effect of benzo[a]pyrene on p-glycoprotein-mediated transport in caco-2 cell monolayer

Toxicology 223 (2006) 156–165

Effect of benzo[a]pyrene on P-glycoprotein-mediatedtransport in Caco-2 cell monolayer

Narumi Sugihara ∗, Kumiko Toyama, Akihiro Michihara,Kenji Akasaki, Hiroshi Tsuji, Koji Furuno

Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Sanzou, Gakuen-cho,Fukuyama, Hiroshima 729-0292, Japan

Received 8 February 2006; received in revised form 16 March 2006; accepted 21 March 2006Available online 28 March 2006

Abstract

The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of thisstudy was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinalepithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the humanCaco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparentpermeability coefficients (Papp) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which areP-gp inhibitors, decreased the Papp of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced
by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor ofRho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expressionof MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonalantibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bapexposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility wassuggested that Bap might affect the disposition of medicines by increasing P-gp expression.© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Caco-2 cells; P-glycoprotein; MDR; Benzo[a]pyrene; Rhodamine-123

1. Introduction

The 170-kDa P-gp is a plasma-membrane-associatedenergy-dependent efflux pump encoded by the MDR 1gene in humans and is considered to be responsible forthe phenomenon of multiple drug resistance, which is

∗ Corresponding author. Tel.: +81 84 936 2112x5135;fax: +81 84 936 2024.

E-mail address: [email protected](N. Sugihara).

observed in tumor cell lines that are able to decrease theintracellular accumulation of cytostatic drugs (Abolhodaet al., 1999; Arora et al., 2005). The existence of P-gpis observed in not only tumor cells, but also epithelialcells of various tissues (Thiebaut et al., 1987; Arboixet al., 1997; Tanigawara, 2000). The gastrointestinaltract is exposed to many potentially toxic agents as wellas medicines. Thus, the role of P-gp expressed on thegastrointestinal tract might be one defense against toxicexogenous substances (Lechapt-Zalcman et al., 1997;Schwab et al., 2003; Bodo et al., 2003). The activity

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N. Sugihara et al. / Toxicology 223 (2006) 156–165 157

and expression of P-gp were reported to be influencedby medicines, environmental pollutants, foods, etc.(Mitsunaga et al., 2000; Boumendjel et al., 2002; Sai etal., 2003; Penzak et al., 2004). The changes in activityof P-gp on the gastrointestinal tract by those substancesare considered to influence the bioavailability ofmedicines.

Benzo[a]pyrene (BaP) is a widespread environmentalcarcinogen, that belongs to polycyclic aromatic hydro-carbons (PAHs) (Sims et al., 1974; Bostrom et al.,2002). Human exposure studies have revealed that BaP isingested by humans rather than inhaled. The food chainis regarded as the dominant pathway in exposure of Bapto humans (Hattermer-Frey and Travis, 1991). Bap maybe biotransformed in humans and animals to numerousphase 1 metabolites including 3-,7-, and 9-hydroxy-Bap,Bap-dihydrodiols, Bap-dihydrodiol-epoxides, and Bap-quinones (Shou et al., 1994; Kleinow et al., 1998; Jameset al., 2001). 7�,8�-Dihydroxy-9�,10�-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) is considered the ulti-mate carcinogenic metabolite of Bap (Shou et al., 1994).The transcriptional induction of the MDR1 gene encoad-ing P-gp by Bap treatment was demonstrated in studieswith rat liver epithelial and mouse hepatoma cell linesHepa-1c1c7 (Fardel et al., 1996; Mathieu et al., 2001;Lampen et al., 2004). On the contrary, there were reportsthat the basal levels of hepatic or intestinal P-gp werenot influenced by Bap treatment in Xennopus laevis andcatfish to a statistically significant extent (Colombo etal., 2003; Doi et al., 2001). The different results on theiemnsrsh2mMeod2

itG2at

et al., 2001; Niemi et al., 2003). On the other, someflavonoids such as quercetin and kaempferol act directlyon the transport-activity of P-gp (Mitsunaga et al., 2000;Boumendjel et al., 2002). Hence, the induction of MDR1does not necessarily lead to an enhancement of P-gpactivity. As stated above, there is no report indicatingthat Bap influences disposition of substances mediatedby P-gp. The Caco-2 cell monolayer is useful for invitro investigations because it contains morphologicaland biochemical characteristics of human enterocytes.In the present study, the effects of Bap exposure on P-gp-mediated transport of rhodamine-123, a well-knownP-gp substrate (Takano et al., 1998), were examinedusing Caco-2 monolayers.

2. Materials and methods

2.1. Materials

Benzo[a]pyrene was purchased from Wako Pure Chemi-cal Co. (Osaka, Japan). Rho-123 was obtained from KantoChemical (Tokyo, Japan). Quinidine and cyclosporine A werefrom Sigma Chemical Co. (MO). A monoclonal antibody for P-glycoprotein, C219, was from Signet Laboratories (MA, USA)and a secondary antibody, peroxidase-labelled affinity-purifiedantibody to mouse immunoglobulin G (H + L), was fromKirkegoard and Perry Laboratories Inc. (Gaithersburg, MD).All other chemicals used were of the highest purity available.

2.2. Culture

nduction of MDR1 may be dependent on metabolicnzyme activity of the biomaterial used in these experi-ents. It was suggested that the metabolites of Bap, but

ot Bap itself, were involved with the enhanced tran-criptional level of MDR1 (Mathieu et al., 2001). Withespect to human intestine, it was indicated that expo-ure to Bap caused induction of MDR1 in Caco-2 cells, auman colonic adenocarcinoma cell line (Lampen et al.,004). Caco-2 cells appear to retain the activity of Bap-etabolizing enzymes. However, only the induction ofDR1 transcription was demonstrated in Caco-2 cells

xposed to Bap. The influence of Bap on the expressionf P-gp encoded by the MDR1 gene, and P-gp-mediatedrug transport have not been reported until now in Caco-cells.Possible mechanisms influencing P-gp function

nclude a change in transcriptional level of MDR1 orhe direct interaction with P-gp (Abolhoda et al., 1999;eick et al., 2001; Niemi et al., 2003; Mitsunaga et al.,000; Boumendjel et al., 2002). For example, antibioticgents such as adriamycin and rifampin are well knowno induce P-gp expression (Abolhoda et al., 1999; Geick

Caco-2 cells were obtained in passage 40 from theRIKEN (NO. RCB0988) and used for the experimentsin passages 43–63. The cell line was cultured in Dul-becco’s modified Eagle’s medium supplemented with 20%foetal bovine serum, 1% non-essential amino acids, andpenicillin–streptomycin–amphotericin B (Cambrex Bio Sci-ence Walkersville Inc., MD) in an atmosphere of 5% CO2–95%air at 37 ◦C. Cells were seeded at a density of 2 × 105 cells/cm2

on collagen-coated polycarbonate petri dishes (60 cm2) andtissue culture-treated Transwell inserts (4.7 cm2 growth areaand 0.4 �m mean pore size polycarbonate membranes; Corn-ing Costar Co., Cambridge, MA). Cells were incubated for16–18 days after seeding and used for experiments.

2.3. Transepithelial transport across Caco-2 cellmonolayers

Transepithelial electric resistance (TEER) of Caco-2 cellmonolayers cultured in a Transwell chamber was monitoredbefore transport studies using a Millicell ERS testing device(Millipore, Bedford, MA). The monolayers with TEER of morethan 250 � cm2 were used for transport studies. The monolay-ers were treated by the addition of Bap dissolved in dimethylsu-foxide (DMSO) to the medium. The control monolayers were

158 N. Sugihara et al. / Toxicology 223 (2006) 156–165

treated with DMSO. Rho-123 was dissolved at a concentrationof 5 �M in Dulbecco’s phosphate-buffered saline containing25 mM HEPES and 25 mM glucose. Rho-123 was placed eitheron the apical (1.5 ml) or basolateral (2.6 ml) side and measuredon the opposite side to the placed site at 37 ◦C periodicallyfor 60 min. The transepithelial transport of Rho-123 acrosscell monolayers treated with or without Bap for 72 h beforeexperiments was determined fluorometrically with Hitachi flu-orescence spectrophotometer F-3000 (Tokyo, Japan). In theexperiment under the co-presence of Bap, Bap was added tonon-treated monolayers cultured in a Transwell chamber afterreplacing the HBSS buffer and kept in the medium during theexperiment.

The apparent permeability coefficient (Papp) values werecalculated using the equation: Papp = (dQ/dt)/(A × C0), wheredQ/dt is the Rho-123 permeation rate (mol/s), A is the filter/cellsurface area (4.7 cm2), and C0 is the initial concentration ofRho-123 (mol/ml) (Tang et al., 2004).

2.4. RT-PCR for MDR

mRNA was prepared from Bap-treated and control Caco-2cells using a QuickPrep micro mRNA Purification Kit (Amer-sham Biosciences, UK) as described by the manufacturer. RNAconcentration was determined with a UV–vis spectrophotome-ter. Reverse transcription of 10 ng RNA using oligo (dT)15 wasperformed for 30 min at 45 ◦C with one unit AMV reversetranscriptase XL (TaKaRa RNA PCR Kit AMV Ver.3.0) in50 mM Tris buffer, pH 7.4, containing 75 mM KCl, 3 mMMgCl2, 0.2 mM each of dATP, dGTP, dCTP and dTTP. Thepolymerase chain reaction was performed on 10 �l of the pre-pared cDNA. Primers for human MDR1 were nt 1740–1759

Caco-2 cells collected were homogenized in a buffer con-taining 250 mM sucrose, 5 mM Tris–HCl (pH 7.4) and pro-tease inhibitors (1 �g/ml pepstatin A, 1 �g/ml leupeptin and1 tablet/10 ml Complete Mini; Roche Diagnostics, IN). Thehomogenates were then centrifuged at 54,000 rpm for 1 hafter 1000 × g centrifugation and the supernatant was usedfor further analysis. The crude membrane (40 �g protein)was loaded onto 7.5% acrylamide–bisacrylamide gels with-out prior heating. The protein was transferred electophoret-ically onto a polyvinylidene difluoride membrane (0.45 mmpore size; Bio-Rad Laboratories, CA), and incubated with amonoclonal antibody C219 (10 �g/mL). A peroxidase-labeledaffinity-purified antibody to mouse IgG was used as the sec-ondary antibody. Detection was made according to an enhancedchemiluminescence technique (ECL Western Blotting Detec-tion System; Amersham Pharmacai Biotech, UK). The blotswere exposed to Hyperfilm ECL (Amersham PharmaciaBiotech).

2.6. Statistical analysis

The data in figures are given as the mean ± S.E.M. of fourexperiments. Differences among mean values were assessedby Dunnett’s test using Stat-100 (BIOSOFT, UK) or Student’st test. A p value of <0.05 was considered significant.

3. Results

3.1. Transepithelial transports of Rho-123 andRho-B in Caco-2 cells treated with and without Bap

for sense and nt 1939–1958 for antisense (Genbank acces-sion no. M14758). Primers for human �-GAPDH were nt246–264 for sense and nt 674–693 for antisense (Genbankaccession no. M33197). PCR was performed with 0.5 unitsof Taq polymerase (TakaRa Ex Taq HS) by adding 50 �l ofa PCR master mixture containing a PCR buffer, MgCl2 (toa final concentration of 1.5 mM), and 0.2 �M of each primerto the cDNA samples. Mixtures were incubated in an auto-matic DNA thermal cycler (PC812, ASTEC) for 30 cyclesunder the following condition: 30 s at 94 ◦C, 30 s at 60 ◦C,and 50 s at 72 ◦C. Under these conditions, all cDNA fragmentamplifications were found to produce single products within alinear range. Aliquots (10 �l) of RT-PCR products were thensubjected to electrophoresis in a 2% agarose gel. RT-PCRfragments were visualized by staining the gel with ethidiumbromide and were analyzed by Bio Image Intelligent Quantifier(GENOMIC SOLUTIONSTM).

2.5. Western blot analysis

The concentration of P-gp in the crude membrane frac-tion of Caco-2 cells was determined by a Western blot per-formed using the procedure modified from that of Takano etal. (Huang et al., 2000). Caco-2 cells were harvested afterwashing out with ice-cold phosphate buffered saline. The

The transport of Rho-123 in the basolateral-to-apicaldirection (B → A) was much higher than that in theapical-to-basolateral direction (A → B), as shownin Fig. 1. The Papp ratio (Papp B to A/Papp A to B) ofRho-123 flux measured in the B → A direction versusthe flux in the A → B direction was 3.19, whichsuggests the involvement of an efflux transporter forRho-123 (Table 1). In Bap-treated Caco-2 monolyers,the transport of Rho-123 in the B → A direction was

Table 1The apparent permeability coefficient (Papp) of rhodamine-123 acrossCaco-2 cell monolayers treated with or without BaP

Papp 10−6 (cm/s)

B to A A to B Ratio(Papp B to A/Papp A to B)

Control 4.13 ± 0.50 1.29 ± 0.07 3.19BaP 10 �M 4.87 ± 0.15 1.14 ± 0.09 4.25BaP 50 �M 7.73 ± 0.78* 1.05 ± 0.09 7.35

Papp entries are mean ± S.E.M (n = 4).* Significantly different from that of control.


Fig. 1. Transepithelial transport of rhodamine-123 in the basolateral (B)-to-apical (A) direction (1) and in the apical (A)-to-basolateral (B) direction(2) across Caco-2 cell monolayers. The monolayers were treated with BaP 10 �M (�); 50 �M (�) or dimethyl sulfoxide (©, control) for 72 h. Beforethe transport experiments, the medium was replaced by an HBSS buffer. The concentration of rhodamine-123 was 5 �M. Each volume representsthe mean ± S.E.M. of five trials. *Significantly different from that of control.

enhanced compared with that in control cells, althoughtransports of Rho-123 in the A → B direction showedno significant difference between cells with and withouttreatment of Bap. As a result, the Papp B to A/Papp A to Bratios of Rho-123 were 4.25 and 7.35 in cells treated atconcentrations of 10 �M or 50 �M Bap, respectively.

The efflux of Rho-123 from Caco-2 cells was facilitatedby the Bap treatment.

Rho-B was not found to be a substrate for the trans-porter in control cells (Fig. 2). Further, the Bap treatmentdid not induce an increase in the Papp B to A/Papp A to Bratio in Rho-B transport (Table 2).

Fig. 2. Transepithelial transport of rhodamine-B in the basolateral (B)-to-apical (A) direction (1) and in the apical (A)-to-basolateral (B) direction(2) across Caco-2 cell monolayers. The monolayers were treated with BaP 50 �M (�) or dimethyl sulfoxide (©, control) for 72 h. The concentrationof rhodamine-B was 5 �M. Other experimental conditions were the same as in Fig. 1. Each volume represents the mean ± S.E.M. of five trials.


Table 2The apparent permeability coefficient (Papp) of rhodamine-B acrossCaco-2 cell monolayers treated with or without BaP

Papp 10−4 (cm/s)


Control 1.43 ± 0.20 1.47 ± 0.09 0.97BaP 1.54 ± 0.20 1.43 ± 0.06 1.09

Papp entries are mean ± S.E.M (n = 5).

3.2. Effects of the P-gp inhibitor on transepithelialtransport of Rho-123 across Caco-2 cell monolayers

The effects of quinidine and cyclosporine A ontransepithelial trasport, which are well-known inhibitorsof P-gp-mediated transport, were investigated in controland Bap-treated Caco-2 cells (Figs. 3 and 4). Transportof Rho-123 in the B → A direction across the mono-layer in control Caco-2 cells was decreased by these P-gpinhibitors. The transport of Rho-123 in the A → B direc-tion tended to increase, although it was not significantlydifferent. The Papp B to A/Papp A to B ratios were 1.21 and1.28 in Caco-2 cells with the quinidine and cyclosporineA treatments, respectively. The efflux activity of Rho-123 in Caco-2 exposed to Bap was decreased markedlyand reached the same level as that of control cells byusing these P-gp inhibitors. In Caco-2 cells exposed

Table 3The apparent permeability coefficient (Papp) of rhodamine-123 acrossCaco-2 cell monolayer treated with or without BaP in the absence orpresence of quinidine or cyclosporin A

Papp 10−6 (cm/s)


Control 4.13 ± 0.50 1.29 ± 0.07 3.19Control + quinidine 1.86 ± 0.26* 1.54 ± 0.22 1.21Control + cyclosporin A 1.48 ± 0.09* 1.15 ± 0.08 1.28BaP 7.73 ± 0.78 1.05 ± 0.09 7.35BaP + quinidine 1.87 ± 0.11* 1.23 ± 0.14 1.59BaP + cyclosporin A 1.69 ± 0.19* 1.16 ± 0.30 1.18

Papp was determined in the absence or presence of quinidine (100 �M)and cyclosporin A (5 �M). Papp entries are mean ± S.E.M. (n = 4).

* Significantly different from that of control or BaP-treated mono-layer without inhibitors at a level of P < 0.05.

to Bap, the Papp B to A/Papp A to B ratios were 1.59 and1.18 with the quinidine and cyclosporine A treatments,respectively (Table 3).

3.3. Transepithelial transport of Rho-123 acrossCaco-2 cell monolayers in the presence of Bap

It was investigated whether Bap enhanced the effluxtransport of Rho-123 through a direct mechanism. Thetransport of Rho-123 was not influenced by the presence

lateral (ubated wl; (�) corom tha

Fig. 3. Effect of quinidine on transport of rhodamine-123 in the basodirection (2) across Caco-2 cell monolayers. The monolayers were incOther experimental conditions were the same as in Fig. 1. (©) controrepresents the mean ± S.E.M. of four trials. *,+Significantly different frespectively.

B)-to-apical (A) direction (1) and in the apical (A)-to-basolateral (B)ith Rho-123 (5 �M) in the absence or presence of quinidine 100 �M.ntrol with quinidine; (�) BaP; (�) BaP with quinidine. Each volumet of control or the BaP-treated monolayer in the absence of quinidine,


Fig. 4. Effect of cyclosporine A on transport of rhodamine-123 in the basolateral (B)-to-apical (A) direction (1) and in the apical (A)-to-basolateral(B) direction (2) across Caco-2 cell monolayers. The monolayers were incubated with Rho-123 (5 �M) in the absence or presence of cyclosporin A5 �M. Other experimental conditions were the same as in Fig. 1. (©) Control; (�) control with cyclosporin A; (�) BaP; (�) BaP with cyclosporinA. Each volume represents the mean ± S.E.M. of four trials. *,+Significantly different from that of control or BaP-treated monolayer in the absenceof cyclosporin A, respectively.

of Bap, compared with that in control cells treated withDMSO (Fig. 5 and Table 4).

3.4. mRNA expression of P-gp in Caco-2 cellexposed to Bap

RT-PCR was performed to determine the relativeexpression of mRNA for the MDR1 gene in Caco-2 cells

Table 4The apparent permeability coefficient (Papp) of rhodamine-123 acrossCaco-2 cell monolayer in the presence and absence of BaP

Papp 10−6 (cm/s)

B to A A to B Ratio (Papp B to A/Papp A to B)

Control 4.06 ± 0.64 1.33 ± 0.14 3.05BaP 4.19 ± 0.56 1.18 ± 0.18 3.55

Papp entries are mean ± S.E.M (n = 3).

(Fig. 6). A house keeping gene, GAPDH, was used as aninternal standard. The expression of GAPDH was of asimilar magnitude in control and in Bap-treated cells.The expression of MDR1 mRNA tended to increasein a dose-dependent manner for Bap. As compared tothat of control cells, a significant increase in expres-sion of MDR1 mRNA was recognized only in Caco-2cells exposed to 50 �M Bap for 72 h under the exper-imental conditions used in this study (Table 5). How-ever, the induction of MDR1 mRNA expression was notobserved in cells exposed to 50 �M Bap for 24 h (data notshown).

3.5. Immunoblot analysis of P-gp in Caco-2 cellsexposed to Bap

A crude membrane fraction which was prepared fromCaco-2 cells exposed to Bap was used to investigateP-gp expression by Western blot analysis. As shown

Table 5Analysis of MDR1 (P-gp) mRNA expression in Caco-2 cells treated with BaP for 72 h

Control 10 �M BaP 20 �M BaP 50 �M BaP

MDR1/GAPDH 0.63 ± 0.06 0.74 ± 0.16 0.92 ± 0.10 1.08 ± 0.135*

BaP/control 1.20 1.45 1.71

Caco-2 cells were treated with BaP for 72 h. RT-PCR signal intensity was normalized against the GAPDH gene.* Significantly different from that of the control at a level of P < 0.05.


