AICR Conference Fenech MF, Am J Clin Nutr.

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

Washington 21-22 October 2010

Dietary reference values of

individual micronutrients and
nutriomes for DNA damage prevention
Current status and a road map to the future

Michael Fenech
CSIRO Food and Nutritional Sciences
GENOME HEALTH NUTRIGENOMICS LABORATORY

michael.fenech@csiro.au
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 AGEING
FROM WOMB TO TOMB
 BIOMARKERS OF DNA DAMAGE

NPB

dicentric

MN

Gene expression arrays γH2AX

 The principal
structural
chromosomal
aberrations which
contribute
acentric fragments
(AF) to form
micronuclei (MN)
and nucleoplasmic
bridges (NPB)

Acknowledgement
John RK Savage
Atlas Genet Cytogenet
Oncol Haematol. July 2000
 Genome damage
Micronuclei

CYTOKINESIS-BLOCK MICRONUCLEUS (CBMN) ASSAY

Human cells
• Oxidative stress • Strand breaks in DNA with damaged
• Nutrient deficiency •Chromosome malsegregation & unstable
• Excess calories •DNA hypomethylation genomes
•Telomere shortening
Increased lymphocyte micronucleus frequency in early pregnancy is
associated prospectively with pre-eclampsia and/or intrauterine
growth restriction (PEIUGR)
RISK GROUPS PREGNANCY OUTCOME
GROUPS
D.L.F FurnesS, G.A Dekker, W.M Hague T .
LRN
Y Khong and M.F Fenech
N=38 Mutagenesis (2010)

LOW RISK ADVERSE PEIUGR

N=45 N=7 N=36

PROSPECTIVE ODDS RATIO

OF PEIUGR FOR ALL COHORT
HIGH RISK ADVERSE OAO
N=91 N=67 N=38 P=0.001
16
14

Odds Ratio
HRN
N=24 12
10
8
6 P=0.006
LYMPHOCYTE 4
P=0.003

MICRONUCLEI 2
MEASURED AT 0
>19.3 >28.0 >36.7
18 WKS
MN-BN ‰
GESTATION
 Risk of cancer increases with
higher MN frequency

6,983 subjects
275 cancer cases
PROBABILITY OF SURVIVING
WITHOUT CANCER

FOLLOW UP TIME IN YEARS

HUMN project (www.humn.org) Bonassi et al (Carcinogenesis 2007)

 Lymphocyte Micronucleus frequency is associated
prospectively with cardiovascular disease mortality in
both the general population and those with known
coronary artery disease

MN<
MN<1‰
1650 subjects
111 deaths
39 due to CVD
MN>1‰
MN>1‰ Mean age 65 y

Federici C et al

MN frequency

178 CAD patients

Mean age 62 y
 GENOME DAMAGE INCREASED IN LYMPHOCYTES OF
ALZHEIMER AND PARKINSON’S DISEASE PATIENTS

DNA DAMAGE IN LYMPHOCYTES

* P<0.001
20 * * DISTRIBUTION OF TYPE OF MICRONUCLEI
MN (CHROM. LOSS) MN (CHROM. BREAK)
MN PER 1000 BNCs

15
p < 0.0001
100
10

75
5
%
50
0
AD PD CONTROL
25

0
AD PD CONTROL

Petrozzi et al. Neurol. Sci. 2002

 DNA damage increases with
age ....or…. poor choices of nutrition, life-style,
physical and socio-psychological environments?

50 What is the
MN per 1000 BN cells

threshold of
40 DNA damage
we should
allow?
30

Can we design
20
“exposomes”
that enable us
10 to stay below
this threshold?
0
46-55

56-65

66-75

76-90
26-35

36-45
18-25

AGE (years)
Fenech et al. 2000
 CAUSES AND EFFECTS OF GENOME DAMAGE
GENOME
DAMAGE IN: HIGHER
RISK
GERMLINE
E MALNUTRITION
X EMBRYO

DEVELOPMENTAL
GENETIC

DEGENERATIVE
P LIFE-STYLE FOETUS

DISEASES
AND
O GENOTOXINS
ACQUIRED
BABY
S PSYCHOLOGICAL SUSCEPT-
O STRESS
IBILITY CHILD
M ENVIRONMENTAL TEENAGER
E GENOTOXINS

