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THE FOUR RULES OF AGING
1) AGING IS UNIVERSAL
2) AGING IS PROGRESSIVE
3) AGING IS ENDOGENOUS
4) AGING-POSTMITOTIC (DELETEREOUS)
SOD, CAT, GPx, GR, GSH, ASC (%)
0 5 15 20 25 35 40
MAXIMUM LONGEVITY (YEARS)
VERTEBRATES
0
100
80
60
40
20
10 30
PIGEON
20 40 60 80 100 1200MAXIMUM 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)
LOW ANTIOXIDANT LEVELS
LOW RATE OF MITOCHONDRIALOXYGEN 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 inJ. Comp. Physiol. In 1998
COMPARATIVE STUDIES OF MITOCHONDRIAL H2O2 PRODUCTION IN MAMMALS AND BIRDS WITH DIFFERENT MAXIMUM LONGEVITIES (MLSP)
PIGEONMLSP= 35 YEARS
RATMLSP= 4 YEARS
H2O2 GENERATION. HEART MITOCHONDRIA
MOUSEMLSP= 3.5 YEARS
PARAKEETMLSP= 21 YEARS
(Melopsittacus undulatus)
CANARYMLSP= 24 YEARS(Serinus canarius)
S
S
S
ROS
ROS
NO ROS
O2
O2
O2
Cx I Cx IVCx III
Cx II
Q
Succinate
Pyruvate/Malate
O2
H2O
c
ROT
TTFA
AA
LOW RATE OF MITOCHONDRIALOXYGEN RADICAL PRODUCTION
LOW OXIDATIVE DAMAGE IN MITOCHONDRIAL DNA
(LONG-LIVED ANIMAL SPECIES)
(HYPOTHESIS)
8-oxoGua
O
H2N
O
NH
HN
HN
N
8-o
xodG
/ 10
5d
G in
hea
rt m
t DN
A
Rat
Mouse
Guinea pig
Horse
Pig
Rabbit
Sheep
Cow
10 403020 50
12
0
10
8
6
4
2
MAXIMUM LONGEVITY (YEARS)
G.Barja & A. Herrero. FASEB J. 14: 312-318 (2000)
r=-0.92p<0.000
(HEART)
(SAME RESULT IN BRAIN)
10 403020 500
2
1
0
8-o
xod
G/
105 d
G i
n n
DN
A
MLSP (YEARS)
Rat
Mouse Guinea Pig
Horse
Rabbit Cow
(BRAIN)
(SIMILAR RESULTS IN HEART)
H2O2 PRODUCTION HEART MITOCHONDRIA
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
nm
ole
s H
2O2/
min
· m
g p
rot AC
OCOR
Pyruvate/ malateLong-term Caloric Restriction (1 year)
0.0
0.5
1.0
1.5
2.0
2.5
H2O2 PRODUCTION HEART MITOCHONDRIA
nm
ole
s H
2O2/
min
· m
g p
rot
Succinate (+ Rotenone)
Long-term Caloric Restriction (1 year)
ACOCOR
Cx I Cx IVCx III
Cx II
Q
Succinate
Pyruvate/Malate
O2
H2O
c
ROT
TTFA
AA
Mitochondrial matrix
Intermembrane space
NADH NAD + +H +
FMN
nxFeSFeCN
O2
O2·-
QH 2
Q
H2O2H+
ROT
Q Complex III
Complex I
Pyr/ mal
Pyr/ mal
H+
e-
Q ·-Fe SN-2
C. Krebs
1,2-NAPHTOQUINONE
ROTENONE
FERRICYANIDE
MENADIONE
p-ClHg BENZOATE
ETHOXYFORMIC ANHYDRIDE
NADH
Q
Cx I
O2 CONSUMPTION HEART MITOCHONDRIA
Pyruvate/ malateLong-term Caloric Restriction (1 year)
0
8
16
24
32
nm
ole
s O
2/ m
in·
mg
pro
t ACOCOR
0.0
0.5
1.0
1.5
2.0
2.5
FREE RADICAL LEAK HEART MITOCHONDRIA
Pyruvate/ malateLong-term Caloric Restriction (1 year)
ACOCOR
(%)
**
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/105dG) 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/105dG in nuclear DNA
8-oxodG in HEART mtDNA
Long-term Caloric Restriction (1 year)
0
1
2
3
4
5
6
7
8-o
xod
G/ 1
05 d
GACOCOR
*
MITOCHONDRIAL ROS PRODUCTION RATE
AGING RATE
RATE OF ACCUMULATION OF mtDNA MUTATIONS
MITOCHONDRIAL DNA
CALORIC RESTRICTION
LONG-LIVEDANIMALS
(-) (-)
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.
8-o
xod
G/1
05d
G i
n m
tDN
A
HEARTBRAIN0.0
1.0
2.0
3.0
4.0
5.0
*
*WILD TYPEAMES DWARF
LOW [8-oxodG]
Low repair ?
Long-lived animals
Restricted animals
Low attack
HIGH [8-oxodG]
High (8-oxo mtDNArepair ? (post.mitot.)
Short-lived animals
Ad libitum animals
High attack
(Low mitoch. ROS product.)
(High mitoch. ROS product.)
Low 8-oxodG mtDNArepair ? (post.mitot.)
MLSP (AÑOS)
10 100
IND
ICE
DE
DO
BL
ES
EN
LA
CE
S (
DB
I)
100
200
300
400
CERDO
RATON
RATA
CONEJOVACA
CABALLO
COBAYA
OVEJA
CORAZON
DOUBLE BOND INDEX OF FATTY ACIDS HEART
MAX. LONGEVITY (YEARS)
(MOUSE)
(G. pig)
(Horse)
(Pig)
(Cow)(Sheep)(Rabbit)
(Rat)
Maximum longevity (years)
% L
INO
LE
IC A
CID
(18
:2n-
6)
Horse
Cow
Pig
Sheep
Rabbit
Guinea Pig
Mouse
10
20
30
40
10010
Rat
0
100
Horse
30
20
10
10
Mouse
Guinea pig
SheepPig
Rabbit Cow
Rat
Maximum longevity (years)%
DO
CO
SA
HE
XA
EN
OIC
AC
ID (
22:6
n-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
MITOCHONDRIAL ROS PRODUCTION RATE
AGING RATE
RATE OF ACCUMULATION OF mtDNA MUTATIONS
MITOCHONDRIAL DNA
CALORIC RESTRICTION
LONG-LIVEDANIMALS
(-) (-) DBI
? MDA
Prot. ox.?
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
Cx I Cx III Cx IV
STATE 4 (RESTING RESPIRATION)
High rateof electron flux
ROS
+ADPSTATE 3 (ACTIVE, ATP is being generated at complex V)
Cx I Cx III Cx IV
Low rate of electron flux
ROS ROS
O2
H2O
O2
H2O
mtc DNA
1/2 O2
e-
S
H2 O2
H2O2
HOMOVANILLIC ACID
HORSERADISH PEROXIDASE
DIMER
FLUORESCENCE312 nm EXC420nm EM
Positive fluorescent metodSpecific for H2O2
SensitiveDoes not alter mitochondriaInstantaneous response to H2O2
No antioxidant interference
(Barja G. (2002) J. Bioenerg. Biomembr. 33:227-233
UNIVERSAL + +
PROGRESSIVE + +
ENDOGENOUS + +
POST-MITOTIC + +
RATE MIT.ROS PROD.
FATTY ACIDUNSATURATION
(DBI)
(Endogenous generation of oxidative damage)
THE RATE OF GENERATION OF
ENDOGENOUS DAMAGE IS THE
CAUSE OF AGING
COLLABORATION
IS MUCH BETTER THAN
COMPETITION
(A L S O I N S C I E N C E)