Better Foods for Better Health, 3rd edition 12 – 14 September 2012

Antioxidants, Nutrition and Ageing – Miracles, Myths and Misunderstandings

Professor Barry Halliwell Tan Chin Tuan Centennial Professor Deputy President (Research & Technology) National University of Singapore Antioxidants, Nutrition and Ageing

Current Research in the Halliwell Lab 1. Antioxidants in Human Health and Disease 2. Mechanisms of Neurodegeneration 3. Artefacts in Cell Culture 4. Ageing in the nematode C.elegans Antioxidants, Nutrition and Ageing Oxidant – Antioxidant Balance AA Metabolism Peroxiredoxins Glutathione system SOD, Catalase Phagocytes

Diet-Derived Antioxidants Mitochondrial Blood Respiration components Other electron Iron chelators Albumin, transport chains NADPH oxidases Caeruloplasmin, DUOxes Transferrin, haptoglobin etc Xanthine oxidase Haem proteins So some oxidative damage is inevitable and repair &/or replacement of damaged molecules is essential Antioxidants, Nutrition and Ageing OXIDATIVE DAMAGE AND LOSS OF COGNITIVE FUNCTION

• There is strikingly-elevated oxidative damage to all types of biomolecules in the brain in Alzheimer’s Disease (AD) • Oxidative damage is already elevated in mild cognitive impairment, by several biomarkers • Thus it may precede AD development • But why have antioxidant therapies to delay onset or progression of AD not been employed?

Halliwell B (2001) Role of free radicals in the neurodegenerative diseases. Therapeutic implications for antioxidant treatment. Drugs & Aging 18, 685 –716.

Halliwell B (2006) Oxidative stress and neurodegeneration; where are we now? J. Neurochem. 97, 1634-58. Antioxidants, Nutrition and Ageing

A ROLE FOR ANTIOXIDANTS? Risk Factors for AD • ApoE status (plus other genetic factors) • Low intake of folic acid • Hypercholesterolaemia (causes oxidative stress) • Repeated brain trauma (e.g. dementia pugilistica) • Low level of education • Poor language skills / lack of mental exercise • Lack of physical exercise • Diets low in certain polysaturated fatty acids??? • Low dietary intake of antioxidants Antioxidants, Nutrition and Ageing

Mechanism of Neurodegeneration CAN IT BE SLOWED BY DIETARY OR SYNTHETIC COMPONENTS?

HOW TO STUDY THIS? 1. Cell culture 2. Animal models 3. Humans Antioxidants, Nutrition and Ageing

When I was young, the Free Radical –Antioxidant Field was SIMPLE

 Free radicals are bad  Antioxidants are good  Taking antioxidants will prevent disease  Since free radicals are implicated in ageing, antioxidants will make you live longer

Halliwell B (2012) Free radicals and antioxidants: updating a personal view. Nutr Reviews. 70:257-265

Halliwell B (2012) The antioxidant paradox: less paradoxical now? Brit J Clin Pharmacol. In press. Antioxidants, Nutrition and Ageing

Protective Plasma Levels

 50 M vit. C

 30 M vit. E (E:CL ratio > 5.2 mol/mmol)

 0.4 M β-carotene

Gey (1995) J Nutr Biochem 6, 206-236

Antioxidants, Nutrition and Ageing

Protective Plasma Levels

BUT BE CAREFUL

THESE ARE A MARKER OF A DIET RICH IN FRUITS AND VEGETABLES, WHICH IS ITSELF PROTECTIVE AGAINST DISEASE

It could be any component or mixture of components in that diet that is protective

Antioxidants, Nutrition and Ageing Plasma levels of E, C and beta- carotene could reflect intake of

• Other tocopherols / tocotrienols • Other carotenoids • Flavonoids • Fibre • Phase II inducers • Inhibition / upregulation of cytochromes P450 • Anti-and pro-apoptotic agents • Any other product in the plant Antioxidants, Nutrition and Ageing When I was young, it was SIMPLE Taking Antioxidants will Prevent Disease

(with several exceptions) Antioxidants, Nutrition and Ageing BUT SEVERAL TRIALS DID SUCCEED!

