Understanding Reactive Oxygen Species and Flavonoids in Aging and Inflammation

1. My Research • Biological Oxidations • Theory of Aging – Chemopreventive Agents • Antioxidants • Role of Ca2+ and Zn2+ in biological oxidations • Destruction of xenobiotics by O2 at RT and 1 atm. • Chemical mechanisms of O2 evolution in plants • All involve Reactive Oxygen Species (ROS) University of Idaho

2. Reactive Oxygen Species • O2 is surprisingly un-reactive • O=O bond is about 120 kcal/mol • Biological systems have learned how to overcome activation barriers • Reduction of O=O bond results in more reactive forms University of Idaho

3. •O=O• b.o. = 2  120 kcal/mol +e- +2e- +2H+ •O-O• b.o. = 1.5  80 kcal/mol G +4e- +4H+ HO-OH b.o. = 1  50 kcal/mol 2H2O Reactive Oxygen Species University of Idaho

4. Reactive Oxygen Species in Biology University of Idaho

5. Oxidative Phosphorylation University of Idaho

6. Oxidative Phosphorylation & ROS

7. ROS and the Aging Hypothesis Mitochondria -Energy producing region of cell oxidative phosphorylation -Contains its own DNA ROS production in Mitochondria -1% of O2 consumed results in ROS University of Idaho

8. ROS and the Aging Hypothesis Mitochondrial DNA -Encodes information for respiration enzymes -Mutates 40x faster than nuclear DNA -Respiration enzymes become less efficient with age University of Idaho

9. ROS and Inflammation Phagocytes Infections -viruses -bacteria -parasites Foreign Substances -smoke -asbestos Activation of Inflammatory Cells Damaged Tissue -heat -mechanical -UV ROS RNS University of Idaho

10. ROS and Inflammation HOCl Cl- O2.- H2O2 NO. ONOO.- University of Idaho

11. The Fenton Reaction -Reduction of HO-OH H2O2 + FeII = FeIII + HO- + HO. HO. + e- = HO- Eo = 1.8 volts -HO. reacts with organics with diffusion limited kinetics -Possible Roles for O2.- 2O2.- + 2H+ H2O2 + O2 O2.- + FeIII O2 + FeII University of Idaho

12. Iron Enzymes and the Fenton Reaction Hemes/Cytochromes Oxygenases Oxidases H2O2 HO. + reducing agent • All Fe-containing enzymes are quite good Fenton Reaction agents. • Oxidative Stress University of Idaho

13. University of Idaho

14. Inflammation • The Good Inflammation protects the body • Destroys invading pathogens • Dissolves damaged tissue • The Bad Chronic or prolong inflammation • Allergies and Autoimmune Diseases • All the many types of allergies • Many of the autoimmune diseases University of Idaho

15. Chronic Inflammation • The Uglycollateral damage results in….. • Asthma • Rheumatoid Arthritis (RA) • Multiple Sclerosis (MS) • Systemic Lupus Erythematosus (SLE) • Chronic Obstructive Pulmonary Disease (COPD) • Huntingdon Disease • Parkinson’s Disease • Alzheimer’s Disease • Circulatory Diseases University of Idaho

16. Antioxidant Therapy • Prevention of cumulative oxidative damage to physiological components • HO. Scavenging – Vitamin E/Tocopherol • Transition Metal Chelation • Flavonoids – Plant coloration agents University of Idaho

17. Flavonoids Are found in most leafy plants, examples…….. Grapes – proanthocyanidins Pycnogenol – bark of French maritime pine tree Green Tea – catehins Citrus – flavones Soy – isoflavones controversial University of Idaho

18. Flavonoids in the News Wine seems protective against dementia By Ed SusmanUPI Science News NEW YORK, Oct. 14 (UPI) -- Something in a glass of wine appears to protect people from the most common forms of dementia -- Alzheimer's disease or stroke-cause mental deterioration -- doctors said Monday………. University of Idaho

19. Flavonoids Plant-borne polyphenols Phytochemical Coloration – Fall Leaves ? UV protection ? Protection against pathogens ? Transition metal transport Average human consumption 100-500 mg per day Mediterranean Diet is Heavy in Flavonoids Medicinal benefits Chemopreventive Agent Antioxidant Anti-inflammatory Agent Anti-cancer Agent

