FEDERAL INSTITUTE FOR RISK ASSESSMENT · The ABC-transporter BCRP is responsible for the transport of B[a]P-sulfates und B[a]P-glucuronides AhR Agonists induce the gene- and protein

FED

ERA

L IN

STIT

UTE

FO

R R

ISK

ASS

ESSM

ENT

Können Pflanzeninhaltsstoffe den biochemischen

Schutz des menschlichen Darmes gegenüber

Lebensmittelkontaminanten verstärken?

Alfonso Lampen, Stefanie Hessel

ABCCOOHH2N

BCRP: Breast cancer resistance protein

Prof. Dr. Dr. A. Lampen, Stefanie Hessel, 08.11.2007, Können Pflanzeninhaltsstoffe d. biochem. Schutz d. menschl. Dünndarms gegenüber Lebensmittelkontaminanten verstärken?“

Page 2

Resorption, Resorption, biotransformationbiotransformation and and eliminationelimination of of B[B[aa]P]P fromfromenterocytesenterocytes

Resorption

Oxidation

Conjugation

ABC-Transporter

Xenobiotica

Transformation product

Conjugate

?

apical membrane

CYP‘s

transferases

1A1,1B1

PAPSGSHUDPGlcUA

blood

Export to lumen

ABC-Transporter:p-GlycoproteinMRP2 (MultidrugResistance Protein)BCRP

H2N ABC BCRP COOH


Page 3

Expression von BCRP

Tissue Functionstomach, duodenum, colon reduction of resorption of xenobiotica, drugs

hepato-biliar excretion, renal excretionliver, kidney

brain protection against xenobiotica?

placenta protection of fetus

mammary gland ???

BCRP


Page 4

Metabolisierung des LebensmittelkontaminantenBenzo[a]pyren; eine Modellsubstanz mit hohem karzinogenen Potential

Benzo(a)pyren

CYPs

Phase II-Enzyme

OH

O-R

1. Lipophiler Fremdstoff:freie Diffusion durch

Membranen

2. Hydroxylierung von B[a]P:Bildung potentieller Substrate für Phase II Enzyme; Bioaktivierung zu mutagenen Derivaten

3. Hydrophiler Phase II-Metabolit:aktiver Transport aus der Zelle notwendig

Phase I: CYPs

Phase II: SULT

ABC


Page 5

Transepithelial efflux of B[a]P-1-sulfate and B[a]P-3-sulfate in human intestinal Caco-2 cells

0

25

50

75

100

125

150

0 2 4 6 8 10 12

unbeh.; apikal

unbeh.; basal

[pmol/cm²]

time [h]

[pmol/cm²]

time [h]

0

10

20

30

40

50

0 2 4 6 8 10 12

unbeh.; apikal

unbeh.; basal

B[a]P-1-SulfatB[a]P-3-Sulfat

Transwell™Caco-2cells


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BCRP is responsible for the transepithelial transport of B[a]P-sulfates in Caco-2 cells

- Inhibitor + Inhibitor

substrate: B[a]P-3-OH

Inhibition of the efflux of BAP-3-sulfate inCaco-2 cells by Ko 143

0

200

400

600

800

1000

1200

1400

0 3 6 9time [h]

[pm

ol/ c

m²]

control, apical

control, basal

Ko 143 (5µM), apical

Ko 143 (5µM), basal

NN

NH

OCH3O

O

O

O

Ko 143

H


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AhAh--Rezeptor Aktivierung durch Rezeptor Aktivierung durch B[a]PB[a]P ffüührt zu einer Induktion hrt zu einer Induktion von CYP1A1/1B1von CYP1A1/1B1Kernrezeptor: Kernrezeptor: ArylhydrocarbonArylhydrocarbon--RezeptorRezeptor AhRAhR

Zellkern

Fremdstoff (z.B. PAK)

Ah-Rezeptor

ARNT

ARNT

EnzymeCYP1B1

mRNA

DNA (DRE)

Gentranskription

EnzymeCYP1A1

AhR/ARNT/Fremd-stoff-Komplex

BCRP?

