Majid Iqbal

Prenatal Synthetic Glucocorticoid Treatment Changes DNA Methylation States in Male Organ Systems: Multigenerational Effects

Prenatal synthetic glucocorticoids (sGC) are administered to pregnant women at risk of delivering preterm, approximately 10% of all pregnancies. Animal studies have demonstrated that offspring exposed to elevated glucocorticoids, either by administration of sGC or as a result of maternal stress, are at increased risk of developing behavioral, endocrine, and metabolic abnormalities. DNA methylation is a covalent modification of DNA that plays a critical role in long-lasting programming of gene expression. Here we tested the hypothesis that prenatal sGC treatment has both acute and long-term effects on DNA methylation states in the fetus and offspring and that these effects extend into a subsequent generation. Pregnant guinea pigs were treated with sGC in late gestation, and methylation analysis by luminometric methylation assay was undertaken in organs from fetuses and offspring across two generations. Expression of genes that modify the epigenetic state were measured by quantitative real-time PCR. Results indicate that there are organ-specific developmental trajectories of methylation in the fetus and newborn. Furthermore, these trajectories are substantially modified by intrauterine exposure to sGC. These sGC-induced changes in DNA methylation remain into adulthood and are evident in the next generation. Furthermore, prenatal sGC exposure alters the expression of several genes encoding proteins that modulate the epigenetic state. Several of these changes are long lasting and are also present in the next generation. These data support the hypothesis that prenatal sGC exposure leads to broad changes in critical components of the epigenetic machinery and that these effects can pass to the next generation.

Transgenerational Effects of Prenatal Synthetic Glucocorticoids on Hypothalamic-Pituitary-Adrenal Function

Approximately 10% of pregnant women are at risk of preterm delivery and receive synthetic glucocorticoids (sGC) to promote fetal lung development. Studies have indicated that prenatal sGC therapy modifies hypothalamic-pituitary-adrenal (HPA) function in first-generation (F1) offspring. The objective of this study was to determine whether differences in HPA function and behavior are evident in the subsequent (F2) generation. Pregnant guinea pigs (F0) received betamethasone (BETA; 1 mg/kg) or saline on gestational d 40/41, 50/51, and 60/61. F1 females were mated with control males to create F2 offspring. HPA function was assessed in juvenile and adult F2 offspring. Locomotor activity was assessed in juvenile offspring. Analysis of HPA-related gene expression was undertaken in adult hippocampi, hypothalami, and pituitaries. Locomotor activity was reduced in F2 BETA males (P < 0.05). F2 BETA offspring displayed blunted cortisol response to swim stress (P < 0.05). After dexamethasone challenge, F2 BETA males and females displayed increased and decreased negative feedback, respectively. F2 BETA females had reduced pituitary levels of proopiomelanocortin (and adrenocorticotropic hormone), and corticotropin-releasing hormone receptor mRNA and protein (P < 0.05). F2 BETA males displayed increased hippocampal glucocorticoid receptor (P < 0.001), whereas in BETA females, hippocampal glucocorticoid receptor and mineralocorticoid receptor mRNA were decreased (P < 0.05). In conclusion, prenatal BETA treatment affects HPA function and behavior in F2 offspring. In F2 BETA females, pituitary function appears to be primarily affected, whereas hippocampal glucocorticoid feedback systems appear altered in both F2 BETA males and females. These data have clinical implication given the widespread use of repeat course glucocorticoid therapy in the management of preterm labour.

Corticosteroid Regulation of P-Glycoprotein in the Developing Blood-Brain Barrier

