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ATP-Binding Cassette Transporter Expression in Human Placenta
as a Function of Pregnancy Condition□
S
Cifford W. Mason,
1
Irina A. Buhimschi, Catalin S. Buhimschi, Yafeng Dong, Carl P. Weiner,
and Peter W. Swaan
Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (C.W.M., P.W.S.); Department of
Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut (I.A.B., C.S.B.); and Department of
Obstetrics and Gynecology, University of Kansas School of Medicine, Kansas City, Kansas (Y.D., C.P.W.)
Received January 12, 2011; accepted March 22, 2011
ABSTRACT:
Fetal drug exposure is determined by the type and concentration of
placental transporters, and their regulation is central to the devel-
opment of new treatments and delivery strategies for pregnant
women and their fetuses. We tested the expression of several
clinically important transporters in the human placenta associated
with various pregnancy conditions (i.e., labor, preeclampsia, and
preterm labor-inflammation). Placentas were obtained from five
groups of women at the time of primary cesarean section: 1) term
no labor; 2) term labor; 3) preterm no labor (delivered for severe
preeclampsia); 4) preterm labor without inflammation (PTLNI); and
5) preterm labor with inflammation (PTLI). Samples were analyzed
by Western blot and immunohistochemistry to identify changes in
protein expression. Relative mRNA expression was determined by
quantitative real-time polymerase chain reaction. A functional
genomic approach was used to identify placental gene expression
and elucidate molecular events that underlie the given condition.
Placental expression of ATP-binding cassette transporters from
women in labor and women with preeclampsia was unaltered.
Multidrug resistance protein 1 (MDR1) and breast cancer resis-
tance protein (BCRP) and mRNA expression increased in placentas
of women with preterm labor with inflammation. Molecular path-
ways of genes up-regulated in PTLI samples included cytokine-
cytokine receptor interactions and inflammatory response com-
pared with those in the PTLNI group. The mRNA expression of
MDR1 and BCRP was correlated with that of interleukin-8, which
also increased significantly in PTLI samples. These data suggest
that the transfer of drugs across the placenta may be altered in
preterm pregnancy conditions associated with inflammation
through changes in MDR1 and BCRP.
Introduction
Drug treatment options during pregnancy and lactation are limited
because few products have been tested for safety and efficacy in these
two patient groups. The placenta is a partially protective barrier that
limits fetal exposure to xenobiotics, which is attributed in part to the
expression of transporter proteins on placental apical and basal mem-
brane surfaces. Among the most abundant of the apically expressed
xenobiotic transporters on the maternal side of the placenta are mul-
tidrug resistance protein (MDR) 1 (P-glycoprotein; ABCB1), multi-
drug resistance-associated protein 2 (MRP2/ABCC2), and breast can-
cer resistance protein (BCRP/ABCG2), which handle the efflux of
xenobiotics and metabolites out of the fetoplacental compartment
(Jonker et al., 2000). The localization of MDR3 (ABCB4) and MRP1
(ABCC1) is less clear, but studies suggest that these transporters may
be positioned on the basolateral membrane of the placenta where they
transport substrates from mother to fetus (Nagashige et al., 2003;
Evseenko et al., 2006). Additional transporters, including BCRP and
MRP1, line the fetal capillaries, providing yet another barrier against
xenobiotic entry (St-Pierre et al., 2000; Yeboah et al., 2006).
Expression of these clinically important transporters is dependent
on gestational age. However, drug transporter expression and regula-
tion in placenta of women with pregnancy pathology require further
definition. Preterm labor is the leading cause of perinatal morbidity
and mortality. Preeclampsia and inflammation, which are often sec-
ondary to uterine infection, are well recognized causes of preterm
birth and, when diagnosed, frequently result in clinically indicated
This work was supported in part by the National Institutes of Health National
Heart, Lung, and Blood Institute [Grant R01-HL049041]; the National Institutes of
Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant
R01-DK61425]; and the Centers for Disease Control and Prevention [Grant U01-
DP000187].
1
Current affiliation: Department of Obstetrics and Gynecology, University of
Kansas School of Medicine, Kansas City, Kansas.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.038166.
□SThe online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: MDR, multidrug resistance protein; ABC, ATP-binding cassette; MRP, multidrug resistance-associated protein; PCR, poly-
merase chain reaction; BCRP, breast cancer resistance protein; TNL, term no labor; TL, term labor; PTSPE, preterm no labor with indications for
spontaneous preeclampsia; GA, gestational age; PTLNI, preterm labor without inflammation; PTLI, preterm labor with inflammation; ANOVA,
analysis of variance; GO, Gene Ontology; FDR, false discovery rate; qRT, quantitative real-time; TNF, tumor necrosis factor; IL, interleukin; LPS,
lipopolysaccharide; C
t
, cycle threshold.
