Protective effect of heme oxygenase induction in ethinylestradiol-induced cholestasis

Abstract

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis.

Keywords: 17α- ethinylestradiol; bile acids; heme; multidrug resistance-associated protein 3; nuclear factor erythroid-2-related factor-2.

Figure 1

Total heme oxygenase (HMOX) activity in primary rat hepatocytes. Primary hepatocytes were incubated with: (A) taurocholic acid (TCA), (B) unconjugated bilirubin (UCB) for 24 hrs or with (C) 10 μM ethinylestradiol (EE) for 24, 48 and 72 hrs. (D) 10 μM EE was added to media 24 hrs before TCA treatment (100 μM, another 24 hrs). *P ≤ 0.05 versus CON, ^†P ≤ 0.05 versus TCA. Hepatocytes treated with heme served as positive controls.

Figure 2

mRNA expression of key hepatic transporters. Relative expression of key sinusoidal (Ntcp, Oatps, Mrp3 and Mrp4) and canalicular (Mrp2, Bsep) bile acid (BA) transporters was measured in the livers of control (CON), ethinylestradiol (EE), heme (HC) or heme + EE (HE)-treated animals. *P ≤ 0.05 versus CON, ^†P ≤ 0.05 versus EE.

Figure 3

Mrp3 expression in primary rat hepatocytes. Relative expression of Mrp3 transporter mRNA was measured in primary hepatocytes with (Nrf−) or without (Nrf+) Nrf2 silencing. Cells were treated with 50 μM taurocholic acid (TCA), 30 μM heme or both for 4 hrs. *P ≤ 0.05 versus CON, ^†P ≤ 0.05 versus corresponding Nrf+ group.

Figure 4

Urinary bile acid (BA) output and mRNA expression of renal BA transporters. (A) BA concentration was measured in the urine collected for 30 min. from control (CON), ethinylestradiol (EE), heme (HC), or heme + EE (HE)-treated animals and expressed as relative changes from CON. (B) Relative expression of key renal BA transporters was measured in kidneys of CON, EE, HC, or HE-treated animals. *P ≤ 0.05 versus CON.

Figure 5

Proposed mechanism of heme oxygenase-1 (HMOX1) induction on liver and kidney transporters in EE-induced cholestasis. EE-induced cholestasis is characterized by either decrease (red ovals) or increase (green ovals) in the expression of key hepatocyte and kidney transporters. Induction of HMOX1 with heme increased (green arrows), brought close to CON values (dashed green arrows) or decreased (red arrows) expression of these transporters. The changes in the expression of liver and kidney transporters result in the redistribution of bile acid pool in cholestatic (EE) and heme pre-treated (HE) animals.