3-nitrotyrosine and Choline-Deficiency

To investigate the effectiveness of antioxidant compounds in modulating mitochondrial oxidative alterations and lipids accumulation in fatty hepatocytes.. Silybin-phospholipid complex containing vitamin E (Realsil(®)) was daily administered by gavage (one pouch diluted in 3 mL of water and containing 15 mg vitamin E and 47 mg silybin complexed with phospholipids) to rats fed a choline-deprived (CD) or a high fat diet [20% fat, containing 71% total calories as fat, 11% as carbohydrate, and 18% as protein, high fat diet (HFD)] for 30 d and 60 d, respectively. The control group was fed a normal semi-purified diet containing adequate levels of choline (35% total calories as fat, 47% as carbohydrate, and 18% as protein). Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, glutathione peroxidase), NO metabolites (nitrosothiols, nitrotyrosine), lipid peroxides [malondialdehyde-thiobarbituric (MDA-TBA)], and pro-inflammatory keratins (K-18) were measured on days 0, 7, 14, 30, and 60. Mitochondrial respiratory chain proteins and the extent of hepatic fatty infiltration were evaluated.. Both diet regimens produced liver steatosis (50% and 25% of liver slices with CD and HFD, respectively) with no signs of necro-inflammation: fat infiltration ranged from large droplets at day 14 to disseminated and confluent vacuoles resulting in microvesicular steatosis at day 30 (CD) and day 60 (HFD). In plasma, thioredoxin and nitrosothiols were not significantly changed, while MDA-TBA, nitrotyrosine (from 6 ± 1 nmol/L to 14 ± 3 nmol/L day 30 CD, P < 0.001, and 12 ± 2 nmol/L day 60 HFD, P < 0.001), and K-18 (from 198 ± 20 to 289 ± 21 U/L day 30 CD, P < 0.001, and 242 ± 23 U/L day 60 HFD, P < 0.001) levels increased significantly with ongoing steatosis. In the liver, glutathione was decreased (from 34.0 ± 1.3 to 25.3 ± 1.2 nmol/mg prot day 30 CD, P < 0.001, and 22.4 ± 2.4 nmol/mg prot day 60 HFD, P < 0.001), while thioredoxin and glutathione peroxidase were initially increased and then decreased. Nitrosothiols were constantly increased. MDA-TBA levels were five-fold increased from 9.1 ± 1.2 nmol/g to 75.6 ± 5.4 nmol/g on day 30, P < 0.001 (CD) and doubled with HFD on day 60. Realsil administration significantly lowered the extent of fat infiltration, maintained liver glutathione levels during the first half period, and halved its decrease during the second half. Also, Realsil modulated thioredoxin changes and the production of NO derivatives and significantly lowered MDA-TBA levels both in liver (from 73.6 ± 5.4 to 57.2 ± 6.3 nmol/g day 30 CD, P < 0.01 and from 27.3 ± 2.1 nmol/g to 20.5 ± 2.2 nmol/g day 60 HFD, P < 0.01) and in plasma. Changes in mitochondrial respiratory complexes were also attenuated by Realsil in HFD rats with a major protective effect on Complex II subunit CII-30.. Realsil administration effectively contrasts hepatocyte fat deposition, NO derivatives formation, and mitochondrial alterations, allowing the liver to maintain a better glutathione and thioredoxin antioxidant activity.

Topics: Animals; Biomarkers; Choline Deficiency; Diet, High-Fat; Disease Models, Animal; Fatty Liver; Glutathione; Glutathione Peroxidase; Keratin-18; Liver; Male; Malondialdehyde; Mitochondria, Liver; Mitochondrial Proteins; Nitroso Compounds; Oxidative Phosphorylation; Oxidative Stress; Phospholipids; Rats; Rats, Wistar; Silybin; Silymarin; Sulfhydryl Compounds; Thiobarbituric Acid Reactive Substances; Thioredoxins; Time Factors; Tyrosine; Vitamin E

The pathogenesis of nonalcoholic steatohepatitis (NASH) is still unclear. Recently, the 2-hit hypothesis was proposed, in which nitric oxide production, representing oxidative stress, was proposed as a very important candidate for the second hit.. The total study period was 10 weeks. A total of 20 rats were randomly divided into 2 groups. Group 1 was administered the Choline-Deficient, l-Amino Acid-Defined diet to produce a NASH model, and Group 2 as control received the Choline-Sufficient, l-Amino Acid-defined diet. The blood and tissue concentrations of nitrate + nitrite were measured using the Griess reagent and the expression levels of inducible nitric oxide synthase (iNOS) proteins and mRNA was determined by Western blotting.. In regard to nitric oxide (NO) and NO metabolites, there were significant differences in the blood (especially portal venous blood) as well as tissue (liver and visceral fat) concentrations between the 2 animal groups; the amounts of NO metabolites in the tissues were much higher in the NASH models. The level of nitrotyrosine was much markedly higher in the NASH models than in the controls. In regard to the tissue expression of iNOS a significant difference between the 2 groups was found in the visceral fat, especially in the mesenterium.. Based on these results, we hypothesize that the iNOS expression and NO levels in the visceral fat increase, with increased diffusion of NO and its metabolites into the liver, resulting in increased nitrotyrosine formation in the liver; this, in turn, induces inflammation, apoptosis, and fibrosis in the liver, which are one of the characteristic features of NASH.

Topics: Amino Acids; Animals; Blotting, Western; Choline; Choline Deficiency; Diet; Disease Models, Animal; Fatty Liver; Intra-Abdominal Fat; Liver; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Rats; Rats, Inbred F344; RNA, Messenger; Tyrosine