Accepted Manuscript

Effect of Oxygen on Multidrug Resistance in Term Human Placenta

M. Javam, M.C. Audette, M. Iqbal, E. Bloise, W. Gibb, S.G. Matthews

PII: S0143-4004(14)00084-8

DOI: 10.1016/j.placenta.2014.02.010

Reference: YPLAC 2949

To appear in: Placenta

Received Date: 3 December 2013

Revised Date: 10 February 2014

Accepted Date: 23 February 2014

Please cite this article as: Javam M, Audette MC, Iqbal M, Bloise E, Gibb W, Matthews SG, Effectof Oxygen on Multidrug Resistance in Term Human Placenta, Placenta (2014), doi: 10.1016/j.placenta.2014.02.010.

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ACCEPTED MANUSCRIPTEffect of Oxygen on Multidrug Resistance in Term Human Placenta M. Javama, M.C. Audettea,d, M. Iqbala, E. Bloisea, W. Gibbb, S.G. Matthewsa,c-e a Department of Physiology, University of Toronto, Toronto, Canada b Depts Ob-Gyn and Cellular & Molecular Medicine, University of Ottawa, Ottawa, Canada c Department of Ob-Gyn, University of Toronto, Toronto, Canada d Department of Medicine, University of Toronto, Toronto, Canada e Fraser Mustard Institute for Human Development, University of Toronto, Toronto, Canada

Short Tile: Oxygen tension and multidrug resistance in the term placenta

Keywords: P-glycoprotein, BCRP, third-trimester placenta, hypoxia, multidrug resistance

Grant Support: This study was funded by the Canadian Institutes for Health Research (grant: FRN-

57746; to S.G.M. and W.G.).

Disclosure Summary: Authors have nothing to declare

Correspondence and reprint requests:

Dr. Stephen G. Matthews, Department of Physiology, Faculty of Medicine, Medical Sciences

Building, Rm 3302, University of Toronto, 1 King's College Circle, Toronto, ON Canada, M5S 1A8.

Tel: (416) 978-2025 Fax:(416) 978-4940 e-mail: stephen.matthews@utoronto.ca

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ACCEPTED MANUSCRIPTAbstract 1

2

Introduction: The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast 3

cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and 4

nutrients from the maternal to the fetal circulation. The expression of these transporters changes with 5

gestational age, yet the mechanisms involved remain unknown. However, the changes in P-gp and 6

BCRP transporter expression coincide with those of oxygen tension in the placenta, and oxygen 7

tension has been shown to modulate P-gp and BCRP expression in other tissues. The objective of this 8

study was to investigate the effects of oxygen tension on P-gp and BCRP expression in the term 9

human placenta. 10

Methods: Following equilibration in culture (96h), term placental explants (n=7) were cultured in 3% 11

or 20% oxygen for 24 and 48 h. Culture medium was collected every 24 h to measure lactate 12

dehydrogenase (LDH; explant viability) and human chorionic gonadotropin (hCG; 13

syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and 14

mRNA, as well as VEGFA mRNA were measured using western blot and qRT-PCR. P-gp localization 15

was determined using immunofluorescence. 16

Results: Oxygen tension had a significant effect on P-gp expression, with ABCB1/P-gp mRNA and 17

protein levels increased in the hypoxic condition (3% O2) after 48 h (p<0.05). VEGFA mRNA was 18

elevated by hypoxia at both 24 and 48h (p<0.05). In contrast, placental ABCG2/BCRP mRNA and 19

protein expression were stable with changes in oxygen tension. We identified profound differences in 20

the glycosylation of P-gp between cultured and non-cultured placental tissue, with cultured explants 21

expressing deglycosylated P-gp. 22

Conclusions: These findings demonstrate that, at term, the expression of placental P-gp, is regulated 23

by oxygen tension. This suggests that changes in oxygenation of the placenta in the third trimester 24

may alter levels of placental P-gp, and in doing so alter fetal exposure to P-gp substrates, including 25

xenobiotics and certain hormones. 26

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ACCEPTED MANUSCRIPT1. Introduction 1

