DMD Fast Forward. Published on July 6, 2010 as doi:10.1124/dmd.110.033571
DMD #33571
The role of the placenta in fetus exposure to xenobiotics: importance of membrane
transporters, human models for transfer studies
Caroline Prouillac, Sylvaine Lecoeur*
Métabolisme et Toxicologie Comparée des Xénobiotiques (UMR 1233 INRA), VetAgroSup,
Campus Vétérinaire de Lyon, 1 avenue Bourgelat, 69280 Marcy l’Etoile, France.
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Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.
DMD #33571
Running title:
Placental membrane transporters and models for transfer studies
Corresponding author: Sylvaine Lecoeur,
Métabolisme et Toxicologie Comparée des Xénobiotiques (UMR 1233 INRA), VetAgroSup,
Campus Vétérinaire de Lyon, 1 avenue Bourgelat, 69280 Marcy l’Etoile, France.
Tel: +33478872636 ; fax: +330478878012
e-mail: [email protected]
Number of text pages: 44
Number of figures: 1
Number of tables: 4
Number of references: 205
Number of words in abstract: 201
Number of words in introduction: 378
A list of nonstandard abbreviations:
ATP Binding Cassette transporters: ABC transporters
Enzymes of Xenobiotic Metabolism: EXM
CYP: cytochrome P450
P-glycoprotein: P-gp, MDR1, ABCB1
Multi-drug Resistance-associated Proteins: MRPs, ABCCs
Breast Cancer Resistance Protein: BCRP, ABCG2
Solute Carrier Transporters: SLC transporters
Organic Anion-Transporting Polypeptides: OATPs
Organic Anion Transporters: OATs
Organic Cation Transporters: OCTs
Organic Cation/Carnitine Transporter: OCTN
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Serotonin Transporter: SERT
Norepinephrin Transporter: NET
Monocarboxylate Transporters: MCTs
Equilibrative Nucleoside Transporters: ENTs
Folate Receptor alpha: FRα
Reduced Folate Carrier 1: RFC-1
Proton-coupled folate transporter/heme carrier protein 1: PCFT/HCP1
Amino acid Transporter: ASCT
Single Nucleotide Polymorphisms: SNPs
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Abstract
The placenta is a key organ in fetal growth and development, as it controls maternal-to-fetal
exchanges of nutrients and hormones. It also interferes with drug delivery to the fetus, by
expressing active membrane transporters and xenobiotic metabolism enzymes. Developing
strategies to understand the role of the placenta in drug delivery is a challenge in toxicology.
Despite common physiological functions, the placentas of different species are heterogeneous
in both their morphology and in their expression of membrane transporters and metabolizing
proteins. This raises the difficulty of obtaining a good representative model of human
placental transfer. Up to now, different in vitro, in vivo and ex vivo tools have been used to
elucidate transport and metabolism processes in the human placenta. After having
recapitulated the typical features of human placenta, this review presents the placental
enzymes of xenobiotic metabolism, ATP Binding Cassette transporters, Solute Carrier
Transporters, and their role in fetus exposure to xenobiotics. It also compares the
characteristics of different models of human placenta, in terms of membrane localization of
transporters, and the expression of xenobiotic metabolism enzymes. The use of these models
for toxicological studies, in particular xenobiotic transfer, is described, and the advantages
and limits of each model are summarized.
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Contents
1. Introduction
2. Physiological function of human placental barrier
3. Enzymes of Xenobiotic Metabolism (EXM) content
4. Membrane transport in placenta
4.1.Transplacental transfer of xenobiotics
4.2. ATP Binding Cassette (ABC) transporters
4.2.1. P-glycoprotein (P-gp, MDR1, ABCB1)
4.2.2. Multi-drug Resistance-associated Proteins (MRPs, ABCCs)
4.2.3. Breast Cancer Resistance Protein (BCRP, ABCG2)
4.3. Solute Carrier (SLC) Transporters
4.3.1. Organic Anion-Transporting Polypeptides (OATPs)
4.3.2. Organic Anion Transporters (OATs)
4.3.3. Organic Cation Transporters (OCTs)
4.3.4. Monoamine Transporters
4.3.5. Monocarboxylate Transporters (MCTs)
4.3.6. Equilibrative Nucleoside Transporters (ENTs)
4.3.7. Folate Transporters
5. Variation of transporters expression during pregnancy
5.1. ABCG2
5.2. ABCB1, ABCB3
5.3. ABCC1
5.4. OATPs
5.5 Folate Transporters
6. The use of cell lines as placental barrier models
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6.1. Human choriocarcinoma cell lines
6.1.1. BeWo cell line
6.1.2. JAR and JEG-3 cell lines
6.2. Human primary cells
7. Other models for placental transfer studies
7.1. Feto-maternal blood concentration ratio
7.2. Human coelocentesis
7.3. Perfused single human placental cotyledon model
7.4. Human trophoblast tissue preparations
7.5. Membrane vesicles.
8. Conclusions and perspectives.
References
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1. Introduction
The placenta is a key organ for the growth and development of the embryo and fetus during
pregnancy: this semi-permeable barrier separates the mother and the fetus and regulates the
exchange of nutrients, gases, wastes, endogenous and foreign molecules between maternal
and fetal circulations. It has traditionally been considered as a highly permeable organ for a
large variety of substances with diverse molecular structures that are readily able to cross it
from the maternal blood to reach the fetus. Indeed, fetal exposure to maternal intake was
proved for the first time with the thalidomide disaster in 1957-1961. Consequently, it is
recommended to limit the use of drugs during pregnancy as much as possible.
The human placenta barrier consists of a single limiting layer of multinuclear cells, known as
syncytiotrophoblasts (Enders et al., 1999). A microvillous brush border membrane, in direct
contact with maternal blood, constitutes the maternal-facing plasma membrane of the
syncytium. On the opposite side, the basal membrane faces the fetal circulation and lacks
microvillar projections. Both sides of the syncytium are not only structurally distinct, but also
differ in the localization of transporters, enzymes, and hormone receptors. Existing
knowledge underlines the existence of different systems, including plasma membrane carriers,
biotransforming enzymes, and export pumps, that determine the selectivity and efficacy of the
so-called placental barrier. Good understanding of the molecular bases of these processes and
their regulation is crucial for predicting the risk of fetal exposure to active agents.
Beside drug exposure, which can be limited voluntarily during pregnancy, fetus exposure to
food contaminants is characterized by chronic exposure to low doses. Very little information
is available on fetal exposure to these pollutants and their long-term effects. Drug transporters
are involved in the regulation of the chemical environment of the fetus by selectively
transporting and removing toxic substrates. Thus placental epithelia expressing xenobiotic
metabolism enzymes and protein transporters are very helpful models for the in vitro study of
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quantitative and qualitative transfers of molecules to the fetus.
Different experimental trophoblast systems are available, including immortalized cell lines,
which are derived from normal and malignant tissues. In this review, we present a synthesis
on xenobiotic metabolism enzymes and transporters in the placenta, and update the different
experimental models potentially available for the study of the barrier function.
