transepithelial transport of 4-chloro-2-methylphenoxyacetic acid (mcpa) across human intestinal...

Transepithelial Transport of 4-Chloro-2-MethylphenoxyaceticAcid (MCPA) across Human Intestinal Caco-2 Cell Monolayers

Osamu Kimura1, Kensuke Tsukagoshi2, Moriaki Hayasaka3 and Tetsuya Endo1,*

1Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan, 2Hijirigaoka Hospital, Hokkaido, Japan and3Sapporo Higashi-Tokushyukai Hospital, Hokkaido, Japan

(Received 12 October 2011; Accepted 5 December 2011)

Abstract: Mechanisms of transcellular transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) across the small intestine wereinvestigated using Caco-2 cells cultured on permeable membranes. The cell monolayers were incubated with MCPA, either fromapical side at pH 6.0 or 7.4, or basolateral side at pH 7.4. The accumulation and apical-to-basolateral transport of MCPA weremarkedly stimulated by the acidic pH on the apical side (inwardly directed H+ gradient), dependent on metabolic energy andinhibited by co-incubation with acetic acid or benzoic acid. Without the H+ gradient, on the other hand, the basolateral-to-apicaltransport of MCPA (secretory transport) was higher than the apical-to-basolateral transport (absorptive transport), although thesecretory transport of MCPA was markedly lower than the absorptive transport under the H+ gradient. Co-incubation of MCPAwith probenecid from the basolateral side significantly inhibited the accumulation and transport of MCPA, whereas co-incubationwith p-aminohippuric acid did not. These results suggest that the absorptive transport of MCPA is mediated by H+-linked mono-carboxylic acid transporters expressed on the apical membranes, while secretory transport is mediated by a probenecid-sensitivetransporter expressed on the basolateral membranes of Caco-2 cell monolayers.

Phenoxyacetic acid herbicides, such as 4-chloro-2-methylphen-oxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid(2,4-D), are extensively used for the control of broad-leavedweeds. Human exposure to phenoxyacetic acid herbicides fre-quently occurs from occupational use and ingestion of contami-nated food. In general, phenoxyacetic acid herbicides arereadily absorbed after oral administration and predominantlyeliminated by the kidney [1–3]. Recently, we investigated theabsorptive mechanisms of phenoxyacetic acid herbicides, suchas MCPA, 2,4-D, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)and 3,5,6-trichloro-2-pyridinyloxyacetic acid, using humanintestinal epithelial Caco-2 cells cultured on plastic dishes andsuggested that these phenoxyacetic acids were taken up fromthe apical membranes of Caco-2 cells via H+-linked monocar-boxylic acid transporters (MCTs) [4–6]. MCTs in Caco-2 cellscan be classified into two types: one type consists of L-lacticacid-sensitive MCTs, defined as MCT1, MCT3 and MCT4[7,8]; and the other consists of L-lactic acid-insensitive (benzoicacid-sensitive) MCTs distinct from MCT1 to MCT4 [4–6,9–11]. We implied previously that the phenoxyacetic acid herbi-cides are taken up mainly via L-lactic acid-insensitive MCTs,because the decreases in herbicide uptake observed on co-incu-bation with benzoic acid were significantly larger than thoseinduced by co-incubation with L-lactic acid [4–6].On the other hand, the renal secretion of 2,4-D appears to be

mediated via organic anion transporters (OAT1 and OAT3)expressed on the basolateral membranes, which is an organic

anion/dicarboxylate exchanger [12–16]. However, OATs arereported not to be expressed in Caco-2 cells or the human intes-tine [17]. The ATP-binding cassette (ABC) gene superfamily ofmembrane transporters, which includes P-glycoprotein (P-gp)and multidrug resistance-associated protein (MRP), act as anefflux pump extruding a wide variety of chemically unrelatedxenobiotics and drugs. P-gp and MRP function as an effluxpump on the apical membranes of Caco-2 as well as other cells.In general, MRP tends to have high affinity for anionic com-pounds, whereas P-gp transports neutral or cationic compounds[16,18]. Despite many studies on ABC transporters, the secre-tion of phenoxyacetic acid herbicides into the intestinal lumenvia ABC transporters has not yet been investigated.Caco-2 cells can grow not only on plastic surfaces but also

