Transcript Omzetting van polluenten in maag

Rôle du microbiote intestinal dans le
métabolisme des composés aromatiques
polycycliques et hétérocycliques
Role of intestinal microbiota in the metabolism of polycyclic and
heterocyclic aromatic compounds
Tom Van de Wiele, PhD
LabMET
Laboratory of Microbial Ecology and Technology
Ghent University, Belgium
6èmes Journées francophone de Nutrition
Nice
29 nov - 1 déc 2006
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Oral exposure to food pollutants
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Polycyclic aromatic hydrocarbons
Heterocyclic aromatic amines from
grilled meat
Mycotoxins
Dioxins, PCB: Belgium 1999
DDT: milk for infants...
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Human health risk assessment
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Biological availability
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What fraction of the
pollutant reaches the blood
circulation?
Biological activity
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What fraction of the
pollutant causes toxicity in
target organs?
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What happens to ingested pollutants?
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2
L
I
V
E
R
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Release from food matrix
Complexation to organic matter
BIOACCESSIBILITY
Intestinal absorption
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6
5
Biotransformation
BIOAVAILABILITY
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What happens to absorbed pollutants ?
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Liver and intestinal epithelium cells:
Biotransformation reactions (phase I and II)
 Make compound more hydrophilic
 Removal from body in urine or bile
DETOXIFICATION
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But:
Biotransformation sometimes goes wrong
 Dead-end metabolite may be formed
 Higher toxicity than parent compound
TOXIFICATION
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What happens to non-absorbed pollutants ?
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Colon ascendens, colon transversum, colon descendens
Non-absorbed pollutants, detoxified pollutants...
enter the large intestine
Vast microbial community
1000 species, 1012 CFU/mL
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SHIME: gastrointestinal in vitro technology
Simulator of the Human Intestinal Microbial Ecosystem
Dynamic model of the human gut
Easy to sample, lots of parameters under control...
Mechanistic research possible !
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Twin SHIME : parallel treatment and control
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Case study. Oral exposure to PAH
Polycyclic Aromatic Hydrocarbons
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Ingestion of contaminated food through badly cleaned
vegetables
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Concentrations on vegetables:
 Root crops: up to 1% of soil onto vegetable
 1.7 - 60 µg PAH/kg vegetable
Daily intake:
 50 mg soil / d (adults)
 200 mg soil / d (children)
Human health risk assessment
Focus on intestinal absorption
and bioactivation by human enzymes
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Colon microbiota contribute to toxicity?
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If so: incorporate in risk assessment !
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Experimental set-up
Incubate in SHIME:
• pure PAH compounds
• PAH contaminated soil
Stomach
Small
Colon
intestine
• Check PAH release from soil matrix along the gut
•If higher release > higher risk ?
• Check biological activation of PAHs
•Screening for hydroxylated PAH metabolites
•Chemical analysis: LC-ESI-MS
•Biological analysis: yeast estrogen bioassay
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SHIME: colon microbiota activate PAHs
Stomach
Small intestine
Colon
Inactivated colon
3,00
nM EE2 equivalence
2,50
2,00
1,50
1,00
0,50
0,00
naphthalene
phenanthrene
pyrene
benzo(a)pyrene
PAH as such are not estrogenic !!!
Hydroxylated PAH metabolites have estrogenic properties
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Chemical analysis
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LC-ESI-MS: hydroxylation of PAHs
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1-OH pyrene: 4.3 µg/L
7-OH B(a)P: 1.9 µg/L
OH
EE2
7-OH B(a)P
Colon microbiota produce hydroxylated PAHs !!!
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Contaminated matrix: 49.1 ppm PAH
PAH release
estrogenicity
% EE2 equivalence
µg PAH/L released
25
20
15
10
5
0
stomach
small intestine
colon
Lower release gives higher biological activity !!!
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Biological activity assessment
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PAH exposure from contaminated soil ingestion
Adult: 5 g PAH/d
Child:50 g PAH/d
Released PAHs lowest in colon, but highest
bioactivity
Colon microbiota convert PAH to pseudoestrogenic metabolites
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Relevant biological activity in vivo ?
Contributes to general PAH toxicity?
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Van de Wiele et al. (2005) Environmental Health Perspectives
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Case study:
Heterocyclic aromatic amines
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Cooked, broiled meats
IQ: most studied (Humblot et al., 2005)
Intestinal bacteria produce 7-OH IQ
Intestinal bacteria are involved in induction of
DNA damage in colon and liver cells
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PHIP: 2-amino-1-methyl-6-phenylimidazopyridine
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PHIP: most abundant
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400 µg/kg meat
10 ng - 10 µg / person.day
What is role of intestinal bacteria towards
PHIP metabolism ?
Risk factor for colorectal cancer ?
Screening of intestinal bacteria from fecal
samples
Determine metabolism and biological activity
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Chemical analysis (Vanhaecke et al., JAFC, 2006)
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HRMS:
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PHIP: 225
M1: 281.1398
Addition of MW 56 !
Inactivation of
bacteria:
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No transformation !
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Microbial conversion
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First time report of PHIP
metabolism
3'
4'
2'
1'
5'
6
7
CH3
8
N
6'
5
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Addition of ring structure is
rare in microbiology
What is biological relevance ?
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NH 2
N
3'
4'
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N
2
2'
1' 6
5'
7
CH 3
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N
2
6'
5
N
9
N
10
NH
12
OH
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Change in bioactivity ?
AMES test
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PHIP
PHIP
with S9
PHIP-M1
PHIP-M1
with S9
ND
toxicity
ND
ND !
Detoxification !!!
Responsible bacteria ?
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Isolated from human fecal sample
Pediococcus sp.
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Vanhaecke et al. (2006)
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Biological activity assessment
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Daily PHIP intake: 10 µg/person.d
>90% conversion to PHIP-M1
Lower toxicity
Increase detoxification through modulation of
intestinal microbial community
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Probiotics, prebiotics...
Responsible Pediococcus:
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Adheres to epithelium...?
What is mechanism ?
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Take home messages
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Metabolic potency from gut microbiota
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Identification of responsible bacteria and
process conditions needed
Interindividual variability !
Modulation of biological activation through
dietary factors, microbial community
composition...
Higher than currently anticipated
Consider this process for risk assessment
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Contact information
[email protected]
http://labMET.ugent.be/
LabMET – Ghent University
Coupure Links 653
B-9000 Gent
www.shimetec.be
www.food2know.be
+32 9 264 59 76
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