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The Basics of Free Radical Chemistry and Their Role in Diseases

Explore the role of free radicals in various diseases such as neurodegeneration, cataracts, cardiovascular disease, and more. Learn how oxidative damage increases with age and the predictions of the oxidative stress theory of aging.

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The Basics of Free Radical Chemistry and Their Role in Diseases

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  1. The Basics of Free Radical Chemistry and Their Biology Barshop Institute for Longevity and Aging Studies

  2. Role of Free Radicals in Diseases • Neurodegeneration (Alzheimer’s, ALS, Parkinson’s) • Cataracts • Systemic Amyloidosis • Muscular Dystrophy • Progeria and Werner’s syndrome • Rheumatoid Arthritis • Respiratory Distress Syndrome • Cardiovascular Disease • Atherosclerosis • Colitis • Diabetes mellitus, • Down’s Syndrome • Multiple Sclerosis • Aging

  3. w Oxidative Damage Increases with Age Oxidative Stress Theory of Aging w Loss of Cellular Function w Increased Pathology and Aging

  4. w Oxidative Damage Increases with Age. Predictions of the Oxidative Stress Theory of Aging Lipid Peroxidation Protein Oxidation DNA Oxidation Problems with Measuring Oxidative Damage: Multiple Products of Oxidative Damage Sensitivity of Assays

  5. DNA Oxidation Over 100 different types of oxidative damage to DNA: 8-Oxo-deoxyguanosine 8-Oxo-deoxyadenosine 5-Hydroxy-2-deoxycytidine (5-HMdU) Thymidine glycol

  6. H O DNA Oxidation O N N H N H N N H 2 Sugar 8-Oxo-deoxy-guanosine (oxo8dG)

  7. HPLC-EC Analysis of DNA Oxidation 2dG 80 DNA Isolation: Phenol Sodium Iodide NAR 29: 2117, 2001 60 Response (µA) 40 20 0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention time (minutes) 1.50 oxo8dG 1.00 Response (nA) 0.50 0.00 -0.50 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Retention time (minutes)

  8. Liver (mtDNA) * oxo8dG /10 0.25 0.2 5 dG 0.15 0.1 0.05 0 Effect of Age on DNA Oxidation Liver (nDNA) * 0.08 0.06 0.04 0.02 0 Kidney Brain * * 0.06 0.05 oxo8dG /105dG 0.04 0.03 0.02 0.01 Heart Muscle * * 0.06 0.05 0.04 0.03 0.02 0.01 0 5 10 20 5 10 20 PNAS 98: 10469, 2001 Age (months)

  9. Lipid Peroxidation

  10. Formation of Isoprostanes From: J.D. Morrow et al., Prog. Lipid Res. Vol. 36, No.1, 1997, pp.1-21

  11. Effect of Age on Isoprostane Levels in F344 Rats Kidney Plasma 12 1.0 * * 10 0.8 8 0.6 F2-isoprostane ng/ml 6 F2-isoprostane ng/g tissue 0.4 4 0.2 2 0 0 Old Old Young Young Ward et al., J. Gerontol,. 60: 847-51, 2005

  12. Amino Acids Sensitive to Oxidation Met Methionine sulfoxide Cys Disulfides, mixed disulfides,, HNE-Cys Tyr Dityrosine, nitrotyrosine, chlorotyrosines, dopa Trp Hydroxy-and nitro-tryptophans, kynurenines Phe Hydroxyphenylalanines Val,Leu Hydroperoxides His 2-Oxohistidine, asasparagine, aspartate, HNE-His Glu Oxalic acid, pyruvic acid Pro Hydroxyproline, pyrrolidone, glutamic semialdehyde Thr 2-Amino-3-ketobutyric acid Arg Glutamic semialdehyde, chloramines Lys MDA-Lys, HNE-Lys, acrolein-Lys, CML Protein Oxidation

  13. Metal-Catalyzed Protein Oxidation

  14. Assay for Carbonyl Groups in Proteins Lysine Arginine Proline Threonine

  15. Levine, Free Radical Biology and Medicine 32: 790-796 2002

  16. w Identify proteins that show increased damage with age. Future Studies in Protein Oxidation

  17. Assay for Carbonyl Groups in Proteins Lysine Arginine Proline Threonine

  18. O N 2 H N NH NO 2 2 2,4 , Dinitrophenylhydrazine O O H H O O H H O O C C - - OH OH O O NH - C - NHNH 2 S S thiosemicarbazide Fluorescein 5 - (FTC) Carbonyl Probes Chaudhuri et al., Mech. Age. Dev.127: 894, 2006

