Alessandro Arduini

Preterm Resuscitation With Low Oxygen Causes Less Oxidative Stress, Inflammation, and Chronic Lung Disease

OBJECTIVE: The goal was to reduce adverse pulmonary adverse outcomes, oxidative stress, and inflammation in neonates of 24 to 28 weeks of gestation initially resuscitated with fractions of inspired oxygen of 30% or 90%. METHODS: Randomized assignment to receive 30% (N = 37) or 90% (N = 41) oxygen was performed. Targeted oxygen saturation values were 75% at 5 minutes and 85% at 10 minutes. Blood oxidized glutathione (GSSG)/reduced glutathione ratio and urinary o-tyrosine, 8-oxo-dihydroxyguanosine, and isoprostane levels, isofuran elimination, and plasma interleukin 8 and tumor necrosis factor α levels were determined. RESULTS: The low-oxygen group needed fewer days of oxygen supplementation (6 vs 22 days; P < .01) and fewer days of mechanical ventilation (13 vs 27 days; P < .01) and had a lower incidence of bronchopulmonary dysplasia at discharge (15.4% vs 31.7%; P < .05). GSSG/reduced glutathione × 100 ratios at day 1 and 3 were significantly higher in the high-oxygen group (day 1: high-oxygen group: 13.36 ± 5.25; low-oxygen group: 8.46 ± 3.87; P < .01; day 3: high-oxygen group: 8.87 ± 4.40; low-oxygen group: 6.97 ± 3.11; P < .05). Urinary markers of oxidative stress were increased significantly in the high-oxygen group, compared with the low-oxygen group, in the first week after birth. GSSG levels on day 3 and urinary isofuran, o-tyrosine, and 8-hydroxy-2′-deoxyguanosine levels on day 7 were correlated significantly with development of chronic lung disease. CONCLUSIONS: Resuscitation of preterm neonates with 30% oxygen causes less oxidative stress, inflammation, need for oxygen, and risk of bronchopulmonary dysplasia.

Antenatal Steroids and Antioxidant Enzyme Activity in Preterm Infants: Influence of Gender and Timing

Antenatal steroids have improved the survival of preterm infants; however, the mechanism of action is not fully understood. We aimed to establish an association between antenatal steroids and antioxidant activity and postnatal oxidative stress. In a prospective cohort study, extremely preterm neonates receiving antenatal steroids (CORT) or not (NOCORT) were enrolled. An association between antenatal steroids and activities of antioxidant enzymes and glutathione cycle enzymes in cord blood was found. In addition, reduced oxidative stress (GSH/GSSG ratio, CORT vs. NOCORT, 35.68 + or - 12.20 vs. 28.38 + or - 9.92; p < 0.01) and, decreased oxidation of proteins (ortho-tyrosine/phenylalanine ratio, CORT vs. NOCORT, 8.66 + or - 2.45 vs. 12.55 + or - 4.41; p < 0.01) and DNA (8oxodG/2dG ratio, CORT vs. NOCORT, 6.73 + or - 2.18 vs. 9.53 + or - 3.83; p < 0.01) also was found. Antenatal steroids were associated with reduced oxygen supplementation, mechanical ventilation, and conditions such as bronchopulmonary dysplasia, intra-periventricular hemorrhage, or retinopathy of prematurity. The maximal effectiveness was when steroids were administered 2-4 days before delivery. Female preterm infants had less oxidative stress and increased antioxidant activity and better clinical outcomes than did male infants, independent of receiving or not antenatal steroids. Antenatal steroids are accompanied by a reduction in postnatal oxidative-stress-derived conditions and increased antioxidant enzyme activity. Both these effects seem to be influenced by specific timing and female gender.

Mitochondrial function in liver disease.

Oxidative stress is involved in the pathogenesis and progression of different liver diseases, such as alcoholic liver disease and biliary cirrhosis. The increased mitochondrial production of O2(-) at complexes I and III, and consequently of H2O2 and other reactive oxygen species (ROS), triggered by NADH overproduction seems the major cause of mitochondrial and cellular oxidative stress and damage in chronic alcoholism. The mitochondrial oxidative stress renders hepatocytes susceptible to ethanol- or acetaldehyde-induced mitochondrial membrane permeability transition (MMPT) and apoptosis. Nitrosative stress contributes to cell death by peroxynitrite formation. The expression of the death receptor ligand CD95 is also up-regulated by acetaldehyde metabolism. Consequently, a dual mechanism, NADH-driven MMPT and CD95-mediated apoptosis, involving in both cases acetaldehyde metabolism and ROS production, operates in ethanol-induced apoptosis. In the biliary cirrhosis induced by chronic cholestasis, liver mitochondria show increased H2O2 production and GSH depletion and oxidation. Dysfunctional hepatocytes, with a loss in mitochondrial cardiolipin and decreased mitochondrial membrane potential evolve during cholestasis to apoptosis. Ursodeoxycholic acid prevents enlargement of this population as well as mitochondrial oxidative stress. Mitochondrial oxidative stress precedes the initiation and execution of hepatocyte apoptosis in chronic alcoholism and biliary cirrhosis. We suggest that overproduction of mitochondrial NADH is the primary cause for the development of alcoholic and non-alcoholic liver disease by a situation of chronic mitochondrial oxidative stress, which should be considered the second hit that renders hepatocytes susceptible to cell injury and apoptosis.

