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. 2009 Jul;110(1):47-60.
doi: 10.1093/toxsci/kfp094. Epub 2009 Apr 30.

Compensatory induction of liver efflux transporters in response to ANIT-induced liver injury is impaired in FXR-null mice

Yue J Cui  1 , Lauren M Aleksunes, Yuji Tanaka, Michael J Goedken, Curtis D Klaassen
Affiliations

Compensatory induction of liver efflux transporters in response to ANIT-induced liver injury is impaired in FXR-null mice

Yue J Cui et al. Toxicol Sci. 2009 Jul.

Abstract

Alpha-naphthyl isothiocyanate (ANIT) is a hepatotoxicant that produces acute intrahepatic cholestasis in rodents. Farnesoid X receptor (FXR) and pregnane X receptor (PXR) are two major bile acid sensors in liver. The purpose of this study was to characterize the regulation of hepatic transporters by FXR and PXR during ANIT-induced liver injury. Wild-type, FXR-null, and PXR-null mice were administered ANIT (75 mg/kg, po) and evaluated 48 h later for hepatotoxicity and messenger RNA (mRNA) expression of basolateral uptake (sodium taurocholate-cotransporting polypeptide, organic anion transporting polypeptide [Oatp] 1a1, Oatp1a4, Oatp1b2) and efflux transporters (organic solute transporter [Ost] alpha, Ostbeta, multidrug resistance-associated protein [Mrp] 3, Mrp4), as well as canalicular transporters (bile salt export pump [Bsep], Mrp2, multidrug resistance protein 2 [Mdr2], ATPase, class I, type 8B, member 1 [Atp8b1]). Livers from wild-type and PXR-null mice had comparable multifocal necrosis 48 h after ANIT. However, ANIT-treated FXR-null mice have fewer and smaller necrotic foci than wild-type mice but had scattered single-cell hepatocyte necrosis throughout the liver. Serum alanine transaminase, alkaline phosphatase (ALP), and direct bilirubin were increased in all genotypes, with higher ALP levels in FXR-null mice. Serum and liver unconjugated bile acids were higher in ANIT-treated FXR-null mice than the other two genotypes. ANIT induced mRNA expression of Mdr2, Bsep, and Atp8b1 in wild-type and PXR-null mice but failed to upregulate these genes in FXR-null mice. mRNA expression of uptake transporters declined in livers of all genotypes following ANIT treatment. ANIT increased Ostbeta and Mrp3 mRNA in livers of wild-type and PXR-null mice but did not alter Ostbeta mRNA in FXR-null mice. In conclusion, FXR deficiency enhances susceptibility of mice to ANIT-induced liver injury, likely a result of impaired induction of hepatobiliary efflux transporters and subsequent hepatic accumulation of unconjugated bile acids.

