1. Oxidants and Aging Rolf J. Mehlhorn Lawrence Berkeley Laboratory e-mail: rjmehlhorn@lbl.gov

2. The Free Radical Theory of Aging “Aging results from the deleterious effects of free radicals produced in the course of cellular metabolism” Harman D., Aging: A theory based on free radical and radiation chemistry, J. Gerontol. 11: 298, 1956

3. What is a free radical? It is a molecule having unpaired electrons but is distinct from transition metal ions A single molecule can have several unpaired electrons, notably oxygen which can be thought of as a biradical Free radicals rarely occur in nature Magnetic properties allow for detection

4. Free Radical Chemistry Reactive radicals attack indiscriminately Can add to unsaturated bonds Can abstract electrons or hydrogen atoms Propagate chain reactions Can cause bond scission Can cause crosslinking Produce secondary toxic agents

5. Implications for Aging Some free radical-induced chemical modifications may have unique impacts Crosslinked products may not be degradable Scission of bonds in DNA, particularly multiple events may erase vital information

6. Oxygen Toxicity At high pressure oxygen is always lethal Convulsions precede death -- the onset time is a direct function of the oxygen pressure Animals have a limited capacity to adapt to oxygen toxicity Microbes from anaerobic environments are killed by traces of oxygen

7. How can oxygen be toxic? It is chemically much more reactive than most other small molecules It can be activated by sequential one- electron reductions to superoxide, hydrogen peroxide or hydroxyl radical (ROS) It can be converted to reactive singlet oxygen

8. What are the Major Oxidants? Hydroxyl radical (OH. ) Hypochlorite (HOCl) Singlet oxygen 1 O 2 Peroxynitrite (OONO - ) Hydrogen peroxide (H 2 O 2 ) Free or loosely-bound iron, copper or heme Superoxide radical (O 2. - ) Nitric oxide (NO. )

9. Lipid Peroxidation PUFAs* contain weakly bonded hydrogen atoms between double bonds Chain reactions are probable because of high local concentrations of double bonds Lipids are also oxidized by enzymes, producing signaling molecules (eicosanoids) PUFAs may protect more vulnerable targets *PUFAs means polyunsaturated fatty acids

10. Oxidant Sources Enzymes involved in cell signaling Immune cells “Leaky” electron transport Damaged proteins and lipids Toxins (food, water) Irradiation (UV) Smoke Regulated Unregulated

11. Major Antioxidants Vitamins E and C Thiols, particularly glutathione Uric acid Superoxide dismutases (Cu/Zn or Mn SOD) Catalase and glutathione peroxidase Heme oxygenases Protein surface groups (Msr)

12. Free Radical Damage Markers Fluorescent age pigments DNA products like hydroxylated bases Protein products like carbonyls & bityrosine Lipid products like isoprostanes

13. Mitochondria Have been regarded as the “pacemakers of aging” by some investigators Superoxide is released as a direct function of the energy state (high ATP/ADP, strongly reducing conditions) Uncoupling proteins dissipate excess energy as heat, are induced by superoxide; some require free fatty acids for activity

14. Immune System Oxidants NAD(P)H oxidases produce superoxide Myeloperoxidase produces hypochlorite Nitric oxide synthase produces nitric oxide Nitric oxide and superoxide combine to form peroxynitrite Free iron or copper react with hypochlorite or peroxynitrite to form hydroxyl radicals

15. Glucose and Oxidants In cell culture models high glucose correlates with oxidant production Three diabetes-linked effects can be correlated with superoxide production Blood glucose and food consumption (next slide) Insulin pathway and life-span extension

16. Effects of Pro- and Anti-Oxidants Implications for Free Radical Theory Knockout of UCPs provides no evidence pro or con High dose IR affects mean life span only SOD knockout studies: Mn vital, Cu/Zn not HO-1 vital LMW compounds not effective (next slide) Life prolongation reported with some agents

17. Life Span Extension Reports