Dietary reference values of individual micronutrients and

nutriomes for DNA damage prevention

Current status and a road map to the future

Michael FenechMichael FenechCSIRO Food and Nutritional SciencesCSIRO Food and Nutritional SciencesGENOME HEALTH NUTRIGENOMICS LABORATORYGENOME HEALTH NUTRIGENOMICS LABORATORY

michael.fenech@csiro.au

AICR ConferenceAICR ConferenceWashington 21Washington 21--22 October 201022 October 2010

Fenech MF, Am J Clin

Nutr. 2010; 91(5):1438S-1454S

Overview

Biomarkers used to study DNA damage in humans

Association of DNA damage with developmental and degenerative disease

Current knowledge of nutritional requirements for genome maintenance and stability

Effect of nutrient-nutrient and nutrient-genotype interaction on DNA integrity

Strategies to determine DRVs

of single micronutrients and micronutrient combinations (nutriomes) for DNA damage prevention

A NUCLEOCENTRIC VIEW OF AGEINGA NUCLEOCENTRIC VIEW OF AGEINGFROM WOMB TO TOMBFROM WOMB TO TOMB

BIOMARKERS OF DNA DAMAGEBIOMARKERS OF DNA DAMAGE

NPB

MN

dicentric

Gene expression arrays γH2AX

AcknowledgementJohn RK SavageAtlas Genet CytogenetOncol

Haematol. July 2000

The principal structural

chromosomal aberrations which

contribute acentric fragments

(AF) to form micronuclei (MN)

and nucleoplasmicbridges (NPB)

Genome damageGenome damageMicronucleiMicronuclei

Human cellswith damaged& unstablegenomes

• Oxidative stress• Nutrient deficiency• Excess calories

• Strand breaks in DNA•Chromosome malsegregation•DNA hypomethylation•Telomere shortening

CYTOKINESISCYTOKINESIS--BLOCK MICRONUCLEUS (CBMN) ASSAYBLOCK MICRONUCLEUS (CBMN) ASSAY

Increased lymphocyte micronucleus frequency in early pregnancy is associated prospectively with pre-eclampsia

and/or intrauterine growth restriction

(PEIUGR)(PEIUGR)

D.L.F FurnesS, G.A Dekker, W.M Hague T .Y Khong and M.F FenechMutagenesis (2010)

ADVERSEN=7

ADVERSEN=67

LOW RISKN=45

HIGH RISKN=91

LRNN=38

PEIUGRN=36

OAON=38

HRNN=24

RISK GROUPS PREGNANCY OUTCOME GROUPS

LYMPHOCYTELYMPHOCYTEMICRONUCLEIMICRONUCLEIMEASURED AT MEASURED AT 18 WKS18 WKSGESTATIONGESTATION

02468

10121416

>19.3 >28.0 >36.7

Odd

s R

atio

Odd

s R

atio

MNMN--BN BN ‰

P=0.003P=0.003 P=0.006P=0.006

P=0.001P=0.001

PROSPECTIVE ODDS RATIOPROSPECTIVE ODDS RATIOOF PEIUGR FOR ALL COHORTOF PEIUGR FOR ALL COHORT

Risk of cancer increases with higher MN frequency

Bonassi et al (Carcinogenesis 2007)

PRO

BA

BIL

ITY

OF

SUR

VIVI

NG

W

ITH

OU

T C

AN

CER

FOLLOW UP TIME IN YEARS

6,983 subjects275 cancer cases

HUMN project (www.humn.org)

Lymphocyte Micronucleus frequency is associated prospectively with cardiovascular disease mortality in

both the general population and those with known coronary artery disease

MN>1MN>1‰‰

MNMN<<11‰‰

MN frequency

178 CAD patients178 CAD patientsMean age 62 yMean age 62 y

1650 subjects1650 subjects111 deaths111 deaths

39 due to CVD39 due to CVDMean age 65 yMean age 65 y

Federici C et al

GENOME DAMAGE INCREASED IN LYMPHOCYTES OF GENOME DAMAGE INCREASED IN LYMPHOCYTES OF ALZHEIMER AND PARKINSONALZHEIMER AND PARKINSON’’S DISEASE PATIENTSS DISEASE PATIENTS

DNA DAMAGE IN LYMPHOCYTES

AD PD CONTROL0

5

10

15

20 * *

* P<0.001

MN

PER

100

0 B

NC

s

DISTRIBUTION OF TYPE OF MICRONUCLEI

AD PD CONTROL0

25

50

75

100

MN (CHROM. LOSS) MN (CHROM. BREAK)

p < 0.0001

%

PetrozziPetrozzi

et al. Neurol. Sci. 2002et al. Neurol. Sci. 2002

18-2

5

26-3

5

36-4

5

46-5

5

56-6

5

66-7

5

76-9

0

0

10

20

30

40

50

MN

per

100

0 B

N c

ells

AGE (years)

DNA damage increases with DNA damage increases with age ....orage ....or……. poor choices of nutrition, life. poor choices of nutrition, life--style, style,

physical and sociophysical and socio--psychological environments?psychological environments?

Fenech et al. 2000

What is theWhat is thethreshold ofthreshold ofDNA damageDNA damagewe should we should allow?allow?

Can we designCan we design““exposomesexposomes””that enable us that enable us to stay belowto stay belowthis threshold?this threshold?

