Semester

Spring

Date of Graduation

2003

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Timothy S. Tracy.

Abstract

The placenta plays an important role in modulating xenobiotic passage from mother to fetus. Smoking is known to induce cytochrome P450 1Al (CYP1A1) expression in the placenta, however its effect on P-glycoprotein (P-gp) and ABC transporter in placenta (ABCP) levels in placenta is not known. In-vitro studies in hepatocytes have reported induction of P-gp mRNA by some polycyclic aromatic hydrocarbons. To examine whether smoking can affect placental P-gp and ABCP, their expression and activity were determined in placentas from smokers and non-smokers. Uptake of [3H]-vinblastine and [3H]-mitoxantrone was used to measure P-gp and ABCP function, respectively and CYP1A1 activity (positive control) was assessed using ethoxyresorufin O-deethylation as the model reaction. P-gp and ABCP expression was measured by immunoblotting. Uptake of [3H]-vinblastine in vesicles was osmotically sensitive, suggesting intravesicular accumulation and was inhibited by verapamil, a P-gp inhibitor. Uptake of [3H]-mitoxantrone was inhibited by fumitremorgin C, an ABCP inhibitor but not by verapamil. Though CYP1A1 activity was significantly higher in smokers, no statistical difference (P > 0.05) was noted in P-gp and ABCP function or expression between smokers and non-smokers suggesting that smoking has no effect on placental P-gp and ABCP activity or expression.;In order to study the induction of P-gp, we used LS174T cells to screen for compounds for their ability to induce P-gp. As CYP3A4 and P-gp are coordinately upregulated, CYP3A activity was also assessed as the formation of 1 '-hydroxy midazolam. Uptake of VBL was used to measure P-gp function and expression was measured by immunochemical methods. CYP3A activity was 4 times higher and P-gp activity was significantly higher after rifampin treatment. Rifampin treatment also produced an increase in protein abundance and surface expression of P-gp. To assess involvement of the Pregnane X Receptor (PXR) in P-gp induction, a set of multiple compounds with varying degrees of PXR activation were evaluated for effect on P-gp expression. PXR activation and P-gp expression were positively correlated, supporting the role of PXR in regulation of P-gp. These data suggest that LS174T cells have the potential to be used in studying the effect of inducers on CYP3A and P-gp simultaneously.

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