3-nitrotyrosine and Liver-Diseases

The analgesic acetaminophen causes a potentially fatal, hepatic centrilobular necrosis when taken in overdose. The initial phases of toxicity were described in Dr. Gillette's laboratory in the 1970s. These findings indicated that acetaminophen was metabolically activated by cytochrome P450 enzymes to a reactive metabolite that depleted glutathione (GSH) and covalently bound to protein. It was shown that repletion of GSH prevented the toxicity. This finding led to the development of the currently used antidote N-acetylcysteine. The reactive metabolite was subsequently identified to be N-acetyl-p-benzoquinone imine (NAPQI). Although covalent binding has been shown to be an excellent correlate of toxicity, a number of other events have been shown to occur and are likely important in the initiation and repair of toxicity. Recent data have shown that nitrated tyrosine residues as well as acetaminophen adducts occur in the necrotic cells following toxic doses of acetaminophen. Nitrotyrosine was postulated to be mediated by peroxynitrite, a reactive nitrogen species formed by the very rapid reaction of superoxide and nitric oxide (NO). Peroxynitrite is normally detoxified by GSH, which is depleted in acetaminophen toxicity. NO synthesis (serum nitrate plus nitrite) was dramatically increased following acetaminophen. In inducible nitric oxide synthase (iNOS) knockout mice, acetaminophen did not increase NO synthesis or tyrosine nitration; however, histological evidence indicated no difference in toxicity. Acetaminophen did not cause hepatic lipid peroxidation in wild-type mice but did cause lipid peroxidation in iNOS knockout mice. These data suggest that NO may play a role in controlling lipid peroxidation and that reactive nitrogen/oxygen species may be important in toxicity. The source of the superoxide has not been identified, but our recent finding that NADPH oxidase knockout mice were equally sensitive to acetaminophen and had equal nitration of tyrosine suggests that the superoxide is not from the activation of Kupffer cells. It was postulated that NAPQI-mediated mitochondrial injury may be the source of the superoxide. In addition, the significance of cytokines and chemokines in the development of toxicity and repair processes has been demonstrated by several recent studies. IL-1beta is increased early in acetaminophen toxicity and may be important in iNOS induction. Other cytokines, such as IL-10, macrophage inhibitory protein-2 (MIP-2), and monocyte che

Topics: Acetaminophen; Acetylcysteine; Analgesics, Non-Narcotic; Antidotes; Chemical and Drug Induced Liver Injury; Cytokines; Humans; Kupffer Cells; Liver Diseases; Mitochondria, Liver; Oxidative Stress; Tyrosine

To estimate oxidative stress and antioxidant components during different stages of autoimmune liver diseases and assess their possible implication on disease progression.. We determined several markers of oxidative injury (isoprostane, aldehydes, protein carbonyls, 3-nitrotyrosine, and myeloperoxidase) and antioxidant components (glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase) in whole blood, serum, and urine in 49 patients with autoimmune cholestatic liver diseases (AC) and 36 patients with autoimmune hepatitis (AIH) and healthy subjects matched for sex and age.. Both AC and AIH patients had increased levels of all lipid and protein oxidative injury products and significantly decreased whole blood glutathione levels compared to controls. AIH patients had significantly higher levels of aldehydes and glutathione peroxidase activity and significantly lower protein carbonyl levels compared to AC patients. Protein carbonyl and isoprostane levels increased and glutathione levels decreased gradually with progression from mild fibrosis to severe fibrosis and cirrhosis in both AC and AIH patients. In addition, both cirrhotic AC and AIH patients had significantly higher protein carbonyls compared to non-cirrhotics.. We provide novel findings in support of a major contribution of oxidant/antioxidant imbalance in the progression of liver injury in AC and AIH.

