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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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1,50

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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 '

5 '

6 '

1 '

2 '

3 '

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N9

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N

2

N

C H3

N H

1 0 1 1

1 2 O H

4 '

5 '

6 '

1 '

2 '

3 '

6

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N9

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7

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

tom.vandewiele@ugent.be

http://labMET.ugent.be/LabMET – Ghent UniversityCoupure Links 653B-9000 Gent

www.shimetec.be www.food2know.be

+32 9 264 59 76