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Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters
Accepted Manuscript Title: Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters Author: Elisabeth Straka Isabella Ellinger Christina Balthasar Matthias Scheinast Jasmin Schatz Tamara Szattler Sonja Bleichert Leila Saleh Martin Kn¨ofler Harald Zeisler Markus Hengstschl¨ager Margit Rosner Hans Salzer Claudia Gundacker PII: DOI: Reference:
S0300-483X(15)30063-9 http://dx.doi.org/doi:10.1016/j.tox.2015.12.005 TOX 51617
To appear in:
Toxicology
Received date: Revised date: Accepted date:
12-10-2015 10-12-2015 23-12-2015
Please cite this article as: Straka, Elisabeth, Ellinger, Isabella, Balthasar, Christina, Scheinast, Matthias, Schatz, Jasmin, Szattler, Tamara, Bleichert, Sonja, Saleh, Leila, Kn¨ofler, Martin, Zeisler, Harald, Hengstschl¨ager, Markus, Rosner, Margit, Salzer, Hans, Gundacker, Claudia, Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters.Toxicology http://dx.doi.org/10.1016/j.tox.2015.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters Short running title: Mercury toxicokinetics of the human placenta
Elisabeth Straka1&, Isabella Ellinger2&, Christina Balthasar1, Matthias Scheinast1, Jasmin Schatz1, Tamara Szattler1, Sonja Bleichert1, Leila Saleh3, Martin Knöfler3, Harald Zeisler3, Markus Hengstschläger1, Margit Rosner1, Hans Salzer4, Claudia Gundacker1§
1
Institute of Medical Genetics, Medical University Vienna, Vienna, Austria
2
Department of Pathophysiology and Allergy Research, Medical University Vienna, Vienna,
Austria 3
Department of Obstetrics and Fetal-maternal Medicine, Medical University Vienna, Vienna,
Austria 4
Clinic for Pediatrics and Adolescent Medicine, University Hospital Tulln, Tulln, Austria
&
shared first authors
§
Corresponding author
Claudia Gundacker: Medical University Vienna, Institute of Medical Genetics, Waehringer Strasse 10, A-1090 Vienna, Austria, Europe, Phone. +43 1 40160 56503, Email. [email protected] Highlights It is known that MeHg is able to pass the placenta and to affect fetal brain development. Uptake and efflux transporters were examined in human primary trophoblast cells and BeWo cells. Involvement in mercury transfer was assessed by measurement of cellular mercury content upon siRNA mediated gene knockdown. Localization of transporters was determined by immunofluorescence microscopy. LAT1 and rBAT at the apical membrane of the syncytiotrophoblast (STB) are involved in MeHg uptake. MRP1 located at basal membrane of STB mediates mercury efflux. Abstract
BACKGROUND: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. OBJECTIVES: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. METHODS: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by cold vapor-Atomic Fluorescence Spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. RESULTS: We found the amino acid transporter subunits L-Type Amino Acid Transporter (LAT)1 and rBAT (related to b0,+ type amino acid transporter) as well as the efflux transporter Multidrug Resistance Associated Protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-Test P<0.05). CONCLUSION: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is effluxed via MRP1 localized to the basal side of the STB. The findings can well explain why mercury is transported primarily towards the fetal side.
Key words: term placenta, primary trophoblast cells, mercury toxicokinetics, amino acid transporter, ABC transporter Introduction
The knowledge on the capacity of the human placenta to handle exogenous stressors is extremely poor. Mercury is an opportune example because it is well-known that the heavy metal in its most toxic form MeHg, is able to pass the placenta and to adversely affect fetal brain development (Grandjean and Landrigan 2014). However, the mechanisms behind the placental transfer are virtually unknown. An active transport has been assumed because cord blood levels are on average 1.9 fold higher than maternal blood levels (Stern and Smith 2003). In the human body mercury ions including MeHg (CH3Hg+) are preferably conjugated to reduced sulfhydryl groups including cysteine and GSH. The disposition of mercury is regulated by availability of the ligands as well as by ability of the resulting complexes to serve as substrates for a variety of transporters (Ballatori 2002). MeHg-L-cysteine has some structural similarity to the amino acid methionine (Hoffmeyer et al. 2006). The amino acid transporters, which carry methionine into cells, actually transport MeHg-L-cysteine across membranes (Kerper et al. 1992; Simmons-Willis et al. 2002). Once MeHg has entered the cell, it binds to GSH. The conjugate is a substrate for ATP-binding cassette (ABC) transporters that mediate cellular efflux of glutathione S-conjugates (Ballatori 2002). In the attempt to characterize mercury transfer across the human placenta, we selected overall 17 uptake and efflux transporters (Table S1). We included those transporters, which 1) were known or assumed to be involved in mercury transport (Bridges and Zalups 2005, Gundacker et al. 2010) and 2) were known to be expressed on mRNA or protein level in the human placenta (e.g., Kudo and Boyd 2001, Okamoto et al. 2002, Atkinson et al. 2003; Bodó et al. 2003; Pascolo et al. 2003; Evseenko et al. 2006; Serrano et al. 2007). Candidate amino acid transporters include heterodimers comprised of a light (LAT1, LAT2, b0,+) and a heavy subunit (4F2hc [4F2 cell-surface antigen heavy chain], rBAT) transporting
neutral amino acids including methionine or cysteine. They are obligatory exchangers, while active and unidirectional transport by ABC transporters is energy-dependent. Evidence for the candidate proteins to be involved in mercury toxicokinetics was partially available. A particular study yielding a comprehensive view on the complex relationships was missing. Notably, the situation in the placenta was poorly researched. Two studies were available, one on human placenta and one on rat placenta, both dealing with initial uptake mechanisms (Iioka et al. 1987; Kajiwara et al. 1996). It has also been shown that expression of amino acid transporters and ABC transporters in the human placenta is influenced by gestational age and cellular differentiation (Meyer zu Schwabedissen et al. 2005a, Berveiller et al. 2015) as well as by genetic variation (Meyer zu Schwabedissen et al. 2005b). Two further studies examined the disposition of mercury in placenta and fetal tissues in Mrp2 deficient rats exposed to MeHg (Bridges et al. 2012) and in Wistar rats exposed to inorganic mercury (Oliveira et al. 2015). The entity ‘placenta barrier’ consists of the syncytiotrophoblast (STB) that lines the intervillous space covering the villous surface, the initially complete, but later on discontinuous cytotrophoblast (CTB) layer, as well as the fetal endothelial cells (FECs) (Benirschke et al. 2012). To obtain a conclusive model for the placental transfer of mercury it is important to know cellular as well as subcellular localization of the candidate proteins along the placental barrier. Upon start of the project, (sub)cellular distribution of the selected proteins in human placental chorionic tissue was only punctually explored. We used human primary trophoblast cells (hTCs) from healthy term placentas and BeWo, a trophoblast-derived choriocarcinoma cell line, in order to (1) confirm protein expression via immunoblot, (2) characterize trophoblast cells with regard to mercury accumulation, and (3) verify involvement of candidate proteins in mercury toxicokinetics of trophoblast cells via siRNA mediated gene knock down. We expected that in relation to controls (non-targeting
siRNA) downregulation of uptake transporters will reduce cellular mercury contents, as well as that knockdown of efflux transporters will enhance mercury levels. Human term placenta sections were used to (4) determine localization of the proteins in chorionic tissue via immunofluorescence microscopy. Two amino acid transporter subunits and one efflux transporters of the ABC transporter family were found to be involved in mercury toxicokinetics of trophoblast cells (LAT1, rBAT, MRP1). According to the data, a model was deduced, which can explain why mercury is so efficiently transported across the placenta towards the fetal side.
