Allocholic acid protects against α-naphthylisothiocyanate-induced cholestasis in mice by ameliorating disordered bile acid homeostasis.

Cholestasis is a pathological condition characterized by disruptions in bile flow, leading to the accumulation of bile acids (BAs) in hepatocytes. Allocholic acid (ACA), a unique fetal BA known for its potent choleretic effects, reappears during liver regeneration and carcinogenesis. In this research, we investigated the protective effects and underlying mechanisms of ACA against mice with cholestasis brought on by α-naphthylisothiocyanate (ANIT). To achieve this, we combined network pharmacology, targeted BA metabolomics, and molecular biology approaches. The results demonstrated that ACA treatment effectively reduced levels of serum AST, ALP, and DBIL, and ameliorated the pathological injury caused by cholestasis. Network pharmacology analysis suggested that ACA primarily regulated BA and salt transport, along with the signaling pathway associated with bile secretion, to improve cholestasis. Subsequently, we examined changes in BA metabolism using UPLC-MS/MS. The findings indicated that ACA pretreatment induced alterations in the size, distribution, and composition of the liver BA pool. Specifically, it reduced the excessive accumulation of BAs, especially cholic acid (CA), taurocholic acid (TCA), and ß-muricholic acid (ß-MCA), facilitating the restoration of BA homeostasis. Furthermore, ACA pretreatment significantly downregulated the expression of hepatic BA synthase Cyp8b1, while enhancing the expression of hepatic efflux transporter Mrp4, as well as the renal efflux transporters Mdr1 and Mrp2. These changes collectively contributed to improved BA efflux from the liver and enhanced renal elimination of BAs. In conclusion, ACA demonstrated its potential to ameliorate ANIT-induced liver damage by inhibiting BA synthesis and promoting both BA efflux and renal elimination pathways, thus, restoring BA homeostasis.

Silymarin protects the liver from α-naphthylisothiocyanate-induced cholestasis by modulating the expression of genes involved in bile acid homeostasis.

Cholestasis is characterized by impaired bile flow which results in inflammation, cirrhosis, and ultimately liver failure. The current study is aimed to evaluate the anti-cholestatic effect of silymarin against α-naphthylisothiocyanate (ANIT) induced cholestasis. Mice were gavaged with various doses of silymarin or ursodeoxycholic acid (UDCA) for 19 days. Then they were challenged with α-naphthylisothiocyanate (ANIT) and after 48 hours the animals were sacrificed to obtain blood and liver sections. Serum levels of bilirubin, aspartate transaminase (AST), alanine transaminase (ALP), and liver histology were analyzed. mRNA expression of selected transporters (Bile salt export pump (BSEP) and sodium taurocholate cotransporting polypeptide (NTCP)) and proteins (farnesoid x receptor (FXR) and Cytochrome P450 Family 7 Subfamily A Member 1 (Cyp7a1)) involved in bile acids biosynthesis, excretion and uptake were also evaluated by quantitative PCR. The results indicated that the serum levels of bilirubin, AST, and ALP were significantly higher in a cholestatic model group as compared to an untreated control group. However, in silymarin groups, the serum level of these parameters is significantly lower than in a cholestatic model group. Liver histology also showed that silymarin prevents ANIT-induced hepatic injury. mRNA expression of FXR, BSEP, and NTCP was downregulated and expression of Cyp7a1 was upregulated in a cholestatic model group as compared to an untreated control group. However, in silymarin treatment groups, the expression of FXR, BSEP and NTCP was upregulated and the expression of Cyp7a1 was downregulated as compared to the cholestatic model group. In conclusion, silymarin could alleviate hepatic injury by modulating the expression of genes involved in bile acid homeostasis.

IL-25 ameliorates acute cholestatic liver injury via promoting hepatic bile acid secretion.

Cholestasis caused by bile secretion and excretion disorders is a serious manifestation of hepatopathy. Interleukin (IL)-25 is a member of the IL-17 cytokine family, which involves in mucosal immunity and type 2 immunity via its receptor-IL-17RB. Our previous studies have shown that IL-25 improves non-alcoholic fatty liver via stimulating M2 macrophage polarization and promotes development of hepatocellular carcinoma via alternative activation of macrophages. These hepatopathy are closely associated with cholestasis. However, whether IL-25 play an important role in cholestasis remains unclear. IL-25 treatment and IL-25 knockout (Il25-/-) mice were injected intragastrically with α-naphthyl isothiocyanate (ANIT) to determine the biological association between IL-25 and cholestasis. Here, we found that IL-25 and IL-17RB decreased in ANIT-induced cholestatic mice. Il25-/- mice showed exacerbated ANIT-induced parenchymal injury and IL-25 treatment significantly alleviated cholestatic liver injury induced by ANIT. We found that IL-25 reduced the level of hepatic total bile acids and increased the expression of multidrug resistance-associated protein 2 (MRP2) and multidrug resistance-associated protein 3 (MRP3) in liver. In conclusion, IL-25 exhibited a protective effect against ANIT-induced cholestatic liver injury in mice, which may be related to the regulation on bile acids secretion. These results provide a theoretical basis for the use of IL-25 in the treatment of cholestatic hepatopathy.

