4-hydroxy-2-nonenal and Carcinoma--Hepatocellular

Topics: Aldehydes; Animals; Antioxidants; Carcinoma, Hepatocellular; Cell Division; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Lipid Peroxidation; Liver Neoplasms; Liver Neoplasms, Experimental; Neoplasms; Signal Transduction

Topics: Aldehydes; Amino Acid Sequence; Animals; Antioxidants; Apoptosis; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Fatty Acid Transport Proteins; Fatty Acids, Unsaturated; Female; Gene Expression Regulation, Neoplastic; Humans; Kelch-Like ECH-Associated Protein 1; Lipid Peroxidation; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Signal Transduction; Sorafenib; Thioredoxin Reductase 1

The aim of this study was to investigate the effects of medium-chain triglycerides (MCTs) on chemically-induced hepatic carcinogenesis (HCC) in mice.. In a first set of experiments, mice were treated with diethylnitrosoamine intraperitoneally at two weeks of age. They were fed chow containing MCT or a normal chow diet and sacrificed after 28 weeks. Incidence of hepatic tumor was compared between the two groups. Expression of oxidative stress, and inflammatory cytokines and chemokines in liver tissues were examined. In a second set of experiments, the histopathological findings of the intraperitoneal adipose tissue were assessed, and expression of adipocytokines in the fat tissue was measured. In a third set of experiments, plasma β-hydroxybutyrate (HB) concentration was measured in both animals fed chow containing MCT and a normal chow diet. Mouse HCC cells were co-cultured with β-HB, and the numbers of tumor cells were counted at days 3 and 7.. In the first set of experiments, the tumor count observed in the control group was significantly blunted in the MCT group. Maximum tumor diameter also decreased in the MCT group compared to the control group. The expression of inflammatory cytokines and chemokines was significantly decreased by MCT. Furthermore, expression of 4-hydroxynonenal was lower in the MCT group compared to the control group. In the second set of experiments, hypertrophy of the adipocytes was suppressed, and the concentration of adiponectin and leptin in the adipose tissue decreased by MCT. In the third set of experiments, plasma β-HB concentration increased in the MCT group as expected. β-HB significantly inhibited the proliferation of HCC cells.. MCT administration markedly suppresses the incidence of chemically-induced HCC by inhibition of inflammation and increase of ketone bodies.

Topics: 3-Hydroxybutyric Acid; Adipocytes; Adipokines; Adiponectin; Adipose Tissue; Aldehydes; Animal Feed; Animals; Carcinogens; Carcinoma, Hepatocellular; Cell Count; Cell Proliferation; Chemokines; Cytokines; Diethylnitrosamine; Hypertrophy; Inflammation; Leptin; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred C3H; Oxidative Stress; Triglycerides

Altered redox status in cancer cells has been linked to lipid peroxidation induced by reactive oxygen species (ROS) and subsequent formation of reactive lipid electrophiles, especially 4-hydroxy-nonenal (4-HNE). Emerging evidence suggests that cancer cells manipulate redox status to acquire anti-apoptotic phenotype but the underlying mechanisms are poorly understood. Cardiolipin (CL), a mitochondria-specific inner membrane phospholipid, is critical for maintaining mitochondrial function. Paradoxically, liver tissues contain tetralinoleoyl cardiolipin (TLCL) as the major CL in mitochondria yet emerging evidence suggests that ROS generated in mitochondria may lead to CL peroxidation and activation of intrinsic apoptosis. It remains unclear how CL oxidation leads to apoptosis and its relevance to the pathogenesis of hepatocellular carcinoma (HCC). We employed a mass spectrometry-based lipidomic approach to profile lipids in human tissues of HCC and found that CL was gradually decreased in tumor comparing to peripheral non-cancerous tissues, accompanied by a concomitant decrease of oxidized CL and its oxidation product, 4-HNE. Incubation of liver cancer cells with TLCL significantly restored apoptotic sensitivity accompanied by an increase of CL and its oxidation products when treated with staurosporine (STS) or Sorafenib (the standard treatment for late stage HCC patients). Our studies uncovered a novel mechanism by which cancer cells adopt to evade apoptosis, highlighting the importance of mitochondrial control of apoptosis through modulation of CL oxidation and subsequent 4-HNE formation in HCC. Thus manipulation of mitochondrial CL oxidation and lipid electrophile formation may have potential therapeutic value for diseases linked to oxidative stress and mitochondrial dysfunctions.