THE ASAP TRIAL Six year trial of E plus slow release C in 520 hypercholesterolaemic men and women, carotid atherosclerosis progression  Significant effect in men (33 % reduction)  Nonsignificant effect in women (14 %)

Circulation. 2003;107:947-953. Antioxidants, Nutrition and Ageing

Int. J. Cancer (2010) 127, 1875-1881

The Supplementation in Vitamins and Mineral Antioxidants Study was a double-blind, placebo-controlled, randomized trial, in which 12,741 French adults (7,713 women aged 35–60 years and 5,028 men aged 45–60 years) received a combination of ascorbic acid (120 mg), vitamin E (30 mg), -carotene (6 mg), selenium (100 lg) and zinc (20 mg), or placebo daily for a median follow-up time of 7.5 years [October 1994 to September 2002]. Antioxidant supplementation decreased total cancer incidence and total mortality in men. Antioxidants, Nutrition and Ageing

Limited efficacy in Alzheimers disease No effect in mild cognitive impairment No effect on cardiovascular disease No beneficial effect on cancer Similar failures with vitamin C and -carotene Suggestions of deleterious effects in some studies Antioxidants, Nutrition and Ageing

BMJ (2010) 341:c5702

Conclusion In this meta-analysis, vitamin E increased the risk for haemorrhagic stroke by 22% and reduced the risk of ischaemic stroke by 10%. This differential risk pattern is obscured when looking at total stroke. Given the relatively small risk reduction of ischaemic stroke and the generally more severe outcome of haemorrhagic stroke, indiscriminate widespread use of vitamin E should be cautioned against. Antioxidants, Nutrition and Ageing Intervention Trials with Vitamin E

• Vitamin E works much better in (at least some) rodent models of neurodegeneration (e.g. APP, ALS) and cardiovascular disease • And it also decreases oxidative

damage (measured as F2-isoprostanes) in these models

VALIDITY OF RODENT MODELS OF STROKE AND NEURODEGENERATIVE DISEASE?

By courtesy of John Milner, National Cancer Institute Antioxidants, Nutrition and Ageing THERE ARE MULTIPLE EXPLANATIONS FOR THIS “FAILURE” OF ANTIOXIDANTS

LET’S RE-EXAMINE OUR STARTING HYPOTHESIS • Steady-state levels of oxidative DNA damage contribute significantly to the major cancers. (PROBABLY TRUE) Halliwell B (2007) Oxidative stress and cancer: have we moved forward? Biochem. J. (2007) 401, 1-11. • Steady-state levels of lipid peroxidation contribute significantly to cardiovascular disease (POSSIBLY TRUE) and to neurodegenerative disease (PROBABLY TRUE) Halliwell B (2006) Oxidative stress and neurodegeneration; where are we now? J. Neurochem. 97, 1634-58. Libby et al (2011) Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317-25.

Antioxidants, Nutrition and Ageing

IN CARRYING OUT THE INTERVENTION TRIAL, DID WE DECREASE OXIDATIVE DAMAGE IN THE SUBJECTS?

Because if we did not, no effect would be predicted This was assumed, but not tested

Halliwell B (1999) Establishing the significance and optimal intake of dietary antioxidants. The biomarker concept. Nutr. Rev. 57: 104-113. Antioxidants, Nutrition and Ageing

Our Studies and Many Others using Biomarkers of Lipid Peroxidation and Oxidative DNA Damage show NO DECREASE on Supplementing Healthy Volunteers with Ascorbate, Beta-Carotene or Vitamin E. BUT effects on “rancid” individuals rarely considered NOR genetic background or racial type Antioxidants, Nutrition and Ageing THERE ARE MULTIPLE EXPLANATIONS FOR THIS “FAILURE” OF ANTIOXIDANTS

