The antiandrogen flutamide is a novel aryl hydrocarbon receptor ligand that disrupts bile acid homeostasis in mice through induction of Abcc4

Abstract

Flutamide (FLU), an oral, nonsteroidal antiandrogen drug used in the treatment of prostate cancer, is associated with idiosyncratic hepatotoxicity that sometimes causes severe liver damage, including cholestasis, jaundice, and liver necrosis. To understand the mechanism of toxicity, a combination of aryl hydrocarbon receptor (Ahr)-deficient (Ahr^-/-) mice, primary hepatocytes, luciferase reporter gene assays, in silico ligand docking and ultra-performance chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics was used. A significant increase of liver weights, and liver and serum bile acid levels was observed after FLU treatment, indicating hepatomegaly and disrupted bile acid homeostasis. Expression of the AhR gene battery was markedly increased in livers of wild-type mice Ahr^+/+ treated with FLU, while no change was noted in Ahr^-/- mice. Messenger RNAs encoded by AhR target genes were induced in primary mouse hepatocytes cultured with FLU, which confirmed the in vivo results. Ligand-docking analysis further predicted that FLU is an AhR agonist ligand which was confirmed by luciferase reporter gene assays. Multivariate data analysis showed that bile acids were responsible for the separation of vehicle- and FLU-treated Ahr^+/+ mice, while there was no separation in Ahr^-/- mice. Expression of mRNA encoding the bile acid transporter ABCC4 was increased and farnesoid X receptor signaling was inhibited in the livers of Ahr^+/+ mice, but not in Ahr^-/- mice treated with FLU, in agreement with the observed downstream metabolic alterations. These findings provide new insights into the mechanism of liver injury caused by FLU treatment involving activation of AhR and the alterations of bile acid homeostasis, which could guide clinical application.

Keywords: ABCC4; Aryl hydrocarbon receptor; Bile acid homeostasis; Flutamide; Metabolomics.

Fig. 1

Xenobiotic response in mice after treatment with vehicle and FLU (200 mg/kg) for 28 days. (A) Light microscopic examination of H&E-stained liver sections. (B) Total bile acids in serum. (C) Total bile acids in liver. (D) Liver weight. (E) Liver weight /body weight ratio. (F) AhR battery gene expression in liver. (G) PXR battery gene expression in liver. (H) CAR battery gene expression in liver. Data are presented as mean ± SEM; n = 6/group. *P < 0.05, and ** P <0.01 versus vehicle group, by two-tailed Student’s t-test test.

Fig. 2

Time course of the response in mice after treatment with vehicle (coin oil) and FLU (200 mg/kg). (A) Changes in liver weights. (B) Changes in liver weight/body weight ratios. (C-G) Ahr (C) and battery genes, Cyp1a1 (D), Cyp1a2 (E), Nqo1 (F), and Gsta1 (G) mRNA expression. Data are presented as mean ± SEM; n = 6/group. *P < 0.05, and **P < 0.01 versus vehicle group, by two-tailed Student’s t-test test.

Fig. 3

FLU stimulates mice AhR battery gene transcription. (A) qPCR analysis for AhR battery gene mRNA expression in mouse primary hepatocytes after FLU exposure. Significance was determined by two-tailed Student’s t-test test (*P < 0.05, **P < 0.01, versus vehicle group). (B and C) Luciferase assays for AhR activation in HepG2 (B) and Hepa-1c1c7 (C) cells. ***P < 0.001, compared with that of AhR-Luci+DMSO, by two-tailed Student’s t-test test. (D and E) Docking orientation of FLU into mouse AhR-LBD (PDB id: 4GHI) (D) and AhR dimer (PDB id: 4EQ1) binding pocket (ICM v3.5-1n, Molsoft) (E). The protein backbone is displayed as ribbon and colored by secondary structure. The residues are displayed as sticks and colored by atom type, with the carbon atoms in green. The ligands are displayed as sticks, colored by atom type, with carbon atoms in yellow or white, oxygen atom in red, fluorine atom in cyan, and nitrogen atom in blue.

