Pregnancy-Related Hormones Increase UGT1A1-Mediated Labetalol Metabolism in Human Hepatocytes

Abstract

Pregnancy-related hormones (PRH) are recognized as important regulators of hepatic cytochrome P450 enzyme expression and function. However, the impact of PRH on the hepatic expression and function of uridine diphosphate glucuronosyltransferases (UGTs) remains unclear. Using primary human hepatocytes, we evaluated the effect of PRH exposure on mRNA levels and protein concentrations of UGT1A1, UGT2B7, and other key UGT enzymes, and on the metabolism of labetalol (a UGT1A1 and UGT2B7 substrate commonly prescribed to treat hypertensive disorders of pregnancy). Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to the PRH estradiol, estriol, estetrol, progesterone, and cortisol individually or in combination. We quantified protein concentrations of UGT1A1, UGT2B7, and four additional UGT1A isoforms in SCHH membrane fractions and evaluated the metabolism of labetalol to its glucuronide metabolites in SCHH. PRH exposure increased mRNA levels and protein concentrations of UGT1A1 and UGT1A4 in SCHH. PRH exposure also significantly increased labetalol metabolism to its UGT1A1-derived glucuronide metabolite in a concentration-dependent manner, which positively correlated with PRH-induced changes in UGT1A1 protein concentrations. In contrast, PRH did not alter UGT2B7 mRNA levels or protein concentrations in SCHH, and formation of the UGT2B7-derived labetalol glucuronide metabolite was decreased following PRH exposure. Our findings demonstrate that PRH alter expression and function of UGT proteins in an isoform-specific manner and increase UGT1A1-mediated labetalol metabolism in human hepatocytes by inducing UGT1A1 protein concentrations. These results provide mechanistic insight into the increases in labetalol clearance observed in pregnant individuals.

Keywords: estradiol; hepatic metabolism; hypertension; labetalol; pregnancy; progesterone; targeted proteomics; uridine diphosphate glucuronosyltransferases.

FIGURE 1

Effect of pregnancy-related hormones (PRH) on mRNA levels of key UGT isoforms in SCHH. Hepatocytes from four donors (HU1880, HC3-26, HU8284, and HC5-40) were exposed to E2, E3, E4, P4, and CRT, individually or in combination as a PRH cocktail [CKTL], or controls (DMSO, CITCO, Rifampin [RIF]) for 72 h (n = 2 per group within each hepatocyte donor). (A) UGT1A1, (B) UGT1A4, and (C) UGT2B7 mRNA levels were normalized to GAPDH and expressed relative to the vehicle control group (DMSO) within each donor. The data were combined for comparison across experimental groups (n = 4 donors per group; mean ± SEM; *p < 0.05 vs. DMSO). Concentration-dependent effects were assessed (open bar: 1 μM, solid bar: 10 μM; ^p < 0.05 1 vs. 10 µM).

FIGURE 2

Effect of pregnancy-related hormones (PRH) on protein concentrations of UGT1A1 and UGT2B7 in SCHH. Following 72 h of hormone exposure, UGT1A1 and UGT2B7 protein concentrations were quantified by quantitative targeted absolute proteomics in SCHH membrane-associated protein fractions isolated from three donors (HC3-26, HU1880, and HU8284). (A) UGT1A1 and (B) UGT2B7 absolute protein concentrations in the DMSO and PRH cocktail (CKTL) groups were compared separately in donor HC3-26 (mean: n = 2 per group) and donors HU1880 and HU8284 (mean ± SEM: n = 3–4 per group; *p < 0.05 vs. DMSO, ^p < 0.05 1 vs. 10 µM). (C) UGT1A1 and (D) UGT2B7 protein concentrations were expressed relative to vehicle control (DMSO) within each hepatocyte donor, and then combined for comparison across groups. Open bars represent 1 µM CKTL (mean ± SEM: n = 3 donors per group) and 1 µM for the individual hormones (mean: n = 2 donors per group). Solid bars represent 10 µM CKTL and 10 µM for the individual PRH (mean ± SEM: n = 3 donors per group). *p < 0.05 vs. DMSO, ^p < 0.05 1 vs. 10 µM. Comparison of individual PRH effects within each hepatocyte donor are provided in Supplementary Figure S2.

FIGURE 3

UGT1A1 and UGT2B7-mediated glucuronidation of labetalol. (A) Overview of labetalol glucuronidation to its phenolic-OH (Gluc-1), benzylic-OH (Gluc-2), and N-glucuronide (Gluc-3) metabolites. Representative chromatograms of labetalol glucuronide (Gluc-1, Gluc-2, and Gluc-3) formation by human recombinant (B) UGT1A1 and (C) UGT2B7. Relative formation of (D) Gluc-1 and (E) Gluc-2 by human recombinant UGT1A1 and UGT2B7. Data are expressed as a percentage of Gluc-1 formed by UGT1A1 and Gluc-2 formed by UGT2B7, respectively (n = 3/group; mean ± SEM).

