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Placental transfer and metabolism: An overview of the experimental models utilizing human placental tissue
Toxicology in Vitro 27 (2013) 507–512
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Review
Placental transfer and metabolism: An overview of the experimental models utilizing human placental tissue Päivi Myllynen a, Kirsi Vähäkangas b,⇑ a b
Department of Pharmacology and Toxicology, Institute of Biomedicine, University of Oulu, Oulu, Finland Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland
a r t i c l e
i n f o
Article history: Received 12 June 2012 Accepted 22 August 2012 Available online 30 August 2012 Keywords: Human placental perfusion Human placental cell lines BeWo cells Placental transporters In vitro models
a b s t r a c t Over the decades several ex vivo and in vitro models which utilize delivered human placenta have been developed to study various placental functions. The use of models originating from human placenta to study transplacental transfer and related mechanisms is an attractive option because human placenta is relatively easily available for experimental studies. After delivery placenta has served its purpose and is usually disposed of. The purpose of this review is to give an overview of the use of human placental models for the studies on human placental transfer and related mechanisms such as transporter functions and xenobiotic metabolism. Human placental perfusion, the most commonly used continuous cell lines, primary cells and tissue culture, as well as subcellular fractions are briefly introduced and their major advantages and disadvantages are discussed. Ó 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ex vivo human placental perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Continuous cell lines originating from human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Transcellular transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human placental primary cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human placental explants and villous explants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subcellular fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Human placenta plays an important role in the development of the fetus, being the major interface between mother and fetus. Placenta provides a gateway to oxygen and nutrients from the mother to the fetus, produces hormones to support the pregnancy and serves as an excretion pathway for various metabolites and carbon dioxide. However, it also plays a role in the exposure of the fetus to potentially toxic xenobiotics via the maternal circulation. Transport processes across the placenta and metabolism in the placenta ⇑ Corresponding author. Address: University of Eastern Finland, Faculty of Health Sciences, School of Pharmacy/Toxicology, P.O. Box 1627, FI-70211 Kuopio, Finland. Tel.: +358 40 745 5254. E-mail address: kirsi.vahakangas@uef.fi (K. Vähäkangas). 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.08.027
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may determine the exposure of the fetus to xenobiotics. Therefore, the studies of these processes are important when trying to predict possible fetal exposure. Our review describes different models that are used to study transplacental transfer and related processes, and discusses their major advantages and disadvantages. Human placenta starts to develop during early pregnancy. Even before the placenta is formed, a few days after fertilization, the first specialized placental cells, trophoblasts, are present forming the outer layer of the blastocyst. Thus, already at this stage barrier of trophoblastic cells may protect the embryo from xenobiotics. During implantation blastocystic trophoblasts attach to and invade into the uterine lining. The trophoblast cells of the implanting embryonic pole differentiate into two trophoblastic layers: a layer of mononuclear cytotrophoblasts and the multinucleated syncytiotrophoblast. These structures will develop into the placenta during
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the first trimester. The villous structure of the human placenta is gradually formed already by 18–20 days after fertilization (Benirschke et al., 2006). However, during the first trimester the cytotrophoblastic plugs partially occlude the lumen of spiral arteries reducing the amount of maternal blood entering the intervillous space. Using the 3-dimensional vaginal ultrasound and power Doppler angiography, maternal intervillous blood flow has been detected from 6th gestational week onwards with a gradual increase during the first trimester (Mercé et al., 2009). From a toxicological point of view it is important that the placenta provides only meager protection for the fetus against various xenobiotics. The mechanisms for transplacental transfer include passive diffusion, active transport, facilitated diffusion, filtration and pinocytosis. Passive diffusion is an important mechanism for transplacental transfer of xenobiotics. Physicochemical properties of xenobiotics such as molecular weight, pKa and lipid solubility affect the transplacental transfer by passive diffusion. Very large chemical compounds with a molecular weight of over 500D are generally transferred incompletely across the placenta (for a review see e.g. Myllynen et al., 2007). Both transporter proteins and xenobiotic metabolism enzymes have been found in human placental tissue, and their significance in transplacental transfer is being actively studied. Human placenta expresses a number of transporter proteins. In syncytiotrophoblast, various transporters have been found both in the brush-border (apical membrane) facing maternal blood and basolateral membrane close to fetal capillaries (for extensive reviews see e.g. Vähäkangas and Myllynen, 2009; Ni and Mao, 2011). Depending on the localization and function, transporters may either increase or decrease xenobiotic transfer through the placenta towards fetal circulation. The expression of the transporters found in the apical and basal membranes of the syncytiotrophoblast differs leading to polarized transport across the structure. Complexity is further increased by the fact that also cells in fetal capillaries harbor transporters. Multiple studies in several species including human suggest that several ABC (ATP binding cassette) transporters decrease fetal exposure to xenobiotics (see e.g. Lankas et al., 1998; Kalabis et al., 2007; Pávek et al., 2001; Smit et al., 1999; Mölsa et al., 2005; Myllynen et al., 2008). In mouse, the inhibition of the ABCB1 transporter (p-glycoprotein, MDR1) has been reported to increase teratogenicity of ivermectin (Lankas et al., 1998). It is important to get more insight in the role of human placental transporters in fetal exposure because the mechanisms of transplacental transfer of many chemicals are insufficiently known. Placental metabolism may also affect fetal exposure to xenobiotics. Compared to the liver the activities of xenobiotic metabolizing enzymes in human placenta are generally low (Hakkola et al., 1998). Still, placental metabolism may lead to metabolites with toxic potential compared to the parent compound. For instance, human placenta can metabolize retinoids (isotretinoin and tretinoin) to both more and less toxic metabolites (see e.g. Miller et al., 1993) and benzo(a)pyrene to active metabolites binding to DNA (e.g. Karttunen et al., 2010). The expression of xenobiotic metabolizing enzymes is dependent on the developmental stage of the placenta (Myllynen et al., 2007). Quite a few CYP (cytochrome P450) enzymes are expressed at the mRNA level, especially during the first trimester (Hakkola et al., 1998; Myllynen et al., 2007). However, of the CYP enzymes from the groups 1–3, only CYP1A1 seems to be functional in human placenta. In term placenta the CYPs 4B1 and 19 (steroid aromatase) are active and contribute to the metabolism of some xenobiotics, e.g. aflatoxin B1 (Storvik et al., 2011; Hakkola et al., 1998). One of the most recently described CYPs, CYP2S1, is also expressed in the placenta and has both endogenous and xenobiotic substrates (Bebenek et al., 2012). Its role in placental toxicology remains to be studied. At
least the following metabolizing enzyme activities have been described in term human placenta: nitric oxide synthase, quinine reductase, nitroquinoline oxidase and alcohol dehydrogenase). Of the transferases, activities of glutathione-S-transferase, glucuronyltransferase as well as sulfotransferase are found from the first trimester on, and N-acetyltransferase at least in term placenta (for a review see e.g. Myllynen et al., 2007). Currently, for ethical and practical reasons animal experiments and testing are being replaced with in vitro models whenever possible. To predict exposure of human fetus to chemicals, several relevant in vitro models for the study of human placental transfer including transporter function and metabolism have been developed over the past decades. The purpose of this review is to give an overview of such models and to compare their usefulness for estimating human placental transfer and metabolism.