Combination of resveratrol and luteolin ameliorates α-naphthylisothiocyanate-induced cholestasis by regulating the bile acid homeostasis and suppressing oxidative stress.

Cholestasis is a common liver injury without any effective therapeutic drugs so far. Resveratrol (RES) and luteolin (LUT) are natural polyphenols that exert protective effects on multiple liver injuries. Coadministration of RES and LUT could significantly improve the bioavailability of LUT and increase the systemic exposure to RES, and the combined treatment could also benefit from their multi-component and multi-target characteristics. Our current aim is to study the protective effects of coadministration of RES and LUT on α-naphthylisothiocyanate (ANIT)-induced cholestasis. Serum biochemical indices and liver histopathology in rats indicated that coadministration of RES and LUT could improve liver function by suppressing oxidative stress. Dysregulated bile acid (BA) homeostasis is a significant pathological feature of cholestasis, which was determined to explore the potential biomarkers and to clarify the protection mechanism of coadministration of RES and LUT. The levels of cholic acid, chenodeoxycholic acid, taurine conjugates and glycine conjugates, and the ratios of taurine conjugates to their free forms could be used as diagnosis indicators for cholestasis in rats. Furthermore, the coadministration of RES and LUT could restore the BA levels and exert better protective effects than administration alone. This study suggested that the coadministration of RES and LUT could protect against ANIT-induced cholestasis and the mechanism was closely related to regulating BA homeostasis and suppressing oxidative stress.

Role of MRP2 and GSH in intrahepatic cycling of toxins.

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. alpha-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR(-) rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 micromol absolute i.v.) to Wistar and Mrp2-deficient TR(-) rats and collected bile. While both arsenite and GSH secretion were absent in TR(-) rats, the total secretion of arsenite into Wistar bile (2.91 micromol) was accompanied by a excess secretion of 24 micromol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR(-) rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.

1-naphthylisothiocyanate-induced elevation of biliary glutathione.

1-Naphthylisothiocyanate (ANIT) has been used for many years to study cholangiolitic hepatotoxicity in laboratory animals. Hallmarks of ANIT hepatotoxicity include portal edema and inflammation with bile duct epithelial and hepatic parenchymal cell necrosis. In rats, ANIT hepatotoxicity is dependent upon hepatic glutathione. Studies in vitro have demonstrated that ANIT combines reversibly with glutathione and suggest that intracellular formation and secretion of this glutathione-ANIT conjugate from hepatic parenchymal cells may be responsible for the efflux of glutathione observed upon exposure to ANIT. In vivo, glutathione conjugates produced within hepatic parenchymal cells are typically transported into bile for elimination. Therefore, large concentrations of ANIT in bile may result from hepatic parenchymal cell secretion of a reversible glutathione-ANIT conjugate. To investigate this hypothesis, bile and plasma concentrations of ANIT were determined in rats 1, 4, 8, 12 and 24 hr after administration (100 mg/kg, p.o.). Liver and bile glutathione concentrations were also evaluated. Plasma ANIT concentrations ranged between 2 and 5 microM at 1, 4, 8 and 12 hr and were 0.9 microM at 24 hr after administration. ANIT concentrations in bile at 1, 4, 8 and 12 hr were 60, 28, 21 and 22 microM, respectively. Thus, ANIT was concentrated in bile. Hepatic glutathione was not affected by ANIT during the first 12 hr after administration; however, a moderate elevation occurred by 24 hr. In contrast, a marked elevation in bile glutathione concentration (two times control) occurred 1, 4 and 8 hr after ANIT administration. Thus, the early accumulation of ANIT in bile was coincident with an elevation in bile glutathione. These findings support the hypothesis that glutathione functions to concentrate ANIT in bile. The large concentration of this toxicant in bile may be injurious to bile epithelium, a primary cellular target in ANIT hepatotoxicity.

Oxidative conversion of isothiocyanates to isocyanates by rat liver.

