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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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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
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General introductionGeneral 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
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Oral exposure to contaminantsOral 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
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What happens to ingested What happens to ingested contaminants?contaminants?
Stomach
Low pH, pepsin
Small intestine
Breakdown of sugars, fats proteins
Absorption across epithelium
Large intestine
Absorption of water
Microorganisms
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What happens to ingested What happens to ingested contaminants?contaminants?
1 2 3
4Release from soil matrix
Complexation to organic matter
BIOACCESSIBILITY
Intestinal absorption
Biotransformation
BIOAVAILABILITY
LIVER
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Bioavailability versus Bioavailability versus BioaccessibilityBioaccessibility
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
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Part 1Part 1
In vitro methods of the human gut to study lead (Pb)
bioaccessibility
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In vitro In vitro models of the human gut models of the human gut (SHIME) (SHIME)
II
r
III
r
Z P
I: StomachII: DuodenumIII: Jejenum/ileum
IV: Caecum/Colon ascendansV: Colon transversumVI: Colon descendens
A: ZuurP: PancreassappH: pH-controler: Roerder
I
r
VoedingIV
r
pH
V
r
pH
VI
r
pH
Effluent
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Comparison study for Pb Comparison study for Pb bioaccessibilitybioaccessibility
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
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In vivo In vivo fasted : 26 % bioavailabilityfasted : 26 % bioavailability
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In vivo In vivo fed : 2.5 % bioavailabilityfed : 2.5 % bioavailability
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Digestion parametersDigestion parameters
L/S (Liquid to Solid) ratio Equilibrium towards release at higher L/S
SHIME: low L/S of 25
pHLow stomach pH solubilizes more Pb
Neutral intestine pH forms complexes
NutritionFed in vivo bioavail. < fasted in vivo bioavail.
Fed in vitro bioacc. > fasted in vitro bioacc.
Except TIM: only correct method
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Bioaccessibility separation methodBioaccessibility 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 2 3
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Part 1: Take home messagesPart 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
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Part 2Part 2
Release of PAH from soil in the human gastrointestinal tract
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Experimental Set-upExperimental 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?
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Results: PAH desorption studyResults: PAH desorption study
<1% free PAH
19% with bile salts
6% on dissolved OM
35% on particulate OM
40% on large aggregates
Partially absorbedLess than 25% of released fraction
Not absorbedMore than 75% of released fraction
Limited PAH release along GI tract>99% remains on soilStomach: 0.44% Small int.: 0.13% Colon: 0.30%
In small intestine: 0.13% release
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0
0,2
0,4
0,6
0,8
1
1,2
-4,00 -3,00 -2,00 -1,00 0,00 1,00 2,00
log solubility(mg/L)
High molecular weight PAHs
High MW PAHs: higher desorption than expectedIntestinal colloids: enhance solubility with factor 50 !!!
Concern: high molecular PAHs are related with genotoxicity and carcinogenicity
Low molecular weight PAHs
Results: PAH desorption study
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Part 2: Take home messagesPart 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
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Part 3Part 3
Human colon microbiota transform PAH to metabolites with estrogenic properties
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Current knowledge on PAH Current knowledge on PAH bioactivationbioactivation
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
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What happens to non-adsorbed PAHs ?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?
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Experimental set-upExperimental 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
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Yeast Estrogen testYeast Estrogen test
Human estrogen receptor in yeast cell
Estrogen responsive elements in plasmid
Reporter gene lacZ
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SHIME: colon microbiota activate SHIME: colon microbiota activate PAHsPAHs
0.00
0.50
1.00
1.50
2.00
2.50
3.00
naphthalene phenanthrene pyrene benzo(a)pyrene
nM EE2 equivalence
Stomach Small intestine Colon Inactivated colon
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Chemical analysisChemical 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
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Urban playground soil sampleUrban playground soil sample
0
5
10
15
20
25
stomach small intestine colon
µg PAH/L released% EE2 equivalence
PAH release estrogenicity
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ConclusionsConclusions
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
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Part 4Part 4
Chemopreventive effect of the prebiotic inulin towards PAH
bioactivation
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PrebioticsPrebiotics
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
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Experimental set-upExperimental 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.
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Ascending colon: inhibitory effectAscending colon: inhibitory effect
0
20
40
60
80
100
120
-12 -11 -10 -9 -8 -7 -6 -5
log mol L-1
% EE2 equivalence
EE2 Ascending colon start-up Ascending colon inulin
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SCFA: colon ascendensSCFA: colon ascendens
26% increase **
Towards propionic and butyric acid
Reversible effect
Start-up
Treat-ment
Con-trol
% AA 57 37 48
% PA 19 33 19
% BA 21 27 29
0
10
20
30
40
50
60
Start-up Treatment Control
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Pearson correlation [0.0%-100.0%]
100
50
0
PCR-DGGE: BifidobacteriaPCR-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: Realtime PCR: BIFIDOBACTERIA stimulationBIFIDOBACTERIA stimulation
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Part 4: Take home messagesPart 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
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General conclusionsGeneral 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
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Future perspectivesFuture 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
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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), CanadaKerry Peru, John Headley
BARGE (BioAvailability Research Group Europe)Agnes Oomen, Mans Minekus,
Joanna Wragg, Mark Cave, Ben Klinck,
Christa Cornelis, Joop Vanwijnen, Adrienne Sips
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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