Hindawi Publishing CorporationISRN ToxicologyVolume 2013 Article ID 892364 9 pageshttpdxdoiorg1011552013892364

Research ArticleCadmium Transport in a Model of Neonatal Intestinal CellsCorrelates to MRP1 and Not DMT1 or FPN1

Helena Oumlhrvik1 Eva Tydeacuten1 Per Artursson2 Agneta Oskarsson1 and Jonas Tallkvist1

1 Department of Biomedical Sciences and Veterinary Public Health Swedish University of Agricultural Sciences Box 702875007 Uppsala Sweden

2Department of Pharmacy Uppsala University Box 580 75123 Uppsala Sweden

Correspondence should be addressed to Jonas Tallkvist jonastallkvistsluse

Received 8 November 2012 Accepted 19 December 2012

Academic Editors D I Bannon G Borbely K M Erikson and G O Rankin

Copyright copy 2013 Helena Ohrvik et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Newborns have a higher gastrointestinal uptake of cadmium than adults In adults the iron transporters DMT1 and FPN1 areinvolved in the intestinal absorption of cadmium while in neonates the mechanisms for cadmium absorption are unknown Wehave investigated possible cadmium transporters in the neonatal intestine by applying a model of immature human intestinalepithelial Caco-2 cells To mimic the continuous cadmium exposure via diet in neonates cells were allowed to differentiate for7 days in medium containing 1 120583M CdCl

2 A dramatic upregulation of the MT1 gene expression followed cadmium pretreatment

indicating a high sensitivity of the immature cells to cadmium Cadmium pretreatment increased the basolateral efflux of 109Cdwithout causing any effects on the passive diffusion of mannitol or the transepithelial electrical resistanceThe augmented transportof cadmiumwas correlated to an upregulation ofMRP1 gene expression and increased activity of the efflux proteinMRP1 No effectswere observed on gene expression of the efflux proteins MRP2 and P-gp or the iron transporters DMT1 DMT1-IRE and FPN1 Inconclusion our data indicate that continuous cadmium exposure increases the absorption of the metal in immature intestinal cellsand that MRP1 is involved in the intestinal cadmium absorption in newborns

1 Introduction

Cadmium is a ubiquitous toxic metal that has no knownbiological functions but causes several adverse health effectsin humans Neonates are more sensitive to cadmium toxicitythan adults due to a higher gastrointestinal absorption [1ndash3] and the potential of the element to interfere in thedevelopment of the central nervous system [4] The mainsource for cadmium exposure in infants is cereal and soy-based infant formulas and weaning foods [3]

The intestinal absorption of cadmium in adults is gener-ally low but increases during iron deficiency [5ndash9] suggestingthat dietary cadmium is absorbed in the intestine by thesame mechanism as non-heme iron The major transportersresponsible for the absorption of nonheme iron are theapical influx divalent metal transporter 1 (DMT1) and thebasolateral efflux ferroportin 1 (FPN1) [10ndash12] DMT1 theisoform DMT1-IRE (containing an iron responsive element)and FPN1 expressions are induced in human adults during

iron deficiency and repressed under iron sufficient conditions[13 14] In addition studies in adult rats have demonstrateda strong correlation between cadmium absorption and duo-denal expressions of DMT1 and FPN1 [15 16] However innewborns the possible role of DMT1 and FPN1 in cadmiumabsorption has not been clarified It has been demonstratedthat the expression and localization of DMT1 and FPN1 induodenal enterocytes is not affected by iron status neither insuckling piglets at postnatal day 16 nor in suckling rat pupsat postnatal day 10 indicating an immature regulation of iron[17 18] Thus cadmium absorption seems to be regulated byother mechanisms at this developmental stage

Gastrointestinal cadmium absorption may also be medi-ated by proteins belonging to the ATP binding cassette(ABC) superfamily of transporters [19] Multidrug resistanceassociated protein 1 (MRP1) is a transporter involved in thebasolateral efflux of compounds out of enterocytes into thesystemic circulation [20] Cadmium upregulates MRP1 andthe protein have been demonstrated to mediate transport

2 ISRN Toxicology

of the metal in functional yeast experiments [21 22] Theefflux proteins MRP2 and P-glycoprotein (P-gp) located atthe apical membrane of enterocytes are also upregulated bycadmium [23 24] and may thus influence the bioavailabilityof the metal

Repeated cadmium treatment has previously been shownto increase cadmium absorption compared to a single treat-ment both in vivo and in intestinal in vitro preparations ofadult rats [25] However even though MT involvement hasbeen suggested the cellular mechanism for how cadmiumenhances its own absorption remains to be elucidated [25]Intestinal MT is induced by cadmium [26] but there arenumerous reports concluding that the impact of MT on theintestinal cadmium absorption is of minor importance [27]It appears that the predominant role of this cytosolic smallmolecular weight metal binding protein is to protect the cellsfrom cytotoxic effects [28]

Twenty-one-day postconfluent Caco-2 cells which arefully differentiated (mature) have been extensively used asan in vitro model of the adult human intestinal epithelium[29] Under normal cell culture conditions Caco-2 cellsspontaneously differentiate into a phenotype that resemblespolarized absorptive enterocytes [30ndash32] The Caco-2 modelhas been widely used to investigate cadmium absorption andeffects [33ndash38] Mature Caco-2 cells treated with iron havereduced uptake and transport of cadmium that correlates toiron-induced decrease in DMT1 levels [37] In concordancea reduced cadmium uptake was demonstrated in Caco-2 cellssubjected to molecular DMT1 knock down [33] The ABCefflux transporters MRP1 MRP2 and P-gp are present inCaco-2 cells [39 40] and the expression of P-gp in thesecells has been shown to be induced by long-term cadmiumtreatment [23]

The present study was undertaken to investigate possibletransporters involved in the absorption of cadmium in theneonatal intestine and the impact of cadmium pretreatmentTo mimic the continuous cadmium exposure in neonates viamilk andor formula confluent Caco-2 cells were allowed todifferentiate during 7 days (referred to as ldquoimmature Caco-2 cellsrdquo) in the presence and absence of cadmium Apical tobasolateral transport studies with cadmium were performedand gene expressions of the transporters MRP1 MRP2 P-gpDMT1 DMT1-IRE FPN1 and of MT were quantified as wellas the MRP1 activity

2 Materials and Methods

21 Chemicals Cadmium chloride and nitrilotriacetic acidwere purchased from Sigma Aldrich Dulbeccosrsquo modi-fied Eagle medium (DMEM) heat inactivated fetal calfserum nonessential amino acids for modified Eagle medium(MEM) N-2-hydroxyethyl-piperazine-N1015840-2-ethanesulfonicacid (HEPES) buffer 1M nonessential amino acids (100x)and gentamycin were purchased from Invitrogen via VWRInternational LLC Apo-transferrin (tissue culture grade)was obtained from Calbiochem via VWR InternationalLLC [14C]-mannitol (specific activity 209GBqmmol) waspurchased from PerkinElmer and 109Cd (specific activity

152 GBqmg Cd2+) from GE Healthcare Ultima Gold (scin-tillation liquid) was obtained from Chemical InstrumentsAB Calcein acetoxymethyl ester (calcein AM) fluorescentdye was purchased from BD Biosciences Hankrsquos balancedsalt solution (HBSS) and all other chemicals used but notspecified in the experiments were of analytical grade andfrom VWR International LLC

22 Cell Culture and Treatment Human intestinal Caco-2cells were obtained from American Cell Culture Collection(Rockville USA) The cells were cultured as previouslydescribed [41] with some minor modifications Briefly theCaco-2 cells were expanded in normal tissue culture flasks inDMEM supplemented with 10 fetal calf serum (Invitrogen)and 1 gentamycin (50mgmL Invitrogen) The cells wereseeded onto permeable polycarbonate filters at a density of25 times 105 cellsfilter support (Oslash 12mm pore radius 04120583mTranswell Corning) and apical medium was changed 8 hafter seeding to remove excess of cells and to reduce the risk ofmultilayer formation [42]TheCaco-2 cells were then allowedto differentiate for 7 days postconfluency The medium waschanged every second day and the cells were examined dailyby reverse phase microscopy for confluence shape of cellsand dome formations Cells of passage numbers 97ndash100 wereused in the experiments

Each cell monolayer was randomly assigned to differen-tiate for 7 days postconfluency in control (C) medium ormedium supplemented with 1 120583M cadmium (CdCl

2times 1H2O)

(Cd) Previous investigations have demonstrated that theviability of the Caco-2 cells at various stages of differentiationis not affected by 1 120583M cadmium treatment [26]

23 Assessment of Monolayer Integrity The integrity of thecontrol and cadmium pretreated monolayers was exam-ined by determining the permeability of the hydrophilicmarker 14C-mannitol mainly as described [42] Briefly cellmonolayers were rinsed with prewarmed Hankrsquos BalancedSalt Solution pH 74 (HBSS) and then incubated in HBSSat 37∘C for 20min The HBSS was discarded from theapical chambers prior to transferring the filters to a freshand prewarmed plate containing 15mL 37∘C HBSS in thebasolateral chambers HBSS (05mL pH 63) prewarmed to37∘C and containing 014 120583CimL 14C-mannitol was added tothe apical chambers The transport experiment was carriedout at 37∘C under gentle shaking (50 rpm) for 3 h Every30min the filters with the monolayers were transferred tonew wells containing 37∘C HBSS and samples from both theapical and basolateral chambers were taken for subsequentdetermination of radioactivity by 120573-spectrometry (Tricarb1900CA Packard) The integrity of the monolayers wasalso verified by measuring the net transepithelial electricalresistance (TEER) across the monolayers as described [42]Resistance across monolayers was measured in control andcadmium supplemented media (pH 74 37∘C) and back-ground resistance was measured across filters without cells

24 109Cd Transport and Uptake Cell monolayers differ-entiated for 7 days postconfluency in control medium or

ISRN Toxicology 3

Table 1 Primer sequences used for quantitative real-time PCR

Primer Oligo sequence NCBI GenBank Accession No Product size (bp)MRP1 forward 51015840-GCAAATCCAGGAGACAGCTC-31015840 NM 004996 113MRP1 reverse 51015840-TGATGTGCCTGAGAACGAAG-31015840

MRP2 forward 51015840-CTGGTTGGGAACCTGACTGT-31015840 NM 000392 172MRP2 reverse 51015840-CAACAGCCACAATGTTGGTC-31015840

P-gp forward 51015840-GCTGTTAAGGAAGCCAATGC-31015840 NM 000927 120P-gp reverse 51015840-AGCAATGGCGATTCTCTGTT-31015840

DMT1 forward 51015840-CGTGGCGGATTGCAGGAGGA-31015840 NM 001174126 124DMT1 reverse 51015840-ACGCTGACCACAGCAGCCAC-31015840

DMT1-IRE forward 51015840-GCCATCAGAGCCAGTGTGTTTCT-31015840 NM 001174125 198DMT1-IRE reverse 51015840-TGTCAGCTTTTCAAAGATCCCACC-31015840

FPN1 forward 51015840-CGAGATGGATGGGTCTCCTA-31015840 NM 014585 219FPN1 reverse 51015840-GGCTACGTCGAAAATGTGGT-31015840