Fig. 5. Transepithelial transport of rhodamine-123 across Caco-2 cell monolayers in the basolateral (B)-to- apical (A) direction (1) and in theapical (A)-to-basolateral (B) direction (2) in the presence and absence of Bap. (©) control; (�) the monolayers in the presence of BaP 50 �M.Bap was added to non-treated monolayers cultured in a transwell chamber after replacing the HBSS buffer and being kept in medium during theexperiment. The concentration of rhodamine-123 was 5 �M. Other experimental conditions were the same as in Fig. 1. Each volume represents themean ± S.E.M. of three trials.

in Fig. 7, a C219 antibody-reactive band of 170 kDacorresponding to P-gp was markedly overexpressedin Bap-treated Caco-2 cells when compared to theiruntreated counterparts.

Fig. 6. Effect of BaP on mRNA expression of MDR1 (P-gp) in Caco-2cells mRNA from Caco-2 cells treated with BaP for 72 h was sub-jected to reverse-transcription (PCR) using primer pairs specific forthe MDR1 gene. The reverse-transciption (PCR) was analyzed by3% Nusieve3:1Agarose (TaKaRa) gel electrophoresis and stained with

4. Discussion

A significant impact on drug disposition caused bychanges in P-gp has been reported for many drugs interms of membrane transport with involvement of P-gp (Terao et al., 1996; Fromm, 2002; Fromm, 2003).However, it has not been established whether Bap, awidespread environmental pollutant, influences drug dis-position related to P-gp in the gastrointestinal epithe-lium. Using the Caco-2 cell line as a model for the smallintestine, we showed that exposure to Bap might influ-ence drug disposition through the induction of P-gp.

Rhodamine-123 was selected as a substrate for P-gpin this experiment. Substrates for P-gp are frequentlymetabolized by CYP3A because substrate recognitionoverlaps between them (Takano et al., 1998; Niemi etal., 2003). However, the Rho-123 used in the presentstudy is a substrate of P-gp, but not CYP3A. ThePapp B to A/Papp A to B ratio of Rho-123 was increased ina Caco-2 monolayer exposed to Bap at 50 �M for 72 h(Fig. 1). There was no difference in the efflux transportactivity between control and Bap-exposed monolayersin the experiment with Rho-B, which is not a substrateof P-gp (Kajikawa et al., 1999) (Fig. 2). Hence, theseresults indicated that the enhanced transport of Rho-123 in the basolateral (B) to apical (A) direction acrossthe Caco-2 monolayer was due to the increase in theefflux transport mediated by P-gp, but not the leakage ofRho-123 due to the damage of the monolayers causedby Bap exposure. The involvement of P-gp in enhanced
ethidium bromide.


Fig. 7. Western blot analysis with a monoclonal antibody for P-gp (C219) of the intestinal crude epithelial membranes prepared from Caco-2 cellswith and without BaP treatment.

Rho-123 efflux was confirmed in the experiments withquinidine and cyclosporin A, inhibitors of P-gp (Kim,2002). The quinidine and cyclosporin A inhibited P-gp-mediated transport competitively because they aresubstrates of P-gp. The enhancement of efflux trans-port of Rho-123 in Bap-exposed Caco-2 monolayersdisappeared with these inhibitors (Figs. 3 and 4). Fur-thermore, besides the decrease in the Rho-123 effluxtransport enhanced by Bap, the decrease caused by theseinhibitors in control monolayers was observed also inBap-exposed monolayers. Hence, the efflux transport inBap-exposed monolayers reached the same level as thatof control monolayers due to these inhibitors.

The expression or the transport-activity of P-gp seemsto be regulated by various substances. It was reportedthat flavonoids such as quercetin or kaempferol caused adecrease or increase in transport of vincristine at low orhigh concentrations of flavonoids due to their involve-ment in the phosphorylation to P-gp (Mitsunaga et al.,2000). The influence of P-gp-mediated transport inducedby these compounds was observed under the presence ofthese compounds, but the present study showed that thetransport of Rho-123 was not influenced by the presenceof Bap (Fig. 5). The efflux transport of Rho-123 seemednot to change immediately after the addition of Bap tothe medium. Hence, Bap does not appear to act directlyon the efflux activity of P-gp or to be a binding site com-petitor of P-gp.

Antibiotic agents such as adriamycin and rifampinare well known to induce P-gp expression and makecAiotaaPb

period after seeding (Hosoya et al., 1996; Hirsch-Ernstet al., 2001). Hence, studies about MDR1 expressionand P-gp protein levels were performed at the sameperiod after seeding as that of transport experiments witha monolayer of Caco-2 cells grown in Transwells. Anincrease in MDR1 mRNA expression coincided with theenhancement of Rho-123 transport activity in Caco-2cells exposed to Bap (Fig. 6). On the protein level, theenhanced P-gp expression was confirmed in those cellsby Western blot analysis (Fig. 7).

The daily intake of Bap in the human diet is esti-mated to range 120–2800 ng/day (Hattermer-Frey andTravis, 1991). The Bap concentration employed in thepresent study is much higher than those estimated tobe taken orally in gastrointestinal fluid. It is doubtfulwhether the phenomenon caused by Bap in Caco-2 cellsis observed in human intestine. However, the inductionof MDR1 by Bap appears to be perhaps due to metabo-lites of Bap rather than Bap itself (Mathieu et al., 2001).The activity of metabolic enzymes in Caco-2 cells wasreported to be lower than those found in human intestine(Prueksaritanont et al., 1996). Therefore, human expo-sure to reactive Bap metabolites may occur over longperiods of time and accumulate due to exposures includ-ing cigarette smoke, environmental pollutants from theburning of fossil fuel, etc. The possibility exists thatexpression of MDR1 may be caused at much lower Bapconcentrations in human intestines than those employedin this study. The transcription of MDR1 enhanced byexposure to Bap is considered to be a logical protective

ancer cells multidrug-resistant (Abolhoda et al., 1999;rora et al., 2005; Geick et al., 2001). It was proposed

n the present study that the enhanced efflux transportf Rho-123 caused by Bap might be due to transcrip-ional enhancement of expression of P-gp like anticancergents, considering that the influence of Bap on the effluxctivity of Rho-123 was not due to a direct effect on Pgp.-gp-expression levels in Caco-2 cells were reported toe dependent on the condition of culture and the culture

function cells possess to prevent toxin accumulation.There are reports that MDR1 was co-induced with

CYP3A4 by drugs such as rifampin (Geick et al., 2001;Niemi et al., 2003). Since it was indicated that xenobi-otic induction of CYP3A4 is associated with the nuclearreceptor PXR (pregnane X receptor), Geick et al. (2001)identified a distinct PXR binding site, a DR4 nuclearreceptor response element, that is essential for MDR1induction by rifampin using the human colon carcinoma


cell line LS174T. On the other hand, Mathieu et al. (2001)demonstrated that the transcriptional induction of MDR1was directly mediated by p53 in hepatoma cell line Hepa-1c1c7. In reference to the induction of MDR1 causedby Bap, the metabolic activation of Bap into reactivespecies appears to be involved in the trigger of p53 acti-vation associated with DNA damage. The metabolismof Bap generates reactive oxygen species, which inducethe transcriptional activity of NF-�B. There were reportsthat activation of NF-�B or transient oxidative stressinduced rat MDR1b expression (Deng et al., 2001; Felixand Barrand, 2002). It was indicated that Bap activatedthe human p53 gene through induction of NF-�B activity(Pei et al., 1999). Hence, Bap metabolites might acti-vate the p53 gene through induction of NF-�B activityin addition to p53 induction resulting from DNA dam-age. Further studies are needed to clarify the mechanisminvolved in the transcriptional enhancement of P-gp-expression induced by Bap in Caco-2 cells and humanintestine.

In conclusion, the increase of Rho-123 efflux trans-port was demonstrated in Caco-2 cells exposed to Bap.The induction of P-gp was responsible for the increasedtransport of Rho-123 in Caco-2 cells exposed to Bap.The present study suggests the possibility that the dispo-sition of drugs mediated by P-gp in efflux transport mightbe influenced by exposure to smoking or contaminatedfood containing Bap.

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effect of benzo[a]pyrene on p-glycoprotein-mediated transport in caco-2 cell monolayer

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