2. Ex vivo human placental perfusion Human placental perfusion of a single cotyledon with separate maternal and fetal circulations was originally introduced by Panigel (1962) and further developed by the groups of Schneider et al. (1972) and Miller et al., (1989), (Table 1). Placental perfusion is the only experimental model to study human transplacental transfer in organized human placental tissue. So far there are no standardized criteria for ex vivo human placental perfusion. Placentas from both vaginal births and C-sections can be used for the perfusion. We reported recently that the method of delivery does not affect the transplacental transfer of antipyrine, ethanol or two food borne carcinogens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5f]quinoline (IQ) (Mose et al., 2012). Ex vivo placental perfusion may be either nonrecirculating (open/single-pass) or recirculating (closed) perfusion. The methodological details, such as composition of maternal and fetal perfusates, flow rates in maternal and fetal circulations, composition of gas mixtures used to oxygenate the tissue and perfusate volumes all vary from one laboratory to another. In most published studies human placental perfusions have been carried out for 2–6 h although it is possible to perfuse placentas for extended periods of time, up to 48 h (Polliotti et al., 1996; Miller et al., 1989; Heikkilä et al., 2002; Woo et al., 2012). Various markers are used to monitor placental viability during the perfusion and the selection of markers may vary between research groups (see e.g. Myllynen et al., 2010; Vähäkangas et al., 2011). Minimal loss of fetal perfusate is the main marker of a successful perfusion. Volume loss of 2–3 ml from fetal circulation is usually considered acceptable. Recently, a loss greater than 3 ml/ h has been associated with higher transfer of the control substance FITC-dextran which in successful perfusions does not cross placental membranes due to its high molecular weight (Mathiesen et al., 2010). Antipyrine or other reference compounds such as creatinine are used to confirm the overlap between maternal and fetal circulations. These markers may also be used to normalize the perfusion data for interindividual variation. Other markers used for tissue viability include pH, oxygen consumption, net oxygen transfer, glucose consumption, lactate production and tissue morphology after perfusion. Hormone production (human chorionic gonadotropin or hCG, human placental lactogen or hPL, leptin) is also a good marker of functional placental tissue. Although placentas from 27–40 weeks have been perfused (Fokina et al., 2011; Nanovskaya et al., 2008), the challenging perfusion method is usually applied to term placenta. Even then the success rate, counting from the placentas good enough to be hooked to the perfusion equipment, is only about 50%. Thus, the major limitation of this technique is that it does not provide data
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Table 1 Commonly used in vitro models for human placental transfer and associated mechanisms (Vähäkangas and Myllynen, 2006; Vähäkangas et al., 2011). Material
Ex vivo/in vitro models
Used for
Whole tissue
Ex vivo human placental perfusion
Tissue pieces
Explant cultures Villous explant cultures Primary trophoblastic cells Choriocarcinoma cell lines (BeWo, JEG-3, JAr) BeWo b30
Transplacental transfer including xenobiotic metabolism and transporter function Mechanisms affecting transplacental transfer such as uptake, efflux and metabolism Mechanisms affecting transplacental transfer such as uptake, efflux and metabolism Transplacental transfer including xenobiotic metabolism and transporter functions Placental xenobiotic metabolism Placental transporter functions
Cells Tight monolayer culture on permeable membrane Tissue fractions
Placental microsomes and cytosolic fraction Placental microvillous and basal membrane vesicles
on placental transfer in the first and second trimester placentas. It is known that the materno-fetal diffusion distance changes significantly during pregnancy decreasing from 50–100 lm in the second month to 4–5 lm at term (Benirschke et al., 2006). Also, the expression of transporter proteins changes dynamically during pregnancy. For example, the expression of ABCB1 in the apical membrane of syncytiotrophoblast decreases as gestation advances (Mathias et al., 2005; Sun et al., 2006). The protein expression of ABCB1 has been reported to be 44.8-fold higher in placentas from early pregnancy (60–90 days) compared to term placentas (Mathias et al., 2005). So far, a couple of human placental perfusion studies have been conducted comparing placental transfer in term and pre-term placentas (Fokina et al., 2011; Nanovskaya et al., 2008). Nanovskaya et al. (2008) showed in dually perfused human placenta that the transfer of a known ABCB1 substrate, methadone, is 30% higher in term (38–41 weeks) than in pre-term (27–34 weeks) placentas agreeing with the observed decrease in the ABCB1 protein expression towards term. On the other hand, the developmental changes in human placenta from 30 weeks of gestation to term did not affect the transfer of bupropione across human placenta (Fokina et al., 2011). As an isolated organ system, placental perfusion naturally does not reflect the in vivo pharmacokinetic balance of the whole maternal-placental-fetal-system. In vivo maternal pharmacokinetics affects the amount of a xenobiotic available for placental transfer. Furthermore, serum albumin concentrations differ between maternal and fetal circulations and change dynamically during pregnancy. Maternal serum albumin concentration changes from 25 to 35 g/L from 12 to 41 weeks of gestation (Krauer et al., 1984). At 12–15 weeks albumin concentration in fetal serum is much lower (7.5–16 g/L) than in maternal serum. With advancing gestation fetal albumin concentration increases linearly and after 35 weeks it is about 20% higher than albumin concentration in maternal serum (Krauer et al., 1984). If the chemical diffuses passively across the placenta, the free, unbound chemical will equilibrate across the membranes. Higher albumin concentration, and thus higher total amount of albumin bound drugs in fetal circulation, may thus lead to increased placental drug transfer (see Hutson et al., 2011). Recently, Mathiesen et al. (2009) have shown that placental transfer of benzo(a)pyrene is dependent on albumin concentration in ex vivo human placental perfusion. Interestingly, human ex vivo placental perfusion may also be used to characterize the factors causing interindividual variation in transplacental transfer. We have reported that the expression of ABCG2 protein in perfused placenta correlates with fetal to maternal concentration ratio (FM-ratio) of PhIP (Myllynen et al., 2008) but not with the FM-ratio of IQ (Immonen et al., 2010). Recently Tertti et al. (2011) suggested that the SLCO1B3 polymorphism affects fetal to maternal transfer of repaglinide in ex vivo human placental perfusion. However, the number of placentas was not sufficient for a statistical analysis. Because human placental perfusion studies are very time-consuming, their usefulness for
the characterization of factors causing interindividual variation in transplacental kinetics is limited. A recent meta-analysis by Hutson et al. (2011) suggests that placental perfusion predicts relatively well placental transfer in vivo. Hutson et al. (2011) analyzed systematically whether ex vivo placental perfusion accurately predicts the in vivo placental drug transfer. They included in their analysis data on 26 drugs studied using ex vivo human placental perfusion and reported a significant correlation between feto-maternal concentration ratios in perfusion and in vivo cord blood to maternal blood concentration ratios (R2 = 0.28, P = 0.004). After adjusting the perfusion results for differences in maternal and fetal/neonatal protein binding and blood pH Hutson et al. (2011) were able to predict in vivo transfer at steady state more accurately (R2 = 0.54, P < 0.001) although there still were six outliers for which the prediction was not accurate. In one case (propranolol) perfusion significantly overestimated (FM-ratio >200% of the in vivo measured cord blood/maternal blood concentration ratio), and in five cases underestimated (FM ratio 50% or under) the in vivo transplacental transfer. When Hutson et al. (2011) excluded four outliers due to problems in the in vivo data (e.g. maternal sample taken within an hour after birth and not necessarily at the same time as the fetal sample), the perfusion results strongly predicted the in vivo results (R2 = 0.85, P < 0.0001). Interlaboratory comparison as a part of prevalidation within the EU-project ReProTect has been carried out, (Myllynen et al., 2010). Transferability of human placental perfusion method from one laboratory to another was shown to be good. Comparison within and between the studied laboratories showed a relatively good correlation for 3 out of 4 compounds. However, in the case of benzo(a)pyrene the comparison could not be done because the curve fitting of the data failed. Although we were not able to compare results using curve fitting, the results regarding transplacental transfer rate of benzo(a)pyrene were clearly different in the laboratories (Mathiesen et al., 2009; Karttunen et al., 2010). Placental transfer of benzo(a)pyrene is greatly affected by perfusion conditions as shown by Mathiesen et al. (2009). What may have, however, affected the results is the fact that Mathiesen et al. (2009) used 14C-labeled benzo(a)pyrene while Karttunen et al. (2010) used 3H-labeled substance.