2. Physiological function of the human placental barrier
As in other primate species, the human placenta is haemochorial and characterized by direct
contact between maternal blood and trophoblast (figure 1a). The human placenta is divided
into functional vascular units called cotyledons. Within the cotyledon, the villus tree contains
a barrier separating maternal circulation from fetal circulation. The layer of tissue separating
maternal and fetal circulation is composed of the endothelium of fetal capillaries and the
trophoblast, containing the villus strom, the cytotrophoblast and the syncytiotrophoblast. The
thickness of the syncytiotrophoblast layer decreases during gestation, the cytotrophoblast
becomes discontinuous, and exchange-processes between mother and fetus are facilitated (van
der Aa et al., 1998, figure 1b).
The major function of the placenta is the transfer of nutrients that support embryonic and fetal
growth and development. During early gestation, the placenta mediates the implantation of
the embryo in the uterus and produces hormones that prevent the end of the ovarian cycle.
The luteinizing hormone and the human chorionic gonadotrophin have a stimulating action on
estrogen and progesterone synthesis by the placenta (Pasqualini et al., 1985). After
implantation, the placenta regulates the supply of nutrients and oxygen to the fetus. It also
produces angiogenic factors, vasodilators, growth hormones, placental lactogens and different
hormones, all of which stimulate maternal blood cell production, increase blood volume,
regulate appetite, etc.). Other functions of the placenta are immune function (immunoglobulin
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transfert), and removal of waste or toxic products from the fetus and metabolism. The
presence of metabolism enzymes and transport systems in the placenta make these functions
possible.
3. Enzymes of Xenobiotic Metabolism (EXM) content. The human placenta contains a
wealth of enzymatic machinery responsible for both phase I and phase II reactions (table 1).
However, data on the contribution of placental biotranformations in the conversion of
xenobiotics into potential toxic metabolites are very scarce. Compared to the liver, placental
metabolism seems to be minor and does not limit the extent of xenobiotic passage across the
placenta (Pasanen, 1999). Several cytochromes P450 (CYP) including CYP1, CYP2 and
CYP3 have been isolated from the placenta. These proteins are largely responsible for the
detoxification of drugs and toxins. However, the members and the quantity of CYPs vary as a
function of placental development, length of gestation, and maternal health status (Hakkola et
al., 1996a,b). Expression and activities of human CYP enzymes decline during gestation from
the first trimester of pregnancy to the second and third trimesters (Syme et al., 2004). In the
placenta, a large number of enzymes are involved in progesterone and estrogen synthesis from
fetal and maternal precursors (Pasqualini, 2005, Smuc et al., 2009). Enzymes involved in
steroid hormone metabolism are sulfatase (Salido et al., 1990), 3β-hydroxysteroid
dehydrogenase (Luu-The et al. 1990), aromatase and 17β-hydroxysteroid dehydrogenase
(type 1 and type 2) (Luu-The et al. 1989). CYP19 and CYP11A are the major isoforms
expressed (mRNA quantification, Nishimura et al., 2003). Human placenta contains a few
forms of phase II enzymes: glutathione S-transferase alpha and pi (Pacifici et al., 1981;
Pasanen et al., 1997), epoxide hydrolase (Pacifici et al., 1983), N-acetyltranferase (Derewlany
et al., 1994) and sulfotransferases isoforms such as SULT1A1 and SULT1A3 (Mitra et al.,
2009). UDP-glucuronyltransferase isoforms such as UGT1A and UGT2B are also expressed
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(Collier et al., 2002a,b). Data on EXM contents in placenta cell lines are very scarce
compared to those for the whole organ: only a few CYP, including CYP19, and
hydroxysteroid deshydrogenase enzymes have been reported (table 1).
4. Membrane transport in placenta
4.1. Transplacental transfer of xenobiotics. The transfer of molecules between maternal and
fetal circulation occurs across the endothelial-syncytial membrane of placenta. Drug
permeation can be influenced by numerous factors: drug properties (degree of ionization,
lipophilicity, protein binding, molecular weight), placental characteristics (blood flow,
concentration gradient of the drug across the barrier, pH gradient, thinning and aging barrier
with advancement of pregnancy, increasing surface area as pregnancy progesses, developing
metabolism, protein gradients), maternal and fetal factors (fetal growth and development, fetal
metabolism, fetal tissue binding, maternal metabolism, maternal health condition) (Poulsen et
al., 2009). Most of the drugs crossing the human placenta diffuse passively. In this case,
placental blood-flow, pH of maternal and fetal blood, physicochemical characteristics of the
compounds and protein binding determine ability to cross placental membranes (Pacifici and
Nottoli, 1995; Audus, 1999). Facilitated diffusion, phagocytosis and pinocytosis are less
important routes of placental drug transfer (Syme et al., 2004).
In 1998, the report of carrier-mediated transport of xenobiotics in the placenta stressed the
role of ATP Binding Cassette (ABC) transporters in this organ (Lankas et al.1998).
Depending on the localization of these transporters in the apical (maternal facing brush border
membrane) and basal (fetal facing basal membrane) membrane of the syncytiotrophoblast, the
substrates are preferentially transported either in maternal circulation or in fetal circulation
(Unadkat et al., 2004). Placental xenobiotic transporters and their polarized distribution are
summarized in table 2.
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4.2. ATP Binding cassette (ABC) transporters.
4.2.1. P-glycoprotein (P-gp, MDR1, ABCB1). The placental barrier had been commonly
considered as a permeable barrier until the publication of the work by Lankas et al. in 1998.
These authors stressed the role of P-gp in the mouse placenta by exposing pregnant dams,
which contained a spontaneous mutation in the mdr1 gene, to the teratogenic isomer of
avermectin (1.5mg/kg/day, oral administration, 6-15 days gestation) which is a substrate of Pgp. They demonstrated that the degree of chemical exposure of fetuses within each litter was
inversely related to the expression of placental P-gp, linking the fetal genotype to
pharmacokinetic changes and subsequently developmental toxicity.
Other studies confirmed the involvement of P-gp in the limited fetal penetration of various
potentially harmful or therapeutic compounds such as digoxin, saquinavir, paclitaxel (study
performed in P-gp deficient mice, Smit et al., 1999) and indinavir (study using an in vitro
model of human placenta perfusion, Sudhakaran et al., 2007). Indeed, Smit et al. (1999) used
mice with a targeted disruption of Mdr1a and Mdr1b genes. Mdr1a+/–/1b+/– females were
mated with Mdr1a+/–/1b+/– males to obtain fetuses of 3 genotypes (Mdr1a+/+/1b+/+,
Mdr1a+/–/1b+/–, and Mdr 1a–/–/1b–/–) in a single mother. Intravenous administration of a
single dose of P-gp substrate digoxin, saquinavir and paclitaxel to pregnant dams (gestation
day 15) revealed that 2.4-, 7-, and 16-times more drug, respectively, crossed the placenta and
entered the mdr1a−/−/1b−/− fetus, when compared to the wild-type (mdr1a+/+/1b+/+) fetus.
This study also demonstrated that P-gp activity was completely inhibited by oral
administration of the P-gp blockers PSC833 and GG918 to heterozygous mothers, indicating
that P-gp function in the placenta can be cancelled by pharmacological means.