ENVIRONMENTALENVIRONMENTALGENOTOXINSGENOTOXINS

LIFELIFE--STYLESTYLEGENOTOXINSGENOTOXINS

MALNUTRITIONMALNUTRITION

GENETICGENETIC

AND AND

ACQUIRED ACQUIRED

SUSCEPTSUSCEPT--

IBILITYIBILITY

GENOMEGENOMEDAMAGE INDAMAGE IN::

GERMLINEGERMLINE

EMBRYOEMBRYO

FOETUSFOETUS

BABYBABY

CHILDCHILD

TEENAGERTEENAGER

YOUNG ADULTYOUNG ADULT

OLDER ADULTOLDER ADULT

HIGHERHIGHERRISKRISK

LOWERLOWERRISKRISK

DE

VE

LOP

ME

NTA

LD

EV

ELO

PM

EN

TAL

DE

GE

NE

RA

TIVE

DE

GE

NE

RA

TIVE

DIS

EA

SE

SD

ISE

AS

ES

CAUSES AND EFFECTS OF GENOME DAMAGE

EEXXPPOOSSOOMMEE

PSYCHOLOGICALPSYCHOLOGICALSTRESSSTRESS

Italy Germany USA

Poland Mexico Ecquador

Bhutan Chad Egypt Dietary patterns & 1 week’s food

The new frontier in nutrition The new frontier in nutrition

NUTRIOME GENOME

HHEEAALLTTHH

Nutrition designed for diverse genetic backgroundsNutrition designed for diverse genetic backgroundsto optimise genome maintenance and prevent diseases to optimise genome maintenance and prevent diseases

caused by DNA damagecaused by DNA damage

The following genomic stability processes are The following genomic stability processes are modulated by vitamins or micronutrientsmodulated by vitamins or micronutrients:

DNA oxidation preventionDNA oxidation prevention Vit C, Vit E, Se, polyphenols

DNA DNA methylationmethylation, synthesis, synthesis folate, Vit B12, Zn, Mg

DNA repairDNA repair niacin, Zn, folate

Gene expressionGene expression folate, Vit D, Vit A

Chromosome segregationChromosome segregation folate, Vit A, Mg

Telomere lengthTelomere length niacin? via PARP, folate

Necrosis/ApoptosisNecrosis/Apoptosis niacin, Zn, Vit E, Vit D, Vit CVit A, Vit K2.

Folate, B12, B6 and B2 and genome maintenance

METHIONINE

HOMOCYSTEINE

SAM

THF

5-MeTHF

5,10-MeTHFDNA

DNA METHYLATION

DHF

FOLIC ACID

dUMP

dTMP

DNA REPAIR & SYNTHESIS

TS

MTHFRVIT B2

SHMT1VIT B6

MTRR

MTRVIT B12 (CoIII)

MTRVIT B12

(CoI)

SeedLymphocytes

44 h

Nutrients

20 h

Mitogen

1 h

+/-Genotoxin

+/-Genotoxin

Cytochalasin

B (cytokinesis

blocking agent)

72h

Nuclear stain/Fix

Image acquisition

and analysis

Minimally invasive HighMinimally invasive High--Throughput Throughput

Nutrient Array screening for GenomeNutrient Array screening for Genome--Protective agentsProtective agents..

Micronucleus

Targeted Cell

SelectedNuclei

Development of automated CBMN Cytome

assay:MN, NPB, NBUDs, Necrosis, Apoptosis, NDI

+ FISH + Protein expression

20ul blood

A B C

D E

F G H

CBMNCBMNCYTOME CYTOME ASSAYASSAY

A ,B, C

NUCLEAR DIVISION

INDEX

D,E

CELL DEATH

F, G, H

DNA DAMAGE

Fenech M (2007) Nature ProtocolsFenech M (2007) Nature Protocols

MICRONUCLEATED CELLS

12 24 60 1200

10

20

30

40

*

**

[A]

folic acid in medium (nM)

MN

ed B

Ns/

1000

BN

s ANOVA P < 0.0001

NUCLEAR BUDS

12 24 60 1200

5

10

15

ANOVA P<0.0001

*

**

[B]

folic acid in medium (nM)

Bud

s/10

00 B

Ns

NUCLEOPLASMIC BRIDGES

12 24 60 1200.0

2.5

5.0

7.5

10.0ANOVA P<0.0001*

**

[C]

[folic acid] in medium (nM)

NPB

/100

0 B

Ns

URACIL

12 24 60 1200

10

20

30

40

50ANOVA P < 0.0001

[folic acid] in medium (nM)

pg u

raci

l / g

DN

A

* *

[D]

Crott et al. Carcinogenesis 2001

0

5

10

15

20

25

30

35

0 5 10 15 20 25

MN

ed B

Ns/

1000

BN

s

0 5 1024

20120 60 12

rad X-rays

Fenech & Morley, 1986

nM folic acid

Genome damage induction by Folic acid deficiency is of a similarGenome damage induction by Folic acid deficiency is of a similar magnitude as that induced by unsafe doses of magnitude as that induced by unsafe doses of ionisingionising radiationradiation

IAEA annual safe exposure limit ie

equivalent to 0.1-0.5 rad

X-rays

Crott & Fenech 2002

“normal”serum folate

concentration

PLACEBOPLACEBO FOLB12FOLB12

ROUND 1ROUND 1 ROUND 1ROUND 1 BASEBASE--LINELINE*CEREAL CEREAL +ONLY 7ug B12 +

700ug FOL

ROUND 2ROUND 2 ROUND 2ROUND 2 12 WEEKS12 WEEKS*TABLET TABLETPLACEBO 20ug B12 +

2000ug FOL

ROUND 3ROUND 3 ROUND 3ROUND 3 24 WEEKS24 WEEKS*

* blood sample

Randomised, placeboRandomised, placebo--controlled dietary interventioncontrolled dietary intervention

Fenech et al. 1998 Carcinogenesis

0

3

6

9

12

Low MNed cell freq.at R1. [N = 17]ANOVA P = 0.65

High MNed cell freq.at R1. [N = 16]ANOVA P < 0.0005

R1 R1 R2 R2 R3 R3

* *

MN

ed c

ells

per

100

0 B

N c

ells

Supplementation with 3.5 times RDI folic acid & vit B12reduces micronucleus index by 25 % in subjects with above average chromosome instability

