Silvano Pinamonti

Xanthine oxidase activity in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a serious respiratory pathology characterized by irreversible limitation of expiratory flow and includes chronic obstructive bronchitis, chronic airflow limitation, and emphysema. To determine whether xanthine oxidase activity increased in the airspaces of COPD patients, we examined bronchoalveolar lavage fluid (BAL) from COPD patients recruited during a 2-year clinical study. Filtered BAL supernatant from COPD patients and healthy nonsmoking controls was examined by fluorometric analysis of DNA unwinding (FADU) and spectrophotometric assays (cytochrome c reduction kinetics and uric acid kinetics). Compared to controls, filtered BAL supernatant of subjects with COPD exhibited a detectable clastogenic activity probably related to superoxide production. The method of BAL preparation as an acellular system strongly suggests that superoxide production may be due to xanthine oxidase activity.

Detection of xanthine oxidase activity products by EPR and HPLC in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease

Xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22), a molybdenum-containing hydroxylase that produces superoxide and uric acid from purine substrates and molecular oxygen, is involved in the oxidative stress underlying several human pathologies including lung diseases. An enzymatic activity similar to xanthine oxidase was previously reported in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease (COPD-BAL), by fluorometric analysis of DNA unwinding and cytochrome c reduction kinetics. Here we report the detection of xanthine oxidase activity products by electron paramagnetic resonance (EPR) in presence of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reversed-phase high-performance liquid chromatography (RP-HPLC) in COPD-BAL (n = 14, average age of patients 65 years, range 38-81) and BAL from healthy nonsmoker controls (n = 6, average age 64 years, range 44-73). Superoxide DMPO adducts were detected in COPD-BAL and in an in vitro system containing xanthine and xanthine oxidase (XA/XO), but not in BAL controls and when superoxide dismutase (SOD, 1000 I.U./ml) was added to COPD-BAL. The HPLC analyses after addition of xanthine showed production of uric acid in COPD-BAL and in the XA/XO system but not in BAL controls. These results support the involvement of xanthine oxidase in the mechanisms of superoxide production by BAL supernatant, which increases oxidative stress in chronic obstructive pulmonary disease.

Reducing agents inhibit rhinovirus-induced up-regulation of the rhinovirus receptor intercellular adhesion molecule-1 (ICAM-1) in respiratory epithelial cells

Rhinoviruses are the major cause of common colds and of asthma exacerbations. Intercellular adhesion molecule‐1 (ICAM‐1) has a central role in airway inflammation and is the receptor for 90% of rhinoviruses. Rhinovirus infection of airway epithelium induces ICAM‐1. Because redox state is directly implicated in inflammatory responses via molecular signaling mechanisms, here we studied the effects of reducing agents on rhinovirus‐induced ICAM‐1 expression, mRNA up‐regulation, promoter activation, and nuclear factor activation. To investigate the effects of rhinovirus infection on the intracellular redox balance, we also studied whether rhinovirus infection triggers the production of reactive oxygen species. We found that reduced (GSH) but not oxidized (GSSG) glutathione (1–100 μM) inhibited in a dose‐dependent manner rhinovirusinduced ICAM‐1 up‐regulation and mRNA induction in primary bronchial and A549 respiratory epithelial cells. GSH but not GSSG also inhibited rhinovirus‐induced ICAM‐1 promoter activation and rhinovirus‐induced NF‐κB activation. In parallel, we found that rhinovirus infection induced a rapid increase of intracellular superoxide anion that was maximal at the time of NF‐κB activation. This oxidant generation was completely inhibited by GSH. We conclude that redox‐mediated intracellular pathways represent an important target for the therapeutic control of rhinovirusinduced diseases.

Role of Xanthine Oxidase Activation and Reduced Glutathione Depletion in Rhinovirus Induction of Inflammation in Respiratory Epithelial Cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-κB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.

Correlation between age and DNA damage detected by FADU in human peripheral blood lymphocytes.

Fluorometric analysis of DNA unwinding (FADU) is a fast and reliable method for detecting single strand DNA breaks as an index of DNA damage induced by clastogenic agents. A study of damage detected by FADU was conducted on DNA extracted from peripheral blood lymphocytes of 128 healthy nonsmoking regular donors (ranging in age from 19 to 67 years) and from 5 umbilical cord blood samples. DNA damage was measured as percentage of unwound DNA after alkalinization. Statistical analyses, both parametric (Pearson r correlation coefficient, b regression coefficient, ANOVA) and nonparametric (Kruskal-Wallis H test, Spearman rs rank correlation coefficient), support a significant correlation between age of donors and amount of DNA damage. The same results are found when adult donors are divided in four age classes and the ANOVA test performed among the mean percentages of unwound DNA of each class. Furthermore, donors of the same age belonging to different blood groups (A, B, AB and O) do not show any difference in DNA damage detected by FADU.

