3-nitrotyrosine and Liver-Diseases--Alcoholic

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the L-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass(+/-) mice (Ass(-/-) are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass(+/-) mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress by way of the L-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol-treated Ass(+/-) mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase alpha (pAMPKα) to total AMPKα ratio, decreased sirtuin-1 (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1α (Pgc1α) messenger RNAs (mRNAs), lower fatty acid β-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense, and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3.. Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β-oxidation are key events.

Topics: Acute Disease; Alcohol Drinking; Alcoholism; Animals; Argininosuccinate Synthase; Chronic Disease; Cytochrome P-450 CYP2E1; Disease Models, Animal; Down-Regulation; Ethanol; Female; Hepatocytes; Immunohistochemistry; Lipid Peroxidation; Liver Diseases, Alcoholic; Male; Mice; Nitric Oxide; Oxidative Stress; Random Allocation; Rats; Tyrosine

Oxidative stress is increasingly suspected to contribute to the initiation and progression of many disease, including those caused by alcohol exposure. Two major products of reactive oxygen and nitrogen species formation are 4OH-nonenal and 3-nitrotyrosine protein adducts, both of which can be detected by immunohistochemistry. In the past, immunohistochemical techniques have served largely as qualitative measures of changes. However, coupled with digital capture and analysis of photomicrographs, one can now quantitate treatment-related changes with immunohistochemistry. This chapter summarizes techniques for immunohistochemical detection of these products of reactive oxygen and nitrogen species and subsequent image-analysis. Although the methods described herein are based on liver, these techniques have been employed successfully in most tissue types with minor modifications and are therefore broadly applicable.

Topics: Aldehydes; Animals; Biomarkers; Disease Models, Animal; Immunohistochemistry; Lipopolysaccharides; Liver; Liver Diseases, Alcoholic; Mice; Mice, Transgenic; Oxidative Stress; Photomicrography; Proteins; Rats; Reactive Nitrogen Species; Reactive Oxygen Species; Reproducibility of Results; Signal Processing, Computer-Assisted; Specimen Handling; Tyrosine

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.

Topics: Aldehydes; Animals; Antioxidants; Blotting, Western; Buthionine Sulfoximine; Catalysis; Cell Line; Cell Survival; Cyclosporine; Cytochrome P-450 CYP2E1; DNA, Complementary; Endoplasmic Reticulum; Ethanol; Flow Cytometry; Glutathione; Humans; Liver; Liver Diseases, Alcoholic; Male; Membrane Potentials; Microscopy, Confocal; Mitochondria; Mitochondria, Liver; Oxidative Stress; Plasmids; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Time Factors; Transfection; Tyrosine