Materials and Methods All study participants gave written informed consent and the study was approved by the ethics committee (EC) of the Medical University Vienna (EC-number 833). Human placental tissue was obtained within 15 minutes after caesarian section of healthy pregnancies at 38-40 weeks of gestation. The organs were transferred to the laboratory at room temperature within 15 minutes and immediately processed to minimize destructive processes. Isolation and cultivation of hTCs Primary trophoblasts were isolated by trypsin/deoxyribonuclease (DNAse) digestion of dissected tissue according to a formerly published protocol (Szlauer et al. 2009). Briefly, about 80 g villous tissue was teased apart from connective tissue and washed in Phosphatebuffered saline (PBS) before digestion in Hanks’ balanced salt solution containing DNAse (Sigma). Two digestion cycles were performed. Resuspended cells were layered on top of a preformed, discontinuous 70-to-5% Percoll (GE Healthcare) density gradient. Material banding at densities between 1.048 and 1.062 g/ml was collected, washed and resuspended in 1x Hanks solution. Contaminating, human leukocyte antigen (HLA) class I-positive cells were removed using mouse-anti HLA class I antibody (W6/32, Sigma) coupled to magnetic
beads coated with goat anti-mouse Immunoglobulin G (IgG) (Novex). Primary cytotrophoblasts transform into syncytia 48h after plating (Szlauer et al. 2009). Cell culture Isolated primary human trophoblast cells were cultured in Keratinocyte-SFM (serum-free medium) (Gibco), supplemented with 10% Fetal Bovine Serum (FBS) (PAN Biotech) and 1% growth supplement (Gibco). BeWo cells (clone 24) and HeLa cells (ATCC, #CCL-2) were cultured in DMEM (Dulbecco’s modified Eagle’s medium) high glucose (Gibco) supplemented with Penicillin (30 mg/L) and Streptomycin (50 mg/L) (Sigma) as well as with 10% FBS (PAN-Biotech) and 1% Glutamax (Gibco) (BeWo) or 10% FBS (Gibco) and 1% LGlutamine (Gibco) (HeLa). Cells were cultured at 37°C with 5% CO2 and 95% humidity. siRNA and MeHg treatment Transfection efficiencies were tested using siGLO Red Transfection Indicator (GE Dharmacon), NupherinTM (Enzo) and various transfection reagents (ViaFect: Promega, DharmaFECT2: GE Dharmacon, all others: Invitrogen). In knockdown experiments cells were treated with non-targeting siRNA (si control) or targetspecific siRNA (GE Dharmacon) (Table S2) mixed with cell-specific transfection reagent and reduced serum media Opti-MEM (Gibco). BeWo cells were seeded in 6-well or 12-well plates at densities of 1x105 and 5x104 cells, respectively. After 8-12 hours, when about 25% confluent, cells were transfected using Lipofectamine RNAiMAX (Invitrogen) (Rosner et al. 2010). hTCs were transfected during seeding (reverse transfection) at densities of 1x106 and 5×105 in 6-well or 12-well plates, respectively, using Lipofectamine2000 (Invitrogen) according to the protocol provided by the manufacturer. MeHg stock solution was prepared with aqueous MeHg chloride (Alfa Aesar). The solutions were directly added to cell media (hereby diluted 1:1000) to finally receive medium concentrations of 0.03, 0.3, 0.9, and 3.0 µM MeHg. The nominal mercury concentrations
were verified by analyses of cells and culture medium. For examining dose- and timedependent mercury accumulation, cells were seeded in 6-well or 12-well plates (same densities as in knockdown experiments). In dosage experiments, 22 hrs after seeding (BeWo) and 72-80 hrs after seeding (hTCs), cells were exposed to MeHg for 24 hrs (BeWo), for 5 hrs (hTCs) or for 2, 4, 5 hrs (hTCs). In knockdown experiments, 72-80 hrs upon transfection, hTCs and BeWo cells were exposed to 0.9 µM MeHg for 5 hrs (hTCs) or 12 hrs (BeWo). Analyses of cell number and protein content After harvesting of BeWo cells, cell number (number of vital cells after live/dead discrimination) was measured on a CASY cell counter and analyzer (Schärfe Systems, Innovatis, Germany). Protein concentration of hTCs was determined with the Qubit 3.0 (Invitrogen) according to the manufacturer’s protocol. Protein Extraction Protein samples from BeWo cells, hTCs and whole placenta were prepared by lysing cell pellets in RIPA (Radio-Immunoprecipitation Assay) buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton, 0.1% SDS (sodium dodecyl sulfate), 0.5% Sodium deoxycolate, supplemented with 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0,3 µg/ml benzamidinchloride, and 10 µg/ml trypsininhibitor (Sigma). Supernatants were collected by centrifugation at 20.879xg for 20 min at 4°C and stored at -80°C. Immunoblotting Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. They were blocked in 5% dry milk, 1X TBS (Tris-buffered saline), 0.1% Tween for one hour at room temperature. Membranes were incubated with diluted antibody in 5% BSA (bovine serum albumin), 1X TBS, 0.1% Tween at 4°C with gentle shaking, overnight. Thereafter, blots were washed and incubated with respective secondary HRP (Horseradish Peroxidase)conjugated antibodies. For a list of used antibodies see Table S1. Signals were visualized
using the enhanced chemiluminescence method (Pierce). Equal loading of lysates was verified by the detection of alpha-tubulin or GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) (BeWo, hTCs) and Cytokeratin 7 (CK7; hTCs). Histone H3 was used as additional loading control for both cell types. Analyses of mercury In dosage experiments and hTC knockdown experiments, cellular mercury concentrations were analyzed per well, while cell medium was analyzed pooled. In experiments with BeWo cells, cells of duplicates or triplicates were pooled and one aliquot was analyzed. Mercury concentrations were normalized to cell number (BeWo, HeLa) or protein content (hTCs). Samples and reference material were acid-digested with nitric acid (65%; suprapur) in a microwave oven (mls 1200 mega, MLS, Germany; MARS6, CEM, Germany). Total mercury concentrations were determined by CV-AFS (‘mercur plus’, Analytik Jena, Germany). Quality assurance was achieved by measuring blank test solutions (limit of detection [LOD]
was 0.4 µg/L) and reference materials. The mean mercury level of reference material Seronorm Trace Elements Human Whole Blood low level (Nycomed LOT 1103129; N=8) was 18.5 µg/L (recovery: 115±7%), that of Seronorm Trace Elements Urine L-2 (Nycomed LOT 1011645; N=10) was 38.3 µg/L (recovery: 96±7%). In order to control for matrix effects in cell material, we also analyzed mercury in HeLa cells (N=9) spiked with 0.1-1.5 ppb MeHg and obtained a mean recovery of 97±8%. Statistical analyses Student’s t-Test was used to compare mean values using IBM SPSS Statistics 20.0. P<0.05 was used to denote statistical significance. Localization of candidate proteins by immunofluorescence microscopy Chorionic tissue was processed by HOPE-fixation (DCS Innovative Diagnostic Systeme) and paraffin-embedding (Blaschitz et al. 2008). 2-4µm sections were de-waxed and rehydrated.