One of the many roles of the placenta throughout gestation is to act as a protective barrier. In 2

addition to acting as a barrier that separates maternal blood from fetal blood, the placenta also 3

contains a variety of efflux transporters that are localized to the syncytiotrophoblast and fetal capillary 4

endothelium, which limit the passage of molecules from the maternal to the fetal circulation [1]. Many 5

of these efflux transporters belong to the ATP-binding cassette (ABC) family of transporters, which 6

decrease intracellular substrate concentrations through an active, energy-dependent mechanism [2]. 7

Two of these important ABC efflux transporters are P-glycoprotein (P-gp encoded by ABCB1) and 8

breast cancer resistance protein (BCRP encoded by ABCG2) [1]. Prior studies have shown these 9

transporters play a critical role in fetal protection throughout pregnancy [3-6]. For example, all the 10

offspring of mice deficient in placental P-gp showed the cleft palate phenotype when exposed to 11

Avermectin, a known substrate of P-gp, whereas the offspring from wild type animals were normal 12

[3]. In BCRP-deficient pregnant mice there was a five-fold increase in fetal concentrations of the 13

BCRP substrate, nitrofurantoin, compared with wild type animals [5]. 14

We have previously shown that the expression of these transporters in the human placenta 15

changes throughout pregnancy [7, 8]. ABCB1 mRNA and P-gp protein expression decrease with 16

advancing gestation [7, 8]. This decrease in P-gp levels has a potential to leave the fetus susceptible to 17

teratogens that may be present in the maternal circulation. The mechanism behind this decrease in P-18

gp expression remains unknown. However, the decrease in P-gp expression coincides with changes in 19

oxygen tension in the placenta. In the first trimester, the fetus is highly sensitive to molecular oxygen 20

and a low oxygen environment helps protect against oxidative stress [9]. At this early stage of 21

pregnancy, the partial pressure of oxygen is about 20 mmHg (~3% O2) due to the occlusion of the 22

terminal portion of maternal spiral arteries by the invading extravillous trophoblast (EVT) [9]. In the 23

early second trimester, the de-plugging of the spiral arteries allows maternal blood to enter the 24

intervillous space, causing an increase in oxygen tension to about 60 mmHg (~8% O2) [9]. However, 25

improper or inadequate conversion of spiral arteries in the first trimester can lead to chronic placental 26

ischemia and hypoxia later in gestation, which can cause deleterious outcomes such as intrauterine 27

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growth restriction (IUGR), preeclampsia, unexplained miscarriage, and pre-term labor [10-13]. 28

Furthermore, these hypoxic placentas may have altered transporter expression patterns compared to 29

placentas from normal pregnancies [14]. 30

Studies in other cell types, especially cancer cells, have provided valuable insight into the 31

relationship between oxygen tension and transporter expression. Both P-gp and BCRP are highly 32

expressed in a number of cancer cells and expression has been shown to increase under conditions of 33

low oxygen tension [15-18]. This increase in expression is mediated through the binding of hypoxia 34

inducible factor (HIF-1α) to the HIF responsive element (HRE) in the ABCB1 and ABCG2 promoters 35

[15, 18]. We have recently shown that oxygen tension affects P-gp and BCRP transporter expression 36

in first trimester human placental explants [19]. An increase in oxygen tension resulted in an increase 37

in ABCG2 expression with no change in ABCB1 mRNA expression. At the protein level, hypoxia 38

induced P-gp and BCRP expression in proliferating cytotrophoblasts [19]. 39

Term placenta is subjected to higher oxygen tension than first trimester placenta and it is not 40

known whether oxygen tension regulates P-gp and BCRP expression at term. Moreover, 41

uteroplacental hypoxia, as a result of occlusion or inadequate trophoblast invasion of maternal spiral 42

arteries, can lead to chronic placental ischemia and hypoxia later in gestation [20]. Altered P-gp and 43

BCRP expression in response to hypoxia would impact fetal exposure to xenobiotics and hormones 44

present in the maternal circulation. The purpose of the present study was to examine the effect of 45

oxygen on the expression of these transporters in term placenta using an in vitro model of intact 46

placental explants. We hypothesized that a decrease in oxygen tension would result in an increase in 47

P-gp and BCRP levels. 48

49

2. Methods 50

2.1 Placental Collection and Tissue Culture 51

Term C-section placentas (n=7) were collected within 30 minutes (min) of delivery from the Research 52

Centre for Women’s and Infants’ Health (RCWIH) BioBank program at Mount Sinai Hospital. 53

Written informed consent was obtained from all subjects and ethical approval was obtained from the 54

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Mount Sinai Hospital and the University of Toronto Research Ethics Board. Placental tissue biopsies 55

(1 cm3) were further dissected in the laboratory into approximately 8 mm3 fragments. Villous explants 56

were then cultured in individual Costar Netwells (Corning, New York) in CMRL-1066 media 57

(Invitrogen, Carlsbad, California, USA), supplemented with NaHCO3 (2.2 mg/ml; Sigma-Aldrich, St. 58

Louis, MO, USA), antibiotics-antimycotics (Invitrogen), insulin (1 mg/ml; Sigma), retinol acetate (1 59

mg/ml; Sigma), L-glutamine (100 mg/ml; Sigma) and fetal bovine serum (5%; Wisent Inc., St. Bruno, 60

Quebec, Canada) as previously described [21]. We and others [21, 22] have shown that in placental 61

explant cultures, the syncytiotrophoblast sheds within the first 2 days of culture and regenerates by 62

days 3-4, using both measures of syncytiotrophoblast function and histological analysis of 63

synctiotrophoblast integrity. Therefore, placental explants were cultured for 4 days (20% O2, 5% CO2, 64

37 °C) as previously described [21, 22], which allowed syncytiotrophoblast shedding and regeneration 65

to take place. On day 4, oxygen tension was changed to 3% (5% CO2, 37 °C) or maintained at 20% 66

for a further 24 and 48h. Culture media was changed every 24h during the culture period and care was 67

taken throughout the culture period for explants to remain out of the incubators for the same length of 68

time. Lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) was measured in the 69

media to assess explant viability and syncytiotrophoblast function, respectively [21, 22]. 70

71

2.2 Lactate Dehydrogenase (LDH) and Human Chorionic Gonadotropin (hCG) 72

LDH and hCG were measured as described previously [21]. Briefly, LDH was quantified using the 73

Cytotoxicity Detection kit (Roche Applied Science, Indianapolis, USA) according to the 74

manufacturer’s instructions [19, 21]. A standard curve for the LDH assay was generated using L-lactic 75

dehydrogenase from rabbit muscle (Sigma) [19, 21]. hCG was measured by ELISA (DRG 76

Diagnostics, Germany) according to the manufactures’ instructions [21]. 77

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2.3 Quantitative real time PCR 79

Total RNA was extracted from the placental explants using TRIzol reagent (Invitrogen) as described 80

in the manufacturer’s protocol. Contaminating genomic DNA was removed from RNA samples using 81

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a DNA-free kit (Ambion, Austin, Texas, USA). RNA purity (A260/A280 ratio of 1.8-2.0) and 82

concentration was assessed using spectrophotometric analysis, and RNA integrity was verified by 83

agarose gel electrophoresis. RNA was reverse-transcribed to cDNA using High Capacity cDNA 84

Reverse Transcription kit (Applied Biosystems, Carlsbad, California, USA) according to the 85

manufacturer’s instructions and stored (-80°C). 86

Levels of ABCB1, ABCG2, and VEGFA mRNA were measured by real time RT-PCR using 87

SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and the CFX 96 Real-Time 88

system C1000 Thermal Cycler (Bio-Rad). Relative gene expression was measured using [∆∆c(t)] 89

method and normalized to the geometric mean of three housekeeping genes: glyceraldehyde 3-90

phosphate dehydrogenase (GAPDH), TATA-binding protein (TBP), and zeta polypeptide (YWHAZ) 91

[19]. Expression of these genes was stable during the experiments (data not shown). Data was 92

analyzed using CFX Manager Software (Bio-Rad). Primer sequences for the genes analyzed are listed 93

in Table 1. 94

95

2.4 Western Blotting 96

Western blotting was performed as described previously [23]. Briefly, placental explants were 97

homogenized in RIPA lysis buffer (Cell Signaling Technology, Inc., Danvers, MA), centrifuged 98

(10000g, 10min, 4°C), and the supernatant collected. Protein (40µg) separation using SDS-PAGE was 99

performed using polyacrylamide gels (7%) and transferred to nitrocellulose membranes using iBlot 100

transfer apparatus (Invitrogen). Membranes were blocked in BSA (5% w/v of tris buffered saline 101

[TBS] with Tween 20: TBS-T, 1h, 25°C). Membranes were then incubated with primary antibody 102

overnight (4°C). The primary antibodies used were: mouse anti-P-gp (1:5000; clone 5A12.2; EMD 103

Millipore Corporation, Billerica, MA, USA), mouse anti-BCRP (1:500; clone BXP-21; EMD 104

Millipore Corporation), and rabbit anti-actin (1:10000; Sigma). Membranes were washed and 105

incubated with horseradish peroxidase conjugated secondary antibody (1:15000; PerkinElmer, 106

Waltham, MA) against the corresponding primary antibody, followed by incubation (1min) in 107

enhanced chemiluminescence substrate (Invitrogen) and exposed to chemiluminescence film (General 108

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Electric Healthcare, Baie d’Urfe, Quebec, Canada). The relative optical density (ROD) of the bands 109

was measured using computerized image analysis and was standardized against the β-actin signal 110

(MCID Core 7.0, Imaging Research Inc, Interfocus Imaging Ltd, Cambridge, England). 111

112

2.5 Immunofluorescence 113

Placental explants were fixed in neutral buffered formalin (10%; Sigma), washed with PBS and 114

cryosectioned (10µm). Sections were mounted onto microscope slides and stored (-80°C). Prior to 115

immunofluorescence, slides were brought to room temperature and washed with PBS. Antigen 116

retrieval was performed using boiling sodium citrate (10mM, 10min). Sections were cooled on ice 117

(10min) and washed with PBS before blocking in normal goat serum (5%, 1h; Invitrogen). Slides 118

were incubated overnight with mouse anti-P-gp primary antibody (1:50, D-11, Santa Cruz 119

Biotechnology, Santa Cruz, CA, USA) [19], then washed and incubated (1h, 25°C) with Alexa Fluor 120

488-conjugated goat secondary antibody (1:500; Invitrogen). Negative controls were stained by 121

substitution of primary antibody with non-immune mouse IgG (Dako, Burlington, ON, Canada). After 122

final washes, slides were mounted and cover-slipped using mounting media with 4′, 6-diamidino-2-123

phenylindole (Vector Laboratories, Burlingame, CA). Imaging was performed using Zeiss spinning 124

disk confocal microscope (Zeiss Observer.Z1). 125

126

2.6 Deglycosylation 127

P-gp deglycosylation was carried out using Peptide-N-Glycosidase F (PNGase F; Sigma) according to 128

the manufacturer’s instructions. Briefly, denatured and non-denatured protein (30ug) from fresh 129

placental tissue was incubated with PNGase F (4h, 37°C). Both denaturing and non-denaturing 130

conditions were used because some proteins require denaturation prior to PNGase F digestion. 131

Laemmli sample buffer (Bio-Rad) was added, and samples boiled (5min) to inactivate the PNGase F. 132

Western-blot analysis was performed as described above. 133

134

2.7 Statistical Analysis 135

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Data are expressed as mean ± SEM and all statistical analyses were performed using Prism (GraphPad 136

Software, Inc., San Diego, CA). hCG and LDH concentrations were log transformed and analyzed 137

using one-way repeated measures ANOVA with Newman-Keuls post-hoc test. Differences in mRNA 138

and protein expression over time of culture and oxygen tension were assessed by two-way ANOVA 139

with Bonferonni post-hoc test. Protein and mRNA expression were also analyzed using one-way 140

ANOVA, followed by Dunnett post-test for comparisons against the control group. Statistical 141

significance was set at p<0.05. 142

143

3. Results 144

3.1 Tissue Viability and Syncytiotrophoblast Function 145

LDH release from placental explants across the 6-day culture period was measured to assess tissue 146

viability [21, 22]. LDH levels in the media decreased gradually, reaching a stable phase on day 4 (Fig. 147

1A). In order to monitor syncytial shedding and regeneration, hCG secretion was measured, a marker 148

of syncytiotrophoblast endocrine function [21, 22]. Between day 1 and day 2 there was a steep decline 149

in hCG release into the media (p<0.05) (Fig. 1B). From day 2 to day 3 there was a rapid increase in 150

hCG levels (p<0.05). The elevated hCG levels were sustained throughout the rest of the culture 151

period. This hCG pattern was indicative of syncytiotrophoblast shedding and regeneration [21, 22]. 152

Therefore, placental explants were cultured for 4 days in 20% oxygen, which allowed 153

syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 154

3% or maintained at 20% for 24 and 48 h. Day 4 was considered the control group. 155

156

3.2 Effects of Oxygen Tension on ABCB1, ABCG2, and VEGFA mRNA Expression 157

ABCB1 and ABCG2 mRNA expression was measured in the control (day 4 of culture), 24h (day 5), 158

and 48h (day 6) samples from the same placentae using qRT-PCR. There was a significant effect of 159

oxygen tension on ABCB1 expression, assessed by two-way ANOVA (p<0.05) (Fig. 2A), with no 160

effects of time in culture or interaction of the two variables. There was a significant increase in 161

ABCB1 expression after 48 hours in 3% oxygen compared to the control group (p<0.05) (Fig. 2A). 162

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Oxygen tension had no effect on ABCG2 expression, assessed by two-way ANOVA (Fig. 2B), with 163

no effects of time in culture or interaction of the two variables. VEGFA mRNA expression was 164

measured to assess activation of hypoxic pathways [19]. There was a significant increase in VEGFA 165

expression in the hypoxic condition (3% O2) (p<0.05) (Fig. 2C), with no effects of time in culture or 166

interaction of the two variables. VEGFA expression was also significantly increased at both 24h and 167

48h hypoxic conditions compared to the control group. 168

169

3.3 Effects of Oxygen on P-gp and BCRP Expression 170

There was no significant overall effect of oxygen tension on P-gp protein levels, assessed by two-way 171

ANOVA (Fig. 3C), with no interaction between the two variables. However, there was a significant 172

effect of time in culture on P-gp levels (p<0.05), such that there was an increase in P-gp levels with 173

increased time in culture. Further analysis using repeated measures one-way ANOVA revealed a 174

significant increase in P-gp levels after 48h of culture in 3% oxygen compared to control (p<0.05) 175

(Fig. 3C). Oxygen tension had no effect on BCRP protein levels, assessed by two-way ANOVA (Fig. 176

3D), with no effects of time in culture or interaction of the two variables. 177

178

3.4 Effect of Tissue Culture on P-gp Glycosylation and Localization 179

Western blot analysis performed using fresh placental tissue, showed a predominant 150 kDa band for 180

P-gp (Fig. 4A). In contrast, P-gp in cultured placental tissue was approximately 120 kDa (Fig. 4A). To 181

assess whether the difference in the molecular weight of P-gp was due to deglycosylation, protein 182

from fresh placental tissue was treated with PNGase F (this cleaves the link between asparagine and 183

N-acetylglucosamines). PNGase F treatment resulted in a shift in the molecular weight of P-gp from 184

150kDa to 120kDa (Fig. 4B). Changes in oxygen tension had no effect on the glycosylation state of P-185

gp (data not shown). 186

To assess whether deglycosylation altered P-gp localization in the tissue, fresh and cultured explants 187

were analyzed using immunofluorescent staining. In both instances P-gp was localized to the 188

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syncytiotrophoblast (Fig. 5) (n=4). Furthermore, decreasing oxygen tension did not alter P-gp 189

localization to the syncytiotrophoblast (Fig. 5). 190

191

4. Discussion 192

We have shown, for the first time, that changes in oxygen tension in the term human placenta 193

have significant but different effects on P-gp and BCRP expression. Decreased oxygen tension 194

resulted in an increase in ABCB1/P-gp mRNA and protein expression in term placental explants but 195

had no effect on ABCG2/BCRP mRNA or protein expression. We also report the novel finding that 196

while P-gp is glycosylated in fresh placental tissue, it becomes fully deglycosylated in cultured 197

placental explants. This de-glycosylation had no effect on the localization of P-gp to the 198

syncytiotrophoblast. This novel observation has important implications for our understanding of the 199

post-translational regulation of P-gp in the placenta and warrants further detailed investigation. 200

In the human placenta, ABCB1/ P-gp mRNA and protein expression decrease with advancing 201

gestation [7], which correlates closely with changes in oxygen tension throughout pregnancy. In the 202

present study, we found that 48hr exposure to low oxygen tension (3%) resulted in a significant 203

increase in ABCB1/ P-gp mRNA and protein levels, compared to the control group (20% O2). This 204

increase in ABCB1 and P-gp expression in response to hypoxia is consistent with studies performed 205

using other cell types [17, 18]. Furthermore, the responsiveness of P-gp expression to a decrease in 206

oxygen tension provides a possible explanation for the high P-gp levels in the low oxygen tension 207

environment of the first trimester placenta compared to term placenta. 208

Studies investigating the expression pattern of BCRP in the human placenta throughout 209

gestation are inconsistent. We have previously shown that ABCG2 mRNA expression does not change 210

throughout pregnancy, while BCRP protein levels increase towards term [8]. On the other hand, 211

Meyer zu Shwabedisson et al. demonstrated that both ABCG2 mRNA and BCRP protein expression 212

increase with advancing gestation [24] but Mathias et al. found that ABCG2 mRNA and BCRP 213

protein expression did not change with gestational age [25]. The reason for the discrepancy in BCRP 214

expression between studies may result from variability in sampling, as BCRP is present multiple 215

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placental cell-types. In the present study oxygen tension had no effect on ABCG2 mRNA or BCRP 216

protein expression. This would be consistent with the stable ABCG2/BCRP expression pattern during 217

pregnancy, despite the increase in oxygen tension that occurs with advancing gestation. However, a 218

steady level of BCRP expression does not necessarily indicate no change in BCRP function, as no 219

study has measured BCRP activity throughout gestation. In fact, there are numerous studies 220

illustrating posttranslational modifications, such as glycosylation and phosphorylation, changing 221

BCRP function [26-28]. The same is true for P-gp [29-31], as Thews et al have shown a more than 222

doubling of P-gp activity in prostate carcinoma cells in acidic environments with no change in P-gp 223

expression [31]. Future studies are required to measure BCRP and P-gp activity in the placenta with 224

advancing gestation. 225

Compared to the third trimester, the placenta in the first trimester is exposed to lower oxygen 226

levels [12]. Early in the first trimester the extra villous trophoblast (EVT) invade and occlude the 227

terminal portion of spiral arteries, inhibiting maternal blood from entering the intervillous space and 228

resulting in a low oxygen environment (~3% O2) [10]. In late first trimester, the endovascular EVT 229

causes physiological changes in placental spiral arteries, forming funnel-shaped flaccid arterioles with 230

increased vascular compliance and circumference [10]. This leads to the flow of maternal blood into 231

the intervillous space and a dramatic increase in oxygen levels for the remainder of pregnancy (~8% 232