ABCB1 mRNA is strongly expressed in the human placenta, comparable with mRNA levels
in the intestine and liver (Bremer et al., 1992). Protein expression, confirmed by
immunohistochemical studies, is located on the maternal side of the trophoblast layer (St-
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Pierre et al., 2000), and on the syncytiotrophoblast microvillous border of the first-trimester
and term placentas (MacFarland et al., 1994; Nakamura et al. 1997).
The mRNA expression of MDR3 (MDR1 homolog, ABCB4) has also been reported in human
term and pre-term placenta by Patel et al. (2003). These results were confirmed by Evseenko
et al. (2006a) who identified MDR3 protein in the basolateral membrane of the syncytium of
the term placenta and suggested that MDR3 mediated the vectorial transport of substrates
toward the fetus. MDR3 functions as a lipid translocator and may also interact with
pharmaceutical drugs (Kimura et al., 2007). Although an overexpression of MDR3 was
reported in resistant tumor cells (Scheffer et al., 2000), the role of MDR3 in multidrug
resistance is probably minor and remains to be confirmed.
4.2.2. Multidrug Resistance-associated Proteins (MRPs, ABCCs). The human term
placenta expresses at least three members of the MRP family: MRP1, MRP2, and MRP3 (StPierre et al., 2000). The localization of MRP1 has been studied using human placental brushborder, basal membrane vesicles and immunohistochemical studies, but the results are
controversial, as both apical (St-Pierre et al., 2000) and basal (Nagashige et al., 2003)
localization have been reported. MRP2 was clearly detected by both immunofluorescence and
western blotting in the apical membranes of syncytiotrophoblasts, while immunoblotting
suggested an apical localization facing the maternal blood of MRP3 (St-Pierre et al., 2000).
MRPs mRNA have been identified in placental cell lines: MRP1 and MRP2 transcripts were
found in the human choriocarcinoma cell line BeWo (Pascolo et al., 2000; Prouillac et al.,
2009). Serrano et al. (2007) also reported the comparative mRNA expression of members of
the MRP family (MRP1, MRP2, MRP3, MRP4) in human trophoblast and choriocarcinoma
cells (BeWo, Jeg-3, JAR). MRP1, MRP2 and MRP3 are known to mediate the transport of
various glucuronides, including estradiol and biblirubin, and handle various xenobiotics and
their metabolites as substrates (as a review see Deeley et al., 2006). However their role in the
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placenta has not been clearly established. It is probably related to the efflux of polar
conjugates of xenobiotics or metabolites of endogenous compounds as steroid conjugates.
Protein and mRNA expression, localization, and the function of MRP5 in human placenta
have been studied by Pascolo et al. (2003) and Zu Schwabedissen et al. (2005). Mostly
located in the basal membrane of syncytiotrophoblasts, and in and around fetal vessels, MRP5
has also been detected in the apical membrane. MRP5 acts as an export pump for cyclic
nucleotides, especially cGMP, which play a major role in cytotrophoboblast differentiation.
Although the role of MRP5 in this process is still unknown, MRP5 has been shown to confer
resistance to nucleobase and nucleoside analogs used in anticancer and antiviral therapy, by
cellular export of nucleoside monophosphate (Wijnholds et al., 2000).
4.2.3. Breast Cancer Resistance Protein (BCRP, ABCG2). Evidence of a BCRP-mediated
transport in the placenta was highlighted by Jonker et al. (2000). Using P-gp-deficient
pregnant mice, the authors demonstrated that the co-administration (at gestation day 15) of
Bcrp1 inhibitor GF120918 (50 mg/kg, oral administration) increased the fetal concentration of
topotecan (a BCRP/Bcrp1 substrate) after drug administration (1 mg/kg, oral administration).
Other in vivo studies using Bcrp1-knockout mice demonstrated the transport activity of
placental bcrp1 (Enokizono et al., 2007; Zhang et al., 2007) and its role in limiting fetal
exposure to phytoestrogen (30 µmol/kg genistein, coumestrol or daidzein, oral administration,
2 weeks gestation) and nitrofurantoin (5mg/kg, retro-orbital injection, 2 weeks of gestation).
The feto-protective function of BCRP was highlighted by studies using a human placental
perfusion model, demonstrating an increase in the fetal to maternal concentration ratio of
glyburide (100 ng/ml perfusion, Pollex E. et al., 2008) and PhIP (2 µM perfusion, Myllinen P.
et al., 2008) after pharmacological inhibition of BCRP. The limitation of fetal glyburide
distribution by BCRP was also confirmed in mice by Zhou et al. (2008), and in vesicles of
human brush border by Gedeon et al. (2008).
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The expression of BCRP mRNA is known to be higher in human placenta than in other
organs (Allikmets et al., 1998). The corresponding protein is expressed in the apical
membrane of syncytiotrophoblasts, and in the fetal vessels of chorionic villi (Maliepaard et
al., 2001; Evseenko et al., 2006a; Yeboah et al., 2006). BCRP structure, substrate selectivity
and localization in the placenta suggest that, like P-gp, it may have a protective role,
removing cytotoxic drugs from fetal tissues.
4.3. Solute Carrier (SLC) transporters
4.3.1. Organic Anion-Transporting Polypeptides (OATPs). Members of the organic aniontransporting polypeptides (OATP/SLCO) are expressed in the placenta (OATP1A2,
OATP1B1, OATP-2B1, OATP-3A1, OATP-4A1). These proteins are involved in the Na+independent uptake of bile acids in hepatocytes. As shown in vitro in transfected epithelial
cells, OATP2B1 could be involved in the transepithelial transport of steroid sulfates in human
placenta (Grube et al., 2007). The role of other OATPs in placental drug transport is not well
known, although they could transport drugs such as methotrexate, antibiotics and NSAID
(Mikkaichi et al., 2004, Ugele et al., 2003).
4.3.2. Organic Anion Transporters (OATs). OAT-4 is the most expressed OAT. It is
located in cytotrophoblast membranes and the basal surfaces of syncytiotrophoblasts (Cha et
al., 2000; Ugele et al., 2003). Involved in the transport of steroid sulfates that allow estrogen
synthesis in the placenta, it is also a multi-specific xenobiotic transporter which could interact
with environmental toxins and drugs (You et al., 2004 a,b), and contribute to fetal exposure to
xenobiotics.
4.3.3. Organic Cation Transporters (OCTs). Different organic cation transporters have
been identified in the human placenta. The organic cation/carnitine transporter 1 (OCTN1), a
member of a subfamily of OCTs (SLC22A subfamily), is expressed on the apical side of
human syncytiotrophoblasts (Tamai et al., 1997; Ganapathy et al., 2005). OCTN2 is also
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present on the maternal side (microvillous membrane) of trophoblast cells, as shown by
Lahjouji et al. (2004). OCTN2 transports L-carnitine in a Na+-dependent manner, from
maternal circulation to fetal circulation. It could also mediate the transport of drugs such as
antidepressants, amphetamines and β-lactam antibiotics (Grube et al., 2005). OCT3 is a Na+and Cl- independent monoamine transporter mostly found in the placenta (Kekuda et al.,
1998). OCT3 transcript and protein have been detected at the basal membrane of human
trophoblast cells (Sata et al., 2005). As it shows a high affinity for monoamines (serotonin,
dopamine, norepinephrine and histamine), human OCT3 is considered as an extraneuronal
monoamine transporter. However, antidepressants (desipramine, imipramine, amphetamines)
could also interact with OCT3.
