Jeffrey A. Keelan

Drug transfer and metabolism by the human placenta

The major function of the placenta is to transfer nutrients and oxygen from the mother to the foetus and to assist in the removal of waste products from the foetus to the mother. In addition, it plays an important role in the synthesis of hormones, peptides and steroids that are vital for a successful pregnancy. The placenta provides a link between the circulations of two distinct individuals but also acts as a barrier to protect the foetus from xenobiotics in the maternal blood.However, the impression that the placenta forms an impenetrable obstacle against most drugs is now widely regarded as false. It has been shown that that nearly all drugs that are administered during pregnancy will enter, to some degree, the circulation of the foetus via passive diffusion. In addition, some drugs are pumped across the placenta by various active transporters located on both the fetal and maternal side of the trophoblast layer. It is only in recent years that the impact of active transporters such as P-glycoprotein on the disposition of drugs has been demonstrated. Facilitated diffusion appears to be a minor transfer mechanism for some drugs, and pinocytosis and phagocytosis are considered too slow to have any significant effect on fetal drug concentrations.The extent to which drugs cross the placenta is also modulated by the actions of placental phase I and II drug-metabolising enzymes, which are present at levels that fluctuate throughout gestation. Cytochrome P450 (CYP) enzymes in particular have been well characterised in the placenta at the level of mRNA, protein, and enzyme activity. CYP1A1, 2E1, 3A4, 3A5, 3A7 and 4B1 have been detected in the term placenta. While much less is known about phase II enzymes in the placenta, some enzymes, in particular uridine diphosphate glucuronosyltransferases, have been detected and shown to have specific activity towards marker substrates, suggesting a significant role of this enzyme in placental drug detoxification.The increasing experimental data on placental drug transfer has enabled clinicians to make better informed decisions about which drugs significantly cross the placenta and develop dosage regimens that minimise fetal exposure to potentially toxic concentrations. Indeed, the foetus has now become the object of intended drug treatment. Extensive research on the placental transfer of drugs such as digoxin and zidovudine has assisted with the safe treatment of the foetus with these drugs in utero. Improved knowledge regarding transplacental drug transfer and metabolism will result in further expansion of pharmacological treatment of fetal conditions.

Critical Paracrine Interactions Between TNF-α and IL-10 Regulate Lipopolysaccharide-Stimulated Human Choriodecidual Cytokine and Prostaglandin E2 Production

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 μg/ml) induced a rapid increase in TNF-α production, peaking at 4 h. In contrast, IL-10, IL-1β, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-α was a primary regulator of IL-1β, IL-10, and PGE2 production, while IL-1β stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-α and PGE2 production. IL-10 treatment markedly decreased TNF-α and IL-1β production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-α may be a potential target for treatment intervention; IL-10 could be one such therapeutic.

UDP-glucuronosyltransferase activity, expression and cellular localization in human placenta at term

The activity, expression and localization of the UDP-glucuronosyltransferases (UGTs) were investigated in human placenta at term. UGT activity (measured with the substrate 4-methylumbelliferone (4-MU)) was observed in all 25 placentas sampled and maximum velocity (V(max)) ranged 13-fold from 5.1+/-0.9 to 66.9+/-17.5 nmol/min/mg protein (mean+/-SD). Substrate affinity (K(m)) ranged 5-fold from 246+/-24 to 1124+/-422 microM. Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of the isoforms UGT2B4, 2B7, 2B10, 2B11 and 2B15 was observed in all (12/12) placentas sampled and expression of UGT2B17 was noted in 8/12 placentas. Northern analysis of the UGT2B7 isoform in 12 placentas revealed a 10-fold difference in expression with RT-PCR variability and the 13-fold variation observed in UGT activity. The presence of UGT2B4 and 2B7 proteins (52 and 56kDa, respectively) was demonstrated by Western blotting. The sites of placental UGT2B transcription (in situ hybridization) and protein expression (immunohistochemistry) were located in the syncytium of the placental trophoblasts bordering the placental villi. UGT1A proteins could not be observed with immunohistochemistry or Western blotting and expression could not be observed with RT-PCR. Our discovery of UGT expression and activity at the site of maternal-fetal exchange is consistent with a role for UGTs in detoxification of exogenous and endogenous ligands and the maintenance of placental function through clearance and regulation of steroid hormones.

The ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction

The efflux pump ATP binding cassette superfamily member G2 (ABCG2)/breast cancer resistance protein (BCRP) is highly expressed in human placenta. We have investigated the role of BCRP in the protection of the human placental trophoblasts from apoptosis and its expression in idiopathic fetal growth restriction, a condition associated with abnormal pla‐cental apoptosis. Inhibition of BCRP activity with the selective inhibitor Ko143 augmented cytokine (tumor necrosis factor‐α/interferon‐γ)‐induced apoptosis and phosphatidylserine externalization in primary tropho‐blast and trophoblast‐like BeWo cells. Silencing of BCRP expression in BeWo cells significantly increased their sensitivity to apoptotic injury in response to cytokines and exogenous C6 and C8 ceramides. BCRP silencing also increased intracellular ceramide levels after cytokine exposure but did not affect cellular protoporphyrin IX concentrations or sensitivity to activators of the intrinsic apoptotic pathway. BCRP expression in placentas from pregnancies complicated by idiopathic fetal growth restriction was decreased compared with controls, suggesting reduced transport of its substrates from the placenta. We conclude that BCRP may play a hitherto unrecognized survival role in the placenta, protecting the trophoblast against cytokine‐induced apoptosis and possibly other extrinsic activators via modulation of ceramide signaling. Decreased placental BCRP expression may result in reduced viability and hence functional deficit, contributing to the fetal growth restriction phenotype.—Evseenko, D. A., Murthi, P., Paxton, J. W., Reid, G., Emerald, B. S., Mohankumar, K. M., Lobie, P. E., Brennecke, S. P., Kalionis, B. Keelan, J. A. The ABC transporter BCRP/ ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction. FASEB J. 21, 3592–3605 (2007)

Active transport across the human placenta: impact on drug efficacy and toxicity

The human placenta expresses a large number of transport proteins. The ATP-binding cassette (ABC) family of active efflux pumps, predominantly localised to the maternal-facing syncytial membrane of placental microvilli, comprise the major placental drug efflux transporters. A variety of other transporters are also expressed in the placenta that can facilitate xenobiotic transfer in both the maternal and fetal directions. Many drugs administered in pregnancy are ABC transporter substrates, and many are either teratogenic or fetotoxic. The invitro, invivo and clinical evidence reviewed in this article argues that active efflux of drugs by placental transporters helps to maintain its barrier function, reducing the incidence of adverse fetal effects. ABC transporter polymorphisms may explain the wide variability observed in fetal drug concentrations, incidence of teratogenesis or drug failure in pregnancies exposed to therapeutic agents. Although our understanding of the molecular mechanics and dynamics of placental drug transfer is advancing, much work is needed to fully appreciate the significance of placental drug transporters in the face of increasing drug administration in pregnancy.

Epithelial Cell-Derived Neutrophil-Activating Peptide-78 Is Present in Fetal Membranes and Amniotic Fluid at Increased Concentrations with Intra-amniotic Infection and Preterm Delivery

Abstract Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased (∼4-fold) with term labor in amnion (n = 15), and with preterm labor (∼30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r2 = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1β and tumor necrosis factor α. Production of ENA-78 by choriodecidual explants was increased modestly after 2–4 h of exposure to lipopolysaccharide (5 μg/ml). An immunoreactive doublet (∼8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.

Activin-A stimulates, while transforming growth factor β1 inhibits,chorionic gonadotrophin production and aromatase activity in cultured human placental trophoblasts

Transforming growth factor-beta (TGF-beta) and activin-A, two members of a ubiquitous family of regulators of growth, differentiation and hormonogenesis, are produced by the human placenta. Their effects on placental hCG, inhibin, and oestrogen production in vitro, either alone or in combination, were investigated using cultured Percoll-purified placental trophoblasts. Inhibin and hCG were measured by immunoassay, while aromatase activity (i.e. oestrogen production) was measured using the tritiated water method. Aromatase activity and production of hCG, but not inhibin, were inhibited (up to approximately 30 per cent) in a dose-dependent fashion by 48 h treatment with TGF-beta. The effects were significant at all doses tested, from 0.1-10 ng/ml. In contrast, activin stimulated hCG production and aromatase activity over the doses tested (0.25-25 ng/ml). The maximum effect (approximately 50 per cent stimulation above control) was seen at the 2.5 ng/ml dose, with lesser effects seen at the lower and higher doses. This characteristic bell-shaped dose-response curve was maintained in the presence of TGF-beta (10 ng/ml) or a maximally-effective dose of forskolin (6.7 microM). This suggests that the actions of activin were independent of those of TGF-beta, and were not mediated by the protein kinase-A pathway. Activin had a weak stimulatory effect on inhibin production. The results indicate that in the placenta activin and TGF-beta have opposing actions on hormonogenesis. Both factors may play a role in regulating placental function and the timing and progression of labour.