Fig. 4

FLU alleviates hepatomegaly in Ahr^-/- mice after treatment with vehicle or FLU (200 mg/kg) for 28 days. (A) Light microscopic examination of H&E-stained liver sections from mice. (B) Liver weights. (C) Liver/body weight ratios. (D) qPCR analysis for AhR battery genes mRNA expression in liver. (E) Total bile acids in serum. (F) Total bile acids in liver. (G) CAR battery gene expression in liver. Data are presented as mean ± SEM; n = 6/group. N.S., not significant. *P < 0.05, or **P < 0.01, by one-way ANOVA test.

Fig. 5

FLU alleviates hepatomegaly in Ahr^-/- mice after treatment with vehicle and flutamide (200 mg/kg) for 3 days. A Liver weights. B Liver/body weight ratios. C qPCR analysis for AhR battery genes mRNA expression in liver. D CAR battery gene expression in liver. Data are presented as mean ± SEM; n = 6/group. N.S., not significant. *P < 0.05, or **P < 0.01, by one-way ANOVA test.

Fig. 6

Multivariate data analysisand metabolite identification in Ahr^+/+ and Ahr^-/- mice by using UPLC ESI QTOFMS analysis. Ahr^+/+ and Ahr^-/- mice were treated with vehicle or FLU (200mg/kg) for 28 days. (A) Scores plot of serum metabolome in WT mice treated with vehicle ( ) and FLU ( ) as determined by PCA. (B) Scores plot of serum metabolome in Ahr^-/- mice dosed with vehicle ( ) and FLU ( ) as determined by PCA. (C) S-plot of OPLS-DA recognized serum metabolome in vehicle- and FLU-treated Ahr^+/+ mice, in which identified metabolites were indicated. (D) S-plot of OPLS-DA recognized serum metabolome in vehicle- and FLU-treated Ahr^-/- mice. Each point represents an individual mouse serum sample (A, B) and a unique ion (C, D). The t[1] and t[2] represent principal components 1 and 2, respectively. The p(corr)[1] represents the interclass difference, and p[1] represents the relative abundance of the ions. (E-H) Quantitation of bile acids in Ahr^+/+ and Ahr^-/- mice. (E) Individual bile acids in serum of Ahr^+/+ mice. (F) Individual bile acids in serum of Ahr^-/- mice. (G) Individual bile acids in livers of Ahr^+/+ mice. (H) Individual bile acids in livers of Ahr^-/- mice. Data are presented as mean ± SEM; n = 6/group. *P < 0.05, or **P < 0.01, versus vehicle group, by two-tailed Student’s t-test.

Fig. 7

TCA/TβMCA ratios in the serum and livers of mice treated with vehicle or FLU (200 mg/kg) for 28 days. Data are presented as mean ± SEM; n = 6/group. *P < 0.05 versus vehicle group, by two-tailed Student’s t-test.

Fig. 8

qPCR analysis for FXR and relative target gene expression mRNAs after FLU exposure. (A) qPCR analysis for Fxr and Shp mRNA expression in the livers of Ahr^+/+ mice treated with FLU for 28 days. (B) qPCR analysis for bile acid synthesis mRNAs in the livers of Ahr^+/+ mice treated with FLU for 28 days. (C) qPCR analysis for bile acid transporter mRNAs in the livers of Ahr^+/+ mice treated with FLU for 28 days. (D) qPCR analysis for Fxr and Shp mRNA expression in the livers of Ahr^-/- mice treated with FLU for 28 days. (E) qPCR analysis for bile acid synthesis mRNAs in the livers of Ahr^-/- mice treated with FLU for 28 days. (F) qPCR analysis for bile acid transporter mRNAs in the livers of Ahr^-/- mice treated with FLU for 28 days. (G) qPCR analysis for Fxr and Shp mRNA expression in the livers of Ahr^+/+ mice treated with FLU for 3 days. (H) qPCR analysis for bile acid synthesis mRNAs in the livers of Ahr^+/+ mice treated with FLU for 3 days. (I) qPCR analysis for bile acid transporter mRNAs in the livers of Ahr^+/+ mice treated with FLU for 3 days. Data are presented as mean ± SEM; n = 6/group. *P < 0.05, or **P < 0.01, versus vehicle group, by two-tailed Student’s t-test.