This report describes the oxidative metabolism of isothiocyanates to isocyanates catalyzed by rat liver microsomes. Incubation of 2-naphthylisothiocyanate, microsomes, and NADPH yielded either N,N'-di-naphthylurea or, on inclusion of 2-aminofluorene in the incubations, N-2-naphthyl-N'-2-fluorenylurea. These ureas were formed by the production of the known genotoxicant, 2-naphthylisocyanate, which reacted with its hydrolysis product, 2-aminonaphthalene, to yield the symmetrical urea, or with 2-aminofluorene to form the mixed urea. Formation of N,N'-di-2-naphthylthiourea was also observed because 2-aminonaphthalene reacted with the substrate. Urea formation was dependent on the microsomes, NADPH, and oxygen. Use of microsomes from rats previously treated with Aroclor 1254 increased urea formation greater than 10-fold. The enzyme activity was inhibited by alpha-napthoflavone, flavone, or CO, and slightly inhibited by metyrapone, 7-ethoxycoumarin, or SKF-525A. It was not inhibited by methimazole or paraoxon, suggesting that neither flavin-containing monooxygenase nor hydrolytic enzyme was involved. These data are consistent with a cytochrome P450-dependent, oxidative desulfuration of 2-naphthylisothiocyanate to yield 2-naphthylisocyanate. Further studies with the isomeric 1-naphthylisothiocyanate and the dietary benzylisothiocyanate showed that they can also be metabolized to their isocyanates, as evidenced by the trapping of isocyanates with 2-aminofluorene to form the mixed ureas.

Oxidative conversion by rat liver microsomes of 2-naphthyl isothiocyanate to 2-naphthyl isocyanate, a genotoxicant.

The present study investigated the oxidative metabolism of 2-naphthyl isothiocyanate catalyzed by rat liver microsomes. Incubation of 2-naphthyl isothiocyanate, microsomes, and NADPH yielded either N,N'-di-2-naphthylurea or, on inclusion of 2-aminofluorene in the incubations, N-2-naphthyl-N'-2-fluorenylurea. These ureas were formed by the production of 2-naphthyl isocyanate, which reacted with its hydrolysis product, 2-aminonaphthalene, to yield the symmetrical urea or, with 2-aminofluorene, to form the mixed urea. Formation of N,N'-di-2-naphthylthiourea was also observed, since 2-aminonaphthalene reacted with the substrate. Urea formation was dependent on microsomes, NADPH, and O2. Use of microsomes from rats previously treated with Aroclor increased urea formation > or = 10-fold. The enzyme activity was inhibited by alpha-naphthoflavone, flavone, or CO and slightly inhibited by metyrapone, 7-ethoxycoumarin, or SKF-525A. It was not inhibited by methimazole or paraoxon. These data are consistent with a cytochrome P-450-dependent, oxidative desulfuration of the isothiocyanate to yield an isocyanate.

Involvement of glutathione in 1-naphthylisothiocyanate (ANIT) metabolism and toxicity to isolated hepatocytes.

1-Naphthylisothiocyanate (ANIT) is a model compound which causes cholestasis in laboratory animals. Various biochemical and morphological changes including biliary epithelial and parenchymal cell necrosis occur in the liver of animals treated with ANIT. Although the mechanism(s) for these effects is not understood, a role for glutathione (GSH) in toxicity has been implicated. The possible role of GSH in hepatocellular toxicity caused by ANIT was investigated in this study. Treatment of freshly isolated rat hepatocytes with ANIT caused a concentration- and time-dependent depletion of cellular GSH that preceded lactate dehydrogenase (LDH) leakage. Analysis of the incubation medium indicated that the majority of the cellular GSH which was lost was present extracellularly as GSH or as a GSH-releasing compound. Mixing ANIT with GSH at pH 7.5 yielded a compound that was characterized by HPLC and fast atom bombardment-mass spectrometry (FAB-MS) S-(N-naphthyl-thiocarbamoyl)-L-glutathione (GS-ANIT). When dissolved in aqueous solutions at neutral pH, 95% of GS-ANIT dissociated to yield free ANIT and GSH. Under conditions designed to maximize formation and stability of GS-ANIT, GS-ANIT was found in the extracellular medium of hepatocytes treated with ANIT. Treatment of hepatocytes with the GS-ANIT caused GSH depletion and LDH leakage similar to that observed with equimolar amounts of ANIT. These data suggest that ANIT depletes hepatocytes of GSH through a reversible conjugation process. Such a process may play a role in the toxicity of ANIT.