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“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