Fenech et al. 1998 Carcinogenesis BaseBase--lineline 7ug B127ug B12700ug FA700ug FA

20ug B1220ug B122mg FA2mg FA

0 5 10 15 200

5

10

15

20

N = 48R = 0.392P = 0.006

MNed cells per 1000 BN cells

plas

ma

HC

(m

ol/l)

0 5 10 15 200

150

300

450

600

750

N = 48R = -0.489P = 0.0004

MNed cells per 1000 BN cells

seru

m B

12 (p

mol

/l)

Fenech et al. 1998 Carcinogenesis

M N frequency vs plasma HC inmen (50-70years) who were not

deficient in B12 or folate

0 10 20 30 400

5

10

15

R = 0.4148 P = 0.0086 N = 39

MN frequency per 1000 BN cells

plas

ma

HC

[m

ol/l]

serum B12 vs MN frequency inmen aged 50-70 years who

were not vitamin B12 deficient

0 10 20 30 400

100

200

300

400

500

600

R = -0.3418 P = 0.0127 N = 62

MN frequency per 1000 BN cells

seru

m B

12 [p

mol

/l]

MICRONUCLEUS FREQUENCY IS MINIMISED WHEN MICRONUCLEUS FREQUENCY IS MINIMISED WHEN HOMOCYSTEINE < 7.5umol/l and B12 > 300pmol/lHOMOCYSTEINE < 7.5umol/l and B12 > 300pmol/l

Fenech et al. (1997) Carcinogenesis

YOUNG ADULTS 18YOUNG ADULTS 18--32Y32Y

N=62N=62R=R=--0.310.31P==0.013P==0.013

MEN AGED 50MEN AGED 50--70Y70Y

300 times recommended intake of B12 is required to 300 times recommended intake of B12 is required to normalise normalise holoTCholoTC, MMA and , MMA and homocysteinehomocysteine in >70 year oldsin >70 year olds

EussenEussen

SJ et al 2005SJ et al 2005

Future Challenges: We have started to determine the impact of common polymorphisms in folate metabolism genes on chromosome damage in

Australians

Dhillon et al Mutation Res Fund Mech

2009

*age & gender adjusted

% variation in genome damage withincreased intake relative to lowest tertile of intake

Vita

min

E

Cal

cium

Fola

te

Ret

inol

Nic

otin

ic a

cid

-Car

oten

e R

ibof

lavi

n

Pant

othe

nic

acid

Bio

tin

-50-40-30-20-10

010203040506070

mid-tertilehighest tertile

*

** * *

*

*

*

**

** P < 0.006

% v

aria

tion

in M

N fr

eq

MICRONUTRIENTS AND GENOME DAMAGEMICRONUTRIENTS AND GENOME DAMAGERESULTS OF ANALYSIS OF FOOD FREQUENCY QUESTIONNAIRE AND GENOME DAMAGE DATABASE

Fenech et al.Fenech et al.CarcinogenesisCarcinogenesis(2005)(2005)

COMBINED EFFECT OF FOLATE AND CALCIUM INTAKE ON MN FREQUENCY

-70

-60

-50

-40

-30

-20

-10

0

low tertile mid tertile high tertile

low tertilemid tertilehigh tertile

CALCIUM INTAKE

FOLATEINTAKE

% V

ARIA

TIO

N

*

** *

**

(a) COMBINED EFFECT OF FOLATE AND RIBOFLAVIN INTAKE ON MN FREQUENCY

-40-30-20

-100

10

203040

50

low tertile mid tertile high tertile

low tertilemid tertilehigh tertile

FOLATE INTAKE

RIBOFLAVININTAKE

% V

ARIA

TIO

N * *

* *

(b)

The combined effect of (a) calcium and folate

intake and (b) riboflavin and folate

intake on MN frequency. Results shown are the % variation relative to the combined lowest tertiles

of intake in the pair of nutrients examined. * P < 0.05 for comparison with the referent value for the combined lowest tertile

of intake for the pair of nutrients examined.

NUTRIENTNUTRIENT--NUTRIENT INTERACTION ON CHROMOSOME DAMAGENUTRIENT INTERACTION ON CHROMOSOME DAMAGE

MORE RIBOFLAVINMORE RIBOFLAVININ A LOW FOLATE IN A LOW FOLATE

BACKGROUND BACKGROUND MAY BE MAY BE

GENOTOXICGENOTOXIC

Fenech et al 2005 Carcinogenesis; Fenech 2010 AJCNFenech et al 2005 Carcinogenesis; Fenech 2010 AJCN

ALMONDSWHEATBRAN

CHEDDAR CHEESE

BROCCOLI (BOILED)

TUNA (CANNED)

BEEF (COOKED)BANANA

0

100

200

300CALCIUMFOLATENIACINVITAMIN EBETA-CAROTENERETINOL

AMOUNTS IN 100g EXPRESSED AS % OF MIMIMUMREQUIREMENT FOR OPTIMUM GENOME HEALTH

Fenech, Food Chem

Tox

2008

Q. Which dietary pattern will work for your genotype ?Q. Which dietary pattern will work for your genotype ?

A. It depends on the A. It depends on the ““nutriomenutriome””

of the foods you prefer to eatof the foods you prefer to eat

. High Folate (HF) Vegetables Low Folate (LF) Vegetables

Pulses Leafy or cruciferous vegetables

Roots or Tubers

“Fruit” Vegetables

Red Kidney beans (130)

Broccoli (93) Onions (16) Tomato (15)

Mung beans (60)

Brussel sprouts (60)

Potato (22) Pumpkin (9)

Chickpeas (171)

Cabbage (43) Turnip (9) Cucumber (6)

Lentils (180) Endive (142) Parsnip (57) Capsicum (11)

Peas (59) Spinach (146) Swede (21) Eggplant (14)

Lima beans (50)

Lettuce (73) Carrot (14) Olives (0)

Mean (108) Mean (93) Mean (23) Mean (10)

Mean (100) Mean (16)

Folate content of vegetables (DFE µg per 100g)*

Eating the“wrong”

vegetablescould lead to folate

deficiency

Folate

RDA 400μgrequires eating:

2.5Kg LF veg/dor0.4Kg HF veg/d

*Data from USDA National Nutrient data base. DFE = dietary folate

equivalent. DFE values are shown in brackets.