O2) [10]. However, improper or inadequate conversion of spiral arteries in the first trimester can lead 233

to chronic placental ischemia and hypoxia later in gestation [10-13]. Moreover, embryogenesis in the 234

first trimester already takes place under anaerobic conditions, and in the second and third trimester 235

oxygen becomes more important for normal fetal organogenesis and growth [32]. Protection from 236

hypoxia at this stage of development is established via up-regulation of various genes, including 237

superoxide dismutase, insulin-like growth factor (IGF)-2, and heat shock protein 70 [33-35]. Our data 238

indicate that ABCB1 may be one of the protective genes activated in response to hypoxia in the third 239

trimester placenta. In fact, P-gp has been suggested to play a critical role in cell protection by 240

extruding toxins and harmful products of oxidative stress [36]. Therefore, P-gp may also play a role in 241

protecting the fetus from toxic products of oxidative stress in third trimester hypoxic placentas. 242

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We have recently shown that in the first trimester placenta, ABCG2 mRNA expression 243

increased significantly in hyperoxic (20%) conditions after 48hrs in culture with no change in ABCB1 244

mRNA expression [19]. At the protein level, hypoxia resulted in increased immunoreactive BCRP in 245

cytotrophoblasts and in the microvillous membrane of the syncytium, and increased P-gp 246

immunoreactivity in proliferating cytotrophoblasts. This contrasts with the results of the current study 247

and suggests that the placenta in the different stages of pregnancy responds quite differently to 248

changes in oxygen tension. This is understandable, as oxygen tension that is physiological in the first 249

trimester would be considered hypoxic at term. 250

Interestingly, we found that cultured placental tissue contained P-gp that was less glycosylated 251

compared to fresh placental tissue. Post-translational modifications, such as glycosylation, have been 252

shown to alter P-gp activity in various ways, depending on the cell line examined [29]. In general, 253

glycosylation can affect a transporter’s activity, folding, stability, subcellular trafficking, localization, 254

and turnover [37, 38]. P-gp is glycosylated on three recognition sites (Asn 91, 94, and 99) in the first 255

extracellular loop [37]. Deglycosylation of P-gp in fresh placenta decreased the molecular weight 256

from approximately 150 kDa to 120 kDa. This 30 kDa decrease is in agreement with previous studies 257

[39, 40]. Since the effect of deglycosylation on P-gp localization is cell type specific [30, 37, 40, 41], 258

we examined whether deglycosylation affects P-gp localization in term placental tissue. Using 259

immunofluorescent staining, the glycosylation state of P-gp was not found to affect P-gp localization 260

in the explants. Furthermore, oxygen tension had no effect on P-gp glycosylation or its localization to 261

the syncytiotrophoblast. Since the impact of glycosylation on P-gp activity is cell-specific [29, 30, 262

41], future studies are required to determine the effects of deglycosylation on P-gp activity in the 263

placenta. It is also important to highlight the potential limitation of the present study. For example, in 264

the immunofluorescent staining we were unable to definitively distinguish between the 265

syncytiotrophoblast and unfused cytotrophoblasts in the outer trophoblast layer. Also, it is possible 266

that molecular changes in syncytiotrophoblast function occur in pathways that indirectly modulate 267

multidrug resistance, following long term explant cultures. Future studies are required to determine 268

the precise molecular pathways involved in P-gp and BCRP regulation. 269

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In conclusion, oxygen tension affects multidrug resistance in term human placenta. These 270

changes could alter fetal exposure to drugs, xenobiotic and endogenous substrates. Notably, improper 271

or inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia 272

and hypoxia later in gestation, which can cause deleterious outcomes such as intrauterine growth 273

