Placental BCRP transporter reduces cadmium accumulation and toxicity in immortalized human trophoblasts.

Cadmium (Cd) is a ubiquitous environmental metal detectable in most pregnant women. Animal and human studies demonstrate that in utero exposure to Cd reduces birth weight and impairs perinatal growth due to placental toxicity. BCRP is a prominent transporter that can efflux xenobiotics from the placenta. This study sought to investigate Cd transport and toxicity in cultured human BeWo trophoblasts with reduced expression and function of the placental barrier transporter BCRP. Knockdown (KD) of BCRP protein expression and function in BeWo trophoblasts increased the intracellular accumulation of Cd by 100% following treatment with 1 µM CdCl2. No change in the expression of Cd uptake transporters was observed between control and BCRP-KD cells. Reduced BCRP expression impaired viability of BeWo cells exposed to CdCl2 for 48 hr (BCRP-KD IC50: 11 µM, control cells IC50: 18 µM). Moreover, BCRP-KD cells were more sensitive to CdCl2-induced cytotoxicity compared to control BeWo cells. CdCl2 treatment strongly induced the expression of the metal-binding protein metallothionein (MT) in both control and BCRP-KD cells, with significantly greater MT upregulation in Cd-treated BCRP-KD cells. These data suggest that the BCRP transporter reduces Cd accumulation in syncytiotrophoblasts, which may be one mechanism to reduce subsequent toxicity to the placenta and developing fetus.

Low oxygen tension differentially regulates the expression of placental solute carriers and ABC transporters.

Low oxygen concentration, or hypoxia, is an important physiological regulator of placental function including chemical disposition. Here, we compared the ability of low oxygen tension to alter the expression of solute carriers (SLC) and ABC transporters in two human placental models, namely BeWo cells and term placental explants. We found that exposure to low oxygen concentration differentially regulates transporter expression in BeWo cells, including downregulation of ENT1, OATP4A1, OCTN2, BCRP, and MRP2/3/5, and upregulation of CNT1, OAT4, OATP2B1, SERT, SOAT, and MRP1. Similar upregulation of MRP1 and downregulation of MRP5 and BCRP were observed in explants, whereas uptake transporters were decreased or unchanged. Furthermore, a screening of transcriptional regulators of transporters revealed that hypoxia leads to a decrease in the mRNA levels of aryl hydrocarbon receptor, nuclear factor erythroid 2-related factor 2, and retinoid x receptor alpha in both human placental models. These data suggest that transporter expression is differentially regulated by oxygen concentration across experimental human placental models.

Transcription factor-mediated regulation of the BCRP/ABCG2 efflux transporter: a review across tissues and species.

Introduction: The breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette superfamily of transporters. Using the energy garnered from the hydrolysis of ATP, BCRP actively removes drugs and endogenous molecules from the cell. With broad expression across the liver, kidney, brain, placenta, testes, and small intestines, BCRP can impact the pharmacokinetics and pharmacodynamics of xenobiotics.Areas covered: The purpose of this review is to summarize the transcriptional signaling pathways that regulate BCRP expression across various tissues and mammalian species. We will cover the endobiotic- and xenobiotic-activated transcription factors that regulate the expression and activity of BCRP. These include the estrogen receptor, progesterone receptor, peroxisome proliferator-activated receptor, constitutive androstane receptor, pregnane X receptor, nuclear factor e2-related factor 2, and aryl hydrocarbon receptor.Expert opinion: Key transcription factors regulate BCRP expression and function in response to hormones and xenobiotics. Understanding this regulation provides an opportunity to improve pharmacotherapeutic outcomes by enhancing the efficacy and reducing the toxicity of drugs that are substrates of this efflux transporter.

Increased MDR1 Transporter Expression in Human Brain Endothelial Cells Through Enhanced Histone Acetylation and Activation of Aryl Hydrocarbon Receptor Signaling.

Multidrug resistance protein 1 (MDR1, ABCB1, P-glycoprotein) is a critical efflux transporter that extrudes chemicals from the blood-brain barrier (BBB) and limits neuronal exposure to xenobiotics. Prior studies in malignant cells demonstrated that MDR1 expression can be altered by inhibition of histone deacetylases (HDAC), enzymes that modify histone structure and influence transcription factor binding to DNA. Here, we sought to identify the mechanisms responsible for the up-regulation of MDR1 by HDAC inhibitors in human BBB cells. Immortalized human brain capillary endothelial (hCMEC/D3) cells were treated with HDAC inhibitors and assessed for MDR1 expression and function. Of the HDAC inhibitors profiled, valproic acid (VPA), apicidin, and suberoylanilide hydroxamic acid (SAHA) increased MDR1 mRNA and protein levels by 30-200%, which corresponded with reduced intracellular accumulation of the MDR1 substrate rhodamine 123. Interestingly, induction of MDR1 mRNA by HDAC inhibitors mirrored increases in the expression of the aryl hydrocarbon receptor (AHR) and its target gene cytochrome P450 1A1. To explore the role of AHR in HDAC inhibitor-mediated regulation of MDR1, a pharmacological activator (ß-naphthoflavone, ßNF) and inhibitor (CH-223191, CH) of AHR were tested. The induction of MDR1 in cells treated with SAHA was amplified by ßNF and attenuated by CH. Furthermore, SAHA increased the binding of acetylated histone H3K9/K14 and AHR proteins to regions of the MDR1 promoter that contain AHR response elements. In conclusion, HDAC inhibitors up-regulate the expression and activity of the MDR1 transporter in human brain endothelial cells by increasing histone acetylation and facilitating AHR binding at the MDR1 promoter.

