1. THE FOUR RULES OF AGING 1) AGING IS UNIVERSAL 2) AGING IS PROGRESSIVE 3) AGING IS ENDOGENOUS 4) AGING-POSTMITOTIC (DELETEREOUS)

2. SOD, CAT, GPx, GR, GSH, ASC (%) 051520253540 MAXIMUM LONGEVITY (YEARS) VERTEBRATES 0 100 80 60 40 20 1030 PIGEON 204060801001200 MAXIMUM LONGEVITY (YEARS) MAMMALS 0 100 80 60 40 20 CAT, GPx, GR, GSH, ASC (%) HUMAN From other four different independent laboratories From: López-Torres et al. Mech Ageing Dev 70 (1993) Barja et al Free Radic Res 21 (1994) Pérez-Campo et al J Comp Physiol [B] 163 (1994) Barja et al Comp Biochem Physiol Biochem Mol Biol 108 (1994)

3. LOW ANTIOXIDANT LEVELS LOW RATE OF MITOCHONDRIAL OXYGEN RADICAL PRODUCTION (LONG-LIVED ANIMAL SPECIES) (HYPOTHESIS) 1993 (Barja & cols.: MAD 1993; CBP1994; JCP, 1994; Free Rad. Res. 94, Review (written in 1994, passed through 4 journals & finally published in J. Comp. Physiol. In 1998

5. COMPARATIVE STUDIES OF MITOCHONDRIAL H 2 O 2 PRODUCTION IN MAMMALS AND BIRDS WITH DIFFERENT MAXIMUM LONGEVITIES (MLSP)

6. PIGEON MLSP= 35 YEARS RAT MLSP= 4 YEARS

7. H2O2 GENERATION. HEART MITOCHONDRIA

8. MOUSE MLSP= 3.5 YEARS PARAKEET MLSP= 21 YEARS (Melopsittacus undulatus) CANARY MLSP= 24 YEARS (Serinus canarius)

10. S S S ROS NO ROS O2O2 O2O2 O2O2

12. Cx I Cx IVCx III Cx II Q Succinate Pyruvate/ Malate O2O2 H2OH2O c ROT TTFA AA

13. LOW RATE OF MITOCHONDRIAL OXYGEN RADICAL PRODUCTION LOW OXIDATIVE DAMAGE IN MITOCHONDRIAL DNA (LONG-LIVED ANIMAL SPECIES) (HYPOTHESIS)

14. 8-oxoGua O H 2 N O N H HN HNHN N

15. 8-oxodG/ 10 5 dG in heart mt DNA Rat Mouse Guinea pig Horse Pig Rabbit Sheep Cow 1040302050 1212 0 1010 8 6 4 2 MAXIMUM LONGEVITY (YEARS) G.Barja & A. Herrero. FASEB J. 14: 312-318 (2000) r=-0.92 p<0.000 (HEART) (SAME RESULT IN BRAIN)

16. 1040302050 0 2 1 0 8-oxodG/ 10 5 dG in nDNA MLSP (YEARS) Rat Mouse Guinea Pig Horse Rabbit Cow (BRAIN) (SIMILAR RESULTS IN HEART)

18. H 2 O 2 PRODUCTION HEART MITOCHONDRIA * 0.0 0.2 0.4 0.6 0.8 1.0 1.2 nmoles H 2 O 2 / min· mg prot AC OC OR Pyruvate/ malate Long-term Caloric Restriction (1 year)

19. 0.0 0.5 1.0 1.5 2.0 2.5 H 2 O 2 PRODUCTION HEART MITOCHONDRIA nmoles H 2 O 2 / min· mg prot Succinate (+ Rotenone) Long-term Caloric Restriction (1 year) AC OC OR

20. Cx I Cx IVCx III Cx II Q Succinate Pyruvate/ Malate O2O2 H2OH2O c ROT TTFA AA

21. Mitochondrial matrix Intermembrane space NADH NAD + +H + FMN nxFeS FeCN O 2 O 2 ·- QH 2 Q H 2 O 2 H + ROT Q Complex III Complex I Pyr/ mal H + e - Q ·- Fe SN-2 C. Krebs

22. 1,2-NAPHTOQUINONE ROTENONE FERRICYANIDE MENADIONE p-ClHg BENZOATE ETHOXYFORMIC ANHYDRIDE NADH Q Cx I

24. O 2 CONSUMPTION HEART MITOCHONDRIA Pyruvate/ malate Long-term Caloric Restriction (1 year) 0 8 16 24 32 nmoles O 2 / min· mg prot AC OC OR

25. 0.0 0.5 1.0 1.5 2.0 2.5 FREE RADICAL LEAK HEART MITOCHONDRIA Pyruvate/ malate Long-term Caloric Restriction (1 year) AC OC OR (%) **

26. Age of animals at time of analysis: adult (7 months), old (24 months). Results are means ± SEM (n). No significant differences due to age or restriction were observed. Effect of long-term caloric restriction (1 year) on the steady-state levels of oxidative damage (8-oxodG/10 5 dG) in rat heart nuclear DNA Adult control 0.61 ± 0.09 (6) Old control 0.49 ± 0.04 (6) Old restricted 0.54 ± 0.05 (6) 8-oxodG/10 5 dG in nuclear DNA

27. 8-oxodG in HEART mtDNA Long-term Caloric Restriction (1 year) 0 1 2 3 4 5 6 7 8-oxodG/ 10 5 dG AC OC OR *

28. MITOCHONDRIAL ROS PRODUCTION RATE AGING RATE RATE OF ACCUMULATION OF mtDNA MUTATIONS MITOCHONDRIAL DNA CALORIC RESTRICTION LONG-LIVED ANIMALS (-)

29. Survival plots of dwarf mice. Left panel: Snell dwarf mice and normal controls. Right panel: Ames dwarf mice and normal controls. Reproduced from Bartke and Turyn 2001.

30. 8-oxodG/10 5 dG in mtDNA HEART BRAIN 0.0 1.0 2.0 3.0 4.0 5.0 * * WILD TYPE AMES DWARF

31. LOW [8-oxodG] Low repair ? Long-lived animals Restricted animals Low attack HIGH [8-oxodG] High (8-oxo mtDNA repair ? (post.mitot.) Short-lived animals Ad libitum animals High attack (Low mitoch. ROS product.) (High mitoch. ROS product.) Low 8-oxodG mtDNA repair ? (post.mitot.)

32. DOUBLE BOND INDEX OF FATTY ACIDS HEART MAX. LONGEVITY (YEARS) (MOUSE) (G. pig) (Horse) (Pig) (Cow) (Sheep) (Rabbit) (Rat)

33. Maximum longevity (years) % LINOLEIC ACID (18:2n-6) Horse Cow Pig Sheep Rabbit Guinea Pig Mouse 10 20 30 40 10010 Rat 0 100 Horse 30 20 10 Mouse Guinea pig Sheep Pig Rabbit Cow Rat Maximum longevity (years) % DOCOSAHEXAENOIC ACID (22:6n-3) Relationship between maximum longevity and linoleic acid (18:2n-6) and docosahexanoic acid (22:6n-3) contents in heart phospholipids of 8 mammalian species

36. MITOCHONDRIAL ROS PRODUCTION RATE AGING RATE RATE OF ACCUMULATION OF mtDNA MUTATIONS MITOCHONDRIAL DNA CALORIC RESTRICTION LONG-LIVED ANIMALS (-)  DBI ?  MDA  Prot. ox. ?

37. OXYGEN RADICAL PRODUCTION IS NOT NECCESSARILY PROPORTIONAL TO OXYGEN CONSUMPTION FRL DECREASES FROM MAMMALS TO BIRDS FROM STATE 4 TO STATE 3 FROM EU- TO HYPERTHYROIDISM

39. Cx ICx IIICx IV STATE 4 (RESTING RESPIRATION) High rate of electron flux ROS +ADP  STATE 3 (ACTIVE, ATP is being generated at complex V) Cx ICx IIICx IV Low rate of electron flux ROS O2O2 H2OH2O O2O2 H2OH2O

40. mtc DNA 1/2 O 2 e-e- S H 2 O 2 H2O2H2O2 HOMOVANILLIC ACID HORSERADISH PEROXIDASE DIMER FLUORESCENCE 312 nm EXC 420nm EM Positive fluorescent metod Specific for H 2 O 2 Sensitive Does not alter mitochondria Instantaneous response to H 2 O 2 No antioxidant interference (Barja G. (2002) J. Bioenerg. Biomembr. 33:227-233

41. UNIVERSAL ++ PROGRESSIVE ++ ENDOGENOUS ++ POST-MITOTIC ++ RATE MIT. ROS PROD. FATTY ACID UNSATURATION (DBI) (Endogenous generation of oxidative damage)

42. THE RATE OF GENERATION OF ENDOGENOUS DAMAGE IS THE CAUSE OF AGING

43. COLLABORATION IS MUCH BETTER THAN COMPETITION (A L S O I N S C I E N C E)