Modulation of Multidrug Resistance Phosphoglycoprotein in the Mouse Placenta and Fetal Brain by the Selective Serotonin Reuptake Inhibitor Sertraline and Maternal Bacterial Infection

Multidrug resistance phosphoglycoprotein (P-gp) is expressed in the placenta and fetal blood-brain barrier (BBB) and plays a critical role in reducing fetal accumulation of xenobiotics. In other tissues, P-gp activity is inhibited by selective serotonin reuptake inhibitors (SSRIs) and by lethal doses of LPS modeling a bacterial infection. However, nothing is known with respect to the effects of SSRIs or nonlethal infection on P-gp activity in the placenta or fetal tissues. In the studies presented in this thesis, we hypothesized that (1) the SSRI sertraline and (2) a nonlethal maternal bacterial infection would decrease P-gp activity in the placenta and fetal BBB. The first study shows that sertraline affects P-gp activity at these barrier sites in a tissue-specific manner. The second study shows that nonlethal infection does not significantly affect P-gp activity at either site. However, nonlethal infection may still influence substrate biodistribution by altering hepatic elimination of these substrates.MAS

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

Placental size and P-gp transport efficiency.

<p>Small, mid-range and larger placentas from each litter were grouped and averaged for placental (A) and fetal (B) weight and fetal to placental (F:P) weight ratio (C). (D) shows the relationship between individual placental weights and [³H]digoxin fetal accumulation at E15.5, for vehicle (r = −0.6657, n = 31 fetuses from 5 litters, <i>p</i><0.0001) and chronic LPS (r = −0.5099, n = 39 fetuses from 6 litters, <i>p</i><0.001) groups. (E) [³H]digoxin fetal accumulation from small, mid-range and larger placentas at E15.5 (vehicle n = 5; LPS n = 6). (A,B,C and E, repeated measures two-way ANOVA followed by Bonferonni’s test, <i>p</i><0.05; Pearson’s correlation test).</p

Rate of fetal death/reabsorption after acute LPS exposure.

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

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

Placental and maternal myocardial P-gp activity after chronic sub-lethal LPS exposure.

<p>(A) fetal units [³H]digoxin accumulation (all fetuses/dam) and (B) maternal myocardial [³H]digoxin accumulation, 4 h after last LPS chronic treatment (daily from E11.5 until E15.5) on E15.5 (vehicle n = 5; chronic LPS n = 6). Values are means±SEM. *P = 0.05 (one-way ANOVA followed by the Tukey’s post-hoc test).</p

Placental and maternal myocardial P-gp activity after acute sub-lethal LPS exposure:

<p>Fetal Units [³H]digoxin accumulation (4 fetal units/dam were randomly harvested and assayed) on E15.5 (A) (n = 5 dams/gp) and E17.5 (B) (4 h n = 5 dams/gp; 24 h n = 4 dams/gp); 4 or 24 h after acute LPS treatment. Maternal myocardial [³H]digoxin accumulation on E15.5 (C) and E17.5 (D) 4 or 24 h after acute LPS treatment. Values are means±SEM. *<i>p<</i>0.05 (one-way ANOVA followed by the Tukey’s post-hoc test on E15.5 and Kruskal-Wallis analysis of variance followed by Dunn’s test on E17.5).</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

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