TELOMERES (TTAGGG TELOMERES (TTAGGG repeats) ARE ESSENTIALrepeats) ARE ESSENTIALFOR CHROMOSOMEFOR CHROMOSOMESTABILITYSTABILITY

TELOMERE SHORTENING OR TELOMERE SHORTENING OR DYSFUNCTION INCREASES DYSFUNCTION INCREASES RISK FOR CANCER ANDRISK FOR CANCER ANDACCELERATED SENESCENCEACCELERATED SENESCENCE

TELOMERESTELOMERES

Wong & Collins Lancet 2003Wong & Collins Lancet 2003

SHORTER TELOMERES ARE ASSOCIATED SHORTER TELOMERES ARE ASSOCIATED PROSPECTIVELY WITH INCREASED RISK FOR CANCER PROSPECTIVELY WITH INCREASED RISK FOR CANCER

Willeit

P et al, JAMA.

2010; 304(1):69-75.

0

0.5

1

1.5

2

2.5

3

3.5

1st Qtr

LONGMEDIUMSHORT

LEUKOCYTE LEUKOCYTE TELOMERE LENGTHTELOMERE LENGTH

MULTIVARIATE HAZARDMULTIVARIATE HAZARDRATIO FOR CANCER INCIDENCE RATIO FOR CANCER INCIDENCE

Shorter telomeres in WBCs

and Buccal cells of Alzheimer’s disease (AD) cases compared to controls

Young contro

lsOld co

ntrols

Younger AD

Older AD

0

25

50

75

100

125

150

175 a

b

bcc

Abs

olut

e te

lom

ere

leng

th(K

b pe

r di

ploi

d ge

nom

e)

Young contro

lsOld co

ntrols

Younger AD

Older AD

0

10

20

30

40

50 aa

ab

b

Abs

olut

e te

lom

ere

leng

th(K

b pe

r di

ploi

d ge

nom

e)

young controls (N=30), old controls (N=26), younger AD (N=14), older AD (N=18).

Thomas et al, Mech Aging Dev, 2008

WBC TL <115 Kb per diploid genome OR of being diagnosed with AD is 10.8specificity 46% sensitivity 92.9%.

Buccal TL <40 kb per diploid genomeOR of being identified with AD is 4.6 specificity 63% sensitivity 72.7%.

FOLATEFOLATE

VITAMIN EVITAMIN E

VITAMIN DVITAMIN D

ΩΩ33--FATTY ACIDSFATTY ACIDS

CEREAL FIBRECEREAL FIBRE

MULTIVITAMIN USEMULTIVITAMIN USE

PUFAPUFA

OXIDATIVE STRESSOXIDATIVE STRESS

OBESITYOBESITY

PSYCHOLOGICAL PSYCHOLOGICAL STRESSSTRESS

PROCESSED MEATPROCESSED MEAT

HOMOCYSTEINEHOMOCYSTEINE

LONGERLONGER SHORTERSHORTERTELOMERESTELOMERES

CURRENTCURRENTKNOWLEDGEKNOWLEDGE

Int

J Obes

(Lond). 2010 Aug;34(8):1345-8; Am J Clin

Nutr. 2010 May;91(5):1273-80. Rejuvenation Res. 2009 Oct;12(5):341-9. ; Rejuvenation Res. 2009 Jun;12(3):169-76. J Nutr. 2009 Jul;139(7):1273-8; Am J Clin

Nutr. 2009 Jun;89(6):1857-63. Am J Clin

Nutr. 2007 Nov;86(5):1420-5.

FOLATE DEFICIENCY

NIACIN ORNICOTINIC

ACIDDEFICIENCY

ANTIOXIDANTDEFICIENCY

OR OXIDATIVESTRESS

8-OHdG

URACIL

HYPO-METHYLATED

SUBTELOMERE

BREAKS IN TELOMERE

REDUCEDTRF1, TRF2

BINDING

LOSS OFTELOMERE

LENGTH CONTROL

DYSFUNCTIONALAND/OR SHORT

TELOMERES;TELOMERE END

FUSIONS;AND CIN

REDUCED TANK1

ACTIVITY

?

?

?

TELOMERE DAMAGE CASCADETELOMERE DAMAGE CASCADE

Bull & Fenech Proc. Br Bull & Fenech Proc. Br NutrNutr

Soc 2008Soc 2008

High protein (TWD) or high carbohydrate (HC) weight-loss diets reverse telomere shortening in rectum in over-weight men

TWDTWD HCHC

Telo

mer

e le

ngth

(kb)

Telo

mer

e le

ngth

(kb)

0 12 52 0 12 520 12 52 0 12 52

2.62.6

5.25.2

7.87.8

10.410.4

13.013.0

15.615.6

18.218.2

0.00.0

2 way ANOVA2 way ANOVAP<0.0001P<0.0001

weeks on dietweeks on dietOO’’Callaghan, Clifton, Callaghan, Clifton, NoakesNoakes, Fenech. , Fenech. Rejuvenation Res. 2009Rejuvenation Res. 2009

IS A COMPREHENSIVE SET OF DNA IS A COMPREHENSIVE SET OF DNA DAMAGE BIOMARKERS NEEDED DAMAGE BIOMARKERS NEEDED

FOR PERSONALISED NUTRITION ANDFOR PERSONALISED NUTRITION ANDDRV DETERMINATION FOR GENOMEDRV DETERMINATION FOR GENOME

DAMAGE PREVENTION?DAMAGE PREVENTION?