Reactive oxygen species in human inner ear perilymph

Conclusions: The results reported here provide the first evidence of the production of superoxide, a biologically relevant reactive oxygen species (ROS), in human inner ear perilymph (hIP) in pathological conditions, by the activity of the xanthine dehydrogenase/xanthine oxidase (XA/XO) enzyme system. Objective: To investigate the presence of ROS in hIP. Methods: Since damage and apoptosis of inner ear hair cells may occur as a result of ROS-mediated injury, we investigated the presence and production of ROS in 105 hIP samples; 98 collected from patients affected by profound sensorineural hearing loss, during surgery for cochlear implantation, and 7 controls, collected from patients affected by otosclerosis, in case of spontaneous leakage after stapedotomy. ROS production was investigated by spectrophotometric analysis and polyacrylamide gel electrophoresis (SDS-PAGE). Results: In hIP samples tested by cytochrome c reduction kinetics, the average superoxide production was 27.34 μmoles per mg of total protein, against 0.36 in controls. Some of these hIP samples, analyzed by cytochrome c reduction kinetics in the presence of xanthine, were found to be positive for ROS-producing XA/XO enzyme. These results were supported by SDS-PAGE analysis.

Oxygen radical scavengers inhibit clastogenic activity induced by sonication of human serum

Clastogenic factors (CF) are diffusible molecules that damage DNA. They are generated within biological media by a variety of physical and chemical stimuli. Their nature and mechanism of action remain largely unknown. Clastogenic activity can be experimentally generated by pulsed ultrasound treatment of human serum. To investigate whether oxygen radicals are involved in the clastogenic activity induced by sonication of human serum, we examined the effects on such clastogenic activity of different oxygen radical scavengers added to human serum before and after sonication. Human serum was sonicated for 50 min at 24 microW/cm2 by pulsed ultrasound. The clastogenic activity of sonicated human serum was examined in the presence or absence of oxygen radical scavengers by measuring the amount of DNA damage induced in autologous human lymphocytes, assessed with the fluorometric analysis of DNA unwinding (FADU). Sonication of human serum generated significant DNA damage in autologous lymphocytes (DNA unwinding averaged 31.79% +/- 2.1 after sonication vs. 12.82% +/- 2.6 in the controls, p < 0.005). Superoxide dismutase (SOD; 500 I.U./ml), catalase (500 I.U./ml), mannitol (50 mM), and glutathione (50 mM) completely prevented DNA damage when added before serum sonication, whereas only mannitol (86%) and glutathione (90%) almost completely inhibited DNA damage when added after sonication. SOD and catalase had only a partial inhibitory effect when added after sonication (49% and 63%, respectively). The prevention of DNA damage was also obtained by an association of subliminal amounts of glutathione (20 mM) and vitamin E (1 I.U./ml). These results suggest that the clastogenic activity generated by sonication of human serum is mediated by oxygen radicals.

Differential responsesin vitroof rice cultivars to Italian lineages of the blast pathogenPyricularia grisea (Cooke) Sacc. 1. Oxidative burst

Suspension cultured cells of six rice cultivars differing in their sensitivity to blast were treated with mycelial wall hydrolysates prepared from seven isolates belonging to different Pyricularia grisea lineages. Soon after elicitor addition, rice cells produced significant amounts of superoxide anion, which was rapidly converted into diffusible peroxide. Maximal effects were achieved at 50 mg L-1 elicitor. In all cases, a 7 to 13-fold increase in the basal rate of reactive oxygen species production was found. Neither differential effects among strains nor clear relationships between lineage and the resulting oxidative burst were evident. Interestingly, a good correlation was found between basal (and elicited) levels of peroxide generation and the overall tolerance of rice cultivars to the pathogen. About two days after elicitation, cell death occurred proportional to the amount of hydrogen peroxide released. Peroxide was required to trigger loss of cell viability, but the latter was not due to a direct toxic effect, suggesting the induction of programmed cell death. Results represent the first data aimed to develop in vitro tests for pathogenicity prediction of Italian blast lineages toward rice cultivars.

Reducing agents inhibit the contractile response of isolated guinea‐pig main bronchi

Background Oxidants are involved in many respiratory disorders, including asthma and chronic obstructive pulmonary diseases. Reduced glutathione (GSH), one of the most important antioxidant compounds against oxidant free radicals, is particularly abundant in the respiratory epithelial lining fluid, where its concentration is increased in inflammatory disorders.

The xanthommatin forming enzyme system of the desert locust, Schistocerca gregaria

Abstract 1. 1. Xanthommatin synthesis was realized in vitro with homogenates and cell fractions of the eyes and integument but not with the fat body or Malpighian tubes of Schistocerca gregaria . 2. 2. The above enzymatic activity, which leads to xanthommatin, was localized in the mitochondria-like fraction of the epidermal cells and seemed to be mainly bound to the membranes. 3. 3. The synthesis is not coupled to the dopa-tyrosinase system, since it was only slightly inhibited by phenylthiourea, and the partially purified enzyme(s) did not show any tyrosinase activity. 4. 4. In the albino insect the xanthommatin synthesis is reduced to a tenth.