Expression and activity of enzymes in the human placenta: pharmacological & toxicological consequences in AZT therapy

Abstract

The aim of this thesis was to study xenobiotic metabolising enzymes (XME) in the human placenta, in particular the uridine diphosphate glucuronosyltransferases' (UGTs) role in the metabolism, transfer and disposition of the drug AZT, and to develop the human placental perfusion model into the first trimester. UGT, β-glucuronidase, Cytochrome p4501A (CYPIA) and CYP reductase were present and active in the human placenta. CYP2E1 protein was expressed but not active. CYPIA, CYP2EI and the UGT2B subfamily were expressed across gestation, but the UGTIA subfamily was only expressed in first trimester placentas. The localisation of XME (the syncytiotrophoblast layer bordering the placental villi) did not change with gestation but enzyme activity and affinity did. Greater activify but lower affinity of UGT and CYPIA were observed in first trimester placentas than at term. In contrast, β-glucuronidase had a high affinity, low activity profile in early gestation but the opposite at term. UGT and CYPIA activities in the first trimester placenta were induced by maternal cigarette smoking and may be synergistically induced by combined alcohol consumption and smoking. A significant correlation with higher UGT activity and earlier gestational age was observed. CYPIA exhibited a significant, negative correlation with maternal age across gestation. Maternal variables had no effect on β-glucuronidase and their effects on the activity of CYP2EI and CYP reductase were not established. AZT caused apoptosis in the placenta and also increased reactive oxygen species and altered XME. The absence of serum enhanced these effects. Alterations in XME expression and activities included a decrease in UGT activity and increases in CYPIA, β-glucuronidase, CYP reductase and glutathione-Ѕ-transferase activity in response to AZT exposure. AZT transport in a perfusion model was bi-directional and reached equilibrium approximately 3 h after addition of AZT into the maternal reservoir. In contrast, transfer of AZT glucuronide (AZT-G) showed significantly greater transport rates out of the fetal compartment resulting in AZT-G concentrations approximately 2-fold higher in the maternal circuit. Transfer of the co-factor UDPGA was significantly greater in the fetal direction and almost complete after 4h of perfusion. The low levels of glucuronidation of AZT catalysed by the human placenta (approximately 2% of a dose) are not due to insufficient transport of the co-factor UDPGA and are unlikely to be significant in terms of maternal whole-body clearance. Therapeutic failure of AZT in protecting the fetus is unlikely to be due to metabolism and clearance performed by the placenta but may be due to placental cytotoxicity. The development of the perfusion model into the first trimester placenta is technically achievable, but was impossible with the tissue available for these studies due to the method used to obtain placentas.