on permeable membranes, and cells grown on these mem-branes can serve as a useful tool to assess cellular uptake andtransport not only from the apical side but also from the baso-lateral side of the cell surface. In the present study, we investi-gated the uptake of MCPA from apical membranes and theapical-to-basolateral transport of MCPA using Caco-2 cellmonolayers cultured on permeable membranes, and confirmedthe apical-to-basolateral transport (absorption) of MCPA viaH+-linked MCTs. Furthermore, we investigated the secretory(basolateral-to-apical) transport of MCPA via OATs, ABC andother types of transporters.

Materials and Methods

Materials. 4-Chloro-2-methylphenoxyacetic acid, L-lactic acid, benzoicacid, acetic acid, succinic acid, p-aminohippuric acid (PAH), verapamilhydrochloride, cyclosporin A, indomethacin, vinblastine sulphate, pro-benecid, glutarate, choline chloride, sodium azide (NaN3) and Dul-becco’s modified Eagle’s medium (DMEM) were purchased from Wako

*Author for correspondence: Tetsuya Endo, Faculty of PharmaceuticalSciences, Health Sciences University of Hokkaido, 1757 Kanazawa,Ishikari-Tobetsu, Hokkaido 061-0293, Japan (fax + 81 133 23 3902,e-mail [email protected]).

© 2011 The AuthorsBasic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society

Basic & Clinical Pharmacology & Toxicology, 2012, 110, 530–536 Doi: 10.1111/j.1742-7843.2011.00850.x

Pure Chemical Industries, Ltd. (Osaka, Japan). a-Cyano-4-hydroxycin-namate (CHC), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS),sulfobromophthalein (BSP) and carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP) were purchased from SigmaChemical Co. (St. Louis, MO, USA). MK571 was obtained from BIO-MOL Research Laboratories, Inc. (Plymouth Meeting, PA, USA). Foetalbovine serum (FBS) and non-essential amino acid (NEAA) wereobtained from Life Technologies (Carlsbad, CA, USA). 4-(2-Hydroxy-ethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-(4-morpholino)ethanesulfonic acid (MES) were purchased from Dojindo Laboratories(Kumamoto, Japan). All other chemicals used were of the highest puritycommercially available. MCPA and some compounds were dissolved inmethanol and added to the incubation medium at a final concentrationof methanol of 1% or lower [4].

Cell culture. Caco-2 cells at passage 40 were obtained from RI-KEN Cell Bank (Tukuba, Japan), and the cells between passage 50and 72 were used in this study. As described previously [5,19], thecells were cultured on permeable membranes (Cell Culture Insert,0.4 lm, 4.2 cm2 growth area; Becton Dickinson, Bedford, MA,USA) in DMEM containing FBS (10%), NEAA (1%), streptomycin(100 lg/mL) and penicillin G (70 lg/mL) at 37°C in a humidifiedatmosphere of 5% CO2–95% air. Each insert chamber was placedin a companion plate (6 wells; Becton Dickinson) with 2.5 mL ofthe basolateral medium and 1.5 mL of the apical medium. The cul-ture medium was replaced every 2 or 3 days after seeding. Conflu-ent culture of Caco-2 cell monolayers, cultured for 21 or 22 dayswith a transepithelial electrical resistance of more than 350 Ω cm2,was used for the experiments.