YOUNG ADULT
LOWER
OLDER ADULT RISK
Italy Germany USA

Poland Mexico Ecquador

Bhutan Chad Egypt Dietary patterns &

1 week’s food
 The new frontier in nutrition

H
E
A
NUTRIOME L GENOME
T
H

Nutrition designed for diverse genetic backgrounds

to optimise genome maintenance and prevent diseases
caused by DNA damage
The following genomic stability processes are
modulated by vitamins or micronutrients:
micronutrients

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

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

DNA repair niacin, Zn, folate

Gene expression folate, Vit D, Vit A

Chromosome segregation folate, Vit A, Mg

Telomere length niacin? via PARP, folate

Necrosis/Apoptosis niacin, Zn, Vit E, Vit D, Vit C

Vit A, Vit K2.
Folate, B12, B6 and B2 and genome maintenance
FOLIC ACID

THF DHF
METHIONINE dUMP

SHMT1
MTR VIT B6
VIT B12
TS
SAM (CoIII)
MTRR 5,10-MeTHF
DNA MTR dTMP
VIT B12
(CoI)
MTHFR
VIT B2

HOMOCYSTEINE
5-MeTHF

DNA METHYLATION DNA REPAIR & SYNTHESIS

 Minimally invasive High-Throughput
Nutrient Array screening for Genome-Protective agents.

20ul blood

Nutrients
Seed
Lymphocytes Mitogen Cytochalasin B
(cytokinesis
blocking agent)

Image
acquisition
1h 20 h 44 h and analysis

+/- +/- 72h

Genotoxin Genotoxin Nuclear Micronucleus
stain/Fix

Development of automated CBMN Cytome assay:

MN, NPB, NBUDs, Necrosis, Apoptosis, NDI
Selected
+ FISH + Protein expression Targeted Nuclei
Cell
 Fenech M (2007) Nature Protocols

A ,B, C
A B C

NUCLEAR
DIVISION
INDEX

D E D,E
CBMN
CYTOME
ASSAY CELL DEATH

F G H F, G, H

DNA DAMAGE
 [A] [B] NUCLEAR BUDS
MICRONUCLEATED CELLS
40 15
ANOVA P < 0.0001
ANOVA P<0.0001
MNed BNs/1000 BNs *

Buds/1000 BNs
30 *
** 10

20
**

5
10

0 0
12 24 60 120 12 24 60 120
folic acid in medium (nM) folic acid in medium (nM)

[D]
URACIL
[C]
NUCLEOPLASMIC BRIDGES
10.0 50
ANOVA P<0.0001 * *
* ANOVA P < 0.0001

pg uracil / g DNA
40
NPB/1000 BNs

7.5
**
30
5.0
20

2.5
10

0.0 0
12 24 60 120 12 24 60 120
[folic acid] in medium (nM) [folic acid] in medium (nM)
Crott et al. Carcinogenesis 2001
Genome damage induction by Folic acid deficiency is of a similar
magnitude as that induced by unsafe doses of ionising radiation
IAEA annual safe exposure limit ie equivalent to 0.1-0.5 rad X-rays

35
Crott & Fenech 2002
30
MNed BNs/1000 BNs

25

20 Fenech & Morley, 1986

15

10

0
0 5 10 15 20 25
0 5 10 20 rad X-rays
120 60 24 12 nM folic acid

“normal”
serum folate
concentration
Randomised, placebo-controlled dietary intervention
PLACEBO FOLB12

ROUND 1 ROUND 1 BASE-LINE*

LINE
CEREAL CEREAL +
ONLY 7ug B12 +
700ug FOL

ROUND 2 ROUND 2 12 WEEKS*

WEEKS
TABLET TABLET
PLACEBO 20ug B12 +
2000ug FOL

ROUND 3 ROUND 3 24 WEEKS*

WEEKS

* blood sample
Fenech et al. 1998 Carcinogenesis
 Supplementation with 3.5 times RDI folic acid & vit B12
reduces micronucleus index by 25 % in subjects with
above average chromosome instability
Low MNed cell freq. High MNed cell freq.
at R1. [N = 17] at R1. [N = 16]
ANOVA P = 0.65 ANOVA P < 0.0005
MNed cells per 1000 BN cells
12

9
* *

0
R1 R1 R2 R2 R3 R3
7ug B12 20ug B12
Fenech et al. 1998 Carcinogenesis Base-line
700ug FA 2mg FA
 serum B12 vs MN
MICRONUCLEUS FREQUENCY IS MINIMISED frequency in
WHEN
men aged 50-70 years who
HOMOCYSTEINE < 7.5umol/l and were
B12not
> 300pmol/l
vitamin B12 deficient

YOUNG ADULTS 18-

18-32Y MEN AGED 50-
50-70Y
R = -0.3418 P = 0.0127 N = 62
750
600 N=62
serum B12 (pmol/l)

N = 48 R=-
R=-0.31

serum B12 [pmol/l]

600 500
R = -0.489 P==0.013
P = 0.0004 400
450
300
300
200

150 100

0 M N frequency vs plasma HC in
0 0 10 20 30
men (50-70years) who were not40
0 5 10 15 20 MN frequency
deficient in per
B121000 BN cells
or folate
MNed cells per 1000 BN cells
R = 0.4148 P = 0.0086 N = 39
20
N = 48
plasma HC (mol/l)

R = 0.392 15
15
P = 0.006

plasma HC [mol/l]
10 10

5
5

0
0 5 10 15 20 0
0 10 20 30 40
MNed cells per 1000 BN cells
MN frequency per 1000 BN cells
Fenech et al. 1998 Carcinogenesis Fenech et al. (1997) Carcinogenesis
 Eussen SJ et al 2005

300 times recommended intake of B12 is required to

normalise holoTC, MMA and homocysteine in >70 year olds
Future Challenges: We have started to determine the impact of common
polymorphisms in folate metabolism genes on chromosome damage in
Australians

*age & gender adjusted

Dhillon et al Mutation Res Fund Mech 2009

MICRONUTRIENTS AND GENOME DAMAGE
RESULTS OF ANALYSIS OF FOOD FREQUENCY QUESTIONNAIRE AND GENOME DAMAGE DATABASE

% variation in genome damage with

increased intake relative to lowest tertile of intake

* P < 0.006
70 * * mid-tertile
% variation in MN freq

60
50 * highest tertile
40 *
30
20
10
0
-10
-20
-30 *
-40 * * *
-50 *
* *
Folate

 -Carotene
Nicotinic acid

Biotin
Riboflavin

Pantothenic acid
Retinol
Vitamin E

Calcium

Fenech et al.
Carcinogenesis
(2005)
 Fenech et al 2005 Carcinogenesis; Fenech 2010 AJCN

NUTRIENT-NUTRIENT INTERACTION ON CHROMOSOME DAMAGE

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

0 50
* *

% VARIATION
40
% VARIATION

-10
FOLATE 30 RIBOFLAVIN
-20 INTAKE
INTAKE 20
-30 * low tertile 10 low tertile
-40
* * mid tertile 0 mid tertile
high tertile -10 high tertile
-50
* -20
-60
* * -30 *
-70 -40
*
low tertile mid tertile high tertile low tertile mid tertile high tertile

CALCIUM INTAKE FOLATE INTAKE

MORE RIBOFLAVIN
The combined effect of (a) calcium and folate intake and (b) riboflavin
IN A LOW FOLATE
and folate intake on MN frequency. Results shown are the % variation
BACKGROUND
relative to the combined lowest tertiles of intake in the pair of nutrients
MAY BE
examined. * P < 0.05 for comparison with the referent value for the
GENOTOXIC
combined lowest tertile of intake for the pair of nutrients examined.
 Q. Which dietary
AMOUNTS pattern
IN 100g will work for
EXPRESSED AS %your
OF genotype
MIMIMUM ?
REQUIREMENT FOR OPTIMUM GENOME HEALTH
A. It depends on the “nutriome” of the foods you prefer to eat
300
CALCIUM
FOLATE
200 NIACIN
VITAMIN E
BETA-CAROTENE
100 RETINOL

0
)

)
EA DS

SE
N

A
ED
ED

ED
A

N
EE
N

A
K
N
IL
O

TB

N
O
EE AN
O
H

A
LM

O
C

B
(C

(C
(
A

LI
H

F
W

O
D

N
C
ED

TU
C

B
O
H

R
C

Fenech, Food Chem Tox 2008

 Folate content of vegetables (DFE µg per 100g)*
*Data from USDA National Nutrient data base. DFE = dietary folate equivalent. DFE values are shown in brackets.

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

Eating the
Pulses Leafy or Roots or “Fruit”
cruciferous Tubers Vegetables “wrong”
vegetables
vegetables
Red Kidney Broccoli (93) Onions (16) Tomato (15)
beans (130) could lead
Mung beans Brussel sprouts Potato (22) Pumpkin (9) to folate
(60) (60)
deficiency
Chickpeas Cabbage (43) Turnip (9) Cucumber (6)
(171)
Lentils (180) Endive (142) Parsnip (57) Capsicum (11) Folate RDA 400μg
Peas (59) Spinach (146) Swede (21) Eggplant (14) requires eating:
Lima beans Lettuce (73) Carrot (14) Olives (0)
(50) 2.5Kg LF veg/d
Mean (108) Mean (93) Mean (23) Mean (10) or
0.4Kg HF veg/d
Mean (100) Mean (16)
TELOMERES
TELOMERES (TTAGGG
repeats) ARE ESSENTIAL
FOR CHROMOSOME
STABILITY

TELOMERE SHORTENING OR
DYSFUNCTION INCREASES
RISK FOR CANCER AND
ACCELERATED SENESCENCE
 SHORTER TELOMERES ARE ASSOCIATED
PROSPECTIVELY WITH INCREASED RISK FOR CANCER

MULTIVARIATE HAZARD
RATIO FOR CANCER INCIDENCE

3.5
3
2.5
2 LONG
1.5 MEDIUM
SHORT
1
0.5
0
1st Qtr
LEUKOCYTE
TELOMERE LENGTH

Wong & Collins Lancet 2003 Willeit P et al, JAMA. 2010; 304(1):69-75.
 Thomas et al, Mech Aging Dev, 2008

Shorter telomeres in WBCs and Buccal cells of

Alzheimer’s disease (AD) cases compared to controls

175 a
a
Absolute telomere length

50 a
(Kb per diploid genome)

Absolute telomere length

(Kb per diploid genome)
150 ab
40
125 b
100
c bc 30
b
75
20
50
10
25
0 0

ls

ls

D
ls

ls

rA

A
ro

ro
rA

A
ro

ro

er
nt

nt
er
nt

nt

ge
ge

ld
co

co
ld
co

co

un
un

O
O

ld

Yo
g

ld

Yo

un

O
un

Yo
Yo

WBC TL <115 Kb per diploid genome Buccal TL <40 kb per diploid genome
OR of being diagnosed with AD is 10.8 OR of being identified with AD is 4.6
specificity 46% sensitivity 92.9%. specificity 63% sensitivity 72.7%.

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

FOLATE PUFA

VITAMIN E OXIDATIVE STRESS

VITAMIN D OBESITY

Ω3-FATTY ACIDS PSYCHOLOGICAL

CURRENT STRESS
CEREAL FIBRE KNOWLEDGE
PROCESSED MEAT
MULTIVITAMIN USE
HOMOCYSTEINE

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.
 Bull & Fenech Proc. Br Nutr Soc 2008
ANTIOXIDANT
DEFICIENCY
8-OHdG
OR OXIDATIVE
STRESS
BREAKS IN
TELOMERE

URACIL
?
DYSFUNCTIONAL
REDUCED AND/OR SHORT
TRF1, TRF2 TELOMERES;
FOLATE TELOMERE END
DEFICIENCY BINDING
FUSIONS;
AND CIN

? HYPO-
METHYLATED
SUBTELOMERE LOSS OF
TELOMERE
LENGTH
CONTROL
NIACIN OR
? REDUCED
NICOTINIC
TANK1
ACID
ACTIVITY
DEFICIENCY
TELOMERE DAMAGE CASCADE
 High protein (TWD) or high carbohydrate
(HC) weight-loss diets reverse telomere
shortening in rectum in over-weight men

18.2
TWD HC
15.6
Telomere length (kb)

13.0 2 way ANOVA

P<0.0001
10.4

7.8

5.2
2.6

0.0
0 12 52 0 12 52
O’Callaghan, Clifton, Noakes,
Noakes, Fenech.
Rejuvenation Res. 2009 weeks on diet
 IS A COMPREHENSIVE SET OF DNA
DAMAGE BIOMARKERS NEEDED
FOR PERSONALISED NUTRITION AND
DRV DETERMINATION FOR GENOME
DAMAGE PREVENTION?
Fenech M (2010) Am. J. Clin.
Clin. Nutr.
Nutr.

LYMPHOCYTE CBMN ASSAY BUCCAL MN ASSAY

RBC MN ASSAY
COMET ASSAY

TELOMERE LENGTH MITOCHONDRIAL DNA DELETION DNA OXIDATION DNA METHYLATION

Validation status of DNA damage biomarkers
Fenech M (2010) Am. J. Clin.
Clin. Nutr.
Nutr.
ASSOCIATION WITH NUTRITIONAL ASSOCIATION WITH DEVELOPMENTAL
STATUS OR DEGENERATIVE DISEASE OR
DNA MORTALITY
DAMAGE CROSS-SECTIONAL PLACEBO- CASE-CONTROL PROSPECTIVE
BIOMARKERS STUDIES CONTROLLED STUDIES COHORT STUDIES
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

  
MITOCHONDRIAL
DNA DELETION ?
PROPOSED ROAD-MAP TO DETERMINE
DRVs FOR GENOME STABILITY

NUTRITION STUDY OUTCOME DRVs FOR

VARIABLES DESIGN MEASURES GENOME
STABILITY
PRIMARY
IN VITRO
SINGLE
MODELS DNA DAMAGE
MICRONUTRIENT DATABASES
BIOMARKERS:
ON VITAMIN
MICRONUTRIENT MICRONUCLEUS & MINERAL
IN VIVO
COMBINATION CYTOME ASSAYS REQUIREMENTS
CROSS- COMET ASSAY
FOR GENOME
SECTIONAL DNA OXIDATION
FUNCTIONAL DNA METHYLATION STABILITY
STUDIES
FOOD TELOMERE LENGTH
IN
mtDNA DELETION
DIVERSE
FOOD GROUP SECONDARY GENETIC
PLACEB0-
CONTROLLED BACKGROUNDS
DIETARY TISSUE AT THE
TRIALS
PATTERN MICRONUTRIENT VARIOUS
CONCENTRATION LIFE-STAGES

Fenech M (2010) Am. J. Clin.

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

LIFE-STYLE IS ALSO AN IMPORTANT DETERMINANT OF DNA DAMAGE

LIFE-STYLE HPI INDEX

 NUTRI- PSYCHO-
GENOMICS GENOMICS
GENOME
HEALTH
STATUS
OF THE
POPULATION LIFESTYLE
TOXICO-
GENOMICS GENOMICS
 AUTOMATED DIAGNOSTICS
RADICAL INNOVATION IN NUTRITION

VERIFICATION OF EFFICACY
GENOME HEALTH - NUTRIENT STATUS - GENOTYPE

NUTRIGENOMICS
EXPERT ADVICE SYSTEM

FEEDBACK TO DATABASE
DATA BASE

INDIVIDUALISED NUTRITION
DIETARY PATTERNS - FUNCTIONAL FOODS - SUPPLEMENTS

GENOME HEALTH OPTIMISED

DEVELOPMENTAL & DEGENERATIVE DISEASE PREVENTED

Funding: Reach 100, NCEFF Fenech M (2005) Mutagenesis 20: 225-

225-269 ABC Catalyst, DNA Doctor Story
 “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 2010