• They ignore individual differences in “rancidity” (which depend on age to some extent) Increased isoprostanes in thalassaemia

Antioxidants, Nutrition and Ageing RESPONSES MAY ALSO CHANGE WITH RACIAL ORIGIN. Vorinostat Histone deacetylase inhibitor under evaluation as anti-cancer drug

Vorinostat Glucuronide Glucuronidation (UGT2B17) (less activity) Vorinostat

Hydrolysis followed 4-Anilino-4-oxobutanoic acid by β-oxidation

UGT2B17*2 (del variant) •Reduced UGT2B17 activity •Homozygotes:  10% Caucasians Wong et al. Pharmacogenetics & Genomics  60-70% Asians 2011, 21(11):760-8 UGT2B17*2 homozygotes present in 62% of our cohort RESPONSE TO NUTRIENTS IS ALSO LIKELY TO BE DIFFERENT Slide courtesy of Prof John Wong Antioxidants, Nutrition and Ageing THEY MAY ALSO VARY WITH GENETIC BACKGROUND AND AGE The gene encoding HAPTOGLOBIN (a haemoglobin binding protein) is polymorphic with 3 genotypes, 2-2, 2-1, 1-1. The 2 allele is a poorer antioxidant against haemoglobin-induced oxidative damage in vitro.

Arterioscler Thromb Vasc Biol. 2008; 28:341-347

Atherosclerosis 2011; 219:240-244

Pharmacogenomics 2010; 11:675-684 Antioxidants, Nutrition and Ageing THE FLAVONOID STORY

• Polyphenols have powerful antioxidant effects in vivo. • Polyphenols are not-fully absorbed and are rapidly metabolised, blocking the antioxidant –OH groups. • Phenolic metabolites usually have lower antioxidant activity • Levels of phenolics are low in the face of all the other plasma antioxidants. • Their bioavailabilty to the brain is poor but consumption of flavonoid-rich foods seems to enhance cognition in some studies

It is unlikely that Flavonoids act as Systemic Antioxidants In Vivo

Halliwell B. (2008) Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch Biochem Biophys. 476, 107-112 Antioxidants, Nutrition and Ageing

FLAVONOIDS AND OTHER ANTIOXIDANTS MAY ACT DIRECTLY IN THE GASTROINTESTINAL TRACT

Halliwell et al (2000) Free Rad Res 33, 819-830 The GI tract: a major site of antioxidant Action?

Jenner AM, Rafter J, Halliwell B (2005) Free Radic Biol Med. 38, 763-772 Human fecal water content of phenolics: The extent of colonic exposure to aromatic compounds.

Antioxidants, Nutrition and Ageing Antioxidants Work in the GI Tract!

STOMACH SMALL COLON/RECTUM INTESTINE Duodenum Ileum Jejenum

High concentration of ascorbate in food Vitamin C completely absorbed* Little vitamin C present* and gastric juice Limited α–tocopherol present*

Carotenoids, tocopherols, tocotrienols, Vitamin E largely absorbed* Considerable amounts of unabsorbed flavonoids, other phenolics in food (if Some tocotrienols and β, γ, δ– flavonoids, other fruit/vegetable/grain – rich diets tocopherols returned to GI tract in bile. phenolics,carotenoids, tocotrienols, β, consumed) γ, δ–tocopherols (if diet rich in these compounds). Scavenge Some carotenoids cleaved to vitamin A Extensive metabolism of polyphenols  RNS from acidified nitrite Some carotenoids, flavonoids and other by colonic flora to generate simpler  OH▪ from Fe or Cu/ascorbate phenolic compounds absorbed, but many phenols interactions are not. Scavenging/metal binding/other ▪ ▪  RO and RO2 from dietary lipid actions of phenols might help delay peroxides colon/rectal cancer development by  Some phenolics bind Fe2+ or Cu2+, exerting external protective effects on to decrease their ability to generate colonic/rectal epithelium RS Inhibitions of LOX, COX–2,  Ferryl species from haem angiogenesis, matrix protein/peroxide interactions metalloproteinases etc by phenols stimulate lipid oxidation; quenched may help to limit cancer development by ascorbate and phenols which are preferentially oxidized  H2O2 in consumed beverages