Topics: Adult; Aged; Aldehydes; Antioxidants; Autoimmune Diseases; Carbon; Catalase; Disease Progression; Female; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Hepatitis, Autoimmune; Humans; Isoprostanes; Liver Diseases; Male; Middle Aged; Oxidative Stress; Peroxidase; Superoxide Dismutase; Tyrosine

This study investigated the effect of dietary fish oil on systemic inflammation and hepatic injury in mice with polymicrobial sepsis. Male ICR mice were assigned to a control group (C, n=30) and a fish oil group (FO, n=30). Mice in the C group were fed a semi-purified diet with 10% soybean oil, and those in the FO group were fed a fish oil diet (2.5% fish oil+7.5% soybean oil; w/w). Three weeks later, sepsis was induced by cecal ligation and puncture (CLP), and mice were sacrificed at 0, 6 and 24 h after CLP, respectively. Results showed that compared with C group, the FO group had lower plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and nitrite at 6 and 24 h after CLP. Also, peritoneal lavage fluid concentrations of TNF-α and prostaglandin (PG) E2 were significantly lower at 24 h in the FO than in the C group. The FO group had lower myeloperoxidase activities at 6 h after CLP in various organs. Plasma aminotransferase and alanine aminotransferase activities revealed significantly decreased in the FO group. The DNA-binding activity of peroxisome proliferators-activated receptor gamma (PPARγ) and mRNA expression of I kappaB alpha (IκBα) were up-regulated while nuclear factor (NF)-κB p65 DNA-binding activity, inducible nitric oxide synthase protein expression and the concentration of nitrotyrosine were significantly decreased in the FO group in liver after CLP. These results indicate that dietary fish oil administration may attenuate systemic inflammation and up-regulate hepatic PPARγ DNA-binding activity, which may consequently have ameliorated liver injury in these septic mice.

Topics: Animals; Biomarkers; Fatty Acids, Omega-3; Fish Oils; Inflammation; Interleukin-10; Interleukin-6; Liver; Liver Diseases; Male; Mice; Mice, Inbred ICR; NF-kappa B; Nitric Oxide Synthase Type II; Peroxidase; PPAR gamma; Sepsis; Tumor Necrosis Factor-alpha; Tyrosine; Up-Regulation

Hydrogen sulfide (H2S) is a naturally occurring gaseous transmitter, which may play important roles in normal physiology and disease. Here, we investigated the effect of endogenously formed H2S in the endotoxemic organ injury.. Male Wistar rats were subjected to acute endotoxemia [Escherichia coli lipopolysaccharide (LPS) 20 mg kg(-1), intraperitoneally (ip)]. A group of animals was injected d,l-propargylglycine (PAG, 50 mg kg(-1), ip), an inhibitor of the H2S-synthesizing enzyme cystathionine-γ-lyase (CSE), 60 min before LPS administration. Six hours after the LPS treatment, animals were sacrificed. Myeloperoxidase (MPO), dimethylarginine dimethylaminohydrolase (DDAH) activities and levels of nitrotyrosine and GSH were measured in the liver. Asymmetric dimethylarginine (ADMA) and arginine levels in both liver and plasma were determined using HPLC.. LPS injections caused liver injury, as evidenced by the activities of serum aspartate aminotransferase and arginase. After LPS injections, increased arginine content and arginine/ADMA ratio were observed in the liver, together with significant decrements in both DDAH activity and GSH levels. Despite the accumulation of ADMA in the plasma, its level remained unchanged in the liver. PAG pretreatment aggravated the LPS-induced increase in the activities of MPO and serum enzymes. The most profound effect of PAG pretreatment was observed in nitrotyrosine levels in the liver, which were increased significantly as compared with the control and LPS-injected groups.. These findings support the view that the suppression of nitrosative stress by endogenous H2S is one of the mechanisms to protect liver against endotoxemic injury.

Topics: Alkynes; Amidohydrolases; Animals; Arginine; Aspartate Aminotransferases; Chromatography, High Pressure Liquid; Cystathionine gamma-Lyase; Endotoxemia; Glutathione; Glycine; Hydrogen Sulfide; Lipopolysaccharides; Liver Diseases; Male; Peroxidase; Rats; Rats, Wistar; Stress, Physiological; Tyrosine

Total parenteral nutrition (TPN) is an artificial way to support daily nutritional requirements by bypassing the digestive system, but long-term TPN administration may cause severe liver dysfunction. Glycyrrhizin is an active component of licorice root that has been widely used to treat chronic hepatitis. The aim of this study is to investigate the hepatoprotective effect of glycyrrhizin on TPN-associated acute liver injury in vivo. Liver dysfunction was induced by intravenous infusion of TPN at a flow rate of 20 mL/kg/h for three h in Sprague Dawley rats. The rats were pretreated with Glycyrrhizin (1, 3 and 10 mg/kg intravenously). After receiving TPN or saline (control group) for three h, the rats were sacrificed, blood samples were collected for biochemical analyses and liver tissue was removed for histopathological and immunohistochemical examination. We found that aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB) and triglyceride (TG) levels were significantly increased in the TPN group without glycyrrhizin pretreatment and decreased in the glycyrrhizin-pretreated TPN group in a dose-dependent manner. The stained liver sections showed that glycyrrhizin relieved acute liver injury. The upregulation of serum protein biomarkers of reactive nitrogen species, including nitrotyrosine and inducible NO synthase (iNOS), were attenuated by glycyrrhizin pretreatment. Levels of endoplasmic reticulum (ER) stress factors, such as phosphorylation of JNK1/2, p38 MAPK and CHOP, were decreased by glycyrrhizin pretreatment. In summary, our results suggest that glycyrrhizin decreases TPN-associated acute liver injury factors by suppressing endoplasmic reticulum stress and reactive nitrogen stress.

Topics: Animals; Apoptosis; Bilirubin; Caspase 3; Cytokines; Endoplasmic Reticulum Stress; Glycyrrhizic Acid; Immunohistochemistry; Inflammation; Inflammation Mediators; JNK Mitogen-Activated Protein Kinases; Liver; Liver Diseases; Male; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Parenteral Nutrition, Total; Phosphorylation; Rats, Sprague-Dawley; Reactive Nitrogen Species; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Transcription Factor CHOP; Triglycerides; Tyrosine

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced during hemorrhagic shock and resuscitation (H/R), which may contribute to multiple organ failure. The Aim of this study was to test the hypothesis that green tea (Camellia sinenesis) extract containing 85% polyphenols decreases injury after H/R in rats by scavenging ROS and RNS.. Female Sprague Dawley rats were given 100 mg polyphenol extract/kg body weight or vehicle 2 h prior to hemorrhagic shock. H/R was induced by two protocols: 1) withdrawal of blood to a mean arterial pressure of 40 mm Hg followed by further withdrawals to decrease blood pressure progressively to 28 mm Hg over 1 h (severe), and 2) withdrawal of blood to a sustained hypotension of 40 mm Hg for 1 h (moderate). Rats were then resuscitated over 1 h with 60% of the shed blood volume plus twice the shed blood volume of lactated Ringer's solution. Serum samples were collected at 10 min and 2 h after resuscitation. At 2 or 18 h, livers were harvested for cytokine and 3-nitrotyrosine quantification, immunohistochemical detection of 4-hydroxynonenol (4-HNE) and inducible nitric oxide synthase (iNOS) protein expression.. After severe H/R, 18-h survival increased from 20% after vehicle to 70% after polyphenols (p < 0.05). After moderate H/R, survival was greater (80%) and not different between vehicle and polyphenols. In moderate H/R, serum alanine aminotransferase (ALT) increased at 10 min and 2 h postresuscitation to 345 and 545 IU/L, respectively. Polyphenol treatment blunted this increase to 153 and 252 IU/L at 10 min and 2 h (p < 0.01). Polyphenols also blunted increases in liver homogenates of TNFalpha (7.0 pg/mg with vehicle vs. 4.9 pg/mg with polyphenols, p < 0.05), IL-1beta (0.80 vs. 0.37 pg/mg, p < 0.05), IL-6 (6.9 vs. 5.1 pg/mg, p < 0.05) and nitrotyrosine (1.9 pg/mg vs. 0.6 pg/mg, p < 0.05) measured 18 h after H/R. Hepatic 4-HNE immunostaining indicative of lipid peroxidation also decreased from 4.8% after vehicle to 1.5% after polyphenols (p < 0.05). By contrast, polyphenols did not block increased iNOS expression at 2 h after H/R.. Polyphenols decrease ROS/RNS formation and are beneficial after hemorrhagic shock and resuscitation.

