The production of superoxide and nitric oxide individually has been associated with the development of several diseases but only recently has it been realised that interactions between them may also be important in disease pathology. The central hypothesis which is emerging is that the balance between nitric oxide and superoxide generation is a critical determinant in the aetiology of many human diseases including atherosclerosis, neurodegenerative disease, ischaemia‐reperfusion and cancer. These ideas are discussed in this short overview and placed in the context of the current and future status of therapies which could modulate the balance between nitric oxide and superoxide.
AbstractIt is thought that 5–10% of human serum copper consists of copper ions complexed with histidine, amino acids or albumin. The phenanthroline assay developed by Gutteridge (Biochem. J. 218, 983–985; 1984) is shown to measure all these forms of copper down to a sensitivity of 0.1μmol/dm3, yet it does not detect any copper ions in freshly-prepared serum or plasma from rats, mice, rabbits or guinea-pigs, or freshlyprepared serum from humans. It is concluded that the “non-caeruloplasmin copper pool” is much smaller than has previously been supposed. No phenanthroline-detectable copper could be measured in serum freshly prepared from four patients with uncomplicated Wilson's disease, but it could be measured in serum from a patient with fulminant hepatic failure. After liver transplantation, concentrations of phenanthroline-detectable copper in this patient fell to zero within two days. Studies on the ferroxidase activity of freshly-prepared serum or plasma samples shows that little ferroxidase II activity is present in samples from healthy adults or from the patient with Wilson's disease and fulminant hepatic failure. In all the patients, ferroxidase I activities are sub-normal. Freshly-prepared plasma or serum samples from several animal species generally show lower ferroxidase I and greater ferroxidase II activities than do human samples, but only in rabbits does ferrroxidase II account for a high proportion of total plasma ferroxidase activity. Storage of biological fluids can cause release of copper from caeruloplasmin and a rise in ferroxidase II activity; these events may have confused some earlier studies.Key Words: CopperPenicillamineWilsons diseaseFerroxidasecaeruloplasmin
The nature of non-transferrin-bound iron in the plasma or serum of iron-overloaded hemochromatosis patients was studied by high performance liquid chromatography (HPLC) and high resolution nuclear magnetic resonance (NMR).500-MHz proton Hahn spinecho NMR spectra of plasma or serum, combined with the use of the iron chelator desferrioxamine, suggests complexation of iron ions with citrate and a possible involvement of acetate.Addition of FeCL to hemochromatosis samples broadened the NMR signals from citrate.HPLC analysis rigorously confirmed the presence of an iron-citrate complex in ultrafiltrates of plasma or serum studies with added FeCls or desferrioxamine supported this conclusion.It is proposed that nontransferrin-bound iron in the plasma of iron-overloaded patients exists largely as complexes with citrate and possibly also as ternary iron-citrate-acetate complexes.The presence of such complexes would account for the ability of non-transferrin-bound iron to be measurable by the bleomycin assay and for its rapid clearance from the circulation by the liver.
The fructose bisphosphatase (EC 3.1.3.11) activity of type A chloroplasts isolated from young (9-day-old) pea (Pisum sativum var. Progress no. 9) plants, assayed at physiological pH, substrate and Mg2+ concentrations, increased rapidly on illumination. The enzyme activity detected was more than sufficient to account for observed rates of Co2 fixation both during the induction period and during steady-state CO2 fixation, whether or not dihydroxyacetone phosphate had been added to the preparation. Omission of catalase from the suspension medium had no effect. On switching off the light, CO2 fixation by the chloroplasts ceased at once, yet fructose bisphosphatase activity decreased much more slowly. Changes in enzyme activity were much less marked if assays were conducted at 3 mM substrate and 10 mM-Mg2+. Chloroplasts from older (13--20-day-old) peas only fixed CO2 rapidly if catalase was present in the assay medium. The fructose bisphosphatase activity detected under physiological assay conditions was again more than sufficient to account for observed rates of Co2 fixation. In the presence of added dihydroxyacetone phosphate, however, the rate of Co2 fixation appeared to be determined by the rate of light activation of fructose bisphosphatase. In general, the rates of Co2 fixation and enzyme activation, and the final enzyme activity achieved, decreased markedly with increasing age of the plants. The role of light activation of fructose bisphosphatase as a means of controlling the rate of CO2 fixation in pea chloroplasts is discussed.
Transition metal ions, especially iron, appear to be important mediators of oxidative damage in vivo. Iron(II) reacts with H 2 O 2 to give more‐reactive radicals. On the basis of ESR spin‐trapping data with DMPO, supported by aromatic hydroxylation studies and patterns of DNA base modification, it is concluded that hydroxyl radical (OH • ) is likely to be the major damaging species formed in Fenton Systems under biologically‐relevant conditions (which include iron concentrations no higher than the micromolar range). Although reactive oxo‐iron species (such as ferryl and perferryl) may also be important, chemical evidence for their formation and identity in biologically relevant Fenton systems is currently lacking. Studies at alkaline pH values show that iron(IV) and iron(V) species are highly oxidizing under those reaction conditions, with a pattern of reactivity different from that of OH • .
ESCODD (European Standards Committee on Oxidative DNA Damage)* Accepted by Prof. B. Halliwell (Received 10 August 1999; In revised form 4 September 1999) We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2'-deoxyguano- sine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within :t:40% of the target, as did two of four GC-MS pro- cedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8- oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be pro- cedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical vari- ation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable pre- cision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, 32p-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision ~ and detection of dose response, cannot at present be recommended. Keywords: Oxidative DNA damage, 8-hydroxy-2'- deoxyguanosine, HPLC, GC-MS, methods validation INTRODUCTION Measurement of oxidative DNA damage in human cells is crucial to an understanding of the consequences of oxidative stress in health and disease, and of the influence of dietary antioxi- dants. 8-Hydroxy-2'-deoxyguanosine (8-oxodG), or the corresponding base 8-oxoguanine (8-oxo- gua), are most commonly measured as a marker of oxidative damage, but estimates of the levels of damage in normal human cells by GC-MS and HPLC range over several orders of magnitude. [11 The European Standards Committee for Oxida- tive DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of oxidative damage in human cells. In phase 1, laboratories participat- ing in ESCODD received samples of calf thymus DNA and liver tissue, as well as standard 8-oxodG and 8-oxodG-containing oligonucleotides for analysis. Results confirmed the existence of wide variations between methods and indicated that even the determination of standard 8-oxodG * Address for Correspondence: Dr. Andrew Collins, DNA Instability Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK. Tel.: +44(0)1224 716634. Fax: +44(0)1224 716629. E-mail: a.collins@rri.sari.ac.uk. 333
No abstract is provided for this article.
Abstract This chapter reviews how reactive species (RS) are involved in the origin and progression of human disorders/diseases. This includes atherosclerosis (especially the role of LDL and HDL oxidation), obesity, diabetes (mechanisms of oxidative damage resulting from hyperglycaemia and hyperlipidaemia and how AGE biomolecules form), metabolic syndrome, cystic fibrosis (including oxidative damage caused by P. aeruginosa infection), H. pylori infection, inflammatory bowel diseases (Crohns disease, ulcerative colitis), Dupuytren contracture, Peyronie disease, rheumatoid arthritis, systemic sclerosis, other autoimmune diseases, multiple organ dysfunction syndrome, systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), viral infections (especially hepatitis B and C, and HIV), sepsis/septic shock, haemorrhagic shock, anxiety, aneurysm, pancreatitis, chronic pain, coeliac disease, schizophrenia, stress, bipolar disorder, periodontal disease, and adverse drug reactions. The roles of RS in ischaemia–reperfusion (and pre-conditioning) are described, e.g. in stroke, brain trauma, sleep apnoea, claudication, myocardial infarction (including thrombolysis), angioplasty, limb reattachment, and organ transplantation. The roles of RS in the initiation, promotion, and progression of cancer, are presented, especially how RS interact with genes and their products (particularly p53) to drive cancer development. The roles that RS play in carcinogen (e.g. benzopyrene) action, cachexia, and in chemotherapy (e.g. with bleomycin and anthracyclines), and its side-effects are presented. RS formation and antioxidant defences in the healthy and diseased brain and nervous system are explored, especially in Parkinson and Alzheimer diseases, but also in Friedreich ataxia, Huntington disease, multiple sclerosis, amyotrophic lateral sclerosis, amyloid diseases, prions (e.g. Creutzfeldt–Jakob disease), aluminium neurotoxicity, and the neuronal ceroid lipofuscinoses.
Activated phagocytic cells produce superoxide (O 2 - ) and hydrogen peroxide (H 2 O 2 ) ; their production is important in bacterial killing by neutrophils and has been implicated in tissue damage by activated phagocytes. H 2 O 2 and O 2 are poorly reactive in aqueous solution and their damaging actions may be related to formation of more reactive species from them. One such species is hydroxyl radical (OH . ), formed from H 2 O 2 in the presence of iron- or copper-ion catalysts. A major determinant of the cytotoxicity of O - 2 and H 2 O 2 is thus the availability and location of metal-ion catalysts of OH* formation. Hydroxyl radical is an initiator of lipid peroxidation. Iron promoters of OH* production present in vivo include ferritin, and loosely bound iron complexes detectable by the ‘bleomycin assay’. The chelating agent Desferal (desferrioxamine B methanesulphonate) prevents iron-dependent formation of OH* and protects against phagocyte-dependent tissue injury in several animal models of human disease. The use of Desferal for human treatment should be approached with caution, because preliminary results upon human rheumatoid patients have revealed side effects. It is proposed that OH* radical is a major damaging agent in the inflamed rheumatoid joint and that its formation is facilitated by the release of iron from transferrin, which can be achieved at the low pH present in the micro-environment created by adherent activated phagocytic cells. It is further proposed that one function of lactoferrin is to protect against iron-dependent radical reactions rather than to act as a catalyst of OH· production.
A number of anti-inflammatory and other drugs used in the treatment of rheumatoid arthritis have been screened for their ability to cause oxidative damage to lipids and proteins in vitro. Although many drugs exhibited an antioxidant profile, a few drugs tested were pro-oxidant, increasing peroxidation of arachidonic acid by mixtures of haem proteins and H2O2. This system may be an appropriate model to use in the inflammatory situation, since microbleeding to release haemoglobin occurs in the inflamed rheumatoid joint, where H2O2 is produced by invading neutrophils. The damaging effects of the pro-oxidant drugs phenylbutazone, meclofenamic acid and flufenamic acid were investigated in some detail using this system. Arachidonic acid peroxidation was accentuated in a dose-dependent manner and in the presence of haem proteins and H2O2, phenylbutazone also causes inactivation of α1-antiproteinase, a major serine proteinase inhibitor in biological fluids. The above drugs may interact with ferryl haemoglobin, produced by the reaction of H2O2 with haemoglobin, to generate drug-derived radicals causing oxidative damage in these systems. If such reactions occur in vivo, they could contribute to the side-effects induced by these drugs on administration to certain rheumatoid arthritis patients.