FIGURE 4

Effect of pregnancy-related hormones (PRH) on labetalol glucuronide (Gluc-1) formation in SCHH. Following 72 h of PRH exposure, SCHH from two donors (HC3-26, HU1880) were incubated with labetalol (1 mM) for 4 h. Labetalol glucuronide (Gluc-1) peak area was measured in both SCHH cell lysates (A, C) and SCHH media (B, D), normalized to internal standard (labetalol-d₃) peak area in each sample, and compared across treatment groups in donor HC3-26 (A: cell lysate, n = 3 per group; B: media, n = 3–6 per group) and donor HU1880 (C: cell lysate, n = 4 per group; D: media, n = 4 per group). In the donor HC3-26 experiment, cell lysates were not harvested for glucuronide measurements in the 1 µM E2, P4, or CRT groups. Data are calculated as the fold-change in Gluc-1 metabolite formation relative to vehicle control (*p < 0.05 vs. DMSO). Concentration-dependent effects were assessed (open bar: 1 μM, solid bar: 10 μM; ^p < 0.05 1 vs. 10 µM). In the donor HC3-26 experiments, itraconazole co-administration (+ITZ, 5 μM) was included in the DMSO-, PRH cocktail (CKTL)-, and rifampin (RIF)-treated groups (n = 1 per group in cell lysates; n = 2 per group in media) to confirm UGT1A1-mediated effects. N.D., experimental group not studied. The correlation between UGT1A1 protein levels and labetalol Gluc-1 formation in (E) SCHH cell lysates and (F) SCHH media in both donors is presented. Each data point represents the mean fold-change value for the various treatment groups, relative to DMSO, within each hepatocyte donor. The Pearson correlation coefficient (r) and p-value are provided.

FIGURE 5

Effect of pregnancy-related hormones (PRH) on labetalol glucuronide (Gluc-2) formation in SCHH. Following 72 h of PRH exposure, SCHH from two donors (HC3-26, HU1880) were incubated with labetalol (1 mM) for 4 h. Labetalol glucuronide (Gluc-2) peak area was measured in both SCHH cell lysates (A, C) and SCHH media (B, D), normalized to internal standard (labetalol-d₃) peak area in each sample, and compared across treatment groups in donor HC3-26 (A: cell lysate, n = 3 per group; B: media, n = 3–6 per group) and donor HU1880 (C: cell lysate, n = 4 per group; D: media, n = 4 per group). In the donor HC3-26 experiment, cell lysates were not harvested for glucuronide measurements in the 1 µM E2, P4, or CRT groups. Data are calculated as the fold-change in Gluc-2 metabolite formation relative to vehicle control (*p < 0.05 vs. DMSO). Concentration-dependent effects were assessed (open bar: 1 μM, solid bar: 10 μM; ^p < 0.05 1 vs. 10 µM). N.D., experimental group not studied.

FIGURE 6

Effect of pregnancy-related hormones (PRH) on protein concentrations of UGT1A4 and other key UGT1A isoforms in SCHH. Following 72 h of PRH exposure, UGT1A4 protein concentrations were quantified by quantitative targeted absolute proteomics in SCHH membrane-associated protein fractions isolated from three donors (HC3-26, HU1880, and HU8284). (A) UGT1A4 absolute protein concentrations in the DMSO and PRH cocktail (CKTL) groups were compared separately in donor HC3-26 (mean: n = 2 per group) and donors HU1880 and HU8284 (mean ± SEM: n = 3–4 per group; *p < 0.05 vs. DMSO, ^p < 0.05 1 vs. 10 µM). Comparisons of PRH CKTL effects on UGT1A3, UGT1A6, and UGT1A9 concentrations within each hepatocyte donor are provided in Supplementary Figure S3. (B) UGT1A4, (C) UGT1A3, (D) UGT1A6, and (E) UGT1A9 protein concentrations were expressed relative to vehicle control (DMSO) within each hepatocyte donor, and then combined for comparison across groups. Open bars represent 1 µM CKTL (mean ± SEM: n = 3 donors per group) and 1 µM for the individual hormones (mean: n = 2 donors per group). Solid bars represent 10 µM CKTL and 10 µM for the individual hormones (mean ± SEM: n = 3 donors per group). *p < 0.05 vs. DMSO, ^p < 0.05 1 vs. 10 µM. Comparison of individual PRH effects on UGT1A4 levels within each hepatocyte donor is provided in Supplementary Figure S2C.