0090-9556/11/3906-1000–1007$25.00
DRUG METABOLISM AND DISPOSITION Vol. 39, No. 6
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 38166/3691996
DMD 39:1000–1007, 2011 Printed in U.S.A.
1000
premature delivery. Treatment for the prevention of preterm birth has
thus far been unsuccessful, and the rate of premature birth has in-
creased over the years. We hypothesize that pregnancy conditions
associated with preterm birth, such as spontaneous preterm labor,
preeclampsia, and preterm labor-inflammation, alter the expression of
drug transporters in human placenta. We applied immunohistochem-
ical analysis, Western blot, and quantitative real-time PCR to deter-
mine the localization and protein and mRNA expression of transport-
ers in a series of human placentas obtained from women with
clinically diagnosed pregnancy conditions. In addition, we applied
functional genomic profiling, an effective approach for obtaining
mechanistic understanding of underlying disease through changes in
gene expression (Mason et al., 2006), to gain insight into the processes
associated with abnormal labor. We postulate that these processes
may mediate the observed changes in transporter expression.
The results from these studies provide evidence for altered expres-
sion of MDR1 and BCRP during inflammation-associated spontane-
ous preterm labor. They also support the involvement of cytokine-
mediated events as a means to explain the observed increase in MDR1
and BCRP expression. Overall, our data suggest that up-regulation of
MDR1 and BCRP could alter drug transfer across the placenta. These
results will help predict human fetal drug toxicity and drug delivery
and offer new insights into the regulation of placental drug transport-
ers and the impact of various pregnancy conditions on them.
Materials and Methods
Study Design. Placenta were obtained with institutional review board
approval and after written consent from five groups of women undergoing
primary cesarean section at Yale University: 1) term no labor (TNL); 2) term
labor (TL); 3) preterm no labor delivered for severe preeclampsia (PTSPE)
[mean gestational age (GA), 30.3 weeks; range, 25.6 –33.0 weeks]; 4) preterm
labor unassociated with inflammation (PTLNI) (mean GA, 30.5 weeks; range,
25.3–36.6 weeks, histological chorioamnionitis, stage 0); and 5) preterm labor
with inflammation (PTLI) (mean GA, 28.7 weeks; range, 28.0 –33.3 weeks;
histological chorioamnionitis, stage III). Labor was defined by the presence of
regular uterine contractions accompanied by progressive cervical dilation. The
diagnosis of intra-amniotic inflammation was based on an amniotic fluid mass
restricted score of 3 or 4 plus ⬎100 white blood cells/
l
3
in the context of a
positive amniotic fluid culture in a sample that was obtained by transabdominal
amniocentesis (Buhimschi et al., 2005). These tests provide the most accurate
tools currently available to maximize the likelihood of sample homogeneity.
The mass restricted score provides qualitative information regarding the pres-
ence or absence of intra-amniotic inflammation. In brief, the score ranges from
0 to 4, depending on the presence (assigned a value of 1) or absence (assigned
a value of 0) of each of four protein biomarkers (Buhimschi et al., 2005). A
score of 3 to 4 indicates inflammation, whereas a score of 0 to 2 excludes it.
This biomarker pattern is predictive of preterm birth, histological chorioam-
nionitis, and adverse neonatal outcome. A detailed description of the mass
restricted method has been published previously (Buhimschi et al., 2005).
Preeclampsia was defined according to established criteria from the American
College of Obstetricians and Gynecologists as systolic blood pressure of 140
mm Hg or diastolic blood pressure of 90 mm Hg and proteinuria of at least ⫹1
on dipstick testing, each on two occasions 4 to 6 h apart. In a 24-h urine
collection, proteinuria was defined as ⱖ300 mg of protein. Indications for
cesarean delivery in the PTLNI group were related to spontaneous preterm
labor. The indication for cesarean delivery in the TNL and TL groups was
related to breech presentation and an arrest of cervical dilation at ⱖ6 cm,
respectively. Clinical data were retrieved from the medical records, and sta-
tistical analysis of patient demographics was performed using one-way anal-
ysis of variance (ANOVA), followed by the Student-Newman-Keuls post hoc
test for multiple comparisons.
RNA Isolation and Microarray Preparation. Total RNA isolation and
gene profiling of placenta were performed in triplicate for term and preterm
samples using the Affymetrix GeneChip Human Genome U133 Plus 2.0
microarray (Affymetrix, Santa Clara, CA) as described previously (Mason et
al., 2010).
Microarray Data Processing and Statistical Analysis. The quality of the
microarray experiment was assessed as described by Chang et al. (2007) using
bioconductor packages for statistical analysis of microarray data. Multidimen-
sional scaling analysis was performed with the signal estimates to assess
sample variability. The quality assessment and multidimensional scaling anal-
yses identified and disqualified discordant sample chips. Signal data were
obtained using the RMA algorithm. Differential gene expression between the
individual pair-wise conditions was assessed by modified ttests as described
previously (Kedziorek et al., 2010). The search for genes varying among the
conditions was made by combining all the pair-wise comparisons above to
construct an F test, which is equivalent to a one-way ANOVA for each gene
except that the residual mean squares have been moderated between genes
(Smyth, 2004). The pvalues for the tests provide a way to rank genes in terms
of the evidence for differential gene expression to obtain the most likely
differentially expressed genes between and among conditions. pⱕ0.05 and a
1.5-fold threshold were used as a cutoff for gene inclusion in our analysis.
Microarray Data Analysis. DAVID (Huang da et al., 2009), an ontology-
based Web tool, was used to evaluate statistical measures of knowledge-based
groups of genes from publications and public resources. The biological func-
tions of the genes in the placental groups were examined in DAVID on the
basis of information from the Gene Ontology (GO) terms, Kyoto Encyclopedia
of Genes and Genomes pathways, and gene descriptions from various public
databases. We distinguished genes that were up-regulated and down-regulated
(differently expressed genes) and used DAVID to determine Gene Ontology
categories that were overrepresented (enriched) with differentially expressed
genes. The false discovery rate (FDR) filter identified categories (biological
processes, pathways, or molecular functions) that were changed by random
chance. The FDR was set at 10%, and GO categories with FDR ⬍10% were
considered significantly enriched.
Quantitative Real-Time PCR. Primer sequences for amplifications were
chosen on the basis of previously published cDNA sequences (Supplemental
Table 1). For normalization of the mRNA data, the endogenous reference gene
18s rRNA was used. All primer sets were tested to ensure efficiency of
amplification over a wide range of template concentrations. SYBR Green
(Bio-Rad Laboratories, Hercules, CA) was used for amplicon detection. A melt
curve was used after amplification to ensure that all samples exhibited a single
amplicon. Each sample was assayed in triplicate. The average C
t
value (cycle
threshold for target or endogenous reference gene amplification) was estimated
using the software associated with the iCycler real-time PCR detection system
(Bio-Rad Laboratories). Relative changes in mRNA expression of the target
genes were analyzed using the ⌬⌬C
t
method (2
⫺⌬⌬C
t) (Livak and Schmittgen,
2001). In this method, the average ⌬C
t
was calculated by subtracting the
average C
t
value of the endogenous reference gene (18s rRNA) from the
average C
t
value of the target gene for the condition and control placental
groups. Fold changes in mRNA expression of target genes in placenta from the
condition groups (TL, PTSPE, PTLNI, and PTLI) were expressed relative to
that of the TNL placental control group.
To validate (biological and statistical) the microarray results, we performed
qRT-PCR on select genes that were differentially expressed and/or signifi-
cantly different in comparisons of either PTLI versus PTLNI or PTLI
versus TL.
Western Blot Analysis. Human placentas were processed according to
methods described previously (Novotna et al., 2004). In brief, placentas were
homogenized in buffer containing 250 mM sucrose, 10 mM Tris, 5 mM EDTA,
and complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN)
supplemented with phenylmethylsulfonyl fluoride (1 mM), pH 7.4. Crude
membrane fractions were obtained through differential centrifugation. The
homogenate was initially centrifuged at 10,000gfor 10 min at 4°C. The
resulting supernatant was then centrifuged at 36,000gfor 70 min at 4°C.
Protein concentration was determined using Bradford (Bio-Rad Laboratories)
assays according to the manufacturer’s protocol. Membrane protein (50 –75
g) was subjected to SDS-polyacrylamide gel electrophoresis on precast
Tris-HCl gels (Bio-Rad Laboratories). The separated proteins were transferred
to polyvinylidene difluoride membranes and blocked using 5% nonfat dry
milk. Placental transporters were detected by incubating the membranes over-
night at 4°C with a 1:500 dilution of the monoclonal primary antibody for
1001TRANSPORTER EXPRESSION AND PREGNANCY CONDITION
MDR1 (F4; Sigma-Aldrich, St. Louis, MO) and BCRP (clone BXP-21; Mil-
lipore Bioscience Research Reagents, Temecula, CA), a 1:500 dilution for
MDR3 (clone P3 II-26; Millipore Bioscience Research Reagents), a 1:400
dilution for MRP1 (MRPr1; Enzo Life Sciences, Inc., Plymouth Meeting, PA),
and a 1:50 dilution for MRP2 (M2 III-6; Millipore Bioscience Research
Reagents). The membranes were immunoblotted using peroxidase-conjugated
secondary antibody and detected using the ECL detection system (GE Health-
care Biosciences, Pittsburgh, PA). Equivalence of protein loading was con-
firmed by secondary immunoblotting with anti-

-actin antibody.
