Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination

Abstract

The authors recently reported the increased oral clearance of labetalol in pregnant women. To elucidate the mechanism of the elevated oral clearance, it was hypothesized that female hormones, at the high concentrations attainable during pregnancy, enhance hepatic metabolism of labetalol. Labetalol glucuronidation, which is the major elimination pathway of labetalol, was characterized by screening six recombinant human UGTs (UGT1A1, 1A4, 1A6, 1A9, 2B4, and 2B7) for their capacity to catalyse labetalol glucuronidation. The effect of female hormones (progesterone, oestradiol, oestriol, or oestrone) on the promoter activities of relevant UDP glucuronosyltransferases (UGT) was investigated using a luciferase reporter assay in HepG2 cells. The involvement of oestrogen receptor alpha (ERalpha) and pregnane X receptor (PXR) was examined by co-transfecting ERalpha- or PXR-constructs. UGT1A1 and UGT2B7 were identified as the major UGT enzymes producing labetalol glucuronides (trace amount of glucuronide conjugate was formed by UGT1A9). The activities of the UGT1A1 promoter containing PXR response elements were enhanced by progesterone, but not by oestrogens, indicating PXR-mediated induction of UGT1A1 promoter activity by progesterone. Results from semi-quantitative real-time polymerase chain reaction (PCR) assays are consistent with the above findings. This effect of progesterone on UGT1A1 promoter activities was concentration dependent. Promoter activities of UGT2B7 were not affected by either oestrogens or progesterone. The results suggest a potential role for progesterone in regulating labetalol elimination by modulating the expression of UGT1A1, leading to enhanced drug metabolism during pregnancy.

Figure 1

Mass spectrum of MS/MS product scan of labetalol glucuronide (m/z = 505) produced by UGT1A1 (A) and UGT2B7 (B) (see text for details). The glucuronide conjugates produced by UGT1A1 and UGT2B7 enzymes eluted at 5.0 and 7.6 min, respectively. Collision energy of 20 V was used to produce the product ions.

Figure 2

Production rates of labetalol glucuronide metabolites over various labetalol concentrations, expressed as % of the largest glucuronide peak area, for UGT1A1 (A), UGT2B7 (B), UGT1A9 (C; inlet figure showing the data range), human microsome (D for late- and E for early-eluting glucuronide peak). Recombinant UGT microsome was incubated in the presence of UDPGA in duplicates. Lines fitted for Mechaelis-Menten equations were shown.

Figure 3

Effect of female hormones on the promoter activity of various 5′-flanking fragments of UGT1A1 promoter in HepG2 cells with exogenously expressed ERα or PXR. Luciferase constructs containing −5.2 to −3.1 (U2K), −3.1 to −2.0 (UB), or −2.0 kbp (UA) regions of UGT1A1 promoter were cotransfected into HepG2 cells along with β-gal and the expression vector (empty pcDNA3, ERα or PXR). The ERα-expressed cells were treated with the vehicle (ethanol) alone or various drugs including estrogen (1 μM) or rifampin (10 μM) for 24 h, and PXR-expressed cells were treated with progesterone (10 μM) additionally. The luciferase activity was determined as described under Materials and Methods. Relative luciferase activity is expressed as ratios relative to vehicle (ethanol) control. Data presented are the mean of a triplicate experiment ± S.D. *: p<0.05 compared to vehicle-treated group.

Figure 4

Effect of various concentrations of progesterone on the promoter activity of U2K, the UGT1A1 promoter region containing PXR response element. U2K, the luciferase construct containing −5.2 to −3.1 kbp region of UGT1A1 promoter, was cotransfected into HepG2 cells with β-gal, and the expression vector for PXR (filled circle) or pcDNA3 empty vector (empty circle). The cells were treated with progesterone (0.5, 1, 5, 10, 50, 100, or 500 μM) or vehicle control. The luciferase activity was determined as described under Materials and Methods. Relative luciferase activity is expressed as ratios relative to vehicle (ethanol) control. Data presented are the mean of a triplicate experiment ± S.D.

Figure 5

Effect of female hormones on the promoter activity of UGT2B7 promoter in HepG2 cells with exogenously expressed ER or PXR. Luciferase constructs containing −2.0 kbp region of UGT2B7 promoter were cotransfected into HepG2 cells with β-gal and the expression vector for ERα or PXR. The ERα-expressed cells were treated with the vehicle (ethanol) alone or drugs such as estrogen (1 μM) or rifampin (10 μM) for 24 h. PXR-transfected cells were treated with progesterone (10 μM) additionally. The luciferase activity was determined as described under Materials and Methods. Relative luciferase activity is expressed as ratios relative to vehicle (ethanol) control. Data presented are the mean of a triplicate experiment ± S.D.

Figure 6

Effect of female hormones on the mRNA levels of UGT1A1 and UGT2B7. The pcDNA3-PXR or control vector (pcDNA3) was transfected into HepG2 cells, and the transfected cells were treated with 1 μM estradiol (E), 50 μM progesterone (P), 10 μM rifampin (R), or vehicle control (V, ethanol) for 72 h. The mRNA levels were semi-quantitatively determined by real-time PCR. Expression levels of UGT1A1 and UGT2B7 were normalized by those of β-actin. Relative expression levels were expressed as ratios relative to vehicle control. Data presented are the mean of a triplicate experiment ± S.D. *: p<0.05 compared to vehicle-treated group.