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1 ORAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS: PROCESSES OF BIOAVAILABILITY AND INTERACTIONS WITH INTESTINAL MICROORGANISMS ORALE BLOOTSTELLING AAN MILIEUCONTAMINANTEN:

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Presentation on theme: "1 ORAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS: PROCESSES OF BIOAVAILABILITY AND INTERACTIONS WITH INTESTINAL MICROORGANISMS ORALE BLOOTSTELLING AAN MILIEUCONTAMINANTEN:"— Presentation transcript:

1 1 ORAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS: PROCESSES OF BIOAVAILABILITY AND INTERACTIONS WITH INTESTINAL MICROORGANISMS ORALE BLOOTSTELLING AAN MILIEUCONTAMINANTEN: PROCESSEN VAN BIOBESCHIKBAARHEID EN INTERACTIES MET INTESTINALE MICRO-ORGANISMEN ir. Tom Van de Wiele Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Toegepaste Biologische Wetenschappen Laboratorium voor Microbiële Ecologie en Technologie Faculteit Bio-ingenieurswetenschappen, Universiteit Gent Decaan: Promotor: prof. dr. ir. H. Van Langenhove prof. dr. S.D. Siciliano prof. dr. ir. W. Verstraete

2 2 Presentation overview General introduction Processes of bioavailability Part 1: In vitro methods of the human gut to study contaminant bioaccessibility Part 2: Release of PAH from soil in the human gastrointestinal tract Interaction with colon microbiota Part 3: Human colon microbiota transform PAH to metabolites with estrogenic properties Part 4: Chemopreventive effect of the prebiotic inulin towards PAH bioactivation General discussion & future perspectives

3 3 General introduction Oral exposure to contaminants Ingestion of contaminated food ‘Dioxin-crisis’ in Belgium 1999 Pesticides and antibiotics in food Flame retardants in human milk Broiled, smoked, grilled meat: HCA … Health risks

4 4 Oral exposure to contaminants Ingestion of contaminated soil Industrial and urban areas PCBs and PAHs 50 g.ha -1.yr -1 Oral uptake Adults: 50 mg.d -1 Children: 200 mg.d -1 Occasionally: 1-20 g.d -1 What are the risks? HUMAN HEALTH RISK ASSESSMENT

5 5 What happens to ingested contaminants? Stomach Low pH, pepsin Small intestine Breakdown of sugars, fats proteins Absorption across epithelium Large intestine Absorption of water Microorganisms

6 6 What happens to ingested contaminants? Release from soil matrix Complexation to organic matter BIOACCESSIBILITY Intestinal absorption Biotransformation BIOAVAILABILITY LIVERLIVER 5 6

7 7 Bioavailability versus Bioaccessibility Bioavailability (in vivo studies) Fraction of a contaminant in the blood compartment Time-consuming, variable, ethical problems Release/complexation processes are a black box Bioaccessibility (in vitro studies) Fraction of a contaminant which releases from soil and which becomes available for intestinal transport Important precursor to bioavailability Estimate Bioavailability by measuring Bioaccessibility

8 8 Part 1 In vitro methods of the human gut to study lead (Pb) bioaccessibility

9 9 In vitro models of the human gut (SHIME) II r III r Z P I: Stomach II: Duodenum III: Jejenum/ileum IV: Caecum/Colon ascendans V: Colon transversum VI: Colon descendens A: Zuur P: Pancreassap pH: pH-controle r: Roerder I r Voeding IV r pH V r VI r pH Effluent

10 10 Comparison study for Pb bioaccessibility Bunker Hill soil (USA): 3066 ± 55 mg Pb.kg DW -1 5 European in vitro models! BGS: PBET Bochum Universität : DIN RIVM LabMET: SHIME TNO : TIM Assess bioaccessibility Relate to in vivo bioavailability FASTED versus FED conditions

11 11 In vivo fasted : 26 % bioavailability

12 12 In vivo fed : 2.5 % bioavailability

13 13 Digestion parameters L/S (Liquid to Solid) ratio Equilibrium towards release at higher L/S SHIME: low L/S of 25 pH Low stomach pH solubilizes more Pb Neutral intestine pH forms complexes Nutrition Fed in vivo bioavail. < fasted in vivo bioavail. Fed in vitro bioacc. > fasted in vitro bioacc. Except TIM: only correct method

14 14 Bioaccessibility separation method 1.Centrifugation (3000 g): Large complexes 2.Microfiltration (0.45 µm): smaller complexes 3.Ultrafiltration (5000 Da): free contaminants + small lipid complexes Small food complexes are not bioaccessible Retained by ultrafiltration, not by other methods 1 23

15 15 Part 1: Take home messages Bioaccessibility should always be higher than Bioavailability Large Pb-food complexes are not available for intestinal absorption ! New! role of separation method in bioaccessibility Contaminant speciation in the gut ! Every in vitro method has its value: proper interpretation needed

16 16 Part 2 Release of PAH from soil in the human gastrointestinal tract

17 17 Experimental Set-up PAH: polycyclic aromatic hydrocarbons Urban playground soil: 50.3 mg PAH.kg DW -1 SHIME: stomach, small intestine, colon Simulate conditions of child gastrointestinal tract Where is PAH release the highest? Which parameters play a role in release process? Which PAHs are released the most?