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Figures

FIG. 1.
FIG. 1.
Levels of serum and liver biomarkers 24 and 48 h after ANIT administration. Serum ALT (A) and ALP (B) are expressed as mean IU/l ± SEM. Serum direct bilirubin (C) is expressed as mean mg/dl ± SEM. Serum total bile acids (D) are expressed as μg/ml ± SEM. Liver total bile acids (E) are expressed as μg/g ± SEM; “*” Represents significant differences (p < 0.05) compared to control mice (n = 5 animals).
FIG. 2.
FIG. 2.
Time-dependent regulation of liver transporters in wild-type mice at 24 and 48 h after ANIT treatment (A, basolateral uptake; B, basolateral efflux; C, canalicular efflux). (D) Activation of FXR- and PXR-dependent pathways in wild-type livers 48 h after ANIT treatment. SHP and Cyp7a1, classic target genes for FXR; Cyp3a11, PXR target gene. Total RNA was isolated from control and ANIT-treated mouse livers and analyzed by the bDNA assay as described in the “Materials and Methods” section. Data are presented as mean relative light units ± SEM (n = 5 animals); “*” Represents significant differences (p < 0.05) compared with control.
FIG. 3.
FIG. 3.
(A) Serum alanine aminotransferase (ALT), ALP, and direct bilirubin 48 h after ANIT administration to wild-type, FXR-null, and PXR-null mice. Serum ALT and ALP are expressed as mean IU/l ± SEM. Serum direct bilirubin is expressed as mean mg/dl ± SEM. “*” Represents significant differences (p < 0.05) compared with vehicle-treated mice. “#” Represents significant differences (p < 0.05) compared with ANIT-treated wild-type mice (n = 5 animals). (B–F) Histopathological analysis of livers from wild-type, FXR-null, and PXR-null mice 48 h after ANIT administration. (B) Mild, multifocal periportal necrosis in wild-type liver (×10); (C) similar periportal necrosis in PXR-null liver (×10); (D) minimal multifocal periportal necrosis and minimal to mild single-cell necrosis in FXR-null liver (×10); (E) FXR-null remaining parenchyma (×10); (F) higher magnification of ANIT-treated FXR-null liver highlighting single-cell necrosis (×20); (G) pale vesicular cytoplasm of periportal hepatocytes (×40). Dashed arrow, focal necrosis; solid arrow, single-cell hepatocellular necrosis.
FIG. 4.
FIG. 4.
Serum and liver unconjugated bile acids in control and ANIT-treated wild-type, FXR-null, and PXR-null mice. Bile acids were quantified by UPLC-MS/MS as described in the “Materials and Methods” section. (A) Serum unconjugated bile acids are expressed as μg/ml ± SEM. (B) Liver unconjugated bile acids are expressed as μg/g ± SEM. “*” Represents significant differences (p < 0.05) compared to control mice (n = 5 animals). “#” Represents significant differences (p < 0.05) compared with ANIT-treated wild-type mice.
FIG. 5.
FIG. 5.
Expression of basolateral transporters Ntcp, Oatp1a1, and Oatp1b2 (uptake), as well as Ostβ and Mrp3 (efflux) in wild-type, FXR-null, and PXR-null mice 48 h after ANIT administration. Total RNA was isolated from control and ANIT-treated mouse livers and analyzed by the bDNA assay as described in the “Materials and Methods” section. Data are presented as mean relative light units ± SEM (n = 5 animals). “*” Represents significant differences (p < 0.05) compared with genotype-matched control mice. “#” Represents significant differences (p < 0.05) compared with ANIT-treated wild-type mice.
FIG. 6.
FIG. 6.
Expression of canalicular efflux transporters Mdr2, Bsep, Mrp2, and Atp8b1 in wild-type, FXR-null, and PXR-null mice 48 h after ANIT administration. Total RNA was isolated from control and ANIT-treated mouse livers and analyzed by the bDNA assay as described in the “Materials and Methods” section. Data are presented as mean relative light units ± SEM (each group, n = 5 animals). “*” Represents statistically significant differences (p < 0.05) compared with control. “#” Represents significant differences (p < 0.05) compared with ANIT-treated wild-type mice.
FIG. 7.
FIG. 7.
Immunofluorescence of Mrp2 protein in livers from wild-type, FXR-null, and PXR-null mice 48 h after ANIT. Immunofluorescence against canalicular Mrp2 (green) was conducted on liver cryosections as described in the “Materials and Methods” section. Portions of images were enlarged and provided as inserts. Representative images are shown. Bar, 50 μm.
FIG. 8.
FIG. 8.
Immunofluorescence of Bsep protein in livers from wild-type, FXR-null, and PXR-null mice 48 h after ANIT. Immunofluorescence against canalicular Bsep (green) was conducted on liver cryosections as described in “Materials and Methods” section. Portions of images were enlarged and provided as inserts. Representative images are shown. Bar, 50 μm.
FIG. 9.
FIG. 9.
Pharmacological activation of FXR pathway by GW4064 induces the prototypical FXR target gene SHP mRNA expression (A) and protects liver from ANIT toxicity in wild-type mice (B–E). “*” Represents statistically significant differences (p < 0.05) compared with control; (B) (vehicle control) and (C) (GW4064 treated) have no lesions; (D) (ANIT treatment only) has mild necrosis, while (E) (ANIT and GW4064) had minimal necrosis. Magnification: ×10.
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References

    1. Aleksunes LM, Slitt AM, Cherrington NJ, Thibodeau MS, Klaassen CD, Manautou JE. Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride. Toxicol. Sci. 2005;83(1):44–52. - PubMed
    1. Alnouti Y, Csanaky IL, Klaassen CD. Quantitative-profiling of bile acids and their conjugates in mouse liver, bile, plasma, and urine using LC-MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008;873:209–217. - PMC - PubMed
    1. Alvarez L, Jara P, Sanchez-Sabate E, Hierro L, Larrauri J, Diaz MC, Camarena C, De la Vega A, Frauca E, Lopez-Collazo E, et al. Reduced hepatic expression of farnesoid X receptor in hereditary cholestasis associated to mutation in ATP8B1. Hum. Mol. Genet. 2004;13(20):2451–2460. - PubMed
    1. Ballatori N, Christian WV, Lee JY, Dawson PA, Soroka CJ, Boyer JL, Madejczyk MS, Li N. OSTalpha-OSTbeta: A major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia. Hepatology. 2005;42(6):1270–1279. - PubMed
    1. Burdelski M, Rogiers X. Liver transplantation in metabolic disorders. Acta Gastroenterol. Belg. 1999;62(3):300–305. - PubMed

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