*May not be true if supplements taken Antioxidants, Nutrition and Ageing And by doing so they may indirectly influence the brain

Genes Nutr (2012) 7:99–109 Antioxidants, Nutrition and Ageing Antioxidant activity equivalents in portions of beverages, vegetables, fruit and chocolate

Comparisons with the ABTS•+ assay (a nitrogen-centred radical)

4.5 Cups of tea 675 ml 6 Apples 960 g 28 White wines 4.2 L

7.5 Blackcurrant Juices 1.1 L 1 bar dark chocolate 50 g 27 Beers 4 L

15 Orange Juices 7 Onions 2 Red wines 2.25 L 1 Kg 300 ml

Slide courtesy of Prof Catherine Rice-Evans Antioxidants, Nutrition and Ageing

TWO QUESTIONS FOLLOW • What components of the food are responsible? • Does it have antioxidant effects in vivo? Antioxidants, Nutrition and Ageing

POWERFUL (ABTS assay) ANTIOXIDANT ACTIVITY OF DARK SOY SAUCE

• Long LH, Kwee DCT and Halliwell B (2000) The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay. Free Rad. Res. 32, 181-186. • Wang HS, Jenner AM, Lee CY, Shui GH, Tang SY, Whiteman M, Wenk MR, Halliwell B (2007) The identification of antioxidants in dark soy sauce. Free Radic Res. 41, 479-488.

Antioxidants, Nutrition and Ageing

WATCH OUT FOR EATING EFFECTS!

Lee CY, Isaac HB, Wang H, Huang SH, Long LH, Jenner AM, Kelly RP, Halliwell B (2006). Cautions in the use of biomarkers of oxidative damage; the vascular and antioxidant effects of dark soy sauce in humans. Biochem Biophys Res Commun 344, 906-911.

Urinary and plasma F2-isoprostanes concentration after placebo and dark soy sauce meal. Paired, two-tailed Student t test indicate ** significance at p<0.01 versus 0 h placebo; † significance at p<0.05 and †† at p<0.01 versus 0 h DSS.

Antioxidants, Nutrition and Ageing

What can alter levels of oxidative damage levels in humans or other animals? • Obesity (Humans, rodents) • Hyperglycaemia (Humans, rodents) • High plasma LDL Cholesterol (Humans, rodents) • High Cholesterol Diet (Rabbits and rats , humans probably not) • Zinc Intake (Rabbits, some other animals, human data inconclusive) • Body Iron Levels (Rabbits, rats, mice, maybe humans) • Certain foods (Humans, e.g. dark soy sauce, tomato, rodents) • Diabetes (in some human studies, not others)*, but probably not the metabolic syndrome • Intake of polyunsaturated fatty acids (PUFAs)  (docosahexaenoic acid, possibly eicosapentaenoic acid) (humans)  It is essential to do appropriate controls in testing effects of foods, because the consumption of any food (antioxidant or not) can sometimes alter levels of certain biomarkers *May depend on how well glucose and lipids have been normalized in the diabetic cohorts studied, or on the degree of obesity, since hyperglycaemia, hyperlipidaemia and obesity can all increase F2-isoprostane levels, i.e. it may not be diabetes per se but its sequelae or predisposing factors that cause the oxidative stress (at least as revealed by studies of F2-isoprostanes) Despite the propensity of PUFAs to oxidise in vitro, growing evidence suggests that they minimise oxidative damage in vivo.

Halliwell B (2012) The antioxidant paradox: less paradoxical now? doi: 10.1111/j.1365-2125.2012.04272.x Halliwell B (2011) Free radicals and antioxidants: - quo vadis? Trends Pharmacol. Sci. 32:125-130.

