Transcript Lecture #4 - Dr. Ames - Molecular and Cell Biology

“Delaying or accelerating the degenerative diseases of aging”
MCB 135K
Lecture 4, Chapters 5 & 6
Dr. Bruce N. Ames
1/30/08
“Research found that a central factor in
aging is the decay of the mitochondria in
cells”
Dr. Ames is a member of the National Academy of Sciences. He was a member of the National
Cancer Advisory Board and Cancer Institute for many years . He received several medals, such as
the Gold Medal Award of the American Institute of Chemists, the Medal of the City of Paris, and
many others.
To read more about Dr. Ames’ accomplishments and contributions to the study of aging and longevity, please
see:
http://www.bruceames.org/
http://www.chori.org/Principal_Investigators/Ames_Bruce/ames_overview.html
Delaying (or Accelerating) the
Degenerative Diseases of Aging
O2
e-
O2-
e-
H2O2
e-
•OH
Bruce N. Ames
Children’s Hospital Oakland Research Institute
Professor, University of California, Berkeley
e-
H 2O
30 Jan ‘08
Timiras class
Estimated oxidative DNA adducts
per rat liver cell
70,000
Old (26-mo)
60,000
67,000
50,000
40,000
30,000
20,000
10,000
0
Young (4-mo)
24,000
carbonyl content
(nmol/mg protein)
carbonyl content
(nmol/mg protein)
6
2
5
4
1
3
0
2
0
20
40
60
Years
80
100
3
12
20
26 *
Months
Source: E. Stadtman, Science 257, 1220-1224 (1992)
160
MDA
(pmol/mg
protein) 140
Young
*
Old
*
120
100
80
60
*
40
20
*
*
0
Brain
Liver
Heart
Kidney
Lung
6
Proc. Natl. Acad. Sci. USA
Vol. 91, pp. 10771-10778, November 1994
Review
Oxidative damage and mitochondrial decay in aging
(bioenergetics / mitochondrial DNA / cardiolipin / acetyl-L-carnitine / neurodegeneration)
Mark K. Shigenaga, Tory M. Hagen, and Bruce N. Ames*
Division of Biochemistry and Molecular Biology,
401 Barker Hall, University of California, Berkeley, CA 94720
Contributed by Bruce N. Ames, July 27, 1994
H+
Cellular Cytoplasm
H+
H+
H+
H+
H+
Mitochondrial Outer Membrane
H+
Intermembrane
Space
Mitochondrial
Matrix
H+
H+
CoQ
I
Inner Membrane
H+
NAD+
H+
H+
FAD
CytC
H+
Succinate
Fumarate
L-Malate
NADH
Pyruvate
NADH
Oxaloacetate
Dehydrogenase
complex
Acetyl-Co-A
Citrate
Synthase
ATP
CITRIC
ACID
CYCLE
Citrate
H+
H+
V
IV
H+
FADH2
H+
H+
III
II
NADH
H+
O2
H2O
H+
ADP
+
ATP H
Succinyl-Co-A
NADH
-Ketoglutarate
Dehydrogenase
Complex
-Ketoglutarate
Isocitrate
NADH
H+
H+
9
Mitochondria from old rats compared
to those from young rats:
1) Lower Cardiolipin
2) Lower Membrane Potential
3) Lower Oxygen Utilization
4) Increased Oxidant Leakage
Cardiolipin (µg per 106 Cells)
Cardiolipin Levels in 3 and 24 Month Old Rat Hepatocytes
30
20
**
10
0
Young
Old
10
R123 Fluorescence in old and young rat hepatocytes
0.03
Normal cell number
Old
0.02
Young
0.01
0.00
10
100
1000
Fluorescence/cell
11
L-Carnitine/Acetyl-L-Carnitine (ALCAR)
• Transports long-chain fatty acids into mitochondria
• Removes short- and medium-chain fatty acids that accumulate
• Mediates the ratio of acetyl-CoA/CoA
• Decreases with age in plasma and in brain
• Improves cognitive function in rats
12
Cardiolipin (µg per 10 cells)
Effect of ALCAR Supplementation on Cardiolipin Levels
30
20
+ ALCAR
**
10
0
Young
Old
14
Normalized Cell Number
Normalized Cell Number
NO
ALCAR
YOUNG
WITH
ALCAR
OLD
NO
ALCAR
WITH
ALCAR
R123 Fluorescence
in Young and Old
Rat Hepatocytes
R--Lipoic Acid (LA) in mitochondria
• LA reduced to dihydrolipoic acid, a potent antioxidant, & chelator of Fe & Cu
• Coenzyme of pyruvate and -ketoglutarate dehydrogenases
• Involved with carbohydrate utilization for ATP production
15
Fl. Units/O2 Consumed per Minute
Lipoic Acid Lowers Mitochondrial Oxidants in Old Rats
**
20
10
+ LA
+ LA
0
Young
Old
80
70
***p<0.001 vs. young rat group
P<0.05
P<0.01
60
50
Old ***
Young
40
30
20
+ ALCAR + LA
+ ALCAR
+ LA
+ ALCAR + LA
0
+ ALCAR
10
+ LA
MDA (pmol/mg protein)
MDA levels in young and old rats with LA, ALCAR, or both
20
800
*
*
vs. young
# vs. old
600
+ LA + ALCAR
400
#
+ LA + ALCAR
Distance Traveled (cm/hour/day)
Ambulatory Activity before and After Supplementation
with Lipoic Acid (LA) + Acetyl-L-Carnitine (ALCAR)
*
200
0
Young
Old
T cell stimulation index
Age-associated decrease in immune function and the effect
of ALCAR (0.2%) + LA (0.1%) treatment for 2 months. Values
are mean + SEM of 10-11 animals.
30
20
10
0
P <0.001
p <0. 01
Morris Water Maze for Testing Spatial Memory
Spatial Memory relies on
intact hippocampal
function.
Treatments improved poor
memory in old rats
22
Spatial Memory Tested With Morris Water Maze
100
P<0.001
P<0.05
Time in Seconds
80
60
40
+ ALCAR
+ LA
+ ALCAR
+ LA
20
0
Young
Old
Old
Old
Old
Peak procedure: for measuring temporal memory.
Associated with striatum, cerebellum, & hippocampus
14
12.00
PEAK RATE:
measures learning
and motivation.
12
10.00
PEAK TIME: measures
internal clock, food is
rewarded only when
animals push lever 40s
after sound or light signal
10
8
Young
Old
Old + ALCAR
Old + LA
Old + ALCAR + LA
8.00
6.00
6
4.00
4.00
2.00
2.00
0.00
0.00
0
50
100
150
SOUND: Time to Signal
200
0
50
100
150
LIGHT: Time to Signal
200
25
Oxidative Damage to Nucleic Acid in Old Rats by mAb to
oxo8G/oxo8dG: Immunohistochemical stain of neurons
26
Staining of oxidized nucleic acid in neurons
(mAb to oxo8dG in DNA/oxo8G in R NA)
RNA is Oxidized
(92% is removed by RNase)
*oxo8G: 8-hydroxyguanosine; oxo8dG: 8-hydroxy-2’-deoxyguanosine
27
Induction of Phase 2 Enzymes
250
Induced
231
Number of Genes
200
150
100
106
50
33
0
31
-50
30
Repressed
6h
24h
7d (diet)
Modulation of Nrf-2-dependent gene expression by D3T in mouse liver.
Kwak, et al. J bio Chem, 2003
The Journal of Biological Chemistry
Vol. 278, pp. 8135-8145, March 7, 2003
Modulation of Gene Expression by Cancer Chemopreventive
Dithiolethiones through the Keap1-Nrf2 Pathway
IDENTIFICATION OF NOVEL GENE CLUSTERS FOR CELL SURVIVAL
Mi-Kyoung Kwak, Nobunao Wakabayashi, Ken Itoh, Hozumi Motohashi,
Masayuki Yamamoto, and Thomas W. Kensler
Proc. Natl. Acad. Sci. USA
Vol. 101, pp. 3381-3386, March 9, 2004
Decline in transcriptional activity of Nrf2 causes age-related loss
of glutathione synthesis, which is reversible with lipoic acid
Jung H. Suh, Swapna V. Shenvi, Brian M. Dixon, Honglei Liu, Anil K. Jaiswal,
Rui-Ming Liu, and Tory M. Hagen
10
10
1/v
1/v
5
5
0
0
-10
0
10
20
30
40
-50
0
1/[ALCAR, mM]
young
100
**
old
800
*
200
* = vs. young
150
#
150
1/[CoA, mM]
#
#
mM
nmol/min/g
1200
50
# = vs. old
#
100
**
400
50
#
0
0
Vmax
Km for
ALCAR
Km for
CoA
“You’re fifty-seven years old.
I’d like to get that down a bit.”
New Yorker, June 6, 2005
Meta-analysis of acetyl-L-carnitine versus placebo for mild
cognitive impairment and mild Alzheimer’s disease
Montgomery, S.A., Thal, L.J., and Amrein, R., Int. Clin. Psychopharmacol 18:61-71 (2003)
Treatment with alpha-lipoic acid significantly improves both
neuropathic symptoms and deficits in diabetic patients with
symptomatic diabetic neuropathy
90
ITT analysis of 4 phase II-III RCTs plus meta-analysis: 600 mg I.v. per day for 3 weeks
Total Symptom Score (TSS): relative improvement at 3 weeks vs baseline
80
-lipoic acid
placebo
79
70
72
66
Percent
60
66
60
61
60
55
50
48
40
30
25
20
10
n= 77
81
338 165
60
60
241 236
716 542
0
ALADIN I
* p<0.05 vs Placebo
ALADIN III
SYDNEY
NATHAN II
Meta-Analysis
Source: Professor Daniel Ziegler of the Diabetes Research Institute, Düsseldorf, Germany:
Meta-Analysis Provides Highest Level of Evidence, Diabetes Monitor (2002, p6)
Micronutrient Undernutrition in Americans
Nutrient
Population Group
% Ingesting
< EAR *
From Food
Minerals
Iron
Women 14 - 50 years
16 %
Magnesium
All
56 %
Zinc
All
12 %
B6
Women > 70 years
49 %
Folate
Adult Women
16 %
E
All
93 %
C
All
31 %
Vitamins
* USDA What we Eat in America (NHANES 2001-2002) Sept. 2005
Methionine
Serine
B6
MTHFR
(polymorphism)
SHMT
B12
CH3-THF
MS
CH2=THF
TS
dUMP
Homocysteine
dTMP
Micronuclei in: RNA positive erythrocytes
RNA negative erythrocytes
Micronuclei per 1000 cells
130
Folic Acid
Folinic Acid
80
40
30
20
Normal
range
0
1 year
preRx
50
100
150
TIME (DAYS)
200
250
300
350
MIN PCEs/1000 PCEs
60
50
40
30
20
x
10
x
0
0
5
10
15
20
PLASMA FOLATE (NG/ML)
25
30
Folate, Vitamin B12, Homocysteine Status and Chromosome Damage Rate in
Lymphocytes of Older Men
Michael Fenech, Ivor Dreostl, and Josephine Rinaldi, Carcinogenesis 13:1329-1336, 1997
Folate, Vitamin B12, Homocysteine Status and DNA Damage in
Young Australian Adults
Michael Fenech, Claire Aitken, and Josephine Rinaldi, Carcinogenesis 19:1163 - 1173, 1998
Micronucleus Frequency in Human Lymphocytes is Related to
Plasma Vitamin B12 and Homocysteine
Michael Fenech, Mutation Research 42: 299 - 304, 1999
In a series of studies, we have been able to confirm that the micronucleus index in cytokinesis-blocked
lymphocytes is significantly negatively correlated with plasma vitamin B12 (B12) concentration and
significantly positively correlated with plasma homocysteine (HC). Furthermore we have shown in a
randomized double-blind placebo-controlled dietary intervention study that intake of 3.5 times the RDI of
folic acid and B12 significantly reduces the micronucleus index only in those with above average levels
of micronucleus frequency. Micronucleus frequency is minimized when plasma HC is below 7.5 µmol/l
and plasma B12 is above 300 pmol/l. Therefore, it is important to take account of the effect of B12 and
HC when using the micronucleus assay for human biomonitoring studies.
Dose-response on micronuclei induction in cultured lymphocytes
Acute exposure to X-rays vs. Folic Acid deficiency
Fenech 2003, Nutrition Research Reviews
Randomized Double-blind placebo-controlled prospective trial
with 3.5X RDA folate and Vitamin B12 supplementation.
Control
Group
Intervention
Group
n=31
n=33
Baseline
Postplacebo
P value
Baseline
postintervention
MNCs PER 1000
BNCs
7.02
(0.70)
6.97
(0.82)
NS
6.92
(0.58)
5.86
(0.51)
0.031
Serum B12 (pmol/l)
336.0
(19.3)
330.8
(25.6)
NS
343.6
(20.3)
382.1
(19.0)
0.004
RBC folate (nmol/l)
455.5
(31.2)
404.2
(24.9)
NS
379.1
(18.9)
712.1
(41.4)
<0.0001
Plasma
homocysteine
(µmol/l)
8.97
(0.36)
8.45
(0.36)
NS
9.36
(0.53)
6.68
(0.39)
<0.0001
Fenech et. al. 1998 Carcinogenesis.
P value
Analysis of nonlinear regression models: comparison of an overall model
and individual models of Z-transformed values vs. ln- nonheme liver iron
3
2.5
Over all
2
DCF-PMNs
Z score
1.5
DCF-Lymph
Rh123-PMNs
1
Rh123-Lymph
0.5
mtDNA damage
0
1/RCR
-0.5
-1
normal
-1.5
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
LN nonheme Fe (µmol/g wet liver)
. Each of the six dependent variables (that were analyzed by nonlinear regression in former figures) were transformed to Z
scores and modeled as a quadratic function of the ln-liver nonheme iron as the independent variable. The equation for the
RCR ratio's Z score was obtained from inverted RCR values (1/RCR) so that normal rats had the lower instead of the higher
values. For presentation purposes each model line was obtained from 9 values of liver iron. All statistics were performed as in
materials and methods.
An overview of evidence for a causal relationship
between iron deficiency
during development and cognitive or behavioral
function in children
Joyce C McCann and Bruce N Ames
(2007) AJCN in press
Is docosahexaenoic acid, an n3 long-chain
polyunsaturated fatty acid, required for development
of normal brain function? An overview of evidence
from cognitive and behavioral tests in humans and
animals
Joyce C McCann and Bruce N Ames
American Journal of Clinical Nutrition (2005)
82:281-95
Is there convincing biological or behavioral evidence linking
Vitamin D deficiency to brain dysfunction?
Joyce C McCann and Bruce N Ames
Faseb J in press
Neurons Contain The Enzyme That Activates Vitamin D
Eyles, DW et al (2005) J Chem Neuroanat 29, 21-30
Calcitriol Target Genes in the Brain
Gene products whose expression in the brain or brain cells has been reported to be affected by calcitriol
Neurotrophins and other growth factors:
NGF; NT-3 & NT-4/5; GDNF; TGF-β2
Calcium-binding proteins:
Calbindin D28K, parvalbumin, calretinin;
Protein sub-units for L-Type Voltage Sensitive Ca++Channels (L-type VSCCs);
Transcription factors or enzymes involved in signal transduction pathways:
N-myc, c-myc, protein kinase C family (PKC);
Other enzymes:
Choline acetyltransferase, responsible for synthesis of the neurotransmitter acetylcholine;
γ-Glutamyltranspeptidase, involved in recycling of the reactive oxygen species scavenger glutathione);
Hormones:
[Oxytocin, the “trust hormone”]
Biochemical or cellular brain functions in which calcitriol target gene products are involved
Synaptogenesis (formation of synaptic connections); Synaptic plasticity (e.g., memory formation); Calcium
signaling and homeostasis; Neurotransmission and neurotransmitter synthesis; Survival and differentiation of
dopaminergic and other neurons; Control of toxic free radicals;
Behavior affected by target gene product dysfunction
Learning and memory; Motor control; Maternal or social behavior; Aging (neuronal density);
McCann, JC, Ames BN (2007) Review Article: Is there convincing biological or behavioral evidence
linking vitamin D deficiency to brain dysfunction? FASEB J, in press.
Zinc Deficiency Induces Increased
Oxidative Stress in C6 Glioma Cells
DCF Fluorescence
Intensity (RFU)
*
140
120
100
80
60
40
20
Control
ZnAD
ZnDF
Zinc Deficiency Induces Fapy Glycosylase (Fpg)-sensitive
Single Strand Breaks in Human Lung Fibroblasts
Comet Score
Control (+Fpg)
ZnAD (+Fpg)
ZnDF (+Fpg)
*
200
160
120
80
40
0
Control
ZnAD
ZnDF
Synthesis of Heme
Cytosol
PBG
Porphyrins
Heme-a
2ALA
ALA
PPIX
FeII
PPGIX
FC
Mitochondria
Heme
Succ-CoA + Gly
PLP
ALA
H+
Cellular Cytoplasm
H+
H+
H+
H+
H+
Mitochondrial Outer Membrane
H+
Intermembrane
Space
Mitochondrial
Matrix
H+
H+
CoQ
I
Inner Membrane
H+
NAD+
H+
H+
FAD
CytC
H+
Succinate
Fumarate
L-Malate
NADH
Pyruvate
NADH
Oxaloacetate
Dehydrogenase
complex
Acetyl-Co-A
Citrate
Synthase
ATP
CITRIC
ACID
CYCLE
Citrate
H+
H+
V
IV
H+
FADH2
H+
H+
III
II
NADH
H+
O2
H2O
H+
ADP
+
ATP H
Succinyl-Co-A
NADH
-Ketoglutarate
Dehydrogenase
Complex
-Ketoglutarate
Isocitrate
NADH
H+
H+
9
Biotin deficiency accelerates cell senescence
Micronutrient deficiency and heme synthesis in human cell culture
Micronutrient
Deficiency
Heme
Deficit
Pyridoxine
[+]
Zinc
Complex IV
Deficit
Oxidative
Stress
DNA
Early
Damage Senescence
++
+
#
#
[+]
++
Riboflavin
Iron
+
+
[+]
Copper
[+]
+
[+]
[+]
+
+
[+]
[+]
[+]
Biotin
Lipoic Acid
Pantothenate
+
+ = Atamna/Ames, ++Askree /Ames, #Ho/Ames [+] Literature
+
Variation in chromosomal DNA damage rates within and between age
groups measured as MNC frequency.
Healthy Non-smoking
Males
Healthy Non-smoking
Females
Fenech 2007, Forum Nutr.
Magnesium Deficiency Shortens Fibroblast Lifespan
Magnesium Deficiency Induces DNA-Protein Crosslinks
Calcium Deficiency
Fenech: chromosome breaks
Lipkin: colon cancer mice
Folate Deficiency
MacGregor/Ames/Fenech: chromosome
breaks mice/humans
Willett: epi colon cancer humans
Vitamin D Deficiency
Holick: epi many types of cancer
Vitamin B12
Fenech: Chromosome breaks
Selenium
Rao: DNA damage
Combs/Trumbo: Cancer humans
Omega-3 FA
Denkins: Cancer
Magnesium Deficiency
Niacin
Bell: chromosome breaks humans
Larsson: epi colorectal cancer humans
Kirkland/Depeint: DNA damage
Zinc Deficiency
Choline
Fong: esophageal cancer humans/rodents
da Costa: DNA damage in humans
Potassium Deficiency
Chang: Cardiovascular Disease
Proc. Natl. Acad. Sci. USA
Vol. 103, pp. 17589-17594, November 2006
Low micronutrient intake may accelerate the degenerative diseases
of aging through allocation of scarce micronutrients by triage
Bruce N. Ames
Children’s Hospital of Oakland Research Institute, Nutrition and Metabolism Center,
5700 Martin Luther King Jr. Way, Oakland, CA 94609
Inadequate dietary intakes of vitamins and minerals are widespread, most likely due to excessive consumption of
energy-rich, micronutrient-poor, refined food. Inadequate intakes may result in chronic metabolic disruption,
including mitochondrial decay. Deficiencies in many micronutrients cause DNA damage, such as chromosome
breaks, in cultured human cells or in vivo. Some of these deficiencies also cause mitochondrial decay with
oxidant leakage and cellular aging, and are associated with late onset diseases such as cancer. I propose DNA
damage and late onset disease are consequences of a triage allocation response to micronutrient scarcity.
Episodic shortages of micronutrients were common during evolution. Natural selection favors short-term
survival at the expense of long-term health. I hypothesize that short-term survival was achieved by
allocating scarce micronutrients by triage, in part through an adjustment of the binding affinity of each
protein for its required micronutrient. If this hypothesis is correct, micronutrient deficiencies that trigger the
triage response would accelerate cancer, aging, and neural decay but would leave critical metabolic functions,
such as ATP production, intact. Evidence that micronutrient malnutrition increases late onset diseases, such as
cancer, is discussed. A multivitamin-mineral supplement is one low-cost way to ensure intake of the
Recommended Dietary Allowance of micronutrients throughout life.
Immune Risk Phenotype of Aging
 Low CD4:CD8 ratio
Increase in anergic effector (CD8+CD28-) T-cells
Low lymphoproliferative response
Decline in antigen-presenting cells
Decreased expression of co-stimulatory molecules
Decline in IL-12 production and Th1 response
Immune Risk Phenotype of Aging
 Low CD4:CD8 ratio
Def: vit A.folate, zinc,
 Increase in anergic effector (CD8+CD28-) T-cells
Def: tryptophan, zinc,
 Low lymphoproliferative response
Def: vit C, vit E, zinc
 Decline in antigen-presenting cells
Def: vit E
 Decreased expression of co-stimulatory molecules
Def: vit E, tryptophan. zinc
 Decline in IL-12 production and Th1 response
Def: vit B6, Vit E, zinc
ADAPTIVE
antigen
capture
epithelial
barrier
dendritic
cell
antigen
presentation
antigen
B cells
CD8+
cells
lymphoid
organ
T-cell
cytokines
T helper
Cells(CD4+)
FOLATE DEFICIENCY
(Courtemanche et al (2004) J Immunol 173:3186)
Life Expectancy of Men and Women at Birth
80
77.5
78.9
74.9
75
71.1
73.2
71.4
69.9
70
66.7
67.1
65.7 65.6
65
61.3 61.4
58
60
56.3
54.5
55
49
50
53.6
50.1
46.4
45
40
1900
1910
1920
1930
1940
1950
1960
1970
1980
1990
SOURCE: National Institute on Aging
END