Accumulation and transport studies. The accumulation and the apical-to-basolateral or basolateral-to-apical transport of MCPA using Caco-2cell monolayers grown on the permeable membrane were studiedunder protein- and amino acid-free conditions [5,19]. The culture med-ium was removed, and the cell monolayers were washed twice withincubation medium (pH 7.4) warmed to 37°C. After washing, the cellmonolayers were pre-incubated for 10 min. at 37°C with 1.5 and 2.2mL of the incubation media (pH 7.4) from the apical and basolateralsides, respectively. After pre-incubation, the cell monolayers wereincubated with 50 lM MCPA for the designated time either from theapical side at pH 6.0 or 7.4, or from the basolateral side at pH 7.4,and the cellular accumulation of MCPA from the medium on one sideand the transport of MCPA to the medium on the other side weremeasured. Hanks’ balanced salt solution (HBSS) containing 25 mM D-glucose and 10 mM HEPES (pH 7.4) or 10 mM MES (pH 6.0)instead of HEPES was used as the incubation medium [5,19].In the metabolic inhibition experiments on the accumulation and

transport of MCPA from the apical side, the cell monolayers werepre-incubated with 10 mM NaN3 or 25 lM FCCP in the incubationmedia of the apical and basolateral sides (pH 7.4) at 37°C for 20min., before the incubation with MCPA. To examine the effect ofinhibitors for MCTs, the cell monolayers were co-incubated with 50lM MCPA and 10 mM L-lactic acid, acetic acid, benzoic acid, CHCor succinic acid for 5 min. from the apical side at pH 6.0.To examine the effect of inhibitors for P-gp and MRP on the accumu-

lation and transport of MCPA from the basolateral side, the cells werepre-incubated with 200 lM verapamil, 20 lM cyclosporine A, 200 lMvinblastine, 200 lM indomethacin, 50 lM MK-571, 5 mM probenecidor 5 mM PAH at 37°C for 30 min. from the apical and basolateral sides(pH 7.4). After pre-incubation, the media were aspirated and the cellularsurface and the permeable membrane were washed once with HBSS,and the cell monolayers were co-incubated with 50 lM MCPA and thesame chemical was used for pre-incubation from the basolateral side ofmonolayers at 37°C for 60 min.

To examine the effects of inhibitors for organic anion or cationtransporters on the accumulation of MCPA from basolateral side, thecell monolayers were co-incubated with 50 lM MCPA and 5 mMprobenecid, 10 mM PAH, glutarate or choline chloride, and 1 mMBSP or DIDS for 5 min. from the basolateral side (pH 7.4).After incubation with MCPA, the incubation media were removed

and the monolayers were quickly washed three times with ice-cold incu-bation medium. The membrane filters were detached from the insertwell, and the cells were then extracted with 1.0 mL of extraction solution(1 N H3PO4/methanol = 1:1) for 60 min. at room temperature. The sus-pension was centrifuged at 13,000 9 g for 10 min., and a 100-lL aliquotof the supernatant was injected onto the HPLC system [4].

Determination of MCPA and protein. Determination of MCPA wascarried out by HPLC as described previously [4]. Protein concentra-tion was determined by the method of Bradford [20] using a Bio-raddye reagent (Richmond, CA, USA) with bovine serum albumin as thestandard.

Statistical analyses. These data were analysed by either Student’s t-test or Scheffe’s multiple comparison test after the analysis of varianceusing the Statcel 2 program, and differences with values of p < 0.05were considered to be significant. Data are shown as mean ± S.E.

Results

Effect of the apical side pH on the accumulation andtranscellular transport of MCPA from apical membranesTo investigate the effect of the apical side pH on the accumu-lation and apical-to-basolateral transport of MCPA, Caco-2cell monolayers were incubated with 50 lM MCPA from theapical side at pH 6.0 or 7.4 and 37°C for up to 60 min. Theaccumulation and transcellular transport of MCPA to the baso-lateral side at pH 7.4 are shown in fig. 1. The initial accumu-lation of MCPA from the apical side at pH 6.0 was rapid andreached a maximum level at 5 min., thereafter graduallydecreasing up to 60 min. The MCPA accumulation at 5 and60 min. was 3.94 ± 0.25 and 1.40 ± 0.09 nmol/mg protein,respectively. In contrast, the MCPA accumulation from theapical side at pH 7.4 reached a plateau at 5 min. (0.86 ± 0.02nmol/mg protein): The MCPA accumulation at pH 7.4 up to30 min. was markedly lower than that at pH 6.0, but the accu-mulation at 60 min. was comparable. On the other hand, theapical-to-basolateral transport of MCPA from the apical sideat pH 6.0 and 7.4 was found to increase with time. However,the MCPA transport at 60 min. from the apical side at pH 6.0(5.20 ± 0.30 nmol/cm2) was more than 4 times larger than thatat pH 7.4 (1.25 ± 0.06 nmol/cm2).To confirm the gradual decrease in MCPA accumulation

from 5 min. after the onset of incubation with 50 lM MCPAand the time-dependent increase in MCPA transport (fig. 1),Caco-2 cell monolayers were incubated with different concen-trations of MCPA (25 and 100 lM) from apical side at pH6.0 for up to 60 min. (fig. 2). The changes in accumulationand transport of MCPA after incubation with 25 and 100 lMMCPA were similar to those with 50 lM MCPA, respectively,and the dose-dependent accumulation and transport of MCPA(25, 50, and 100 lM) were observed.


TRANSEPITHELIAL TRANSPORT OF 4-CHLORO-2-METHYLPHENOXYACETIC ACID 531

Effects of low temperature and metabolic inhibitors on theaccumulation and transcellular transport of MCPA fromapical membranesEffects of low temperature and metabolic inhibitors on theaccumulation and the apical-to-basolateral transport of MCPAfrom apical side at pH 6.0 and 5 min. were investigated (table1). Incubation at low temperature (4°C) significantly reducedthe accumulation and transcellular transport of MCPA.Pre-treatment with NaN3 (metabolic inhibitor) or FCCP(protonophore) also significantly decreased the accumulationand transcellular transport of MCPA. The order of MCPAaccumulation (4°C < NaN3 < FCCP) was the reverse of thatfor MCPA transport (FCCP < NaN3 < 4°C).

Effects of carboxylic acids on the accumulation andtranscellular transport of MCPA from apical membranesTo investigate whether the apical-to-basolateral transport ofMCPA is mediated via H+-linked MCTs, Caco-2 cell mono-

layers were co-incubated with 50 lM MCPA and carboxylicacid compound for 5 min. from the apical side at pH 6.0 (fig.3). Co-incubation of MCPA with acetic acid or benzoic acid(a substrate of some MCT isoforms) from the apical side sig-nificantly decreased the accumulation and transcellular trans-port of MCPA. Co-incubation with L-lactic acid slightly butsignificantly decreased the accumulation and transport ofMCPA. However, co-incubation with CHC (an inhibitor of L-lactic acid-sensitive MCTs) or succinic acid (a dicarboxylicacid) did not decrease the accumulation or transport ofMCPA.

Comparison of the accumulation and transcellular transportof MCPA from basolateral and apical membranesThe accumulation and transcellular transport of MCPA fromthe basolateral side at pH 7.4 were compared with those fromthe apical side at pH 7.4 (fig. 4). The accumulation of MCPAfrom the basolateral side reached a plateau level at 10 min.

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Fig. 1. Time courses of 4-chloro-2-methylphenoxyacetic acid (MCPA)accumulation and transcellular transport from the apical side under aH+-gradient. Caco-2 cells were incubated at 37°C with 50 lM MCPAadded to the apical medium at pH 6.0 (●) or 7.4 (○). The pH of thebasolateral medium was 7.4. Each value represents mean with S.E. forthree to six monolayers.

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Fig. 2. Dose dependency of 4-chloro-2-methylphenoxyacetic acid(MCPA) accumulation and transcellular transport from the apical sideunder a H+ gradient. Caco-2 cells were incubated at 37°C with 25(▲), 50 (●) or 100 lM (■) MCPA added to the apical medium atpH 6.0. The pH of the basolateral medium was 7.4. Each value repre-sents mean with S.E. for four monolayers.


532 OSAMU KIMURA ET AL.

This plateau level was about half of that from the apical sideat pH 7.4 (without H+ gradient). In contrast, the transcellulartransport of MCPA from the basolateral side increased withtime and was markedly greater than the transport from the api-cal side at pH 7.4. A decrease in the pH of apical side frompH 7.4 to 6.0 did not alter the accumulation of MCPA fromthe basolateral side at pH 7.4, while it marginally decreasedthe transport of MCPA.

