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The Basics of Free Radical
Chemistry and Their Biology
Barshop Institute for
Longevity and Aging Studies
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
Oxidative Stress Theory of Aging
w Oxidative Damage Increases
with Age
w Loss of Cellular Function
w Increased Pathology and Aging
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage Increases with Age.
Lipid Peroxidation
Protein Oxidation
DNA Oxidation
Problems with Measuring Oxidative Damage:
Multiple Products of Oxidative Damage
Sensitivity of Assays
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
DNA Oxidation
O
H
N
NH
O
NH
N
NH2
Sugar
8-Oxo-deoxy-guanosine (oxo8dG)
Response (µA)
HPLC-EC Analysis of DNA Oxidation
2dG
80
DNA Isolation:
60
Phenol
Sodium Iodide
40
NAR 29: 2117, 2001
20
0
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Response (nA)
Retention time (minutes)
1.50
1.00
oxo8dG
0.50
0.00
-0.50
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Retention time (minutes)
14.0
Effect of Age on DNA Oxidation
Liver (mtDNA)
*
*
0.25
0.06
0.2
0.15
0.04
0.1
0.05
0.02
oxo8dG /105dG
0.08 Liver (nDNA)
oxo8dG /105dG
0
0
Kidney
*
0.06
0.05
0.04
0.03
0.02
0.01
Heart
0.06
0.05
0.04
0.03
0.02
0.01
0
5
*
10
20
Brain
*
Muscle
5
Age (months)
10
*
20
PNAS 98: 10469, 2001
Lipid Peroxidation
Formation of Isoprostanes
From: J.D. Morrow et al., Prog. Lipid Res.
Vol. 36, No.1, 1997, pp.1-21
Effect of Age on Isoprostane
Levels in F344 Rats
Kidney
1.0
0.8
0.6
0.4
0.2
0
*
F2-isoprostane ng/g tissue
F2-isoprostane ng/ml
Plasma
12
10
*
8
6
4
2
0
Ward et al., J. Gerontol,. 60: 847-51, 2005
Protein Oxidation
Amino Acids Sensitive to Oxidation
Met
Cys
Tyr
Trp
Methionine sulfoxide
Disulfides, mixed disulfides,, HNE-Cys
Dityrosine, nitrotyrosine, chlorotyrosines, dopa
Hydroxy-and nitro-tryptophans, kynurenines
Phe
Val,Leu
His
Glu
Pro
Thr
Arg
Lys
Hydroxyphenylalanines
Hydroperoxides
2-Oxohistidine, asasparagine, aspartate, HNE-His
Oxalic acid, pyruvic acid
Hydroxyproline, pyrrolidone, glutamic semialdehyde
2-Amino-3-ketobutyric acid
Glutamic semialdehyde, chloramines
MDA-Lys, HNE-Lys, acrolein-Lys, CML
Metal-Catalyzed Protein
Oxidation
Assay for Carbonyl Groups in Proteins
Lysine
Arginine
Proline
Threonine
Levine, Free Radical Biology and Medicine 32: 790-796 2002
Future Studies in Protein
Oxidation
w Identify proteins that show increased
damage with age.
Assay for Carbonyl Groups in Proteins
Lysine
Arginine
Proline
Threonine
Carbonyl Probes
HO
OH
O
O2N
H2N
NH
C-OH
NO2
2,4, Dinitrophenylhydrazine
O
NH-C-NHNH2
S
Fluorescein 5 - thiosemicarbazide
(FTC)
Chaudhuri et al., Mech. Age. Dev.127: 894, 2006
Carbonyl Probes
0.25
Intensity (F/C)
HO
OH
O
0.20
C-OH
0.15
O
0.10
NH-C-NHNH2
S
0.05
Fluorescein 5 - thiosemicarbazide
(FTC)
0
4-6
22-23
Age (months)
Chaudhuri et al., Mech. Age. Dev.127: 894, 2006
Effect of Age on Protein Oxidation
(carbonyl groups) in Liver
Young
Old
Fluorscence (carbonly levels)
Effect of Age on Protein Oxidation
(carbonyl groups) in Liver
Young
Old
Sypro Ruby (protein levels)
Effect of Age on Protein Oxidation
(carbonyl groups) in Liver
Young
Old
Albumin
(precusor)
Aldolase2B
Regucalcin
21 mole/mole protein
Cu/ZnSOD
Chaudhuri et al., Mech. Age. Dev.127: 894, 2006
Future Studies in Protein
Oxidation
w Identify proteins that show increased
damage with age.
w Identify the effect of age on oxidative
damage other than carbonyl groups.
Surrogate Marker of Oxidative
Damage
Sensitivity to Oxidative Stress
Paraquat
NADP+ + H+
+
.
O2
NADPH
+
O2
Diquat
Sensitivity to Paraquat Toxicity
Young
% Survival
100
80
60
40
Old
20
0
0
1
2
3
4
5
Days after Paraquat (50 mg/kg)
6
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage and Sensitivity to
Oxidative Stress Increases with Age.
w
Manipulations that Increase Life Span will
Reduce Oxidative Damage and Sensitivity
to Oxidative Stress.
Caloric Restriction
Effect of Caloric Restriction on Lifespan
100
PERCENT ALIVE
90
80
70
60
Lifespan (mos)
50
Median
n
40
Maximum
Ad Libitum 77
30.9
33
Caloric Restriction 79
38.3
49
30
20
10
0
0
5
10
15
20
25
30
35
Age in Months
40
45
50
Effect of Caloric Restriction
on Oxidative Damage
Liver
Kidney
Isoprostanes
Isoprostanes
DNA Oxidation
14
6
4
2
0
*
*
*
20
0.8
0.4
0
15
10
5
0
oxo8dG/2dG x 105
8
1.2
8-isoPGF2a ng/ml
10
0.8
25
oxo8dG/2dG x 105
8-isoPGF2a ng/ml
12
DNA Oxidation
*
0.6
0.4
0.2
0
J. Gerontol. 60: 847, 2005
% Survival
Sensitivity to Oxidative Stress
(Paraquat Toxicity)
100
80
60
Caloric Restriction
40
20
0
Ad Libitum
0
1
2
3
4
5
6
7
8
Days After Treatment
Clin. Endocrinol. Metab., 2004
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage and Sensitivity to
Oxidative Stress Increases with Age.
w
Manipulations that Increase Life Span will
Reduce Oxidative Damage and Sensitivity
to Oxidative Stress.
Caloric Restriction
Long-Lived Invertebrate Mutants
Longevity Mutants of Caenorhabditis elegans
Fraction Survival (%)
100
Age-1
Parental Stock
Parental Stock
80
60
40
20
0
0
10
20
30
40
50
Age (days)
Thomas E. Johnson, 1987
60
Longevity Mutants of Caenorhabditis elegans
LD50 (mM H 2O 2)
25
20
fer-15 age-1
15
fer-15
10
5
0
0
5
10
15
20
25
30
Adult Age (days)
P. L. Larsen, PNAS 90:8905, 1993
Protein Oxidation in Longevity
Mutants of C. elegans
Life Span
(100%)
(70%)
(160%)
Ishi et al., Free Radical Biol. Med. 33: 1021, 2002
Survival of Long-Lived Drosophila
Percent Survival
100
80
Methuselah
60
Parental
Strain
40
20
% Surviving
0
0
20
40
60
80
Days at 25°C
100
120
Percent Surviving at 48 hr.
Paraquat Toxicity
100
80
60
40
20
0
% Surviving
at 48 hrs
wt mth
Males
wt mth
Females
Lin et al. (October, 1998)
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage and Sensitivity to
Oxidative Stress Increases with Age.
w
Manipulations that Increase Life Span will
Reduce Oxidative Damage and Sensitivity
to Oxidative Stress.
Caloric Restriction
Long-Lived Invertebrate Mutants
Dwarf, p66shc-/-, and Igf1r+/- Mice
IGF1 Receptor Heterozygous Mice
Survival
Paraquat Toxicity
100
100
-/+
lgf1r
(n = 19)
80
70
60
+/+
lgf1r
(n = 17)
50
40
30
lgf1r
90
Fraction alive (%)
Percent Survival
90
20
-/+
80
70
lgf1r
+/+
60
50
40
10
30
0
6
12
18
24
Age (months)
30
36
24
36
48
60
72
Time after injection (h)
Nature, Holzenberger et al., 4 Dec 2002
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage and Sensitivity to
Oxidative Stress Increases with Age.
w
Manipulations that Increase Life Span will
Reduce Oxidative Damage and Sensitivity
to Oxidative Stress.
w
Manipulations that Alter Oxidative Damage/
Oxidative Stress will Alter Life Span.
Drosophila
Antioxidant Defense System
Peroxisome
Mitochondria
H+
cytc
H+
III
IV
I
eNADH
ADP + Pi
2
O2
.
OH + OHH2O
Fe2+ (Fenton reaction)
MnSOD
H2O2 + O2
2 O2-
ATP
ATP
ADP
O2- + NO.
GPX
2H2O
2GSH GSSG
V
e-
NAD+
1/
-Oxidation
pathway

H2O2
 Catalase
H2O
+
O2
H+
O2- +
O2-
CuZnSOD
H2O2
2GSH
2H2O
GPX
ONOO-
Cytosol
GSSG
Effect of Overexpressing Cu/ZnSOD on
the Life Span of Drosophila
100
% Alive
80
+ HS
Control
60
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.
Overexpression of Methionine
Sulfoxide Reductase in Drosophila
O
O
H2N
CH
C H2
C
H2N
OH
[ROS]
C H3
C
C H2
C H2
C H2
S
CH
Methionine
Sulfoxide
Reductase
S
C H3
O
OH
Overexpression of Methionine
Sulfoxide Reductase in Drosophila
Ruan et al., PNAS 99: 2748-53, 2002
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage Increases with Age.
w
Manipulations that Increase Life Span will
Reduce Oxidative Damage or Increase
Resistance to Oxidative Stress.
w
Manipulations that Alter Oxidative Damage/
Oxidative Stress will Alter Life Span.
Drosophila
Transgenic/Knockout Mice
Transgenic Mice Overxpressing Cu/ZnSOD
protection againt ischemia damage in brain
nTg
SOD1 Tg
Chan et al., J. Neurosci. 18: 8292, 1998
Life Span of Cu/ZnSOD Transgenic Mice
1.0
0.9
WT
0.8
hTgSOD1+/o
0.7
hTgSOD1+/+
0.6
0.5
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
Antioxidant Defense System
Peroxisome
Mitochondria
H+
cytc
H+
III
IV
I
eNADH
ADP + Pi
2
O2
.
OH + OHH2O
Fe2+ (Fenton reaction)
MnSOD
H2O2 + O2
2 O2-
ATP
ATP
ADP
O2- + NO.
GPX
2H2O
2GSH GSSG
V
e-
NAD+
1/
-Oxidation
pathway

H2O2
 Catalase
H2O
+
O2
H+
O2- +
O2-
CuZnSOD
H2O2
2GSH
2H2O
GPX
ONOO-
Cytosol
GSSG
Characterization of Sod2+/- Mice
J. Biol. Chem. 273: 28510-28515, 1998
Arch. Biochem. Biophys. 363: 91-97, 1999
Physiol. Genomics 16: 29-37, 2003
w
Reduced MnSOD Activity in all Tissues
w
No Compensation from other Antioxidant Enzymes
w
Altered Mitochondria Function
w
Increased Sensitivity to Oxidative Stress
Effect of Age on nDNA and
mtDNA Oxidation
Nuclear
0.08
Liver
d
0.07
b
0.06
0.04
b
c
0.05
0.9
Brain c
b
a
0.03
a
0.02
0.01
0
oxo8dG/2dG x105
oxo8dG/2dG x105
0.09
Mitochondrial
0.8
Liver d
Brain
0.7
0.6
c
0.5
b
0.4
0.3
a
0.2
0.1
0
3-6
26
3-6
26
3-6
26
3-6
26
Age (months)
Age (months)
Sod2+/+
Sod2+/Physiol. Genomics 16: 29-37, 2003
Lifespan of Sod2+/- Mice
Percent Survival
100
Sod2+/+
Sod2+/-
(n = 69)
(n = 70)
80
Mean
(SEM)
894
(19)
918
(19)
60
Maximum
Survival
1189
1239
40
WT
Sod2+/-
20
0
0
200
400
600
800
1000
1200
Age (days)
Physiol. Genomics 16: 29-37, 2003
Lifespan of Sod2+/- Mice
Percent Survival
100
Biomarkers of aging
not altered in the
Sod2+/- mice
80
60
40
WT
Sod2+/-
20
0
0
200
400
600
800
1000
1200
Age (days)
Physiol. Genomics 16: 29-37, 2003
Predictions of the
Oxidative Stress Theory of Aging
w
Oxidative Damage Increases with Age.
w
Manipulations that Increase Life Span will
Reduce Oxidative Damage or Increase
Resistance to Oxidative Stress.
w
Manipulations that Alter Oxidative Damage/
Oxidative Stress will Alter Life Span.
?
Oxidative Stress Theory of Aging
w Oxidative Damage Increases
with Age
w Loss of Cellular Function
w Increased Pathology and Aging
Methionine Sulfoxide Reductase
Knockout Mice
O
O
H2N
CH
C H2
C
H2N
OH
[ROS]
C H3
C
C H2
C H2
C H2
S
CH
Methionine
Sulfoxide
Reductase
S
C H3
O
OH
Methionine Sulfoxide Reductase
Knockout Mice
WT
MsrA+/MsrA -/-
1.0
Cumulative survival
0.8
n
Mean (d)
14
8
680
680
17
409
0.6
0.4
Cumulative survival
0.2
0.0
0
150
300
450
600
750
900
Time (days)
Moskovitz et al., PNAS 98: 12920-25, 2001
Explanation for Contradictory
Data
• Not all types of oxidative damage are
equal.
Enzyme Inactivation
Enzyme Inactivation
Protein Aggregation
AGING
Enzyme Inactivation
Protein Aggregation
Protein Oxidation in Sod2+/- Mice
Carbonyl Groups
12
0.12
10
0.10
Carbonyl Levels
% Metionine Sulfoxide
Methionine Sulfoxide
8
6
4
2
0.08
0.06
0.04
0.02
0
0
Liver Kidney Cortex
4–6
24 – 28
Age (months)
Sod2+/+
Sod2+/-
Stadtman et al., Biochim. Biophys. Acta, 2004
Oxidative Stress Theory of Aging
w Oxidative Damage Increases
with Age
w Loss of Cellular Function
w Increased Pathology and Aging
Oxidative Stress Theory of Aging
w Oxidative Damage Increases
with Age
w Loss of Cellular Function
w Increased Pathology and Aging
Protein
Protection
ROS
(Msr, Trx, GSH)
Protein
Oxidation
Protein
Turnover
Heat
Shock
Proteins
Research on the Role of Oxidative
Stress in a Disease
w Increase in Oxidative Damage
w Alteration in Cellular Function
w Increased Pathology