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ir. Tom Van de Wiele Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Toegepaste Biologische Wete

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

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ir. Tom Van de Wiele Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Toegepaste Biologische Wete

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  1. ORAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS: PROCESSES OF BIOAVAILABILITY AND INTERACTIONS WITH INTESTINAL MICROORGANISMSORALE 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. 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 2

  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 3

  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 4

  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 5

  6. L I V E R 6 1 2 3 4 5 What happens to ingested contaminants? Release from soil matrix Complexation to organic matter BIOACCESSIBILITY Intestinal absorption Biotransformation BIOAVAILABILITY 6

  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 7

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

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

  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 10

  11. In vivo fasted : 26 % bioavailability 11

  12. In vivo fed : 2.5 % bioavailability 12

  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 13

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

  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 15

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

  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? 17

  18. Results: PAH desorption study 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 <1% free PAH Partially absorbed Less than 25% of released fraction 19% with bile salts 6% on dissolved OM 35% on particulate OM Not absorbed More than 75% of released fraction 40% on large aggregates 18

  19. Results: PAH desorption study High molecular weight PAHs Low 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 19

  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 20

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

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

  23. What happens to non-adsorbed PAHs ? • Large fraction of ingested PAHs becomes available to colon micro-organisms • 400 different species, 1014 organisms cfr. 1 kg active yeast • Are colon microbiota capable of biotransforming PAHs? • Are microbial PAH metabolites bioactive? 23

  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 24

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

  26. SHIME: colon microbiota activate PAHs 26

  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 EE2 7-OH B(a)P 27

  28. Urban playground soil sample 28

  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 29

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

  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 • b(2-1) glycosidic bond: Bifidobacteria 31

  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. 32

  33. Ascending colon: inhibitory effect 33

  34. SCFA: colon ascendens 26% increase ** Towards propionic and butyric acid Reversible effect 34

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

  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 36

  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 37

  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 38

  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 39

  40. ORAL EXPOSURE TO ENVIRONMENTAL CONTAMINANTS: PROCESSES OF BIOAVAILABILITY AND INTERACTIONS WITH INTESTINAL MICROORGANISMSORALE 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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