Topics: Alanine Transaminase; Animals; Blood Transfusion; Camellia sinensis; Cytokines; Female; Flavonoids; Lipid Peroxidation; Liver; Liver Diseases; Nitric Oxide Synthase Type II; Phenols; Phytotherapy; Plant Extracts; Polyphenols; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Reactive Oxygen Species; Resuscitation; Shock, Hemorrhagic; Tyrosine

The aim of this study was to determine the effect of bicyclol against cisplatin-induced hepatotoxicity and the influence on the antitumour capacity of cisplatin in hepatocarcinoma 22 (H22) tumour-bearing mice. ICR mice were treated with bicyclol (250 mg/kg, orally) 2 hr before the injection of cisplatin (5 mg/kg, intraperitoneally) for 5 days (once daily) after H22 tumour cells were implanted. All animals were killed on the fifth day after cisplatin treatment and tumour weight of each animal was measured. Liver pathological changes were examined by light microscopy and biochemical assay. The expressions of liver inducible nitric oxide synthase (iNOS and nitric oxide synthase 2) and 3-nitrotyrosine were assessed by Western blotting. Bicyclol showed a significant protection as evidenced by the decrease of elevated serum aminotransferases and lactate dehydrogenase, and improvement of histopathological injury induced by cisplatin. The formation of liver malondialdehyde with a concomitant reduction of reduced glutathione was also inhibited by bicyclol, while the activities of liver superoxide dismutase, catalase and glutathione peroxidase were all increased, respectively. In addition, the over expressions of liver iNOS and 3-nitrotyrosine were suppressed by bicyclol. The administration of bicyclol had no affect on the anti-tumour capacity of cisplatin in mice bearing H22 tumour. The hepatoprotective action of bicyclol provides a new approach for preventing the hepatotoxicity induced by cisplatin in the clinic.

Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Cisplatin; Drug Interactions; Gene Expression Regulation; L-Lactate Dehydrogenase; Liver; Liver Diseases; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred ICR; Nitric Oxide Synthase Type II; Transaminases; Tyrosine

Although trauma-hemorrhage produces tissue hypoxia, systemic inflammatory response and organ dysfunction, the mechanisms responsible for these alterations are not clear. Using a potent selective inducible nitric oxide (NO) synthase inhibitor, N-[3-(aminomethyl) benzyl]acetamidine (1400W), and a nonselective NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), we investigated whether inducible NO synthase plays any role in producing hepatic injury, inflammation, and changes of protein expression following trauma-hemorrhage. To investigate this, male Sprague-Dawley rats were subjected to midline laparotomy and hemorrhagic shock (mean blood pressure 35-40 mmHg for approximately 90 min) followed by fluid resuscitation. Animals were treated with either vehicle (DMSO) or 1400W (10 mg/kg body wt ip), or L-NAME (30 mg/kg iv), 30 min before resuscitation and killed 2 h after resuscitation. Trauma-hemorrhage/resuscitation induced a marked hypotension and increase in markers of hepatic injury (i.e., plasma alpha-glutathione S-transferase, tissue myeloperoxidase activity, and nitrotyrosine formation). Hepatic expression of iNOS, hypoxia-inducible factor-1alpha, ICAM-1, IL-6, TNF-alpha, and neutrophil chemoattractant (cytokine-induced neutrophil chemoattractant-1 and macrophage inflammatory protein-2) protein levels were also markedly increased following trauma-hemorrhage/resuscitation. Administration of the iNOS inhibitor 1400W significantly attenuated hypotension and expression of these mediators of hepatic injury induced by trauma-hemorrhage/resuscitation. However, administration of L-NAME could not attenuate hepatic dysfunction and tissue injury mediated by trauma-hemorrhage, although it improved mean blood pressure as did 1400W. These results indicate that increased expression of iNOS following trauma-hemorrhage plays an important role in the induction of hepatic damage under such conditions.

Topics: Amidines; Animals; Benzylamines; Blood Pressure; Chemokines; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Fluid Therapy; Glutathione Transferase; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Adhesion Molecule-1; Isoenzymes; Liver; Liver Diseases; Male; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Peroxidase; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic; Tyrosine; Wounds and Injuries

