Methods to study expression and function of drug transporters in the placenta

Due to ethical restrictions and technical limitations to perform studies in pregnant women, great deal of current knowledge on ABCG2 expression and function in the fetoplacental unit relies on alternative approaches; these include in vitro cell line models, ex vivo perfused placenta and in vivo transgenic animals. Here we briefly summarize experimental techniques to study ABC efflux transporters in the fetoplacental unit.

In vitro methods

Cells derived from the placental trophoblast or cell lines generated from malignant tissues of human or animal origin (e.g. BeWo, Jeg-3, JAr, HRP-1 etc.) present an extensively used model of the materno-fetal barrier (see Table 1).

Before using any cell-based model, verification of transporter expression is imperative as not all placenta-derived cell lines express the same profile of efflux transporters as the native organ. Actually, even different clones of the same cell line, such as human choriocarcinoma BeWo, show various expression and activity of efflux transporters (Atkinson et al., 2003; Ceckova et al., 2006). We assessed that BeWo cell line (Ceckova et al., 2006) as well as rat placenta HRP-1 cells (Staud et al., 2006) express prominent levels of functional ABCG2 and may, therefore, present a useful tool for in vitro studies. Recently, Beghin et al. (2009) have developed and characterized a primoculture of Wistar rat trophoblast from the labyrinth zone of placenta and evaluated stable expression of Abcg2 throughout the culture period. Therefore, these and other trophoblast and choriocarcinoma cell lines, in which ABCG2/Abcg2 expression has been detected (Bailey-Dell et al., 2001; Evseenko et al., 2006; Serrano et al., 2007) offer suitable in vitro models to study the transport of ABCG2 substrates through the placental barrier.

Although in vitro models help reduce the need of experimental animals in pharmacological research, data obtained from these studies cannot fully reflect the physiological and biochemical changes in placenta; they do, however, offer a valuable tool especially for carrying out pilot experiments.

In situ methods

The methods of dually perfused term human or animal placenta belong to technically demanding yet invaluable approaches for detailed studies of transplacental pharmacokinetics. This technique enables to observe transport of substances in both maternal-to-fetal and fetal-to-maternal directions; with the use of specific substrates and/or inhibitors and concentration-dependent experiments, this approach allows for investigation of individual transport proteins or metabolic enzymes in the placental barrier. The main drawback of the perfused placental system is that it is restricted to the last days of gestation and cannot be used to study processes occurring earlier in the course of pregnancy. Nevertheless, both human (Myllynen et al., 2008) and animal (Staud et al., 2006; Cygalova et al., 2009) placenta perfusions have significantly contributed to the knowledge of ABC drug efflux transporters in the placenta. Click here for more details about dually perfused rat placenta.

In vivo methods

A valuable in vivo approach is represented by mice lacking the transporter of interest either due to natural deficiency (Lankas et al., 1998; Smit et al., 1999) or due to targeted knock-out of the transporter encoding gene(s) (Lagas et al., 2009; Vlaming et al., 2009; DeGorter and Kim, 2011). Mating of transporter-deficient pregnant dams with heterozygous males gives rise to fetuses (and their genetically identical placentas) of three different genotypes; the protective role of the placental transporter can be then evaluated by comparing the rate of drug penetrations to the fetuses across placentas of no, middle and high expression of the transporters (Lagas et al., 2009). To investigate the activity of placental and fetal transporters and their fetoprotective role at different stages of gestation, we introduced a model in which pregnant rats are infused with a substrate and the fetal organs are subsequently collected and processed (Cygalova et al., 2008).

Transferability of data obtained from rodent models to human conditions is often questioned due to interspecies differences in placental anatomy. Although both human and rodent placentas are of hemochorial type (Carter, 2001) they show certain structural differences; whilst trophoblast invasion in rodents is shallow and placenta comprises of three trophoblast layers, invasion of trophoblasts into uterine arteries in human placenta is more extensive with a single layer of syncytial trophoblast (Carter, 2007). Consequently, rodent placenta would not be an optimal model for studies of e.g. trophoblast invasion and vascular remodelling; however, it is a well justified and  frequently used model for assessing transplacental pharmacokinetics because of similar ABC efflux transporters expression, localization and function in human and rodent placentas (Mathias et al., 2005; Meyer Zu Schwabedissen et al., 2005; Aleksunes et al., 2008). Recently, several studies using placentas of different mammal species have presented very similar results regarding transplacental pharmacokinetics of glyburide; ABCG2 was found to significantly limit maternal-to-fetal transport of this compound across human (Gedeon et al., 2008; Pollex et al., 2008), rat (Cygalova et al., 2009) and mouse (Zhou et al., 2008) placenta, thus providing an example to justify the use of rodent models in the research of transplacental pharmacokinetics.

Nevertheless, since placenta is a very complex organ, none of the above mentioned models can exactly reflect all the functions of the human placental tissue in vivo. It is, therefore, highly advisable to use several alternative approaches to investigate the transplacental pharmacokinetics and functions of transporters expressed in the placental trophoblast and then treat the obtained data in a complex manner.

For further information see: (Hahnova-Cygalova et al., 2011; Vahakangas et al., 2011; Staud et al., 2012).

Table 1. Overview of in vitro cell culture models used to investigate transport systems across the placental trophoblast layer (adapted from Cygalova et al 2010) 

^*Expression and function of ABCB1 has been confirmed only on the c30 subclone of BeWo cells (Utoguchi et al., 2000; Atkinson et al., 2003; Ceckova et al., 2006).

^**Transcripts of mRNA ABCB1 has been detected in the study of Serrano et al (Serrano et al., 2007), but no expression of ABCB1 has been previously detected on the mRNA nor protein level (Atkinson et al., 2003).