4.3.4. Monoamine transporters
The serotonin transporter (SERT) and the norepinephrin transporter (NET) have been
identified in the placenta at the maternal facing membrane (Prasad et al., 1996; Bzoskie et al.,
1997). Both SERT and NET proteins are functionally active in the human placenta at term.
Since these transporters critically regulate extracellular monoamine concentrations, they could
play a role in maintaining homeostasis in amniotic fluid and fetal circulation (Prasad et al.,
1994a): they clear the maternal blood of excessive amounts of catecholamines and serotonine,
preventing pre-eclampsia (Bottalico et al., 2004). In addition, the monoamine oxidases A
(MAO-A), which allow the intracellular degradation of monoamines after uptake from the
extracellular space, are less expressed and less active in placentas from pre-eclamptic
pregnancies (Kaaja R.J. et al., 1999; Carrasco G. et al., 2000; Sivasubramaniam S.D. et al.,
2002).
Monoamine transporters have been involved in the adverse effects of cocaine and
amphetamines, because they are cellular targets for these abusable drugs that block
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catecholamine transport. (Ramamoorthy J.D. et al., 1995; Prasad et al., 1996, Bzoskie L.,
1997).
4.3.5. Monocarboxylate Transporters
(MCTs).
There are several isoforms
of
monocarboxylate transporters (MCT1, MCT3, MCT4, MCT5, MCT7, MCT8) (Price et al.
1998, Settle et al., 2004) and dicarboxylate transporters (NaDC3) (Wang et al., 2000) in the
placenta. These proton-coupled transporters are essential for the transport of lactate, ketone
bodies and other monocarboxylates through the plasma membrane. They may contribute to
the net transport of lactate through the placental barrier (Nagai et al., 2010). MCT8 could be
involved in the transport of thyroid hormones and implicated in the development of
trophoblast cells and the fetus (Chan et al., 2006). Other substrates are benzoic acid, acetic
acid, acetylsalicylic acid and antibiotic cefdinir (Utoguchi et al., 1999).
4.3.6. Equilibrative Nucleoside Transporters (ENTs). Both Na+-independent equilibrative
nucleoside transporters ENT1 and ENT2 are expressed in placenta (Barros et al., 1995).
Physiological substrates of these transporters are purine and pyrimidine nucleosides but a
number of anticancer nucleoside analogs are also substrates (Griffiths et al., 1997).
4.3.7. Folate Transporters. As folates are essential nutrients for cell division and growth,
folates deficiencies by insufficient dietary intake could impair fetal development. Besides
their involvement in folate transport, these transporters could interact with a variety of
antifolates used in the treatment of cancer and immune disorders.
Folate receptor alpha (FRα), localized on the apical side of human placental villi, could be
invoved in folate uptake from maternal blood to the placenta (Prasad et al., 1994b; Yasuda et
al., 2008). Reduced Folate Carrier (RFC-1, SLC19A1) is distributed to both the microvillous
plasma membrane of the syncytiotrophoblast and basal syncytiotrophoblast in term-placenta
(Solanky et al., 2010; Yasuda et al., 2008), and mediates bidirectional reduced folate
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transport. Recently, a heme carrier protein 1 (PCFT/HCP1, SLC46A1) implicated in folate
transport has been characterized in the apical side of the trophoblast.
Most studies on the placental expression of transporters have been performed in various
models representating different stages of gestation. Therefore it is very difficult to compare
the levels of expression from one stage of gestation to another. Data on the expression of
transporters throughout gestation are necessary to understand their importance in fetal
development. Indeed, the expression of numerous transporters varies during pregnancy.
5. Variation of transporter expression during pregnancy
The continuous development of placenta defines different stages of pregnancy and results in
modifications of permeability, potentially leading to modulation of fetus exposure.
Among ABC transporters, ABCG2, ABCB1, ABCB33 and ABCC1 have been shown to vary
during pregnancy.
5.1. ABCG2. Different in vivo studies have shown a clear variation in ABCG2 expression as
gestation progresses. Indeed BCRP protein expression and mRNA increased 2-fold in a group
of pre-term placentas (28±1 week of pregnancy, 15 placentas), compared to term placentas
(39±2 week of pregnancy, 29 placentas) (Zu Schwabedissen et al., 2006). On the contrary,
Yeboah et al. (2006) examined BCRP in placenta-tissues at week 6-41 of pregnancy and
reported an increase in protein expression at term, although mRNA levels did not change as
gestation progressed. In the rat placenta, a decrease in the expression of Bcrp (mRNA and
protein) from the mid-stage to the end of gestation was observed (Yasuda et al., 2005). Bcrp
mRNA expression increased 3-fold on the 15th day of gestation compared to the 21st
(Cygalova et al., 2008). In agreement with the latter study, two recent studies on mouse
placenta reported peaked Bcrp1 levels on the 15th gestation day (Wang et al., 2006) and a
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gestation-dependent decrease in the mRNA expression of Bcrp1 from the 9th to the 18th day of
gestation (Kalabis et al., 2007).
5.2. ABCB1, ABCB3. The mean expression of P-gp measured by the Western blotting
method in early (13–14 gestation week) compared to late (full-term) placentas was found to
be 2-fold higher (Sun et al., 2005; Gil et al., 2005). In agreement with this finding, Mathias et
al (2005) demonstrated that the P-gp expression protein decreased dramatically as pregnancy
proceeded and was lowest at term. On the other hand, Novotna et al. (2004) showed
increasing expression of P-gp in rat syncytiotrophoblasts from the 11th to the 18th day of
pregnancy. This was in agreement with a possible role in fetal protection soon after the
establishment of chorioallantoic placenta and until the end of pregnancy.
Unlike human MDR1, human MDR3 was more abundantly expressed in the last stage of
gestation (Patel et al., 2003).
Existing data strongly suggest that ABCG2 and ABCB1 play an important role in protecting
the fetus against the potential toxicity of drugs, xenobiotics, and metabolites. The decrease in
ABCG2 and ABCB1 at the end of gestation could be related to higher fetal susceptibility to
xenobiotic toxicity in the last part of gestation, and higher fetal protection against xenobiotics
early in pregnancy. Indeed, the fetus is most vulnerable to xenobiotic toxicity during
organogenesis.
5.3. ABCC1. In a study using placental samples (cytotrophoblasts and endothelial cells),
MRP1 expression was increased 4-fold in the third trimester, compared to the first trimester
(Pascolo et al., 2003). MRP1 was also increased 20-fold in polarized BeWo cells, compared
to non-polarized cells.
5.4. OATPs. Among SLC transporters, OATP1A2 and OATP3A1 were down-regulated
between the first and third trimester of pregnancy (Patel et al., 2003).
The discrepancies observed in observations in rats, mice and humans suggested different
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mechanisms in the regulation of transporter expression among these species. However the
molecular mechanisms underlying the modulation of placental ABC and SLC transporters by
pregnancy have not been elucidated yet.
5.5. Folate transporters
An increase in the expression levels of FRα, RFC, and PCFT with the progress of gestation in
rat placenta has been shown and could play an important role in the response to increased
need by placenta and fetus for folate during development (Yasuda et al., 2008).