18 18 Results: PAH desorption study <1% free PAH 19% with bile salts 6% on dissolved OM 35% on particulate OM 40% on large aggregates Partially absorbed Less than 25% of released fraction Not absorbed More than 75% of released fraction Limited PAH release along GI tract >99% remains on soil Stomach: 0.44% Small int.: 0.13% Colon: 0.30% In small intestine: 0.13% release

19 19 High molecular weight PAHs High MW PAHs: higher desorption than expected Intestinal colloids: enhance solubility with factor 50 !!! Concern: high molecular PAHs are related with genotoxicity and carcinogenicity Low molecular weight PAHs Results: PAH desorption study

20 20 Part 2: Take home messages Organic matter in the gut increases PAH desorption New! intestinal colloids enhance solubilization of more hydrophobic PAHs SHIME allows mechanistic study of the intestinal lumen

21 21 Part 3 Human colon microbiota transform PAH to metabolites with estrogenic properties

22 22 Current knowledge on PAH bioactivation 1. PAH release from soil / nutrition 2. Intestinal absorption Intestine or liver cells 3. Gene expression Cytoplasm AhR Nucleus mRNA Arnt Translate proteins DRE 4. Possible bioactivation to toxic compounds

23 23 What happens to non-adsorbed PAHs ? Large fraction of ingested PAHs becomes available to colon micro-organisms 400 different species, organisms cfr. 1 kg active yeast Are colon microbiota capable of biotransforming PAHs? Are microbial PAH metabolites bioactive?

24 24 Experimental set-up Incubate PAH in samples from SHIME reactor Screen for PAH metabolites Estrogen receptor bioassay: estrogenicity LC-ESI-MS: hydroxy-PAH Negative control samples Pure PAH compounds PAH contaminated soil samples

25 25 Yeast Estrogen test Human estrogen receptor in yeast cell Estrogen responsive elements in plasmid Reporter gene lacZ

26 26 SHIME: colon microbiota activate PAHs

27 27 Chemical analysis LC-ESI-MS: hydroxylation of PAHs 1-OH pyrene: 4.3 µg/L 7-OH B(a)P: 1.9 µg/L OH EE27-OH B(a)P

28 28 Urban playground soil sample

29 29 Conclusions New! colon microbiota are able to convert PAHs to compounds with estrogenic properties This bioactivation potency is not yet considered in current risk assessment Current risks may be underestimated

30 30 Part 4 Chemopreventive effect of the prebiotic inulin towards PAH bioactivation

31 31 Prebiotics Stimulation of endogenous beneficial bacteria Suppress pathogens or harmful microbial metabolism Inulin Fructo-oligosaccharides, … Not digested in stomach or small intestine Total transfer to the colon  (2-1) glycosidic bond: Bifidobacteria

32 32 Experimental set-up Prebiotic inulin: add to SHIME reactor Evaluate inulin as chemopreventive agent Start-up, inulin treatment (2.5 g/d) Incubate SHIME suspension with 40 µM B(a)P Monitor PAH bioactivation with yeast estrogen bioassay Relate to prebiotic effects Metabolic analysis PCR-DGGE-sequencing Real-time PCR quantification Bifidobacterium sp.

33 33 Ascending colon: inhibitory effect

34 34 SCFA: colon ascendens 26% increase ** Towards propionic and butyric acid Reversible effect Start- up Treat- ment Con- trol % AA % PA % BA212729

35 35 PCR-DGGE: Bifidobacteria Sequencing results: 1.Bifidobacterium sp. 2.Bifidobacterium infantis (96% sim.) 3.Bifidobacterium longum (95% sim.) Start-up and control samples Inulin treatment samples 123 Realtime PCR: BIFIDOBACTERIA stimulation

36 36 Part 4: Take home messages Inulin has prebiotic / bifidogenic effect in all colon vessels New! Inulin exerts chemopreventive activity towards PAH bioactivation in the colon Prebiotic inulin has an added-value

37 37 General conclusions Bioaccessibility measurements need to be conservative estimators of bioavailability In vitro methods must be tuned to consider contaminant speciation Human colon microbiota are able to directly convert PAHs into compounds with estrogenic properties If this significantly contributes to the total risk of ingested PAHs  take up in risk assessment Prebiotic inulin has an added-value by its chemopreventive activity towards PAH bioactivation

38 38 Future perspectives Food contaminants: heterocyclic aromatic amines (HCA): PHIP, IQ… Investigate more in detail metabolic potency of colon microbiota Investigate interaction of microbial groups and metabolites with colon epithelium: adhesion, transport, immune system

39 39 Acknowledgements Laboratory of Microbial Ecology and Technology Els, Siska, Greet Charlotte, Lynn, Yourri, Kasper Patrick, Roel, Vanessa, Sam, Karel, Kristof Nico, Sylvie, Roeland, Wim, Han, Korneel, Frederik, Joris, Hendrik, Sofie… Christine, Regine, Veronique, Annelies All the other collaborators National Water Research Institute (NWRI), Canada Kerry Peru, John Headley BARGE (BioAvailability Research Group Europe) Agnes Oomen, Mans Minekus, Joanna Wragg, Mark Cave, Ben Klinck, Christa Cornelis, Joop Vanwijnen, Adrienne Sips

40 40 ORAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS: PROCESSES OF BIOAVAILABILITY AND INTERACTIONS WITH INTESTINAL MICROORGANISMS ORALE BLOOTSTELLING AAN MILIEUCONTAMINANTEN: PROCESSEN VAN BIOBESCHIKBAARHEID EN INTERACTIES MET INTESTINALE MICRO-ORGANISMEN ir. Tom Van de Wiele Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Toegepaste Biologische Wetenschappen Laboratorium voor Microbiële Ecologie en Technologie Faculteit Bio-ingenieurswetenschappen, Universiteit Gent Decaan: Promotor: prof. dr. ir. H. Van Langenhove prof. dr. S.D. Siciliano prof. dr. ir. W. Verstraete


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