Overexpression of Manganese Superoxide Dismutase Prevents Alcohol-induced Liver Injury in the Rat

Mitochondria are thought to play a major role in hepatic oxidative stress associated with alcohol-induced liver injury. Thus, the hypothesis that delivery of the mitochondrial isoform of superoxide dismutase (Mn-SOD) via recombinant adenovirus would reduce alcohol-induced liver injury was tested. Rats were given recombinant adenovirus containing Mn-SOD (Ad.SOD2) or beta-galactosidase (Ad.lacZ) and then fed alcohol enterally for 4 weeks. Mn-SOD expression and activity of Ad.SOD2 in liver mitochondria of infected animals was increased nearly 3-fold compared with Ad.lacZ-infected controls. Mitochondrial glutathione levels in Ad.lacZ-infected animals were decreased after 4 weeks of chronic ethanol, as expected, but were unchanged in Ad.SOD2-infected animals. Alanine aminotransferase was elevated significantly by ethanol, an effect that was prevented by Ad.SOD2. Moreover, pathology (e.g. the sum of steatosis, inflammation, and necrosis) was elevated dramatically by ethanol in Ad.lacZ-treated rats. This effect was also blunted in animals infected with Ad.SOD2. Neutrophil infiltration was increased about 3-fold in livers from both Ad.lacZ- and Ad.SOD2-infected rats by ethanol treatment. Moreover, ESR-detectable free radical adducts in bile were increased about 8-fold by ethanol. Using (13)C-labeled ethanol, it was determined that nearly 60% of total adducts were due to the alpha-hydroxyethyl radical adduct. This increase in radical formation was blocked completely by Ad.SOD2 infection. Furthermore, apoptosis of hepatocytes was increased about 5-fold by ethanol, an effect also blocked by Ad.SOD2. Interestingly, tumor necrosis factor-alpha mRNA was elevated to the same extent in both Ad.lacZ- and Ad.SOD2-infected animals follows ethanol exposure. These data suggest that hepatocyte mitochondrial oxidative stress is involved in alcohol-induced liver damage and likely follows Kupffer cell activation, cytokine production, and neutrophil infiltration. These results also support the hypothesis that mitochondrial oxidant production is a critical factor in parenchymal cell death caused by alcohol

Upregulation of MIR21 and Repression of GRHL3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4-5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH

Sustained formation of POBN radical adducts in mouse liver by peroxisome proliferators is dependent upon PPARα, but not NADPH oxidase

Reactive oxygen species are thought to be crucial for peroxisome proliferator-induced liver carcinogenesis. Free radicals have been shown to mediate the production of mitogenic cytokines by Kupffer cells and cause DNA damage in rodent liver. Previous in vivo experiments demonstrated that acute administration of the peroxisome proliferator di-(2-ethylhexyl) phthalate (DEHP) led to an increase in production of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) radical adducts in liver, an event that was dependent on Kupffer cell NADPH oxidase, but not peroxisome proliferator activated receptor (PPAR)α. Here, we hypothesized that continuous treatment with peroxisome proliferators will cause a sustained formation in POBN radical adducts in liver. Mice were fed diets containing either 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643, 0.05% w/w), or DEHP (0.6% w/w) for up to three weeks. Liver-derived radical production was assessed in bile samples by measuring POBN-radical adducts using electron spin resonance. Our data indicate that WY-14,643 causes a sustained increase in POBN radical adducts in mouse liver and that this effect is greater than that of DEHP. To understand the molecular source of these radical species, NADPH oxidase-deficient (p47 phox-null) and PPARα-null mice were examined after treatment with WY-14,643. No increase in radicals was observed in PPARα-null mice that were treated with WY-14,643 for 3 weeks, while the response in p47 phox-nulls was similar to that of wild-type mice. These results show that PPARα, not NADPH oxidase, is critical for a sustained increase in POBN radical production caused by peroxisome proliferators in rodent liver. Therefore, peroxisome proliferator-induced POBN radical production in Kupffer cells may be limited to an acute response to these compounds in mouse liver

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

none6siCeruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification.mixedGanini D.; Canistro D.; Jang J.; Stadler K.; Mason R.P.; Kadiiska M.B.Ganini D.; Canistro D.; Jang J.; Stadler K.; Mason R.P.; Kadiiska M.B