Effects of several compounds on the accumulation andtranscellular transport of MCPA in Caco-2 cell monolayersAs the secretory transport of MCPA was greater than theabsorptive transport of MCPA under non-H+-gradient condi-tions (fig. 4), the secretory transport of MCPA in the presenceof P-gp and MRP inhibitors was investigated under non-H+-gradient conditions (the pH of both the apical and basolateralside was 7.4). Treatment with verapamil (200 lM), cyclosporineA (20 lM) or vinblastine (200 lM), inhibitors of P-gp-mediatedtransport, slightly but significantly decreased the basolateral-to-apical transport of MCPA at 60 min. by 16%, 15% and17%, respectively (fig. 5). In contrast, treatment with MK-571(50 lM) or indomethacin (200 lM), inhibitors of MRP-mediated transport, had no inhibitory effect on MCPAtransport. None of the inhibitors tested had any effect on theMCPA accumulation from the basolateral side. Treatment withprobenecid, a non-specific inhibitor of anion transport, signifi-cantly inhibited the accumulation and transport of MCPA fromthe basolateral side by 40% and 31%, respectively, whileco-incubation with PAH, a typical substrate of OATs, did not.

Effects of several compounds on the initial accumulation ofMCPA from basolateral membranesTo characterize the basolateral-to-apical transport of MCPA indetail, the initial accumulation of MCPA across the basolateralmembranes of Caco-2 cell monolayers was investigated.Co-incubation with probenecid significantly inhibited the accu-mulation of MCPA by 37% (fig. 6). In contrast, PAH andglutarate (typical substrates of OATs), BSP (a substrate of the

organic anion transporting polypeptide family, OATPs), DIDS(an inhibitor of anion exchangers) and choline (an inhibitor oforganic cation transport systems, OCTs) did not affect theaccumulation of MCPA from the basolateral membranes. Asdata not shown, co-incubation with probenecid significantlydecreased the transport of MCPA, while co-incubation withthe other compounds did not.

Discussion

The inwardly directed H+ gradient drastically increased the initialaccumulation from the apical membranes as well as theapical-to-basolateral transport of MCPA compared with the

Table 1.Effects of temperature and metabolic inhibitors on the accumulationand transport of 4-chloro-2-methylphenoxyacetic acid (MCPA) inCaco-2 cells.

CompoundConcentration

(mM)Accumulation(% of control)

Transport(% of control)

Control 100 ± 3.8 100 ± 4.64°C 61.9 ± 1.0* 4.5 ± 0.5*NaN3

1 10 44.2 ± 4.2* 58.4 ± 6.5*FCCP1 0.025 22.1 ± 1.0* 83.4 ± 4.9*

Caco-2 cells were incubated at 37°C (control) or 4°C for 5 min. with50 lM MCPA on the apical side at pH 6.0.1Caco-2 cell monolayers were pre-incubated with sodium azide(NaN3) or carbonylcyanide p-trifluoromethoxyphenylhydrazone(FCCP) for 20 min., and then Caco-2 cells were incubated with 50lM MCPA on the apical side at pH 6.0. Each value represents meanwith S.E. for four to six monolayers.*Significantly different from the control.

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Fig. 3. Effects of carboxylic acids on the 4-chloro-2-methylphenoxy-acetic acid (MCPA) accumulation and transcellular transport from theapical side under a H+ gradient. Caco-2 cells were incubated at 37°Cwith 50 lM MCPA added to the apical medium at pH 6.0 for 5 min.in the presence or absence of carboxylic acids. Each value representsmean with S.E. for four to six monolayers. *Significantly differentfrom the control.



accumulation and transport without a H+ gradient (fig. 1).Incubation at low temperature and pre-treatment with NaN3 orFCCP significantly decreased the accumulation and transportof MCPA from the apical side (table 1). Co-incubation withacetic acid or benzoic acid markedly inhibited the accumula-tion and the transport of MCPA from the apical side, whilethat with succinic acid did not (fig. 3). Co-incubation withL-lactic acid (a substrate of MCT1–MCT4) marginallydecreased the accumulation and transport of MCPA from theapical side, while that with CHC (a non-specific inhibitor ofMCT1, MCT2 and MCT4) did not (fig. 3). These results forMCPA using Caco-2 cells cultured on permeable membranesare in agreement with our previous results using Caco-2 cellscultured on plastic surfaces [4], which suggested that theuptake of MCPA from the apical membranes of Caco-2 cellsis mainly mediated via MCTs distinct from L-lactic acid-sensi-tive MCTs.

The accumulation of MCPA at 5 min. from the apical side at50 lM and pH 6.0 was drastically greater than that at pH 7.4,while the accumulation at 60 min. was comparable (fig. 1). It isworthy of note that the accumulation of MCPA from the apicalmembranes at 50 lM gradually decreased after the initial rapidaccumulation observed at 5 min. The gradual decrease inMCPA accumulation was also found for incubation at 25 and100 lM MCPA (fig. 2). These decreases in MCPA lead us tothe hypothesis that once accumulated in Caco-2 cells, theMCPA is then secreted through the apical membranes (secretarydirection) via specific mechanisms. As expected, without the H+

gradient, the basolateral-to-apical transport (secretary direction)of MCPA was larger than the apical-to-basolateral transport(absorptive direction) of MCPA (fig. 4), although the degrees ofboth transport without the H+ gradient were markedly smallerthan the apical-to-basolateral transport of MCPA under a H+

gradient (fig. 1). As discussed later, a probenecid-sensitive

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**

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Fig. 5. Effects of several compounds on the accumulation and trans-cellular transport of 4-chloro-2-methylphenoxyacetic acid (MCPA)from the basolateral side. Caco-2 cells were treated for 30 min. with200 lM verapamil, 20 lM cyclosporin A, 200 lM vinblastine, 50 lMMK-571, 200 lM indomethacin, 5 mM probenecid or PAH. The trea-ted cells were incubated at 37°C with 50 lM MCPA added to thebasolateral medium at pH 7.4 for 60 min. in the presence of the inhib-itors. Each value represents mean with S.E. for four to six monolayers.*Significantly different from the control (without treatment).

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Fig. 4. Time courses of 4-chloro-2-methylphenoxyacetic acid (MCPA)accumulation and transcellular transport from the apical or basolateralside. Caco-2 cells were incubated with 50 lM MCPA either from theapical side at pH 7.4 (○) or from the basolateral side at pH 7.4 (●)under non-H+-gradient conditions. Caco-2 cells were incubated with50 lM MCPA from the basolateral side at pH 7.4 (▲) under a H+

gradient (apical side at pH 6.0). Each value represents mean withS.E. for four to six monolayers.



transporter and P-gp are possible candidates for the possibletransporters of MCPA secretion.Co-incubation with probenecid from the basolateral side

significantly inhibited the accumulation and transport ofMCPA, while co-incubation with PAH and glutarate (typicalsubstrates of OATs), BSP (a substrate of OATPs), DIDS (aninhibitor of anion exchangers) and choline (an inhibitor ofOCTs) did not decrease MCPA accumulation or transport.Although phenoxyacetic acid herbicides such as 2,4-D arereported to be secreted via OAT1 in the kidney [14,15],OAT1 is not expressed in Caco-2 cells or the human intestine[17]. Despite many secretory studies using the kidney, littleinformation is available on the secretion of anionic com-pounds in the intestine. Naruhashi et al. [21] suggested thatthe basolateral-to-apical transport of PAH was mediated by atransporter on the basolateral membranes distinct from theOAT family as well as by a MRP2-like transporter on theapical membranes of Caco-2 cells, and probenecid inhibitedboth transporters on the basolateral and apical membranes, asthat probenecid decreased the accumulation and basolateral-to-apical transport of PAH. The probenecid-sensitive transporterpostulated by Naruhashi et al. [21] may be a candidate forthe transporter of MCPA through the basolateral membranes.Further study is necessary for clarification of the probenecid-sensitive transport of MCPA.Caco-2 cell monolayers retain P-gp on the apical mem-

