1 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

2 Oral exposure to food pollutants Polycyclic aromatic hydrocarbons Heterocyclic aromatic amines from grilled meat Mycotoxins Dioxins, PCB: Belgium 1999 DDT: milk for infants...

3 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?

4 What happens to ingested pollutants? Release from food matrix Complexation to organic matter BIOACCESSIBILITY Intestinal absorption Biotransformation BIOAVAILABILITY LIVERLIVER 5 6

5 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 bile DETOXIFICATION  But: Biotransformation sometimes goes wrong Dead-end metabolite may be formed Higher toxicity than parent compound TOXIFICATION

6 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, CFU/mL

7 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 !

8 Twin SHIME : parallel treatment and control

9 Case study. Oral exposure to PAH Polycyclic Aromatic Hydrocarbons  Ingestion of contaminated food through badly cleaned vegetables Concentrations on vegetables:  Root crops: up to 1% of soil onto vegetable  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 !

10 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

11 SHIME: colon microbiota activate PAHs PAH as such are not estrogenic !!! Hydroxylated PAH metabolites have estrogenic properties

12 Chemical analysis  LC-ESI-MS: hydroxylation of PAHs 1-OH pyrene: 4.3 µg/L 7-OH B(a)P: 1.9 µg/L EE27-OH B(a)P Colon microbiota produce hydroxylated PAHs !!! OH

13 Contaminated matrix: 49.1 ppm PAH Lower release gives higher biological activity !!!

14 Biological activity assessment  PAH exposure from contaminated soil ingestion  Adult: 5 g PAH/dChild: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

15 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

16 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

17 Chemical analysis (Vanhaecke et al., JAFC, 2006)  HRMS: PHIP: 225 M1: Addition of MW 56 !  Inactivation of bacteria: No transformation !

18 Microbial conversion  First time report of PHIP metabolism  Addition of ring structure is rare in microbiology  What is biological relevance ?

19 Change in bioactivity ?  Isolated from human fecal sample  Pediococcus sp.  Vanhaecke et al. (2006) PHIP with S9 PHIP-M1PHIP-M1 with S9 AMES testNDtoxicityNDND ! Responsible bacteria ?  Detoxification !!!

20 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 ?

21 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

22 Contact information LabMET – Ghent University Coupure Links 653 B-9000 Gent