Transcript Isolation, characterisation and applications of 3

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:
prof. dr. ir. H. Van Langenhove
Promotor:
prof. dr. S.D. Siciliano
prof. dr. ir. W. Verstraete
1
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
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
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
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
What happens to ingested contaminants?
1
2
L
I
V
E
R
3
4
Release from soil matrix
Complexation to organic matter
BIOACCESSIBILITY
Intestinal absorption
Biotransformation
BIOAVAILABILITY
6
5
6
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
Part 1
In vitro methods of the human gut to study
lead (Pb) bioaccessibility
8
In vitro models of the human gut
(SHIME)
pH
Z
pH
P
pH
Voeding
Effluent
I
II
III
IV
r
r
r
r
V
VI
r
r
I: Stomach
IV: Caecum/Colon ascendans
II: Duodenum
V: Colon transversum
III: Jejenum/ileum VI: Colon descendens
9
A:
P:
pH:
r:
Zuur
Pancreassap
pH-controle
Roerder
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
In vivo fasted : 26 % bioavailability
11
In vivo fed : 2.5 % bioavailability
12
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
Bioaccessibility separation method
1
2
3
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
14
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
Part 2
Release of PAH from soil in the human
gastrointestinal tract
16
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
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
40% on large aggregates
18
Not absorbed
More than 75% of
released fraction
1,2
Results: PAH desorption study
PAH release(%)
1
0,8
High molecular weight
PAHs
Low molecular weight
PAHs
0,6
0,4
0,2
0
-4,00
-3,00
-2,00
-1,00
0,00
1,00
log solubility(mg/L)
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
2,00
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
Part 3
Human colon microbiota transform PAH to
metabolites with estrogenic properties
21
Current knowledge on PAH bioactivation
3. Gene expression
Cytoplasm
AhR
1. PAH release from
soil / nutrition
Translate
proteins
Arnt
mRNA
DRE
Nucleus
2. Intestinal absorption
Intestine or liver cells
4. Possible bioactivation to toxic compounds
22
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
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
Yeast Estrogen test
Human estrogen receptor in yeast cell
Estrogen responsive elements in plasmid
Reporter gene lacZ
25
SHIME: colon microbiota activate PAHs
Stomach
Small intestine
Colon
Inactivated colon
3.00
nM EE2 equivalence
2.50
2.00
1.50
1.00
0.50
0.00
naphthalene
phenanthrene
26
pyrene
benzo(a)pyrene
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
Urban playground soil sample
PAH release
estrogenicity
% EE2 equivalence
µg PAH/L released
25
20
15
10
5
0
stomach
small intestine
28
colon
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
Part 4
Chemopreventive effect of the prebiotic
inulin towards PAH bioactivation
30
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
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
Ascending colon: inhibitory effect
EE2
Ascending colon start-up
Ascending colon inulin
% EE2 equivalence
120
100
80
60
40
20
0
-12
-11
-10
-9
-8
log mol L-1
33
-7
-6
-5
SCFA: colon ascendens
colon ascendens
26% increase **
Start-up
Towards propionic and
butyric acid
50
µmol/g
40
30
Startup
Treatment
Control
% AA
57
37
48
10
% PA
19
33
19
0
21
27
Control
60
Reversible effect
% BA
Treatment
20
id
c
ca
eti
Ac
29
34
o
Pr
id
ni
o
i
p
c
ca
t
Bu
id
yri
c
ca
r
he
t
O
ids
c
a
To
ta
CF
S
l
A
PCR-DGGE: Bifidobacteria
Sequencing results:
3
2
1
1. Bifidobacterium sp.
2. Bifidobacterium
infantis (96% sim.)
Inulin treatment samples
3. Bifidobacterium
longum (95% sim.)
Start-up and control samples
Realtime PCR: BIFIDOBACTERIA stimulation
35
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
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
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
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
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:
prof. dr. ir. H. Van Langenhove
Promotor:
prof. dr. S.D. Siciliano
prof. dr. ir. W. Verstraete
40