tom van de wiele, phd labmet laboratory of microbial ecology and technology
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R ôle 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 - PowerPoint PPT PresentationTRANSCRIPT
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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
LabMETLaboratory of Microbial Ecology and Technology
Ghent University, Belgium
6èmes Journées francophone de NutritionNice
29 nov - 1 déc 2006
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Oral exposure to food pollutants
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
Biological availability What fraction of the pollutant reaches the blood circulation?
Biological activity What fraction of the pollutant causes toxicity in target organs?
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What happens to ingested pollutants?
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4Release from food matrixComplexation to organic matterBIOACCESSIBILITYIntestinal absorption
Biotransformation
BIOAVAILABILITY
LIVER
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What happens to absorbed pollutants ?
Liver and intestinal epithelium cells: Biotransformation reactions (phase I and II)
Make compound more hydrophilic Removal from body in urine or bileDETOXIFICATION
But: Biotransformation sometimes goes wrong Dead-end metabolite may be formed Higher toxicity than parent compoundTOXIFICATION
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What happens to non-absorbed pollutants ?
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 EcosystemDynamic model of the human gutEasy 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 PAHPolycyclic Aromatic Hydrocarbons
Ingestion of contaminated food through badly cleaned vegetables 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
Colon microbiota contribute to toxicity? If so: incorporate in risk assessment !
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Experimental set-up
Stomach Small intestine
Colon
Incubate in SHIME:• pure PAH compounds• PAH contaminated soil
• 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
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0,50
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naphthalene phenanthrene pyrene benzo(a)pyrene
nM EE2 equivalence
Stomach Small intestine Colon Inactivated colon
PAH as such are not estrogenic !!!
Hydroxylated PAH metabolites have estrogenic properties
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Chemical analysis
LC-ESI-MS: hydroxylation of PAHs 1-OH pyrene: 4.3 µg/L 7-OH B(a)P: 1.9 µg/L
EE2 7-OH B(a)P
Colon microbiota produce hydroxylated PAHs !!!
OH
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Contaminated matrix: 49.1 ppm PAH
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stomach small intestine colon
µg PAH/L released% EE2 equivalence
PAH release estrogenicity
Lower release gives higher biological activity !!!
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Biological activity assessment
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 pseudo-estrogenic metabolites
Relevant biological activity in vivo ? Contributes to general PAH toxicity?
Van de Wiele et al. (2005) Environmental Health Perspectives
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Case study: Heterocyclic aromatic amines
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
PHIP: most abundant 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)
HRMS: PHIP: 225 M1: 281.1398 Addition of MW 56 !
Inactivation of bacteria: No transformation !
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Microbial conversion
First time report of PHIP metabolism
Addition of ring structure is rare in microbiology
What is biological relevance ?
4 '
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N9
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N
2
N
C H3
N H
1 0 1 1
1 2 O H
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1 '
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N9
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N
2
N
N H2
C H3
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Change in bioactivity ?
Isolated from human fecal sample Pediococcus sp. Vanhaecke et al. (2006)
PHIP PHIP with S9
PHIP-M1 PHIP-M1 with S9
AMES test
ND toxicity ND ND !
Responsible bacteria ?
Detoxification !!!
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Biological activity assessment
Daily PHIP intake: 10 µg/person.d >90% conversion to PHIP-M1 Lower toxicity
Increase detoxification through modulation of intestinal microbial community Probiotics, prebiotics...
Responsible Pediococcus: Adheres to epithelium...? What is mechanism ?
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Take home messages
Metabolic potency from gut microbiota 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
http://labMET.ugent.be/LabMET – Ghent UniversityCoupure Links 653B-9000 Gent
www.shimetec.be www.food2know.be
+32 9 264 59 76