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Chemopreventive effects of pro- and prebiotics towards

microbial bioactivation of ingested contaminants

Inulin and Lactobacillus amylovorus supplemented to human gut microbiota lower the microbial bioactivation of

dietary aromatic contaminants to estrogenic metabolites

Tom Van de Wiele, Lynn Vanhaecke, Charlotte Boeckaert, Heidi Jacobs, Willy Verstraete

Laboratory Microbial Ecology and TechnologyGhent University - Belgium

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Colonmicrobiota and health

Aid in digestion Fermentation to useful

VFA

Immunostimulation Vitamin production

Colonization resistance

Production of health-promoting metabolites

Production of toxins Pathogen colonization

Aetiology in colorectal cancer

(geno-)toxic compounds from food (contaminants)

Recent (!): microbiota stimulate fat uptake and synthesis

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Metabolic potency

Microbiota 500 species, 1011 CFU/mL High enzymatic diversity

Saccharolytic and proteolytic fermentation Conversion of food components

Conversion of xenobiotics Deconjugation of phase II metabolites from liver and

enterocytes Reductive, hydrolytic, degradative, even oxidative

Detoxification <-> Toxification More metabolic conversions than in liver !?

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Example 1: phytoestrogens

Gut bacteria convert isoxanthohumol to hoppein

Most powerful phytoestrogen Food supplements Hormone substitution therapy Prevention of hormone related

cancers (breast/prostate)

Possemiers et al. (2006) Journal of Nutrition

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Example 2: Heterocyclic aromatic amines

Intestinal bacteria convert IQ to hydroxylated metabolite

Rumney et al. (1993)

Intestinal bacteria convert procarcinogen PHIP

Vanhaecke et al. (2006) Journal of Agricultural and Food Chemistry

4 '

5 '

6 '

1 '

2 '

3 '

6

5

N9

8

7

N

2

N

C H3

N H

1 0 1 1

1 2 O H

4 '

5 '

6 '

1 '

2 '

3 '

6

5

N9

8

7

N

2

N

N H2

C H3

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Example 3: mycotoxins

Conversion of zearalenone to zearalenol

Increase in estrogenic properties

Relationship with aetiology of cancer development

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Colon microbiota may toxify xenobiotics

Oral exposure to food and environmental contaminants

Enormous microbial metabolic potency

If toxification significantly contributes to total risk from ingested contaminant...

Can we do something about it ? Manipulate microbial community ? Functional foods: probiotics, prebiotics

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Case study: polycyclic aromatic hydrocarbons

Food: Grilled, broiled... meat

0.35 - 99 g B(a)P / kg

Environment: Atmospheric PAH deposition on soil: 50 g.ha-1.yr-1

Soil ingestion (EPA): Adults: 50 mg.d-1

Children: 200 mg.d-1

Occasionally: 1-20 g.d-1

Inhalation of dust and subsequent ingestion

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Incubation of PAH in SHIME

Stomach Small intestine

Colon

Simulator of Human Intestinal Microbial Ecosystem• pure PAH compounds• PAH contaminated soil

• Colon microbiota bioactivate PAH

• Hydroxylated PAH metabolites

• Estrogenic property

• Not considered in current risk assessment !!!

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SHIME: colon microbiota activate PAHs

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

LC-ESI-MS: hydroxylation of PAHs in colon 1-OH pyrene: 4.3 µg/L 7-OH B(a)P: 1.9 µg/L

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Playground soil : 50 ppm PAH

0

5

10

15

20

25

stomach small intestine colon

µg PAH/L released% EE2 equivalence

PAH release estrogenicity

Lowest concentration in colon Highest estrogenic activity Risk assessment !!!

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Manipulate bioactivation with pro- and prebiotics Probiotic: Lactobacillus amylovorus

Incubate 40 µM B(a)P in colon suspension With / without 107 CFU/mL L. amylovorus Short term effect

Prebiotic: oligofructose DP 3-60 (inulin) SHIME run:

Start-up: 2 weeks Treatment: 3 weeks inulin (2.5 g/d)

Incubate 40 µM B(a)P in colon suspension Compare start-up with treatment period Long term effect

Chemopreventive effect as added-value ?

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Chemopreventive effect: probiotic

L. amylovorus lowers estrogenicity from B(a)P incubated colon samples

0

5

10

15

20

25

30

35

40

Colon Colon + B(a)P Colon + B(a)P + L.amylovorus

% equivalence to 6.96 nM EE2

a

ab

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Chemopreventive effect: prebiotic

0

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5

log mol L-1

% EE2 equivalence

EE2 Proximal colon start-up Proximal colon inulin

0

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5

log mol L-1

% EE2 equivalence

EE2 Distal colon start-up Distal colon inulin

Proximal colon: 40% decrease in estrogenicity Distal colon: no significant decrease Similar effects with phenanthrene and pyrene Inulin exerts chemopreventive effect in proximal

colon

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Prebiotic effects from inulin 26% increase in SCFA (towards propionic and

butyric acid) Significant increase in Bifidobacteria More pronounced effects in proximal colon

0

10

20

30

40

50

60

Start-up Treatment ControlRealtime PCR Bifidobacteria

0

1

2

3

4

5

6

7

8

9

Proximal colon Distal colon

log copy nr. / mL

Start-upInulin

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Chemopreventive effect from functional food

Incubation of B(a)P in fecal microbiota

0

5

10

15

20

25

30

35

40

45

Infant (10months)

Adult (29 yr) Senior (80yr)

Vegetarian Inactivatedcolon

microbiota(29 yr adult)

% equivalence to 6,96 nM EE2

Take into account interindividual variability Steer microbial community to health-promoting

composition Minimize hazardous bioactivation of ingested

contaminants

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Take home messages Metabolic potency from gut microbiota

Consider this process in risk assessment Bacterial players in biological activation Process conditions

Modulate hazardous bioactivation through diet Chemopreventive process: how does it work Inhibition of bioactivation reactions Inhibition of responsible microorganism Sorption to dietary fibres lower bioavailability

Chemopreventive effects are an added-value Manipulation of community through functional food

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Contact information

LabMET – Ghent UniversityCoupure Links 653B-9000 Gent+32 9 264.59.76

tom.vandewiele@ugent.be

http://labMET.ugent.be http://www.shimetec.be http://www.food2know.be