Acknowledgements THE GENOME HEALTH
NUTRIGENOMICS
CRC Diagnostics TEAM

CHORI (USA)
Bruce Ames,
Susan Mashiyama

Natl. Ins. Hlth & Nutr Japan Felicia Bulman

Keizo Umegaki, Michiyo Kimura Julie Turner
Carolyn Salisbury
WCH Adelaide Philip Thomas
Graeme Suthers Jimmy Crott
Peter Baghurst Will Greenrod
RAH Adelaide Josy Rinaldi
Ian Olver, Eric Yeoh Clare Aitken
Sally Record
FUNDING Maryam Hor
•Kellogs Pty Ltd, Theodora Hua Haen
•MLA
•Blackmore’s,
HUMN
International Collaborative Project on
Jing Wu
Micronucleus frequency in human populations
Caroline Bull
•Nutrilite/Amway Coordinating Group: Nathan O’Callaghan
•NHMRC, Michael Fenech (Australia) Chairman
Stefano Bonassi (Italy)
Wayne Leifert
Wushou Chang (Taiwan)
•Cancer Council SA, Nina Holland (USA)
Errol Zeiger (USA) Glen Patten
•NIH/NIAID Micheline Kirsch-Volders (Belgium)
Erin Symonds
•EUCancerRiskBio- Founded Toulouse 1997
Bianca Benassi
markers Program Sasja Beetstra
•DPI-Victoria •40 labs
•16 countries
•>12,000 subjects
NO CONFLICT OF •>70,000 person years

INTEREST
 O’Callaghan, Clifton, Noakes,
Noakes, Fenech. Rejuvenation Res. 2009

REDUCTION IN FAT CORRELATES WITH INCREASE IN TELOMERE LENGTH

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

Telomere length is negatively Telomere length is positively

correlated with plasma correlated with plasma folate
homocysteine in older men. in males but not in females.

Males; r = -0.57, p = 0.004 (n = 24). Males; r = 0.42, p = 0.04 (n = 24).

Females; r = 0.092, p = 0.68 (n = 23) Females; r = -0.11, p = 0.61 (n = 23)

Bull, O’Callaghan, Mayrhofer & Fenech Rejuvenation Res (2009)

Prof. Michael Fenech
Minimally invasive High-
High-Throughput
michael.fenech@csiro.au Nutrient Array screening for Genome- agents.
Genome-Protective agents.

Nutritional Genomics and

DNA Damage Diagnostics Laboratory Nutrients
Seed
CSIRO Food & Nutritional Sciences cells Mitogen Cytochalasin B
(cytokinesis
blocking agent)

Image
acquisition
and analysis
Nuclear
9 day culture stain/Fix
Micronucleus

Development of automated CBMN Cytome assay:

MN, NPB, NBUDs, Necrosis, Apoptosis, NDI
+ FISH + Protein expression
Selected
Targeted Nuclei
Cell
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
 FOLATE: A DOUBLE-EDGED SWORD IN CANCER
DEPENDING ON DOSE AND TIMING

FOLATE DEFICIENCY FOLATE DEFICIENCY

<300 mcg/day* <300 mcg/day*

promotes inhibits??

NORMAL ADENOMA CANCER

inhibits promotes

ADEQUATE FOLATE EXCESSIVE FOLATE

300-500mcg /day > 1000mcg/day

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

 Metabolism of Folate and Methionine
Cell Proliferation Apoptosis
+
+ + + Methylthioribose-1-P Methional

Spermidine MTA DNA Synthesis

& Repair
MTOB
Salvage Pathway

DHF
Spermine
dTMP
Protein
Putrescine dcSAM Synthesis

Vit. B12 THF

SAM Methionine Cob(III)
Ornithine TS

Vit. B12 Catalytic

Pathway
De Novo Pathway MTR 5,10-Methylene
CH3 SAH MTRR dUMP
THF

MTHFR
Vit. B2
Vit. B12
DNA & Protein Homocysteine Cob(I)
5-Methyl THF
Methylation CBS
RFC-1 Cytoplasm
Extra Cellular
Trans-Sulphuration
Pathway Folic Acid
monoglutamate
Diet

monoglutamate FGCP Folate

polyglutamate
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
 REGENERATIVE POTENTIAL REGENERATIVE POTENTIAL
OF THE HIPPOCAMPUS OF 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 days
for cells
to migrate
from basal
layer to
keratinised
layer

Acknowledgement S Thuret (University College London)

Buccal Micronucleus Cytome is a powerful
diagnostic of accelerated ageing Fenech & Thomas 2007 Mutagenesis

MICRONUCLEI KARYORRHEXIS
3 60
p = 0.0003
p<0.0001
50

2 40

30

1 20

10

0 0

ER
LS

LS

LS

ER
S

LS
N

N
O

M
W

M
W
TR

TR

TR
EI

TR
O

EI
O
D

H
N

N
LZ

LZ
O

O
C

C
A

A
ER

ER

ER

ER
G

LD

LD
N

N
O

O
U

U
YO

YO
CONDENSED CHROMATIN BASAL CELLS
75 100
p<0.0001
p<0.0001
75
50

50
25
25

0 0

ER
LS

LS
LS

ER
S

LS

N
N

O
W

M
O

M
W

TR

TR
TR

EI
TR

O
EI
O

H
D

N
N

LZ
LZ

O
O

C
C

A
A

ER

ER
ER

ER

LD
G

LD

N
N

O
O

U
U

YO
YO
 Buccal Micronucleus Cytome Assay &
Alzheimer Disease Risk
PPV = 98%; NPV = 77%;
Sensitivity = 82%, Specificity = 97%
LR = 25, OR = 140 for Biomarker 1+2 < 41
150
AD
AD & MCI
CONTROLS
CONTROLS
BIOMARKER 2
karyorrhexis

100

50

0
0 50 100 150 200
BIOMARKER 1

Basal cells
Fenech & Thomas 2007 Mutagenesis
 Thomas P et al 2008; Wang J et al 2009

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

One way ANOVA p<0.0001

Micronucleated cells at 9 months

Frequency of buccal cells with MN

0.65 a

(% of total cells sampled)

3a) Basal cell 3b) Normal differentiated Cell
0.60
0.55
0.50
0.45
0.40 a 3c) Basal with MN 3d) Differentiated with MN
0.35
0.30
a
0.25
0.20 3e) Early karyolytic 3f) Mid karyolytic
0.15
0.10
b b
0.05
0.00
3g) late karyolytic

R
N

SE

SE
O

G
C
C

M
D

D
D
T
DNA damage in

A
W

D
A
Buccal cells

Effects of polyphenol consumption on Ab levels in the brain

and serum of APPSwe/PS1dE9 transgenic mice

One way ANOVA p=0.004

Micronucleated PCE at 9 months
Micronuclei in Mature and immature erythrocytes

Frequency of micronucleate PCE's

a
5
a
a) Polychromatic erythrocyte b) Polychromatic erythrocyte with
with micronuclei micronuclei

4
ab

per 1000 PCE's

ab
3 PCE

b c) Mature erythrocytes with

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

2
DNA damage in
1 bone marrow cells

R
N

SE

SE
U
CO

G
C
C

M
D
AD
T

AD

A
W

D
A
 Fenech M 2007 Nature Protocols

A B C

D E

CYTOME

F G H

NUTRIOME CYTOME &

DNA DAMAGE
Fenech 2008
 Folate and mtDNA deletions

Accumulated lymphocytic mtDNA deletions depend upon

(a) dietary folate deprivation,
(b) depletion of cellular folate storage

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

Lymphocyte mtDNA deletions are +vely correlated Department of Nutritional Science, Fu-Jen University,
Hsinchuang 242, Taipei County, Taiwan.
with brain mtDNA deletions (r = 0.917, P < 0.0001).
POSSIBLE MECHANISMS BY WHICH MICRONUTRIENT DEFICIENCY
COULD CAUSE DAMAGE TO THE GENOME

Centromere
dysfunction Chromosome
Cytosine Loss or
Hypome-
Malsegregation,
thylation Telomere
Folate/B12 Micronucleus
dysfunction formation
choline
deficiency Uracil