(DRE = Dioxin response element, Promoter)


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The role of AhR: expression of BCRP-protein in MCF-7 cells

WT AhR200

B[k]F - + - +

BCRP

K

WT AhR200

BCRP

PCB 77, 1 µM

TBHQ, 50 µM

Quercetin, 25 µM

DBM, 50 µM

AhR200 : AhR-deficient(Yeh et al., 1999): smallbasal expression of BCRP

DBM = DibenzoylmethanTBHQ= tert-Butyl-hydrochinon


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Induction of BCRP by AhR-agonists in Caco-2 cells

BaP

BkF

TCDD

HNN

HBCRP

1

2,44

1,75

2,172,30

0

0,5

1

1,5

2

2,5

3

Kontrolle

(0,1%

DMSO)

TCDD 50 nM

DIM 25 µ

M

BAP 10 µM

BkF 5

µM

BC

RP-

Prot

einl

evel

(x-fa

che

Erhö

hung

)

K TCDD DIM BAP B[k]F

DIM = 3,3`-Diindolylmethan

O

O

Cl

Cl

Cl

Cl


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Induction of BCRP expression by phytochemicals in Caco-2 cells

1

2,462,34

1,841,69 1,67 1,74

2,19

0

0,5

1

1,5

2

2,5

3

Kontrolle

(0,1%

DMSO)

TCDD 50 nM

Querceti

n 25 µM

Curcumin 25

µMSem

pervirin

25 µM

Juglon 10

µM

DMB 25 µM

DBM 50 µM

Behandlung (3 Tage)

BC

RP-

Prot

einl

evel

(x-fa

che

Erhö

hung

)

M K TCDD 4 5 6 7 8 9

75 kDa

OHOH

OHO

OH O

OH

Quercetin

Curcumin

O

HO

H3CO OCH3

OH

O

O O

Dibenzoylmethan

O

O

OH

Juglon


Page 11

37

3436

51

42

4949

42 43

0

10

20

30

40

50

60

2 4 6

Time [h]

Api

cally

tran

spor

ted

BP3

S [%

of t

otal

am

ount

]control, apicalDBM (50 µM), apicalQuercetin (25 µM), apical

* *

* *

**

Effect of DBM and Quercetin on the transport of B[a]P-sulfatein Caco-2 cells

DBM = Dibenzoylmethan(aus Süßholzpflanze)


Page 12

Conclusions

-

blood (basal)

enterocyte (Caco-2)

benzo[a]pyrene

Phase 2Phase 1

benzo[a]pyrene metabolite

B[a]P-1-sulfate; B[a]P-3-sulfate B[a]P-3-glucuronide

ABC BCRPABC

ABC: ABC-transportproteins

Phase 1 und 2: enzymes of phase 1 and 2

ABC

phytochemicals

phytochemicals

There is a functional interplay between metabolism of CYP1A1/CYP1B1 substrates (PAHs), Phase II and the transport of metabolites by BCRP

The ABC-transporter BCRP is responsible for the transport of B[a]P-sulfates und B[a]P-glucuronides

AhR Agonists induce the gene- and protein expression of the responsible CYPs and BCRP.

Regulation of BCRP-transporter is most likely AhR dependent.

Phytochemicals that are AhR-agonist do have an impact on gene/protein expression of BCRP and on its functonal transport of potential toxic phase II metabolites.

Phytochemicals may influence bioavailability of potential toxic compounds and protect the bodyagainst toxic metabolites.


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Dr. Bettina EbertDr. Lutz StumkatYan Li (Ch), Ph.DRoland Büsen,Ph.DMelissa Mock (AUS) Ph.DFachgruppe 56 des BfR

PD Dr. Albrecht SeidelBiochemisches Institut für Umweltkarzinogene, Prof. G. Grimmer-Foundation, Großhansdorf

Prof. A. Schinkel, University ofAmsterdam, Netherlands

Prof. H.R. GlattDIFE, Rehbrücke, Potsdam

Prof. Dr. Dr. Alfonso LampenDepartment of Food SafetyFederal Institute for Risk Assessment (BfR), [email protected]

Thank youfor your attention

Acknowledgment

Documents

FEDERAL INSTITUTE FOR RISK ASSESSMENT · The ABC-transporter BCRP is responsible for the transport of B[a]P-sulfates und B[a]P-glucuronides AhR Agonists induce the gene- and protein