A new pathway to generate nitric oxide by xanthine oxidase
Z. Zhang, D. Naughton, P. Winyard, D. Blake, and M. Symons
Bone and Joint Research Unit, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London E1 2AD, UK
Nitric oxide (NO) synthesis is now well-known to result from the oxidation of L-arginine by a family of NO synthases (NOS). This L-arginine:NO pathway catalysed by NOS exclusively require oxygen to form NO. However, recent studies suggested that under hypoxic conditions, this mechanism of NO synthesis is impaired and NO is likely formed by acid catalysed degradation of nitrite, a mechanism independent of NOS. The nitrite producing bacterial nitrate reductase has close structural similarity to mammalian xanthine oxidase (XO). Although nitrate reductase activity of XO has already been demonstrated with a Km of 140 mM for nitrite generation, the nitrite reductase activity of XO has not been studied. The present study was designed to examine further reduction of nitrite to NO by XO under hypoxic conditions. Using a chemiluminescent NO meter, we have shown that nitrite is readily reduced to NO by XO, in the presence of NADH as an electron donor. Heparin binding, which is known to modify the behaviour of XO, caused an increase in catalytic activity of nitrite reduction without altering NADH oxidation by XO. Inhibitors of XO, allopurinol and diphenyleneiodonium (DPI), blocked the synthesis of NO by XO, nitrite and NADH. Inflamed rheumatoid synovial tissue, which is known to contain elevated levels of XO, also generates NO from nitrite in the presence of NADH, and this activity was not inhibited by the NOS inhibitor, L-NNA. We propose that a major function of XO is to generate NO from nitrite in the presence of NADH. This mechanism would be important in redistribution of blood flow to ischaemic tissue by virtue of the vasodilator effect of NO.
Ben-Zhan Zhu and Mordechai Chevion
Department of Cellular Biochemistry, Hebrew University-Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel
The effect of 1,10-Phenanthroline (OP) on H2O2-mediated toxicity to E. coli was found to be dependent on the concentration of OP. At low concentrations, OP markedly enhanced H2O2-induced bacterial killing but shifted to protection at high concentrations. This was correlated with the effect of OP on iron-mediated DNA damage induced by H2O2. It is known that iron and OP could form three distinct complexes: Fe2+-(OP)1, Fe2+-(OP)2 and Fe2+(OP)3 complex. Moreover, the mono and bis complexes react with H2O2 to produce hydroxyl radicals, whereas the tris complex is stable towards H2O2. Therefore, the enhancing effect at low OP concentration could be explained by the reaction of OP with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+, while the protective effect at high OP concentration could be attributed to the exclusive formation of the tris complex.
Ben-Zhan Zhu and Mordechai Chevion
Department of Cellular Biochemistry, Hebrew University-Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel
Peroxidase enzymes were found to be inactivated during pentachlorophenol (PCP) oxidation by peroxidases in the presence of hydrogen peroxide (H2O2). Tetrachloro-1,4-benzoquinone (TCBQ) has been observed as the oxidation product and implicated in genotoxic effects associated with PCP. In this report we studied the interaction between TCBQ and H2O2 by using ESR spin-trapping technique, to determine whether hydroxyl free radical (.OH) was produced or not. We found that in the presence of H2O2, TCBQ could produce DMPO-OH signal with concomitant formation of tetrachlorosemiquinone radical (TCSQ.). Superoxide dismutase (SOD) had no effect on both DMPO-OH and TCSQ. signal, while catalase could eliminate DMPO-OH without affecting TCSQ. signal. The production of .OH was further supported by using .OH scavenging agents such as dimethyl sulphoxide (DMSO) and ethanol, to form the characteristic DMPO-CH3 and DMPO-CH(OH)CH3 signals, respectively. The iron chelator desferrioxamine (DFO) and DTPA, and the copper- specific chelator bathocuprione disulfonic acid (BCS) were employed to study the possible role of transition metals. We found that both DTPA and BCS had no effect, while DFO totally abolished both TCSQ. and DMPO- OH signal with a concomitant formation of DFO nitroxide radical. Other hydroxamates such as rhodotorulic acid and benzohydroxamic acid (BHA) showed similar effects with DFO. Based on the above results, we concluded that the interaction of TCBQ and H2O2 could produce .OH and this might be responsible for the inactivation of peroxidase enzymes. The mechanism of the production of .OH by TCBQ and H2O2 was proposed through an organic Fenton reaction, i.e. TCSQ + H2O2 Þ .OH.
Ben-Zhan Zhu, Svetlana Schechtman, and Mordechai Chevion
Department of Cellular Biochemistry, Hebrew University-Hadassah Schools of Medicine and Dental Medicine, Jerusalem 91120, Israel
The evaluation of the toxicity of environmental pollutants has been based mainly on the effect of the pure substance. However, environmental pollutants generally appear as complex mixtures in air, water and soil. These substances may interact within the mixture to produce unexpected combined effects. There is only little knowledge on these effects especially when substances occur at sub-toxic concentrations.
Pentachlorophenol (PCP) is one of the most widely used biocides, ubiquitously present in our environment and even occurs in body fluids and tissues of people who are not occupationally exposed to it. Because of its toxicity and carcinogenicity, PCP has been listed as a priority pollutant by the U.S. Environmental Protection Agency. Copper, chromium and arsenic, which are the active ingredients of the chromated- copper-arsenate inorganic wood preservative, are often found in association with PCP contamination.
Incubation of bacteria with PCP and metal complex, showed a dramatic synergistic toxic effect as measured by both inhibition of growth and loss of colony-forming ability, while either PCP or metal complex alone had only marginal effect. The synergistic toxic effects of PCP in combination with metal complexes were found to be due to the formation of a lipophilic adduct, which leads to the synergistic transport of both compounds into the cells. This kind of interaction and lipophilic adduct formation could be of relevance as a general mechanism of toxicity. Such interaction should be of much concern when evaluating the toxicity of environmental pollutants, which are found at currently considered non- or sub-lethal concentrations but could show a synergistic action and lead to manifested toxicity when present together.
Tetrachlorohydroquinone (TCHQ) has been identified as the main toxic metabolite of both pentachlorophenol and hexachloro-benzene, which, in turn, are highly ubiquitous as environmental pollutants. TCHQ could covalently bind to DNA and induce DNA single strand breaks. The trihydroxamate iron chelator desferri-oxamine (DFO) could protect against DNA scission caused by TCHQ, while other iron chelators such as DTPA do not. The autooxidation process of TCHQ yielding the tetrachlorosemiquinone radical (TCSQ.) intermediate, was studied in the presence of chelators. DFO led to a marked reduction in both concentration and life span of TCSQ. via formation of DFO- nitroxide radical (DFO.). In contrast, DTPA had no detectable effect on TCHQ autooxidation. Present studies indicate that the protective effects of DFO on TCHQ-induced DNA damage were not due to the binding of iron, but rather to scavenging of the reactive TCSQ. and the formation of the less reactive DFO.. DFO also stimulate the hydrolysis (dechlorination) of tetrachloro-1,4-benzoquinone, the oxidation product of TCHQ. The results demonstrate two new modes of action for DFO: the scavenging of deleterious semiquinone radical, and the stimulation of the hydrolysis of halogenated substituents on the quinone structure. Both modes might prove highly relevant to the biological activities of DFO.
Hemoglobin catalyses LDL-Apo B modification during extracorporeal circulation of blood and plasma
Ouliana Ziouzenkova1, Liana Asatryan1, Mary Lou Wratten2, Ciro Tetta2, and Alex Sevanian1
1 Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, USA, and 2Clinical and Laboratory Research Department, Bellco S.p.A., Italy
The current mechanism(s) describing lipid peroxidation in LDL cannot adequately explain the formation of a modified LDL bearing a higher electronegative charge (LDL-) in human plasma. We show that ferrylhemoglobin catalyses the crosslinking of LDL and converts up to 30% of LDL to LDL- in dose dependent manner during 1h incubation under an argon atmosphere. A similarly high (10 fold) increase in LDL- production was observed during the 4 hours incubation of LDL with methemoglobin. A 2-fold increase in LDL- levels was evident even in presence of 5% plasma, however, increasing plasma concentrations inhibited, but never completely prevented, this process. LDL modification was accompanied by enhanced fluorescence at ex/em 327/400 nm, which may be indicative of bityrosyl formation. In contrast plasma incubation with 10mM MDA leads to the MDA incorporation of LDL but not the the significant production of LDL-. LDL- production is significantly potentiated during the circulation of plasma containing the nmolar concentrations of methemoglobin, through hemodialysis (HD) filters used clinically for the treatment of uremic patients. The 200% increase in LDL- in these experiments was not accompanied by enhanced malondialdehyde (MDA) levels. Circulation of blood for 4 hours in vivo or in a model HD system lead to the release of micromolar concentrations of hemoglobin species accompanied by an approximately 2-fold increase in LDL- levels. LDL- proportions in hemodialysis patients increased from 1.3-18.8% of total LDL as compared to 1.0-3.3 % in healthy subjects. MDA levels were not correlated with LDL- levels. Our results suggest that LDL can be modified by hemoglobin in plasma and that this is prevalent during HD. Hemolysis and oxidative modification of LDL during HD represents a potential mechanism for atherogenic modification of LDL ApoB without pronounced lipid peroxi dation, and may account for the increased risk to atherosclerosis in HD patients.
Jay L. Zweier, Penghai Wang, Emad A. Mikhail, and Howard Elford*
EPR Center and Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, and *Molecules For Health, Richmond, VA
Free radicals generated by neutrophil (PMN) activation are important mediators of reperfusion injury in the postischemic heart. We have demonstrated that the free radical scavenging drug ethyl 3,4,5-trihydroxybenzimidate (Imidate) blocks radical generation from (PMN) NADPH oxidase with an IC50 of approximately 10 µM. In the present study, we evaluate the efficacy of Imidate in reducing myocardial infarct size in an in-vivo rat model of regional myocardial ischemia and reflow. Rats were subjected to 35 minutes of left anterior descending coronary artery occlusion followed by 2 hours of reperfusion and infarct size was measured as percent of risk region using TTC staining and monastral blue infusion into the non-risk region. Two groups of animals were studied, untreated control and Imidate treated, 100 mg/kg. Imidate treatment dramatically reduced infarct size from 57 ± 3% to 37 ± 3% of the risk region. In a separate group of isolated hearts studied with 20 minutes of global ischemia and reperfusion in the presence of blood factors, Imidate enhanced the recovery of contractile function by more than three fold and quenched the reperfusion associated burst of free radical generation as measured by EPR spin trapping. Imidate also prevented the reperfusion associated increase in CD18 expression and PMN adhesion. Thus, Imidate was highly effective at salvaging myocardium at risk of infarction and preserving contractile function in the postischemic heart.
Evidence for chronic oxidative stress in Down syndrome
Slobodan V. Jovanovic and Kent MacLeod
International Center for Metabolic Testing, 1305 Richmond Road, Ottawa, ON, Canada K2B 7Y4
There is convincing indirect evidence that individuals with Down syndrome suffer from chronic oxidative stress. Build up of b-amyloid plaques is visible in the brain even at 30 years of age, cardiovascular diseases are frequent in childhood, incidence of cataracts is significantly higher in Downs. Increased oxidative damage has been attributed to a genetic overdose of Cu,Zn-superoxide dismutase, which is believed to generate harmful excesses of hydrogen peroxide. In addition, recent in vitro study demonstrates that oxidative damage to Down syndrome neurons may be averted by simple chemical antioxidants. In spite of the importance of the understanding of the role of oxidative stress in Down syndrome, especially regarding the meaningful intervention to delay the onset of deleterious biological consequences of oxidative stress, the in vivo measurements have not been done.
To investigate the in vivo oxidative stress status, we measured urinary 8-hydroxy-2¹-deoxyguanosine in individuals with Down syndrome and their siblings. 168 individuals participated in the study, 85 Downs and 81 controls. The concentration of urinary 8-hydroxy-2¹ deoxyguanosine normalized to creatinine was found to be significantly elevated (P = 0.00025) in individuals with Down syndrome, that is 2.35 ± 1.69 in Downs versus 1.40 ± 1.09 nmol/mmol creatinine in controls. The dietary habits and lifestyle (second-hand smoking, exercise) have little influence on the DNA damage expressed as concentration of 8 hydroxy-2¹-deoxyguanosine. The implications of these measurements for the prevention of oxidative damage related pathologies in individuals with Down syndrome are discussed.