Reactive oxygen and nitrogen species have been implicated in ischemia-reperfusion (I/R) injury. Metalloporphyrins (MP) are stable catalytic antioxidants that can scavenge superoxide, hydrogen peroxide, peroxynitrite and lipid peroxyl radicals. Studies were conducted with three manganese-porphyrin (MnP) complexes with varying superoxide dimutase (SOD) and catalase catalytic activity to determine if the MnP attenuates I/R injury in isolated perfused mouse livers. The release of the hepatocellular enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) was maximal at 1 min reperfusion, decreased rapidly and increased gradually by 90 min. Manganese tetrakis-(N-ethyl-2 pyridyl) porphyrin (MnTE-2-PyP) decreased ALT, AST, LDH at 1-90 min reperfusion, while manganese tetrakis-(N-methyl-2 pyridyl) porphyrin (MnTM-2-PyP) and manganese tetrakis-(ethoxycarbonyl) porphyrin (MnTECP) decreased ALT and LDH from 5 to 90 min reperfusion. The release of thiobarbituric acid-reacting substances (TBARS) was diminished by MnTE-2-PyP and MnTM-2-PyP at 90 min. The extent of protein nitration (nitrotyrosine, NT) was decreased in all three MnPs treated livers. These results demonstrate that MnP complexes can attenuate hepatic I/R injury and may have therapeutic implications in disease states involving oxidants.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Ischemia; L-Lactate Dehydrogenase; Lipid Peroxidation; Liver; Liver Diseases; Male; Metalloporphyrins; Mice; Mice, Inbred C57BL; Nitrosation; Reactive Oxygen Species; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Tyrosine

Cytochrome P450 2E1 (CYP2E1) activates several hepatotoxins and contributes to alcoholic liver damage. Obesity is a growing health problem in the United States. The aim of the present study was to evaluate whether acetone- or pyrazole-mediated induction of CYP2E1 can potentiate liver injury in obesity. CYP2E1 protein and activity were elevated in acetone- or pyrazole-treated obese and lean mice. Acetone or pyrazole induced distinct histological changes in liver and significantly higher aminotransferase enzymes in obese mice compared to obese controls or acetone- or pyrazole-treated lean mice. Higher caspase-3 activity and numerous apoptotic hepatocytes were observed in the acetone- or pyrazole-treated obese mice. Increased protein carbonyls, malondialdehyde, 4-hydroxynonenal-protein adducts, elevated levels of inducible nitric oxide synthase, and higher 3-nitrotyrosine protein adducts were found in livers of acetone- or pyrazole-treated obese animals, suggesting elevated oxidative and nitrosative stress. Liver tumor necrosis factor alpha levels were higher in pyrazole-treated animals. The CYP2E1 inhibitor chlormethiazole and iNOS inhibitor N-(3-(aminomethyl)-benzyl) acetamidine abrogated the toxicity and the oxidative/nitrosative stress elicited by the induction of CYP2E1.. These results show that obesity contributes to oxidative stress and liver injury and that induction of CYP2E1 enhances these effects.

Topics: Acetone; Animals; Apoptosis; Caspase 3; Catalysis; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP2E1; DNA Adducts; DNA Fragmentation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Lipid Peroxidation; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Nitric Oxide Synthase Type II; Obesity; Proteins; Pyrazoles; RNA, Messenger; Solvents; Tumor Necrosis Factor-alpha; Tyrosine

In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion.. Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide).. Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia.. During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.

Topics: Animals; Blotting, Western; Disease Models, Animal; Fluorescent Antibody Technique; Ischemia; Liver; Liver Diseases; Liver Function Tests; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Nitrosative stress resulting from increased nitric oxide (NO) synthesis contributes to the pathogenesis of chronic inflammatory diseases, including chronic viral hepatitis. Our goal was to assess the expression of inducible nitric oxide synthase (iNOS) and the formation of nitrotyrosine (NTY), as a marker of nitrosative stress, in liver biopsies from primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) patients.. Intrahepatic expression of iNOS and NTY was measured immunohistochemically and compared to histological scores of the severity of liver disease.. Hepatocellular iNOS expression was observed in liver sections from PBC patients (with a diffuse lobular distribution) and from AIH patients (marked staining in areas of pronounced inflammation and necrosis), but not in control liver sections, including non-autoimmune cholestatic liver disease. Liver samples from PBC and AIH patients, but not from controls, showed NTY accumulation in clusters of hepatocytes and Kupffer cells. Increased iNOS expression and NTY accumulation correlated with the histological severity of PBC or AIH, especially with the degree of inflammation.. Patients with PBC and AIH showed an enhanced intrahepatic iNOS expression and NTY accumulation, related to the histological severity of liver disease, consistent with NO-mediated nitration of hepatocellular proteins contributing to liver damage in both diseases.

Topics: Adult; Chronic Disease; Female; Hepatitis, Autoimmune; Humans; Liver; Liver Cirrhosis, Biliary; Liver Diseases; Male; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Severity of Illness Index; Tyrosine