LYMPHOCYTE CBMN ASSAYLYMPHOCYTE CBMN ASSAY

RBC MN ASSAYRBC MN ASSAY

BUCCAL MN ASSAYBUCCAL MN ASSAY

COMET ASSAYCOMET ASSAY

TELOMERE LENGTH MITOCHONDRIAL DNA DELETION TELOMERE LENGTH MITOCHONDRIAL DNA DELETION DNA OXIDATION DNA METHYLATIONDNA OXIDATION DNA METHYLATION

Fenech M (2010) Am. J. Fenech M (2010) Am. J. ClinClin. . NutrNutr..

Validation status of DNA damage biomarkersASSOCIATION WITH NUTRITIONAL

STATUSASSOCIATION WITH DEVELOPMENTAL

OR DEGENERATIVE DISEASE OR MORTALITY

CROSS-SECTIONAL STUDIES

PLACEBO- CONTROLLED

STUDIES

CASE-CONTROL STUDIES

PROSPECTIVE COHORT STUDIES

CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY

IN LYMPHOCYTES

MICRONUCLEUS ASSAY IN BUCCAL CELLS ?MICRONUCLEUS

ASSAY IN ERYTHROCYTES

?DNA STRAND BREAKS IN LYMPHOCYTES BY

COMET ASSAY ?

DNA OXIDATION ?DNA METHYLATION ?

TELOMERE LENGTH IN LEUKOCYTES OR LYMPHOCYTES

?

MITOCHONDRIALDNA DELETION ?

DNA DNA DAMAGE DAMAGE

BIOMARKERSBIOMARKERS

Fenech M (2010) Am. J. Fenech M (2010) Am. J. ClinClin. . NutrNutr..

NUTRITIONVARIABLES

SINGLEMICRONUTRIENT

MICRONUTRIENTCOMBINATION

FUNCTIONALFOOD

FOOD GROUP

DIETARY PATTERN

STUDY DESIGN

IN VITRO MODELS

IN VIVOCROSS-

SECTIONALSTUDIES

PLACEB0-CONTROLLED

TRIALS

OUTCOME MEASURES

PRIMARY

DNA DAMAGEBIOMARKERS:

MICRONUCLEUS CYTOME ASSAYS

COMET ASSAYDNA OXIDATION

DNA METHYLATIONTELOMERE LENGTH

mtDNA DELETION

SECONDARY

TISSUEMICRONUTRIENTCONCENTRATION

DRVs FORGENOME STABILITY

DATABASESON VITAMIN & MINERAL

REQUIREMENTSFOR GENOME

STABILITYIN

DIVERSEGENETIC

BACKGROUNDSAT THE

VARIOUS LIFE-STAGES

PROPOSED ROAD-MAP TO DETERMINE DRVs FOR GENOME STABILITY

Fenech M (2010) Am. J. Fenech M (2010) Am. J. ClinClin. . NutrNutr..

LIFELIFE--STYLE HPI INDEXSTYLE HPI INDEX

LIFELIFE--STYLE IS ALSO AN IMPORTANT DETERMINANT OF DNA DAMAGESTYLE IS ALSO AN IMPORTANT DETERMINANT OF DNA DAMAGE

Huang et al Preventive Medicine 48 (2009) 383-388

NUTRINUTRI--GENOMICSGENOMICS

TOXICOTOXICO--GENOMICSGENOMICS

GENOMEGENOMEHEALTH HEALTH STATUSSTATUSOF THE OF THE

POPULATIONPOPULATION

PSYCHOPSYCHO--GENOMICSGENOMICS

LIFESTYLELIFESTYLEGENOMICSGENOMICS

AUTOMATED DIAGNOSTICSAUTOMATED DIAGNOSTICSGENOME HEALTH -

NUTRIENT STATUS -

GENOTYPE

NUTRIGENOMICSNUTRIGENOMICSEXPERT ADVICE SYSTEM

DATA BASE

INDIVIDUALISED NUTRITIONINDIVIDUALISED NUTRITIONDIETARY PATTERNS -

FUNCTIONAL FOODS -

SUPPLEMENTS

RA

DIC

AL IN

NO

VATIO

N IN

NU

TRITIO

N

VE

RIFIC

ATIO

N O

F EFFIC

AC

Y

GENOME HEALTH OPTIMISEDGENOME HEALTH OPTIMISEDDEVELOPMENTAL & DEGENERATIVE DISEASE PREVENTED

FEE

DB

ACK

TO D

ATAB

ASE

Funding: Reach 100, NCEFFFunding: Reach 100, NCEFF ABC Catalyst, DNA Doctor StoryABC Catalyst, DNA Doctor StoryFenech M (2005) Mutagenesis 20: 225Fenech M (2005) Mutagenesis 20: 225--269269

“We are at the threshold of a new era in which harm to the genome, which is the most fundamental pathology, can

be efficiently diagnosed and prevented.

A person’s DNA damage profile is likely to become the ultimate routine biomarker of health status.

Prevention of DNA damage will soon achieve its rightful place as one of the most important objectives of global

health strategies.”