Following antigen retrieval with 0.05% (v/v) citraconic anhydride solution, pH 7.4, for 20 min (Leong 2010), sections were incubated with blocking buffer (5% (v/v) goat serum (Jackson ImmunoResearch Laboratories) in PBS containing 0.05% (w/v) saponin (Sigma)) for 1h at room temperature. Primary antibodies and corresponding Alexa-Fluor®-conjugated secondary antibodies, diluted in blocking buffer, were applied overnight at 4°C or for 1h at room temperature, respectively. Antibody dilutions are detailed in Table S1. In control incubations (negative control), primary antibodies were omitted. Nuclei were stained with 4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Roche Diagnostics GmbH, 50 ug/mL in PBS). After each incubation step, sections were washed intensively with PBS. Fluoromount-G (SouthernBiotech) was used as mounting medium. Grayscale images of individual fluorescence channel were acquired using an Imager Z1 (Zeiss) equipped with a 63x/0.5 oil objective lens (Plan-Apochromat, Zeiss) and Chroma Technology Corp. filter sets in combination with TissueFAXS - Image Acquisition and Management Software (Version 4.2) (TissueGnostic GmbH, Vienna, Austria). Pseudocolors were assigned to the individual images and images were merged using TissueFAXS software. Representative pictures of all placentas were selected and presented with Adobe Photoshop (Version 11.0).
Results Isolation of hTCs Upon isolation of hTCs (Figure 1A), purity of preparations was determined by immunoblotting. Presence of strong Cytokeratin 7 bands and nearly absent Vimentin signals (Figure 1B) indicate absence of non-epithelial cells.
Trophoblast cells have different capacity for mercury accumulation
BeWo cells and hTCs accumulated mercury to clearly detectable amounts, when exposed to MeHg in a range between 0.3 and 3.0 µM (Figures 2A,C). The concentration of 0.9 µM MeHg was furthermore applied in knockdown experiments. Mercury levels increased with dose and time in a similar manner in hTCs prepared from two placentas (Figures 2C,E), however, to variable overall amounts; this is also visible in culture medium concentrations (Figure 2F). This effect was dose- and time-independent. Cell number of BeWo cells decreased with increased MeHg dose, while protein content of hTCs appeared to be unchanged (Figures 2B,D). Non-exposed hTCs (Figure 2C) showed higher mercury levels than non-exposed BeWo cells (Figure 2A) indicating that some mercury exposure has occurred during pregnancy. Transfection efficiencies and knockdown experiments We examined transfection efficiencies of BeWo cells and hard-to-transfect hTCs under various conditions using siGLO Red Transfection Indicator. The effects of forward transfection (siRNA treatment, when cells are 25-30% confluent) and reverse transfection (siRNA treatment at seeding) in combination with Nupherin, which promotes lipofection, were compared (Figure S1C). We also tested a variety of transfection reagents (Figure S1D). We found that reverse knockdown leads to higher transfection efficiency in hTCs than forward knockdown. Nupherin, however, did not enhance transfection efficiency in hTCs to a large extent and was not used in further experiments. Viafect and Lipofecamine2000 were identified as best transfection reagents for hTCs. The experimental protocols are outlined in Figures 3D (BeWo), 3E (hTCs) and the Methods section. Out of 17 candidate proteins, we found two amino acid transporter subunits and one ABC transporter significantly involved in mercury kinetics of BeWo cells (LAT1, rBAT) and hTCs (MRP1). For many proteins, the commercially available antibodies did not provide specific
signals in immunofluorescence microscopy and/or immunoblotting as validated by gene knockdown (results summarized in Table S1). Amino acid transporters are involved in uptake of mercury Upon downregulation of the light chain LAT1 and the heavy chain rBAT, mercury contents in BeWo cells were significantly decreased to 68% and 71%, relative to controls (Figure 3A). For LAT2 a decrease was also observed, however only as a trend (Fig. 3A,B). This trend for decreasing mercury accumulation was visible in hTCs following LAT1 downregulation (78%) (Figure 3B) as well as in HeLa cells (67% and 75%) (Figure S1A). Efflux transporters are involved in mercury efflux In hTCs, downregulation of MRP1 resulted in significant increase of cellular mercury contents (124%) of trophoblast cells (Figure 3C). The antibodies for most ABC transporters (although recommended for use in immunofluorescence) did not provide specific signals (Table S1). Due to low expression levels of ABC transporters in BeWo cells (the only exception is Multidrug resistance protein (MDR)1; Figure 4A), we did not perform knockdown of ABC transporters in BeWo cells. Trophoblast cells show differences in protein expression We observed large variability in protein levels between BeWo cells and hTCs, as well as among hTCs prepared from different placentas, notably for LAT1, 4F2hc, MDR1, and MDR3 (Figure 4A) (our specific observations on MRP2 localization and highly variable MRP2 expression are prepared for a separate publication; Ellinger et al., unpublished). Protein localization The predominant cell type in chorionic tissue expressing amino acid transporters was found to be the trophoblast, which mediates uptake of amino acids as well as of MeHg from maternal blood at the apical plasma membrane (Figure 4B). Low expression was found in stromal cells including endothelial cells. While the predominant subcellular localization of
the heavy chains 4F2hc and rBAT was at the plasma membrane (apical and basolateral), the light chains LAT1, LAT2 and b0,+ showed a higher extent of intracellular, vesicular localization.
Discussion Trophoblast cells have different capacity for mercury accumulation The varying capacities for mercury accumulation of human primary trophoblast cells is in line with observations that genetic polymorphisms in ABC transporters affect placental protein expression and drug transport (Ni and Mao 2011; Meyer zu Schwabedissen et al. 2005b). Additionally, the different capacities for mercury accumulation can well explain the observations from epidemiological studies on mercury levels of mother-child-pairs. A metaanalysis generated a mean ratio of cord blood to maternal blood of 1.9 (central tendency estimate: 1.7, 95th percentile: 3.4). The clearly higher cord blood levels indicate active mercury transport from the mother to the fetus. The ratios however strongly varied between and among populations (Stern and Smith 2003) suggesting that the amounts of mercury transported and retained by the human placenta differ in quite large dimensions. This raises the question on the involved proteins, their expression levels and the regulatory factors behind. Amino acid transporters are involved in mercury uptake Our findings on system L light chains LAT1 (and probably also LAT2) involved in uptake of MeHg into human trophoblast cells are new. Experiments on rat placenta already indicated active MeHg uptake as cysteine conjugate via neutral amino acid carriers (Kajiwara et al. 1996). Similarly, Xenopus laevis oocytes overexpressing LAT1 and LAT2 had increased methionine and MeHg uptake compared to control oocytes with low endogenous LAT1 and LAT2 levels (Simmons‐Willis et al. 2002) and downregulation of LAT1 reduced methionine
uptake into lung cancer cells (Dann et al. 2015). In line with our findings, system L activity was reduced by 17% (si LAT1) and 13% (si LAT2) in experiments on hTCs (Gacciolo et al. 2015). MeHg-cysteine also seems to be a substrate of b0,+-rBAT, another heterodimeric amino acid transporter (Wang et al. 2012). While BeWo cells respond to downregulated rBAT with reduced mercury accumulation, hTCs were unaffected by this treatment. The finding indicates that rBAT participates in mercury uptake into BeWo cells. Because b0,+ downregulation had no such effect, we assume that another light chain may be responsible for mercury uptake into cells. It is known that rBAT, which is present in excess over b0,+, is able to bind other light chain(s) (Fernandez et al. 2002). Currently, it remains unclear whether trophoblast cells can compensate for reduced rBAT protein. We found the predominant subcellular localization of the heavy chains 4F2hc and rBAT at the membrane, while the light chains LAT1, LAT2 and b0,+ show a higher extent of intracellular, vesicular localization. According to Rosario (2013) the internalization of light chains could be an effective measure to regulate amino acid transport. This localization of the transporter light subunits is in agreement with previous reports demonstrating LAT1 and LAT2 presence at the apical membrane of STB (Gaccioli et al. 2015; Kudo and Boyd 2001; Okamoto et al. 2002). Others found 4F2hc expressed at the apical STB but no evidence for system b0,+ in term placenta (Ayuk et al. 2000). ABC transporters are involved in mercury efflux The ABC transporters MDR1, MDR3 and MRP1-MRP5 are present in BeWo and trophoblast cells but there are many discrepancies regarding their expression levels (Atkinson et al. 2003; Bodó et al. 2003; Evseenko et al. 2006; Pascolo et al. 2003; Serrano et al. 2007). Our findings on ABC transporter expression confirm that expression varies between hTCs and BeWo cells, but as well among hTCs, particularly with regard to MDR1, MDR3, and MRP2. MRP1 is
more evenly expressed in both cell types, which is in accordance to the literature (Atkinson et al. 2003; Evseenko et al. 2006; Serrano et al. 2007). Likely candidates for efflux of mercury-GSH conjugates are MRP1 and MRP2 (Gundacker et al. 2010) known to transport GSH, GSSG (glutathione disulfide) and GSH conjugates with different affinities (Deeley and Cole 2006). Upon downregulation of MRP1, hTCs accumulated significantly more mercury than controls. This is in line with previous findings, where Nrf2 activation suppressed mercury accumulation and intoxication in primary mouse hepatocytes through upregulation of downstream proteins among them MRP1 and MRP2 (Toyama et al. 2007). In another study MRP1 overexpressing leukemia cells showed higher resistance to mercury than wildtype cells (Kim et al. 2005). In polarized kidney and liver cells, MRP1 is expressed at the basolateral membrane (Deeley and Cole 2006). In the human placenta, localization of MRP1 was found at the basolateral side of the STB as well as in FECs. The transporter might also have some expression at the apical STB membrane (Nagashige et al. 2003, Ni and Mao 2011). Together with the published localization data, our findings suggest that MRP1 is involved in transport of mercury towards the fetal circulation. Unexpectedly, MRP2 downregulation did not significantly affect cellular mercury concentrations, which is contrary to previous reports (Bridges et al. 2012; Madejczyk et al. 2007). It is possible that MRP2 downregulation is compensated by MRP3. A reciprocal relationship between the two transporters in cholestasis and Dubin-Johnson syndrome has been described (Bodó et al. 2003; Deeley and Cole 2006). Limitations of the study It was not possible to confirm siRNA mediated knockdowns for all 17 candidate proteins on protein level due to lack of proper antibodies (rBAT and MRP3). Although a specific LAT2 antibody was available upon start of the project, it was later on withdrawn from the market.
Conclusions We found two amino acid transporter subunits (LAT1, rBAT) and one ABC transporter (MRP1) to be involved in mercury toxicokinetics of trophoblast cells. According to our data, a model can be deduced (Figure 5) which can explain why mercury is efficiently transported towards the fetal side: It uses LAT1 (probably also LAT2) and rBAT dimerized to another light chain than b0,+ at the apical side of the STB to enter trophoblast cells. Intracellular mercury dissociates from cysteine, binds to GSH and is therefore no longer a substrate of amino acid transporters. Mercury conjugated to GSH is effluxed by MRP1. We made two interesting observations. Human trophoblast cells show strong variation in mercury accumulation capacity and in transporter expression levels. It seems likely that, e.g., LAT1 or MRP1 levels determine mercury transfer rates across the STB. Due to limited amount of trophoblast cells per preparation, we could not keep enough cell material to retrospectively clarify these relationships. Many of the transporters involved in mercury toxicokinetics (system L, ABC transporters) are likely involved in placental dysfunctions such as intrauterine growth restriction, gestational diabetes mellitus, or preeclampsia (Burton and Jauniaux 2004; Johnstone et al. 2011; Regnault et al. 2007). As seen in our study, MeHg uses both amino acid transporters and ABC transporters to pass through the STB. This unique feature suggests the use of MeHg in future studies as a model substrate to investigate placental pathophysiology.
Acknowledgments We are grateful to study participants and medical staff at the clinics as well as to Hana Uher for technical assistance. The study was supported by LifeScience2010 (Project LS10026), NFB (Niederösterreichische Forschungs- und Bildungsgesellschaft).
Financial interest declaration The authors declare no competing interests including financial interests.
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Figure caption Figure 1. Isolation of human primary trophoblast cells (hTCs). (A) Procedure of trophoblast isolation from healthy human term placenta. (B) Purity of trophoblast preparation indicated by Vimentin‐negative and Cytokeratin (CK) 7‐positive cells on immunoblot showing five hTC samples and one Rc sample (i.e., cells remaining in top layer). *The lower band in Rc is an unspecific signal.
Figure 2. Comparison of mercury accumulation in BeWo cells and hTCs. (A) Mercury content and (B) cell number of BeWo cells upon exposure to MeHg for 24 hrs as indicated. (C) Dose‐dependent mercury accumulation upon MeHg treatment for 5 hrs as indicated and (E) time‐dependent mercury accumulation upon exposure to 0.9 µM MeHg in hTCs from two placentas (PI and PII). (D) Protein content following MeHg exposure in hTCs from two placentas. (F) Mercury concentrations in pooled cell medium upon MeHg treatment. Data are mean±SD from two independent experiments made in duplicates or triplicates.░
Figure 3. Effect of siRNA-mediated knockdown on mercury accumulation. (A) Cellular mercury content and cell number in BeWo cells upon siRNA‐mediated knockdown of uptake transporters. (B) Cellular mercury and protein content in hTCs upon siRNA‐mediated knockdown of uptake transporters. (C) Cellular mercury and protein content in hTCs upon siRNA‐ mediated knockdown of efflux transporters. (A‐C) Gene knockdown was confirmed by immunoblotting. Representative immunoblots are shown. (D) Experimental protocol on BeWo cells. (E) Experimental protocol on hTCs. Data are mean±SD from three to four independent experiments made in duplicates or triplicates. *t‐ Test P<0.05, **P<0.01 for comparison with controls (non‐targeting siRNA) Figure 4. Protein expression and localization. (A) Protein expression of uptake and efflux transporters in BeWo cells, two hTC (hTCI, hTCII) and two whole placenta samples (wP1, wP2). Representative immunoblots are shown. MDR1, MDR3, MRP2, and 4F2hc as well as LAT1 and MRP5 were detected on the same blots; the respective GAPDH and Histone H3 bands are therefore identical. Note that here the Aviva 4F2hc antibody was used (68kDa). (B) Representative images for the localization of 4F2hc, LAT1, LAT2, CK7 (secondary antibody: Alexa Fluor® 568), rBAT and b0,+ (Alexa Fluor® 647). A representative image for Alexa Fluor® 568 is shown (negative control). Images were acquired using a wide‐field fluorescence microscope equipped with a 63x objective. Arrows indicate the STB layer, which can be identified by Cytokeratin (CK) 7 expression. (a) indicates the apical plasma membrane of the STB. Bars represent 20 µm.
Figure 5. The proposed model for placental mercury toxicokinetics enabling active and efficient transfer from the maternal to the fetal side via the STB. The amino acid transporters LAT1‐4F2hc (perhaps also LAT2‐4F2hc) and X‐rBAT ("X" indicates possible interaction partners of rBAT besides b0,+) located at the apical side of the STB manage uptake of MeHg‐cysteine. Intracellularly, mercury (Hg2+ or MeHg+) is readily conjugated to GSH. The GSH‐Hg conjugates (i.e., GS‐Hg‐SG, MeHg‐SG) are then effluxed from the STB via MRP1 predominantly localized to the basal side of the STB. The model shows only transporters, which, according to our data, are involved in transfer of mercury across the STB. Proteins involved in mercury transfer across FECs have not been explored in this study and remain currently unknown.
S0300-483X(15)30063-9 http://dx.doi.org/doi:10.1016/j.tox.2015.12.005 TOX 51617
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Toxicology
Received date: Revised date: Accepted date:
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Please cite this article as: Straka, Elisabeth, Ellinger, Isabella, Balthasar, Christina, Scheinast, Matthias, Schatz, Jasmin, Szattler, Tamara, Bleichert, Sonja, Saleh, Leila, Kn¨ofler, Martin, Zeisler, Harald, Hengstschl¨ager, Markus, Rosner, Margit, Salzer, Hans, Gundacker, Claudia, Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters.Toxicology http://dx.doi.org/10.1016/j.tox.2015.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters Short running title: Mercury toxicokinetics of the human placenta
Elisabeth Straka1&, Isabella Ellinger2&, Christina Balthasar1, Matthias Scheinast1, Jasmin Schatz1, Tamara Szattler1, Sonja Bleichert1, Leila Saleh3, Martin Knöfler3, Harald Zeisler3, Markus Hengstschläger1, Margit Rosner1, Hans Salzer4, Claudia Gundacker1§
1
Institute of Medical Genetics, Medical University Vienna, Vienna, Austria
2
Department of Pathophysiology and Allergy Research, Medical University Vienna, Vienna,
Austria 3
Department of Obstetrics and Fetal-maternal Medicine, Medical University Vienna, Vienna,
Austria 4
Clinic for Pediatrics and Adolescent Medicine, University Hospital Tulln, Tulln, Austria
&
shared first authors
§
Corresponding author
Claudia Gundacker: Medical University Vienna, Institute of Medical Genetics, Waehringer Strasse 10, A-1090 Vienna, Austria, Europe, Phone. +43 1 40160 56503, Email. [email protected] Highlights It is known that MeHg is able to pass the placenta and to affect fetal brain development. Uptake and efflux transporters were examined in human primary trophoblast cells and BeWo cells. Involvement in mercury transfer was assessed by measurement of cellular mercury content upon siRNA mediated gene knockdown. Localization of transporters was determined by immunofluorescence microscopy. LAT1 and rBAT at the apical membrane of the syncytiotrophoblast (STB) are involved in MeHg uptake. MRP1 located at basal membrane of STB mediates mercury efflux. Abstract
BACKGROUND: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. OBJECTIVES: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. METHODS: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by cold vapor-Atomic Fluorescence Spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. RESULTS: We found the amino acid transporter subunits L-Type Amino Acid Transporter (LAT)1 and rBAT (related to b0,+ type amino acid transporter) as well as the efflux transporter Multidrug Resistance Associated Protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-Test P<0.05). CONCLUSION: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is effluxed via MRP1 localized to the basal side of the STB. The findings can well explain why mercury is transported primarily towards the fetal side.
Key words: term placenta, primary trophoblast cells, mercury toxicokinetics, amino acid transporter, ABC transporter Introduction
The knowledge on the capacity of the human placenta to handle exogenous stressors is extremely poor. Mercury is an opportune example because it is well-known that the heavy metal in its most toxic form MeHg, is able to pass the placenta and to adversely affect fetal brain development (Grandjean and Landrigan 2014). However, the mechanisms behind the placental transfer are virtually unknown. An active transport has been assumed because cord blood levels are on average 1.9 fold higher than maternal blood levels (Stern and Smith 2003). In the human body mercury ions including MeHg (CH3Hg+) are preferably conjugated to reduced sulfhydryl groups including cysteine and GSH. The disposition of mercury is regulated by availability of the ligands as well as by ability of the resulting complexes to serve as substrates for a variety of transporters (Ballatori 2002). MeHg-L-cysteine has some structural similarity to the amino acid methionine (Hoffmeyer et al. 2006). The amino acid transporters, which carry methionine into cells, actually transport MeHg-L-cysteine across membranes (Kerper et al. 1992; Simmons-Willis et al. 2002). Once MeHg has entered the cell, it binds to GSH. The conjugate is a substrate for ATP-binding cassette (ABC) transporters that mediate cellular efflux of glutathione S-conjugates (Ballatori 2002). In the attempt to characterize mercury transfer across the human placenta, we selected overall 17 uptake and efflux transporters (Table S1). We included those transporters, which 1) were known or assumed to be involved in mercury transport (Bridges and Zalups 2005, Gundacker et al. 2010) and 2) were known to be expressed on mRNA or protein level in the human placenta (e.g., Kudo and Boyd 2001, Okamoto et al. 2002, Atkinson et al. 2003; Bodó et al. 2003; Pascolo et al. 2003; Evseenko et al. 2006; Serrano et al. 2007). Candidate amino acid transporters include heterodimers comprised of a light (LAT1, LAT2, b0,+) and a heavy subunit (4F2hc [4F2 cell-surface antigen heavy chain], rBAT) transporting
neutral amino acids including methionine or cysteine. They are obligatory exchangers, while active and unidirectional transport by ABC transporters is energy-dependent. Evidence for the candidate proteins to be involved in mercury toxicokinetics was partially available. A particular study yielding a comprehensive view on the complex relationships was missing. Notably, the situation in the placenta was poorly researched. Two studies were available, one on human placenta and one on rat placenta, both dealing with initial uptake mechanisms (Iioka et al. 1987; Kajiwara et al. 1996). It has also been shown that expression of amino acid transporters and ABC transporters in the human placenta is influenced by gestational age and cellular differentiation (Meyer zu Schwabedissen et al. 2005a, Berveiller et al. 2015) as well as by genetic variation (Meyer zu Schwabedissen et al. 2005b). Two further studies examined the disposition of mercury in placenta and fetal tissues in Mrp2 deficient rats exposed to MeHg (Bridges et al. 2012) and in Wistar rats exposed to inorganic mercury (Oliveira et al. 2015). The entity ‘placenta barrier’ consists of the syncytiotrophoblast (STB) that lines the intervillous space covering the villous surface, the initially complete, but later on discontinuous cytotrophoblast (CTB) layer, as well as the fetal endothelial cells (FECs) (Benirschke et al. 2012). To obtain a conclusive model for the placental transfer of mercury it is important to know cellular as well as subcellular localization of the candidate proteins along the placental barrier. Upon start of the project, (sub)cellular distribution of the selected proteins in human placental chorionic tissue was only punctually explored. We used human primary trophoblast cells (hTCs) from healthy term placentas and BeWo, a trophoblast-derived choriocarcinoma cell line, in order to (1) confirm protein expression via immunoblot, (2) characterize trophoblast cells with regard to mercury accumulation, and (3) verify involvement of candidate proteins in mercury toxicokinetics of trophoblast cells via siRNA mediated gene knock down. We expected that in relation to controls (non-targeting
siRNA) downregulation of uptake transporters will reduce cellular mercury contents, as well as that knockdown of efflux transporters will enhance mercury levels. Human term placenta sections were used to (4) determine localization of the proteins in chorionic tissue via immunofluorescence microscopy. Two amino acid transporter subunits and one efflux transporters of the ABC transporter family were found to be involved in mercury toxicokinetics of trophoblast cells (LAT1, rBAT, MRP1). According to the data, a model was deduced, which can explain why mercury is so efficiently transported across the placenta towards the fetal side.