MT1 forward 51015840-GCAAATGCAAAGAGTGCAAA-31015840 NM 005946 213MT1 reverse 51015840-ATGGGTCAGGGTTGTATGGA-31015840

medium supplemented with cadmium were used for uptake(from apical chamber into the cells) and transport (fromapical chamber via the cells into the basolateral chamber)experiments The experiments were performed at 37∘C pH74 in sterile filtered HBSS buffered with 25mM HEPESmainly as previously described [41] Briefly the monolayerswere first rinsed in prewarmed HBSS and then incubated inHBSS at 37∘C for 20min before being transferred to newwellswhere fresh 37∘C HBSS was added to the apical (05mL pH63) and basolateral (15mL pH 74 supplementedwith 15120583Mhuman apo-transferrin) chambers The apical solution wassupplemented with 30 nM 109Cd (1120583CimL) To determinethe transport of 109Cd across the monolayers 14mL sampleswere taken from the basolateral chambers at 30min intervalsfor 3 h and at the same time the cell monolayers weretransferred to new wells containing prewarmed HBSS asdescribed above The radioactivity in the collected sam-ples was measured by 120574-spectrometry (Cobra Auto-gammaPackard) To determine the cellular uptake of 109Cd fromthe apical chamber following the 3 h incubation period thefilters with the monolayers were rinsed in 4∘C HBSS for5min and then dissolved in 1mL 05M NaOH for 48 hfollowed by 120574-spectrometry of an aliquote of the lysate Inorder to reduce the unstirred water layer during the transportand uptake experiments a plate shaker was used to agitatethe monolayers at a rate of 50 rpm The experiments werereproduced two times

25 Quantitative Gene Expression Total RNA from Caco-2 cells treated as described above prior to the transportexperiments was isolated by the use of NucleoSpin RNAII kit containing Dnase I according to the instructionsof the manufacturer (Macherey-Nagel Germany) The cellswere rinsed with ice cold PBS prior to the addition oflysis buffer and the isolation of RNA The integrity of theRNA was confirmed by gel electrophoresis and quantifi-cation of the RNA was performed according to the RNA-specificQuant-iTRiboGreen protocol (Invitrogen) RNAwasstored at minus70∘C until used for quantitative gene expressionanalyses

Quantification of gene expressions of the proteins pre-sented in Table 1 was performed by one step real-timeRT-PCR by applying a Rotorgene RG 3000 instrument(Corbett Research Australia) and QuantiTect SYBR GreenRT-PCR (Qiagen) reagents mainly according to the manu-facturerrsquos instructions Gene expression was normalized tototal RNA Primer concentration was 04 120583M and 200 ngtotal RNAwas used as template Gene-specific primers for thehuman genes were designed by using NCBI primer designand all gene products were sequenced and compared againstthe human genome by applying the UCSC Human GenomeBrowser (Table 1) Triplicate 25120583L volume RT-PCR reactionswere used under the following PCR cycling conditions Step1 50∘C 30min Step 2 95∘C 15min Step 3 94∘C 60 secStep 4 55∘C 60 sec Step 5 68∘C 45 s Step 6 35 or 40repeats of steps 3ndash5 Step 7 68∘C 7min In the analyses fiveknown concentrations of the human genes and a nontemplatecontrol served as internal controls Melt curve analysis wasperformed for each sample to check the specificity of theobtained PCR products Relative quantification of mRNAexpressions was performed as previously described [43]Quantification of gene expressions as well as analyses of meltcurves were carried out by the use of Rotorgene Software(Corbett Research Australia)

26 Functional Activity MRP-Mediated Efflux of Calcein Tomeasure the functional activity of MRP transporter proteinsfollowing cadmium pretreatment an efflux assay with calceinwas performed Calcein AM is a lipophilic nonfluorescentdye that rapidly enters cells through the plasma membraneOnce inside the cell endogenous esterases cleave the AMester bond releasing calcein a hydrophilic highly fluores-cent dye that becomes trapped inside the cell The ABCtransporters MRP1 and MRP2 have been shown to transportboth calcein AM and calcein In contrast P-gp transporterproteins efflux the nonfluorescent calcein AM but not thefluorescent calcein from the cells into the apical chamber[44] MRP1 transport proteins efflux both the nonfluorescentcalcein AM and the fluorescent calcein from the cells acrossthe basolateral membrane andMRP2 transports both calcein

4 ISRN Toxicology

AMand calcein although across the apicalmembrane into theapical chamber [45]

Cells were seeded onto filters and allowed to differentiatein medium supplemented with Cd as described above Themonolayers were washed as described above in 37∘C HBSSbuffered with 25mM HEPES before loading the cells with13 120583Mcalcein AM solved in 1DMSO and added both to theapical and basolateral chambersThe loading of the cells withthe permeable calcein AM was conducted at 7∘C to reducethe active efflux by P-gp and MRPs of the compound for 1 hin HBSS Two quick washes in 7∘C HBSS were performedafter loading to remove excessive calcein AM and then themonolayers were transferred to new wells with 37∘C HBSSstarting the efflux study During the 2 h efflux study sampleswere withdrawn every 30min from the basolateral chambersAt the end of the experiment samples were withdrawn alsofrom the apical chambers The samples were immediatelytransferred to black 96 plate wells and fluorescence wasmeasured instantly at 490 nm (excitation) and at 520 nm(emission) by a multiplate reader During the loading andefflux experiment a plate shaker was used to gently agitatethe monolayers at a rate of 50 rpm as described aboveThe functional assay of MRP-mediated calcein efflux wasreproduced two times

27 Statistical Analyses The data are presented as meanplusmn SEM unless otherwise stated The apparent perme-ability coefficient for mannitol was calculated as 119875app =(dQdt)(1(AC

0)) where dQdt is the steady state flux

(dpms) 119860 is the filter area (cm2) and 1198620is the initial

concentration in the apical chamber (120583M) The statisticalsoftware Minitab release 15 (Minitab Inc) was used forcalculations of basic descriptive statistics and analysis ofvariances (ANOVA) Data were tested for homogeneity ofvariances by Bartlettrsquos test Data on cadmium transport werelog transformed before statistical analysis ANOVA was usedto investigate the effects of cadmium on the dependentvariables (cadmium uptake and transport MRP activityDMT1 DMT1-IRE FPN1 MRP1 MRP2 P-gp and MT1gene expressions) followed by Tukeyrsquos test to determinedifferences between means The level of significance was setat 119875 le 005 two-sided

3 Results

31 Assessment of Monolayer Integrity and Histology ofthe Immature Caco-2 Cells The passive diffusion of 14C-mannitol across the monolayers was linear during the 3 hexperimental period The apparent permeability coefficient(119875app) for mannitol across the control monolayers was 115 plusmn03 times 10

minus6 cms (mean plusmn SD 119899 = 3) demonstrating that the 7days postconfluent cells had developed a barrier Cadmiumpretreatment did not influence the apparent permeabilityfor mannitol (119875 = 060) The TEER across the controlmonolayers was 275 plusmn 18Ωlowastcm2 (mean plusmn SEM 119899 = 6)Background resistance across filters in control and cadmiumsupplemented media (pH 74 37∘C) without cells was about

80Ωlowastcm2 Cadmium pretreatment did not affect the TEERvalue for the monolayers (119875 = 070)

The histological examination of the sectioned immatureCaco-2 monolayers did not reveal any difference betweencontrols and cadmium pretreated cells Dome formationswere observed in all monolayers at the end of the 7 daysdifferentiation period The nuclei of the immature cellsappeared larger and more centrally located as compared tomature cells which have smaller nuclei with a more basallocalization [31]

32 Transepithelial Transport and Uptake of 109Cd Thetransport of cadmium from the apical to the basolateralchamber across the Caco-2 monolayers was linear after aninitial one-hour lag phase (Figure 1(a)) At each 30min timeinterval the transport of cadmium was higher across thecadmium pretreated monolayers as compared to the controls(Figure 1(a)) The cumulative cadmium transport from theapical to the basolateral chamber was increased by 170in cells pretreated with cadmium (Figure 1(b)) Intracellularcadmium levels in cadmium pretreated cells was reduced byapproximately 20 as compared to the controls at the end ofthe 3 h incubation period (Figure 1(c))

33 Gene Expressions The MRP1 gene expression in cellspretreated with cadmium was increased by 55 (Figure 2)whereas the MRP2 and P-gp mRNA levels were not signifi-cantly affected by cadmium pretreatment The gene expres-sions of the iron transporters DMT1 DMT1-IRE and FPN1were not affected in theCaco-2 cells pretreatedwith cadmium(Figure 2) Gene expression of MT1 was increased 30 timesafter cadmium pretreatment (control 10 plusmn 01 cadmium315 plusmn 21 mean plusmn SEM 119899 = 3 119875 le 0001)

34 Functional Activity MRP-Mediated Efflux of CalceinThe basolateral efflux of calcein fluorescent dye was mea-sured during two hours after one hour initial loading ofthe nonfluorescent dye calcein AM Cadmium pretreatmentsignificantly increased the efflux of calcein into the basolat-eral chamber (Figure 3(a)) The efflux of calcein into apicalchamber was approximately four times higher than the effluxinto the basolateral chamber However the pretreatment withcadmium did not affect the calcein efflux into the apicalchamber (Figure 3(b))

4 Discussion

The role of transporters in the high gastrointestinal uptake ofcadmium in newborns is not fully understood [2 17] In addi-tion the cellular mechanism behind the increased intestinalabsorption of cadmium following continuous exposure thanafter a single dose of the metal remains to be elucidated [25]In the present study we have demonstrated an upregulationof the basolateral efflux protein MRP1 in immature Caco-2cells after cadmium pretreatment both at the gene and at thefunctional level The increased MRP1 gene expression andactivity was correlated to an increased cadmium transportacross the basolateral membrane indicating that MRP1 is

ISRN Toxicology 5

0

000005

00001

000015

00002

000025

0 50 100 150 200

Frac

tiona

l tra

nspo

rt

Time (min)

Cd

C

(a)

0

100

200

300

400

500

600

700

800

900

1000

C Cd

Cum

ulat

ive

109 C

d tr

ansp

ort (

fg C

dm

g pr

ot3

h)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

500

C Cd

Cum

ulat

ive

109 C

d up

take

(pg

Cd

mg

prot

ein

3 h)

lowast

(c)

Figure 1 Fraction of 109Cd transported across immature Caco-2 cells from the apical to the basolateral chamber (a) Cumulative transportof 109Cd across immature Caco-2 cells (b) Apical uptake of 109Cd across into immature Caco-2 cells (c) Data are presented as mean plusmn SEM119899 = 6 statistical significant difference compared to control lowast119875 le 005 lowastlowastlowast119875 le 0001 Legends to the pretreatments C control Cd cadmium

involved in this process The augmented transport of themetal was not due to disrupted tight junctions as neitherthe passive diffusion of the paracellular marker mannitol northe TEER was affected by cadmium Cadmium is knownto bind with high affinity to the thiol (SH) groups in GSH[28 46] and it has been demonstrated in yeast that cadmium-GSH complexes can be transported by the MRP1 analogueYCF1 [21 22] Cadmium-GSH has also been shown to betransported by MRP1 in lung epithelial fibroblasts [47] Wesuggest that cadmium bound to GSH is transported byMRP1 across the basolateral membrane in the immatureintestinal cells a transport that is upregulated by cadmiumpretreatment

Our results showed a 30-fold increase in intracellu-lar level of MT mRNA after cadmium pretreatment ofthe immature Caco-2 cells Previously a 5-fold increasein MT has been reported after long-term cadmium treat-ment of mature Caco-2 cells [26] indicating that imma-ture cells are more sensitive to cadmium treatment andtherefore induces MT expression more strongly In con-cordance Cardin et al [35] demonstrated a higher induc-tion of MT in immature Caco-2 cells compared to matureones These authors also reported that cadmium treatmentdecreased the thiol content by 65 in Caco-2 cells differ-entiated for 7 days while no effects were observed on thiolcontent in mature Caco-2 cells indicating developmental

