RESUMO
The chemokine receptor CXCR3 is functionally pleiotropic, not only recruiting immune cells to the inflamed liver but also mediating the pathological process of cholestatic liver injury (CLI). However, the mechanism of its involvement in the CLI remains unclear. Both alpha-naphthylisothiocyanate (ANIT) and triptolide are hepatotoxicants that induce CLI by bile acid (BA) dysregulation, inflammation, and endoplasmic reticulum (ER)/oxidative stress. Through molecular docking, CXCR3 is a potential target of ANIT and triptolide. Therefore, this study aimed to investigate the role of CXCR3 in ANIT- and triptolide-induced CLI and to explore the underlying mechanisms. Wild-type mice and CXCR3-deficient mice were administered with ANIT or triptolide to compare CLI, BA profile, hepatic recruitment of IFN-γ/IL-4/IL-17+CD4+T cells, IFN-γ/IL-4/IL-17+iNKT cells and IFN-γ/IL-4+NK cells, and the expression of ER/oxidative stress pathway. The results showed that CXCR3 deficiency ameliorated ANIT- and triptolide-induced CLI. CXCR3 deficiency alleviated ANIT-induced dysregulated BA metabolism, which decreased the recruitment of IFN-γ+NK cells and IL-4+NK cells to the liver and inhibited ER stress. After triptolide administration, CXCR3 deficiency ameliorated dysregulation of BA metabolism, which reduced the migration of IL-4+iNKT cells and IL-17+iNKT cells and reduced oxidative stress through inhibition of Egr1 expression and AKT phosphorylation. Our findings suggest a detrimental role of CXCR3 in ANIT- and triptolide-induced CLI, providing a promising therapeutic target and introducing novel mechanisms for understanding cholestatic liver diseases.
Assuntos
1-Naftilisotiocianato , Colestase , Diterpenos , Fenantrenos , Animais , Camundongos , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Interleucina-17/toxicidade , Interleucina-17/metabolismo , Interleucina-17/uso terapêutico , Interleucina-4/toxicidade , Interleucina-4/metabolismo , Interleucina-4/uso terapêutico , Simulação de Acoplamento Molecular , Fígado/metabolismo , Colestase/induzido quimicamente , Ácidos e Sais Biliares , Compostos de EpóxiRESUMO
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.
Assuntos
Ácidos e Sais Biliares , Colestase , Camundongos , Animais , Ácidos e Sais Biliares/metabolismo , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Colestase/induzido quimicamente , Colestase/prevenção & controle , Fígado , Ácidos Cólicos/metabolismo , Ácidos Cólicos/farmacologia , Ácidos Cólicos/uso terapêutico , Proteínas de Membrana Transportadoras/metabolismo , HomeostaseRESUMO
The aim of this study was to determine the effect of tauroursodeoxycholic acid (TUDCA) on the alpha-naphthylisothiocyanate (ANIT)-induced model of cholestasis in mice. Wild-type and farnesoid X receptor (FXR)-deficient (Fxr-/- ) mice were used to generate cholestasis models by gavage with ANIT. Obeticholic acid (OCA) was used as a positive control. In wild-type mice, treatment with TUDCA for 7 days resulted in a dramatic increase in serum levels of alanine aminotransferase (ALT), with aggravation of bile infarcts and hepatocyte necrosis with ANIT-induction. TUDCA activated FXR to upregulate the expression of bile salt export pump (BSEP), increasing bile acids (BAs)-dependent bile flow, but aggravating cholestatic liver injury when bile ducts were obstructed resulting from ANIT. In contrast, TUDCA improved the liver pathology and decreased serum ALT and alkaline phosphatase (ALP) levels in ANIT-induced Fxr-/- mice. Furthermore, TUDCA inhibited the expression of cleaved caspase-3 and reduced the area of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in the model mice. TUDCA also upregulated anion exchanger 2 (AE2) protein expression, protecting cholangiocytes against excessive toxic BAs. Our results showed that TUDCA aggravated cholestatic liver injury via the FXR/BSEP pathway when bile ducts were obstructed, although TUDCA inhibited apoptotic activity and protected cholangiocytes against excessive toxic BAs.
