Interindividual Regulation of the Breast Cancer Resistance Protein/ABCG2 Transporter in Term Human Placentas.

The breast cancer resistance protein (BCRP/ABCG2) is a maternally-facing efflux transporter that regulates the placental disposition of chemicals. Transcription factors and gene variants are important regulatory factors that influence transporter expression. In this study, we sought to identify the genetic and transcriptional mechanisms underlying the interindividual expression of BCRP mRNA and protein across 137 term placentas from uncomplicated pregnancies. Placental expression of BCRP and regulatory transcription factor mRNAs was measured using multiplex-branched DNA analysis. BCRP expression and ABCG2 genotypes were determined using Western blot and Fluidigm Biomark genetic analysis, respectively. Placentas were obtained from a racially and ethnically diverse population, including Caucasian (33%), African American (14%), Asian (14%), Hispanic (15%), and mixed (16%) backgrounds, as well as unknown origins (7%). Between placentas, BCRP mRNA and protein varied up to 47-fold and 14-fold, respectively. In particular, BCRP mRNA correlated significantly with known transcription factor mRNAs, including nuclear factor erythroid 2-related factor 2 and aryl hydrocarbon receptor. Somewhat surprisingly, single-nucleotide polymorphisms (SNPs) in the ABCG2 noncoding regions were not associated with variation in placental BCRP mRNA or protein. Instead, the coding region polymorphism (C421A/Q141K) corresponded with 40%-50% lower BCRP protein in 421C/A and 421A/A placentas compared with wild types (421C/C). Although BCRP protein and mRNA expression weakly correlated (r = 0.25, P = 0.040), this relationship was absent in individuals expressing the C421A variant allele. Study results contribute to our understanding of the interindividual regulation of BCRP expression in term placentas and may help to identify infants at risk for increased fetal exposure to chemicals due to low expression of this efflux protein.

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.

Inherited disorders of bilirubin clearance.

Inherited disorders of hyperbilirubinemia may be caused by increased bilirubin production or decreased bilirubin clearance. Reduced hepatic bilirubin clearance can be due to defective (i) unconjugated bilirubin uptake and intrahepatic storage, (ii) conjugation of glucuronic acid to bilirubin (e.g., Gilbert syndrome, Crigler-Najjar syndrome, Lucey-Driscoll syndrome, breast milk jaundice), (iii) bilirubin excretion into bile (Dubin-Johnson syndrome), or (iv) conjugated bilirubin re-uptake (Rotor syndrome). In this review, the molecular mechanisms and clinical manifestations of these conditions are described, as well as current approaches to diagnosis and therapy.

Suppression of Bile Acid Synthesis in a Preterm Infant Receiving Prolonged Parenteral Nutrition.

Bile acid metabolism is altered in neonates on parenteral nutrition (PN), predisposing them to parenteral nutrition-associated liver disease. Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the bile acid synthesis pathway, is repressed by fibroblast growth factor 19 (FGF19) and phytosterols (PS). We describe a case of a preterm infant who developed necrotizing enterocolitis (NEC) and received exclusive PN for over 2 months. Our objective was to serially assess CYP7A1 activity and plasma FGF19 and PS concentrations in this infant case compared to five healthy preterm infants. We found that CYP7A1 activity increased during the first 2 weeks of life in control infants but was undetectable in the infant case. FGF19 concentrations were high at birth in all infants and subsequently declined and did not differ between the case and control infants. As expected, PS concentrations were elevated in the infant case and continued to increase despite lipid minimization. In conclusion, CYP7A1 activity was gradually upregulated in healthy preterm infants but remained suppressed in the infant requiring prolonged PN. Preterm infants also had elevated FGF19 concentrations at birth, which decreased with advancing postnatal age.

Developmental regulation of the gut-liver (FGF19-CYP7A1) axis in neonates.

Introduction: Fibroblast growth factor 19 (FGF19) is a gut-derived hormone that regulates the expression of CYP7A1, the rate-limiting enzyme in bile acid (BA) synthesis pathway. Dysregulation of the FGF19-CYP7A1 (gut-liver) axis is associated with cholestatic liver disease. Infants, especially preterm infants and those with intestinal failure are at high risk for developing cholestatic liver disease. The activity of the gut-liver axis has not been characterized in this population. Our objective was to assess relationships between circulating FGF19 concentrations and CYP7A1 activity in neonates.Materials and methods: Plasma samples were obtained longitudinally from term and preterm infants (22-41-week gestation) hospitalized in a neonatal intensive care unit. Infants with congenital and acquired gastrointestinal disorders were excluded. Plasma levels of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, were quantified using HPLC-MS/MS. Plasma FGF19 concentrations were quantified by ELISA. Data were analyzed using linear regression models and structural equation modeling.Results: One hundred eighty-one plasma samples were analyzed from 62 infants. C4 concentrations were undetectable prior to 30 weeks' gestation and, thereafter, increased with advancing gestational age and with volume of enteral feeds. They did not correlate with serum FGF19 concentrations, which decreased with advancing gestational age and volume of enteral feeds.Discussion: The activity of CYP7A1, the rate-limiting BA synthetic enzyme in adults, is developmentally regulated and undetectable in newborns less than 30 weeks' gestation. FGF19 concentrations do not correlate with CYP7A1 activity, suggesting that the gut-liver axis is not functional in infants. High FGF19 concentrations at birth in infants less than 37 weeks' gestation is a novel finding, and its source and role in preterm infants warrants further investigation.Rationale: The intestinal hormone, fibroblast growth factor 19 (FGF19), is a major regulator of CYP7A1, the rate limiting enzyme in bile acid (BA) synthesis. Recently, dysregulation of the gut-liver (FGF19-CYP7A1) axis has been implicated in adult cholestatic liver disease, and animal studies have shown that exogenous FGF19 protects against liver injury. Given the therapeutic potential related to this signaling pathway, we sought to characterize the association between CYP7A1 and FGF19 in term and preterm infants. We conducted a prospective, observational study that measured in vivo CYP7A1 activity and FGF19 concentrations in 62 term and preterm infants (n = 181 samples). We found that CYP7A1 activity is developmentally regulated; its activity is undetectable prior to 30 weeks' gestation and increases with advancing gestational age and volume of enteral feeds. Contrary to expectation, we demonstrated that FGF19 is expressed at birth in preterm infants and decreases over time, even as enteral feeds increase. Using structural equation modeling, we were able to show that CYP7A1 activity does not correlate with FGF19 concentrations. Our results suggest that the gut-liver axis is not upregulated in preterm and term infants and that neonates with cholestatic liver disease will unlikely benefit from supplemental FGF19. We also report novel findings of elevated FGF19 concentrations in preterm infants at birth and speculate that there may be an extra-intestinal source of FGF19 that is developmentally expressed in these infants.

Regional expression of the BCRP/ABCG2 transporter in term human placentas.

The breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter that removes xenobiotics that cross the placenta back to the maternal circulation, thereby limiting exposure of the fetus to drugs and chemicals. Currently, variability of BCRP expression within the placenta is not known. Ten placentas were collected from healthy women undergoing elective Cesarean sections at term. Villous samples were dissected in defined regions (medial, intermediate, and peripheral) and BCRP mRNA and protein were quantified. There were no regional differences in mRNA expression of housekeeping genes (GAPDH, RPL13a, PRL, 18S). GAPDH had the lowest correlation with BCRP Ct values and was used for BCRP mRNA normalization. No differences in placental BCRP mRNA and protein were observed among the sample sites (<20% variability). Sampling site does not affect the expression of BCRP, supporting the utility of single site sampling protocols to assess the interindividual regulation of this transporter in human placentas.