6 ISRN Toxicology

MRP1 MRP2 P-gp DMT1 DMT1-IRE FPN10

02

04

06

08

1

12

14

16

18

2

Relat

ive g

ene e

xpre

ssio

n

CCd

lowastlowast

Figure 2 Relative gene expressions of MRP1 MRP2 P-gp DMT1 DMT1-IRE and FPN1 in immature Caco-2 cells Data are expressedrelative to controls set at 1 mean plusmn SEM 119899 = 3 statistical significant difference compared to control lowast119875 le 005 lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

0

02

04

06

08

1

12

14

16

18

Relat

ive b

asol

ater

al ca

lcei

n effl

ux

C Cd

lowastlowast

(a)

0

02

04

06

08

1

12

14

16

18

C Cd

Relat

ive a

pica

l cal

cein

efflux

(b)

Figure 3 Relative basolateral (a) and apical (b) efflux of fluorescent calcein in immature Caco-2 cells The MRP1 activity was examined bydetermining the cumulative level of fluorescent calcein effluxed into the basolateral chamber following two hours of incubation Data areexpressed relative to controls set at 1 mean plusmn SD 119899 = 6 statistical significant difference compared to control lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

differences in the cell defense against cadmium-inducedtoxicity [35] The reduced thiol content is in line with oursuggestion of cadmium-induced formation of cadmium-GSH complexes which are transported out of the cells byMRP1

Several studies have demonstrated that the iron trans-porters DMT1 and FPN1 upregulated at iron deficiencyand repressed at iron sufficient conditions are involved incadmium transport in adult mammals [15 16] It has beensuggested that there are ontogenic changes in the intestinalabsorption and handling of iron in neonates and that iron

absorption and expressions of DMT1 and FPN1 are notregulated until the time of weaning [18 48] Both irontransporters are exclusively located intracellularly at midinfancy and not until weaning present at the membranes ofthe duodenal enterocytes in rats [49 50] and in pigs [17]In suckling piglets neither iron nor cadmium pretreatmentshave resulted in any effect on expression or localization ofduodenal DMT1 or FPN1 [17] In the present study cadmiumpretreatment did not affect the gene expression of DMT1or FPN1 in the Caco-2 cells Thus the increased apical tobasolateral cadmium transport in cadmium pretreated cells

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

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Volume 2014

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Advances in Pharmacological Sciences

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

2 ISRN Toxicology

of the metal in functional yeast experiments [21 22] Theefflux proteins MRP2 and P-glycoprotein (P-gp) located atthe apical membrane of enterocytes are also upregulated bycadmium [23 24] and may thus influence the bioavailabilityof the metal

Repeated cadmium treatment has previously been shownto increase cadmium absorption compared to a single treat-ment both in vivo and in intestinal in vitro preparations ofadult rats [25] However even though MT involvement hasbeen suggested the cellular mechanism for how cadmiumenhances its own absorption remains to be elucidated [25]Intestinal MT is induced by cadmium [26] but there arenumerous reports concluding that the impact of MT on theintestinal cadmium absorption is of minor importance [27]It appears that the predominant role of this cytosolic smallmolecular weight metal binding protein is to protect the cellsfrom cytotoxic effects [28]

Twenty-one-day postconfluent Caco-2 cells which arefully differentiated (mature) have been extensively used asan in vitro model of the adult human intestinal epithelium[29] Under normal cell culture conditions Caco-2 cellsspontaneously differentiate into a phenotype that resemblespolarized absorptive enterocytes [30ndash32] The Caco-2 modelhas been widely used to investigate cadmium absorption andeffects [33ndash38] Mature Caco-2 cells treated with iron havereduced uptake and transport of cadmium that correlates toiron-induced decrease in DMT1 levels [37] In concordancea reduced cadmium uptake was demonstrated in Caco-2 cellssubjected to molecular DMT1 knock down [33] The ABCefflux transporters MRP1 MRP2 and P-gp are present inCaco-2 cells [39 40] and the expression of P-gp in thesecells has been shown to be induced by long-term cadmiumtreatment [23]

The present study was undertaken to investigate possibletransporters involved in the absorption of cadmium in theneonatal intestine and the impact of cadmium pretreatmentTo mimic the continuous cadmium exposure in neonates viamilk andor formula confluent Caco-2 cells were allowed todifferentiate during 7 days (referred to as ldquoimmature Caco-2 cellsrdquo) in the presence and absence of cadmium Apical tobasolateral transport studies with cadmium were performedand gene expressions of the transporters MRP1 MRP2 P-gpDMT1 DMT1-IRE FPN1 and of MT were quantified as wellas the MRP1 activity

2 Materials and Methods

21 Chemicals Cadmium chloride and nitrilotriacetic acidwere purchased from Sigma Aldrich Dulbeccosrsquo modi-fied Eagle medium (DMEM) heat inactivated fetal calfserum nonessential amino acids for modified Eagle medium(MEM) N-2-hydroxyethyl-piperazine-N1015840-2-ethanesulfonicacid (HEPES) buffer 1M nonessential amino acids (100x)and gentamycin were purchased from Invitrogen via VWRInternational LLC Apo-transferrin (tissue culture grade)was obtained from Calbiochem via VWR InternationalLLC [14C]-mannitol (specific activity 209GBqmmol) waspurchased from PerkinElmer and 109Cd (specific activity

152 GBqmg Cd2+) from GE Healthcare Ultima Gold (scin-tillation liquid) was obtained from Chemical InstrumentsAB Calcein acetoxymethyl ester (calcein AM) fluorescentdye was purchased from BD Biosciences Hankrsquos balancedsalt solution (HBSS) and all other chemicals used but notspecified in the experiments were of analytical grade andfrom VWR International LLC

22 Cell Culture and Treatment Human intestinal Caco-2cells were obtained from American Cell Culture Collection(Rockville USA) The cells were cultured as previouslydescribed [41] with some minor modifications Briefly theCaco-2 cells were expanded in normal tissue culture flasks inDMEM supplemented with 10 fetal calf serum (Invitrogen)and 1 gentamycin (50mgmL Invitrogen) The cells wereseeded onto permeable polycarbonate filters at a density of25 times 105 cellsfilter support (Oslash 12mm pore radius 04120583mTranswell Corning) and apical medium was changed 8 hafter seeding to remove excess of cells and to reduce the risk ofmultilayer formation [42]TheCaco-2 cells were then allowedto differentiate for 7 days postconfluency The medium waschanged every second day and the cells were examined dailyby reverse phase microscopy for confluence shape of cellsand dome formations Cells of passage numbers 97ndash100 wereused in the experiments

Each cell monolayer was randomly assigned to differen-tiate for 7 days postconfluency in control (C) medium ormedium supplemented with 1 120583M cadmium (CdCl

2times 1H2O)

(Cd) Previous investigations have demonstrated that theviability of the Caco-2 cells at various stages of differentiationis not affected by 1 120583M cadmium treatment [26]

23 Assessment of Monolayer Integrity The integrity of thecontrol and cadmium pretreated monolayers was exam-ined by determining the permeability of the hydrophilicmarker 14C-mannitol mainly as described [42] Briefly cellmonolayers were rinsed with prewarmed Hankrsquos BalancedSalt Solution pH 74 (HBSS) and then incubated in HBSSat 37∘C for 20min The HBSS was discarded from theapical chambers prior to transferring the filters to a freshand prewarmed plate containing 15mL 37∘C HBSS in thebasolateral chambers HBSS (05mL pH 63) prewarmed to37∘C and containing 014 120583CimL 14C-mannitol was added tothe apical chambers The transport experiment was carriedout at 37∘C under gentle shaking (50 rpm) for 3 h Every30min the filters with the monolayers were transferred tonew wells containing 37∘C HBSS and samples from both theapical and basolateral chambers were taken for subsequentdetermination of radioactivity by 120573-spectrometry (Tricarb1900CA Packard) The integrity of the monolayers wasalso verified by measuring the net transepithelial electricalresistance (TEER) across the monolayers as described [42]Resistance across monolayers was measured in control andcadmium supplemented media (pH 74 37∘C) and back-ground resistance was measured across filters without cells

24 109Cd Transport and Uptake Cell monolayers differ-entiated for 7 days postconfluency in control medium or

ISRN Toxicology 3

Table 1 Primer sequences used for quantitative real-time PCR

Primer Oligo sequence NCBI GenBank Accession No Product size (bp)MRP1 forward 51015840-GCAAATCCAGGAGACAGCTC-31015840 NM 004996 113MRP1 reverse 51015840-TGATGTGCCTGAGAACGAAG-31015840

MRP2 forward 51015840-CTGGTTGGGAACCTGACTGT-31015840 NM 000392 172MRP2 reverse 51015840-CAACAGCCACAATGTTGGTC-31015840

P-gp forward 51015840-GCTGTTAAGGAAGCCAATGC-31015840 NM 000927 120P-gp reverse 51015840-AGCAATGGCGATTCTCTGTT-31015840

DMT1 forward 51015840-CGTGGCGGATTGCAGGAGGA-31015840 NM 001174126 124DMT1 reverse 51015840-ACGCTGACCACAGCAGCCAC-31015840

DMT1-IRE forward 51015840-GCCATCAGAGCCAGTGTGTTTCT-31015840 NM 001174125 198DMT1-IRE reverse 51015840-TGTCAGCTTTTCAAAGATCCCACC-31015840

FPN1 forward 51015840-CGAGATGGATGGGTCTCCTA-31015840 NM 014585 219FPN1 reverse 51015840-GGCTACGTCGAAAATGTGGT-31015840

MT1 forward 51015840-GCAAATGCAAAGAGTGCAAA-31015840 NM 005946 213MT1 reverse 51015840-ATGGGTCAGGGTTGTATGGA-31015840

medium supplemented with cadmium were used for uptake(from apical chamber into the cells) and transport (fromapical chamber via the cells into the basolateral chamber)experiments The experiments were performed at 37∘C pH74 in sterile filtered HBSS buffered with 25mM HEPESmainly as previously described [41] Briefly the monolayerswere first rinsed in prewarmed HBSS and then incubated inHBSS at 37∘C for 20min before being transferred to newwellswhere fresh 37∘C HBSS was added to the apical (05mL pH63) and basolateral (15mL pH 74 supplementedwith 15120583Mhuman apo-transferrin) chambers The apical solution wassupplemented with 30 nM 109Cd (1120583CimL) To determinethe transport of 109Cd across the monolayers 14mL sampleswere taken from the basolateral chambers at 30min intervalsfor 3 h and at the same time the cell monolayers weretransferred to new wells containing prewarmed HBSS asdescribed above The radioactivity in the collected sam-ples was measured by 120574-spectrometry (Cobra Auto-gammaPackard) To determine the cellular uptake of 109Cd fromthe apical chamber following the 3 h incubation period thefilters with the monolayers were rinsed in 4∘C HBSS for5min and then dissolved in 1mL 05M NaOH for 48 hfollowed by 120574-spectrometry of an aliquote of the lysate Inorder to reduce the unstirred water layer during the transportand uptake experiments a plate shaker was used to agitatethe monolayers at a rate of 50 rpm The experiments werereproduced two times

25 Quantitative Gene Expression Total RNA from Caco-2 cells treated as described above prior to the transportexperiments was isolated by the use of NucleoSpin RNAII kit containing Dnase I according to the instructionsof the manufacturer (Macherey-Nagel Germany) The cellswere rinsed with ice cold PBS prior to the addition oflysis buffer and the isolation of RNA The integrity of theRNA was confirmed by gel electrophoresis and quantifi-cation of the RNA was performed according to the RNA-specificQuant-iTRiboGreen protocol (Invitrogen) RNAwasstored at minus70∘C until used for quantitative gene expressionanalyses

