Effect of Oxygen on Multidrug Resistance in Term Human Placenta

The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics and certain hormones from the maternal to the fetal circulation. The expression of these transporters changes with gestational age, yet the mechanism(s) regulating this change in expression remains unknown. However, the changes in P-gp and BCRP transporter expression coincide with changes in oxygen tension in the placenta and studies in other tissues have shown that oxygen tension regulates P-gp and BCRP expression. Here, we investigate the effects of oxygen tension on P-gp and BCRP expression in the term human placenta. We demonstrate that the expression of placental P-gp is regulated by oxygen tension, which suggests that changes in oxygenation of the placenta in the third trimester have the potential to alter fetal drug exposure.MAS

TGF-β1 regulation of multidrug resistance P-glycoprotein in the developing male blood-brain barrier

P-glycoprotein (P-gp), an efflux transporter encoded by the abcb1 gene, protects the developing fetal brain. Levels of P-gp in endothelial cells of the blood-brain barrier (BBB) increase dramatically during the period of peak brain growth. This is coincident with increased release of TGF-β1 by astrocytes and neurons. Although TGF-β1 has been shown to modulate P-gp activity in a number of cell types, little is known about how TGF-β1 regulates brain protection. In the present study, we hypothesized that TGF-β1 increases abcb1 expression and P-gp activity in fetal and postnatal BBB in an age-dependent manner. We found TGF-β1 to potently regulate abcb1 mRNA and P-gp function. TGF-β1 increased P-gp function in brain endothelial cells (BECs) derived from fetal and postnatal male guinea pigs. These effects were more pronounced earlier in gestation when compared with BECs derived postnatally. To investigate the signaling pathways involved, BECs derived at gestational day 50 and postnatal day 14 were exposed to ALK1 and ALK5 inhibitors and agonists. Through inhibition of ALK5, we demonstrated that ALK5 is required for the TGF-β1 effects on P-gp function. Activation of ALK1, by the agonist BMP-9, produced similar results to TGF-β1 on P-gp function. However, TGF-β1 signaling through the ALK1 pathway is age-dependent as dorsomorphin, an ALK1 inhibitor, attenuated TGF-β1-mediated effects in BECs derived at postnatal day 14 but not in those derived at gestational day 50. In conclusion, TGF-β1 regulates P-gp at the fetal and neonatal BBB and both ALK5 and ALK1 pathways are implicated in the regulation of P-gp function. Aberrations in TGF-β1 levels at the developing BBB may lead to substantial changes in fetal brain exposure to P-gp substrates, triggering consequences for brain development.This study was funded by the Canadian Institutes for Health Research (FRN-57746 to S.G.M. and W.G.)

Impact of bacterial and viral challenge on multidrug resistance in first- and third-trimester human placenta

The ABC transporters P-glycoprotein (P-gp, official gene symbol ABCB1) and breast cancer resistance protein (BCRP, official gene symbol ABCG2) protect the conceptus from exposure to toxins and xenobiotics present in the maternal circulation. Viral or bacterial challenges alter expression of placental multidrug transporters in rodents. We hypothesized that exposure to lipopolysaccharide (LPS, bacterial antigen) and polyinosinic-polycytidylic acid (poly(I:C), viral antigen) would decrease P-gp and BCRP in the human placenta. Placental explants from first and third trimesters were challenged with 0.1 to 10 μg/mL LPS or 1 to 50 μg/mL poly(I:C) for 4 or 24 hours; mRNA levels, protein expression, and localization were assessed by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, respectively. Toll-like receptor (TLR)-3 and TLR-4 mRNA expression increased from the first to third trimester (P < 0.01), and the receptors localized to cytotrophoblasts in the first trimester and to syncytiotrophoblasts in the third trimester. LPS exposure in first-trimester explants decreased (P < 0.001) ABCB1 and ABCG2 mRNA and protein levels. In contrast, poly(I:C) decreased (P < 0.05) ABCB1, TLR-3, and TLR-4 mRNA levels in the third trimester but not first trimester. LPS and poly(I:C) treatments increased (P < 0.01) IL-8 and chemokine ligand 2. Results suggest that bacterial infections likely alter exposure of the conceptus to toxins and drugs during early pregnancy, whereas viral infections may disrupt fetal protection in later stages of pregnancy

Prenatal endotoxemia and placental drug transport in the mouse: placental size-specific effects.

Lipopolysaccharide (LPS) in high doses inhibits placental multidrug resistance P-glycoprotein (P-gp--Abcb1a/b) and breast cancer resistance protein (BCRP--Abcg2). This potentially impairs fetal protection against harmful factors in the maternal circulation. However, it is unknown whether LPS exposure, at doses that mimic sub-lethal clinical infection, alters placental multidrug resistance. We hypothesized that sub-lethal (fetal) LPS exposure reduces placental P-gp activity. Acute LPS (n = 19;150 µg/kg; ip) or vehicle (n = 19) were given to C57BL/6 mice at E15.5 and E17.5. Placentas and fetal-units were collected 4 and 24 h following injection. Chronic LPS (n = 6; 5 µg/kg/day; ip) or vehicle (n = 5) were administered from E11.5-15.5 and tissues were collected 4 h after final treatment. P-gp activity was assessed by [³H]digoxin accumulation. Placental Abcb1a/b, Abcg2, interleukin-6 (Il-6), Tnf-α, Il-10 and toll-like receptor-4 (Tlr-4) mRNA were measured by qPCR. Maternal plasma IL-6 was determined. At E15.5, maternal IL-6 was elevated 4 h after single (p<0.001) and chronic (p<0.05) LPS, but levels had returned to baseline by 24 h. Placental Il-6 mRNA was also increased after acute and chronic LPS treatments (p<0.05), whereas Abcb1a/b and Abcg2 mRNA were unaffected. However, fetal [³H]digoxin accumulation was increased (p<0.05) 4 h after acute LPS, and maternal [³H]digoxin myocardial accumulation was increased (p<0.05) in mice exposed to chronic LPS treatments. There was a negative correlation between fetal [³H]digoxin accumulation and placental size (p<0.0001). Acute and chronic sub-lethal LPS exposure resulted in a robust inflammatory response in the maternal systemic circulation and placenta. Acute infection decreased placental P-gp activity in a time- and gestational age-dependent manner. Chronic LPS decreased P-gp activity in the maternal myocardium and there was a trend for fetuses with smaller placentas to accumulate more P-gp substrate than their larger counterparts. Collectively, we demonstrate that acute sub-lethal LPS exposure during pregnancy impairs fetal protection against potentially harmful xenobiotics in the maternal circulation

Maternal IL-6 plasma levels and spleen weight in mice exposed to chronic LPS treatments.

<p>Chronic LPS (5 µg/Kg) treatment was performed daily from E11.5 until E15.5. (A) Maternal plasma was extracted 4 h after last LPS treatment on E15.5 (Vehicle n = 9; LPS n = 10). (B) Maternal splenic weight from animals exposed to chronic LPS treatment (Vehicle n = 9; LPS n = 10). Values are means±SEM. *P<0.05, **<i>p<</i>0.001 (one-way ANOVA followed by the Tukey’s post-hoc test).</p

Rate of fetal death/reabsorption after acute LPS exposure.

<p>Rate of fetal death/reabsorption after acute LPS exposure.</p

Rate of of fetal death/reabsorption after multiple LPS exposure.

*<p>Dams received daily Veh/LPS treatments from E11.5 until E15.5 and were euthanized 4 hs after last treatment on E15.5.</p

Placental mRNA expression of the multidrug resistance genes (Abcb1a, Abcb1b, and Abcg2) and the pro-inflammatory cytokine Il-6 after acute sub-lethal LPS exposure: one placenta per group was randomly harvested 4 h after LPS insult and processed for qPCR analyses (n = 6 dams/gp).

<p>Values are fold increase of the means±SEM. *<i>p<</i>0.05 (one-way ANOVA followed by the Tukey’s post-hoc test). Relative gene expression normalized to Tbp Gapdh.</p

Maternal IL-6 plasma levels in mice exposed to acute LPS treatment.

<p>Vehicle and acute LPS (150 µg/Kg) treatments were performed and maternal plasma was extracted: 4 h (n = 6/gp) or 24 h (n = 5/gp) after single LPS exposure. Values are means±SEM. ***<i>p<</i>0.0001 (one-way ANOVA followed by the Tukey’s post-hoc test).</p

Placental mRNA expression of the multidrug resistance genes (Abcb1a, Abcb1b, and Abcg2), Il-6, Tnf-α, Il-10 and Tlr-4 after chronic LPS exposure.

<p>The small, mid-range and larger placentas from each litter were grouped and assayed for mRNA expression. (Veh, n = 7 dams; LPS n = 7). (Repeated measures two-way ANOVA followed by Bonferonni’s test, <i>p</i><0.05). Relative gene expression normalized to <i>Tbp, Gapdh</i> and <i>Hprt</i>.</p