Antioxidants, Nutrition and Ageing Hydrogen Peroxide Generation by Beverages Measured by FOX assay and O2 electrode

Long LH, Lan ANB, Hsuan, FTY and Halliwell B (1999) Generation of hydrogen peroxide by "antioxidant" beverages and the effect of milk addition. Is cocoa the best beverage? Free Radic Res. 31, 67-71.

Antioxidants, Nutrition and Ageing

Green tea, black tea and red wine are supposed to be good for you, but they can make hydrogen peroxide.

THESE DATA ILLUSTRATE THE READY OXIDATION OF POLYPHENOLS! SO ARE THEY PRO-OXIDANTS RATHER THAN ANTIOXIDANTS??

Antioxidants,Oxidant Nutrition and Ageing – Antioxidant Balance AA Metabolism Peroxiredoxins Glutathione system SOD, Catalase Phagocytes

Diet-Derived Antioxidants Mitochondrial Blood Respiration components Other electron Iron chelators Albumin, transport chains NADPH oxidases Caeruloplasmin, DUOxes Transferrin, haptoglobin etc Xanthine oxidase Haem proteins

DO POLYPHENOLS ACT AS ANTIOXIDANTS &/OR PRO-OXIDANTS IN THE GASTROINTESTINAL TRACT, AND DOES IT MATTER WHICH? Artefacts in Cell Culture Antioxidants, Nutrition and Ageing Cell Culture is an Oxidative Stress

• Trypsinization  GSH • Serum deprivation  ROS • The major factor – OXYGEN

Intracellular pO2 (except skin, respiratory tract, cornea) 1 – 10 mmHg

95% air / 5% CO2 is ~150mmHg Therefore more ROS are made! • Oxygen levels also fluctuate wildly during culture and cell handling and with depth of medium Antioxidants, Nutrition and Ageing Cell Culture is an Oxidative Stress Second major factor – cell culture media are poor in antioxidants • vitamin C • vitamin E (little lipid) • Selenium

Antioxidants, Nutrition and Ageing Cell Killing by Ascorbate in HL-60 Cells: PROPIDIUM IODIDE

Clement, MV, Ramalingam, J, Long, LH and Halliwell B. The in vitro cytotoxicity of ascorbate depends on the culture medium used to perform the assay and involves hydrogen peroxide. Antiox. Redox Signaling, 3, 157-163 Antioxidants, Nutrition and Ageing

H2O2 Generation by Ascorbate Antioxidants, Nutrition and Ageing Other compounds that generate H2O2 include Delphinidin, Rosmarinic acid, Hydroxytyrosol, Quercetin, Catechin and Gallic Acid

Long, LH, Clement, MV, and Halliwell B. (2000) Artifacts in cell culture: Rapid generation of hydrogen peroxide on addition of (-) – epigallocatechin, (-) – epigallocatechin gallate, (+) – catechin and quercetin to commonly used cell culture media. Biochem. Biophys. Res. Commun. 273, 50-53

Long LH, Kirkland D, Whitwell J, Halliwell B. (2007) Different cytotoxic and clastogenic effects of epigallocatechin gallate in various cell culture media due to variable rates of its oxidation in the culture medium. Mutat Res/Genet. Toxicol. Environ. Mutagen. 634, 177-183 Antioxidants, Nutrition and Ageing Rapid loss of polyphenols upon incubation in DMEM at 37oC. Data are mean ± SD, n ≥ 3.