Cross-Talk between Oxidative Stress and Pro-Inflammatory Cytokines in Acute Pancreatitis: A Key Role for Protein Phosphatases

Acute pancreatitis is an acute inflammatory process localized in the pancreatic gland that frequently involves peripancreatic tissues. It is still under investigation why an episode of acute pancreatitis remains mild affecting only the pancreas or progresses to a severe form leading to multiple organ failure and death. Proinflammatory cytokines and oxidative stress play a pivotal role in the early pathophysiological events of the disease. Cytokines such as interleukin 1beta and tumor necrosis factor alpha initiate and propagate almost all consequences of the systemic inflammatory response syndrome. On the other hand, depletion of pancreatic glutathione is an early hallmark of acute pancreatitis and reactive oxygen species are also associated with the inflammatory process. Changes in thiol homestasis and redox signaling decisively contribute to amplification of the inflammatory cascade through mitogen activated protein kinase (MAP kinase) pathways. This review focuses on the relationship between oxidative stress, pro-inflammatory cytokines and MAP kinase/protein phosphatase pathways as major modulators of the inflammatory response in acute pancreatitis. Redox sensitive signal transduction mediated by inactivation of protein phosphatases, particularly protein tyrosin phosphatases, is highlighted.

Amniotic Fluid Oxidative and Nitrosative Stress Biomarkers Correlate with Fetal Chronic Hypoxia in Diabetic Pregnancies

Background: In spite of improvement in obstetrical care, pregnancy in women with type 1 diabetes mellitus is associated with increased perinatal morbidity and mortality. Hyperglycemia during pregnancy causes excessive fetal growth and chronic fetal hypoxia as reflected in increased erythropoietin (EPO) levels in amniotic fluid (AF). Objectives: We hypothesized that the degree of fetal hypoxia would correlate with fetal oxidative and nitrosative stress as evidenced by the concentration of specific biomarkers in AF. Material and Methods: 19 pregnant women with type 1 or insulin-treated gestational diabetes mellitus were studied. AF samples were collected and processed for EPO, meta-tyrosine, nitro-tyrosine and 8-hydroxy-2-deoxiguanosine by chemiluminescent immunoassay and high-performance liquid chromatography coupled to tandem mass spectrometry methods, respectively. Results: The mean (SD) of the last HbA1c concentration before delivery was 7.7% (1.1). Median gestational age was 258 days (range 231–268). Birth weight was 3,868 ± 695 g with a z-score >2 SD in 47% of the cases. A significant correlation was found between the concentrations of AF EPO and meta-tyrosine/phenylalanine ratio (p < 0.001), nitro-tyrosine (p < 0.01) and 8-oxo-dG/2dG ratio (p < 0.001). Conclusions: We confirmed that fetuses of type 1 diabetes or insulin-treated gestational diabetes pregnancies experience chronic hypoxia as reflected by increased EPO concentrations in AF near term. Moreover, EPO levels significantly correlated with the concentration of oxidative and nitrosative stress biomarkers in AF. This pro-oxidant status may predispose newborn infants to poor postnatal adaptation and early neonatal complications.

Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age.