20. Flavonoids are Chemo-preventive Agents University of Idaho

21. Flavonoid Structure • 200-300 Related Polyphenols • Substitution on the C ring distinguishes the classes flavonoids • Substitution on the A and B rings distinguish structures within a class • Three potential metal binding sites exist University of Idaho

22. My Interests with Flavonoids Molecular level basis for its antioxidant action. Ability to moderate the Fenton Reaction Structure-Activity Relationship 300 flavonoids which might be most active University of Idaho

23. Hypothesis – Flavonoid Action Known to bind transition metals Interfere with oxidations – Fenton Reaction -Moderation of p450 activity -LMW Iron Structure-Activity Relationships -Catechol Containing Flavonoids University of Idaho

24. Modes of Antioxidant Action Radical Scavenging – may not be the only action Flavonoid + OH. = Flavonoid-OH Metal Binding Thermodynamic/Kinetic Inhibition of Fenton Reaction University of Idaho

25. Iron-Flavonoid Complexes • Likely metal binding sites • Does the iron-flavonoid complex drive the Fenton Reaction • Metal binding constants • M + nL  MLn Quercetin University of Idaho

26. LMW Fe University of Idaho

27. Antioxidant Chelate University of Idaho

28. FeIIIATP Cyclic Voltammetric Studies FeIIIATP+Querectin Top) Moderation of the Fenton reaction characteristic of 0.25 mM FeIIIATP, 8.6 mM H2O2 in 0.0625 M HEPES buffer pH 7.4 by quercetin. The top graph are the cyclic voltammograms take at 20 mV/s for various quercetin/iron ratios. The quercetin/FeIIIATP ratios (I-VII) are indicated on the x-axis of the bottom graph. The catalytic wave at -0.400 volts are plotted versus the quercetin/iron ratio in the bottom graph. The current of point VII at –0.400 volts is the same as the background current (0.00 mM H2O2)

29. Results of CV studies • Flavonoid-Quercetin • Inhibits the Fenton Reaction of FeIIATP complex • Maximum Inhibition 1:1 [Q]:[FeIIATP] • First demonstration of a molecular-level antioxidant action University of Idaho

30. Metal Binding Studies University of Idaho

33. Summary of Metal Binding Studies University of Idaho

34. Summary of LMW Fe Work • Electrochemical Evidence • -Ability to inhibit the Fenton Reaction • Thermodynamic Binding Constants • -Relatively High • -Catechol Containing University of Idaho

35. Cytochrome p450 • Involved with many pro-oxidant actions • Redox is based on an Fe-porphryin complex • Hydroxylation is a key step in the detoxification of xenobiotics • Problems arise in that p450 action sometimes increases toxicity of some xenobiotics University of Idaho

36. H C C H 3 3 + + N N N N Fe N N + + N N C H H C 3 3 Inhibition of p450 Action Fe rric T etra M ethyl Py ridyl P orphyrin ( FeTMPyP ) Model System: University of Idaho

37. Quercetin and FeIII-TMPyP Spectroscopic data -binding constants Electrochemical Data -evidence for binding -inhibition of the Fenton reaction University of Idaho

38. Spectroscopic Evidence Weak Interaction University of Idaho

39. Spectroscopic Evidence Stronger University of Idaho

40. Spectroscopic Evidence Strongest University of Idaho

41. Electrochemical Evidence University of Idaho

42. Q Q FeIII FeIII Binding of Q to FeIIITMPyP OOH University of Idaho

43. O H O H O H O H H O O O O H Quercetin 3'4'-dihydroxyflavonone O H O O O O H Flavonol O Summary of Flavonoids+p450 • Q is a relatively strong binder to FeIII-TMPyP >> > • Q is able to moderate the Fenton Reaction of Fe-TMPyP without directly binding to the metal center University of Idaho

44. Acknowledgements Funding NIH NSF NASA BLM Mark D. Engelmann Tina Nordoun Ryan Hutcheson Rob Bobier Terry Hiatt University of Idaho

45. UI Stipend goes along way in Moscow, Idaho TA+Renfrew Scholarship 12 months $17,000 Effective Fees -$1,900 Health Insurance -$800 Average One Bedroom Apt $350-550 Average Two Bedroom Apt $450-650 University of Idaho

46. University of Idaho

47. Visit Our Web Site! www.chem.uidaho.edu University of Idaho