  19. O O H H O O H H O O C C - - OH OH O O NH - C - NHNH 2 S S thiosemicarbazide Fluorescein 5 - (FTC) Carbonyl Probes 0.25 0.20 0.15 Intensity (F/C) 0.10 0.05 0 4-6 22-23 Age (months) Chaudhuri et al., Mech. Age. Dev.127: 894, 2006

  20. Effect of Age on Protein Oxidation (carbonyl groups) in Liver Young Old Fluorscence (carbonly levels)

  21. Effect of Age on Protein Oxidation (carbonyl groups) in Liver Young Old Sypro Ruby (protein levels)

  22. Aldolase2B Albumin (precusor) Regucalcin Cu/ZnSOD Effect of Age on Protein Oxidation (carbonyl groups) in Liver Young Old 21 mole/mole protein Chaudhuri et al., Mech. Age. Dev.127: 894, 2006

  23. w Identify proteins that show increased damage with age. w Identify the effect of age on oxidative damage other than carbonyl groups. Future Studies in Protein Oxidation

  24. Paraquat NADP+ + H+ + O2 NADPH + O2 Diquat . - Surrogate Marker of Oxidative Damage Sensitivity to Oxidative Stress

  25. Sensitivity to Paraquat Toxicity Young 100 80 60 % Survival 40 Old 20 0 0 1 2 3 4 5 6 Days after Paraquat (50 mg/kg)

  26. w Oxidative Damage and Sensitivity to Oxidative Stress Increases with Age. Manipulations that Increase Life Span will Reduce Oxidative Damage and Sensitivity to Oxidative Stress. w Predictions of the Oxidative Stress Theory of Aging Caloric Restriction

  27. Effect of Caloric Restriction on Lifespan 100 90 80 70 60 Median Maximum PERCENT ALIVE Lifespan (mos) 50 n 77 30.9 33 Ad Libitum 40 38.3 79 49 Caloric Restriction 30 20 10 0 0 5 50 15 20 25 30 35 45 10 40 Age in Months

  28. Effect of Caloric Restriction on Oxidative Damage Liver Liver Kidney Kidney Isoprostanes DNA Oxidation Isoprostanes DNA Oxidation 14 14 0.8 0.8 25 25 * * * * * * 12 12 1.2 1.2 * * 20 20 0.6 0.6 10 10 15 15 8 8 0.8 0.8 0.4 0.4 oxo8dG/2dG x 105 oxo8dG/2dG x 105 8-isoPGF2a ng/ml 6 6 8-isoPGF2a ng/ml 10 10 4 4 0.4 0.4 0.2 0.2 5 5 2 2 0 0 0 0 0 0 0 0 AL AL AL AL AL AL AL AL AL AL AL AL AL AL AL AL CR CR CR CR CR CR CR CR - - - - - - - - - - - - - - - - - - - - - - - - Y Y Y Y Y Y Y Y O O O O O O O O O O O O O O O O J. Gerontol. 60: 847, 2005

  29. 100 80 % Survival 60 Caloric Restriction 40 20 Ad Libitum 0 0 1 2 3 4 5 6 7 8 Days After Treatment Clin. Endocrinol. Metab., 2004 Sensitivity to Oxidative Stress (Paraquat Toxicity)

  30. w Oxidative Damage and Sensitivity to Oxidative Stress Increases with Age. Manipulations that Increase Life Span will Reduce Oxidative Damage and Sensitivity to Oxidative Stress. w Predictions of the Oxidative Stress Theory of Aging Predictions of the Oxidative Stress Theory of Aging Caloric Restriction Long-Lived Invertebrate Mutants

  31. Longevity Mutants of Caenorhabditis elegans 100 Age-1 80 Parental Stock Parental Stock 60 Fraction Survival (%) 40 20 0 0 10 20 30 40 50 60 Age (days) Thomas E. Johnson, 1987

  32. 25 ) 20 2 O 2 fer-15 age-1 15 fer-15 (mM H 10 50 5 LD 0 0 5 10 15 20 25 30 Adult Age (days) Longevity Mutants of Caenorhabditis elegans P. L. Larsen, PNAS 90:8905, 1993

  33. Protein Oxidation in Longevity Mutants of C. elegans Life Span (100%) (70%) (160%) Ishi et al., Free Radical Biol. Med. 33: 1021, 2002

  34. Survival of Long-Lived Drosophila Paraquat Toxicity 100 100 80 80 Methuselah 60 60 Percent Survival Percent Surviving at 48 hr. Parental 40 40 Strain 20 20 % Surviving 0 0 % Surviving at 48 hrs wt mth wt mth 0 20 40 60 80 100 120 Males Females Days at 25°C

  35. w Oxidative Damage and Sensitivity to Oxidative Stress Increases with Age. Manipulations that Increase Life Span will Reduce Oxidative Damage and Sensitivity to Oxidative Stress. w Predictions of the Oxidative Stress Theory of Aging Predictions of the Oxidative Stress Theory of Aging Caloric Restriction Long-Lived Invertebrate Mutants Dwarf, p66shc-/-, and Igf1r+/- Mice

  36. Paraquat Toxicity 100 -/+ lgf1r 90 80 70 +/+ lgf1r Fraction alive (%) 60 50 40 30 24 18 30 36 24 36 48 60 72 Time after injection (h) IGF1 Receptor Heterozygous Mice Survival 100 90 -/+ lgf1r (n = 19) 80 70 60 Percent Survival +/+ lgf1r 50 (n = 17) 40 30 20 20 10 0 6 12 Age (months) Nature, Holzenberger et al., 4 Dec 2002

  37. w Oxidative Damage and Sensitivity to Oxidative Stress Increases with Age. Manipulations that Alter Oxidative Damage/ Oxidative Stress will Alter Life Span. Manipulations that Increase Life Span will Reduce Oxidative Damage and Sensitivity to Oxidative Stress. w w Predictions of the Oxidative Stress Theory of Aging Drosophila

  38. H+ O2- + O2- + ONOO- NO. O2- H+ H+ Antioxidant Defense System Peroxisome Mitochondria -Oxidation pathway  H2O2  Catalase H2O + O2 cytc III IV I OH. + OH- e- e- 1/2 O2 H2O NADH NAD+ Fe2+ (Fenton reaction) MnSOD ADP + Pi H2O2 2 O2- O2 + H+ V ATP 2GSH GPX ATP GSSG CuZnSOD 2H2O H2O2 2H2O GPX ADP 2GSH GSSG Cytosol

  39. Effect of Overexpressing Cu/ZnSOD on the Life Span of Drosophila 100 80 + HS Control 60 % Alive FLP + Cu/ZnSOD3A1 40 20 0 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 Days at 250C Sun and Tower (1999) Mol.Cell Biol. 19, 216-228.

  40. Methionine Sulfoxide Reductase Overexpression of Methionine Sulfoxide Reductase in Drosophila O O H N C H C O H H N C H C O H 2 2 C H C H 2 [ROS] 2 C H C H 2 2 S O S C H C H 3 3

  41. Overexpression of Methionine Sulfoxide Reductase in Drosophila Ruan et al., PNAS 99: 2748-53, 2002

  42. w Oxidative Damage Increases with Age. Manipulations that Alter Oxidative Damage/ Oxidative Stress will Alter Life Span. Manipulations that Increase Life Span will Reduce Oxidative Damage or Increase Resistance to Oxidative Stress. w w Predictions of the Oxidative Stress Theory of Aging Drosophila Transgenic/Knockout Mice

  43. Transgenic Mice Overxpressing Cu/ZnSOD protection againt ischemia damage in brain nTg SOD1 Tg Chan et al., J. Neurosci. 18: 8292, 1998

  44. Life Span of Cu/ZnSOD Transgenic Mice 1.0 0.9 WT 0.8 hTgSOD1+/o hTgSOD1+/+ 0.7 0.6 0.5 Survival 0.4 0.3 0.2 0.1 0.0 5 10 15 20 25 30 35 Age (months) Huang et al., Journal of Gerontology, January 2000

  45. H+ O2- + O2- + ONOO- NO. O2- H+ H+ Antioxidant Defense System Peroxisome Mitochondria -Oxidation pathway  H2O2  Catalase H2O + O2 cytc III IV I OH. + OH- e- e- 1/2 O2 H2O NADH NAD+ Fe2+ (Fenton reaction) MnSOD ADP + Pi H2O2 2 O2- O2 + H+ V ATP 2GSH GPX ATP GSSG CuZnSOD 2H2O H2O2 2H2O GPX ADP 2GSH GSSG Cytosol

  46. Characterization of Sod2+/- Mice J. Biol. Chem. 273: 28510-28515, 1998 Arch. Biochem. Biophys. 363: 91-97, 1999 Physiol. Genomics 16: 29-37, 2003 wReduced MnSOD Activity in all Tissues wNo Compensation from other Antioxidant Enzymes wAltered Mitochondria Function wIncreased Sensitivity to Oxidative Stress

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