“Delaying or accelerating the degenerative diseases of aging”

MCB 135KLecture 4, Chapters 5 & 6Dr. Bruce N. Ames1/30/08

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

“Research found that a central factor in aging is the decay of the mitochondria in cells”

Bruce N. AmesChildren’s Hospital Oakland Research InstituteProfessor, University of California, Berkeley

Delaying (or Accelerating) the Degenerative Diseases of Aging

O2 O2-

e- e- e- e-

H2O2 •OH H2O

30 Jan ‘08Timiras class

Estimated oxidative DNA adducts per rat liver cell

0

Old (26-mo)

Young (4-mo)

70,000

60,000

50,000

40,000

30,000

20,000

10,00024,000

67,000

Source: E. Stadtman, Science 257, 1220-1224 (1992)

carbonyl content(nmol/mg protein)

carbonyl content(nmol/mg protein)

6

5

4

3

2

2

1

0

1000

Years

20 40 60 80

Months

3 12 20 26 *

MDA (pmol/mg

protein)

Young

Old

160

140

120

100

80

60

40

20

0

Brain Liver Heart Kidney Lung

* *

*

**

6

Proc. Natl. Acad. Sci. USAVol. 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

MitochondrialMatrix

Cellular Cytoplasm

Mitochondrial Outer MembraneMitochondrial Outer Membrane

Inner MembraneInner Membrane

IntermembraneSpace

VV

IIIIII

IIIIIIIVIV

CytCCytCCoQCoQ

HH++

PyruvateDehydrogenasecomplex

Citrate Synthase

-Ketoglutarate Dehydrogenase

Complex

Fumarate

L-Malate

Oxaloacetate

Acetyl-Co-A Citrate Isocitrate

-Ketoglutarate

Succinyl-Co-A

Succinate

FADFADH2

CITRICACID

CYCLE

H2OO2 ADP

ATP

ATP

NADH

NADH

NADH

NAD+

NADHNADH

HH++

HH++

HH++ HH++

HH++HH++HH++

HH++

HH++HH++

HH++ HH++HH++

HH++

HH++

HH++

HH++ HH++HH++

HH++

HH++

HH++

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 Levels in 3 and 24 Month Old Rat Hepatocytes

Ca

rdio

lipin

(µ

g p

er 1

06 C

ells

) 30

20

10

0Young Old

10

**

R123 Fluorescence in old and young rat hepatocytes

Fluorescence/cell

No

rma

l ce

ll n

umbe

r

0.03

Young

1000100100.00

0.01

0.02

Old

11

L-Carnitine/Acetyl-L-Carnitine (ALCAR)

• Mediates the ratio of acetyl-CoA/CoA

• Decreases with age in plasma and in brain

• Improves cognitive function in rats12

• Transports long-chain fatty acids into mitochondria

• Removes short- and medium-chain fatty acids that accumulate

Effect of ALCAR Supplementation on Cardiolipin Levels

Young

Ca

rdio

lipin

(µ

g p

er 1

0 c

ells

) 30

20

10

0

14

+ ALCAR

Old

**

R123 Fluorescence in Young and Old Rat Hepatocytes

YOUNG

OLD

WITHALCAR

WITHALCAR

NOALCAR

NOALCAR

No

rma

lize

d C

ell

Nu

mb

er

No

rma

lize

d C

ell

Nu

mb

er

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

Lipoic Acid Lowers Mitochondrial Oxidants in Old Rats

Fl.

Un

its

/O2 C

on

sum

ed p

er M

inu

te

20

10

0Young Old

+ LA

+ LA

**

MDA levels in young and old rats with LA, ALCAR, or both

Young Old

MD

A (

pm

ol/m

g p

rote

in)

60

0

50

10

40

30

20

70

80

P<0.01P<0.05

***

+ L

A

+ A

LC

AR

+ A

LC

AR

+ L

A

+ L

A

+ A

LC

AR

+ A

LC

AR

+ L

A

20

***p<0.001 vs. young rat group

Ambulatory Activity before and After Supplementation with Lipoic Acid (LA) + Acetyl-L-Carnitine (ALCAR)

0

200

400

600

800

+ L

A +

AL

CA

R

OldYoung

+ L

A +

AL

CA

R

*

Dis

tan

ce

Tra

vel

ed

(c

m/h

ou

r/d

ay

)

*#

#

vs. young

vs. old

*

0

10

20

30

p <0.01

T c

ell

sti

mu

lati

on

ind

ex

Young

Young Tre

ated

Old T

reat

edOld

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.

P <0.001

Spatial Memory relies on intact hippocampal function.

Treatments improved poor memory in old rats

Morris Water Maze for Testing Spatial Memory

22

P<0.001

20

100

80

60

40

Spatial Memory Tested With Morris Water Maze

Young Old Old Old Old 0

P<0.05

+ ALCAR + LA

+ ALCAR + LA

Tim

e in

Se

con

ds

0.00

2.00

4.00

6

8

10

12

14

SOUND: Time to Signal

0 50 100 150 200

Young

Old

Old + ALCAROld + LA

Old + ALCAR + LA

0.00

2.00

4.00

6.00

8.00

10.00

12.00

LIGHT: Time to Signal

0 50 100 150 200

Peak procedure: for measuring temporal memory. Associated with striatum, cerebellum, & hippocampus

PEAK RATE: measures learning and motivation.

PEAK TIME: measures internal clock, food is rewarded only when animals push lever 40s after sound or light signal

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 RNA)

RNA is Oxidized

(92% is removed by RNase)

*oxo8G: 8-hydroxyguanosine; oxo8dG: 8-hydroxy-2’-deoxyguanosine27

106

231

33

3031

Induced

Repressed

250

200

150

100

50

0

-50

Num

ber

of G

enes

6h 24h 7d (diet)

Modulation of Nrf-2-dependent gene expression by D3T in mouse liver.

Kwak, et al. J bio Chem, 2003

Induction of Phase 2 Enzymes

Proc. Natl. Acad. Sci. USAVol. 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

The Journal of Biological ChemistryVol. 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

* #

nm

ol/m

in/g

1200

800

400

0

#

M

150

100

50

0Km for ALCAR

Km for CoA

**

##

#**

403020100-10

5

10

0

5

10

0200150100500-50

1/[ALCAR, mM] 1/[CoA, mM]

Vmax

young old = vs. young

# = vs. old

1/v 1/v

*

New Yorker, June 6, 2005

“You’re fifty-seven years old. I’d like to get that down a

bit.”

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

Source: Professor Daniel Ziegler of the Diabetes Research Institute, Düsseldorf, Germany: Meta-Analysis Provides Highest Level of Evidence, Diabetes Monitor (2002, p6)

61

79

60

72

6666

60

25

48

55

0

10

20

30

40

50

60

70

80

90

ALADIN I ALADIN III SYDNEY NATHAN II Meta-Analysis

Pe

rcen

t

-lipoic acidplacebo

* p<0.05 vs Placebo

ITT analysis of 4 phase II-III RCTs plus meta-analysis: 600 mg I.v. per day for 3 weeksTotal Symptom Score (TSS): relative improvement at 3 weeks vs baseline

n= 77 81 338 165 60 60 241 236 716 542

Micronutrient Undernutrition in Americans

Nutrient Population Group

% Ingesting < EAR * From Food

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 %

Minerals

Vitamins

* USDA What we Eat in America (NHANES 2001-2002) Sept. 2005

dUMP dTMP

Methionine

SHMT

B6

Serine

MTHFR

Homocysteine

(polymorphism)

TS

MS

CH2=THF

B12CH3-THF

Micronuclei in: RNA positive erythrocytes RNA negative erythrocytes

Folic AcidFolinic Acid

TIME (DAYS)

1 yearpreRx

Normalrange

Mic

ron

ucl

ei p

er 1

000

cell

s

20

50 100 150 200 250 300 350

0

30

40

80

130

xx

PLASMA FOLATE (NG/ML)

MIN

PC

Es/

1000

PC

Es 60

50

40

30

20

10

00 5 10 15 20 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

  BaselinePost-placebo P value Baseline

post-intervention P value

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.

. 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.

Analysis of nonlinear regression models: comparison of an overall model and individual models of Z-transformed values vs. ln- nonheme liver iron

Over all

DCF-PMNs

DCF-Lymph

Rh123-PMNs

Rh123-Lymph

mtDNA damage

1/RCR

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

LN nonheme Fe (µmol/g wet liver)

Z score

normal

An overview of evidence for a causal relationship between iron deficiencyduring 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 AmesAmerican Journal of Clinical Nutrition (2005) 82:281-95

Is there convincing biological or behavioral evidence linkingVitamin D deficiency to brain dysfunction?

Joyce C McCann and Bruce N AmesFaseb 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 BrainGene 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.

20

40

6080

100

120

140

Control ZnAD ZnDF

DC

F F

luo

resc

ence

In

ten

sity

(R

FU

)

Zinc Deficiency Induces IncreasedOxidative Stress in C6 Glioma Cells

*

Zinc Deficiency Induces Fapy Glycosylase (Fpg)-sensitive Single Strand Breaks in Human Lung Fibroblasts

0

40

80

120

160

200

Control ZnAD ZnDF

Co

met

Sco

re

Control (+Fpg) ZnAD (+Fpg) ZnDF (+Fpg)

*

Succ-CoA + Gly ALA

2ALA

PBG Porphyrins

Cytosol

PPIX PPGIX

Heme

FeII

MitochondriaFC

Synthesis of Heme

Heme-a

PLP

ALA

MitochondrialMatrix

Cellular Cytoplasm

Mitochondrial Outer MembraneMitochondrial Outer Membrane

Inner MembraneInner Membrane

IntermembraneSpace

VV

IIIIII

IIIIIIIVIV

CytCCytCCoQCoQ

HH++

PyruvateDehydrogenasecomplex

Citrate Synthase

-Ketoglutarate Dehydrogenase

Complex

Fumarate

L-Malate

Oxaloacetate

Acetyl-Co-A Citrate Isocitrate

-Ketoglutarate

Succinyl-Co-A

Succinate

FADFADH2

CITRICACID

CYCLE

H2OO2 ADP

ATP

ATP

NADH

NADH

NADH

NAD+

NADHNADH

HH++

HH++

HH++ HH++

HH++HH++HH++

HH++

HH++HH++

HH++ HH++HH++

HH++

HH++

HH++

HH++ HH++HH++

HH++

HH++

HH++

9

Effect of Biotin on Cell Senescence

0.0 2.5 5.0 7.5 10.0 12.5 15.020253035404550556065

cdFBS-BD

nFBS-BSnFBS-+B--BS

Weeks

cdFBS+B-BS

Biotin deficiency accelerates cell senescence

MicronutrientDeficiency

HemeDeficit

Complex IVDeficit

Oxidative Stress

DNA Damage

Early Senescence

Pyridoxine [+] ++ ++

Zinc + # #

Riboflavin

Iron + + [+] [+]

Copper [+] [+] [+]

Biotin + + + + +

Lipoic Acid [+]Pantothenate [+] [+]

Micronutrient deficiency and heme synthesis in human cell culture

+ = Atamna/Ames, ++Askree /Ames, #Ho/Ames [+] Literature

Fenech 2007, Forum Nutr.

Healthy Non-smoking Males

Healthy Non-smoking Females

Variation in chromosomal DNA damage rates within and between age groups measured as MNC frequency.

Magnesium Deficiency Shortens Fibroblast Lifespan

Magnesium Deficiency Induces DNA-Protein Crosslinks

Calcium Deficiency Vitamin B12

Fenech: chromosome breaks Fenech: Chromosome breaks

Lipkin: colon cancer mice

Folate Deficiency Selenium

MacGregor/Ames/Fenech: chromosome Rao: DNA damage

breaks mice/humans Combs/Trumbo: Cancer humans

Willett: epi colon cancer humans

Vitamin D Deficiency Omega-3 FA

Holick: epi many types of cancer Denkins: Cancer

Magnesium Deficiency Niacin

Bell: chromosome breaks humans Kirkland/Depeint: DNA damage

Larsson: epi colorectal cancer humans

Zinc Deficiency Choline

Fong: esophageal cancer humans/rodents da Costa: DNA damage in humans

Potassium Deficiency Chang: Cardiovascular Disease

Proc. Natl. Acad. Sci. USAVol. 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. AmesChildren’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 ratioDef: vit A.folate, zinc,

Increase in anergic effector (CD8+CD28-) T-cellsDef: tryptophan, zinc,

Low lymphoproliferative responseDef: vit C, vit E, zinc

Decline in antigen-presenting cellsDef: vit E

Decreased expression of co-stimulatory moleculesDef: vit E, tryptophan. zinc

Decline in IL-12 production and Th1 responseDef: vit B6, Vit E, zinc

epithelialbarrier

antigen

antigencapture

T-cell

antigenpresentation

lymphoidorgan

T helperCells(CD4+)

B cells CD8+cells

dendriticcell

ADAPTIVE

cytokines

FOLATE DEFICIENCY

(Courtemanche et al (2004) J Immunol 173:3186)

80

75

70

65

60

55

50

45

401900 1910 1920 1930 1940 1950 1960 1970 1980 1990

46.4

4950.1

53.654.5

56.3

5861.3 61.4

65.7 65.666.7

71.173.2

74.9

77.578.9

71.469.9

67.1

Life Expectancy of Men and Women at Birth

SOURCE: National Institute on Aging

END