Due to obvious ethical reasons, studies of fetal risk from maternal exposure to chemicals and
pharmaceuticals are not performed in humans. Besides in vivo/ex vivo models, cell lines have
been developped to provide important knowledge on the transplacental transfer in humans of
new chemical substances and on environmental exposures to hazardous compounds.
The use of non differentiated models, such as choriocarcinoma cell lines, is also an alternative
in the study of the expression of transporters during early stages of gestation.
6. The use of cell lines as placental barrier models
The properties and main uses of these cell lines are summarized in table 3.
6.1. Human choriocarcinoma cell lines
6.1.1. BeWo cell line. Developed from a malignant gestational choriocarcinoma of the fetal
placenta, the BeWo cell line (Patillo et al., 1968) displays morphological and biochemical
enzymes of trophoblasts, showing hormonal secretion properties (hCG, progesterone,
placental lactogen) (Pattillo et al., 1979; Nickel et al., 1991) and cytokine expression (IL4,
Sacks et al., 2001; IL6 and IL8, Fujisawa et al., 2000; IFNα, IFNβ and IL10, Bennett et al.,
1996).
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The BeWo cell line is widely used as a model system to study trophoblast differentiation
(Hussa et al., 1997; Vogt et al., 1997; Rao et al., 2003; Xu et al., 1999; Rote, 2005), placental
metabolism, and nutrient and drug distribution across the placental barrier (Furesz et al.,
1993; Moe et al., 1994; Way et al., 1998; Shah et al., 1999; Vardhana et al., 2002; Schmidt et
al., 2003; Takahashi et al., 2001; van der Ende et al., 1989; Prasad et al., 1996; Eaton et al.,
1998; Ellinger et al., 1999, Ushigome et al., 2000). BeWo cells form a confluent polarized
monolayer on a permeable support, as shown by the development of a transepithelial electric
resistance and a high density of microvilli on the apical surface of the monolayer (Liu et al.,
1997). The b24 and b30 BeWo clones (developed by Dr. Alan Schwartz) have shown better
monolayer-forming ability than the original BeWo clone available from the American Type
Culture Collection (ATCC) (Bode et al., 2006). This contradicts studies by Parry et al. (2006)
and Mark and Waddell (2006) who used the BeWo clone from ATCC as a representative
model of transepithelial barrier. The ability of cells to form a monolayer on a permeable
support depends on the formation of tight junctions. Many factors such as the choice of media
(essential nutrients and growth factors), seeding density, extent of cell confluency, exposure
time, environmental conditions, and choice of permeable insert (diameter, pore size,
supporting matrix) may influence tight junction development. Validity of the monolayer is
assessed by the measuring trans-epithelial electrical resistance (TEER) and determining the
transfer of paracellular markers (inulin, sucrose, fluorescein, FITC-dextran, Lucifer yellow)
(Liu at al., 1997, Konsoula et al., 2005). BeWo monolayers have been used for transport
studies but the latter all report different cell culture conditions (Saunders et al, 2009).
The BeWo cells consist of undifferentiated cytotrophoblasts with few syncytialized cells
(Wice et al., 1990). Their differentiation into syncytiotrophoblasts does not occur
spontaneously, but the syncytialization process can be induced by forskolin or 8bromoadenosine 38,58-cyclic monophosphate treatment (Borges et al., 2003). For this reason,
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BeWo cells are used for studies on the maturation of trophoblasts: they can model either
cytotrophoblasts of early gestation or syncytiotrophoblasts of late gestation. Treatments with
syncytium inducers result in an increase of monolayer permeability (Liu et al., 1997), which
can be a disadvantage for transfer studies.
BeWo cells constitute a good model for studying the fusion process that occurs during
syncytium formation. Envelope proteins such as Syncytin-1, derived from human endogenous
retroviruses, seem to play an essential role in these cellular fusion events (see the review by
Pötgens et al., 2004). Syncytin-1 expression is up-regulated after stimulating the fusion of
primary cytotrophoblast into syncytia by forskolin, a cAMP analogue. Recently, Vargas et al.
(2009) demonstrated a more important role for syncytin-2 in trophoblast fusion, showing that
syncytin-1 expression changed slightly in BeWo on stimulation, compared to syncytin-2
fluctuation. Moreover, using two different targeted siRNAs for each transcript, the authors
demonstrated that a reduction of syncytin-2 expression led to a significant decrease in the
number of BeWo cell fusions. Two amino acid transporters ASCT1 and ASCT2 expressed in
trophoblast are potential receptors of syncytin-1. The receptor of Syncytin-2 protein is also
placenta-specific (Esnault et al., 2008).
ABC efflux transporters including P-gp, MRPs and BCRP are expressed in BeWo cells (table
2). The pattern of expression is similar to that of syncytiotrophoblasts isolated from human
placenta. BCRP is highly expressed in BeWo cells, whereas P-gp transcript and protein are
minimally expressed or even absent. Both transporters are localized predominantly in the
apical plasma membrane and show efflux activity. mRNA of MRP1, MRP2, MRP3, MRP5
and MRP8 are variably expressed, depending on differentiation status (Pascolo et al., 2000,
Pascolo et al., 2003, Serrano et al., 2007). Folate transporters (Yasuda et al., 2008) and
OCTN2 (Rytting et al., 2005; Rytting et al., 2007) are present, while OATs and MCTs
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expression has not been reported up to now. Expression of ABC transporters (BCRP, MRP1,
MRP2) is modulated by cell differentiation (Evseenko et al., 2006b; Prouillac et al., 2009).
BeWo cells are stable, easily maintained by trypsinization, and form a confluent monolayer
on a permeable support in a relatively short period of time (4–5 days). These advantages make
this cell line an attractive in vitro model for studying trans-trophoblast transport and drug
metabolism (Poulsen et al., 2009), as shown by numerous studies on the transport of digoxin,
vinblastine, vincristine (Ushigome et al., 2000), opioides peptides (Ampasavate et al., 2002),
iron (Heaton et al., 2008), and cholesterol (Schmidt et al., 2003) (table 4).
6.1.2. JAR and JEG-3 cell lines. Derived from choriocarcinoma cell lines, they are used in
studies of placental function including differentiation, invasion, hypoxia/oxidative stress
response, endocrinology, maternal fetal immunology and transplacental transfer. The JEG-3
cells possess many of the biological and biochemical characteristics of syncytiotrophoblasts,
although they are mononucleated and proliferative (Matsuo et al., 1994). They produce
placental hormones (progesterone, hCG, steroids) (Chou, 1982; Kato et al., 1991) and express
various enzymes (Sun et al., 1998; Tremblay et al., 1999). Although they show biochemical
differentiation, such as increased hCG secretion, JEG-3 cells do not morphologically
differentiate with forskolin treatment (Al-Naisiry et al., 2006). However, JEG-3 cells have a
lower rate of proliferation and a higher degree of differentiation than BeWo and JAR cells
(Borges et al., 2003; Kitano et al., 2004; White et al., 1988; Aplin et al., 1992).
