Saurabh Chatterjee

Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis

BACKGROUND & AIMS Progression from steatosis to steatohepatitic lesions is hypothesized to require a second hit. These lesions have been associated with increased oxidative stress, often ascribed to high levels of leptin and other proinflammatory mediators. Here we have examined the role of leptin in inducing oxidative stress and Kupffer cell activation in CCl4-mediated steatohepatitic lesions of obese mice. METHODS Male C57BL/6 mice fed with a high-fat diet (60%kcal) at 16 weeks were administered CCl₄ to induce steatohepatitic lesions. Approaches included use of immuno-spin trapping for measuring free radical stress, gene-deficient mice for leptin, p47 phox, iNOS and adoptive transfer of leptin primed macrophages in vivo. RESULTS Diet-induced obese (DIO) mice, treated with CCl4 increased serum leptin levels. Oxidative stress was significantly elevated in the DIO mouse liver, but not in ob/ob mice, or in DIO mice treated with leptin antibody. In ob/ob mice, leptin supplementation restored markers of free radical generation. Markers of free radical formation were significantly decreased by the peroxynitrite decomposition catalyst FeTPPS, the iNOS inhibitor 1400W, the NADPH oxidase inhibitor apocynin, or in iNOS or p47 phox-deficient mice. These results correlated with the decreased expression of TNF-alpha and MCP-1. Kupffer cell depletion eliminated oxidative stress and inflammation, whereas in macrophage-depleted mice, the adoptive transfer of leptin-primed macrophages significantly restored inflammation. CONCLUSIONS These results, for the first time, suggest that leptin action in macrophages of the steatotic liver, through induction of iNOS and NADPH oxidase, causes peroxynitrite-mediated oxidative stress thus activating Kupffer cells.

Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH.

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H(2)O(2) outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H(2)O(2) further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H(2)O(2) to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.

Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite

Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical (·SO3−). This free radical further reacts with oxygen to form peroxymonosulfate anion radical (−O3SOO·) and the very reactive sulfate anion radical (SO4̇̄), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H2O2 is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO4̇̄), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H2O2 in the presence of bisulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H2O2 induced the formation of radical-derived DMPO spin-trapped human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in bisulfite-exacerbated eosinophilic inflammatory disorders.

P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice.

While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.

Environmental toxin-linked nonalcoholic steatohepatitis and hepatic metabolic reprogramming in obese mice.

Obesity is associated with strong risks of development of chronic inflammatory liver disease and metabolic syndrome following a second hit. This study tests the hypothesis that free radical metabolism of low chronic exposure to bromodichloromethane (BDCM), a disinfection byproduct of drinking water, causes nonalcoholic steatohepatitis (NASH), mediated by cytochrome P450 isoform CYP2E1 and adipokine leptin. Using diet-induced obese mice (DIO), mice deficient in CYP2E1, and mice with spontaneous knockout of the leptin gene, we show that BDCM caused increased lipid peroxidation and increased tyrosine nitration in DIO mice, events dependent on reductive metabolism by CYP2E1. DIO mice, exposed to BDCM, exhibited increased hepatic leptin levels and higher levels of proinflammatory gene expression and Kupffer cell activation. Obese mice exposed to BDCM also showed profound hepatic necrosis, Mallory body formation, collagen deposition, and higher alpha smooth muscle actin expression, events that are hallmarks of NASH. The absence of CYP2E1 gene in mice that were fed with a high-fat diet did not show NASH symptoms and were also protected from hepatic metabolic alterations in Glut-1, Glut-4, phosphofructokinase and phosphoenolpyruvate carboxykinase gene expressions (involved in carbohydrate metabolism), and UCP-1, PGC-1α, SREBP-1c, and PPAR-γ genes (involved in hepatic fat metabolism). Mice lacking the leptin gene were significantly protected from both NASH and metabolic alterations following BDCM exposure, suggesting that higher levels of leptin induction by BDCM in the liver contribute to the development of NASH and metabolic alterations in obesity. These results provide novel insights into BDCM-induced NASH and hepatic metabolic reprogramming and show the regulation of obesity-linked susceptibility to NASH by environmental factors, CYP2E1, and leptin.

Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice

Post-translational modification of proteins due to exposure to radicals and other reactive species are markers of metabolic and inflammatory oxidative stress such as sepsis. This study uses the nitrone spin-trap DMPO and a combination of immuno-spin trapping and mass spectrometry to identify in vivo products of radical reactions in mice. We report the detection of dose-dependent production of DMPO-carboxypeptidase B1 (CPB1) adducts in the spleens of mice treated with lipopolysaccharide (LPS). Additionally, we report significant detection of DMPO-CPB1 adducts in mice experiencing normal physiological conditions. Treatments with inhibitors and experiments with knock-out mice indicate that xanthine oxidase and endothelial nitric oxide synthase are important sources of the reactive species that lead to CPB1 adduct formation. We also report a significant loss of CPB1 activity following LPS challenge in conjunction with an increase in CPB1 protein accumulation. This suggests the presence of a possible mechanism for CPB1 activity loss with compensatory protein production.

Site-specific radical formation in DNA induced by Cu(II)–H2O2 oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H₂O₂ oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.

Immuno-spin trapping from biochemistry to medicine: Advances, challenges, and pitfalls. Focus on protein-centered radicals

BACKGROUND Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Oxidative stress induces protein and DNA radical formation in follicular dendritic cells of the germinal center and modulates its cell death patterns in late sepsis

Profound depletion of follicular dendritic cells (FDCs) is a hallmark of sepsis-like syndrome, but the exact causes of the ensuing cell death are unknown. The cell death-driven depletion contributes to immunoparalysis and is responsible for most of the morbidity and mortality in sepsis. Here we have utilized immuno-spin trapping, a method for detection of free radical formation, to detect oxidative stress-induced protein and DNA radical adducts in FDCs isolated from the spleens of septic mice and from human tonsil-derived HK cells, a subtype of germinal center FDCs, to study their role in FDC depletion. At 24h post-lipopolysaccharide administration, protein radical formation and oxidation were significantly elevated in vivo and in HK cells as shown by ELISA and confocal microscopy. The xanthine oxidase inhibitor allopurinol and the iron chelator desferrioxamine significantly decreased the formation of protein radicals, suggesting the role of xanthine oxidase and Fenton-like chemistry in radical formation. Protein and DNA radical formation correlated mostly with apoptotic features at 24h and necrotic morphology of all the cell types studied at 48h with concomitant inhibition of caspase-3. The cytotoxicity of FDCs resulted in decreased CD45R/CD138-positive plasma cell numbers, indicating a possible defect in B cell differentiation. In one such mechanism, radical formation initiated by xanthine oxidase formed protein and DNA radicals, which may lead to cell death of germinal center FDCs.