Induction of cells differentiation and ABC transporters expression by a myco-estrogen, zearalenone, in human choriocarcinoma cell line (BeWo)
Introduction
Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin biosynthesized by a variety of Fusarium spp. which are common soil fungi, in temperate and warm countries, and are regular contaminants of cereal crops worldwide (Bennett and Klich, 2003). ZEA have been shown to competitively bind to estrogen receptors (Kuiper et al., 1998, Eriksen et al., 2002, Le Guevel and Pakdel, 2001, Minervini et al., 2005) with an affinity of 0.01 relative to 17β-estradiol (E2) in an immortalized pituitary cell line (Stahl et al., 1998). Numbers of in vivo studies report its toxicological effect on reproductive system: the hormonal effects of ZEA exposure include decrease in the rate of ovulation, decrease in the size of the litter in small ruminant females, penis abnormalities and degenerating sperm in males (Zinedine et al., 2007). No teratogenic effects were found in animals in spite of the evidence of a transplacental transfer of ZEA and its major metabolites (Bernhoft et al., 2001, Kumagai and Shimizu, 1982).
During gestation, the ability of villous trophoblast to differentiate to the advanced stage of scyncytiotrophoblast phenotype is a major determinant for placental and fetal growth and development. The differentiation process consists on aggregation process followed by fusion of their plasma membrane. In vitro, this phenomenon is modulated by the balanced actions of extracellular matrices and of various soluble factors like (epidermal growth factor, human chorionic gonadotropin (hCG)). Moreover estrogen stimulates morphological and functional differentiation of human trophoblast (Cronier et al., 1999). The placenta has also 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. Interest in the ability of the placenta to reduce the passage of drugs has increased following the identification of drug efflux transporters located on apical or basal surface of polarized trophoblast cells. Depending on their location some of these proteins act as efflux pumps expelling xenobiotics from the placenta to maternal plasma for example. But other pumps also function as importers, bringing in xenobiotics from the maternal or blood compartment, and may enhance fetal or infant exposure.
Among the xenobiotic transporters expressed at the maternal-facing apical surface of the placental syncytiotrophoblast membrane, the most abundant are multidrug resistance gene product 1 (MDR1/ABCB1), also known as P-glycoprotein, multidrug resistance protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) (Evseenko et al., 2006a). These proteins play prominent roles in rejecting drugs and toxic metabolites from the fetoplacental compartment (Lankas et al., 1998). The two predominantly basolateral (i.e., fetal-facing) ABC transporters, MDR3/ABCB4 and multidrug resistance protein 1 (MRP1/ABCC1), transport their substrates from mother to fetus, vectorially opposite to MDR1 and BCRP. Net transfer across the placenta is then determined by the balance of activity of maternal- and fetal-facing transporters (Evseenko et al., 2006a). Little is known about the factors that regulate ABC transporter expression and activity in the placenta. There is some evidence that their expression changes with gestational age, hormonal influence (estrogen, progesterone) (Mathias et al., 2005) trophoblast differentiation (Pascolo et al., 2003). However, data are very scarce about the effect of environmental contaminants, particularly endocrine disruptors, on placental function and ABC transporter expression.
In this work, we investigated the effect of ZEA on trophoblast differentiation and ABC transporter expression by using an in vitro model of transplacental barrier, the BeWo cell line. This choriocarcinoma cell line, developed from a malignant gestational choriocarcinoma of the fetal placenta (Pattillo and Gey, 1968), exhibits morphological and biochemical characteristics similar to the human trophoblast cells, and presents transporters for multidrug resistance (Evseenko et al., 2006a, Utoguchi et al., 2000). The BeWo cell line has the capacity to non spontaneously differentiate into syncytiotrophoblast, and display several functions of normal human trophoblast cells, including hormone production (progesterone, hCG). Cronier et al. (1999) reported that estradiol stimulated morphological and functional differentiation of human villous cytotrophoblast. Thus, we compared the respective effects of ZEA, 17β-estradiol (E2) and forskolin, an inducer of syncytialisation (Wice et al., 1990), on morphological and biochemical differentiation (hCG secretion in culture media). Effects of combination of ZEA or E2 with the anti-estrogen ICI-182780 were also studied. ABC transporter expression, as well as trophoblast differentiation, has been shown to be under hormonal influence (Evseenko et al., 2006b, Cronier et al., 1999). Therefore, in this study, we evaluated the potential effect of estrogen (ZEA, E2) influence and trophoblast differentiation on ABC transporter expression (P-gp, MRP1, MRP2, BCRP) mRNA and protein expression.
Section snippets
Chemicals and reagents
ZEA, FITC-conjugated goat anti-mouse IgG F(ab′)2 fragment, Hoescht 33442, fulvestrant, E2, forskolin and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) kit were purchased from Sigma–Aldrich (St. Quentin Fallavier, France). Saponin came from Acros organics, Noisy-le-Grand, France. Primary antibodies (JSB-1, M2I-4, BXP-34) were obtained from Alexis Biochemicals Corp. (San Diego, CA, USA). Mouse IgG1 isotype (MOPC 31C) came from Ancell Corp. (Bayport, MN, USA). HPLC grade
Cytotoxicity
BeWo cell monolayers were exposed to ZEA concentrations ranging from 0.1 to 200 μM for 24, 48 and 72 h (Fig. 1). A significant effect on cell viability was observed from 20 μM ZEA, whatever time of exposure. The IC50 was 80 μM at 24 h, 20 μM at 48 h and 15 μM at 72 h. In accordance with these results, ZEA effects were studied at concentrations of 0.1–10 μM. Concentrations of E2, forskolin and ICI-182780 were chosen according literature data, absence of cytotoxicity being verified for each compound (data
Discussion
It is still commonly believed that the placenta protects the fetus from harmful agents. However, historical incident like thalidomide example (McBride, 1961) demonstrate that drugs can cross the placenta and produce serious developmental toxicity. Environmental chemicals (methylmercure, cadmium, lead) also cross the placenta, leading to untoward effects on the developing fetus (Domingo, 1994). The defining cell type of human placenta is the trophoblast, a highly specific type of epithelium that
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
The authors thank Dr. François Guiguen for his fruitful advices in flow cytometry.
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