When compared to cytotrophoblast cells stemming from isolated from human placenta, JAR
cells are more similar to non-differentiated cytotrophoblasts (Evseenko et al., 2006a). The
JAR cell line is probably an unsuitable model for studies of transepithelial transport (Mitchell
et al., 1995), exactly like JEG-3 cells, if one refers to the few studies reporting the use of JEG3 as a model for vectorial transepithelial transport (Hardman et al., 2007). Indeed, the ability
of both cell lines to form a polarized monolayer is controversial (Bode et al., 2006). The
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transcriptional profile and protein expression of many transporters, in particular export pumps
(P-gp, MRPs and BCRP) and organic anion transporters (OATPs), show quantitative variation
in both cell lines (table 2, Serrano et al., 2007).
6.2. Human primary cells
Primary cell lines of undifferentiated cytotrophoblasts can be produced from human placenta.
Unless primary cells are allowed to differentiate, they represent early stages of gestation.
These nonproliferative, multinucleated cells are able to syncytialize spontaneously. They form
aggregates with large intercellular spaces, making studies of polarized transport of
xenobiotics impossible when they are grown on semi-permeable membranes (Liu et al., 1997,
Yui et al., 1994). Hemmings et al. (2001) reported that primary cytotrophoblasts formed tight
junctions after three successive cycles of seeding and differentiation. Although this method
produced multiple overlapping layers of syncytialized cells, areas of microvillar projections
were formed on the apical surface and functioned as a barrier to low and high-molecularweight molecules. Besides the fact that obtaining a reproducible cell population may be
tricky, primary cell lines also have high contamination levels and are not viable for many
passages. They are mostly used in investigations involving parasites or virus (Abbasi et al.,
2003).
7. Other models for placental transfer studies
Experimental models have to replicate the full features and functionality of in vivo trophoblast
cells. For obvious ethical reasons, in vivo experimentation of placental drug transport in
humans cannot be performed. Therefore the risk assessment of fetal injury (teratogenic and
fetotoxic potential of xenobiotics) from maternal exposure to xenobiotics is based on results
from animal studies. Animal models provide the advantages of a complete physiological
system. However, interspecies differences in placental morphology and length of gestation
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demand caution when assuming that the maternal-to-fetal transfer of substances observed in
animal models will be observed in the same way in humans (Enders et al., 1999; Moe et al.,
1995). For these reasons, mechanisms of placental transport, metabolism and placental
toxicity are best investigated in models of human origin (Myllynen et al., 2002). Primates
such as rhesus macaques and baboons have also been used to evaluate the transfer of
compounds since their haemochorial placentation is similar to that of humans (Patterson et al.,
2000). Recently, an original study has reported the use of positron emission tomography
imaging to monitor the placental P-gp function during pregnancy in non-human primates
(Chung et al., 2010).
7.1. Feto-maternal blood concentration ratio. Human maternal and umbilical cord blood
are simultaneously sampled during labor. Further analysis for the presence of compounds
constitutes a simple and ethical method that illustrates the drug concentration between both
circulations. However, the lack of comparison with a proper reference compound, the
difficulties in evaluating the influence of placental metabolism on drug transfer and the lack
of information on drug distribution and accumulation in tissue are definite drawbacks
(Waddell et al., 1981; Levy, 1981; Chamberlain, 1986; Simone et al., 1994).
7.2. Human coelocentesis. This alternative to fetal blood sampling was introduced in 1991
(Jauniaux et al., 1991). It consists of sampling from human exocoelomic and amniotic fluid by
transvaginal puncture at week 6-10 and week 7-12 respectively (Jauniaux et al., 2000). This
method does not allow building a kinetic model which is necessary to extrapolate quantitative
drug transfer.
7.3. Perfused single human placental cotyledon model. This technique of ex vivo perfusion
of human placenta was first described in 1967 by Panigel et al., then modified by Schneider et
al. (1972). It is commonly used to investigate the mechanism and predict the rate of transfer
of drugs through the placenta in vivo (Bourget et al., 1995). This model has been used to
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elucidate the mechanisms of placental transfer of HIV protease inhibitors and anti-epileptic
drugs (Forestier et al., 2001; Myllynen et al., 2003). Ethical problems are minimal because
this technique is non invasive and placentas are discarded or incinerated after birth. The
critical point of the model is that it does not represent the first trimester placenta, when the
fetus’ susceptibility to toxic hazards is high. However, since placental thickness and the
number of cell layers decrease at the end of pregnancy, it is possible that the term placenta
might be more sensitive to environmental agents than first anticipated (Vähäkangas et al.,
2006). Another disadvantage is possible underestimation of the contribution made by
transporters and metabolism during pregnancy, since transporters and metabolism differ
between first trimester and full term placenta. Inter-individual variation also raises the
question of the number of placentas that have to be perfused to validate the method. However,
the influence of placental metabolism, the preferential direction of transport and the presence
of overall active transport can be evaluated. Results are generally compared with data on
reference compounds such as inulin or antipyrine.
7.4. Human trophoblast tissue preparations. Tissue explants can be used to study the
transport of substances from the maternal circulation into the syncytiotrophoblast, as well as
the metabolism, endocrine function, enzyme function, cellular proliferation and differentiation
in the placenta at early gestation or near term (Miller et al., 2005). This method requires
taking into careful consideration the potential contribution of mesenchymal and endothelial
cells to the metabolic process (Syme et al., 2004). Moreover, a recent publication has reported
the limits of this model: the functional characterization of individual transporters is difficult,
due to the lack of selective substrates and/or specific inhibitors. Furthermore, the authors
suggested the presence of compensatory regulatory mechanisms, which could explain the
difficulty of performing a good kinetic transfer study (Vaidya et al., 2009).
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7.5. Membrane vesicles. Isolated membrane vesicles can be obtained from either the
membrane of the brush border, or the basal surface of the trophoblast. Therefore the study of
transport mechanisms is possible in both fetal and maternal plasma membranes (Bissonnette
et al., 1982; Murer et al., 1980). This model enables characterizing the expression and
functionality (susceptibility to inhibitors, specificity, saturability) of transporters, but does not
reflect the in vivo situation, due to a lack of regulatory factors.
8. Conclusions and perspectives
The expression of both ATP and SLC transporters has been demonstrated in many polarized
epithelial barriers (i.e. kidney, liver, blood-brain barrier, intestine, lung, etc.) and many
different species. Cellular localization combined with knowledge of the protein function gives
insight into the role of these proteins in the accumulation and removal of specific substrates
from the cells at each barrier. However, despite the considerable efforts invested in studying
these transporters, few works have been dedicated to the cellular localization of these
transporters in the placenta. Moreover, too little information is available on the mechanisms
of fetal exposure to pollutants and the role of the placenta in this exposure.
Firstly, placental models efficient for studying xenobiotic transport are far from being fully
characterized. Better knowledge of cell properties (content in xenobiotic metabolism enzymes
and transporters, number of passage, phenotype, differentiation) is required and numerous
external factors must be considered and optimized (Audus et al., 1990):
- culture conditions (culture medium composition, cell seeding density, stage of
differentiation);
- permeable support characteristics (diameter, pore size, supporting matrix like collagen);
- transport study conditions (transport medium composition, pH, temperature, etc.);
- diffusion apparatus (stirring or not).