3. Continuous cell lines originating from human placenta The most commonly used continuous human placental cell lines in toxicological research are the oldest placental cell lines, BeWo, JAr and JEG-3, which have been available for over 30 years (Pattillo and Gey, 1968; Pattillo et al., 1971; Kohler and Bridson, 1971). BeWo cells originate from choriocarcinoma which was first transferred to the cheek-pouch of a hamster and later placed in co-culture with human decidual explants. From this co-culture Pattillo and Gey (1968) isolated the BeWo cell line. The same serially
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transferred choriocarcinoma tissues (the same strain as Pattillo and co-workers had used) were also used to create the JEG-3 choriocarcinoma cell line (Kohler and Bridson, 1971). The JAr choriocarcinoma cells were directly established by Pattillo and coworkers from a trophoblastic tumor of human placenta (see Azizkhan et al., 1979). BeWo cells maintain many of the morphological characteristics of human trophoblasts (Wolfe, 2006). They resemble undifferentiated cytotrophoblasts with a few syncytialized cells (Wice et al., 1990). Because these cell lines have been used for such a long time, different strains of the cell lines have been described. For instance, in the case of BeWo cells variation between the fusion rates for syncytialization have been reported by different laboratories (Orendi et al., 2011). Unlike BeWo cells, which resemble cytotrophoblasts, JEG-3 cells possess many of the biological and biochemical characteristics of syncytiotrophoblast, although in contrast to syncytiotrophoblast they are single mononucleated cells which proliferate (for a review see e.g. Prouillac and Lecoeur, 2010). BeWo, JEG-3, and JAr cells have been used widely to study the fate and effects of xenobiotics in the placental cells such as effects on hormone production. They can be used to study various aspects of transplacental transfer such as cellular uptake or efflux of xenobiotics from the cells, and the role of transporters in these processes, as well as xenobiotic metabolism. Also, they can be used to study the effect of xenobiotics on the expression of transporter proteins. However, ABCB1 expression in all three cell lines is much lower (or even not detected at all) compared to primary trophoblasts isolated from term placentas (Evseenko et al., 2006) which is a limitation for these models. On the other hand, similarly to human placenta, ABCG2 is highly expressed in BeWo and JAr cells. Also, JEG-3 cells express ABCG2 although the expression level is not as high as in BeWo cells (Myllynen et al., unpublished results). ABCC2 protein expression has been reported to be low in BeWo and JAr cells (Evseenko et al., 2006) while ABCC1 expression is higher in BeWo and JAr cells than in primary trophoblasts. BeWo, JEG-3 and JAr cells also express xenobiotic metabolizing enzymes. Similarly to human placenta, CYP1A1 enzyme is present and inducible in BeWo and JEG-3 cell lines (Wójtowicz et al., 2011; Augustowska et al., 2007; Avery et al., 2003). In addition, the human placental JEG-3 and JAr choriocarcinoma cell lines have been used as models for the study of CYP19 (aromatase) activity and endocrine functions of human placenta (Letcher et al., 1999). BeWo cells express also aromatase although basal aromatase activity in JEG-3 cells has been reported to be at least 10-fold greater than in BeWo cells (Klempan et al., 2011). Other CYP enzymes such as CYP3A4 and CYP2C9 have been detected at mRNA level in JEG-3 and BeWo cells (Pavek et al., 2007). Thus, these cell lines may serve as a model for xenobiotic metabolism of human placenta. 3.1. Transcellular transport Human placental choriocarcinoma cell lines have been grown on Transwell inserts as an in vitro model for the placental barrier. In the Transwell system trophoblastic cells are cultured in a double chamber, on a filter support. If the cells are capable of forming a tight polarized monolayer on the filter, the chambers are fully separated by the cell layer and transport through the layer is expected to reflect the physiological transport through the trophoblast. The most commonly used human cell line in these experiments has been BeWo b30, a clone of BeWo cells (Liu et al., 1997). In addition to BeWo b30 cells, Ikeda et al. (2011) recently suggested that JEG-3 choriocarcinoma cells can be grown under certain culture conditions into a tight monolayer which is suitable as an in vitro model for placental xenobiotic transfer. The BeWo b30 based cell model is a not a validated test system and as far as we know comparisons between this model and
transplacental transfer in vivo have not been done. Interestingly, data produced by this model and ex vivo placental perfusion seem to agree rather well for several substrates. A recent study suggests a good correlation between the results from human placental perfusion and BeWo b30 cells in the Transwell model of four model substrates, caffeine, antipyrine, benzoic acid and glyphosate (Poulsen et al., 2009). Placental transfer of non-dioxin-like PCBs in BeWo Transwell model and ex vivo human placental perfusion have been reported to be in agreement (Correia-Carreira et al., 2011). There are also data comparing placental perfusion and Transwell model using two mycotoxins, deoxynivalenol (DON; Nielsen et al., 2011) and Ochratoxin A (OTA; Woo et al., 2012). In the study by Nielsen et al. (2011) DON was relatively slowly transported in the Transwell model which was supported by the ex vivo perfusion data. In our recent study (Woo et al., 2012), after a 20-h exposure only 3% of OTA was transferred in the Transwell model in the presence of protein which agreed well with the ex vivo human placental perfusions suggesting minimal transplacental transfer in human term placenta. 4. Human placental primary cells Human placental villous trophoblasts are currently routinely isolated in many laboratories. However, compared to the use of immortalized or cancer cell lines establishing cultures of primary placental cells is laborious. The classical isolation procedure includes a trypsin digestion of placental villuses followed by purification steps (see e.g. Orendi et al., 2011). Once the primary villous cytotrophoblasts have been isolated they may be stored frozen to use the cells from a single placenta for several experiments. However, isolated primary trophoblasts do not proliferate in culture, which is a major disadvantage of this model. On the other hand, primary cells clearly have the advantage of representing normal cells, while cancer cell lines have been malignantly transformed. In addition, in vitro culture of primary trophoblast cells affects the transporter expression profile. Evseenko et al. (2006) reported that in term trophoblast p-glycoprotein expression significantly decreases during a 120-h incubation. In contrast, the level of ABCG2 increases (Evseenko et al., 2006). Similarly to continuous cell lines, primary trophoblasts are useful when studying the mechanisms affecting transplacental transfer such as uptake, efflux and xenobiotic metabolism. Because primary trophoblasts can be isolated at different stages of gestation it is possible to compare first trimester and term cells. However, these cells do not form a tight-junctioned monolayer when cultured on semi-permeable membranes, but rather aggregate with large intercellular spaces (Bode et al., 2006). Hemmings et al. (2001) did overcome some of the problems using highly purified primary cultures of cytotrophoblasts and multiple cycles of seeding and differentiation. They were able to grow a culture which consisted of multiple overlapping layers of syncytialized cells which functioned as a barrier for low- and high-molecular weight markers. Overall, although some attempts have been made to grow these cells on semi-permeable membranes there is so far no established model, which could be used routinely in transplacental transfer studies. 5. Human placental explants and villous explants Human placental explant cultures have been widely used in placental research. Earliest studies focused on placental transport, metabolism and endocrine function but later this technique has been used also for other purposes, like to study cellular proliferation and differentiation (Miller et al., 2005; Orendi et al., 2011). Placental tissues used for experiments have mainly been from the first
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or third trimester. Similarly to in vivo situation, villous explants contain multiple cell types in addition to trophoblastic cells including mesenchymal stromal cells (fibroblasts, myofibroblasts, smooth muscle cells), endothelial cells, blood cells and placental immune cells, which is an advantage for this system (Miller et al., 2005). Unlike isolated primary trophoblasts, villous explants are not regularly frozen. Thus, for each experiment fresh placental tissue is needed and cultures cannot be repeated using the same placenta. Similarly to primary trophoblasts these villous explants can be used to study the mechanisms of transplacental transfer as well as the regulation of transporters and xenobiotic metabolizing enzymes. However, because there is no continuous membrane, but separate tissue pieces, they are not useful for studying transplacental transport. 6. Subcellular fractions Microsomal fractions are prepared from endoplasmic reticulum using differential centrifugation. Microsomal preparations, which contain the membrane bound cytochromes P450 (CYP enzymes), from different tissues including placenta, have been extensively used in studies on xenobiotic metabolism (e.g. Vähäkangas et al., 1989). In addition, placental metabolism has been studied using cytosolic fractions (e.g. Partanen et al., 2010) because some of the metabolizing enzymes are present in the cytosol rather than in microsomes. Other subcellular fractions include human placental membrane vesicles, which are useful for studying basic transporter mechanisms. Both apical brush border and basal membrane vesicles have been prepared from human placenta to study the expression and function of transporters. The purity of isolated membrane vesicles can be determined with specific markers (Glazier and Sibley, 2006). It is also possible to prepare membrane vesicles overexpressing different transporters to further study their role in transport of potential substrates (Vaidya et al., 2009). The membrane preparations are useful in studying transfer mechanisms such as active uptake and efflux. Also, apical and basal membranes can be studied separately. 7. Conclusions Human placental tissue is relatively easily available for experimental studies because after delivery placenta has served its purpose and it is usually disposed of. Ex vivo and in vitro models originating from human placenta provide useful information related to chemical transfer and possible fetal exposure to chemicals. Human placental perfusion and trophoblastic cells grown as a tight monolayer on a semipermeable membrane can be used to study transfer across trophoblastic cell layer while other models provide data on metabolism and transporter function which may modify transplacental transfer (Table 1). So far, studies on different experimental models as well as characterization of born placentas have given valuable information of human placental transporters and xenobiotic metabolizing enzymes. Human placental perfusion retains the structure, including the polarized syncytiotrophoblast, and some prevalidation studies have been carried out within the EU-project ReProTect (Myllynen et al., 2010). Based on the results by Hutson et al. (2011) adjusting the placental perfusion results for protein binding and blood pH improves the accuracy of data provided by ex vivo placental perfusion. Although ex vivo placental perfusion is very time and labor consuming, and thus could not be applied in large scale screening, it may be useful at final stages of risk assessment when information of fetal exposure of a fetotoxic compound is needed. Cell-based placental model systems have been shown to be useful tools to study protein expression and function of placental transporters, as well as xenobiotic metabolism. The restrictions
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naturally are the characteristics of the cell lines, like the lack of ABCB1 in BeWo cells. Some cell lines, e.g. BeWo b30, form a tight monolayer in the Transwell system enabling their use to study transport across trophoblast cell layers. Interestingly, continuous cell lines grown on semi-permeable membranes seem to provide data which is in good agreement with data from human placental perfusion. Importantly, none of the substrates tested so far is a substrate for ABCB1/p-glycoprotein which is believed to be a major efflux transporter in human placenta and which is not significantly expressed in BeWo cells. Obviously further studies are needed with a wider selection of substrates to further evaluate the existing cell models. Employing tight monolayer cultures of trophoblastic cells as an initial screening tool for placental transfer and fetal exposure may be feasible. Data from in vitro experiments may be sufficient if the results are consistent with expectations based on physical and chemical characteristics and comparison to chemically similar compounds. Additional studies using e.g. ex vivo dually perfused human placenta may be necessary in the case of divergent results, or when more complete information is required in a system more closely simulating the in vivo situation. So far none of these models have been validated as a routine test system for transplacental transfer of chemicals across human placenta. Recent reports suggest that human placental perfusion can produce valid data on transplacental transfer at term especially after adjusting the results with protein binding data. It is currently not known how well the placental perfusion and the current in vitro Tanswell models with trophoblastic cells represent fetal exposure at early human pregnancy because in humans there are only very limited possibilities available to study transplacental transfer of xenobiotics at early stages of pregnancy when major structural malformations may occur. On the other hand, transferrelated mechanisms, like expression of transporter proteins, and activities and expression of xenobiotic metabolizing enzymes can be studied in villous explants and primary cells from early placentas giving useful information. Acknowledgements Authors are grateful for the financial support and collaborations within the EU networks NewGeneris (FOOD-CT: 2005-016320) and ReProTect (LSHB-CT-2004-503257). Especially the collaboration with Professor Lisbeth Knudsen (University of Copenhagen) and her research group in these EU-projects has been valuable. References Augustowska, K., Magnowska, Z., Kapiszewska, M., Gregoraszczuk, E.L., 2007. Is the natural PCDD/PCDF mixture toxic for human placental JEG-3 cell line? The action of the toxicants on hormonal profile, CYP1A1 activity, DNA damage and cell apoptosis. Hum. Exp. Toxicol. 26, 407–417. Avery, M.L., Meek, C.E., Audus, K.L., 2003. The presence of inducible cytochrome P450 types 1A1 and 1A2 in the BeWo cell line. Placenta 24, 45–52. Azizkhan, J.C., Speeg Jr., K.V., Stromberg, K., Goode, D., 1979. Stimulation of human chorionic gonadotropin by JAr line choriocarcinoma after inhibition of DNA synthesis. Cancer Res. 39, 1952–1959. Bebenek, I.G., Solaimani, P., Bui, P., Hankinson, O., 2012. CYP2S1 is negatively regulated by corticosteroids in human cell lines. Toxicol. Lett. 209, 30–34. Benirschke, K., Kaufmann, P., Baergen, R.N., 2006. Pathology of the Human Placenta, fifth. Springer, New York. Bode, C.J., Jin, H., Rytting, E., Silverstein, P.S., Young, A.M., Audus, K.L., 2006. In vitro models for studying trophoblast transcellular transport. Methods Mol. Med. 122, 225–239. Correia-Carreira, S., Cartwright, L., Mathiesen, L., Knudsen, L.E., Saunders, M., 2011. Studying placental transfer of highly purified non-dioxin-like PCBs in two models of the placental barrier. Placenta 32, 283–291. Evseenko, D.A., Paxton, J.W., Keelan, J.A., 2006. ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1357–R1365. Fokina, V.M., Patrikeeva, S.L., Zharikova, O.L., Nanovskaya, T.N., Hankins, G.V.D., Ahmed, M.S., 2011. Transplacental transfer and metabolism of buprenorphine in preterm human placenta. Am. J. Perinatol. 28, 25–32.
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Review
Placental transfer and metabolism: An overview of the experimental models utilizing human placental tissue Päivi Myllynen a, Kirsi Vähäkangas b,⇑ a b
Department of Pharmacology and Toxicology, Institute of Biomedicine, University of Oulu, Oulu, Finland Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Kuopio, Finland
a r t i c l e
i n f o
Article history: Received 12 June 2012 Accepted 22 August 2012 Available online 30 August 2012 Keywords: Human placental perfusion Human placental cell lines BeWo cells Placental transporters In vitro models
a b s t r a c t Over the decades several ex vivo and in vitro models which utilize delivered human placenta have been developed to study various placental functions. The use of models originating from human placenta to study transplacental transfer and related mechanisms is an attractive option because human placenta is relatively easily available for experimental studies. After delivery placenta has served its purpose and is usually disposed of. The purpose of this review is to give an overview of the use of human placental models for the studies on human placental transfer and related mechanisms such as transporter functions and xenobiotic metabolism. Human placental perfusion, the most commonly used continuous cell lines, primary cells and tissue culture, as well as subcellular fractions are briefly introduced and their major advantages and disadvantages are discussed. Ó 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ex vivo human placental perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Continuous cell lines originating from human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Transcellular transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human placental primary cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human placental explants and villous explants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subcellular fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Human placenta plays an important role in the development of the fetus, being the major interface between mother and fetus. Placenta provides a gateway to oxygen and nutrients from the mother to the fetus, produces hormones to support the pregnancy and serves as an excretion pathway for various metabolites and carbon dioxide. However, it also plays a role in the exposure of the fetus to potentially toxic xenobiotics via the maternal circulation. Transport processes across the placenta and metabolism in the placenta ⇑ Corresponding author. Address: University of Eastern Finland, Faculty of Health Sciences, School of Pharmacy/Toxicology, P.O. Box 1627, FI-70211 Kuopio, Finland. Tel.: +358 40 745 5254. E-mail address: kirsi.vahakangas@uef.fi (K. Vähäkangas). 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.08.027
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may determine the exposure of the fetus to xenobiotics. Therefore, the studies of these processes are important when trying to predict possible fetal exposure. Our review describes different models that are used to study transplacental transfer and related processes, and discusses their major advantages and disadvantages. Human placenta starts to develop during early pregnancy. Even before the placenta is formed, a few days after fertilization, the first specialized placental cells, trophoblasts, are present forming the outer layer of the blastocyst. Thus, already at this stage barrier of trophoblastic cells may protect the embryo from xenobiotics. During implantation blastocystic trophoblasts attach to and invade into the uterine lining. The trophoblast cells of the implanting embryonic pole differentiate into two trophoblastic layers: a layer of mononuclear cytotrophoblasts and the multinucleated syncytiotrophoblast. These structures will develop into the placenta during
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the first trimester. The villous structure of the human placenta is gradually formed already by 18–20 days after fertilization (Benirschke et al., 2006). However, during the first trimester the cytotrophoblastic plugs partially occlude the lumen of spiral arteries reducing the amount of maternal blood entering the intervillous space. Using the 3-dimensional vaginal ultrasound and power Doppler angiography, maternal intervillous blood flow has been detected from 6th gestational week onwards with a gradual increase during the first trimester (Mercé et al., 2009). From a toxicological point of view it is important that the placenta provides only meager protection for the fetus against various xenobiotics. The mechanisms for transplacental transfer include passive diffusion, active transport, facilitated diffusion, filtration and pinocytosis. Passive diffusion is an important mechanism for transplacental transfer of xenobiotics. Physicochemical properties of xenobiotics such as molecular weight, pKa and lipid solubility affect the transplacental transfer by passive diffusion. Very large chemical compounds with a molecular weight of over 500D are generally transferred incompletely across the placenta (for a review see e.g. Myllynen et al., 2007). Both transporter proteins and xenobiotic metabolism enzymes have been found in human placental tissue, and their significance in transplacental transfer is being actively studied. Human placenta expresses a number of transporter proteins. In syncytiotrophoblast, various transporters have been found both in the brush-border (apical membrane) facing maternal blood and basolateral membrane close to fetal capillaries (for extensive reviews see e.g. Vähäkangas and Myllynen, 2009; Ni and Mao, 2011). Depending on the localization and function, transporters may either increase or decrease xenobiotic transfer through the placenta towards fetal circulation. The expression of the transporters found in the apical and basal membranes of the syncytiotrophoblast differs leading to polarized transport across the structure. Complexity is further increased by the fact that also cells in fetal capillaries harbor transporters. Multiple studies in several species including human suggest that several ABC (ATP binding cassette) transporters decrease fetal exposure to xenobiotics (see e.g. Lankas et al., 1998; Kalabis et al., 2007; Pávek et al., 2001; Smit et al., 1999; Mölsa et al., 2005; Myllynen et al., 2008). In mouse, the inhibition of the ABCB1 transporter (p-glycoprotein, MDR1) has been reported to increase teratogenicity of ivermectin (Lankas et al., 1998). It is important to get more insight in the role of human placental transporters in fetal exposure because the mechanisms of transplacental transfer of many chemicals are insufficiently known. Placental metabolism may also affect fetal exposure to xenobiotics. Compared to the liver the activities of xenobiotic metabolizing enzymes in human placenta are generally low (Hakkola et al., 1998). Still, placental metabolism may lead to metabolites with toxic potential compared to the parent compound. For instance, human placenta can metabolize retinoids (isotretinoin and tretinoin) to both more and less toxic metabolites (see e.g. Miller et al., 1993) and benzo(a)pyrene to active metabolites binding to DNA (e.g. Karttunen et al., 2010). The expression of xenobiotic metabolizing enzymes is dependent on the developmental stage of the placenta (Myllynen et al., 2007). Quite a few CYP (cytochrome P450) enzymes are expressed at the mRNA level, especially during the first trimester (Hakkola et al., 1998; Myllynen et al., 2007). However, of the CYP enzymes from the groups 1–3, only CYP1A1 seems to be functional in human placenta. In term placenta the CYPs 4B1 and 19 (steroid aromatase) are active and contribute to the metabolism of some xenobiotics, e.g. aflatoxin B1 (Storvik et al., 2011; Hakkola et al., 1998). One of the most recently described CYPs, CYP2S1, is also expressed in the placenta and has both endogenous and xenobiotic substrates (Bebenek et al., 2012). Its role in placental toxicology remains to be studied. At
least the following metabolizing enzyme activities have been described in term human placenta: nitric oxide synthase, quinine reductase, nitroquinoline oxidase and alcohol dehydrogenase). Of the transferases, activities of glutathione-S-transferase, glucuronyltransferase as well as sulfotransferase are found from the first trimester on, and N-acetyltransferase at least in term placenta (for a review see e.g. Myllynen et al., 2007). Currently, for ethical and practical reasons animal experiments and testing are being replaced with in vitro models whenever possible. To predict exposure of human fetus to chemicals, several relevant in vitro models for the study of human placental transfer including transporter function and metabolism have been developed over the past decades. The purpose of this review is to give an overview of such models and to compare their usefulness for estimating human placental transfer and metabolism.
2. Ex vivo human placental perfusion Human placental perfusion of a single cotyledon with separate maternal and fetal circulations was originally introduced by Panigel (1962) and further developed by the groups of Schneider et al. (1972) and Miller et al., (1989), (Table 1). Placental perfusion is the only experimental model to study human transplacental transfer in organized human placental tissue. So far there are no standardized criteria for ex vivo human placental perfusion. Placentas from both vaginal births and C-sections can be used for the perfusion. We reported recently that the method of delivery does not affect the transplacental transfer of antipyrine, ethanol or two food borne carcinogens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5f]quinoline (IQ) (Mose et al., 2012). Ex vivo placental perfusion may be either nonrecirculating (open/single-pass) or recirculating (closed) perfusion. The methodological details, such as composition of maternal and fetal perfusates, flow rates in maternal and fetal circulations, composition of gas mixtures used to oxygenate the tissue and perfusate volumes all vary from one laboratory to another. In most published studies human placental perfusions have been carried out for 2–6 h although it is possible to perfuse placentas for extended periods of time, up to 48 h (Polliotti et al., 1996; Miller et al., 1989; Heikkilä et al., 2002; Woo et al., 2012). Various markers are used to monitor placental viability during the perfusion and the selection of markers may vary between research groups (see e.g. Myllynen et al., 2010; Vähäkangas et al., 2011). Minimal loss of fetal perfusate is the main marker of a successful perfusion. Volume loss of 2–3 ml from fetal circulation is usually considered acceptable. Recently, a loss greater than 3 ml/ h has been associated with higher transfer of the control substance FITC-dextran which in successful perfusions does not cross placental membranes due to its high molecular weight (Mathiesen et al., 2010). Antipyrine or other reference compounds such as creatinine are used to confirm the overlap between maternal and fetal circulations. These markers may also be used to normalize the perfusion data for interindividual variation. Other markers used for tissue viability include pH, oxygen consumption, net oxygen transfer, glucose consumption, lactate production and tissue morphology after perfusion. Hormone production (human chorionic gonadotropin or hCG, human placental lactogen or hPL, leptin) is also a good marker of functional placental tissue. Although placentas from 27–40 weeks have been perfused (Fokina et al., 2011; Nanovskaya et al., 2008), the challenging perfusion method is usually applied to term placenta. Even then the success rate, counting from the placentas good enough to be hooked to the perfusion equipment, is only about 50%. Thus, the major limitation of this technique is that it does not provide data
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Table 1 Commonly used in vitro models for human placental transfer and associated mechanisms (Vähäkangas and Myllynen, 2006; Vähäkangas et al., 2011). Material
Ex vivo/in vitro models
Used for
Whole tissue
Ex vivo human placental perfusion
Tissue pieces
Explant cultures Villous explant cultures Primary trophoblastic cells Choriocarcinoma cell lines (BeWo, JEG-3, JAr) BeWo b30
Transplacental transfer including xenobiotic metabolism and transporter function Mechanisms affecting transplacental transfer such as uptake, efflux and metabolism Mechanisms affecting transplacental transfer such as uptake, efflux and metabolism Transplacental transfer including xenobiotic metabolism and transporter functions Placental xenobiotic metabolism Placental transporter functions
Cells Tight monolayer culture on permeable membrane Tissue fractions
Placental microsomes and cytosolic fraction Placental microvillous and basal membrane vesicles
on placental transfer in the first and second trimester placentas. It is known that the materno-fetal diffusion distance changes significantly during pregnancy decreasing from 50–100 lm in the second month to 4–5 lm at term (Benirschke et al., 2006). Also, the expression of transporter proteins changes dynamically during pregnancy. For example, the expression of ABCB1 in the apical membrane of syncytiotrophoblast decreases as gestation advances (Mathias et al., 2005; Sun et al., 2006). The protein expression of ABCB1 has been reported to be 44.8-fold higher in placentas from early pregnancy (60–90 days) compared to term placentas (Mathias et al., 2005). So far, a couple of human placental perfusion studies have been conducted comparing placental transfer in term and pre-term placentas (Fokina et al., 2011; Nanovskaya et al., 2008). Nanovskaya et al. (2008) showed in dually perfused human placenta that the transfer of a known ABCB1 substrate, methadone, is 30% higher in term (38–41 weeks) than in pre-term (27–34 weeks) placentas agreeing with the observed decrease in the ABCB1 protein expression towards term. On the other hand, the developmental changes in human placenta from 30 weeks of gestation to term did not affect the transfer of bupropione across human placenta (Fokina et al., 2011). As an isolated organ system, placental perfusion naturally does not reflect the in vivo pharmacokinetic balance of the whole maternal-placental-fetal-system. In vivo maternal pharmacokinetics affects the amount of a xenobiotic available for placental transfer. Furthermore, serum albumin concentrations differ between maternal and fetal circulations and change dynamically during pregnancy. Maternal serum albumin concentration changes from 25 to 35 g/L from 12 to 41 weeks of gestation (Krauer et al., 1984). At 12–15 weeks albumin concentration in fetal serum is much lower (7.5–16 g/L) than in maternal serum. With advancing gestation fetal albumin concentration increases linearly and after 35 weeks it is about 20% higher than albumin concentration in maternal serum (Krauer et al., 1984). If the chemical diffuses passively across the placenta, the free, unbound chemical will equilibrate across the membranes. Higher albumin concentration, and thus higher total amount of albumin bound drugs in fetal circulation, may thus lead to increased placental drug transfer (see Hutson et al., 2011). Recently, Mathiesen et al. (2009) have shown that placental transfer of benzo(a)pyrene is dependent on albumin concentration in ex vivo human placental perfusion. Interestingly, human ex vivo placental perfusion may also be used to characterize the factors causing interindividual variation in transplacental transfer. We have reported that the expression of ABCG2 protein in perfused placenta correlates with fetal to maternal concentration ratio (FM-ratio) of PhIP (Myllynen et al., 2008) but not with the FM-ratio of IQ (Immonen et al., 2010). Recently Tertti et al. (2011) suggested that the SLCO1B3 polymorphism affects fetal to maternal transfer of repaglinide in ex vivo human placental perfusion. However, the number of placentas was not sufficient for a statistical analysis. Because human placental perfusion studies are very time-consuming, their usefulness for
the characterization of factors causing interindividual variation in transplacental kinetics is limited. A recent meta-analysis by Hutson et al. (2011) suggests that placental perfusion predicts relatively well placental transfer in vivo. Hutson et al. (2011) analyzed systematically whether ex vivo placental perfusion accurately predicts the in vivo placental drug transfer. They included in their analysis data on 26 drugs studied using ex vivo human placental perfusion and reported a significant correlation between feto-maternal concentration ratios in perfusion and in vivo cord blood to maternal blood concentration ratios (R2 = 0.28, P = 0.004). After adjusting the perfusion results for differences in maternal and fetal/neonatal protein binding and blood pH Hutson et al. (2011) were able to predict in vivo transfer at steady state more accurately (R2 = 0.54, P < 0.001) although there still were six outliers for which the prediction was not accurate. In one case (propranolol) perfusion significantly overestimated (FM-ratio >200% of the in vivo measured cord blood/maternal blood concentration ratio), and in five cases underestimated (FM ratio 50% or under) the in vivo transplacental transfer. When Hutson et al. (2011) excluded four outliers due to problems in the in vivo data (e.g. maternal sample taken within an hour after birth and not necessarily at the same time as the fetal sample), the perfusion results strongly predicted the in vivo results (R2 = 0.85, P < 0.0001). Interlaboratory comparison as a part of prevalidation within the EU-project ReProTect has been carried out, (Myllynen et al., 2010). Transferability of human placental perfusion method from one laboratory to another was shown to be good. Comparison within and between the studied laboratories showed a relatively good correlation for 3 out of 4 compounds. However, in the case of benzo(a)pyrene the comparison could not be done because the curve fitting of the data failed. Although we were not able to compare results using curve fitting, the results regarding transplacental transfer rate of benzo(a)pyrene were clearly different in the laboratories (Mathiesen et al., 2009; Karttunen et al., 2010). Placental transfer of benzo(a)pyrene is greatly affected by perfusion conditions as shown by Mathiesen et al. (2009). What may have, however, affected the results is the fact that Mathiesen et al. (2009) used 14C-labeled benzo(a)pyrene while Karttunen et al. (2010) used 3H-labeled substance.
3. Continuous cell lines originating from human placenta The most commonly used continuous human placental cell lines in toxicological research are the oldest placental cell lines, BeWo, JAr and JEG-3, which have been available for over 30 years (Pattillo and Gey, 1968; Pattillo et al., 1971; Kohler and Bridson, 1971). BeWo cells originate from choriocarcinoma which was first transferred to the cheek-pouch of a hamster and later placed in co-culture with human decidual explants. From this co-culture Pattillo and Gey (1968) isolated the BeWo cell line. The same serially
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transferred choriocarcinoma tissues (the same strain as Pattillo and co-workers had used) were also used to create the JEG-3 choriocarcinoma cell line (Kohler and Bridson, 1971). The JAr choriocarcinoma cells were directly established by Pattillo and coworkers from a trophoblastic tumor of human placenta (see Azizkhan et al., 1979). BeWo cells maintain many of the morphological characteristics of human trophoblasts (Wolfe, 2006). They resemble undifferentiated cytotrophoblasts with a few syncytialized cells (Wice et al., 1990). Because these cell lines have been used for such a long time, different strains of the cell lines have been described. For instance, in the case of BeWo cells variation between the fusion rates for syncytialization have been reported by different laboratories (Orendi et al., 2011). Unlike BeWo cells, which resemble cytotrophoblasts, JEG-3 cells possess many of the biological and biochemical characteristics of syncytiotrophoblast, although in contrast to syncytiotrophoblast they are single mononucleated cells which proliferate (for a review see e.g. Prouillac and Lecoeur, 2010). BeWo, JEG-3, and JAr cells have been used widely to study the fate and effects of xenobiotics in the placental cells such as effects on hormone production. They can be used to study various aspects of transplacental transfer such as cellular uptake or efflux of xenobiotics from the cells, and the role of transporters in these processes, as well as xenobiotic metabolism. Also, they can be used to study the effect of xenobiotics on the expression of transporter proteins. However, ABCB1 expression in all three cell lines is much lower (or even not detected at all) compared to primary trophoblasts isolated from term placentas (Evseenko et al., 2006) which is a limitation for these models. On the other hand, similarly to human placenta, ABCG2 is highly expressed in BeWo and JAr cells. Also, JEG-3 cells express ABCG2 although the expression level is not as high as in BeWo cells (Myllynen et al., unpublished results). ABCC2 protein expression has been reported to be low in BeWo and JAr cells (Evseenko et al., 2006) while ABCC1 expression is higher in BeWo and JAr cells than in primary trophoblasts. BeWo, JEG-3 and JAr cells also express xenobiotic metabolizing enzymes. Similarly to human placenta, CYP1A1 enzyme is present and inducible in BeWo and JEG-3 cell lines (Wójtowicz et al., 2011; Augustowska et al., 2007; Avery et al., 2003). In addition, the human placental JEG-3 and JAr choriocarcinoma cell lines have been used as models for the study of CYP19 (aromatase) activity and endocrine functions of human placenta (Letcher et al., 1999). BeWo cells express also aromatase although basal aromatase activity in JEG-3 cells has been reported to be at least 10-fold greater than in BeWo cells (Klempan et al., 2011). Other CYP enzymes such as CYP3A4 and CYP2C9 have been detected at mRNA level in JEG-3 and BeWo cells (Pavek et al., 2007). Thus, these cell lines may serve as a model for xenobiotic metabolism of human placenta. 3.1. Transcellular transport Human placental choriocarcinoma cell lines have been grown on Transwell inserts as an in vitro model for the placental barrier. In the Transwell system trophoblastic cells are cultured in a double chamber, on a filter support. If the cells are capable of forming a tight polarized monolayer on the filter, the chambers are fully separated by the cell layer and transport through the layer is expected to reflect the physiological transport through the trophoblast. The most commonly used human cell line in these experiments has been BeWo b30, a clone of BeWo cells (Liu et al., 1997). In addition to BeWo b30 cells, Ikeda et al. (2011) recently suggested that JEG-3 choriocarcinoma cells can be grown under certain culture conditions into a tight monolayer which is suitable as an in vitro model for placental xenobiotic transfer. The BeWo b30 based cell model is a not a validated test system and as far as we know comparisons between this model and
transplacental transfer in vivo have not been done. Interestingly, data produced by this model and ex vivo placental perfusion seem to agree rather well for several substrates. A recent study suggests a good correlation between the results from human placental perfusion and BeWo b30 cells in the Transwell model of four model substrates, caffeine, antipyrine, benzoic acid and glyphosate (Poulsen et al., 2009). Placental transfer of non-dioxin-like PCBs in BeWo Transwell model and ex vivo human placental perfusion have been reported to be in agreement (Correia-Carreira et al., 2011). There are also data comparing placental perfusion and Transwell model using two mycotoxins, deoxynivalenol (DON; Nielsen et al., 2011) and Ochratoxin A (OTA; Woo et al., 2012). In the study by Nielsen et al. (2011) DON was relatively slowly transported in the Transwell model which was supported by the ex vivo perfusion data. In our recent study (Woo et al., 2012), after a 20-h exposure only 3% of OTA was transferred in the Transwell model in the presence of protein which agreed well with the ex vivo human placental perfusions suggesting minimal transplacental transfer in human term placenta. 4. Human placental primary cells Human placental villous trophoblasts are currently routinely isolated in many laboratories. However, compared to the use of immortalized or cancer cell lines establishing cultures of primary placental cells is laborious. The classical isolation procedure includes a trypsin digestion of placental villuses followed by purification steps (see e.g. Orendi et al., 2011). Once the primary villous cytotrophoblasts have been isolated they may be stored frozen to use the cells from a single placenta for several experiments. However, isolated primary trophoblasts do not proliferate in culture, which is a major disadvantage of this model. On the other hand, primary cells clearly have the advantage of representing normal cells, while cancer cell lines have been malignantly transformed. In addition, in vitro culture of primary trophoblast cells affects the transporter expression profile. Evseenko et al. (2006) reported that in term trophoblast p-glycoprotein expression significantly decreases during a 120-h incubation. In contrast, the level of ABCG2 increases (Evseenko et al., 2006). Similarly to continuous cell lines, primary trophoblasts are useful when studying the mechanisms affecting transplacental transfer such as uptake, efflux and xenobiotic metabolism. Because primary trophoblasts can be isolated at different stages of gestation it is possible to compare first trimester and term cells. However, these cells do not form a tight-junctioned monolayer when cultured on semi-permeable membranes, but rather aggregate with large intercellular spaces (Bode et al., 2006). Hemmings et al. (2001) did overcome some of the problems using highly purified primary cultures of cytotrophoblasts and multiple cycles of seeding and differentiation. They were able to grow a culture which consisted of multiple overlapping layers of syncytialized cells which functioned as a barrier for low- and high-molecular weight markers. Overall, although some attempts have been made to grow these cells on semi-permeable membranes there is so far no established model, which could be used routinely in transplacental transfer studies. 5. Human placental explants and villous explants Human placental explant cultures have been widely used in placental research. Earliest studies focused on placental transport, metabolism and endocrine function but later this technique has been used also for other purposes, like to study cellular proliferation and differentiation (Miller et al., 2005; Orendi et al., 2011). Placental tissues used for experiments have mainly been from the first
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or third trimester. Similarly to in vivo situation, villous explants contain multiple cell types in addition to trophoblastic cells including mesenchymal stromal cells (fibroblasts, myofibroblasts, smooth muscle cells), endothelial cells, blood cells and placental immune cells, which is an advantage for this system (Miller et al., 2005). Unlike isolated primary trophoblasts, villous explants are not regularly frozen. Thus, for each experiment fresh placental tissue is needed and cultures cannot be repeated using the same placenta. Similarly to primary trophoblasts these villous explants can be used to study the mechanisms of transplacental transfer as well as the regulation of transporters and xenobiotic metabolizing enzymes. However, because there is no continuous membrane, but separate tissue pieces, they are not useful for studying transplacental transport. 6. Subcellular fractions Microsomal fractions are prepared from endoplasmic reticulum using differential centrifugation. Microsomal preparations, which contain the membrane bound cytochromes P450 (CYP enzymes), from different tissues including placenta, have been extensively used in studies on xenobiotic metabolism (e.g. Vähäkangas et al., 1989). In addition, placental metabolism has been studied using cytosolic fractions (e.g. Partanen et al., 2010) because some of the metabolizing enzymes are present in the cytosol rather than in microsomes. Other subcellular fractions include human placental membrane vesicles, which are useful for studying basic transporter mechanisms. Both apical brush border and basal membrane vesicles have been prepared from human placenta to study the expression and function of transporters. The purity of isolated membrane vesicles can be determined with specific markers (Glazier and Sibley, 2006). It is also possible to prepare membrane vesicles overexpressing different transporters to further study their role in transport of potential substrates (Vaidya et al., 2009). The membrane preparations are useful in studying transfer mechanisms such as active uptake and efflux. Also, apical and basal membranes can be studied separately. 7. Conclusions Human placental tissue is relatively easily available for experimental studies because after delivery placenta has served its purpose and it is usually disposed of. Ex vivo and in vitro models originating from human placenta provide useful information related to chemical transfer and possible fetal exposure to chemicals. Human placental perfusion and trophoblastic cells grown as a tight monolayer on a semipermeable membrane can be used to study transfer across trophoblastic cell layer while other models provide data on metabolism and transporter function which may modify transplacental transfer (Table 1). So far, studies on different experimental models as well as characterization of born placentas have given valuable information of human placental transporters and xenobiotic metabolizing enzymes. Human placental perfusion retains the structure, including the polarized syncytiotrophoblast, and some prevalidation studies have been carried out within the EU-project ReProTect (Myllynen et al., 2010). Based on the results by Hutson et al. (2011) adjusting the placental perfusion results for protein binding and blood pH improves the accuracy of data provided by ex vivo placental perfusion. Although ex vivo placental perfusion is very time and labor consuming, and thus could not be applied in large scale screening, it may be useful at final stages of risk assessment when information of fetal exposure of a fetotoxic compound is needed. Cell-based placental model systems have been shown to be useful tools to study protein expression and function of placental transporters, as well as xenobiotic metabolism. The restrictions
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naturally are the characteristics of the cell lines, like the lack of ABCB1 in BeWo cells. Some cell lines, e.g. BeWo b30, form a tight monolayer in the Transwell system enabling their use to study transport across trophoblast cell layers. Interestingly, continuous cell lines grown on semi-permeable membranes seem to provide data which is in good agreement with data from human placental perfusion. Importantly, none of the substrates tested so far is a substrate for ABCB1/p-glycoprotein which is believed to be a major efflux transporter in human placenta and which is not significantly expressed in BeWo cells. Obviously further studies are needed with a wider selection of substrates to further evaluate the existing cell models. Employing tight monolayer cultures of trophoblastic cells as an initial screening tool for placental transfer and fetal exposure may be feasible. Data from in vitro experiments may be sufficient if the results are consistent with expectations based on physical and chemical characteristics and comparison to chemically similar compounds. Additional studies using e.g. ex vivo dually perfused human placenta may be necessary in the case of divergent results, or when more complete information is required in a system more closely simulating the in vivo situation. So far none of these models have been validated as a routine test system for transplacental transfer of chemicals across human placenta. Recent reports suggest that human placental perfusion can produce valid data on transplacental transfer at term especially after adjusting the results with protein binding data. It is currently not known how well the placental perfusion and the current in vitro Tanswell models with trophoblastic cells represent fetal exposure at early human pregnancy because in humans there are only very limited possibilities available to study transplacental transfer of xenobiotics at early stages of pregnancy when major structural malformations may occur. On the other hand, transferrelated mechanisms, like expression of transporter proteins, and activities and expression of xenobiotic metabolizing enzymes can be studied in villous explants and primary cells from early placentas giving useful information. Acknowledgements Authors are grateful for the financial support and collaborations within the EU networks NewGeneris (FOOD-CT: 2005-016320) and ReProTect (LSHB-CT-2004-503257). Especially the collaboration with Professor Lisbeth Knudsen (University of Copenhagen) and her research group in these EU-projects has been valuable. References Augustowska, K., Magnowska, Z., Kapiszewska, M., Gregoraszczuk, E.L., 2007. Is the natural PCDD/PCDF mixture toxic for human placental JEG-3 cell line? The action of the toxicants on hormonal profile, CYP1A1 activity, DNA damage and cell apoptosis. Hum. Exp. Toxicol. 26, 407–417. Avery, M.L., Meek, C.E., Audus, K.L., 2003. The presence of inducible cytochrome P450 types 1A1 and 1A2 in the BeWo cell line. Placenta 24, 45–52. Azizkhan, J.C., Speeg Jr., K.V., Stromberg, K., Goode, D., 1979. Stimulation of human chorionic gonadotropin by JAr line choriocarcinoma after inhibition of DNA synthesis. Cancer Res. 39, 1952–1959. Bebenek, I.G., Solaimani, P., Bui, P., Hankinson, O., 2012. CYP2S1 is negatively regulated by corticosteroids in human cell lines. Toxicol. Lett. 209, 30–34. Benirschke, K., Kaufmann, P., Baergen, R.N., 2006. Pathology of the Human Placenta, fifth. Springer, New York. Bode, C.J., Jin, H., Rytting, E., Silverstein, P.S., Young, A.M., Audus, K.L., 2006. In vitro models for studying trophoblast transcellular transport. Methods Mol. Med. 122, 225–239. Correia-Carreira, S., Cartwright, L., Mathiesen, L., Knudsen, L.E., Saunders, M., 2011. Studying placental transfer of highly purified non-dioxin-like PCBs in two models of the placental barrier. Placenta 32, 283–291. Evseenko, D.A., Paxton, J.W., Keelan, J.A., 2006. ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1357–R1365. Fokina, V.M., Patrikeeva, S.L., Zharikova, O.L., Nanovskaya, T.N., Hankins, G.V.D., Ahmed, M.S., 2011. Transplacental transfer and metabolism of buprenorphine in preterm human placenta. Am. J. Perinatol. 28, 25–32.
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