Antioxidants, Nutrition and Ageing Miracles, Myths and ... · Antioxidants, Nutrition and Ageing – Miracles, ... Therapeutic implications for ... antioxidants. • Their bioavailabilty

Professor Barry Halliwell Tan Chin Tuan Centennial Professor

Deputy President (Research & Technology)

National University of Singapore

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

Antioxidants, Nutrition and Ageing –

Miracles, Myths and

Misunderstandings

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


Oxidant – Antioxidant Balance

Phagocytes

AA Metabolism

Mitochondrial

Respiration

Xanthine oxidase

Peroxiredoxins

Glutathione system SOD, Catalase

Blood

components

Albumin,

Caeruloplasmin,

Transferrin,

haptoglobin etc

Diet-Derived

Antioxidants

Iron chelators

So some oxidative damage is inevitable and repair &/or replacement of

damaged molecules is essential

NADPH oxidases

DUOxes

Other electron

transport chains

Haem proteins


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.


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


HOW TO STUDY THIS?

1. Cell culture

2. Animal models

3. Humans

Mechanism of

Neurodegeneration CAN IT BE SLOWED BY DIETARY OR SYNTHETIC

COMPONENTS?


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

When I was young, the Free

Radical –Antioxidant Field

was SIMPLE

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.


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


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


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


Taking Antioxidants will

Prevent Disease

When I was young, it was SIMPLE

(with several exceptions)


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 %)

BUT SEVERAL TRIALS DID SUCCEED!

Circulation.

2003;107:947-953.


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.

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


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


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.

BMJ (2010) 341:c5702


• 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

Intervention Trials with Vitamin E

VALIDITY OF RODENT

MODELS OF STROKE AND

NEURODEGENERATIVE

DISEASE? By courtesy of John Milner, National Cancer

Institute


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.


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.


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


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


Vorinostat Histone deacetylase inhibitor under evaluation as anti-cancer drug

Wong et al. Pharmacogenetics & Genomics

2011, 21(11):760-8

Vorinostat

Vorinostat Glucuronide

(less activity)

4-Anilino-4-oxobutanoic acid

Glucuronidation (UGT2B17)

Hydrolysis followed

by β-oxidation

UGT2B17*2 homozygotes present in 62% of our cohort

UGT2B17*2 (del variant)

•Reduced UGT2B17 activity

•Homozygotes: 10% Caucasians

60-70% Asians

Slide courtesy of Prof John Wong

RESPONSES MAY ALSO CHANGE WITH

RACIAL ORIGIN.

RESPONSE TO NUTRIENTS IS ALSO LIKELY

TO BE DIFFERENT


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.

THEY MAY ALSO VARY WITH GENETIC

BACKGROUND AND AGE

Arterioscler Thromb Vasc Biol.

2008; 28:341-347

Atherosclerosis 2011;

219:240-244

Pharmacogenomics 2010;

11:675-684


• 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

THE FLAVONOID STORY

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


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 Work in the GI Tract!

STOMACH SMALL

INTESTINE

COLON/RECTUM

High concentration of ascorbate in food

and gastric juice

Vitamin C completely absorbed* Little vitamin C present*

Limited α–tocopherol present*

Carotenoids, tocopherols, tocotrienols,

flavonoids, other phenolics in food (if

fruit/vegetable/grain – rich diets

consumed)

Vitamin E largely absorbed*

Some tocotrienols and β, γ, δ–

tocopherols returned to GI tract in bile.

Considerable amounts of unabsorbed

flavonoids, other

phenolics,carotenoids, tocotrienols, β,

γ, δ–tocopherols (if diet rich in these

compounds).

Scavenge

RNS from acidified nitrite

OH▪ from Fe or Cu/ascorbate

interactions

RO▪ and RO2▪ from dietary lipid

peroxides

Some phenolics bind Fe2+ or Cu2+,

to decrease their ability to generate

RS

Ferryl species from haem

protein/peroxide interactions

stimulate lipid oxidation; quenched

by ascorbate and phenols which

are preferentially oxidized

H2O2 in consumed beverages

Some carotenoids cleaved to vitamin A

Some carotenoids, flavonoids and other

phenolic compounds absorbed, but many

are not.

Extensive metabolism of polyphenols

by colonic flora to generate simpler

phenols

Scavenging/metal binding/other

actions of phenols might help delay

colon/rectal cancer development by

exerting external protective effects on

colonic/rectal epithelium

Inhibitions of LOX, COX–2,

angiogenesis, matrix

metalloproteinases etc by phenols

may help to limit cancer development

*May not be true if supplements taken

Duodenum Ileum Jejenum


And by doing so they may indirectly influence

the brain

Genes Nutr (2012) 7:99–109


Antioxidant activity equivalents in portions of

beverages, vegetables, fruit and chocolate

7.5 Blackcurrant Juices

1.1 L

7 Onions

1 Kg 2 Red wines

300 ml

27 Beers

4 L

15 Orange Juices

2.25 L

6 Apples

960 g

Slide courtesy of Prof Catherine Rice-Evans

Comparisons with the ABTS•+ assay

(a nitrogen-centred radical)

4.5 Cups of tea

675 ml

28 White wines

4.2 L

1 bar dark chocolate

50 g


• What components of the

food are responsible?