Topics: Aldehydes; Apoptosis; Carcinoma, Hepatocellular; Cardiolipins; Humans; Lipid Peroxidation; Lipids; Liver; Liver Neoplasms; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species

Increased thioredoxin reductase (TrxR) levels in serum were recently identified as possible prognostic markers for human prostate cancer or hepatocellular carcinoma. We had earlier shown that serum levels of TrxR protein are very low in healthy mice, but can in close correlation to alanine aminotransferase (ALT) increase more than 200-fold upon chemically induced liver damage. We also found that enzymatic TrxR activity in serum is counteracted by a yet unidentified oxidase activity in serum. In the present study we found that mice carrying H22 hepatocellular carcinoma tumors present highly increased levels of TrxR in serum, similarly to that reported in human patients. In this case ALT levels did not parallel those of TrxR. We also discovered here that the TrxR-antagonistic oxidase activity in serum is due to the presence of quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We furthermore found that the chemotherapeutic agents cisplatin or auranofin, when given systemically to H22 tumor bearing mice, can further inhibit TrxR activities in serum. The TrxR serum activity was also inhibited by endogenous electrophilic inhibitors, found to increase in tumor-bearing mice and to include protoporphyrin IX (PpIX) and 4-hydroxynonenal (HNE). Thus, hepatocellular carcinoma triggers high levels of serum TrxR that are not paralleled by ALT, and TrxR enzyme activity in serum is counteracted by several different mechanisms. The physiological role of TrxR in serum, if any, as well as its potential value as a prognostic marker for tumor progression, needs to be studied further.

Topics: Alanine Transaminase; Aldehydes; Animals; Antineoplastic Agents; Auranofin; Carboplatin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cisplatin; Liver Neoplasms; Male; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Oxidoreductases Acting on Sulfur Group Donors; Protoporphyrins; Thioredoxin-Disulfide Reductase

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

Dysregulation of cell signaling by electrophiles such as 4-hydroxynonenal (4-HNE) is a key component in the pathogenesis of chronic inflammatory liver disease. Another consequence of inflammation is the perpetuation of oxidative damage by the production of reactive oxidative species such as hydrogen peroxide. Previously, we have demonstrated Akt2 as a direct target of 4-HNE in hepatocellular carcinoma cells. In the present study, we used the hepatocellular carcinoma cell line HepG2 as model to understand the combinatorial effects of 4-HNE and hydrogen peroxide. We demonstrate that 4-HNE inhibits hydrogen peroxide-mediated phosphorylation of Akt1 but not Akt2. Pretreatment of HepG2 cells with 4-HNE prevented hydrogen peroxide stimulation of Akt-dependent phosphorylation of downstream targets and intracellular Akt activity compared with untreated control cells. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment resulted in carbonylation of Akt1, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt1 (rAkt1) with 20 or 40 μΜ 4-HNE inhibited rAkt1 activity by 29 and 60%, respectively. We further demonstrate that 4-HNE activates Erk via a PI3 kinase and PP2A-dependent mechanism leading to increased Jnk phosphorylation. At higher concentrations, 4-HNE decreased both cell survival and proliferation as evidenced by MTT assays and EdU incorporation as well as decreased expression of cyclin D1 and β-catenin, an effect only moderately increased by the addition of hydrogen peroxide. The ability of 4-HNE to exert combinatorial effects on Erk, Jnk, and Akt-dependent cell survival pathways provides additional insight into the mechanisms of cellular damage associated with chronic inflammation.

Topics: Aldehydes; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Humans; Hydrogen Peroxide; Liver Neoplasms; Oxidants; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Cells, Cultured

There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells overexpressing CYP2E1 were subjected to arachidonic acid, iron and 100mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down-regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up-regulated by ethanol alone and betaine tended to prevent this. All 4 agents down-regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up-regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.

Topics: Aldehydes; Anti-Infective Agents, Local; Antioxidants; Betaine; Blotting, Western; Carcinoma, Hepatocellular; Catechin; Cells, Cultured; Cytochrome P-450 CYP2E1; Ethanol; Gastrointestinal Agents; Hepatocytes; Humans; In Vitro Techniques; Liver Neoplasms; Malondialdehyde; Oxidative Stress; Quercetin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Chronic inflammatory processes induce oxidative and nitrative stress that trigger lipid peroxidation (LPO), whereby DNA-reactive aldehydes such as trans-4-hydroxy-2-nonenal (HNE) are generated. Miscoding etheno-modified DNA adducts including 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) are formed by reaction of HNE with DNA-bases which are excreted in urine, following elimination from tissue DNA. An ultrasensitive and specific immunoprecipitation/HPLC-fluorescence detection method was developed for quantifying epsilondA excreted in urine. Levels in urine of Thai and European liver disease-free subjects were in the range of 3-6 fmol epsilondA/micromol creatinine. Subjects with inflammatory cancer-prone liver diseases caused by viral infection or alcohol abuse excreted massively increased and highly variable epsilondA-levels. Groups of Thai subjects (N=21) with chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC) due to HBV infection had 20, 73 and 39 times higher urinary epsilondA levels, respectively when compared to asymptomatic HBsAg carriers. In over two thirds of European patients (N=38) with HBV-, HCV- and alcohol-related liver disease, urinary epsilondA levels were increased 7-10-fold compared to healthy controls. Based on this pilot study we conclude: (i) high urinary epsilondA-levels, reflecting massive LPO-derived DNA damage in vivo may contribute to the development of HCC; (ii) epsilondA-measurements in urine and target tissues should thus be further explored as a putative risk marker to follow malignant progression of inflammatory liver diseases in affected patients; (iii) etheno adducts may serve as biomarkers to assess the efficacy of (chemo-)preventive and therapeutic interventions.

Topics: Adult; Aged; Alcohol Drinking; Aldehydes; Carcinoma, Hepatocellular; Case-Control Studies; Deoxyadenosines; DNA Adducts; DNA Damage; Europe; Hepatitis B; Hepatitis B Surface Antigens; Humans; Lipid Peroxidation; Liver Cirrhosis, Alcoholic; Liver Neoplasms; Male; Middle Aged; Pilot Projects

The transcription factors that bind to EpRE's play a key role in the regulation of phase II genes. In this study, we examined whether c-Jun, a partner of Nrf2 in binding to EpRE's, requires phosphorylation by JNK for binding and transcriptional activation. We used chromatin immunoprecipitation assays to measure the recruitment of transcription factors to EpRE sequences in NQO2, GCLC, and GCLM; Western analysis for phosphorylation of JNK; and EpRE-driven reporters along with a JNK-specific inhibitor peptide to determine the potential importance of c-Jun phosphorylation. Human bronchial epithelial (HBE1) and human hepatoma (HepG2) cells were exposed to 4-hydroxy-2-nonenal (HNE), and differences in the regulation of the same EpRE sequences were examined. We found that binding of c-Jun to EpRE sequences increased subsequent to HNE exposure in HepG2 cells; however, in HNE-exposed HBE1 cells, the binding of only phosphorylated c-Jun to the three EpRE sequences increased. Despite the increase in binding of phosphorylated c-Jun, reporter assays for EpRE's showed that inhibition of c-Jun phosphorylation had variable effects on basal and HNE-induced transcription of GCLC and GCLM in HBE1 cells. Thus, in terms of its role in mediating HNE induction of EpRE-mediated transcription, c-Jun seems to be a partner of Nrf2 and, whereas its phosphorylated form may predominate in one cell type versus another, the effects of phosphorylation of c-Jun on transcription can vary with the gene. This contrasts markedly with the well-established requirement for phosphorylation of c-Jun in the activation of AP-1/TRE-mediated transcription.

Topics: Aldehydes; Binding Sites; Blotting, Western; Bronchi; Carcinoma, Hepatocellular; Cells, Cultured; Chromatin Immunoprecipitation; Cysteine Proteinase Inhibitors; Fluorescent Antibody Technique; Genes, Dominant; Glutamate-Cysteine Ligase; Humans; Immunoprecipitation; Liver Neoplasms; Luciferases; NF-E2-Related Factor 2; Phosphorylation; Promoter Regions, Genetic; Protein Transport; Proto-Oncogene Proteins c-jun; Quinone Reductases; Response Elements; Transcriptional Activation

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.

Topics: Adenocarcinoma; Aldehydes; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Cells, Cultured; Gene Expression Regulation; Glutamate-Cysteine Ligase; Humans; Kidney; Lipid Peroxidation; Liver Neoplasms; Male; MAP Kinase Kinase 1; Mice; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-E2-Related Factor 2; Response Elements; Retinoid X Receptor alpha; Retinoid X Receptor beta; RNA, Small Interfering; Tretinoin

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

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

Trans-4-hydroxy-2-nonenal (4-HNE), a major electrophilic by-product of lipid peroxidation, is able to interact with DNA to form exocyclic guanine adducts. 4-HNE is a mutagen and a significant amount of 4-HNE-guanine adduct has been detected in normal cells. Recently, it has been reported that exposure of the wild-type p53 human lymphoblastoid cell line to 4-HNE causes a high frequency of G to T transversion mutations at the third base of codon 249 (-AGG*-) in the p53 gene, a mutational hotspot in human cancers, particularly hepatocellular carcinoma. These findings raise a possibility that 4-HNE could be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene. However, to date, the sequence specificity of 4-HNE-DNA binding remains unclear due to the lack of methodology. To address this question, we have developed a method, using UvrABC nuclease, a nucleotide excision repair enzyme complex isolated from Escherichia coli, to map the distribution of 4-HNE-DNA adducts in human p53 gene at the nucleotide sequence level. We found that 4-HNE-DNA adducts are preferentially formed at the third base of codon 249 in the p53 gene. The preferential binding of 4-HNE was also observed at codon 174, which has the same sequence and the same nearest neighbor sequences (-GAGG*C-) as codon 249. These results suggest that 4-HNE may be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene.

Topics: Aldehydes; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Codon; CpG Islands; DNA Adducts; DNA Damage; DNA Methylation; DNA, Bacterial; DNA, Neoplasm; DNA, Superhelical; Endodeoxyribonucleases; Escherichia coli Proteins; Genes, p53; Humans; Lipid Peroxidation; Liver Neoplasms; Neoplasms; Plasmids

Induction of cytochrome P450 2E1 (CYP2E1) and the formation of reactive oxygen species (ROS) appear to be one of the mechanisms by which ethanol is hepatotoxic. Glutathione peroxidase and catalase detoxify H(2)O(2). Glutathione S-transferases (GST) provide protection from membrane lipid peroxidation, have GSH peroxidase activity, and reduce lipid hydroperoxides. Previous studies showed an up-regulation of GSH synthesis in CYP2E1 expressing HepG2 cells; this finding prompted an evaluation of the levels of other antioxidant exzymes. In CYP2E1 expressing cells, the alpha and microsomal GST messenger RNA (mRNA) are increased by 4- and 2-fold, respectively, and catalase protein and mRNA is increased by 2-fold. The increase in alpha and microsomal GST mRNA correlates with increased total enzymatic activity and is caused by increased transcription as evidenced by run-on transcription assays. In HepG2 cells transfected to express a different cytochrome P450, CYP3A4, there was an increase in alpha GST. However, in contrast to the CYP2E1 expressing cells, neither microsomal GST nor catalase were induced, suggesting some specificity for CYP2E1. In agreement with an increased antioxidant defense system, the sensitivity to added prooxidants such as menadione, antimycin A, H(2)O(2), and 4-hydroxynonenal was lower in the CYP2E1 expressing cells as compared with control cells. In conclusion, overexpression of CYP2E1 in HepG2 cells, besides elevating total GSH levels, also induces expression of catalase and alpha and microsomal GST. This induction confers resistance to the cells against several prooxidants and is suggested to reflect an adaptive response by the cells against CYP2E1-mediated oxidative stress.

Topics: Aldehydes; Carcinoma, Hepatocellular; Catalase; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Enzyme Induction; Extracellular Space; Glutathione Transferase; Humans; Hydrogen Peroxide; Isoenzymes; Liver Neoplasms; Microsomes, Liver; Mixed Function Oxygenases; Oxidants; Oxidative Stress; RNA, Messenger; Tumor Cells, Cultured; Vitamin E

The cellular metabolism of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic and genotoxic product of oxidative stress-induced lipid peroxidation, was investigated in rat H35 hepatoma cells. Previous studies from our laboratory (1) have characterized the degree to which oxidative, reductive, and conjugative metabolic pathways function simultaneously during hepatocellular metabolism of 4-HNE to rapidly eliminate the compound from suspensions of freshly isolated rat hepatocytes. In the current studies, we have extended the investigation of 4-HNE metabolism to examine the pharmacokinetic parameters of 4-HNE elimination and export in a hepatoma cell line and determined that the ensuing oxidative and conjugative metabolites of 4-HNE are rapidly and efficiently transported out the cell. Low concentrations of 4-HNE (25 microM) were used in an attempt to simulate physiologically relevant conditions. The H35 hepatoma cell line studied was first evaluated for enzymes known to play important roles in the metabolism of 4-HNE and were found to possess activities for glutathione S-transferase, aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase of 24.00 +/- 1.12, 3. 45 +/- 0.17, and 6.44 +/- 0.29 nmol min-1 mg-1 protein, respectively. Hepatoma cells were incubated with 25 microM 4-HNE and metabolites in intra- and extracellular fractions were quantitated by reversed-phase HPLC over the time course of treatment. Reduced glutathione (GSH) and the GSH metabolites of 4-HNE were quantitated by reversed-phase HPLC as the dinitrobenzene derivatives. Uptake of 4-HNE from the extracellular medium occurred with an estimated rate of 0.398 +/- 0.181 min-1 10(6) hepatoma cells-1. The oxidative metabolite of 4-HNE, 4-hydroxy-2-nonenoic acid (HNA), produced by ALDH, appeared rapidly in the intracellular fraction achieving concentrations of 0.28 HNA nmol 10(6) hepatoma cells-1 and was efficiently eliminated with a first-order rate constant of 0.988 min-1. The GST-mediated conjugative metabolite, 3-glutathionyl-4-hydroxy-2-nonanal (4-HNE-SG), rapidly reached maximal intracellular concentrations of 1.88 +/- 0.44 nmol 10(6) hepatoma cells-1 and was eliminated at a rate of 0.101 +/- 0.033 min-1. Extracellular rates of formation, representing export, for HNA and 4-HNE-SG were 0.247 +/- 0.045 and 0.044 +/- 0.009 min-1 10(6) hepatoma cells-1, resulting in maximal extracellular concentrations for HNA and 4-HNE-SG of 0.70 +/- 0.10 and 3.03 +/- 0. 84 nmol 10(6) hepatoma cells-1. Approximately 75% o

Topics: Aldehydes; Animals; Biological Transport; Carcinoma, Hepatocellular; Extracellular Space; Glutathione; Glutathione Transferase; Intracellular Fluid; Rats; Tumor Cells, Cultured

Topics: Acrolein; Aldehydes; Animals; Antioxidants; Base Sequence; Carcinoma, Hepatocellular; Gene Expression Regulation, Enzymologic; Glutathione Transferase; Isoenzymes; Molecular Sequence Data; Rats; Response Elements; Tumor Cells, Cultured

Topics: Aldehyde Reductase; Aldehydes; Animals; Carcinoma, Hepatocellular; Male; Rats; Rats, Inbred F344; Tumor Cells, Cultured

A series of alpha,beta-unsaturated aldehydes was evaluated to determine if these compounds could mediate inducible expression of glutathione S-transferase (GST) through the 5'-flanking antioxidant response element (ARE). The ARE from rGST A1 was subcloned into a luciferase reporter construct and used to transiently transfect rat Clone 9 hepatoma cells. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4-HNE), trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethacrynic acid (EA), a control compound also containing an alpha,beta-unsaturated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC50 values for growth inhibition were measured using 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. IC50 values in Clone 9 cells were: 4-HNE, 6.3 +/- 0.7 microM; t-2-HE, 16.0 +/- 0.7 microM; 2-PE, 2.2 +/- 0.4 microM; and EA, 38.0 +/- 1.6 microM. A dose-dependent increase in luciferase activity was observed in transfected cells with all four compounds tested, indicating that alpha, beta-unsaturated aldehydes function as direct activators of the ARE. To determine whether or not the observed promoter activation led to increased transcriptional and translational induction of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in Clone 9 cells revealed increased steady-state message for GST A1 and A4, increased GST A4-4 protein by Western blotting, and increased GST activity toward 1-chloro-2,4-dinitrobenzene in response to treatment with all four compounds evaluated. Collectively, these studies demonstrate that EA and certain alpha,beta-unsaturated aldehydes produced as a result of cellular membrane lipid peroxidation are activators of the ARE and efficient inducers of GST A1-1 and A4-4.

Topics: Acrolein; Aldehydes; Animals; Antioxidants; Blotting, Northern; Blotting, Western; Carcinoma, Hepatocellular; Cross-Linking Reagents; Enzyme Activation; Ethacrynic Acid; Glutathione Transferase; Rats; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Transfection; Tumor Cells, Cultured

Hep G2 cells do not synthesize heat-shock proteins when incubated with ADP-iron, under conditions that can trigger lipoperoxidation. However, exposure of these cells to added 4-hydroxynonenal (HNE), one of the main products of lipoperoxidation, induces the synthesis of hsp70, the most conserved among heat-shock proteins. HNE acts mainly on transcription: in Hep G2 cells the increase in the steady-state level of hsp70 mRNA is detectable by two different hybridization techniques.

Topics: Adenosine Diphosphate; Aldehydes; Animals; Blotting, Northern; Carcinoma, Hepatocellular; DNA Probes; Electrophoresis; Gene Expression; Heat-Shock Proteins; Humans; Immunoblotting; Iron Chelating Agents; Liver Neoplasms; Liver Neoplasms, Experimental; Neoplasm Proteins; Nucleic Acid Hybridization; Rats; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured