1Chapt. 24 Oxygen toxicityCh. 24 Oxygen toxicity, free-radical injuryStudent Learning Outcomes:• Explain how O2is both essential to life and toxic• Oxidative phosphorylation, Reactive Oxygen radicals• Explain formation of the major ROS• Enzymatic by products, non-enzymatic• Describe toxic effects of ROS on cells• Describe nitrogen NO and RNOS radicals• Explain cell protective mechanisms• Enzymes, antioxidants, compartmentalization• Describe association between ROS and diseasesTable 1 diseases associated with free-radical injuryFig. 2; Cell has defenses against damage by ROS and RNOS (Reactive oxygen species, Reactive nitrogen-oxygen species):Antioxidants, enzymesTable 1 diseases associated with free-radical injury:• Amyotrophoic lateral sclerosis (ALS) • Ischemia/reperfusion injury• OXPHOS diseases (mitochondria)• Alzheimer’s disease• Parkinson’s disease• Diabetes• AgingReactive Oxygen SpeciesReactive Oxygen Species (ROS) are a natural occurrence:• Accidental products of nonenzymaticand enzymatic processes• Deliberate production by immunecells killing pathogens• UV irradiation, pollutants• Cells have many defensesFig. 1 O2has 2 antibondinge- with parallel spins; tendency to form toxic ROSRadical nature of OxygenRadical nature of oxygen:• Radical is an unpaired e-• Free radical has independent existence (not bound to enzyme)• Free radical extracts e- from other molecules• O2-accepts e- from strong reducer such as CoQH•in ETCFig. 3, O2can accept total of 4 e- to form H2O2Table 2 ROS speciesTable 2 Some Reactive Oxygen species (ROS)O2-Superoxide anion Produced by ETC and other sites;does not diffuse far, generates other ROS, such as by reaction with H2O2in Haber-Weiss reactionH2O2Hydrogen peroxide Not a free radical, but generates freeradicals by reaction with transition metal (e.g. Fe2+); diffuses into and through cell membranesOH• Hydroxyl radical The most reactive in attacking biological molecules; produced from H2O2in Fenton reaction in presence of Fe2+ or Cu+HOCl Hypochlorous acid Produced from H2O2by neutrophilsto destroy invading organisms (OCl-)Hydroxyl radicalFig. 4Formation of very reactive oxygen species OH•• Two nonenzymatic reactions can form OH• by transfer of single e-• Metals Fe2+ or Cu+are kept sequesteredC. Major sources of ROSFig. 5; ETCFe-H is Fe-heme of cytochromesC. Major source of ROS:• COQH• in electron transport chainaccidental interaction with O2(COQH• is free in membrane)Sources of ROSOxidases, oxygenases, peroxidases generate ROS:• Enzymes bind O2, transfer 1 e- via metals; • Accidental leakage of free-radical intermediates• Ex. Cyt P450 mono-oxygenases detoxify many organic compounds (alcohol, drugs, toxic chemicals like CCl4)• Peroxidases generate H2O2(ex. VLCFA in peroxisomes)Fig. 19.12, 19.13 cytochrome P450 mono-oxygenase3Sources of ROSFig. 6Ionizing Radiation generates OH•• Cosmic rays• Radioactive chemicals• X-raysMay also generate organic radicalsfrom contact biomoleculesOxygen radicals react with cell componentsFig. 7*Oxygen radicals react with cell components:• Lipid peroxidation of membranes• Increased permeability → influx Ca2+ → mitochondrial damage• Cys SH and other aa of proteins oxidized and degraded• DNA oxidized → breakageLipid peroxy radicalsFig. 8Lipid peroxidation: free-radical chain reaction:A. Initiation by OH• attack of poylunsaturated lipid → lipid•B. free-radical chain reaction by reaction with O2 C. Lipid peroxy radical propagates, lipid peroxide degradesD. Terminate by vitE or lipid-soluble antioxidantsMajor contribution toROS-induced injuryROS attack proteins, peptides, DNAFig. 9ROS attack proteins, peptides and DNA• Pro, his, arg, cys, met most susceptible• Protein fragment, cross-link, may aggregate, also will be degraded• Glutathionine (γ-glu-cys-gly) is anti-oxidant, cell defense• DNA oxidized bases mispairat replication (G-C → T-A)• DNA backbone broken• repair mechanisms exist4Reactive Nitrogen-oxygen speciesFig. 10Nitric Oxide and Reactive Nitrogen-oxygen speciesNO is both essential for life and toxic:• Gas, diffuses through membranes• Low concentrations: neurotransmitter, hormone (vasodilation)• Nitroglycerin tablets release NO, vasodilator for heart • Binds Fe-heme in receptor guanylyl cyclase, cGMP activates signal cascadeNO works short distance from source• nNOS and eNOS regulated by Ca2+• iNOS inducible in immune cells,prouces high levels of NONO is toxicFig. 11At high concentrations,NO is toxic, RNOS form• RNOS can cause as much damage as ROS, plus also do nitrating, nitrosylating• RNOS damage proteins, cause lipid peroxidation, DNA breaksRNOS are involved in:• neuro-degenerative diseases like Parkinson’s, • chronic inflammation like rheumatoid arthritis (RA)Phagocytosis uses free radicalsFig. 12Phagocytic cells of immune system do respiratory burst: O2→ ROS, RNOS• Part of antimicrobial defense, also anti-tumor (~ 30-60 min)• NADPH oxidase forms O2-→ H2O2and OH•• Myeloperoxidase forms HOCl → OCl-• iNOS activated, makes NO → RNOSFree-radical release insome disease states contributes to injury; chronic inflammationV. Cellular DefensesV. Cells have defenses against oxygen toxicity:• Antioxidant scavenging enzymes (red)• Nonenzymatic antioxidants (free radical scavengers)• Compartmentalization• Repair of damaged components• Metal sequestrationFig. 13* compartmentalizationSOD = superoxide dismutaseGSH = glutathione5Antioxidant scavenging enzymesFig. 14Antioxidant scavenging enzymes:• Superoxide dismutase (SOD)• Converts O2- to H2O2 • 3 isoforms:  Cytosol, mitochondria, extracellular• Catalase• Reduces H2O2to H2O• Prevents OH• formation• mostly peroxisomeAntioxidant enzymes• Glutathione peroxidase, glutathione reductase:• GSH = glutathione (γ-glu-cys-gly) • Peroxidase reduces H2O2, oxidizes two SH groups → GSSG• Peroxidase in cytosol, mitochondria, have selenium• Reductase recycles the glutathione, reduces with NADPHFig. 15Nonenzymatic antioxidants Fig. 16Vitamin E (αααα-tocopherol) is antioxidant:• Lipid-soluble, protects against lipid peroxidation in membranes• Nonenzymatic terminator of free-radical chain reaction• Lipid fraction of vegetable oils,liver, egg yolks, cereals• Lipoprotein particles in bloodAntioxidantsFig. 17Vitamin C (ascorbate) is antioxidant:• Can donate e- to vitamin E to