• Does it have antioxidant

effects in vivo?

TWO QUESTIONS FOLLOW


POWERFUL

ANTIOXIDANT

ACTIVITY OF DARK

SOY SAUCE

(ABTS assay)

• 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.

http://apps.isiknowledge.com.libproxy1.nus.edu.sg/WoS/CIW.cgi?SID=T1gdgc6lgGfm5oMadGL&Func=OneClickSearch&field=AU&val=Halliwell+B&curr_doc=1/1&Form=FullRecordPage&doc=1/1


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.

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.


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.


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.


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??


Oxidant – Antioxidant Balance

Phagocytes

AA Metabolism

Mitochondrial

Respiration

Xanthine oxidase

Peroxiredoxins

Glutathione system SOD, Catalase

Blood

components

Albumin,

Caeruloplasmin,

Transferrin,

haptoglobin etc

Diet-Derived

Antioxidants

Iron chelators

NADPH oxidases

DUOxes

Other electron

transport chains

Haem proteins

DO POLYPHENOLS ACT AS ANTIOXIDANTS &/OR PRO-OXIDANTS

IN THE GASTROINTESTINAL TRACT,

AND DOES IT MATTER WHICH?

Artefacts in

Cell Culture


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


Cell Culture is an Oxidative

Stress

Second major factor – cell culture

media are poor in antioxidants

• vitamin C

• vitamin E (little lipid)

• Selenium


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


H2O2 Generation by Ascorbate


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

Other compounds that generate H2O2 include Delphinidin, Rosmarinic acid, Hydroxytyrosol, Quercetin, Catechin

and Gallic Acid


Rapid loss of polyphenols upon incubation in DMEM at 37oC.

Data are mean ± SD, n ≥ 3.

Delphinidin chloride

Hydroxytyrosol

Rosmarinic acid

Curcumin

Resveratrol

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

not all generate H2O2!


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

Examples of artefacts caused by oxidation of

compounds added to cell culture media


REUTERS/CORBIS

Centenarians now constitute the

fastest-growing age group owing to

advances in health care.

Source – Nature 467 (2010), 274-

275

0 5 10 15 20 25 30 35

CH

JP

SG

AUST

DK

UK

2030

2005

1980

Proportion of population aged 65+ in

selected IARU countries

Slide by courtesy of Dr Kenneth Howse, Oxford

University

Source: UN Population database The International Alliance of Research Universities

(IARU) is a collaboration between ten of the world’s leading research-intensive universities who share 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.

A COMING PROBLEM FOR SINGAPORE


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


C.elegans as a Model System

• Soil dwelling nematode worm

• Eats bacteria

• 99% Self-fertilizing hermaphrodites

• => Isogenetic clones !

Adult:

length: 1mm Ø: 100m

Volume: approx 1nL

Metabolic rate: 45nW

Offspring: ~300 in < 8 days

Lifespan: max 40 days

WORK WORM OF AGEING RESEARCH


Control 10μg/ml 100μg/ml

C-

PC 10μg/ml

Some more advantages

• Extensive set of mutants and tools

- 1000s of mutant strains

- GFP reporter gene system

- RNAi is relatively easy

• Very “simple” nervous systems

• Total complement of 302 neurons

– Essentially invariant structure

– Complete wiring diagram

known

• There is still randomness in the

ageing process.

PC

C


Lifespan and Healthspan

Histogram for day of death for 200 isogenic C.

elegans clones, born on the same day in our lab and

grown under identical culture conditions. Even

though their genome as well as environment are

identical, their individual lifespan ranges from 10

days to 34 days, probably due to intrinsic stochastic

factors inherent in ageing.

Motility phenotype distribution in the same

isogenic C. elegans population on different

days of life. Even thought both genome and

environment are identical, surviving worms

of the same age may be found to have

dramatically different motility class, an

indicator of health.


The effects of plant-derived

“antioxidants” are dose-variable and

independent of antioxidant activity.

Biogerontology (2010) 11:17-30


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.

Several effective compounds isolated


• 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.

Major Challenges in the ROS field


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


Documents

Antioxidants, Nutrition and Ageing Miracles, Myths and ... · Antioxidants, Nutrition and Ageing – Miracles, ... Therapeutic implications for ... antioxidants. • Their bioavailabilty