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DMD #33571
In vitro placental transfer studies show high variability, occasionally leading to controversial
results. Further investigation into transporter activity and membrane localization in the
polarized placental epithelium will help to define the physiological and pharmacological
functions of transporters in placental cells.
Secondly, the modulation of the expression of placental transporters may influence their role.
Besides variation of expression due to the gestation stage, in vivo genetic variability may also
exist in xenobiotic transport by placenta. This is due to the presence of single nucleotide
polymorphisms (SNPs), as has been shown for ABC transporter expression. This genetic
polymorphism has been described for placental P-gp (Tanabe et al., 2001) and BCRP
(Kobayashi et al., 2005), and associated with altered P-glycoprotein expression (Hitzl et al.,
2004). However, it remains to be determined whether these SNPs influence the
pharmacokinetic and dynamic properties of clinically useful drugs. Rahi et al., (2008)
reported that although altering the expression levels of P-gp in the human placenta, SNPs did
not have any consequences on P-gp-mediated placental transfer of saquinavir. On the
contrary, recent studies have shown that some homozygous P-gp variants (C1236T, C3245T)
are associated with an increase in placental P-gp efflux of paclitaxel (Hemauer et al., 2010).
Transfected cell lines expressing the polymorphic variant (Q141K) of ABCG2 exhibit altered
placental pharmacokinetics of glyburide (Pollex et al., 2010). Further investigations are
needed to define the significance of SNPs in the placental transport of xenobiotics.
Thirdly, in addition to the trophoblast cells, human fetal membranes such as the amnion,
chorion and yolk sac, may constitute an additional site of drug transfer in pregnant humans
and rodents. Data are very scarce on transporter expression and function in these fetal
annexes. The amnion can be considered as a barrier separating maternal and fetal
compartments and expresses functional transporter proteins such as MRPs and BCRP (Aye et
al., 2007), as well as the chorion, which expresses BCRP (Yeboah et al., 2008). RFC protein
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DMD #33571
is expressed in the yolk sac (Maddox et al., 2003), and the existence of xenobiotic efflux
transporters in extraembryonic fetal membranes has recently been demonstrated in mice
(Aleksunes et al., 2008; Kalabis et al., 2007). It is now necessary to study the contribution of
fetal membranes in the transfer of xenobiotics between the mother and the fetus, opening up a
new field of research.
ABC and SLC proteins are able to transport nutrients as well as xenobiotics. These
xenobiotics may compete with the physiological substrates of the placental transporters, and
interfere with the delivery of nutrients to the fetus. Future studies should deal with the
possibility of an interaction that alters exchanges between fetus and mother. Exchange
modulation may have consequences on the growth and development of the fetus, a crucial
stage where small alterations may lead to long-term modulation of metabolic regulation and
programming.
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FIGURE LEGEND
Figure 1: Schematic drawing of human placental structure. a) Transverse section through a
full-term placenta. b) Localization and structure of human placental barrier
45
DMD #33571
Table 1: Metabolism enzyme expression as a function of pregnancy stage and placental
models (+: positive signal, ++: strong positive signal, -: negative result; nd: no data available)
CYP isoform
First trimester
of pregnancy
Full term
placenta
JAR cells
Jeg-3 cells
BeWo cells
46
CYP1A1
CYP1A2
CYP2A
CYP2B
CYP2D6
CYP3A4
CYP3A5
CYP3A7
CYP1A1
CYP1A2
CYP2A6/7
CYP 2B6/7
CYP2C8-19
CYP2D6
CYP2E1
CYP2F1
CYP3A4
CYP3A5
CYP3A7
CYP4B1
CYP11A1
CYP19
CYP19
mRNA
level
++
+
+
+
+
+
++
+
+
+
+
+
++
++
+
Protein
level
nd
nd
+
+
+
+
nd
nd
nd
nd
nd
nd
nd
nd
+
Enzyme
Activity
nd
+
nd
nd
nd
nd
nd
nd
+
nd
nd
nd
nd
nd
nd
+
+
+
No data for other cytochromes
nd
+
CYP1B1
nd
+
CYP2C9
nd
+
CYP3A4
+
+
CYP19
nd
+
3beta-HSD1
nd
+
17beta-HSD112
CYP1A1
+
+
CYP1A2
+
+
CYP2C9
+
nd
CYP3A4
+
nd
CYP19
+
+
Reference
Hakkola et al. (1996a)
Schuetz et al. (1993)
Pasanen et al. (1997)
Bièche et al. (2007)
Hakkola et al. (1996b)
Pasanen et al. (1997)
Nishimura et al. (2003)
Nakanishi et al. (2002)
nd
nd
nd
+
+
+
Peikonen et al. (1984)
Pavek et al. (2007)
Nakanishi et al. (2002)
Samson et al. (2009)
Smuc et al. (2009)
+
+
nd
nd
+
Avery et al. (2003)
Pavek et al. (2007)
Nakanishi et al. (2002)
DMD #33571
Table 2: Comparison of the polarized distribution and relative expression at mRNA/protein level (immunohistochemistry, Western blot and
functionality) of placental transporters relevant to drug disposition across the maternal-fetal interface. +: positive signal; ++: strong positive
signal; -: negative result; nd: no data available; alimit detection.