Delphinidin chloride Resveratrol

Hydroxytyrosol Curcumin Long LH, Hoi A, Halliwell B (2010) Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media (2010) Archives of Biochemistry and Biophysics. 501:162-169 Many are unstable but

Rosmarinic acid not all generate H2O2! Antioxidants,Examples Nutrition and of Ageing artefacts caused by oxidation of compounds added to cell culture media

Halliwell B. (2008) Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch Biochem Biophys. 476, 107-112 Antioxidants, Nutrition and Ageing A COMING PROBLEM FOR SINGAPORE

Proportion of population aged 65+ in selected IARU countries

UK

DK

AUST 2030 2005 SG 1980

JP

CH

0 5 10 15 20 25 30 35

REUTERS/CORBIS Slide by courtesy of Dr Kenneth Howse, Oxford Centenarians now constitute the University fastest-growing age group owing to Source: UN Population database advances in health care. The International Alliance of Research Universities (IARU) is a collaboration between ten of the world’s Source – Nature 467 (2010), 274- leading research-intensive universities who share 275 similar visions for higher education, in particular the education of future leaders. IARU comprises ANU, ETH Zurich, NUS, Peking, Berkeley, Cambridge, University of Copenhagen, Oxford, University of Tokyo and Yale University. Antioxidants, Nutrition and Ageing Ageing

• Progressive decline in organ function (but much redundancy) • Lowered ability to respond to environmental changes and injury (e.g. wound healing) • Increased risk of age-related disease, including - Cancer - Osteoporosis - Cardiovascular disease - Neurodegenerative disease (Parkinson, Alzheimer) - Type 2 diabetes - Cataract and macular degeneration • A big random element (Very old people >100 have everything wrong with them but nothing in particular)

RESPONSE TO NUTRIENTS ARE LIKELY TO VARY WITH AGE Antioxidants, Nutrition and Ageing C.elegans as a Model System

• Soil dwelling nematode worm • Eats bacteria • 99% Self-fertilizing hermaphrodites

• => Isogenetic clones !

Adult: length: 1mm Ø: 100m Volume: approx 1nL

Metabolic rate: 45nW Offspring: ~300 in < 8 days

Lifespan: max 40 days

WORK WORM OF AGEING RESEARCH Antioxidants, Nutrition and Ageing Some more advantages • Extensive set of mutants and tools - 1000s of mutant strains PC - GFP reporter gene system - RNAi is relatively easy • Very “simple” nervous systems • Total complement of 302 neurons – Essentially invariant structure – Complete wiring diagram known Control 10μg/ml 100μg/ml • There is still randomness in the ageing process.

PC 10μg/ml C

C- Antioxidants, Nutrition and Ageing

Lifespan and Healthspan

Histogram for day of death for 200 isogenic C. Motility phenotype distribution in the same elegans clones, born on the same day in our lab and isogenic C. elegans population on different grown under identical culture conditions. Even days of life. Even thought both genome and though their genome as well as environment are environment are identical, surviving worms identical, their individual lifespan ranges from 10 of the same age may be found to have days to 34 days, probably due to intrinsic stochastic dramatically different motility class, an factors inherent in ageing. indicator of health. Antioxidants, Nutrition and Ageing The effects of plant-derived “antioxidants” are dose-variable and independent of antioxidant activity.

Biogerontology (2010) 11:17-30 Antioxidants, Nutrition and Ageing

Several effective compounds isolated

The molecular mechanism of Psoralea corylifolia extract is under further investigation - likely to involve activation of endogenous stress response pathways - possibly through hormesis.

Several further compounds have also since been tested and their effects on lifespan, markers of oxidative damage, ROS production, mitochondrial function, growth and development is being investigated. Antioxidants, Nutrition and Ageing Major Challenges in the ROS field

• Developing antioxidants that work, especially in the brain (slowing neurodegeneration) • Establishing the true role of ROS in ageing • Doing something about it! • Understanding redox signalling at the cellular and whole organism level • If I escape dementia perhaps my next lecture will explain how these things were achieved. Antioxidants, Nutrition and Ageing Acknowledgements Biomedical Research Council National Medical Research Council Academic Research Fund, National University of Singapore (NUS) Ministry of Education (Tier 2) Office of Life Sciences, NUS Singapore Totalisator Board Tan Chin Tuan Centennial Professorship Funds, NUS

‘Money won’t buy happiness, but it will pay the salaries of a large research staff to study the problem.’ Bill Vaughan, American chemist and author, 1915-1977 Antioxidants, Nutrition and Ageing