Placental BCRP/ABCG2 Transporter Prevents Fetal Exposure to the Estrogenic Mycotoxin Zearalenone.

In the placenta, the breast cancer resistance protein (BCRP)/ABCG2 efflux transporter limits the maternal-to-fetal transfer of drugs and chemicals. Previous research has pointed to the estrogenic mycotoxin zearalenone as a potential substrate for BCRP. Here, we sought to assess the role of the BCRP transporter in the transplacental disposition of zearalenone during pregnancy. In vitro transwell transport assays employing BCRP/Bcrp-transfected Madine-Darby canine kidney cells and BeWo trophoblasts with reduced BCRP expression were used to characterize the impact of BCRP on the bidirectional transport of zearalenone. In both models, the presence of BCRP protein increased the basolateral-to-apical transport and reduced the apical-to-basolateral transport of zearalenone over a 2-h period. In vivo pharmacokinetic analyses were then performed using pregnant wild-type and Bcrp-/- mice after a single tail vein injection of zearalenone. Zearalenone and its metabolite α-zearalenol were detectable in serum, placentas, and fetuses from all animals, and ß-zearalenol was detected in serum and fetuses, but not placentas. There were no significant differences in the maternal serum concentrations of any analytes between the two genotypes. In Bcrp-/- mice, the free fetal concentrations of zearalenone, α-zearalenol, and ß-zearalenol were increased by 115%, 84%, and 150%, respectively, when compared with wild-type mice. Concentrations of free zearalenone and α-zearalenol were elevated 145% and 78% in Bcrp-/- placentas, respectively, when compared with wild-type placentas. Taken together, these data indicate that the placental BCRP transporter functions to reduce the fetal accumulation of zearalenone, which may impact susceptibility to developmental toxicities associated with in utero zearalenone exposure.

Down-regulation of the placental BCRP/ABCG2 transporter in response to hypoxia signaling.

INTRODUCTION: The BCRP/ABCG2 efflux transporter protects the developing fetus by limiting the transplacental transfer of drugs and chemicals and prevents the apoptosis of trophoblasts. The purpose of this study was to determine whether hypoxia-related signaling alters placental BCRP expression and function in vitro and in human pregnancies. METHODS: Human BeWo choriocarcinoma cells were treated with the hypoxia mimetic, cobalt chloride (CoCl2), or 3% oxygen for 24-48 h. Activation of HIF-1α signaling and regulation of BCRP was assessed using qPCR, ELISA, western blotting and a fluorescent substrate transport assay. In addition, healthy term placentas from high altitude pregnancies with chronic hypoxia were assessed for BCRP expression. RESULTS: CoCl2 and 3% oxygen increased HIF-1α protein signaling and decreased the mRNA and protein expression of BCRP by 30-75% in BeWo cells. Reduced BCRP expression corresponded with impaired efflux activity during hypoxia as evidenced by accumulation of the substrate Hoechst 33342. A number of transcription factors known to regulate BCRP, including AHR, NRF2 and PPARγ, were also coordinately down-regulated by 3% oxygen in BeWo cells. Moreover, women who gave birth at a high altitude (3100 m) exhibited signs of chronic placental hypoxia, including enhanced protein expression of the HIF-1α target GLUT1, and had reduced BCRP levels in microvillous membranes compared to women at a moderate altitude (1600 m). DISCUSSION: This study provides novel insight into the regulation of the placental BCRP transporter by hypoxia, which may be important for exposure of the fetus to chemicals during early development and in hypoxia-related pregnancy disorders.

Regulation of the placental BCRP transporter by PPAR gamma.

Identifying regulators of placental breast cancer resistance protein (BCRP) expression is critical as downregulation of this transporter may increase exposure of the fetus to xenobiotics. Here, we sought to test whether the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) regulates BCRP expression in the placenta. To test this, human BeWo placental choriocarcinoma cells were cultured with the PPARγ agonist rosiglitazone or the PPARγ antagonist T0070907 for 24 h. Messenger RNA (mRNA) expression of syncytialization markers, GCM1 and hCGß, as well as BCRP increased with PPARγ agonist treatment. Conversely, BCRP mRNA and protein expression decreased 30%-50% with PPARγ antagonist treatment. Rosiglitazone enhanced BCRP protein expression and transport activity, resulting in a 20% greater efflux of the substrate Hoechst 33342 compared with control cells. These results suggest that PPARγ can upregulate BCRP expression in the placenta, which may be important in understanding mechanisms that protect the fetus from xenobiotic exposure during development.

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter.

Glyburide is frequently used to treat gestational diabetes owing to its low fetal accumulation resulting from placental efflux by the breast cancer resistance protein (BCRP)/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells, and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of (3)H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of (3)H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 minutes increased the accumulation of (3)H-glyburide 30%-70% at concentrations relevant to dietary exposure (IC50 ∼180 nM). Continuous exposure of BeWo cells to genistein for 48 hours reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacologic antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 hours did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that whereas genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to downregulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide.