration of D-penicillamine, a copper-chelating agent, prevented acute hepatitis in Long-Evans Cinnamon rats. In this study we investigated whether long-term administration of D-penicillamine could also prevent chronic hepatitis and subsequent hepatocellular carcinoma in Long-Evans Cinnamon rats. During long-term observation, which was continued from 11 to 70 wk after birth, no elevation of serum transaminase levels was observed in the Long-Evans Cinnamon rats treated with D-penicillamine. Moreover, no histological changes characteristic of the chronic hepatitis were observed in D-penicillamine-treated Long-Evans Cinnamon rats, which were killed at 70 wk of age. Furthermore, placental glutathione S-transferase-positive foci, described as a marker for preneoplastic lesions in the liver, were not detected, and thus hepatocarcinogenesis was completely prevented in D-penicillamine-treated Long-Evans Cinnamon rats. We also found that the amount of 8-hydroxy-deoxyguanosine, one of oxidative DNA damage products in the liver, was decreased in the Long-Evans Cinnamon rats treated with D-penicillamine. These findings suggest that a process of the prolonged liver-cell injury and regeneration was essential for spontaneous development of hepatocellular carcinoma in Long-Evans Cinnamon rats with abnormal copper metabolism.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alanine Transaminase; Animals; Anticarcinogenic Agents; Aspartate Aminotransferases; Carcinoma, Hepatocellular; Copper; Deoxyguanosine; DNA; Female; Hepatitis; Liver; Liver Neoplasms; Male; Penicillamine; Rats; Rats, Inbred Strains; Time Factors