Predicting preterm delivery: comparison of cervicovaginal interleukin (IL)-1β, IL-6 and IL-8 with fetal fibronectin and cervical dilatation

OBJECTIVE To compare the cervicovaginal cytokines IL-1beta, IL-6 and IL-8 with fetal fibronectin (fFN) and cervical dilatation in the prediction of preterm delivery. STUDY DESIGN Cervicovaginal cytokine concentration and fFN status were measured in 104 women with symptoms of preterm labour and intact membranes between 24(0) and 33(6) weeks and related to delivery within 2 and 7 days. RESULTS A group of 18% had cervical dilatation > or = 1cm and 18% were positive for fFN. Preterm delivery within 2 and 7 days occurred in 5 and 12%, respectively. Only IL-6 demonstrated any ability to predict delivery within 2 and 7 days (area under the ROC curve = 0.63 and 0.75, respectively). Using 35pg/ml (75th centile) as a cut-off, IL-6 had a sensitivity and specificity of 60 and 77% for predicting delivery within 2 days, and 62 and 80% for predicting delivery within 7 days. This is similar to the performance of cervical dilatation or fFN status. CONCLUSIONS Measurement of cervicovaginal cytokines has limited ability to predict imminent delivery apart from cervicovaginal IL-6 concentrations, which, in this population, is equivalent to that of fFN status and cervical dilatation > or = 1cm.

Key enzymes of prostaglandin biosynthesis and metabolism. Coordinate regulation of expression by cytokines in gestational tissues: a review

Preterm labor is frequently associated with ascending intrauterine infection, accompanied by leukocytes infiltration and enhanced local production of cytokines and other inflammatory mediators. The resulting amplification of the inflammatory response, and of prostanoid production in particular, is postulated to be a principal mechanism of infection-driven preterm labor. In this review the effects of pro- and anti-inflammatory cytokines are discussed with respect to the expression of enzymes involved in three key steps of prostanoid biosynthesis and metabolism: liberation of arachidonic acid (AA), conversion of AA to bioactive prostanoids, and prostanoid catabolism. We suggest that by exerting coordinate actions on all three key steps, through multiple molecular mechanisms, inflammatory cytokines acutely up-regulate prostanoid production in intrauterine tissues.

Serum activin A, inhibin A, and follistatin concentrations in preeclampsia or small for gestational age pregnancies

OBJECTIVE To determine whether maternal serum activin A, inhibin A, and follistatin concentrations in idiopathic small for gestational age (SGA) pregnancies are similar to those in normal pregnancies or elevated as in preeclampsia. METHODS Maternal serum activin A, inhibin A, and follistatin concentrations were determined in 1) nulliparous women with idiopathic SGA (birth weight <10th percentile; n = 18), preeclampsia (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg plus proteinuria ≥2+or >0.3 g/24h; n = 22), and normotensive controls, matched for gestational age at sampling (n = 22), and 2) a longitudinal series of samples collected at five intervals throughout pregnancy from nulliparous women with idiopathic SGA (n = 19), preeclampsia (n = 22), preeclampsia plus SGA (n = 15), or who had uncomplicated pregnancies (n = 20). RESULTS Serum concentrations of activin A and inhibin A were similar in idiopathic SGA pregnancies to controls. In preeclampsia, activin A and inhibin A levels were markedly increased compared with controls or women with idiopathic SGA (P < .001), particularly in those with early‐onset disease. Follistatin concentrations were only modestly (<twofold) elevated in preeclampsia (P < .001). In the longitudinal study, serum activin A or inhibin A concentrations were increased in women who later developed preeclampsia, whereas in women with idiopathic SGA pregnancy, a small overall increase in activin A levels was observed. CONCLUSIONS In contrast to women with preeclampsia, normotensive women with SGA pregnancies do not have markedly elevated circulating levels of activin A and inhibin A. These data support the hypothesis that increased serum activin A concentrations in preeclampsia may be a manifestation of maternal disease rather than just a marker of abnormal placentation.