restriction (IUGR) and preeclampsia. Glycosylation is an important regulatory mechanism of P-gp 274

function and expression, and should be considered so that we can better understand the various roles 275

of P-gp both in normal pregnancy progression and in disease states. Moreover, future studies should 276

focus on the expression profile and function of drug transporters in oxygen-related placental 277

pathologies since changes in the activity of these transporters could have a profound impact on fetal 278

drug exposure. 279

280

References 281

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Table 1. Primers used for real-time PCR. Fig. 1. Placental explant viability and syncytiotrophoblast function. (A) Lactate dehydrogenase (LDH) and (B) Human chorionic gonadotropin (hCG) released from explants into the culture media over the 6-day culture period. Results shown as mean ± S.E.M. Three separate tissues assayed in triplicate. hCG and LDH concentrations were log transformed and analyzed using one-way repeated measures ANOVA with Newman-Keuls post-hoc test. # represents significant difference from day 1 (p<0.05). * represents significant difference from all other days (p<0.05). Fig. 2. Effects of oxygen tension on ABCB1, ABCG2, and VEGF expression. (A) ABCB1 (B) ABCG2 (C) VEGFA mRNA expression in term placental explants normalized to the geometric mean of three housekeeping genes. Placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 3% or continued at 20% for 24 and 48 h. Day 4 was considered the control group. There was a significant effect of oxygen tension on ABCB1 and VEGFA expression. Solid bars represent 3% oxygen tension, while open bars represent 20% oxygen tension. * Represents significant difference from control (p<0.05). Results shown as mean ± S.E.M. (n=7) Fig. 3. Effects of oxygen tension on P-gp and BCRP protein expression. Representative western blot of (A) P-gp and (B) BCRP protein from a single placenta. Protein expression of (C) P-gp and (D) BCRP in term placental explants normalized to β-actin. Placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 3% or continued at 20% for 24 and 48 h. Day 4 was considered the control group. There was a significant effect of oxygen tension on P-gp expression. Solid bars represent 3% oxygen tension, while open bars represent 20% oxygen tension. * Represents significant difference from control (p<0.05). Results shown as mean ± S.E.M. of relative optical density (ROD). (n=7; *p < 0.05) Fig. 4. Deglycosylation of placental P-gp. (A) Representative western blot comparing P-gp expression in fresh term placental tissue and cultured placental explants. (B) Deglycosylation of fresh placental tissue. (i) Fresh term placental tissue (ii) Denatured protein from fresh placental tissue treated with PNGase F (iii) Non-denatured protein from fresh placental tissue treated with PNGase F (iv) cultured placental explant on Day 6. Fig. 5. Representative immunofluorescent images showing P-gp (green) localization to the syncytiotrophoblast in placental explants exposed to different oxygen tensions. Nuclei were stained by 4’,6-diamidino-2-phenylindole (blue) (n=4). Negative controls were stained by substitution of primary antibody with non-immune mouse IgG. Arrows point to syncytiotrophoblast.

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Table 1. Primers used for real-time PCR. Gene Symbol Sequence References ABCB1 Forward: AGC AGA GGC CGC TGT TCG TT 19 Reverse: CCA TTC CGA CCT CGC GCT CC ABCG2 Forward: TGG AAT CCA GAA CAG AGC TGG GGT 19 Reverse: AGA GTT CCA CGG CTG AAA CAC TGC VEGFA Forward: CGG GCC TCC GAAA CCA TGA ACT T 19 Reverse: CCC TCC TCC TTC TGC CAT GGG T TBP Forward: CAC GAA CCA CGG CAC TGA TT 19 Reverse: TTT TCT TGC TGC CAG TCT GGA C GAPDH Forward: AGA TCA TCA GCA ATG CCT CC 19 Reverse: CAT GAG TCC TCC CAC GAT AC YWHAZ Forward: ACT TTT GGT ACA TTG TGG CTT CAA 19 Reverse: CCG CCA GGA CAA ACC AGT AT

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