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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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6

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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

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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