Quantification of gene expressions of the proteins pre-sented in Table 1 was performed by one step real-timeRT-PCR by applying a Rotorgene RG 3000 instrument(Corbett Research Australia) and QuantiTect SYBR GreenRT-PCR (Qiagen) reagents mainly according to the manu-facturerrsquos instructions Gene expression was normalized tototal RNA Primer concentration was 04 120583M and 200 ngtotal RNAwas used as template Gene-specific primers for thehuman genes were designed by using NCBI primer designand all gene products were sequenced and compared againstthe human genome by applying the UCSC Human GenomeBrowser (Table 1) Triplicate 25120583L volume RT-PCR reactionswere used under the following PCR cycling conditions Step1 50∘C 30min Step 2 95∘C 15min Step 3 94∘C 60 secStep 4 55∘C 60 sec Step 5 68∘C 45 s Step 6 35 or 40repeats of steps 3ndash5 Step 7 68∘C 7min In the analyses fiveknown concentrations of the human genes and a nontemplatecontrol served as internal controls Melt curve analysis wasperformed for each sample to check the specificity of theobtained PCR products Relative quantification of mRNAexpressions was performed as previously described [43]Quantification of gene expressions as well as analyses of meltcurves were carried out by the use of Rotorgene Software(Corbett Research Australia)

26 Functional Activity MRP-Mediated Efflux of Calcein Tomeasure the functional activity of MRP transporter proteinsfollowing cadmium pretreatment an efflux assay with calceinwas performed Calcein AM is a lipophilic nonfluorescentdye that rapidly enters cells through the plasma membraneOnce inside the cell endogenous esterases cleave the AMester bond releasing calcein a hydrophilic highly fluores-cent dye that becomes trapped inside the cell The ABCtransporters MRP1 and MRP2 have been shown to transportboth calcein AM and calcein In contrast P-gp transporterproteins efflux the nonfluorescent calcein AM but not thefluorescent calcein from the cells into the apical chamber[44] MRP1 transport proteins efflux both the nonfluorescentcalcein AM and the fluorescent calcein from the cells acrossthe basolateral membrane andMRP2 transports both calcein

4 ISRN Toxicology

AMand calcein although across the apicalmembrane into theapical chamber [45]

Cells were seeded onto filters and allowed to differentiatein medium supplemented with Cd as described above Themonolayers were washed as described above in 37∘C HBSSbuffered with 25mM HEPES before loading the cells with13 120583Mcalcein AM solved in 1DMSO and added both to theapical and basolateral chambersThe loading of the cells withthe permeable calcein AM was conducted at 7∘C to reducethe active efflux by P-gp and MRPs of the compound for 1 hin HBSS Two quick washes in 7∘C HBSS were performedafter loading to remove excessive calcein AM and then themonolayers were transferred to new wells with 37∘C HBSSstarting the efflux study During the 2 h efflux study sampleswere withdrawn every 30min from the basolateral chambersAt the end of the experiment samples were withdrawn alsofrom the apical chambers The samples were immediatelytransferred to black 96 plate wells and fluorescence wasmeasured instantly at 490 nm (excitation) and at 520 nm(emission) by a multiplate reader During the loading andefflux experiment a plate shaker was used to gently agitatethe monolayers at a rate of 50 rpm as described aboveThe functional assay of MRP-mediated calcein efflux wasreproduced two times

27 Statistical Analyses The data are presented as meanplusmn SEM unless otherwise stated The apparent perme-ability coefficient for mannitol was calculated as 119875app =(dQdt)(1(AC

0)) where dQdt is the steady state flux

(dpms) 119860 is the filter area (cm2) and 1198620is the initial

concentration in the apical chamber (120583M) The statisticalsoftware Minitab release 15 (Minitab Inc) was used forcalculations of basic descriptive statistics and analysis ofvariances (ANOVA) Data were tested for homogeneity ofvariances by Bartlettrsquos test Data on cadmium transport werelog transformed before statistical analysis ANOVA was usedto investigate the effects of cadmium on the dependentvariables (cadmium uptake and transport MRP activityDMT1 DMT1-IRE FPN1 MRP1 MRP2 P-gp and MT1gene expressions) followed by Tukeyrsquos test to determinedifferences between means The level of significance was setat 119875 le 005 two-sided

3 Results

31 Assessment of Monolayer Integrity and Histology ofthe Immature Caco-2 Cells The passive diffusion of 14C-mannitol across the monolayers was linear during the 3 hexperimental period The apparent permeability coefficient(119875app) for mannitol across the control monolayers was 115 plusmn03 times 10

minus6 cms (mean plusmn SD 119899 = 3) demonstrating that the 7days postconfluent cells had developed a barrier Cadmiumpretreatment did not influence the apparent permeabilityfor mannitol (119875 = 060) The TEER across the controlmonolayers was 275 plusmn 18Ωlowastcm2 (mean plusmn SEM 119899 = 6)Background resistance across filters in control and cadmiumsupplemented media (pH 74 37∘C) without cells was about

80Ωlowastcm2 Cadmium pretreatment did not affect the TEERvalue for the monolayers (119875 = 070)

The histological examination of the sectioned immatureCaco-2 monolayers did not reveal any difference betweencontrols and cadmium pretreated cells Dome formationswere observed in all monolayers at the end of the 7 daysdifferentiation period The nuclei of the immature cellsappeared larger and more centrally located as compared tomature cells which have smaller nuclei with a more basallocalization [31]

32 Transepithelial Transport and Uptake of 109Cd Thetransport of cadmium from the apical to the basolateralchamber across the Caco-2 monolayers was linear after aninitial one-hour lag phase (Figure 1(a)) At each 30min timeinterval the transport of cadmium was higher across thecadmium pretreated monolayers as compared to the controls(Figure 1(a)) The cumulative cadmium transport from theapical to the basolateral chamber was increased by 170in cells pretreated with cadmium (Figure 1(b)) Intracellularcadmium levels in cadmium pretreated cells was reduced byapproximately 20 as compared to the controls at the end ofthe 3 h incubation period (Figure 1(c))

33 Gene Expressions The MRP1 gene expression in cellspretreated with cadmium was increased by 55 (Figure 2)whereas the MRP2 and P-gp mRNA levels were not signifi-cantly affected by cadmium pretreatment The gene expres-sions of the iron transporters DMT1 DMT1-IRE and FPN1were not affected in theCaco-2 cells pretreatedwith cadmium(Figure 2) Gene expression of MT1 was increased 30 timesafter cadmium pretreatment (control 10 plusmn 01 cadmium315 plusmn 21 mean plusmn SEM 119899 = 3 119875 le 0001)

34 Functional Activity MRP-Mediated Efflux of CalceinThe basolateral efflux of calcein fluorescent dye was mea-sured during two hours after one hour initial loading ofthe nonfluorescent dye calcein AM Cadmium pretreatmentsignificantly increased the efflux of calcein into the basolat-eral chamber (Figure 3(a)) The efflux of calcein into apicalchamber was approximately four times higher than the effluxinto the basolateral chamber However the pretreatment withcadmium did not affect the calcein efflux into the apicalchamber (Figure 3(b))

4 Discussion

The role of transporters in the high gastrointestinal uptake ofcadmium in newborns is not fully understood [2 17] In addi-tion the cellular mechanism behind the increased intestinalabsorption of cadmium following continuous exposure thanafter a single dose of the metal remains to be elucidated [25]In the present study we have demonstrated an upregulationof the basolateral efflux protein MRP1 in immature Caco-2cells after cadmium pretreatment both at the gene and at thefunctional level The increased MRP1 gene expression andactivity was correlated to an increased cadmium transportacross the basolateral membrane indicating that MRP1 is

ISRN Toxicology 5

0

000005

00001

000015

00002

000025

0 50 100 150 200

Frac

tiona

l tra

nspo

rt

Time (min)

Cd

C

(a)

0

100

200

300

400

500

600

700

800

900

1000

C Cd

Cum

ulat

ive

109 C

d tr

ansp

ort (

fg C

dm

g pr

ot3

h)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

500

C Cd

Cum

ulat

ive

109 C

d up

take

(pg

Cd

mg

prot

ein

3 h)

lowast

(c)

Figure 1 Fraction of 109Cd transported across immature Caco-2 cells from the apical to the basolateral chamber (a) Cumulative transportof 109Cd across immature Caco-2 cells (b) Apical uptake of 109Cd across into immature Caco-2 cells (c) Data are presented as mean plusmn SEM119899 = 6 statistical significant difference compared to control lowast119875 le 005 lowastlowastlowast119875 le 0001 Legends to the pretreatments C control Cd cadmium

involved in this process The augmented transport of themetal was not due to disrupted tight junctions as neitherthe passive diffusion of the paracellular marker mannitol northe TEER was affected by cadmium Cadmium is knownto bind with high affinity to the thiol (SH) groups in GSH[28 46] and it has been demonstrated in yeast that cadmium-GSH complexes can be transported by the MRP1 analogueYCF1 [21 22] Cadmium-GSH has also been shown to betransported by MRP1 in lung epithelial fibroblasts [47] Wesuggest that cadmium bound to GSH is transported byMRP1 across the basolateral membrane in the immatureintestinal cells a transport that is upregulated by cadmiumpretreatment

Our results showed a 30-fold increase in intracellu-lar level of MT mRNA after cadmium pretreatment ofthe immature Caco-2 cells Previously a 5-fold increasein MT has been reported after long-term cadmium treat-ment of mature Caco-2 cells [26] indicating that imma-ture cells are more sensitive to cadmium treatment andtherefore induces MT expression more strongly In con-cordance Cardin et al [35] demonstrated a higher induc-tion of MT in immature Caco-2 cells compared to matureones These authors also reported that cadmium treatmentdecreased the thiol content by 65 in Caco-2 cells differ-entiated for 7 days while no effects were observed on thiolcontent in mature Caco-2 cells indicating developmental

6 ISRN Toxicology

MRP1 MRP2 P-gp DMT1 DMT1-IRE FPN10

02

04

06

08

1

12

14

16

18

2

Relat

ive g

ene e

xpre

ssio

n

CCd

lowastlowast

Figure 2 Relative gene expressions of MRP1 MRP2 P-gp DMT1 DMT1-IRE and FPN1 in immature Caco-2 cells Data are expressedrelative to controls set at 1 mean plusmn SEM 119899 = 3 statistical significant difference compared to control lowast119875 le 005 lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

0

02

04

06

08

1

12

14

16

18

Relat

ive b

asol

ater

al ca

lcei

n effl

ux

C Cd

lowastlowast

(a)

0

02

04

06

08

1

12

14

16

18

C Cd

Relat

ive a

pica

l cal

cein

efflux

(b)

Figure 3 Relative basolateral (a) and apical (b) efflux of fluorescent calcein in immature Caco-2 cells The MRP1 activity was examined bydetermining the cumulative level of fluorescent calcein effluxed into the basolateral chamber following two hours of incubation Data areexpressed relative to controls set at 1 mean plusmn SD 119899 = 6 statistical significant difference compared to control lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

differences in the cell defense against cadmium-inducedtoxicity [35] The reduced thiol content is in line with oursuggestion of cadmium-induced formation of cadmium-GSH complexes which are transported out of the cells byMRP1

Several studies have demonstrated that the iron trans-porters DMT1 and FPN1 upregulated at iron deficiencyand repressed at iron sufficient conditions are involved incadmium transport in adult mammals [15 16] It has beensuggested that there are ontogenic changes in the intestinalabsorption and handling of iron in neonates and that iron

absorption and expressions of DMT1 and FPN1 are notregulated until the time of weaning [18 48] Both irontransporters are exclusively located intracellularly at midinfancy and not until weaning present at the membranes ofthe duodenal enterocytes in rats [49 50] and in pigs [17]In suckling piglets neither iron nor cadmium pretreatmentshave resulted in any effect on expression or localization ofduodenal DMT1 or FPN1 [17] In the present study cadmiumpretreatment did not affect the gene expression of DMT1or FPN1 in the Caco-2 cells Thus the increased apical tobasolateral cadmium transport in cadmium pretreated cells