Immunohistochemical Analysis. Immunohistochemical detection of MDR1
and BCRP was performed on frozen sections of placenta from each of the five
groups of women (n⫽3/group). Placental sections were blocked and incubated
overnight at 4°C with the MDR1 and BCRP monoclonal antibodies and dilutions
used for Western blots. Biotin-labeled secondary antibodies were visualized using
peroxidase-conjugated streptavidin (Vectastain ABC kit; Vector Laboratories,
Burlingame, CA) with diaminobenzidine (Sigma-Aldrich) as the substrate. Slides
were then counterstained with hematoxylin followed by dehydration in a graded
series of ethanol dilutions, cleared by xylene substitute, and mounted with DPX
mountant (Sigma-Aldrich). Control incubations did not include primary antibody.
Statistical Analysis. Statistical analysis was done with GraphPad Prism
(version 4.0; GraphPad Software Inc., San Diego, CA). Quantitative real-time
PCR results were reported as fold change in mRNA expression of target genes
(mean ⫾S.E.M.) for each placental group relative to the mRNA expression
found in the TNL control placental group. Mean fold changes in mRNA
expression in all the groups were compared by ANOVA followed by the post
hoc Student-Newman-Keuls multiple comparison test. Pearson coefficient
analysis was used to determine the correlation between the fold changes in
mRNA expression of the target genes. Statistical significance was set at p⬍
0.05.
Results
Clinical Characteristics of Placental Samples. There were no
pathological changes in placentas from TNL, TL, and PTLNI groups
on the basis of histological evaluation. The samples from the PTLI
group were associated with histological stage III chorioamnionitis
(full-thickness inflammation of both chorion and amnion). This was
complemented by inflammation of the amnion (range, grades 1–3;
mode, grade 3), inflammation of the chorion-decidua (range, grades
3– 4; mode, grade 3), and funisitis (range, grade 1– 4; mode, grade 3).
Histological grading was based on the four-grade system devised by
Salafia et al. (1989). Pathological abnormalities associated with pla-
centas from the PTSPE group consisted of placental infarcts less than
3 cm, fibrin deposition, decidual vascular thrombosis, decidual hem-
orrhage, necrosis, and hyperplastic arteriosclerosis. Furthermore,
there were no statistically significant differences in gestational age or
fetal birth weight among those women who delivered term (no labor
versus labor) or preterm (preeclampsia, inflammation, labor, and no
labor) or in maternal age (data not shown). Thus, the observed
differences in the ABC transporters among the placental groups are
probably the result of the pregnancy condition rather than any varia-
tion in maternal age or variation in gestational age within term or
preterm groups (i.e., PTLNI versus PTLI).
Expression and Localization of Placental Drug Transporters.
Levels of MDR1, MDR3, MRP1, MRP2, and BCRP were determined
from immunoblot analyses. Greater expression of MDR1 and BCRP
was observed in placentas of women with preterm labor (Fig. 1, A and
B) than in placentas of those with term labor. Furthermore, protein
expression was higher in the placentas of women in the PTLI group
than in those of women in the PTLNI group (Fig. 1, A and B).
Other laboratories have shown that MDR1 expression is not de-
pendent on the region of the sample or on cesarean versus vaginal
delivery (Camus et al., 2006; Sun et al., 2006). MRP1, MRP2, and
MDR3 were present in all samples, but their expression was
variable and did not appear to be dramatically affected by preg-
nancy condition (Fig. 1, C–E).
The proper cellular localization is essential for transporters to
perform their transport function. Immunohistochemical analysis ver-
ified that the observed changes were due to MDR1 and BCRP (Fig. 2)
expression at the membrane of the syncytiotrophoblast cells. BCRP
was also localized to fetal blood vessel endothelial cells (Fig. 2).
In many instances, the regulation of these transporters occurs at
transcription. Given the range of gestational ages in each placental
group (condition), we increased the number of placental samples (n⫽
6 –10), after Western blot analysis and immunohistochemical analysis,
used for semiquantitative real-time PCR. We found significant in-
creases in MDR1 and BCRP gene expression in the PTLI samples
(Fig. 3), which corresponds with their observed protein levels in this
condition. There were no changes in MRP1 and MRP2 gene expres-
sion among the given conditions. MDR3 mRNA levels were signifi-
cantly increased in the PTLI group, and there were higher levels in the
PTSPE group than in the TL and TNL groups.
Functional Characteristics of Genes Overexpressed in PTLI.
PTLI compared with PTLNI. We identified 127 genes that were
overexpressed (ⱖ1.5; pⱕ0.05) in PTLI compared with PTLNI. The
enrichment of these genes was categorized by GO (pathways, biolog-
ical processes, and molecular functions). Pathway analysis using the
Kyoto Encyclopedia of Genes and Genomes database revealed only
one significantly enriched pathway, cytokine-cytokine receptor inter-
action (FDR 7.7). Significantly up-regulated genes were further cat-
egorized by biological processes and molecular functions. We iden-
tified seven biological processes that were significantly enriched:
1) response to wounding (FDR 0.6); 2) inflammatory response (FDR
0.8); 3) regulation of cell motion (FDR 3.9); 4) regulation of cell
FIG. 1. Immunoblot analysis of protein expression of MDR1 (A), BCRP (B),
MRP2 (C), MRP1 (D), and MDR3 (E) in human placentas from women after
primary cesarean section during TNL, TL, PTSPE, PTLNI, and PTLI.
1002 MASON ET AL.
proliferation (FDR 5.7); 5) defense response (FDR 6.4); 6) positive
regulation of signal transduction (FDR 7.7); and 7) positive regulation
of cell motion (FDR 8.0). Three molecular functions were signifi-
cantly enriched: 1) growth factor binding (FDR 0.1); 2) cytokine
binding (1.3); and 3) cytokine receptor activity (FDR 8.4).
PTLI compared with TL. We identified 137 genes that were over-
expressed (ⱖ1.5; pⱕ0.05) in PTLI compared with TL. We found
only the focal adhesion pathway (FDR 9.9) to be significantly en-
riched. Nine biological processes enriched: 1) female pregnancy (FDR
0.4); 2) tube development (FDR 3.3); 3) ossification (FDR 3.8);
4) positive regulation of kinase activity (FDR 4.3); 5) bone develop-
ment (FDR 5.1); 6) positive regulation of transferase activity (FDR
5.2); 7) wound healing (FDR 7.08); 8) regulation of locomotion (FDR
7.3); ans 9) anion transport (FDR 9.4); two molecular functions were
enriched: 1) growth factor binding (FDR 1.95); and 2) actin binding
(FDR 5.7).
Functional Characteristics of Genes Underexpressed in PTLI.
PTLI compared with PTLNI. We identified 216 genes that were
underexpressed (ⱖ1.5; pⱕ0.05) in PTLI compared with PTLNI.
There was only one significantly enriched pathway: extracellular
matrix-receptor interaction (FDR 1.7). Ten biological processes were
found to be significantly enriched: 1) unsaturated fatty acid metabolic
process (FDR 0.6); 2) fatty acid metabolic process (FDR 1.1);
3) branching morphogenesis of a tube (FDR 1.8); 4) morphogenesis of
a branching structure (FDR 3.2); 5) eicosanoid metabolic process
(FDR 3.9); 6) tube morphogenesis (FDR 6.8); 7) negative regulation
of binding (FDR 8.7); 8) lipid biosynthesis process (FDR 8.9); 9)
positive regulation of cell adhesion (FDR 9.2); and 10) eicosanoid
biosynthesis process (FDR 9.4). Six molecular functions were signif-
icantly enriched: 1) lipid binding (FDR 0.4); 2) coenzyme binding
(FDR 1.8); 3) cofactor binding (FDR 3.6); 4) actin binding (FDR 6.7);
5) peroxidase activity (FDR 8.1); and 6) oxidoreductase activity,
acting on peroxide as acceptor (FDR 8.1).
PTLI compared with TL. There were 140 genes underexpressed
(ⱖ1.5; pⱕ0.05) in PTLI compared with TL. No pathways or
biological processes were significantly enriched (FDR ⬍10%). Lipid
binding (FDR 1.8) was the only molecular function that was signifi-
cantly enriched.
Biological Validation of Microarray Gene Expression. To verify
the microarray results, highly differentially expressed genes including

1
adrenergic receptor (ADRB1), eosinophil major basic protein, also
referred to as proteoglycan 2 (MBP or PRG2), stanniocalcin 1 (STC1),
and hydroxysteroid (11-

) dehydrogenase 2 (HSD11

2) were se-
lected and analyzed by qRT-PCR. We confirmed changes in expres-
sion (direction and magnitude) of these genes between PTLI and
PTLNI (Supplemental Table 2) and PTLI and TL (Supplemental
Table 3). Overall, the direction of change in gene expression by
qRT-PCR was consistent with the microarray analysis of these four
genes. Additional genes encoding human chorionic gonadotropin

polypeptide (

hCG), retinoid X receptor
␣
(RXR
␣
), and GATA bind-
ing protein 2 (GATA2) were used to confirm statistical significance of
microarray genes.
Changes in the mRNA Expression of Proinflammatory Cyto-
kines in Various Placental Conditions. Previous reports have indi-
cated inverse correlations between MDR1 and proinflammatory cy-
tokines. However, neither TNF-
␣
nor IL-6 mRNA expression was
altered, and fold changes in IL-8 mRNA expression were significantly
increased (12.1-fold, p⬍0.001) in PTLI compared with TNL (Fig. 4).
IL-8 mRNA expression in PTLI was greater than that in other con-
ditions including PTSPE, in which the fold change in IL-8 mRNA
expression (6.3-fold, p⬍0.05) was greater than that of TL but not that
of PTL with and without inflammation (Fig. 4). The fold changes in
FIG. 2. Immunohistochemical localization of MDR1 and BCRP in human placen-
tas. Results show MDR1 and BCRP localization to the membrane of the syncy-
tiotrophoblast cells (arrows) in all tissue conditions (n⫽3/tissue group). BCRP was
also localized to the fetal blood vessel endothelial cells (arrowheads). The MDR1
and BCRP controls are indicative of immunostaining without primary antibody.
Original magnification, 100⫻. Scale bars (in control), 120
m.
1003TRANSPORTER EXPRESSION AND PREGNANCY CONDITION
mRNA expression of IL-8 were correlated with that of MDR1 (Pear-
son r⫽0.50, p⬍0.05, respectively) and that of BCRP (Pearson r⫽
0.65, p⬍0.00) among the placental groups.
Discussion
Expression patterns of placental ABC transporters vary with ges-
tational age and medical condition during pregnancy. The general
consensus is that MDR1 and BCRP expression decline (Gil et al.,
2005; Mathias et al., 2005; Sun et al., 2006; Meyer zu Schwabedissen
et al., 2006), whereas MRP2 and MDR3 levels increase with gesta-
tional age toward term (Patel et al., 2003; Meyer zu Schwabedissen et
al., 2005). These changes may reflect a physiological adaptation to the
changing requirements for fetal protection, especially in the preterm
period. However, several discrepancies have been observed, particu-
larly in humans. For example, Mathias et al. (2005) reported that
BCRP expression in human placenta does not change significantly
with gestational age, whereas Yeboah et al. (2006) showed that
placental BCRP levels increased toward term, whereas mRNA ex-
pression remained unchanged. The placental samples used here en-
compass two very distinct gestational time points: a preterm preg-
nancy period (28 –31 weeks) and a term pregnancy period (38 – 41
weeks). Because this is not a continuous time course analysis, we
cannot infer gestational regulation of the transporters inspected. How-
ever, we do observe relatively higher expression of MDR1 and BCRP
in placental samples from preterm women compared with those from
term women (Fig. 1, A and B).
There were no apparent changes in expression levels of the ABC
transporters in response to labor (term or preterm), which is consistent
with prior reports in which expression levels of BCRP in human
reproductive tissues (fetal membranes and attached deciduas) (Ye-
boah et al., 2008) and MDR1 in human placenta (Sun et al., 2006)
were not altered by labor at term. Our data further support the fact that
MDR1 and BCRP expression does not change with preterm labor.
Changes in MRP1, MRP2, and MDR3 expression were less apparent
in crude membrane fractions of placental tissue. These preparations
differ from isolated syncytiotrophoblasts in purity and may explain
potential differences with other results, specifically in the extent of
BCRP and MRP1 expression, which is also localized to the fetal
capillary endothelial cells. However, immunohistochemical analysis
revealed that the cellular localization of MDR1 and BCRP was not
altered in the placental groups. Furthermore, mRNA expression ap-
pears to parallel that of protein expression. We suspect that the
observed differences in protein expression are due to the specific
pregnancy condition rather than to variation in experimental design.
Preterm birth is the leading cause of perinatal morbidity and mor-
tality. A large proportion of preterm births are associated with pre-
eclampsia and inflammation, often secondary to infection. It is in-
creasingly clear that inflammation (outside of that associated with
pregnancy) affects the expression of drug transporters (Petrovic et al.,
2007). We found that both MDR1 and BCRP (protein and mRNA
expression) are highest in placentas from women with inflammation
(Figs. 1, A and B, and 3, A and B). Given their high white blood cell
FIG. 3. Relative changes in the gene expression (A, MDR1; B, BCRP; C, MRP2; D, MRP1; and E, MDR3) of ABC transporters was determined by real-time PCR in human
placenta (n⫽6 –10) from women with various pregnancy conditions. The mean fold change in ABC transporter genes, normalized to the endogenous reference gene, 18s
rRNA and relative to the expression of the TNL control, was calculated in each sample by the 2
⫺⌬⌬C
tmethod. Differences between all possible pairs of group means were
determined by one-way ANOVA followed by a Student-Newman-Keuls multiple comparison post hoc test. Data are presented as mean ⫾S.E.M. ⴱ,p⬍0.05; ⴱⴱ,p⬍
0.01; ⴱⴱⴱ,p⬍0.001; NS, no significant difference. There were no differences in the mean fold changes of MRP1 and MRP2. Relative mRNA expression in PTLI was
significantly higher for MDR1 (p⬍0.01), BCRP (p⬍0.001), and MDR3 (p⬍0.05), by 6.4-, 3.7-, and 12.7-fold, respectively, than that in the TNL placental group.
1004 MASON ET AL.
counts, it is probable that inflammation (i.e., stage III chorioamnio-
nitis) is a response to uterine infection. These data represent the first
evidence of direct infection-mediated transporter regulation.
Our findings differ from prior literature reports noting transporter
down-regulation during inflammation caused by inflammatory cyto-
kines such as TNF-
␣
, IL-6, and endotoxin (i.e., LPS) in rats (Sukhai
et al., 2001; Chen et al., 2005; Wang et al., 2005) and human primary
placental cells (Evseenko et al., 2007). We offer several possible
explanations for these differences: the impact of LPS-induced inflam-
mation on drug transporters has yet to be evaluated at different
gestational stages, and previous reports have indicated that preterm
placentas respond differently to LPS than those at term, specifically in
their patterns of cytokine release (Holcberg et al., 2007). More im-
portantly, common clinical infections of the reproductive compart-
ments are associated with microorganisms that lack LPS, such as
Ureaplasma species, Mycoplasma hominis, and group B Streptococ-
cus. It is evident that different pathogens or pathogen components
elicit diverse patterns of gene expression and cytokine release (Flad et
al., 1993; Ueyama et al., 2005). For example, IL-8 was significantly
elevated in amniotic fluid and umbilical cord blood in cases of
intrauterine Ureaplasma infection, which was not observed with other
pathogens (Witt et al., 2005). Taken together, these results indicate
that stimulation of alternative cytokines or inflammatory mediators
could have contrasting affects on ABC transporters. Thus, observed
differences in transporter regulation among various experimental
models are not surprising. This is evident in cases of patients with
inflammation from rheumatoid arthritis in whom an increase, rather
than a decrease, in MDR1 expression is observed (Llorente et al.,
2000). It is clear that the impact of inflammation on drug transporters
in the human placenta is still in a nascent stage. The development of
models that more closely mimic the human pathological pregnancy
condition will expound differences in transporter regulation, including
the need to evaluate various inflammatory pathogens and or stimuli
during pregnancy.
Hence, we adopted a functional genomic approach to identify
potential mechanisms driving changes in gene expression during
PTLI. We hypothesized that underlying inflammatory events may
account for the observed MDR1 and BCRP regulation. Because the
PTLI group is defined, in part, by labor, it was logical to compare this
placental group with those also associated with labor, specifically
PTLNI and TL. In general, functional pathways and biological pro-
cesses associated with pregnancy and development were found to be
enriched (overrepresented) with genes overexpressed in PTLI com-
pared with TL. Of interest, these events appeared to be similar in
comparisons of genes up-regulated in PTLNI compared with TL
(supplemental data). When we compared PTLI with PTLNI, we found
that genes were up-regulated in processes associated with inflamma-
tion and cellular regulation, in particular, the cytokine-cytokine re-
ceptor interaction pathway, as were molecular functions related to
cytokine activity. These results provide biological relevance for the
given PTLI condition and further suggest that proinflammatory cyto-
kines may be involved in the pathways regulating MDR1 and BCRP.
Thus, we evaluated the correlation between expression of well rec-
ognized proinflammatory cytokines, IL-6, IL-8, and TNF-
␣
and
MDR1 and BCRP.
IL-8 is a potent chemotactic agent and activates neutrophils, po-
tentiating the host defense mechanism against inflammation. It is
thought to be constitutively produced by the human placenta (Shi-
moya et al., 1992) independent of preterm versus term delivery
(Keelan et al., 1999). IL-8 is increased in placental tissue during
FIG. 4. Relative changes in mRNA expression of proinflammatory cytokines. A, mRNA levels of IL-6, TNF-
␣
, and IL-8 were analyzed by quantitative real-time PCR.
The mean fold change in ABC transporter genes, normalized to the endogenous reference gene, 18s rRNA, and relative to the expression of the TNL control, was calculated
in each sample by the 2
⫺⌬⌬C
tmethod. Differences between all possible pairs of group means were determined by one-way ANOVA followed by a Student-Newman-Keuls
multiple comparison post hoc test. Data shown are the mean ⫾S.E.M. from 6 to 10 independent placentas from each of the five groups. ⴱ,p⬍0.05; ⴱⴱ,p⬍0.01;
ⴱⴱⴱ,p⬍0.001; NS, no significant difference. There were no differences in the mean fold changes of TNF-
␣
and IL-6. Relative mRNA expression in PTSPE and PTLI
was significantly higher for IL-8 by 6.3-fold (p⬍0.05) and 12.1-fold (p⬍0.001), respectively, than that in the TNL placental group. B, fold changes in mRNA expression
of IL-8 transcripts were correlated with those for MDR1 and BCRP in the human placentas of woman with pregnancy conditions. Correlation analysis was performed using
Pearson correlation.
1005TRANSPORTER EXPRESSION AND PREGNANCY CONDITION
chorioamnionitis (Lockwood et al., 2006) as well as in amniotic fluid
and cord blood from women with intrauterine infection. We found a
significant fold increase in IL-8 mRNA expression in placentas in
PTLI, whereas there were no differences in placentas in preterm
versus term pregnancy as demonstrated in comparisons between
PTLNI and TL and TNL (Fig. 4). These results are consistent with the
literature. Fold changes in IL-8 mRNA expression were correlated
with that of MDR1 and that of BCRP. On the basis of the aforemen-
tioned association between IL-8 and inflammation-infection, these
data support altered expression of MDR1 and BCRP in placentas of
women with preterm labor and inflammation. Of interest, there were
no changes in mRNA expression of other proinflammatory cytokines,
TNF-
␣
and IL-6. However, changes in these cytokines may be more
apparent in the amniotic fluid or the maternal or fetal serum.
We observed elevated placental mRNA expression of IL-8 in
women with PTSPE compared with women with term labor (TNL and
TL). These results are consistent with reports of increased IL-8
production in trophoblasts (Bowen et al., 2005) and elevated IL-8
levels in maternal and umbilical cord serum as well as amniotic fluid
of preeclamptic women (Nakabayashi et al., 1998; Laskowska et al.,
2007). In contrast, Wang et al. (1999) found a decrease in placental
IL-8 production in preeclampsia. Additional experiments may be
required to determine the association of preeclampsia and cytokine-
specific production.
In this study, we did not detect significant changes in protein or
mRNA expression of the multidrug-associated proteins, MRP1 and
MRP2. LPS and proinflammatory cytokines have been shown to
down-regulate MRP2 expression in the liver of rodents (Teng and
Piquette-Miller, 2008); however, there are currently no data to support
inflammatory-induced changes in MRP2 and MRP1 expression in
humans and in placental tissue. Although MDR3 has generally been
considered a liver-specific transporter, MDR3 expression in human
term and preterm placentas has been described previously (Patel et al.,
2003); however, its physiologic function in syncytiotrophoblasts re-
mains speculative. We observed that MDR3 levels were not altered to
the same extent as its mRNA expression. Others have also indicated
discrepancies in MDR3 and its mRNA expression in trophoblasts,
which may be attributed, in part, to translational regulation (Evseenko
et al., 2006).
In the present study, we found that MDR1 and BCRP are signifi-
cantly regulated in human placenta. Prior studies have shown that
MDR1 and BCRP are coregulated in various tissue barriers to enhance
tissue protection from xenobiotics. For example, de Vries et al. (2007)
showed that these two transporters act in concert to limit the pene-
tration of topotecan at the blood-brain barrier. Like the blood-brain
barrier, the placenta protects against harmful toxic substances and
restricts the entry of therapeutic agents. Therefore, changes in placen-
tal expression of these transporters could have a profound impact on
drug efficacy or toxicity. We further demonstrated that both MDR1
and BCRP expression increase in association with underlying inflam-
mation. Up-regulation of MDR1 and BCRP in placenta during pre-
term inflammation and/or labor could significantly impair therapeutic
intervention. For example, MDR1 and BCRP transport a variety of
drugs including drugs necessary for fetal therapy. BCRP/Bcrp1 sig-
nificantly limits the fetal level of nitrofurantoin, an antibiotic com-
monly used to treat urinary tract infections during pregnancy (Zhang
et al., 2007), whereas MDR1/Mdr1a/b transports antibiotics such as
azithromycin, erythromycin, clarithromycin, levofloxacin, and rifam-
pin (Thuerauf and Fromm, 2006), agents currently used to prevent
maternofetal infections. MDR1 may also limit the transplacental
transfer of protease inhibitors such as nelfinavir, ritonavir, saquinavir,
and lopinavir, which are used in human immunodeficiency virus-
infected women to prevent transmission to the fetus. At the present
time, perinatal drug therapy in an inflamed and/or infected maternal-
fetal milieu is secondary to clinical premature fetal delivery. Further
studies will be needed to demonstrate that placental MDR1 and BCRP
expression during preterm inflammatory conditions directly correlates
with drug exposure and outcome. A variety of placental transporters
localize to the maternal interface of the placenta, the fetal membrane
surface, or both. Additional studies should be focused on other im-
portant placental transporter proteins and their regulation under var-
ious pregnancy conditions.
Authorship Contributions
Participated in research design: Mason, Weiner, and Swaan.
Conducted experiments: Mason and Dong.
Contributed new reagents or analytic tools: I.A. Buhismschi and C.S.
Buhismschi.
Performed data analysis: Mason and Swaan.
Wrote or contributed to the writing of the manuscript: Mason, Weiner, and
Swaan.
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Address correspondence to: Dr. Peter W. Swaan, Department of Pharma-
ceutical Sciences, University of Maryland, 20 Penn St., HSF2-621, Baltimore, MD
21201. E-mail: pswaan@rx.umaryland.edu
1007TRANSPORTER EXPRESSION AND PREGNANCY CONDITION
