Phytochemicals and Genomic Stability Group

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Dietary salicylates nutri'omic approaches to
investigate impact on gut health
Janice E. Drew
Molecular Nutrition Group, Gut Health Division,
Rowett Research Institute, Greenburn Road,
Bucksburn, Aberdeen, AB21 9SB, Scotland.
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Plant-based diet reduces colon cancer risk
years
5 or more servings fruit/day - 40 decreased
risk of adenomas 4 or more servings legumes/day -
33 decreased risk of adenomas Michels et al,
Cancer Research 2006, 66, 3942
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RECOMMENDATION 4 PLANT FOODS Eat mostly foods of
plant origin PUBLIC HEALTH GOALS Population
average consumption of non-starchy vegetables and
of fruits to be at least 600 g (21 oz) daily
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Dietary Phenolics
Compounds in plants not recognised as nutrients
(ie. Not essential dietary factors such as
vitamins, minerals, amino acids and fatty
acids) No deficiency syndromes May affect
mammalian biological functions
/- Health
Many are basis for drugs
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Salicylic acid

• Salicylic acid, a plant secondary metabolite
  involved in normal defence mechanisms
• Present in wide range of plant based foods
• Metabolite of aspirin contributing factor in
  fruit and vegetable protection against
  development of colon cancer?

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Role in colon cancer prevention ?

• 6 epidemiological studies suggest aspirin
  (acetylsalicylic acid) gives 15-72 protection
  against colorectal cancer

• 3 recent randomised intervention studies indicate
  protective effect of aspirin (Plt0.004).
  Terminated early

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Aspirin is a pro-drug of salicylic acid

• Aspirin is unstable
• Quickly hydrolyses to salicylic acid

• Therefore likely that any bioactivity, including
  anti-cancer effect of aspirin potentially due to
  salicylic acid!

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In vitro evidence for colon cancer prevention

• In vitro studies indicate number of potential
  mechanisms
• Modulation of inflammatory pathways - COX 2 and
  NFkB
• Antioxidant enzyme up-regulation reduction of
  oxidative stress
• Moderation of apoptosis (par-4, Bcl-XL, caspase,
  p38)
• Upregulation of tumour suppressor genes (p53,
  p22, Bax)
• Enhances DNA mis-match repair

Reviewed in Aspirin, salicylates and cancer
evidence suggestive of benefit, possible
mechanisms and clinical implications. Elwood PC,
Gallagher A, Duthie GG et al Lancet, In Press
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Low dietary antioxidant/ plant-derived
anti-inflammatory dietary phenolic intake High
intake of pro-inflammatory dietary fats
(omega-6) High calorie intake Low physical
activity Increased age

• Western style diet
• hi fat, lo fibre, lo fruit and veg
• OBESITY

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MECHANISTIC FOCUS
Inflammation/Oxidative stress
Pro/Anti-inflammatory dietary components
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Rodent Models Strategies and Mechanisms
Chemical induced CRC
Dietary induced oxidative stress
Mutant mice/Diet-induced obese rodents
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Diet induced oxidative stress model
Vitamin E depletion
OXIDATIVE STRESS PROSTAGLANDINS cyGLUTATHIONE
PEROXIDASE
Drew et al., 2005 Biochem Pharm 70888-93
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cyGpx gene expression
Gpx1
Gpx2
Drew et al., 2005 FEBS 2596135-9
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Proteomic Analysis

• Proteomic Analysis
• soluble protein extract distal colon
• 1000 resolved spots
• statistical analysis to select altered
• expression patterns
• LC/MS/MS

Drew et al., 2006 Biochem Pharm 72204-216
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Mechanism of Salicylic Acid Modulation
Cytoskeleton
REDOX
Metabolism
Salicylic Acid
Protein Folding
Drew et al., 2006 Biochem Pharm 72204-216
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Thioredoxin peroxidase 1 MPEFs
Estimated 20.58 21.60 21.23 Mass
(kDa) Estimated pI 5.3 5.5 5.5
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Altered Mitochondrial Proteins in Colon
Drew et al., 2006 Biochem Pharm 72204-216
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Salicylic acid reduces oxidative stress in obese
rats
Diet-induced obese resistant Diet-induced obese
sensitive


Saline Dimethylhydrazine
Dimethylhydrazine Salicylic Acid
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Salicylic acid reduces plasma insulin rats
susceptible to diet induced obesity
Diet-induced obese sensitive
Diet-induced obese resistant

saline
DMH
DMHSA
DMHSA
saline
DMH
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What is the dietary intake?
Mean intake 9 mg/day Range 1-30 mg/day N 66
Cf Vit E 8 mg/day Flavonols 18 mg/day
Prof Garry Duthie, RRI
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How bioavailable is it from complex food matrices?
Dietary intervention - from strawberries,
salicylate rich breakfast, cranberry juice and
vegetable curry. Also effects of delivery vehicle
(cumin yoghurt, cumin soya yoghurt, cumin
moller rice). Bedevilled by large individual
variation!

• Food matrix?
• Genetic variation in enzymes required to
  metabolise salicylates?
• Gut microflora?

Prof Garry Duthie, RRI
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Is intake related to colon cancer risk?
Need to do more, if possible!
SA Intakes cases and controls
Collaboration Prof Emad El Omar, Dr. Geraldine
McNeill
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Is SA also present in colon?
Endogenous synthesis?
M
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P
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Total SA in 100g fresh faeces 1.66 /- 0.32mg
(n4) Total phenolic acids in 100g fresh faeces
28.08 /- 8.12 mg (n4)
Colonic Bugs
Breakdown of complex polyphenols?
Dr Wendy Russell, RRI
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Anti-inflammatory Dietary Phenolics
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Determination of anti/pro cancer properties
faecal water
Antioxidant activity
P lt 0.05
volunteer 1 volunteer 2
Inflammatory activity
Dr Wendy Russell, RRI Dr Charles Bestwick, RRI
volunteer 1 volunteer 2
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Human Colon Gene Expression Profiling
Pro/Anti-inflammatory targets
14 gene targets
Chemokines and cytokines Chemokine and Cytokine
Receptors Interleukins Receptors TNF Ligands
Receptors Other factors involved in
Inflammatory Response
Collaboration Prof. Emad El Omar, Dr. Mairi
McLean, IMS, University of Aberdeen.
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Research Strategy
Food bioavailability metabolism pro/anti-inflammat
ory
In vitro
Colon Cancer insulin/adipokine
signalling pro/anti-inflammatory
cytokines mitochondria function and health
Genomics Proteomics Biochemistry Metabolomics
Rodent models
In vivo
Human
Mechanisms
colon cancer risk Interactions with dietary
phytochemicals
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Acknowledgements
Molecular Nutrition Group, Gut Health, RRI Prof
Garry Duthie, Dr Wendy Russell, Dr Charles
Bestwick Andrew Farquharson, Sara Padidar, Phil
Morrice, Lorraine Scobbie, Adrian Wood Microbial
Ecology, Gut Health, RRI Dr. Sylvia
Duncan Metabolic Health, RRI BIOSS, RRI
Proteomics Unit, RRI Dr. Julian Mercer, Dr.
Graham Horgan Dr.Garry Rucklidge, Dr. Zoe
Archer Dr. Claus Mayer Martin Reid,
Gary Duncan Medicine and Therapeutics,
RRI/Aberdeen University Aberdeen University
Dr. Geraldine McNeil Prof. Emad El Omar, Dr.
Mairi McLean Funding Chief
Scientists Office, Scotland Scottish Government
SGRERAD