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

ISRN Toxicology 3

Table 1 Primer sequences used for quantitative real-time PCR

Primer Oligo sequence NCBI GenBank Accession No Product size (bp)MRP1 forward 51015840-GCAAATCCAGGAGACAGCTC-31015840 NM 004996 113MRP1 reverse 51015840-TGATGTGCCTGAGAACGAAG-31015840

MRP2 forward 51015840-CTGGTTGGGAACCTGACTGT-31015840 NM 000392 172MRP2 reverse 51015840-CAACAGCCACAATGTTGGTC-31015840

P-gp forward 51015840-GCTGTTAAGGAAGCCAATGC-31015840 NM 000927 120P-gp reverse 51015840-AGCAATGGCGATTCTCTGTT-31015840

DMT1 forward 51015840-CGTGGCGGATTGCAGGAGGA-31015840 NM 001174126 124DMT1 reverse 51015840-ACGCTGACCACAGCAGCCAC-31015840

DMT1-IRE forward 51015840-GCCATCAGAGCCAGTGTGTTTCT-31015840 NM 001174125 198DMT1-IRE reverse 51015840-TGTCAGCTTTTCAAAGATCCCACC-31015840

FPN1 forward 51015840-CGAGATGGATGGGTCTCCTA-31015840 NM 014585 219FPN1 reverse 51015840-GGCTACGTCGAAAATGTGGT-31015840

MT1 forward 51015840-GCAAATGCAAAGAGTGCAAA-31015840 NM 005946 213MT1 reverse 51015840-ATGGGTCAGGGTTGTATGGA-31015840

medium supplemented with cadmium were used for uptake(from apical chamber into the cells) and transport (fromapical chamber via the cells into the basolateral chamber)experiments The experiments were performed at 37∘C pH74 in sterile filtered HBSS buffered with 25mM HEPESmainly as previously described [41] Briefly the monolayerswere first rinsed in prewarmed HBSS and then incubated inHBSS at 37∘C for 20min before being transferred to newwellswhere fresh 37∘C HBSS was added to the apical (05mL pH63) and basolateral (15mL pH 74 supplementedwith 15120583Mhuman apo-transferrin) chambers The apical solution wassupplemented with 30 nM 109Cd (1120583CimL) To determinethe transport of 109Cd across the monolayers 14mL sampleswere taken from the basolateral chambers at 30min intervalsfor 3 h and at the same time the cell monolayers weretransferred to new wells containing prewarmed HBSS asdescribed above The radioactivity in the collected sam-ples was measured by 120574-spectrometry (Cobra Auto-gammaPackard) To determine the cellular uptake of 109Cd fromthe apical chamber following the 3 h incubation period thefilters with the monolayers were rinsed in 4∘C HBSS for5min and then dissolved in 1mL 05M NaOH for 48 hfollowed by 120574-spectrometry of an aliquote of the lysate Inorder to reduce the unstirred water layer during the transportand uptake experiments a plate shaker was used to agitatethe monolayers at a rate of 50 rpm The experiments werereproduced two times

25 Quantitative Gene Expression Total RNA from Caco-2 cells treated as described above prior to the transportexperiments was isolated by the use of NucleoSpin RNAII kit containing Dnase I according to the instructionsof the manufacturer (Macherey-Nagel Germany) The cellswere rinsed with ice cold PBS prior to the addition oflysis buffer and the isolation of RNA The integrity of theRNA was confirmed by gel electrophoresis and quantifi-cation of the RNA was performed according to the RNA-specificQuant-iTRiboGreen protocol (Invitrogen) RNAwasstored at minus70∘C until used for quantitative gene expressionanalyses

Quantification of gene expressions of the proteins pre-sented in Table 1 was performed by one step real-timeRT-PCR by applying a Rotorgene RG 3000 instrument(Corbett Research Australia) and QuantiTect SYBR GreenRT-PCR (Qiagen) reagents mainly according to the manu-facturerrsquos instructions Gene expression was normalized tototal RNA Primer concentration was 04 120583M and 200 ngtotal RNAwas used as template Gene-specific primers for thehuman genes were designed by using NCBI primer designand all gene products were sequenced and compared againstthe human genome by applying the UCSC Human GenomeBrowser (Table 1) Triplicate 25120583L volume RT-PCR reactionswere used under the following PCR cycling conditions Step1 50∘C 30min Step 2 95∘C 15min Step 3 94∘C 60 secStep 4 55∘C 60 sec Step 5 68∘C 45 s Step 6 35 or 40repeats of steps 3ndash5 Step 7 68∘C 7min In the analyses fiveknown concentrations of the human genes and a nontemplatecontrol served as internal controls Melt curve analysis wasperformed for each sample to check the specificity of theobtained PCR products Relative quantification of mRNAexpressions was performed as previously described [43]Quantification of gene expressions as well as analyses of meltcurves were carried out by the use of Rotorgene Software(Corbett Research Australia)

26 Functional Activity MRP-Mediated Efflux of Calcein Tomeasure the functional activity of MRP transporter proteinsfollowing cadmium pretreatment an efflux assay with calceinwas performed Calcein AM is a lipophilic nonfluorescentdye that rapidly enters cells through the plasma membraneOnce inside the cell endogenous esterases cleave the AMester bond releasing calcein a hydrophilic highly fluores-cent dye that becomes trapped inside the cell The ABCtransporters MRP1 and MRP2 have been shown to transportboth calcein AM and calcein In contrast P-gp transporterproteins efflux the nonfluorescent calcein AM but not thefluorescent calcein from the cells into the apical chamber[44] MRP1 transport proteins efflux both the nonfluorescentcalcein AM and the fluorescent calcein from the cells acrossthe basolateral membrane andMRP2 transports both calcein

4 ISRN Toxicology

AMand calcein although across the apicalmembrane into theapical chamber [45]

Cells were seeded onto filters and allowed to differentiatein medium supplemented with Cd as described above Themonolayers were washed as described above in 37∘C HBSSbuffered with 25mM HEPES before loading the cells with13 120583Mcalcein AM solved in 1DMSO and added both to theapical and basolateral chambersThe loading of the cells withthe permeable calcein AM was conducted at 7∘C to reducethe active efflux by P-gp and MRPs of the compound for 1 hin HBSS Two quick washes in 7∘C HBSS were performedafter loading to remove excessive calcein AM and then themonolayers were transferred to new wells with 37∘C HBSSstarting the efflux study During the 2 h efflux study sampleswere withdrawn every 30min from the basolateral chambersAt the end of the experiment samples were withdrawn alsofrom the apical chambers The samples were immediatelytransferred to black 96 plate wells and fluorescence wasmeasured instantly at 490 nm (excitation) and at 520 nm(emission) by a multiplate reader During the loading andefflux experiment a plate shaker was used to gently agitatethe monolayers at a rate of 50 rpm as described aboveThe functional assay of MRP-mediated calcein efflux wasreproduced two times

27 Statistical Analyses The data are presented as meanplusmn SEM unless otherwise stated The apparent perme-ability coefficient for mannitol was calculated as 119875app =(dQdt)(1(AC

0)) where dQdt is the steady state flux

(dpms) 119860 is the filter area (cm2) and 1198620is the initial

concentration in the apical chamber (120583M) The statisticalsoftware Minitab release 15 (Minitab Inc) was used forcalculations of basic descriptive statistics and analysis ofvariances (ANOVA) Data were tested for homogeneity ofvariances by Bartlettrsquos test Data on cadmium transport werelog transformed before statistical analysis ANOVA was usedto investigate the effects of cadmium on the dependentvariables (cadmium uptake and transport MRP activityDMT1 DMT1-IRE FPN1 MRP1 MRP2 P-gp and MT1gene expressions) followed by Tukeyrsquos test to determinedifferences between means The level of significance was setat 119875 le 005 two-sided

3 Results

31 Assessment of Monolayer Integrity and Histology ofthe Immature Caco-2 Cells The passive diffusion of 14C-mannitol across the monolayers was linear during the 3 hexperimental period The apparent permeability coefficient(119875app) for mannitol across the control monolayers was 115 plusmn03 times 10

minus6 cms (mean plusmn SD 119899 = 3) demonstrating that the 7days postconfluent cells had developed a barrier Cadmiumpretreatment did not influence the apparent permeabilityfor mannitol (119875 = 060) The TEER across the controlmonolayers was 275 plusmn 18Ωlowastcm2 (mean plusmn SEM 119899 = 6)Background resistance across filters in control and cadmiumsupplemented media (pH 74 37∘C) without cells was about

80Ωlowastcm2 Cadmium pretreatment did not affect the TEERvalue for the monolayers (119875 = 070)

The histological examination of the sectioned immatureCaco-2 monolayers did not reveal any difference betweencontrols and cadmium pretreated cells Dome formationswere observed in all monolayers at the end of the 7 daysdifferentiation period The nuclei of the immature cellsappeared larger and more centrally located as compared tomature cells which have smaller nuclei with a more basallocalization [31]

32 Transepithelial Transport and Uptake of 109Cd Thetransport of cadmium from the apical to the basolateralchamber across the Caco-2 monolayers was linear after aninitial one-hour lag phase (Figure 1(a)) At each 30min timeinterval the transport of cadmium was higher across thecadmium pretreated monolayers as compared to the controls(Figure 1(a)) The cumulative cadmium transport from theapical to the basolateral chamber was increased by 170in cells pretreated with cadmium (Figure 1(b)) Intracellularcadmium levels in cadmium pretreated cells was reduced byapproximately 20 as compared to the controls at the end ofthe 3 h incubation period (Figure 1(c))

33 Gene Expressions The MRP1 gene expression in cellspretreated with cadmium was increased by 55 (Figure 2)whereas the MRP2 and P-gp mRNA levels were not signifi-cantly affected by cadmium pretreatment The gene expres-sions of the iron transporters DMT1 DMT1-IRE and FPN1were not affected in theCaco-2 cells pretreatedwith cadmium(Figure 2) Gene expression of MT1 was increased 30 timesafter cadmium pretreatment (control 10 plusmn 01 cadmium315 plusmn 21 mean plusmn SEM 119899 = 3 119875 le 0001)

34 Functional Activity MRP-Mediated Efflux of CalceinThe basolateral efflux of calcein fluorescent dye was mea-sured during two hours after one hour initial loading ofthe nonfluorescent dye calcein AM Cadmium pretreatmentsignificantly increased the efflux of calcein into the basolat-eral chamber (Figure 3(a)) The efflux of calcein into apicalchamber was approximately four times higher than the effluxinto the basolateral chamber However the pretreatment withcadmium did not affect the calcein efflux into the apicalchamber (Figure 3(b))

4 Discussion

The role of transporters in the high gastrointestinal uptake ofcadmium in newborns is not fully understood [2 17] In addi-tion the cellular mechanism behind the increased intestinalabsorption of cadmium following continuous exposure thanafter a single dose of the metal remains to be elucidated [25]In the present study we have demonstrated an upregulationof the basolateral efflux protein MRP1 in immature Caco-2cells after cadmium pretreatment both at the gene and at thefunctional level The increased MRP1 gene expression andactivity was correlated to an increased cadmium transportacross the basolateral membrane indicating that MRP1 is

ISRN Toxicology 5

0

000005

00001

000015

00002

000025

0 50 100 150 200

Frac

tiona

l tra

nspo

rt

Time (min)

Cd

C

(a)