branes, which functions as an energy-dependent and pH-inde-pendent efflux pump of xenobiotics and drugs [22–25].Treatment with verapamil, cyclosporine A or vinblastine, asubstrate for P-gp, slightly but significantly decreased the ba-solateral-to-apical transport of MCPA and showed no effecton the accumulation of MCPA from the basolateral side under

non-H+-gradient conditions. These results suggest that P-gphas only a small contribution to the secretory transport ofMCPA. Caco-2 cell monolayers also retain MRP2 on the api-cal membranes, where it excretes anionic compounds from theintestine and cells [16,25,26]. Wielandt et al. [27] reportedthat orally administered 2,4,5-T, a structural analogue ofMCPA, increased expression of Mrp2 mRNA levels in themouse liver. As MCPA is an organic anion, MRPs wereexpected to mediate the secretory transport of MCPA throughthe apical membranes. However, treatment with MK-571 orindomethacin, non-specific inhibitors of MRPs, had no inhibi-tory effect on the accumulation and transport of MCPA (fig.5). Beside P-gp and MRPs, Caco-2 cells retain the ABC half-transporter of breast cancer resistance protein (BCRP) [24].However, treatment with novobiocin, an inhibitor of BCRP,had no effect on the accumulation and transcellular transportof MCPA from the basolateral side of Caco-2 cells (data notshown). These results suggest that MRPs and BCRP do notcontribute to the secretory transport of MCPA.The order of the inhibitory effects of low temperature,

NaN3 and FCCP on MCPA accumulation was the reverseof that on MCPA transport (table 1). The reason for this isunclear because of the complicated transport mechanisms ofMCPA on the apical and basolateral membranes. Althoughsome isoforms (MCT4 and MCT5) are expressed on the ba-solateral membranes of human intestine [28], basolateralmembrane expression of MCTs was not assessed in Caco-2cells. The transport of MCPA through the basolateral mem-branes via those MCTs cannot be ruled out as the explana-tion for the gradual decrease in MCPA accumulation (fig.1). Further study is necessary to elucidate the transcellulartransport mechanisms of MCPA in Caco-2 cells.In summary, the present study confirmed that MCPA is taken

up from the apical membranes of Caco-2 cell monolayers viaH+-linked and L-lactic acid-insensitive MCTs. In the absence ofa H+ gradient, the secretory transport of MCPA was greater thanthe absorptive transport of MCPA, although the degree of thissecretion was markedly smaller than that of absorption in thepresence of the H+ gradient. Co-incubation with probenecidinhibited the accumulation and transport of MCPA from the ba-solateral side, while co-incubation with PAH did not. Treatmentwith P-gp inhibitors slightly but significantly decreased the ba-solateral-to-apical transport of MCPA from the basolateral side.These results suggest that probenecid-sensitive transportersexpressed on the basolateral and apical membranes, and P-gpexpressed on the apical membranes may mediate the secretorytransport of MCPA in Caco-2 cell monolayers.

References

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2 Lappin GJ, Hardwick TD, Stow R, Pigott GH, van Ravenzwaay B.Absorption, metabolism and excretion of 4-chloro-2-metylphenoxy-acetic acid (MCPA) in rat and dog. Xenobiotica 2002;32:153–63.

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Glutarate BSP

DIDS

Choline

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PA A

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Accumulation

Fig. 6. Effects of several compounds on the accumulation frombasolateral membranes. Caco-2 cells were incubated with 50 lM 4-chloro-2-methylphenoxyacetic acid (MCPA) at 37°C for 5 min. in thepresence of 5 mM probenecid, 10 mM p-aminohippuric acid (PAH),glutarate or choline, 1 mM sulfobromophthalein (BSP) or 4,4′-diisothi-ocyanostilbene-2,2′-disulfonic acid (DIDS). Each value representsmean with S.E. for four to six monolayers. *Significantly differentfrom the control.



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transepithelial transport of 4-chloro-2-methylphenoxyacetic acid (mcpa) across human intestinal...

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