Michael Fenech October 2010Michael Fenech October 2010

Felicia BulmanJulie Turner

Carolyn SalisburyPhilip ThomasJimmy Crott

Will GreenrodJosy RinaldiClare AitkenSally RecordMaryam Hor

Theodora Hua HaenJing Wu

Caroline BullNathan O’Callaghan

Wayne LeifertGlen Patten

Erin SymondsBianca BenassiSasja Beetstra

CHORI (USA)CHORI (USA)Bruce Ames, Susan Mashiyama

Natl. Ins. Natl. Ins. HlthHlth & & NutrNutr JapanJapanKeizo Umegaki, Michiyo Kimura

HUMNInternational Collaborative Project on Micronucleus frequency in human populations

•40 labs•16 countries•>12,000 subjects•>70,000 person years

Coordinating Group:Michael Fenech (Australia) ChairmanStefano Bonassi (Italy)Wushou Chang (Taiwan)Nina Holland (USA)Errol Zeiger (USA)Micheline Kirsch-Volders (Belgium)

Founded Toulouse 1997

WCH AdelaideGraeme SuthersPeter BaghurstRAH AdelaideIan Olver, Eric Yeoh

AcknowledgementsAcknowledgements

FUNDING •Kellogs Pty Ltd,•MLA•Blackmore’s,•Nutrilite/Amway•NHMRC,•Cancer Council SA, •NIH/NIAID•EUCancerRiskBio- markers Program•DPI-Victoria

THE GENOME HEALTH THE GENOME HEALTH NUTRIGENOMICS NUTRIGENOMICS

TEAMTEAMCRC DiagnosticsCRC Diagnostics

NO CONFLICT OF NO CONFLICT OF INTEREST INTEREST

OO’’Callaghan, Clifton, Callaghan, Clifton, NoakesNoakes, Fenech. Rejuvenation Res. 2009, Fenech. Rejuvenation Res. 2009

REDUCTION IN FAT CORRELATES WITH INCREASE IN TELOMERE LENGTHREDUCTION IN FAT CORRELATES WITH INCREASE IN TELOMERE LENGTH

Telomere length in older men is significantly associated with plasma folate and homocysteine

Males; r = -0.57, p = 0.004 (n = 24). Females; r = 0.092, p = 0.68 (n = 23)

Males; r = 0.42, p = 0.04 (n = 24). Females; r = -0.11, p = 0.61 (n = 23)

Telomere length is negatively correlated with plasma homocysteine in older men.

Telomere length is positively correlated with plasma folatein males but not in females.

Bull, O’Callaghan, Mayrhofer

& Fenech Rejuvenation Res (2009)

Nutritional Genomics and Nutritional Genomics and DNA Damage Diagnostics LaboratoryDNA Damage Diagnostics LaboratoryCSIRO Food & Nutritional Sciences CSIRO Food & Nutritional Sciences Seed

cells

Nutrients

Mitogen Cytochalasin B (cytokinesisblocking agent)

Nuclear stain/Fix

Image acquisition

and analysis

Minimally invasive HighMinimally invasive High--Throughput Throughput

Nutrient Array screening for GenomeNutrient Array screening for Genome--Protective agentsProtective agents..

Micronucleus

Targeted Cell

SelectedNuclei

Development of automated CBMN Cytome assay:MN, NPB, NBUDs, Necrosis, Apoptosis, NDI

+ FISH + Protein expression

9 day culture

Prof. Michael FenechProf. Michael Fenechmichael.fenech@csiro.au

Is it possible to identify the nutriome

that prevents the growth of each cancer?

One of the greatest challenges in ageing populations is the need

to prevent the proliferation of cancers which accumulate with age.

Currently there is no rational advice on the appropriate diet to

adopt once a person is diagnosed with cancer because our knowledge on nutrient-gene interaction with respect to cancers is rudimentary.

Furthermore there is concern that supplementation with certain nutrients that are required for genome maintenance and cell growth (e.g. folate, methionine) may stimulate the cancer growth

NORMAL ADENOMA NORMAL ADENOMA CANCERCANCER

FOLATE DEFICIENCYFOLATE DEFICIENCY<300 mcg/day*<300 mcg/day*

ADEQUATE FOLATEADEQUATE FOLATE300300--500mcg /day500mcg /day

EXCESSIVE FOLATEEXCESSIVE FOLATE> 1000mcg/day> 1000mcg/day

inhibits??inhibits??

inhibitsinhibits

promotespromotes

promotespromotes

FOLATE DEFICIENCYFOLATE DEFICIENCY<300 mcg/day*<300 mcg/day*

* folate deficiency increases risk of cancer initiation, dementia, stroke, osteoporosis

FOLATE: A DOUBLEFOLATE: A DOUBLE--EDGED SWORD IN CANCER EDGED SWORD IN CANCER DEPENDING ON DOSE AND TIMINGDEPENDING ON DOSE AND TIMING

Metabolism of Folate and MethionineMetabolism of Folate and Methionine

DHF

THF

5,10-Methylene THF

5-Methyl THF

MTHFRVit. B2

Homocysteine

MethionineSAM

SAHCH3

DNA & Protein Methylation

Methylthioribose-1-P

MTOB

Methional

MTA

dcSAMPutrescine

Ornithine

Spermidine

Spermine

Cell Proliferation

+ + +

Protein Synthesis

Salvage Pathway

Apoptosis

+

Vit. B12Cob(III)

Vit. B12Cob(I)

Trans-Sulphuration Pathway

De Novo Pathway

Vit. B12 CatalyticPathway

MTR MTRR

CBS

Folatepolyglutamate

monoglutamate

Diet

RFC-1

Folic Acidmonoglutamate

FGCP

dTMP

dUMP

DNA Synthesis & Repair

TS

CytoplasmExtra Cellular

A nutrient array system could interrogate which nutrient restrictions or supplementations could control any cancer

Cancers are genetically and epigenetically very different from normal tissue in the same person and from each other across persons.

The multiple mutations in a cancer may make it difficult to rely

on genotyping or gene expression patterns to work out an appropriate dietary control strategy.

Some cancers amplify the high affinity folate

receptor and may benefit from folate

restriction.

Other cancers may have defects in methionine

metabolism making them susceptible to methionine

restriction

A nutrient array system could identify the appropriate nutriome

to control a cancer without knowing its genotype/epigenotype

PERSONALISED NUTRIOMES FOR GENOME STABILITY OF STEM CELL & iPS

CELL CULTURES

We now live in an era when stem cells are taken out of the body and expanded in vitro before being returned to the body

Stem cell or iPS

cell cytogenetic abnormalities constitute a roadblock to regenerative therapies because they increase the rate of senescence and the risk of oncogenic

transformation.

Determining the optimal nutriome

in culture medium that prevent chromosomal instability for each stem cell or iPS

culture is therefore important not only to predict in vivo requirements but also in vitro nutrient requirements for DNA damage prevention

Acknowledgement S Acknowledgement S ThuretThuret

(University College London) (University College London)

REGENERATIVE POTENTIAL REGENERATIVE POTENTIAL OF THE HIPPOCAMPUSOF THE HIPPOCAMPUS

REGENERATIVE POTENTIAL REGENERATIVE POTENTIAL OF THE BUCCAL MUCOSAOF THE BUCCAL MUCOSA

The oral epithelium is a stratified squamous epithelium. It consists of four layers:

The keratinised layer at the surface

The prickle cell layer

The basal layer

Lamina propria

7-10 daysfor cells to migrate from basal layer to keratinised layer

MICRONUCLEI

YOUNGER CONTROLS

DOWNSOLDER C

ONTROLSALZHEIM

ER

0

1

2

3 p = 0.0003

KARYORRHEXIS

YOUNGER CONTROLS

DOWNSOLDER C

ONTROLSALZHEIM

ER

0

10

20

30

40

50

60p<0.0001

CONDENSED CHROMATIN

YOUNGER CONTROLS

DOWNSOLDER C

ONTROLSALZHEIM

ER

0

25

50

75 p<0.0001

BASAL CELLS

YOUNGER CONTROLS

DOWNSOLDER C

ONTROLSALZHEIM

ER

0

25

50

75

100

p<0.0001

Buccal Micronucleus Cytome is a powerfulBuccal Micronucleus Cytome is a powerfuldiagnostic of accelerated ageingdiagnostic of accelerated ageing Fenech & Thomas 2007 Mutagenesis

0 50 100 150 2000

50

100

150AD & MCICONTROLS

BIOMARKER 1

BIO

MA

RK

ER 2

Basal cells

kary

orrh

exis

PPV = 98%; NPV = 77%; Sensitivity = 82%, Specificity = 97%LR = 25, OR = 140 for Biomarker 1+2 < 41

ADCONTROLS

Fenech & Thomas 2007 Mutagenesis Fenech & Thomas 2007 Mutagenesis

Buccal Micronucleus Cytome Assay &Alzheimer Disease Risk

a) Polychromatic erythrocyte with micronuclei

b) Polychromatic erythrocyte with micronuclei

c) Mature erythrocytes with micronuclei

d) Polychromatic erythrocyte (bright orange) and mature erythrocyte.

PCE

Micronuclei in Mature and immature erythrocytes

WT CON

AD CON

AD CUR

AD GSE

AD MGSE

0

1

2

3

4

5

Micronucleated PCE at 9 monthsOne way ANOVA p=0.004

a

a

b

ab ab

Freq

uenc

y of

mic

ronu

clea

te P

CE'

spe

r 10

00 P

CE'

sDNA damage inbone marrow cells

Micronucleated cells at 9 months

WT CON

ADCONAD C

UR

AD GSE

AD MGSE

0.000.050.100.150.200.250.300.350.400.450.500.550.600.65

One way ANOVA p<0.0001

a

a

b b

a

Freq

uenc

y of

buc

cal c

ells

with

MN

(% o

f tot

al c

ells

sam

pled

) 3a) Basal cell 3b) Normal differentiated Cell

3c) Basal with MN 3d) Differentiated with MN

3e) Early karyolytic 3f) Mid karyolytic

3g) late karyolytic

DNA damage in Buccal cells

Effects of polyphenol consumption on Ab levels in the brain and serum of APPSwe/PS1dE9 transgenic mice

Effects of polyphenol consumption on Ab plaque burden in brain section spanning hippocampus of APPSwe/PS1dE9 transgenic mice

Protective effects of grape seed polyphenol and curcumin consumption on Aβ plaque burden in brain section spanning hippocampus and DNA damage in blood and buccal cells of APPSwe/PS1dE9 transgenic mice

Thomas P et al 2008; Wang J et al 2009Thomas P et al 2008; Wang J et al 2009

A B C

D E

F G H

CYTOME CYTOME

Fenech M 2007 Nature ProtocolsFenech M 2007 Nature Protocols

CYTOME &DNA DAMAGE

NUTRIOMENUTRIOME

Fenech 2008Fenech 2008

Folate

and mtDNA deletions

Chou and Huang (2009) EJN 48:429-436

Accumulated lymphocytic mtDNA deletions depend upon (a) dietary folate deprivation,(b) depletion of cellular folate storage

Lymphocyte mtDNA

deletions are +vely

correlatedwith brain mtDNA

deletions (r = 0.917, P < 0.0001).Department of Nutritional Science, Fu-Jen University, Hsinchuang

242, Taipei County, Taiwan.