The early fetal brain is susceptible to teratogens in the maternal circulation, because brain microvessel expression of drug efflux transporter, P-glycoprotein (P-gp), is very low. However, there is a dramatic up-regulation of brain microvessel P-gp in late gestation. This study investigated the role of cortisol and dexamethasone in this up-regulation of fetal brain microvessel P-gp expression. Primary brain endothelial cell (BEC) cultures derived from gestational d (GD)40, GD50, GD65 (term, ∼68 d) and postnatal d 14 male guinea pigs were treated with varying doses (10(-8) to 10(-5) m) of cortisol, dexamethasone, and aldosterone. After treatment, P-gp function was assessed using calcein-acetoxymethyl ester (P-gp substrate; 1 μm for 1 h) and measuring BEC accumulation of calcein. Corticosteroid treatment of BECs derived from postnatal d 14 resulted in increased P-gp activity. BECs derived from GD65 (near term) responded similarly, but these cells were extremely sensitive to the effects of mineralocorticoid receptor agonists (cortisol and aldosterone). BECs derived from GD50 displayed dose-dependent increases in P-gp function with dexamethasone (P < 0.05) and a trend towards increased function with cortisol. Cells derived from GD40 were unresponsive to all treatments. In conclusion, P-gp function in BECs is more responsive to glucocorticoids (GCs) in late gestation. Therefore, the late gestational surge in fetal plasma GCs, which parallels the increase in brain microvessel P-gp expression, may contribute to this P-gp up-regulation. Further, synthetic GCs (administered to pregnant women at risk of preterm delivery) may increase the protective capacity of the developing fetal blood-brain barrier, depending on the timing of GC exposure.

The ontogeny of P-glycoprotein in the developing human blood–brain barrier: implication for opioid toxicity in neonates

Background:Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults. The limited development of P-glycoprotein (P-gp) may limit the ability of the neonate to efflux morphine from the brain back to the systemic circulation. The objective of the study was to determine the ontogeny of P-gp in the human brain.Methods:Postmortem cortex samples from gestational age (GA) 20–26 wk, GA 36–40 wk, postnatal age (PNA) 0–3 mo, PNA 3–6 mo, and adults were immunostained for P-gp.Results:The intensity of P-gp staining in adults was significantly higher compared to at GA 20–26 wk (P < 0.05), GA 36–40 wk (P < 0.05), and PNA 0–3 mo (P < 0.05). P-gp intensity at GA 20–26 wk (P < 0.05), GA 36–40 wk (P < 0.05), and PNA 0–3 mo (P < 0.05) was significantly lower compared to at PNA 3–6 mo.Conclusion:P-gp expression in the brain is limited at birth, increases with postnatal maturation, and reaches adult levels at ~3–6 mo of age. Given the immaturity of blood–brain barrier (BBB) P-gp after birth, morphine may concentrate in the brain. This provides mechanistic support to life threatening opioid toxicity seen with maternal codeine use during breastfeeding.

Pro-Inflammatory Cytokine Regulation of P-glycoprotein in the Developing Blood-Brain Barrier

Placental P-glycoprotein (P-gp) acts to protect the developing fetus from exogenous compounds. This protection declines with advancing gestation leaving the fetus and fetal brain vulnerable to these compounds and potential teratogens in maternal circulation. This vulnerability may be more pronounced in pregnancies complicated by infection, which is common during pregnancy. Pro-inflammatory cytokines (released during infection) have been shown to be potent inhibitors of P-gp, but nothing is known regarding their effects at the developing blood-brain barrier (BBB). We hypothesized that P-gp function and expression in endothelial cells of the developing BBB will be inhibited by pro-inflammatory cytokines. We have derived brain endothelial cell (BEC) cultures from various stages of development of the guinea pig: gestational day (GD) 50, 65 (term ∼68 days) and postnatal day (PND) 14. Once these cultures reached confluence, BECs were treated with various doses (100–104 pg/mL) of pro-inflammatory cytokines: interleukin-1β (IL-1β), interleukin-6 (IL-6) or tumor necrosis factor- α (TNF-α). P-gp function or abcb1 mRNA (encodes P-gp) expression was assessed following treatment. Incubation of GD50 BECs with IL-1β, IL-6 or TNF-α resulted in no change in P-gp function. GD65 BECs displayed a dose-dependent decrease in function with all cytokines tested; maximal effects at 42%, 65% and 34% with IL-1β, IL-6 and TNF-α treatment, respectively (P<0.01). Inhibition of P-gp function by IL-1β, IL-6 and TNF-α was even greater in PND14 BECs; maximal effects at 36% (P<0.01), 84% (P<0.05) and 55% (P<0.01), respectively. Cytokine-induced reductions in P-gp function were associated with decreased abcb1 mRNA expression. These data suggest that BBB P-gp function is increasingly responsive to the inhibitory effects of pro-inflammatory cytokines, with increasing developmental age. Thus, women who experience infection and take prescription medication during pregnancy may expose the developing fetal brain to greater amounts of exogenous compounds – many of which are considered potentially teratogenic.