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n=40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions

Chronic cholestasis is characterized by mitochondrial dysfunction, associated with loss of mitochondrial membrane potential, decreased activities of respiratory chain complexes, and ATP production. Our aim was to determine the molecular mechanisms that link long-term cholestasis to mitochondrial dysfunction. We studied a model of chronic cholestasis induced by bile duct ligation in rats. Key sensors and regulators of the energetic state and mitochondrial biogenesis, mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio (mtDNA/nDNA) relative copy number, mtDNA deletions, and indexes of apoptosis (BAX, BCL-2, and cleaved caspase 3) and cell proliferation (PCNA) were evaluated. Our results show that long-term cholestasis is associated with absence of activation of key sensors of the energetic state, evidenced by decreased SIRT1 and pyruvate dehydrogenase kinase levels and lack of AMPK activation. Key mitochondrial biogenesis regulators (PGC-1α and GABP-α) decreased and NRF-1 was not transcriptionally active. Mitochondrial transcription factor A (TFAM) protein levels increased transiently in liver mitochondria at 2 wk after bile duct ligation, but they dramatically decreased at 4 wk. Reduced TFAM levels at this stage were mirrored by a marked decrease (65%) in mtDNA/nDNA relative copy number. The blockade of mitochondrial biogenesis should not be ascribed to activation of apoptosis or inhibition of cell proliferation. Impaired mitochondrial turnover and loss of the DNA stabilizing effect of TFAM are likely the causative event involved in the genetic instability evidenced by accumulation of mtDNA deletions. In conclusion, the lack of stimulation of mitochondrial biogenesis leads to mtDNA severe depletion and deletions in long-term cholestasis. Hence, long-term cholestasis should be considered a secondary mitochondrial hepatopathy.

Mitochondrial dysfunction in cholestatic liver diseases

Cholestatic liver diseases are characterized by blockade of bile flow from the liver to the intestine, and accumulation of hydrophobic bile acids in the liver and plasma. As a consequence an inflammatory response evolves associated with increased apoptosis, oxidative stress, and eventually fibrosis. Cholestasis is associated with profound metabolic changes, alterations in the mitochondrial function, decreased fatty acid oxidation, and increased glycolisis. Mitochondria play a central role in the development of this liver disease because they mediate death receptor signaling - triggered by inflammatory cytokines or bile acids - and contribute to oxidative damage, metabolic disorder, and onset of fibrosis. During the pathogenesis of biliary cirrhosis mitochondrias need for renewal is hampered by a blunted mitochondrial biogenesis. Lack of stimulation of mitochondrial renewal helps to explain mitochondrial impairment in long-term cholestasis. The marked depletion of mitochondrial DNA and occurrence of mitochondrial DNA deletions are probably relevant contributors to the progression of this severe disease. All these findings certainly support the consideration of long-term cholestasis as a secondary mitochondrial hepatopathy.

Oxidative and nitrosative stress in acute pancreatitis. Modulation by pentoxifylline and oxypurinol.

Reactive oxygen species are considered mediators of the inflammatory response and tissue damage in acute pancreatitis. We previously found that the combined treatment with oxypurinol - as inhibitor of xanthine oxidase- and pentoxifylline - as inhibitor of TNF-α production-restrained local and systemic inflammatory response and decreased mortality in experimental acute pancreatitis. Our aims were (1) to determine the time-course of glutathione depletion and oxidation in necrotizing pancreatitis in rats and its modulation by oxypurinol and pentoxifylline; (2) to determine whether TNF-α is responsible for glutathione depletion in acute pancreatitis; and (3) to elucidate the role of oxidative stress in the inflammatory cascade in pancreatic AR42J acinar cells. We report here that oxidative stress and nitrosative stress occur in pancreas and lung in acute pancreatitis and the co-treatment with oxypurinol and pentoxifylline prevents oxidative stress in both tissues. Oxypurinol was effective in preventing glutathione oxidation, whereas pentoxifylline abrogated glutathione depletion. This latter effect was independent of TNF-α since glutathione depletion occurred in mice deficient in TNF-α or its receptors after induction of pancreatitis. The beneficial effects of oxypurinol in the inflammatory response may also be ascribed to a partial inhibition of MEK1/2 activity. Pentoxifylline markedly reduced the expression of Icam1 and iNos induced by TNF-α in vitro in AR42J cells. Oxidative stress significantly contributes to the TNF-α-induced up-regulation of Icam and iNos in AR42J cells. These results provide new insights into the mechanism of action of oxypurinol and pentoxifylline as anti-inflammatory agents in acute pancreatitis.

Liver-specific p38α deficiency causes reduced cell growth and cytokinesis failure during chronic biliary cirrhosis in mice†

p38α mitogen‐activated protein kinases (MAPK) may be essential in the up‐regulation of proinflammatory cytokines and can be activated by transforming growth factor β, tumor necrosis factor‐α, interleukin‐1β, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α‐deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver‐specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life‐span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α‐deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α‐deficient mice during chronic cholestasis through down‐regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up‐regulated in liver of p38α‐deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down‐regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α‐deficient mice. p38α‐knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α‐deficient mice. Conclusion: Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis. (HEPATOLOGY 2013)