Telomere Telomere
Oxidised shortening end fusion
Deficiency DNA
of antioxidant bases CHROMOSOME
INSTABILITY
Vitamins and mtDNA ACCELERATED BFB PHENOTPYE
Cofactors of deletion SENESCENCE CYCLES &
Antioxidant Unrepaired ABERRANT
enzymes DNA KARYOTYPE
adducts Base
Sequence Anaphase
mutation bridge
DNA break or deletion formation
Deficiency of misrepair
Cofactors Dicentric
of DNA chromosomes
Repair Micronucleus
enzymes Unrepaired formation
DNA breaks Acentric
Chromosome
fragments
 Ageing causes hypomethylation of satellite DNA which
leads to loss of ch 1, 9, 16 and micronucleus formation

50
45
40
35
30
Mned cells
25 (%)
20
Bright Mni
15
(%)
10
5
0
YOUNG OLD AZAC 33
32PDLs 71PDLs PDLs

Suzuki et al. 2002 Exp. Gerontology

 THE HORMESIS HYPOTHESIS OF
CALORIC/NUTRIENT RESTRICTION

Protein/ Caloric
Methionine Restriction
restriction Mimetics

VISFATIN

SIRT1

KNOWN
CALORIC
RESTRICTION
MIMETICS:
XENOHORMETIC AGENTS??
 DISCOVERY OF CALORIC RESTRICTION MIMETICS
DECELERATED AGEING

HEALTHY OPTIMAL
GENOME RESVERATROL FAT LEVEL
•DNA repair
•Oncogenes silenced
•Compact chromatin
+

SIRTUIN DEACETYLASE
ACTIVATION

•Oxidants
•Excess Calories - PPAR-γ
•DNA misrepair NAD
EXCESS
DAMAGED
NICOTINIC ACID FAT
GENOME
TRYPTOPHAN

ACCELERATED AGEING
Howitz et al Nature 2004
ENVIRONMENTAL STRESSORS MAJOR LIFE EVENTS TRAUMA, ABUSE
(work, home, neighbourhood)

PERCEIVED STRESS
INDIVIDUAL DIFFERENCES BEHAVIOURAL RESPONSES
(genes,development, experiences) (fight or flight, smoking, drink,
diet, exercise)

PHYSIOLOGICAL RESPONSE

ALLOSTASIS ADAPTATION

ALLOSTATIC LOAD

McEwen BS Physiol Rev (2006)

 2010 May 25;5(5):e10826

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

LIFE-STYLE IS ALSO AN IMPORTANT DETERMINANT OF DNA DAMAGE

LIFE-STYLE HPI INDEX

•DNA DAMAGE IS THE MOST FUNDAMENTAL DISEASE

•DNA DAMAGE CAN BE EFFICIENTLY DIAGNOSED

•DNA DAMAGE CAN BE PREVENTED

•DNA DAMAGE DIAGNOSTICS SHOULD BECOME ROUTINE

IN INTEGRATIVE & PREVENTIVE MEDICINE PRACTICES
 AUTOMATED DIAGNOSTICS
RADICAL INNOVATION IN NUTRITION

VERIFICATION OF EFFICACY
GENOME HEALTH - NUTRIENT STATUS - GENOTYPE

NUTRIGENOMICS
EXPERT ADVICE SYSTEM

FEEDBACK TO DATABASE
DATA BASE

INDIVIDUALISED NUTRITION
DIETARY PATTERNS - FUNCTIONAL FOODS - SUPPLEMENTS

GENOME HEALTH OPTIMISED

DEVELOPMENTAL & DEGENERATIVE DISEASE PREVENTED

Funding: Reach 100, NCEFF Fenech M (2005) Mutagenesis 20: 225-

225-269 ABC Catalyst, DNA Doctor Story
WHAT IS THE GENE EXPRESSION PATTERN ASSOCIATED
WITH MICRONUCLEUS FORMATION?
NETWORK OF GENES ASSOCIATED WITH MN
FORMATION BASED ON DATA IN LITERATURE
Dedicated network showing
customised MN-related gene–gene
interactions with p53 as central hub
and validated against actual
occurrence of MN in exposed
individuals has been constructed

Van Leeuwen DM et al Mutagenesis (in press)