Localization
Brush border
(maternal
facing)
membrane
Basal (fetal
facing)
membrane
47
Transporter
Placenta
BeWo
Jeg-3
P-gp (MDR1)
++/+
+/+
+/+
++/+
MRP2
+/+
++/+
+/nd
++/-
MRP8
BCRP
+/nd
+/+
+/nd
++/++
++/nd
+/+
+/nd
++/+
+/nd
+/nd
+/nd
nd/+
+/+
+/+
nd/+
nd/+
+/+
+/+
+/+
+/+
+/+
+/+
+/++
++/+
++/++
++/+
+/+
+/+
+/+
+/nd
nd/nd
nd/nd
nd/nd
nd/nd
+/+
nd/nd
+/+
nd/nd
+/+
+/nd
+/nd
+/+
nd/nd
+a/+
+a/nd
+/nd
nd/nd
+/+
-/nd
nd/nd
+/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
+a/nd
nd/nd
+/nd
nd/nd
nd/nd
nd/nd
nd/nd
+/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
nd/nd
+/nd
nd/nd
nd/nd
+/+
nd/nd
+a/++
nd/nd
+/nd
nd/nd
nd/nd
nd/nd
nd/nd
OATP1A2 (OATP-A)
OATP1B1 (OATP-C)
OATP3A1 (OATP-D)
OATP4A1 (OATP-E)
OCTN1/SLC22A4
OCTN2/SLC22A5
NaDC3
hENT1/SLC29A1
MCT-4/SLC16A4
FRα
PCFT (SLC46A1)
RFC-1 (SLC19A1)
SERT
NET
MDR3
MRP5
OATP2B1 (OATP-B)
OAT4
RFC-1 (SLC19A1)
OCT3
MCT-1/SLC16A1
JAR
Reference
St Pierre et al., 2000; Patel et al., 2003 ; Evseenko et al.,
2006
St Pierre et al., 2000; Pascolo et al., 2000; Evseenko et al.,
2006; Serrano et al., 2007; Prouillac et al., 2009
Serrano et al., 2007
Maliepaard et al., 2001; Evseenko et al., 2006; Yeboah et
al., 2006
Ugele et al., 2003; Patel et al., 2003; Serrano et al., 2007
Ugele et al., 2003, Patel et al., 2003; Serrano et al., 2007
Ugele et al., 2003, Patel et al., 2003; Serrano et al., 2007
Sato et al., 2003
Tamai et al., 1997; Ganapathy et al., 2005
Lahjouji et al., 2004; Rytting et al. 2005; Rytting et al. 2007
Wang et al., 2000;
Barros et al., 1995; Yasuda et al., 2008
Settle et al. 2004
Yasuda et al., 2008; Solanky et al., 2009
Yasuda et al., 2008; Solanky et al., 2009
Yasuda et al., 2008; Solanky et al., 2009
Prasad et al., 1996
Bzoskie L. et al., 1997
Patel et al., 2003; Evseenko et al., 2006
Pascolo et al. 2003; Zu Schwabedissen et al., 2005;
St Pierre et al., 2002; Serrano et al., 2007 Grube et al., 2007
Cha et al., 2000; Ugele et al., 2003
Yasuda et al., 2008; Solanky et al., 2009
Sata et al. 2005
Settle et al. 2004
DMD #33571
Unknown or
controversial
localization
48
MRP1
+/+
++/++
++/+
+/+
MRP3
MRP4
MCT-8
MCT3, MCT5, MCT7
hENT2/SLC29A2
++/+
+/nd
+/+
+/nd
nd/+
+/nd
+/nd
nd/+
nd/nd
nd/nd
+/nd
++/nd
nd/nd
nd/nd
nd/nd
+/nd
+/nd
nd/nd
nd/nd
nd/nd
St Pierre et al., 2000; Nagashige et al., 2003 ; Evseenko et
al., 2006
St Pierre et al., 2000
Serrano et al., 2007
Utoguchi et al., 1999; Chan et al. 2006
Price et al., 1998; Settle et al. 2004
Barros et al., 1995
DMD #33571
Table 3: Properties and use of placental cell lines
Origin
Differentiation
Hormonal
secretion
Ability to form
confluent
monolayer on
permeable support
Use
49
BeWo
Choriocarcinoma cells
undifferentiated
cytotrophoblasts
Mononucleated and
proliferative
Not Spontaneously
(forskolin, c-AMP
analog)
Biochemical and
morphological
differentiation
(Al-Nasiry et al., 2006)
Progesterone, β-hCG,
Steroids, placental
lactogen
(Pattillo et al., 1979;
Nickel et al., 1991 ;
Prouillac et al., 2009)
Yes
(Liu et al., 1997)
Transplacental transport
of drug
(Saunders, 2009)
Trophoblast
differentiation
(Nampoothiri L. et al.,
2007)
Jeg-3
Choriocarcinoma cells
derived from BeWo
Mononucleated and
proliferative
JAR
Choriocarcinoma cells
derived from BeWo
Early placental
trophoblast
Primary placental cells
Normal trophopblast
Contamination by other cell
types (endothelial cells,etc.)
Not spontaneously
(forskolin, c-AMP
analog)
Only biochemical
differentiation
(Al-Nasiry et al., 2006)
Morphological
differentiation
(Borges et al., 2003)
Spontaneously
(Morrish D.W. et al., 1997)
Progesterone, β-hCG,
Steroids
(Chou, 1982)
Progesterone, β-hCG,
Steroids
(Kato et al., 1991)
Progesterone, β-hCG, steroids
(Morrish D.W. et al., 1997)
Controversial
(Hardman at al., 2007;
Bode et al. 2006)
Controversial
(Mitchell et al.,1995;
Bode et al. 2006)
Controversial
(Hemming et al., 2001)
Gene expression
(Rena et al., 2009)
Apoptosis
(Saulsbury, 2008)
Gene expression
Physiological stimulis,
hormone secretion
Apoptosis, parasites, virus
(Abbasi et al., 2003 ; Kilani et
al, 2007)
DMD #33571
Table 4: Studies of therapeutic agents, abused drugs and food contaminants transfer using in vivo and in vitro models of placenta
Perfused human
placenta
Physiological
molecules
ND
Abused agents
Cocaine, alcohol,
nicotine, morphine
HIV inhibitors,
Antidepressants,
Antibiotics, Anti-acid,
Antidiabetic
Anaesthesics, Antiepileptic, Heart
condition
Pregnancy related
treatment
Medicinal
drugs
(1,2)
Food
contaminant
50
Caffeine , Glyphosate,
Benzoic acid (19), 2amino-1-methyl-6phenylimidazo[4,5-
In vivo
transplacental
transfer
BeWo
Jeg-3
Intracellular
accumulation
Vectorial transport
ND
ND
Amino acid (5),
Glucose (6),
Glucocorticoids (7),
Folic acid (7), Fatty
acid (8),
Transferrin(9),
Serotonin(10),
Bilirubin (10),
Choline(11), IgG (11),
Cholesterol (12),
Leptin (13)
Antiviral drugs,
Antimitotic,
colchicines, HIV
inhibitors,
Antidepressants,
Antibiotics, Antiacid,
Antidiabetic,
Anaesthesics,
Anti-epileptic,
Heart condition (1)
ND
Levofloxacin (3)
Riboflavin (4)
Valproic acid (14),
Opioid peptides (15),
Vincristine,
vinblastine, digoxin
(16)
, Monocarboxylic
acid (17)
Caffeine,
Glyphosate,
Benzoic acid
(18)
ND
Intracellular
accumulatio
n
ND
JAR
Vectorial
transport
Copper (21)
Intracellular
accumulation
Folic acid (22)
ND
Nitrofurantoin
ND
(23)
ND
Vectorial
transport
ND
DMD #33571
b]pyridine (PhIP) (20)
1
Rama Sastry, 1999, 2Myren et al., 2007, 3 Polachek et al., 2010, 4 Huang et al., 2001,5 Jones et al., 2006, 6 Vardhana et al., 2002, 7 Takahashi et al., 2001, 8 Liu et al., 1997, 9
Van der Ende, 1990, 10 Pascolo et al., 2001, 11 Ellinger et al., 1999, 12 Schmidt et al., 2003, Wyrwoll et al., 2005, 14 Utoguchi et al., 2000a, 15 Ampasavate et al., 2002, 16
Ushigome et al., 2000, 17 Utoguchi et al., 1999, 18 Poulsen et al., 2009, 19 Mose et al., 2008, 20 Myllinen et al., 2008, 21 Hardman et al., 2007, 22 Keating et al., 2006, 23
Feinshtein et al., 2010.
51
Umbilical cord
Fetus
Fetal portion
Umbilical artery
Umbilical vein
Amniotic
cavity
Chorionic villous
Chorion
Uterine myometrium
uterus
Amniotic fluid
Intervillous space
Uterine endometrium
Maternal portion
Figure 1a
Chorionic villous
Maternal tissue
Conjonctival tissue
SCT
CT
Intervillous
space
Intervillous
space
Syncytotrophoblast
(SCT)
Maternal
erythrocyte
Fetal capillary
Cytotrophoblast (CT)
Umbilical artery
Umbilical vein
Figure 1b
chorion