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.

Effect of oxygen on multidrug resistance in term human placenta

INTRODUCTION The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and nutrients from the maternal to the fetal circulation. The expression of these transporters changes with gestational age, yet the mechanisms involved remain unknown. However, the changes in P-gp and BCRP transporter expression coincide with those of oxygen tension in the placenta, and oxygen tension has been shown to modulate P-gp and BCRP expression in other tissues. The objective of this study was to investigate the effects of oxygen tension on P-gp and BCRP expression in the term human placenta. METHODS Following equilibration in culture (96 h), term placental explants (n = 7) were cultured in 3% or 20% oxygen for 24 and 48 h. Culture medium was collected every 24 h to measure lactate dehydrogenase (LDH; explant viability) and human chorionic gonadotropin (hCG; syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and mRNA, as well as VEGFA mRNA were measured using western blot and qRT-PCR. P-gp localization was determined using immunofluorescence. RESULTS Oxygen tension had a significant effect on P-gp expression, with ABCB1/P-gp mRNA and protein levels increased in the hypoxic condition (3% O2) after 48 h (p < 0.05). VEGFA mRNA was elevated by hypoxia at both 24 and 48 h (p < 0.05). In contrast, placental ABCG2/BCRP mRNA and protein expression were stable with changes in oxygen tension. We identified profound differences in the glycosylation of P-gp between cultured and non-cultured placental tissue, with cultured explants expressing deglycosylated P-gp. CONCLUSIONS These findings demonstrate that, at term, the expression of placental P-gp, is regulated by oxygen tension. This suggests that changes in oxygenation of the placenta in the third trimester may alter levels of placental P-gp, and in doing so alter fetal exposure to P-gp substrates, including xenobiotics and certain hormones.

Effects of Sertraline and Fluoxetine on P-Glycoprotein at Barrier Sites: In Vivo and In Vitro Approaches

Background and Purpose Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo. Experimental Approach The P-gp substrate, tritiated digoxin ([3H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects. Key Results In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [3H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo. Conclusions and Implications Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.

The multidrug resistance 1 gene Abcb1 in brain and placenta: comparative analysis in human and guinea pig.

The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain.

Regulation of Multidrug Resistance P-Glycoprotein in the Developing Blood-Brain Barrier: Interplay between Glucocorticoids and Cytokines.

P‐glycoprotein (P‐gp) encoded by Abcb1 provides protection to the developing brain from xenobiotics. P‐gp in brain endothelial cells (BECs) derived from the developing brain microvasculature is up‐regulated by glucocorticoids and inhibited by pro‐inflammatory cytokines in vitro. However, little is known about how prenatal maternal glucocorticoid treatment can affect Abcb1/P‐gp function and subsequent cytokine regulation in foetal BECs. We hypothesised that glucocorticoid exposure increases Abcb1/P‐gp in the foetal brain microvasculature and enhances the sensitivity of Abcb1/P‐gp in BECs to the inhibitory effects of cytokines. BECs isolated from dexamethasone‐ or vehicle‐exposed foetal guinea pigs were cultured and treated with interleukin‐1β, interleukin‐6 or tumour necrosis factor‐α, and Abcb1/P‐gp expression and function were assessed. Prenatal dexamethasone exposure significantly increased Abcb1/P‐gp expression/activity and cytokine receptor levels in BECs of the foetal brain microvasculature. Foetal dexamethasone exposure in vivo also increased the subsequent responsiveness of BECs to pro‐inflammatory cytokines in vitro. In conclusion, maternal treatment with synthetic glucocorticoids appears to prematurely mature P‐gp mediated drug resistance at the foetal BBB in vivo and profoundly impact the subsequent responsiveness of P‐gp to pro‐inflammatory cytokines in the foetal BEC. The significance of these findings to foetal brain protection against xenobiotics and other P‐gp substrates in vivo requires further elaboration. However, the results of the present study may have implications for human pregnancy and foetal brain protection, particularly in cases of preterm birth combined with infection.