0

100

200

300

400

500

600

700

800

900

1000

C Cd

Cum

ulat

ive

109 C

d tr

ansp

ort (

fg C

dm

g pr

ot3

h)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

500

C Cd

Cum

ulat

ive

109 C

d up

take

(pg

Cd

mg

prot

ein

3 h)

lowast

(c)

Figure 1 Fraction of 109Cd transported across immature Caco-2 cells from the apical to the basolateral chamber (a) Cumulative transportof 109Cd across immature Caco-2 cells (b) Apical uptake of 109Cd across into immature Caco-2 cells (c) Data are presented as mean plusmn SEM119899 = 6 statistical significant difference compared to control lowast119875 le 005 lowastlowastlowast119875 le 0001 Legends to the pretreatments C control Cd cadmium

involved in this process The augmented transport of themetal was not due to disrupted tight junctions as neitherthe passive diffusion of the paracellular marker mannitol northe TEER was affected by cadmium Cadmium is knownto bind with high affinity to the thiol (SH) groups in GSH[28 46] and it has been demonstrated in yeast that cadmium-GSH complexes can be transported by the MRP1 analogueYCF1 [21 22] Cadmium-GSH has also been shown to betransported by MRP1 in lung epithelial fibroblasts [47] Wesuggest that cadmium bound to GSH is transported byMRP1 across the basolateral membrane in the immatureintestinal cells a transport that is upregulated by cadmiumpretreatment

Our results showed a 30-fold increase in intracellu-lar level of MT mRNA after cadmium pretreatment ofthe immature Caco-2 cells Previously a 5-fold increasein MT has been reported after long-term cadmium treat-ment of mature Caco-2 cells [26] indicating that imma-ture cells are more sensitive to cadmium treatment andtherefore induces MT expression more strongly In con-cordance Cardin et al [35] demonstrated a higher induc-tion of MT in immature Caco-2 cells compared to matureones These authors also reported that cadmium treatmentdecreased the thiol content by 65 in Caco-2 cells differ-entiated for 7 days while no effects were observed on thiolcontent in mature Caco-2 cells indicating developmental

6 ISRN Toxicology

MRP1 MRP2 P-gp DMT1 DMT1-IRE FPN10

02

04

06

08

1

12

14

16

18

2

Relat

ive g

ene e

xpre

ssio

n

CCd

lowastlowast

Figure 2 Relative gene expressions of MRP1 MRP2 P-gp DMT1 DMT1-IRE and FPN1 in immature Caco-2 cells Data are expressedrelative to controls set at 1 mean plusmn SEM 119899 = 3 statistical significant difference compared to control lowast119875 le 005 lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

0

02

04

06

08

1

12

14

16

18

Relat

ive b

asol

ater

al ca

lcei

n effl

ux

C Cd

lowastlowast

(a)

0

02

04

06

08

1

12

14

16

18

C Cd

Relat

ive a

pica

l cal

cein

efflux

(b)

Figure 3 Relative basolateral (a) and apical (b) efflux of fluorescent calcein in immature Caco-2 cells The MRP1 activity was examined bydetermining the cumulative level of fluorescent calcein effluxed into the basolateral chamber following two hours of incubation Data areexpressed relative to controls set at 1 mean plusmn SD 119899 = 6 statistical significant difference compared to control lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

differences in the cell defense against cadmium-inducedtoxicity [35] The reduced thiol content is in line with oursuggestion of cadmium-induced formation of cadmium-GSH complexes which are transported out of the cells byMRP1

Several studies have demonstrated that the iron trans-porters DMT1 and FPN1 upregulated at iron deficiencyand repressed at iron sufficient conditions are involved incadmium transport in adult mammals [15 16] It has beensuggested that there are ontogenic changes in the intestinalabsorption and handling of iron in neonates and that iron

absorption and expressions of DMT1 and FPN1 are notregulated until the time of weaning [18 48] Both irontransporters are exclusively located intracellularly at midinfancy and not until weaning present at the membranes ofthe duodenal enterocytes in rats [49 50] and in pigs [17]In suckling piglets neither iron nor cadmium pretreatmentshave resulted in any effect on expression or localization ofduodenal DMT1 or FPN1 [17] In the present study cadmiumpretreatment did not affect the gene expression of DMT1or FPN1 in the Caco-2 cells Thus the increased apical tobasolateral cadmium transport in cadmium pretreated cells

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

4 ISRN Toxicology

AMand calcein although across the apicalmembrane into theapical chamber [45]

Cells were seeded onto filters and allowed to differentiatein medium supplemented with Cd as described above Themonolayers were washed as described above in 37∘C HBSSbuffered with 25mM HEPES before loading the cells with13 120583Mcalcein AM solved in 1DMSO and added both to theapical and basolateral chambersThe loading of the cells withthe permeable calcein AM was conducted at 7∘C to reducethe active efflux by P-gp and MRPs of the compound for 1 hin HBSS Two quick washes in 7∘C HBSS were performedafter loading to remove excessive calcein AM and then themonolayers were transferred to new wells with 37∘C HBSSstarting the efflux study During the 2 h efflux study sampleswere withdrawn every 30min from the basolateral chambersAt the end of the experiment samples were withdrawn alsofrom the apical chambers The samples were immediatelytransferred to black 96 plate wells and fluorescence wasmeasured instantly at 490 nm (excitation) and at 520 nm(emission) by a multiplate reader During the loading andefflux experiment a plate shaker was used to gently agitatethe monolayers at a rate of 50 rpm as described aboveThe functional assay of MRP-mediated calcein efflux wasreproduced two times

27 Statistical Analyses The data are presented as meanplusmn SEM unless otherwise stated The apparent perme-ability coefficient for mannitol was calculated as 119875app =(dQdt)(1(AC

0)) where dQdt is the steady state flux

(dpms) 119860 is the filter area (cm2) and 1198620is the initial

concentration in the apical chamber (120583M) The statisticalsoftware Minitab release 15 (Minitab Inc) was used forcalculations of basic descriptive statistics and analysis ofvariances (ANOVA) Data were tested for homogeneity ofvariances by Bartlettrsquos test Data on cadmium transport werelog transformed before statistical analysis ANOVA was usedto investigate the effects of cadmium on the dependentvariables (cadmium uptake and transport MRP activityDMT1 DMT1-IRE FPN1 MRP1 MRP2 P-gp and MT1gene expressions) followed by Tukeyrsquos test to determinedifferences between means The level of significance was setat 119875 le 005 two-sided

3 Results

31 Assessment of Monolayer Integrity and Histology ofthe Immature Caco-2 Cells The passive diffusion of 14C-mannitol across the monolayers was linear during the 3 hexperimental period The apparent permeability coefficient(119875app) for mannitol across the control monolayers was 115 plusmn03 times 10

minus6 cms (mean plusmn SD 119899 = 3) demonstrating that the 7days postconfluent cells had developed a barrier Cadmiumpretreatment did not influence the apparent permeabilityfor mannitol (119875 = 060) The TEER across the controlmonolayers was 275 plusmn 18Ωlowastcm2 (mean plusmn SEM 119899 = 6)Background resistance across filters in control and cadmiumsupplemented media (pH 74 37∘C) without cells was about

80Ωlowastcm2 Cadmium pretreatment did not affect the TEERvalue for the monolayers (119875 = 070)

The histological examination of the sectioned immatureCaco-2 monolayers did not reveal any difference betweencontrols and cadmium pretreated cells Dome formationswere observed in all monolayers at the end of the 7 daysdifferentiation period The nuclei of the immature cellsappeared larger and more centrally located as compared tomature cells which have smaller nuclei with a more basallocalization [31]

32 Transepithelial Transport and Uptake of 109Cd Thetransport of cadmium from the apical to the basolateralchamber across the Caco-2 monolayers was linear after aninitial one-hour lag phase (Figure 1(a)) At each 30min timeinterval the transport of cadmium was higher across thecadmium pretreated monolayers as compared to the controls(Figure 1(a)) The cumulative cadmium transport from theapical to the basolateral chamber was increased by 170in cells pretreated with cadmium (Figure 1(b)) Intracellularcadmium levels in cadmium pretreated cells was reduced byapproximately 20 as compared to the controls at the end ofthe 3 h incubation period (Figure 1(c))

33 Gene Expressions The MRP1 gene expression in cellspretreated with cadmium was increased by 55 (Figure 2)whereas the MRP2 and P-gp mRNA levels were not signifi-cantly affected by cadmium pretreatment The gene expres-sions of the iron transporters DMT1 DMT1-IRE and FPN1were not affected in theCaco-2 cells pretreatedwith cadmium(Figure 2) Gene expression of MT1 was increased 30 timesafter cadmium pretreatment (control 10 plusmn 01 cadmium315 plusmn 21 mean plusmn SEM 119899 = 3 119875 le 0001)

34 Functional Activity MRP-Mediated Efflux of CalceinThe basolateral efflux of calcein fluorescent dye was mea-sured during two hours after one hour initial loading ofthe nonfluorescent dye calcein AM Cadmium pretreatmentsignificantly increased the efflux of calcein into the basolat-eral chamber (Figure 3(a)) The efflux of calcein into apicalchamber was approximately four times higher than the effluxinto the basolateral chamber However the pretreatment withcadmium did not affect the calcein efflux into the apicalchamber (Figure 3(b))

4 Discussion

The role of transporters in the high gastrointestinal uptake ofcadmium in newborns is not fully understood [2 17] In addi-tion the cellular mechanism behind the increased intestinalabsorption of cadmium following continuous exposure thanafter a single dose of the metal remains to be elucidated [25]In the present study we have demonstrated an upregulationof the basolateral efflux protein MRP1 in immature Caco-2cells after cadmium pretreatment both at the gene and at thefunctional level The increased MRP1 gene expression andactivity was correlated to an increased cadmium transportacross the basolateral membrane indicating that MRP1 is

ISRN Toxicology 5

0

000005

00001

000015

00002

000025

0 50 100 150 200

Frac

tiona

l tra

nspo

rt

Time (min)

Cd

C

(a)

0

100

200

300

400

500

600

700

800

900

1000

C Cd

Cum

ulat

ive

109 C

d tr

ansp

ort (

fg C

dm

g pr

ot3

h)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

500

C Cd

Cum

ulat

ive

109 C

d up

take

(pg

Cd

mg

prot

ein

3 h)

lowast

(c)

Figure 1 Fraction of 109Cd transported across immature Caco-2 cells from the apical to the basolateral chamber (a) Cumulative transportof 109Cd across immature Caco-2 cells (b) Apical uptake of 109Cd across into immature Caco-2 cells (c) Data are presented as mean plusmn SEM119899 = 6 statistical significant difference compared to control lowast119875 le 005 lowastlowastlowast119875 le 0001 Legends to the pretreatments C control Cd cadmium

involved in this process The augmented transport of themetal was not due to disrupted tight junctions as neitherthe passive diffusion of the paracellular marker mannitol northe TEER was affected by cadmium Cadmium is knownto bind with high affinity to the thiol (SH) groups in GSH[28 46] and it has been demonstrated in yeast that cadmium-GSH complexes can be transported by the MRP1 analogueYCF1 [21 22] Cadmium-GSH has also been shown to betransported by MRP1 in lung epithelial fibroblasts [47] Wesuggest that cadmium bound to GSH is transported byMRP1 across the basolateral membrane in the immatureintestinal cells a transport that is upregulated by cadmiumpretreatment

Our results showed a 30-fold increase in intracellu-lar level of MT mRNA after cadmium pretreatment ofthe immature Caco-2 cells Previously a 5-fold increasein MT has been reported after long-term cadmium treat-ment of mature Caco-2 cells [26] indicating that imma-ture cells are more sensitive to cadmium treatment andtherefore induces MT expression more strongly In con-cordance Cardin et al [35] demonstrated a higher induc-tion of MT in immature Caco-2 cells compared to matureones These authors also reported that cadmium treatmentdecreased the thiol content by 65 in Caco-2 cells differ-entiated for 7 days while no effects were observed on thiolcontent in mature Caco-2 cells indicating developmental

6 ISRN Toxicology

MRP1 MRP2 P-gp DMT1 DMT1-IRE FPN10

02

04

06

08

1

12

14

16

18

2

Relat

ive g

ene e

xpre

ssio

n

CCd

lowastlowast

Figure 2 Relative gene expressions of MRP1 MRP2 P-gp DMT1 DMT1-IRE and FPN1 in immature Caco-2 cells Data are expressedrelative to controls set at 1 mean plusmn SEM 119899 = 3 statistical significant difference compared to control lowast119875 le 005 lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

0

02

04

06

08

1

12

14

16

18

Relat

ive b

asol

ater

al ca

lcei

n effl

ux

C Cd

lowastlowast

(a)

0

02

04

06

08

1

12

14

16

18

C Cd

Relat

ive a

pica

l cal

cein

efflux

(b)

Figure 3 Relative basolateral (a) and apical (b) efflux of fluorescent calcein in immature Caco-2 cells The MRP1 activity was examined bydetermining the cumulative level of fluorescent calcein effluxed into the basolateral chamber following two hours of incubation Data areexpressed relative to controls set at 1 mean plusmn SD 119899 = 6 statistical significant difference compared to control lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

differences in the cell defense against cadmium-inducedtoxicity [35] The reduced thiol content is in line with oursuggestion of cadmium-induced formation of cadmium-GSH complexes which are transported out of the cells byMRP1

Several studies have demonstrated that the iron trans-porters DMT1 and FPN1 upregulated at iron deficiencyand repressed at iron sufficient conditions are involved incadmium transport in adult mammals [15 16] It has beensuggested that there are ontogenic changes in the intestinalabsorption and handling of iron in neonates and that iron

absorption and expressions of DMT1 and FPN1 are notregulated until the time of weaning [18 48] Both irontransporters are exclusively located intracellularly at midinfancy and not until weaning present at the membranes ofthe duodenal enterocytes in rats [49 50] and in pigs [17]In suckling piglets neither iron nor cadmium pretreatmentshave resulted in any effect on expression or localization ofduodenal DMT1 or FPN1 [17] In the present study cadmiumpretreatment did not affect the gene expression of DMT1or FPN1 in the Caco-2 cells Thus the increased apical tobasolateral cadmium transport in cadmium pretreated cells

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

ISRN Toxicology 5

0

000005

00001

000015

00002

000025

0 50 100 150 200

Frac

tiona

l tra

nspo

rt

Time (min)

Cd

C

(a)

0

100

200

300

400

500

600

700

800

900

1000

C Cd

Cum

ulat

ive

109 C

d tr

ansp

ort (

fg C

dm

g pr

ot3

h)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

500

C Cd

Cum

ulat

ive

109 C

d up

take

(pg

Cd

mg

prot

ein

3 h)

lowast

(c)

Figure 1 Fraction of 109Cd transported across immature Caco-2 cells from the apical to the basolateral chamber (a) Cumulative transportof 109Cd across immature Caco-2 cells (b) Apical uptake of 109Cd across into immature Caco-2 cells (c) Data are presented as mean plusmn SEM119899 = 6 statistical significant difference compared to control lowast119875 le 005 lowastlowastlowast119875 le 0001 Legends to the pretreatments C control Cd cadmium

involved in this process The augmented transport of themetal was not due to disrupted tight junctions as neitherthe passive diffusion of the paracellular marker mannitol northe TEER was affected by cadmium Cadmium is knownto bind with high affinity to the thiol (SH) groups in GSH[28 46] and it has been demonstrated in yeast that cadmium-GSH complexes can be transported by the MRP1 analogueYCF1 [21 22] Cadmium-GSH has also been shown to betransported by MRP1 in lung epithelial fibroblasts [47] Wesuggest that cadmium bound to GSH is transported byMRP1 across the basolateral membrane in the immatureintestinal cells a transport that is upregulated by cadmiumpretreatment

Our results showed a 30-fold increase in intracellu-lar level of MT mRNA after cadmium pretreatment ofthe immature Caco-2 cells Previously a 5-fold increasein MT has been reported after long-term cadmium treat-ment of mature Caco-2 cells [26] indicating that imma-ture cells are more sensitive to cadmium treatment andtherefore induces MT expression more strongly In con-cordance Cardin et al [35] demonstrated a higher induc-tion of MT in immature Caco-2 cells compared to matureones These authors also reported that cadmium treatmentdecreased the thiol content by 65 in Caco-2 cells differ-entiated for 7 days while no effects were observed on thiolcontent in mature Caco-2 cells indicating developmental

6 ISRN Toxicology

MRP1 MRP2 P-gp DMT1 DMT1-IRE FPN10

02

04

06

08

1

12

14

16

18

2

Relat

ive g

ene e

xpre

ssio

n

CCd

lowastlowast

Figure 2 Relative gene expressions of MRP1 MRP2 P-gp DMT1 DMT1-IRE and FPN1 in immature Caco-2 cells Data are expressedrelative to controls set at 1 mean plusmn SEM 119899 = 3 statistical significant difference compared to control lowast119875 le 005 lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

0

02

04

06

08

1

12

14

16

18

Relat

ive b

asol

ater

al ca

lcei

n effl

ux

C Cd

lowastlowast

(a)

0

02

04

06

08

1

12

14

16

18

C Cd

Relat

ive a

pica

l cal

cein

efflux

(b)

Figure 3 Relative basolateral (a) and apical (b) efflux of fluorescent calcein in immature Caco-2 cells The MRP1 activity was examined bydetermining the cumulative level of fluorescent calcein effluxed into the basolateral chamber following two hours of incubation Data areexpressed relative to controls set at 1 mean plusmn SD 119899 = 6 statistical significant difference compared to control lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

differences in the cell defense against cadmium-inducedtoxicity [35] The reduced thiol content is in line with oursuggestion of cadmium-induced formation of cadmium-GSH complexes which are transported out of the cells byMRP1

Several studies have demonstrated that the iron trans-porters DMT1 and FPN1 upregulated at iron deficiencyand repressed at iron sufficient conditions are involved incadmium transport in adult mammals [15 16] It has beensuggested that there are ontogenic changes in the intestinalabsorption and handling of iron in neonates and that iron

absorption and expressions of DMT1 and FPN1 are notregulated until the time of weaning [18 48] Both irontransporters are exclusively located intracellularly at midinfancy and not until weaning present at the membranes ofthe duodenal enterocytes in rats [49 50] and in pigs [17]In suckling piglets neither iron nor cadmium pretreatmentshave resulted in any effect on expression or localization ofduodenal DMT1 or FPN1 [17] In the present study cadmiumpretreatment did not affect the gene expression of DMT1or FPN1 in the Caco-2 cells Thus the increased apical tobasolateral cadmium transport in cadmium pretreated cells

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 ISRN Toxicology

MRP1 MRP2 P-gp DMT1 DMT1-IRE FPN10

02

04

06

08

1

12

14

16

18

2

Relat

ive g

ene e

xpre

ssio

n

CCd

lowastlowast

Figure 2 Relative gene expressions of MRP1 MRP2 P-gp DMT1 DMT1-IRE and FPN1 in immature Caco-2 cells Data are expressedrelative to controls set at 1 mean plusmn SEM 119899 = 3 statistical significant difference compared to control lowast119875 le 005 lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

0

02

04

06

08

1

12

14

16

18

Relat

ive b

asol

ater

al ca

lcei

n effl

ux

C Cd

lowastlowast

(a)

0

02

04

06

08

1

12

14

16

18

C Cd

Relat

ive a

pica

l cal

cein

efflux

(b)

Figure 3 Relative basolateral (a) and apical (b) efflux of fluorescent calcein in immature Caco-2 cells The MRP1 activity was examined bydetermining the cumulative level of fluorescent calcein effluxed into the basolateral chamber following two hours of incubation Data areexpressed relative to controls set at 1 mean plusmn SD 119899 = 6 statistical significant difference compared to control lowastlowast119875 le 001 Legends to thepretreatments C control Cd cadmium

differences in the cell defense against cadmium-inducedtoxicity [35] The reduced thiol content is in line with oursuggestion of cadmium-induced formation of cadmium-GSH complexes which are transported out of the cells byMRP1

Several studies have demonstrated that the iron trans-porters DMT1 and FPN1 upregulated at iron deficiencyand repressed at iron sufficient conditions are involved incadmium transport in adult mammals [15 16] It has beensuggested that there are ontogenic changes in the intestinalabsorption and handling of iron in neonates and that iron

absorption and expressions of DMT1 and FPN1 are notregulated until the time of weaning [18 48] Both irontransporters are exclusively located intracellularly at midinfancy and not until weaning present at the membranes ofthe duodenal enterocytes in rats [49 50] and in pigs [17]In suckling piglets neither iron nor cadmium pretreatmentshave resulted in any effect on expression or localization ofduodenal DMT1 or FPN1 [17] In the present study cadmiumpretreatment did not affect the gene expression of DMT1or FPN1 in the Caco-2 cells Thus the increased apical tobasolateral cadmium transport in cadmium pretreated cells

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

ISRN Toxicology 7

cannot be explained by an increased expression of these irontransporters

Our results demonstrate that reduced apical uptake ofcadmium into cadmiumpretreated Caco-2 cells does not cor-relate to a reducedDMT1 or DMT1-IRE gene expressionThisindicates that cadmiummay be taken up by some other non-DMT1-mediated mechanism across the apical membraneof immature enterocytes and that cadmium pretreatmentnegatively affects this process The reduced cadmium uptakeafter cadmium pretreatment may also in part be explainedby the slightly increased gene expression of the apical effluxprotein P-gp It has been reported that long-term cadmiumexposure increases P-gp expression and activity in matureCaco-2 cells [23] In the present study cadmiumpretreatmentdid not affect gene expression or function of MRP2 whichotherwise could have explained the reduced intracellularcadmium levels following exposure to the element on theapical side The reduced cadmium uptake in the cadmium-treated immature cells did not correlate to the transport ofthe element into the basolateral chamber However it shouldbe pointed out that the amount of cadmium transportedacross the basolateral membrane of the cells only constitutesa minor fraction of the total cellular uptake Thus even if theintracellular cadmium levels are lower in cadmiumpretreatedCaco-2 monolayers the amount of metal for example MRP1is still sufficient to saturate the transporter for extracellularefflux out of the cell into the basolateral compartment

Despite the strong MT induction in vivo studies havedemonstrated that MT only plays minimal roles in the gas-trointestinal absorption of cadmium though the retention ofthe metal in other tissues is MT dependent [27] Accordinglyuptake of cadmium in the immature Caco-2 cells pretreatedwith cadmium was significantly reduced indicating that MTis not involved in the retention of cadmium in neonatalenterocytes

In summary our results showed that cadmium pretreat-ment of immature Caco-2 cells upregulated MRP1 both atthe gene expression and functional levels which correlatedto an increased transport of the metal across the basolateralmembrane In addition the two iron transporters DMT1 andFPN1 were not affected by cadmium and appeared not to beinvolved in the intestinal transport of cadmium in immatureCaco-2 cells In conclusion our data indicate that continuouscadmium exposure increases the absorption of the metalin immature intestinal cells and that MRP1 conceivably isinvolved in the intestinal cadmium absorption in neonates

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors wish to thank PhD Lucia Lazarova for valuablesupport regarding cell culture This study was supported bythe Swedish Research Council for Environment AgriculturalSciences and Spatial Planning (FORMAS)

References

[1] H M Crews L M Owen N Langford et al ldquoUse of thestable isotope 106Cd for studying dietary cadmium absorptionin humansrdquo Toxicology Letters vol 112-113 pp 201ndash207 2000

[2] G Eklund K Petersson Grawe and A Oskarsson ldquoBioavail-ability of cadmium from infant diets in newborn ratsrdquo Archivesof Toxicology vol 75 no 9 pp 522ndash530 2001

[3] G Eklund and A Oskarsson ldquoExposure of cadmium frominfant formulas and weaning foodsrdquo Food Additives and Con-taminants vol 16 no 12 pp 509ndash519 1999

[4] K P Grawe J Pickova P C Dutta and A Oskarsson ldquoFattyacid alterations in liver and milk of cadmium exposed rats andin brain of their suckling offspringrdquo Toxicology Letters vol 148no 1-2 pp 73ndash82 2004

[5] A Akesson M Berglund A Schutz P Bjellerup K BremmeandM Vahter ldquoCadmium exposure in pregnancy and lactationin relation to iron statusrdquoAmerican Journal of Public Health vol92 no 2 pp 284ndash287 2002

[6] M Berglund A Akesson B Nermell andMVahter ldquoIntestinalabsorption of dietary cadmium inwomendepends on body ironstores and fiber intakerdquo Environmental Health Perspectives vol102 no 12 pp 1058ndash1066 1994

[7] E Barany I A Bergdahl L E Bratteby et al ldquoIron statusinfluences trace element levels in human blood and serumrdquoEnvironmental Research vol 98 no 2 pp 215ndash223 2005

[8] P R Flanagan J S McLellan J Haist G Cherian M JChamberlain and L S Valberg ldquoIncreased dietary cadmiumabsorption in mice and human subjects with iron deficiencyrdquoGastroenterology vol 74 no 5 I pp 841ndash846 1978

[9] I M Olsson I Bensryd T Lundh H Ottosson S Skerfvingand A Oskarsson ldquoCadmium in blood and urinemdashimpact ofsex age dietary intake iron status and former smokingmdashassociation of renal effectsrdquo Environmental Health Perspectivesvol 110 no 12 pp 1185ndash1190 2002

[10] F Canonne-Hergaux S Gruenheid P Ponka and P GrosldquoCellular and subcellular localization of the Nramp2 irontransporter in the intestinal brush border and regulation bydietary ironrdquo Blood vol 93 no 12 pp 4406ndash4417 1999

[11] A T McKie P Marciani A Rolfs et al ldquoA novel duodenaliron-regulated transporter IREG1 implicated in the basolateraltransfer of iron to the circulationrdquo Molecular Cell vol 5 no 2pp 299ndash309 2000

[12] N C Andrews and P J Schmidt ldquoIron homeostasisrdquo AnnualReview of Physiology vol 69 pp 69ndash85 2007

[13] H Zoller R O Koch I Theurl et al ldquoExpression of theduodenal iron transporters divalent-metal transporter 1 andferroportin 1 in iron deficiency and iron overloadrdquo Gastroen-terology vol 120 no 6 pp 1412ndash1419 2001

[14] J E Nelson V R Mugford E Kilcourse R S Wang and K VKowdley ldquoRelationship between gene expression of duodenaliron transporters and iron stores in hemochromatosis subjectsrdquoAmerican Journal of Physiology vol 298 no 1 pp G57ndashG622010

[15] D W Kim K Y Kim B S Choi et al ldquoRegulation of metaltransporters by dietary iron and the relationship between bodyiron levels and cadmium uptakerdquo Archives of Toxicology vol 81no 5 pp 327ndash334 2007

[16] D Y Ryu S J Lee D W Park B S Choi C D Klaassen and JD Park ldquoDietary iron regulates intestinal cadmium absorptionthrough iron transporters in ratsrdquo Toxicology Letters vol 152no 1 pp 19ndash25 2004

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 ISRN Toxicology

[17] H OhrvikAOskarsson T Lundh S Skerfving and J TallkvistldquoImpact of iron status on cadmium uptake in suckling pigletsrdquoToxicology vol 240 no 1-2 pp 15ndash24 2007

[18] W I Leong C L Bowlus J Tallkvist and B Lonnerdal ldquoDMT1and FPN1 expression during infancy developmental regulationof iron absorptionrdquoAmerican Journal of Physiology vol 285 no6 pp G1153ndashG1161 2003

[19] F Thevenod ldquoCatch me if you can novel aspects of cadmiumtransport inmammalian cellsrdquoBioMetals vol 23 no 5 pp 857ndash875 2010

[20] R Beedholm-Ebsen K van de Wetering T Hardlei E NexoslashP Borst and S K Moestrup ldquoIdentification of multidrugresistance protein 1 (MRP1ABCC1) as a molecular gate forcellular export of cobalaminrdquo Blood vol 115 no 8 pp 1632ndash1639 2010

[21] Z S Li Y P Lu R G Zhen M Szczypka D J Thiele andP A Rea ldquoA new pathway for vacuolar cadmium seques-tration in Saccharomyces cerevisiae YCF1-catalyzed transportof bis(glutathionato)cadmiumrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no1 pp 42ndash47 1997

[22] R Tommasini R Evers E Vogt et al ldquoThe human multidrugresistance-associated protein functionally complements theyeast cadmium resistance factorrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 93 no13 pp 6743ndash6748 1996

[23] C Huynh-Delerme H Huet L Noel A Frigieri and M Kolf-Clauw ldquoIncreased functional expression of P-glycoprotein inCaco-2 TC7 cells exposed long-term to cadmiumrdquo ToxicologyIn Vitro vol 19 no 4 pp 439ndash447 2005

[24] S A Terlouw C Graeff P H E Smeets et al ldquoShort andlong term influences of heavy metals on anionic drug effluxfrom renal proximal tubulerdquo Journal of Pharmacology andExperimental Therapeutics vol 301 no 2 pp 578ndash585 2002

[25] K H Flaig K Schumann and B Elsenhans ldquoJejunal transferrates of 109cadmium chloride increase in rats in vitro and invivo after oral pretreatment with cadmium or zinc chloriderdquoToxicology vol 183 no 1ndash3 pp 199ndash209 2003

[26] A Blais S Lecoeur G Milhaud D Tome and M Kolf-ClauwldquoCadmium uptake and transepithelial transport in control andlong-term exposed Caco-2 cells the role of metallothioneinrdquoToxicology and Applied Pharmacology vol 160 no 1 pp 76ndash851999

[27] C D Klaassen J Liu and B A Diwan ldquoMetallothioneinprotection of cadmium toxicityrdquo Toxicology and Applied Phar-macology vol 238 no 3 pp 215ndash220 2009

[28] C D Klaassen J Liu and S Choudhuri ldquoMetallothioneinan intracellular protein to protect against cadmium toxicityrdquoAnnual Review of Pharmacology andToxicology vol 39 pp 267ndash294 1999

[29] P Artursson K Palm and K Luthman ldquoCaco-2 monolayersin experimental and theoretical predictions of drug transportrdquoAdvanced Drug Delivery Reviews vol 46 no 1ndash3 pp 27ndash432001

[30] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[31] I Chantret A Barbat E Dussaulx M G Brattain and AZweibaum ldquoEpithelial polarity villin expression and entero-cytic differentiation of cultured human colon carcinoma cells

a survey of twenty cell linesrdquo Cancer Research vol 48 no 7 pp1936ndash1942 1988

[32] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[33] D I Bannon R Abounader P S J Lees and J P Bressler ldquoEffectof DMT1 knockdown on iron cadmium and lead uptake inCaco-2 cellsrdquoAmerican Journal of Physiology vol 284 no 1 ppC44ndashC50 2003

[34] M Boveri P Pazos A Gennari J Casado T Hartung and PPrieto ldquoComparison of the sensitivity of different toxicologicalendpoints in Caco-2 cells after cadmium chloride treatmentrdquoArchives of Toxicology vol 78 no 4 pp 201ndash206 2004

[35] G B Cardin M Mantha and C Jumarie ldquoResistance tocadmium as a function of Caco-2 cell differentiation role ofreactive oxygen species in cadmium- but not zinc-inducedadaptation mechanismsrdquo BioMetals vol 22 no 5 pp 753ndash7692009

[36] C Jumarie P G C Campbell A Berteloot M Houde and FDenizeau ldquoCaco-2 cell line used as an in vitro model to studycadmium accumulation in intestinal epithelial cellsrdquo Journal ofMembrane Biology vol 158 no 1 pp 31ndash48 1997

[37] J Tallkvist C L Bowlus and B Lonnerdal ldquoDMT1 geneexpression and cadmium absorption in human absorptiveenterocytesrdquo Toxicology Letters vol 122 no 2 pp 171ndash177 2001

[38] P Carriere MMantha S Champagne-Paradis and C JumarieldquoCharacterization of basolateral to apical transepithelial trans-port of cadmium in intestinal TC7 cell monolayersrdquo BioMetalsvol 24 no 5 pp 857ndash874 2011

[39] G Englund F Rorsman A Ronnblom et al ldquoRegional levelsof drug transporters along the human intestinal tract co-expression of ABC and SLC transporters and comparison withCaco-2 cellsrdquo European Journal of Pharmaceutical Sciences vol29 no 3-4 pp 269ndash277 2006

[40] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo Journal of Pharmacology andExperimental Therapeutics vol 311 no 2 pp 476ndash484 2004

[41] J Tallkvist and H Tjalve ldquoTransport of nickel across monolay-ers of human intestinal Caco-2 cellsrdquo Toxicology and AppliedPharmacology vol 151 no 1 pp 117ndash122 1998

[42] I Hubatsch E G E Ragnarsson and P Artursson ldquoDetermi-nation of drug permeability and prediction of drug absorptionin Caco-2 monolayersrdquo Nature Protocols vol 2 no 9 pp 2111ndash2119 2007

[43] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[44] L Homolya Z Hollo U A Germann I Pastan M MGottesman and B Sarkadi ldquoFluorescent cellular indicators areextruded by the multidrug resistance proteinrdquo The Journal ofBiological Chemistry vol 268 no 29 pp 21493ndash21496 1993

[45] N Feller H J Broxterman D C R Wahrer and H M PinedoldquoATP-dependent efflux of calcein by the multidrug resistanceprotein (MRP) no inhibition by intracellular glutathione deple-tionrdquo FEBS Letters vol 368 no 2 pp 385ndash388 1995

[46] M S Dıaz-Cruz J Mendieta R Tauler and M EstebanldquoCadmium-binding properties of glutathione a chemometricalanalysis of voltammetric datardquo Journal of Inorganic Biochem-istry vol 66 no 1 pp 29ndash36 1997

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

ISRN Toxicology 9

[47] S H Oh S Y Lee C H Choi S H Lee and S C LimldquoCadmium adaptation is regulated by multidrug resistance-associated protein-mediated Akt pathway and metallothioneininductionrdquo Archives of Pharmacal Research vol 32 no 6 pp883ndash891 2009

[48] M Domellof R J Cohen K G Dewey O Hernell L LRivera and B Lonnerdal ldquoIron supplementation of breast-fedHonduran and Swedish infants from 4 to 9 months of agerdquoJournal of Pediatrics vol 138 no 5 pp 679ndash687 2001

[49] S L Kelleher and B Lonnerdal ldquoZinc supplementation reducesiron absorption through age-dependent changes in small intes-tine iron transporter expression in suckling rat pupsrdquo Journal ofNutrition vol 136 no 5 pp 1185ndash1191 2006

[50] V Lopez Y A Suzuki and B Lonnerdal ldquoOntogenic changesin lactoferrin receptor and DMT1 in mouse small intestineimplications for iron absorption during early liferdquo Biochemistryand Cell Biology vol 84 no 3 pp 337ndash344 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of