Free Radicals in Biology and Medicine 0 \ Second Edition BARRY HALLIWELL Professor of Medical Biochemistry, University of London King's College and JOHN M.C. GUTTERIDGE Senior Scientist, National Institute for Biological Standards and Control and Visiting Professor, Oklahoma Medical Research Foundation Q0 CLARENDON PRESS 1989 OXFORD
Contents 1 Oxygen is poisonous an introduction to oxygen toxicity and free radicals 1 1.1 Oxygen and the Earth 1 1.2 Oxygen and anaerobic organisms 2 1.3 Oxygen and aerobes 3 1.4 What causes the toxic effects of oxygen? 8 1.5 What is a free radical? 10 1.5.1 Oxygen and its derivatives 12 1.5.2 Ozone and oxides of nitrogen 14 1.5.3 Transition metals 15 1.5.4 Other free radicals 19 1.6 Further reading 20 2 The chemistry of oxygen radicals and other oxygen-derived species 22 2.1 Reaction rates and rate constants 22 2.2 Measurement of reaction rates for radical reactions 24 2.2.1 Pulse radiolysis 25 2.2.2 Stopped-flow methods 28 2.3 Reactions of the hydroxyl radical 28 2.4 Production of hydroxyl radicals in living systems 31 2.4.1 Ionizing radiation 31 2.4.2 Ultrasound, lithotripsy, and freeze-drying 33 2.4.3 Reaction of metal ions with hydrogen peroxide 33 2.4.4 Formation of hydroxyl radicals from ozone 46 2.4.5 Ethanol metabolism 47 2.5 Detection of hydroxyl radicals in biological systems 47 2.5.1 Electron spin resonance and spin-trapping 47 2.5.2 Aromatic hydroxylation 53 2.5.3 Measurement of products of radical attack upon DNA 56 2.5.4 Conversion of methional and related compounds into ethene (ethylene) gas (C 2 H 4 ) 57 2.5.5 Other methods 58 2.6 Production of singlet oxygen 58 2.7 Reactions of singlet oxygen 66 2.8 Detection of singlet oxygen in biological systems 70 2.8.1 Use of scavengers 70
xii Contents 2.8.2 Deuterium oxide 70 2.8.3 Light emission 71 2.9 Reactions of the superoxide radical 71 2.10 Hydrogen peroxide in biological systems 79 2.11 Further reading 81 3 Protection against oxidants in biological systems: the superoxide theory of oxygen toxicity 86 3.1 Protection by enzymes 86 $ 3.1.1 Protection against hydrogen peroxide by catalase and peroxidases 86 3.1.2 Superoxide dismutase 106 3.2 Protection by small molecules 123 3.2.1 Ascorbic acid (vitamin C) 123 3.2.2 Glutathione 126 3.2.3 Uric acid 130 9 3.3 Protection by sequestration of metal ions 131 3.3.1 Metallothioneins 133 3.4 Repair systems 134 3.4.1 Methionine sulphoxide reductase 134 3.4.2 DNA repair 134 3.4.3 Degradation of abnormal proteins 136 3.5 The superoxide theory of oxygen toxicity 136 3.5.1 Is superoxide formed in vivo} 137 3.5.2 Is the true function of superoxide dismutase that of removing superoxide radicals? 148 3.5.3 Evidence bearing on the superoxide theory of oxygen toxicity 151 3.5.4 Is superoxide a damaging species? 158 3.5.5 Challenges to the superoxide theory 173 3.5.6 Regulation of bacterial SOD synthesis by metal ions 176 3.6 Oxygen radicals and radiation damage 177 3.6.1 Food irradiation 178 3.7 Further reading 179 4 Lipid peroxidation: a radical chain reaction 188 o 4.1 Membrane structure 189 * 4.2 The peroxidation process 196 4.2.1 Initiation and propagation 196 @ 4.2.2 The importance of iron in lipid peroxidation 200 4.2.3 Products of the reaction of metal complexes with lipid peroxides 204
Contents xiii * 4.3 Enzymic and non-enzymic lipid peroxidation 210 4.4 Acceleration of lipid peroxidation by species other than oxygen radicals 214 4.4.1 Singlet oxygen 'A g 214 4.4.2 Ozone 216 4.4.3 Halogen radicals 217 4.5 Peroxidation of other molecules 217 4.6 Measurement of lipid peroxidation 218 4.6.1 Uptake of oxygen 218 4.6.2 Measurement of peroxides 218 4.6.3 Diene conjugation 220 4.6.4 Measurement of hydrocarbon gases 223 4.6.5 Loss of fatty acids 224 4.6.6 Light emission 224 4.6.7 Measurement of fluorescence 225 tf 4.6.8 The thiobarbituric acid (TBA) test 228 4.6.9 Measurement of aldehydes other than MDA 233 6 4.6.10 Summary which is the method of choice? 233 4.7 Protection against lipid peroxidation 234 ' 4.7.1 Protection by structural organization of the lipids 234, 4.7.2 Protection by 'antioxidants': general principles 236, 4.7.3 Vitamin E 237 4.7.4 Synthetic antioxidants 245 4.7.5 Glutathione peroxidase, a peroxide-decomposing antioxidant 247 4.7.6 Antioxidants of extracellular fluids 253 4.8 Erythrocyte lipid peroxidation 260 4.9 The biomedical significance of lipid peroxidation 263 4.10 Further reading 266 5 Protection against radical damage: systems with problems 277 5.1 The chloroplasts of higher plants 277 5.1.1 The problems 281 5.1.2 The solutions 284 5.2 The eye 289 5.2.1 Diabetic cataract 293 5.3 Further reading 296 6 Free radicals and toxicology 299 6.1 Bipyridyl herbicides 301 6.1.1 Paraquat toxicity to plants 302 6.1.2 Toxicity to bacteria 305
xiv Contents 6.1.3 Toxicity to animals 306 6.1.4 Paraquat, lipid peroxidation, and hydroxyl radical formation 308 6.2 Alloxan and streptozotocin 310 6.3 Substituted dihydroxyphenylalanines and other phenolic compounds 314 6.4 Cigarette smoke 317 6.4.1 Lung defences against cigarette smoke 321 6.5 Other air pollutants 321 6.6 Haemolytic and anti-malarial drugs 323 6.6.1 Hydrazines 324 6.6.2 Sulphur-containing haemolytic drugs 326 6.6.3 Quinones 327 6.6.4 Oxidant stress, favism, and malaria 329 6.7 Ethanol 332 6.8 Paracetamol and phenacetin 334 6.9 Halogenated hydrocarbons 335 6.9.1 Carbon tetrachloride and chloroform 335 6.9.2 Other halogenated hydrocarbons 340 6.10 Hypoxic cell sensitizers and related compounds 341-6.11 Antibiotics 345 6.12 MPTP and Parkinson's disease 347 6.13 Aluminium and Alzheimer's disease 350 6.13.1 Aluminium and lipid peroxidation 353 6.14 Toxicity of other metals 354 6.15 Sporidesmin 355 6.16 The 'Spanish cooking-oil' syndrome 355 6.17 Ultraviolet light 356 6.18 Further reading 357 7 Free radicals as useful species 366 7.1 Reduction of ribonucleosides 366 7.2 Oxidation, carboxylation, and hydroxylation reactions 368 7.2.1 Dioxygenases 368 7.2.2 Pyruvate-metabolizing enzymes 369 7.2.3 Hydroxylases 370 7.2.4 Carboxylation of glutamic acid 371 7.3 Phagocytosis 372 7.3.1 The bacterial killing mechanism 377 7.3.2 Significance of extracellular oxidant production by phagocytes 388 7.4 Peroxidase and NADH oxidase enzymes 390 7.5 Fruit ripening and the'wound response'of plant tissues 394
Contents xv 7.6 Eicosanoids: prostaglandins and leukotrienes 397 7.6.1 Prostaglandins and thromboxanes 398 7.6.2 Leukotrienes and other lipoxygenase products 404 7.7 Endothelial-derived relaxing factor (EDRF) 407 7.8 Further reading 408 Free radicals, ageing, and disease 416 8.1 General principles 416 8.2 Atherosclerosis 417 8.3 Chronic inflammation and the autoimmune diseases 422 8.3.1 Autoimmune diseases and phagocyte action 426 8.3.2 Oxidants and human rheumatoid disease 427 8.3.3 Are oxidants important in autoimmune diseases? 432 8.3.4 Anti-inflammatory drugs and oxidant damage 433 8.4 Ischaemia/reoxygenation injury 438 8.4.1 Intestinal injury 438 0 8.4.2 Cardiac injury 442 8.4.3 Cerebral injury 444 8.4.4 Preservation of organs for transplantation: kidney, liver, and skin 445 8.4.5 Rheumatoid arthritis 446 8.4.6 Pancreas 447 8.4.7 Limb ischaemia 447 8.5 Lung damage and the adult respiratory distress syndrome 448 8.6 Exercise-induced oxidant damage 449 8.7 Ageing 450 e 8.7.1 Programmed ageing 451 «8.7.2 Damage accumulation ageing 454 t 8.7.3 Free-radical theory of ageing 454 8.7.4 Fluorescent tissue pigments 458 8.7.5 Progeroid syndromes 462 8.7.6 Neuronal ceroid lipofuscinosis (NCL) 463 8.7.7 Amyloid 465 8.7.8 Model systems 466 8.8 Cancer 466 8.8.1 Oxidants and carcinogenesis 469 8.8.2 Immune surveillance 473 8.8.3 Mechanism of action of carcinogens 476 8.8.4 Antitumour drugs and oxidant production 481 8.8.5 Footprinting of DNA by using hydroxyl radical and other active complexes 493 8.9 Further reading 494
xvi Contents Appendix I: A consideration of atomic structure and bonding 509 A. 1 Atomic structure 509 A.2 Bonding between atoms 517 A.2.1 Ionic binding 517 A.2.2 Covalent bonding 518 A.2.3 Non-ideal character of bonds 521 A.2.4 Hydrocarbons and electron delocalization 522 A.3 Further reading 524 Appendix II: A simple guide to lymphocytes 525 Index 527