Folate/B12choline deficiency Uracil

CytosineHypome-thylation

OxidisedDNAbases

DNA breakmisrepair

UnrepairedDNA breaks

UnrepairedDNA adducts

Dicentricchromosomes

Acentric Chromosomefragments

Telomeredysfunction

Centromeredysfunction

Telomere shortening

Telomereend fusion

BaseSequencemutationor deletion

ChromosomeLoss orMalsegregation, Micronucleusformation

mtDNAdeletion

Micronucleus formation

Anaphase bridge formation

BFBCYCLES

CHROMOSOMEINSTABILITYPHENOTPYE&ABERRANTKARYOTYPE

ACCELERATEDSENESCENCE

Deficiencyof antioxidantVitamins andCofactors ofAntioxidantenzymes

Deficiency ofCofactorsof DNARepair enzymes

POSSIBLE MECHANISMS BY WHICH MICRONUTRIENT DEFICIENCY POSSIBLE MECHANISMS BY WHICH MICRONUTRIENT DEFICIENCY COULD CAUSE DAMAGE TO THE GENOMECOULD CAUSE DAMAGE TO THE GENOME

Ageing causes Ageing causes hypomethylationhypomethylation

of satellite DNA which of satellite DNA which leads to loss of leads to loss of chch

1, 9, 16 and micronucleus formation1, 9, 16 and micronucleus formation

05

101520253035404550

YOUNG32PDLs

OLD71PDLs

AZAC 33PDLs

Mned cells(%)Bright Mni(%)

Suzuki et al. 2002 Exp. GerontologySuzuki et al. 2002 Exp. Gerontology

THE HORMESIS HYPOTHESIS OF THE HORMESIS HYPOTHESIS OF CALORIC/NUTRIENT RESTRICTIONCALORIC/NUTRIENT RESTRICTION

VISFATINVISFATIN

SIRT1SIRT1

Caloric Caloric RestrictionRestrictionMimeticsMimetics

Protein/Protein/MethionineMethioninerestrictionrestriction

KNOWN KNOWN CALORICCALORICRESTRICTION RESTRICTION MIMETICS:MIMETICS:

XENOHORMETIC AGENTS??XENOHORMETIC AGENTS??

DISCOVERY OF CALORIC RESTRICTION MIMETICSDISCOVERY OF CALORIC RESTRICTION MIMETICSDECELERATED AGEING

ACCELERATED AGEING

DAMAGEDGENOME

HEALTHYGENOME

EXCESSFAT

OPTIMALFAT LEVEL

SIRTUIN DEACETYLASEACTIVATION

NAD

RESVERATROL

PPAR-γ

+

-

NICOTINIC ACIDTRYPTOPHAN

•DNA repair•Oncogenes silenced•Compact chromatin

•Oxidants•Excess Calories•DNA misrepair

Howitz

et al Nature 2004

ENVIRONMENTAL STRESSORS(work, home, neighbourhood)

MAJOR LIFE EVENTS TRAUMA, ABUSE

INDIVIDUAL DIFFERENCES(genes,development, experiences)

PERCEIVED STRESSBEHAVIOURAL RESPONSES(fight or flight, smoking, drink,diet, exercise)

PHYSIOLOGICAL RESPONSE

ALLOSTASIS ADAPTATION

ALLOSTATIC LOAD

McEwen BS McEwen BS PhysiolPhysiol

Rev (2006)Rev (2006)

2010 May 25;5(5):e10826

CorrelationsCorrelations

LIFELIFE--STYLE HPI INDEXSTYLE HPI INDEX

LIFELIFE--STYLE IS ALSO AN IMPORTANT DETERMINANT OF DNA DAMAGESTYLE IS ALSO AN IMPORTANT DETERMINANT OF DNA DAMAGE

Huang et al Preventive Medicine 48 (2009) 383-388

••DNA DAMAGE IS THE MOST FUNDAMENTAL DISEASEDNA DAMAGE IS THE MOST FUNDAMENTAL DISEASE

••DNA DAMAGE CAN BE EFFICIENTLY DIAGNOSEDDNA DAMAGE CAN BE EFFICIENTLY DIAGNOSED

••DNA DAMAGE CAN BE PREVENTEDDNA DAMAGE CAN BE PREVENTED

••DNA DAMAGE DIAGNOSTICS SHOULD BECOME ROUTINE DNA DAMAGE DIAGNOSTICS SHOULD BECOME ROUTINE IN INTEGRATIVE & PREVENTIVE MEDICINE PRACTICESIN INTEGRATIVE & PREVENTIVE MEDICINE PRACTICES

AUTOMATED DIAGNOSTICSAUTOMATED DIAGNOSTICSGENOME HEALTH -

NUTRIENT STATUS -

GENOTYPE

NUTRIGENOMICSNUTRIGENOMICSEXPERT ADVICE SYSTEM

DATA BASE

INDIVIDUALISED NUTRITIONINDIVIDUALISED NUTRITIONDIETARY PATTERNS -

FUNCTIONAL FOODS -

SUPPLEMENTS

RA

DIC

AL IN

NO

VATIO

N IN

NU

TRITIO

N

VE

RIFIC

ATIO

N O

F EFFIC

AC

Y

GENOME HEALTH OPTIMISEDGENOME HEALTH OPTIMISEDDEVELOPMENTAL & DEGENERATIVE DISEASE PREVENTED

FEE

DB

ACK

TO D

ATAB

ASE

Funding: Reach 100, NCEFFFunding: Reach 100, NCEFF ABC Catalyst, DNA Doctor StoryABC Catalyst, DNA Doctor StoryFenech M (2005) Mutagenesis 20: 225Fenech M (2005) Mutagenesis 20: 225--269269

NETWORK OF GENES ASSOCIATED WITH MN NETWORK OF GENES ASSOCIATED WITH MN FORMATION BASED ON DATA IN LITERATURE FORMATION BASED ON DATA IN LITERATURE Dedicated network showing Dedicated network showing

customised MNcustomised MN--related generelated gene––genegene interactions with p53 as central hub interactions with p53 as central hub

and validated against actual and validated against actual occurrence of MN in exposed occurrence of MN in exposed

individuals has been constructedindividuals has been constructed

Van Van LeeuwenLeeuwen

DM et al Mutagenesis (in press)DM et al Mutagenesis (in press)

WHAT IS THE GENE EXPRESSION PATTERN ASSOCIATED WHAT IS THE GENE EXPRESSION PATTERN ASSOCIATED WITH MICRONUCLEUS FORMATION?WITH MICRONUCLEUS FORMATION?