Materials and Methods All study participants gave written informed consent and the study was approved by the ethics committee (EC) of the Medical University Vienna (EC-number 833). Human placental tissue was obtained within 15 minutes after caesarian section of healthy pregnancies at 38-40 weeks of gestation. The organs were transferred to the laboratory at room temperature within 15 minutes and immediately processed to minimize destructive processes. Isolation and cultivation of hTCs Primary trophoblasts were isolated by trypsin/deoxyribonuclease (DNAse) digestion of dissected tissue according to a formerly published protocol (Szlauer et al. 2009). Briefly, about 80 g villous tissue was teased apart from connective tissue and washed in Phosphatebuffered saline (PBS) before digestion in Hanks’ balanced salt solution containing DNAse (Sigma). Two digestion cycles were performed. Resuspended cells were layered on top of a preformed, discontinuous 70-to-5% Percoll (GE Healthcare) density gradient. Material banding at densities between 1.048 and 1.062 g/ml was collected, washed and resuspended in 1x Hanks solution. Contaminating, human leukocyte antigen (HLA) class I-positive cells were removed using mouse-anti HLA class I antibody (W6/32, Sigma) coupled to magnetic
beads coated with goat anti-mouse Immunoglobulin G (IgG) (Novex). Primary cytotrophoblasts transform into syncytia 48h after plating (Szlauer et al. 2009). Cell culture Isolated primary human trophoblast cells were cultured in Keratinocyte-SFM (serum-free medium) (Gibco), supplemented with 10% Fetal Bovine Serum (FBS) (PAN Biotech) and 1% growth supplement (Gibco). BeWo cells (clone 24) and HeLa cells (ATCC, #CCL-2) were cultured in DMEM (Dulbecco’s modified Eagle’s medium) high glucose (Gibco) supplemented with Penicillin (30 mg/L) and Streptomycin (50 mg/L) (Sigma) as well as with 10% FBS (PAN-Biotech) and 1% Glutamax (Gibco) (BeWo) or 10% FBS (Gibco) and 1% LGlutamine (Gibco) (HeLa). Cells were cultured at 37°C with 5% CO2 and 95% humidity. siRNA and MeHg treatment Transfection efficiencies were tested using siGLO Red Transfection Indicator (GE Dharmacon), NupherinTM (Enzo) and various transfection reagents (ViaFect: Promega, DharmaFECT2: GE Dharmacon, all others: Invitrogen). In knockdown experiments cells were treated with non-targeting siRNA (si control) or targetspecific siRNA (GE Dharmacon) (Table S2) mixed with cell-specific transfection reagent and reduced serum media Opti-MEM (Gibco). BeWo cells were seeded in 6-well or 12-well plates at densities of 1x105 and 5x104 cells, respectively. After 8-12 hours, when about 25% confluent, cells were transfected using Lipofectamine RNAiMAX (Invitrogen) (Rosner et al. 2010). hTCs were transfected during seeding (reverse transfection) at densities of 1x106 and 5×105 in 6-well or 12-well plates, respectively, using Lipofectamine2000 (Invitrogen) according to the protocol provided by the manufacturer. MeHg stock solution was prepared with aqueous MeHg chloride (Alfa Aesar). The solutions were directly added to cell media (hereby diluted 1:1000) to finally receive medium concentrations of 0.03, 0.3, 0.9, and 3.0 µM MeHg. The nominal mercury concentrations
were verified by analyses of cells and culture medium. For examining dose- and timedependent mercury accumulation, cells were seeded in 6-well or 12-well plates (same densities as in knockdown experiments). In dosage experiments, 22 hrs after seeding (BeWo) and 72-80 hrs after seeding (hTCs), cells were exposed to MeHg for 24 hrs (BeWo), for 5 hrs (hTCs) or for 2, 4, 5 hrs (hTCs). In knockdown experiments, 72-80 hrs upon transfection, hTCs and BeWo cells were exposed to 0.9 µM MeHg for 5 hrs (hTCs) or 12 hrs (BeWo). Analyses of cell number and protein content After harvesting of BeWo cells, cell number (number of vital cells after live/dead discrimination) was measured on a CASY cell counter and analyzer (Schärfe Systems, Innovatis, Germany). Protein concentration of hTCs was determined with the Qubit 3.0 (Invitrogen) according to the manufacturer’s protocol. Protein Extraction Protein samples from BeWo cells, hTCs and whole placenta were prepared by lysing cell pellets in RIPA (Radio-Immunoprecipitation Assay) buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton, 0.1% SDS (sodium dodecyl sulfate), 0.5% Sodium deoxycolate, supplemented with 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0,3 µg/ml benzamidinchloride, and 10 µg/ml trypsininhibitor (Sigma). Supernatants were collected by centrifugation at 20.879xg for 20 min at 4°C and stored at -80°C. Immunoblotting Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. They were blocked in 5% dry milk, 1X TBS (Tris-buffered saline), 0.1% Tween for one hour at room temperature. Membranes were incubated with diluted antibody in 5% BSA (bovine serum albumin), 1X TBS, 0.1% Tween at 4°C with gentle shaking, overnight. Thereafter, blots were washed and incubated with respective secondary HRP (Horseradish Peroxidase)conjugated antibodies. For a list of used antibodies see Table S1. Signals were visualized
using the enhanced chemiluminescence method (Pierce). Equal loading of lysates was verified by the detection of alpha-tubulin or GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) (BeWo, hTCs) and Cytokeratin 7 (CK7; hTCs). Histone H3 was used as additional loading control for both cell types. Analyses of mercury In dosage experiments and hTC knockdown experiments, cellular mercury concentrations were analyzed per well, while cell medium was analyzed pooled. In experiments with BeWo cells, cells of duplicates or triplicates were pooled and one aliquot was analyzed. Mercury concentrations were normalized to cell number (BeWo, HeLa) or protein content (hTCs). Samples and reference material were acid-digested with nitric acid (65%; suprapur) in a microwave oven (mls 1200 mega, MLS, Germany; MARS6, CEM, Germany). Total mercury concentrations were determined by CV-AFS (‘mercur plus’, Analytik Jena, Germany). Quality assurance was achieved by measuring blank test solutions (limit of detection [LOD]
was 0.4 µg/L) and reference materials. The mean mercury level of reference material Seronorm Trace Elements Human Whole Blood low level (Nycomed LOT 1103129; N=8) was 18.5 µg/L (recovery: 115±7%), that of Seronorm Trace Elements Urine L-2 (Nycomed LOT 1011645; N=10) was 38.3 µg/L (recovery: 96±7%). In order to control for matrix effects in cell material, we also analyzed mercury in HeLa cells (N=9) spiked with 0.1-1.5 ppb MeHg and obtained a mean recovery of 97±8%. Statistical analyses Student’s t-Test was used to compare mean values using IBM SPSS Statistics 20.0. P<0.05 was used to denote statistical significance. Localization of candidate proteins by immunofluorescence microscopy Chorionic tissue was processed by HOPE-fixation (DCS Innovative Diagnostic Systeme) and paraffin-embedding (Blaschitz et al. 2008). 2-4µm sections were de-waxed and rehydrated.
Following antigen retrieval with 0.05% (v/v) citraconic anhydride solution, pH 7.4, for 20 min (Leong 2010), sections were incubated with blocking buffer (5% (v/v) goat serum (Jackson ImmunoResearch Laboratories) in PBS containing 0.05% (w/v) saponin (Sigma)) for 1h at room temperature. Primary antibodies and corresponding Alexa-Fluor®-conjugated secondary antibodies, diluted in blocking buffer, were applied overnight at 4°C or for 1h at room temperature, respectively. Antibody dilutions are detailed in Table S1. In control incubations (negative control), primary antibodies were omitted. Nuclei were stained with 4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Roche Diagnostics GmbH, 50 ug/mL in PBS). After each incubation step, sections were washed intensively with PBS. Fluoromount-G (SouthernBiotech) was used as mounting medium. Grayscale images of individual fluorescence channel were acquired using an Imager Z1 (Zeiss) equipped with a 63x/0.5 oil objective lens (Plan-Apochromat, Zeiss) and Chroma Technology Corp. filter sets in combination with TissueFAXS - Image Acquisition and Management Software (Version 4.2) (TissueGnostic GmbH, Vienna, Austria). Pseudocolors were assigned to the individual images and images were merged using TissueFAXS software. Representative pictures of all placentas were selected and presented with Adobe Photoshop (Version 11.0).
Results Isolation of hTCs Upon isolation of hTCs (Figure 1A), purity of preparations was determined by immunoblotting. Presence of strong Cytokeratin 7 bands and nearly absent Vimentin signals (Figure 1B) indicate absence of non-epithelial cells.
Trophoblast cells have different capacity for mercury accumulation
BeWo cells and hTCs accumulated mercury to clearly detectable amounts, when exposed to MeHg in a range between 0.3 and 3.0 µM (Figures 2A,C). The concentration of 0.9 µM MeHg was furthermore applied in knockdown experiments. Mercury levels increased with dose and time in a similar manner in hTCs prepared from two placentas (Figures 2C,E), however, to variable overall amounts; this is also visible in culture medium concentrations (Figure 2F). This effect was dose- and time-independent. Cell number of BeWo cells decreased with increased MeHg dose, while protein content of hTCs appeared to be unchanged (Figures 2B,D). Non-exposed hTCs (Figure 2C) showed higher mercury levels than non-exposed BeWo cells (Figure 2A) indicating that some mercury exposure has occurred during pregnancy. Transfection efficiencies and knockdown experiments We examined transfection efficiencies of BeWo cells and hard-to-transfect hTCs under various conditions using siGLO Red Transfection Indicator. The effects of forward transfection (siRNA treatment, when cells are 25-30% confluent) and reverse transfection (siRNA treatment at seeding) in combination with Nupherin, which promotes lipofection, were compared (Figure S1C). We also tested a variety of transfection reagents (Figure S1D). We found that reverse knockdown leads to higher transfection efficiency in hTCs than forward knockdown. Nupherin, however, did not enhance transfection efficiency in hTCs to a large extent and was not used in further experiments. Viafect and Lipofecamine2000 were identified as best transfection reagents for hTCs. The experimental protocols are outlined in Figures 3D (BeWo), 3E (hTCs) and the Methods section. Out of 17 candidate proteins, we found two amino acid transporter subunits and one ABC transporter significantly involved in mercury kinetics of BeWo cells (LAT1, rBAT) and hTCs (MRP1). For many proteins, the commercially available antibodies did not provide specific
signals in immunofluorescence microscopy and/or immunoblotting as validated by gene knockdown (results summarized in Table S1). Amino acid transporters are involved in uptake of mercury Upon downregulation of the light chain LAT1 and the heavy chain rBAT, mercury contents in BeWo cells were significantly decreased to 68% and 71%, relative to controls (Figure 3A). For LAT2 a decrease was also observed, however only as a trend (Fig. 3A,B). This trend for decreasing mercury accumulation was visible in hTCs following LAT1 downregulation (78%) (Figure 3B) as well as in HeLa cells (67% and 75%) (Figure S1A). Efflux transporters are involved in mercury efflux In hTCs, downregulation of MRP1 resulted in significant increase of cellular mercury contents (124%) of trophoblast cells (Figure 3C). The antibodies for most ABC transporters (although recommended for use in immunofluorescence) did not provide specific signals (Table S1). Due to low expression levels of ABC transporters in BeWo cells (the only exception is Multidrug resistance protein (MDR)1; Figure 4A), we did not perform knockdown of ABC transporters in BeWo cells. Trophoblast cells show differences in protein expression We observed large variability in protein levels between BeWo cells and hTCs, as well as among hTCs prepared from different placentas, notably for LAT1, 4F2hc, MDR1, and MDR3 (Figure 4A) (our specific observations on MRP2 localization and highly variable MRP2 expression are prepared for a separate publication; Ellinger et al., unpublished). Protein localization The predominant cell type in chorionic tissue expressing amino acid transporters was found to be the trophoblast, which mediates uptake of amino acids as well as of MeHg from maternal blood at the apical plasma membrane (Figure 4B). Low expression was found in stromal cells including endothelial cells. While the predominant subcellular localization of
the heavy chains 4F2hc and rBAT was at the plasma membrane (apical and basolateral), the light chains LAT1, LAT2 and b0,+ showed a higher extent of intracellular, vesicular localization.
Discussion Trophoblast cells have different capacity for mercury accumulation The varying capacities for mercury accumulation of human primary trophoblast cells is in line with observations that genetic polymorphisms in ABC transporters affect placental protein expression and drug transport (Ni and Mao 2011; Meyer zu Schwabedissen et al. 2005b). Additionally, the different capacities for mercury accumulation can well explain the observations from epidemiological studies on mercury levels of mother-child-pairs. A metaanalysis generated a mean ratio of cord blood to maternal blood of 1.9 (central tendency estimate: 1.7, 95th percentile: 3.4). The clearly higher cord blood levels indicate active mercury transport from the mother to the fetus. The ratios however strongly varied between and among populations (Stern and Smith 2003) suggesting that the amounts of mercury transported and retained by the human placenta differ in quite large dimensions. This raises the question on the involved proteins, their expression levels and the regulatory factors behind. Amino acid transporters are involved in mercury uptake Our findings on system L light chains LAT1 (and probably also LAT2) involved in uptake of MeHg into human trophoblast cells are new. Experiments on rat placenta already indicated active MeHg uptake as cysteine conjugate via neutral amino acid carriers (Kajiwara et al. 1996). Similarly, Xenopus laevis oocytes overexpressing LAT1 and LAT2 had increased methionine and MeHg uptake compared to control oocytes with low endogenous LAT1 and LAT2 levels (Simmons‐Willis et al. 2002) and downregulation of LAT1 reduced methionine
uptake into lung cancer cells (Dann et al. 2015). In line with our findings, system L activity was reduced by 17% (si LAT1) and 13% (si LAT2) in experiments on hTCs (Gacciolo et al. 2015). MeHg-cysteine also seems to be a substrate of b0,+-rBAT, another heterodimeric amino acid transporter (Wang et al. 2012). While BeWo cells respond to downregulated rBAT with reduced mercury accumulation, hTCs were unaffected by this treatment. The finding indicates that rBAT participates in mercury uptake into BeWo cells. Because b0,+ downregulation had no such effect, we assume that another light chain may be responsible for mercury uptake into cells. It is known that rBAT, which is present in excess over b0,+, is able to bind other light chain(s) (Fernandez et al. 2002). Currently, it remains unclear whether trophoblast cells can compensate for reduced rBAT protein. We found the predominant subcellular localization of the heavy chains 4F2hc and rBAT at the membrane, while the light chains LAT1, LAT2 and b0,+ show a higher extent of intracellular, vesicular localization. According to Rosario (2013) the internalization of light chains could be an effective measure to regulate amino acid transport. This localization of the transporter light subunits is in agreement with previous reports demonstrating LAT1 and LAT2 presence at the apical membrane of STB (Gaccioli et al. 2015; Kudo and Boyd 2001; Okamoto et al. 2002). Others found 4F2hc expressed at the apical STB but no evidence for system b0,+ in term placenta (Ayuk et al. 2000). ABC transporters are involved in mercury efflux The ABC transporters MDR1, MDR3 and MRP1-MRP5 are present in BeWo and trophoblast cells but there are many discrepancies regarding their expression levels (Atkinson et al. 2003; Bodó et al. 2003; Evseenko et al. 2006; Pascolo et al. 2003; Serrano et al. 2007). Our findings on ABC transporter expression confirm that expression varies between hTCs and BeWo cells, but as well among hTCs, particularly with regard to MDR1, MDR3, and MRP2. MRP1 is
more evenly expressed in both cell types, which is in accordance to the literature (Atkinson et al. 2003; Evseenko et al. 2006; Serrano et al. 2007). Likely candidates for efflux of mercury-GSH conjugates are MRP1 and MRP2 (Gundacker et al. 2010) known to transport GSH, GSSG (glutathione disulfide) and GSH conjugates with different affinities (Deeley and Cole 2006). Upon downregulation of MRP1, hTCs accumulated significantly more mercury than controls. This is in line with previous findings, where Nrf2 activation suppressed mercury accumulation and intoxication in primary mouse hepatocytes through upregulation of downstream proteins among them MRP1 and MRP2 (Toyama et al. 2007). In another study MRP1 overexpressing leukemia cells showed higher resistance to mercury than wildtype cells (Kim et al. 2005). In polarized kidney and liver cells, MRP1 is expressed at the basolateral membrane (Deeley and Cole 2006). In the human placenta, localization of MRP1 was found at the basolateral side of the STB as well as in FECs. The transporter might also have some expression at the apical STB membrane (Nagashige et al. 2003, Ni and Mao 2011). Together with the published localization data, our findings suggest that MRP1 is involved in transport of mercury towards the fetal circulation. Unexpectedly, MRP2 downregulation did not significantly affect cellular mercury concentrations, which is contrary to previous reports (Bridges et al. 2012; Madejczyk et al. 2007). It is possible that MRP2 downregulation is compensated by MRP3. A reciprocal relationship between the two transporters in cholestasis and Dubin-Johnson syndrome has been described (Bodó et al. 2003; Deeley and Cole 2006). Limitations of the study It was not possible to confirm siRNA mediated knockdowns for all 17 candidate proteins on protein level due to lack of proper antibodies (rBAT and MRP3). Although a specific LAT2 antibody was available upon start of the project, it was later on withdrawn from the market.
Conclusions We found two amino acid transporter subunits (LAT1, rBAT) and one ABC transporter (MRP1) to be involved in mercury toxicokinetics of trophoblast cells. According to our data, a model can be deduced (Figure 5) which can explain why mercury is efficiently transported towards the fetal side: It uses LAT1 (probably also LAT2) and rBAT dimerized to another light chain than b0,+ at the apical side of the STB to enter trophoblast cells. Intracellular mercury dissociates from cysteine, binds to GSH and is therefore no longer a substrate of amino acid transporters. Mercury conjugated to GSH is effluxed by MRP1. We made two interesting observations. Human trophoblast cells show strong variation in mercury accumulation capacity and in transporter expression levels. It seems likely that, e.g., LAT1 or MRP1 levels determine mercury transfer rates across the STB. Due to limited amount of trophoblast cells per preparation, we could not keep enough cell material to retrospectively clarify these relationships. Many of the transporters involved in mercury toxicokinetics (system L, ABC transporters) are likely involved in placental dysfunctions such as intrauterine growth restriction, gestational diabetes mellitus, or preeclampsia (Burton and Jauniaux 2004; Johnstone et al. 2011; Regnault et al. 2007). As seen in our study, MeHg uses both amino acid transporters and ABC transporters to pass through the STB. This unique feature suggests the use of MeHg in future studies as a model substrate to investigate placental pathophysiology.
Acknowledgments We are grateful to study participants and medical staff at the clinics as well as to Hana Uher for technical assistance. The study was supported by LifeScience2010 (Project LS10026), NFB (Niederösterreichische Forschungs- und Bildungsgesellschaft).
Financial interest declaration The authors declare no competing interests including financial interests.
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Figure caption Figure 1. Isolation of human primary trophoblast cells (hTCs). (A) Procedure of trophoblast isolation from healthy human term placenta. (B) Purity of trophoblast preparation indicated by Vimentin‐negative and Cytokeratin (CK) 7‐positive cells on immunoblot showing five hTC samples and one Rc sample (i.e., cells remaining in top layer). *The lower band in Rc is an unspecific signal.
Figure 2. Comparison of mercury accumulation in BeWo cells and hTCs. (A) Mercury content and (B) cell number of BeWo cells upon exposure to MeHg for 24 hrs as indicated. (C) Dose‐dependent mercury accumulation upon MeHg treatment for 5 hrs as indicated and (E) time‐dependent mercury accumulation upon exposure to 0.9 µM MeHg in hTCs from two placentas (PI and PII). (D) Protein content following MeHg exposure in hTCs from two placentas. (F) Mercury concentrations in pooled cell medium upon MeHg treatment. Data are mean±SD from two independent experiments made in duplicates or triplicates.░
Figure 3. Effect of siRNA-mediated knockdown on mercury accumulation. (A) Cellular mercury content and cell number in BeWo cells upon siRNA‐mediated knockdown of uptake transporters. (B) Cellular mercury and protein content in hTCs upon siRNA‐mediated knockdown of uptake transporters. (C) Cellular mercury and protein content in hTCs upon siRNA‐ mediated knockdown of efflux transporters. (A‐C) Gene knockdown was confirmed by immunoblotting. Representative immunoblots are shown. (D) Experimental protocol on BeWo cells. (E) Experimental protocol on hTCs. Data are mean±SD from three to four independent experiments made in duplicates or triplicates. *t‐ Test P<0.05, **P<0.01 for comparison with controls (non‐targeting siRNA) Figure 4. Protein expression and localization. (A) Protein expression of uptake and efflux transporters in BeWo cells, two hTC (hTCI, hTCII) and two whole placenta samples (wP1, wP2). Representative immunoblots are shown. MDR1, MDR3, MRP2, and 4F2hc as well as LAT1 and MRP5 were detected on the same blots; the respective GAPDH and Histone H3 bands are therefore identical. Note that here the Aviva 4F2hc antibody was used (68kDa). (B) Representative images for the localization of 4F2hc, LAT1, LAT2, CK7 (secondary antibody: Alexa Fluor® 568), rBAT and b0,+ (Alexa Fluor® 647). A representative image for Alexa Fluor® 568 is shown (negative control). Images were acquired using a wide‐field fluorescence microscope equipped with a 63x objective. Arrows indicate the STB layer, which can be identified by Cytokeratin (CK) 7 expression. (a) indicates the apical plasma membrane of the STB. Bars represent 20 µm.
Figure 5. The proposed model for placental mercury toxicokinetics enabling active and efficient transfer from the maternal to the fetal side via the STB. The amino acid transporters LAT1‐4F2hc (perhaps also LAT2‐4F2hc) and X‐rBAT ("X" indicates possible interaction partners of rBAT besides b0,+) located at the apical side of the STB manage uptake of MeHg‐cysteine. Intracellularly, mercury (Hg2+ or MeHg+) is readily conjugated to GSH. The GSH‐Hg conjugates (i.e., GS‐Hg‐SG, MeHg‐SG) are then effluxed from the STB via MRP1 predominantly localized to the basal side of the STB. The model shows only transporters, which, according to our data, are involved in transfer of mercury across the STB. Proteins involved in mercury transfer across FECs have not been explored in this study and remain currently unknown.