Assuntos
Colagogos e Coleréticos , Colestase , Camundongos , Animais , Colagogos e Coleréticos/efeitos adversos , Colagogos e Coleréticos/metabolismo , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fígado , Colestase/induzido quimicamente , Ácidos e Sais Biliares/metabolismoRESUMO
Liquiritin (LQ) is an important monomer active component in flavonoids of licorice. The objective of this study was to evaluate the hepatoprotective effects of LQ in cholestatic mice. LQ (40 or 80 mg/kg) was intragastrically administered to mice once daily for 6 days, and mice were treated intragastrically with a single dosage of ANIT (75 mg/kg) on the 5th day. On the 7th day, mice were sacrificed to collect blood and livers. The mRNA and protein levels were determined by qRT-PCR and western blot assay. We also conducted systematical assessments of miRNAs expression profiles in the liver. LQ ameliorated ANIT-induced cholestatic liver injury, as evidenced by reduced serum biochemical markers and attenuated pathological changes in liver. Pretreatment of LQ reduced the increase of malondialdehyde, TNF-α, and IL-1ß induced by ANIT. Moreover, ANIT suppressed the expression of Sirt1 and FXR in liver tissue, which was weakened in the LQ pre-treatment group. LQ enhanced the nuclear expression of Nrf2, which was increased in the ANIT group. LQ also increased the mRNA expressions of bile acid transporters Bsep, Ntcp, Mrp3, and Mrp4. Furthermore, a miRNA deep sequencing analysis revealed that LQ had a global regulatory effect on the hepatic miRNA expression. Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis showed that the differentially expressed miRNAs were mainly related to metabolic pathways, endocytosis, and MAPK signaling pathway. Collectively, LQ attenuated hepatotoxicity and cholestasis by regulating the expression of Sirt1/FXR/Nrf2 and the bile acid transporters, indicating that LQ might be an effective approach for cholestatic liver diseases.
Assuntos
Colestase Intra-Hepática , Colestase , MicroRNAs , Camundongos , Animais , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/tratamento farmacológico , Colestase Intra-Hepática/genética , Fígado , Colestase/induzido quimicamente , Colestase/tratamento farmacológico , Ácidos e Sais Biliares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Colestase , Hepatopatias , 1-Naftilisotiocianato/efeitos adversos , 1-Naftilisotiocianato/metabolismo , Animais , Ácidos e Sais Biliares/farmacologia , Colestase/metabolismo , Interleucina-17/metabolismo , Fígado/metabolismo , Hepatopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Colestase , Silimarina , Camundongos , Animais , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Ácidos e Sais Biliares/metabolismo , Silimarina/farmacologia , Colestase/induzido quimicamente , Colestase/tratamento farmacológico , Colestase/genética , Fígado/metabolismo , Aspartato Aminotransferases , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Yinchenwuling Fang (YCWLF), a famous traditional Chinese medicine, has been used clinically for cholestatic liver disease treatment. However, quantification analysis for YCWLF components and their pharmacological effects remains largely unknown. Therefore, we aimed to determine the YCWLF components and their activities. Quantification analysis of 12 YCWLF components was performed using a comprehensive ultra-performance liquid chromatography (UPLC) coupled with the triple-quadrupole mass spectrometry method. Then, the anti-cholestasis effect and potential mechanism of YCWLF were performed in a mouse model induced by alpha-naphthyl isothiocyanate (ANIT). YCWLF decreased serum biochemical indicators (ALT, AST, ALP, TBA, TBIL, and DBIL) and ameliorated liver tissue damage in cholestatic mice. Mechanically, YCWLF increased the expression of the farnesoid X receptor (FXR) and its downstream efflux transporters and metabolic enzyme genes, reversed the disordered homeostasis of bile acids, and decreased cholestatic liver injury. Based on the important role of FXR in YCWLF amelioration on cholestasis, a dual-luciferase assay was used to screen the potential agonist of FXR from 12 YCWLF components. Chlorogenic acid, 4-hydroxyacetophenone, scoparone, atractylenolide â , atractylenolide â ¡, and alisol B 23-acetate exhibited an activity effect of FXR. This study provides novel a therapeutic mechanism and potential active compounds of YCWLF on cholestatic liver injury.
Assuntos
Colestase , Hepatopatias , Camundongos , Animais , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Colestase/induzido quimicamente , Colestase/tratamento farmacológico , Colestase/metabolismo , Fígado/metabolismo , Hepatopatias/metabolismo , Isotiocianatos/farmacologia , Ácidos e Sais Biliares/metabolismoRESUMO
Bile acid (BA) homeostasis throughout the enterohepatic circulation system is a guarantee of liver physiological functions. BA circulation disorders is one of the characteristic clinical manifestations of cholestasis, and have a closely relationship with intestinal barrier function, especially ileum. Here, our in vivo and in vitro studies showed that intestinal tight junctions (TJs) were disrupted by α-naphthylisothiocyanate (ANIT), which also down-regulated the protein expression of sphingosine-1-phosphate receptor 1 (S1PR1) in the top of villus of mice ileum. Activating S1PR1 by specific agonist SEW2871 could improve TJs via inhibiting ERK1/2/LKB1/AMPK signaling pathway in the ileum of ANIT-treated mice and ANIT-cultured Caco-2 cells. SEW2871 not only regained ileum TJs by activating S1PR1 in the epithelial cells of ileum mucosa, but also recovered ileum barrier function, which was further verified by the recovered BA homeostasis in mice ileum (content and tissue) by using of high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). Subsequently, the improved intestinal injury and inflammation further strengthened that SEW2871 modulated intestinal barrier function in ANIT-treated mice. Finally, our data revealed that along with the down-regulated levels of serum lipopolysaccharides (LPS), SEW2871 improved liver function and relieved hepatitis via blocking TLR4/MyD88/NF-kB signaling pathway in ANIT-treated mice. In conclusion, these results demonstrated that activating intestinal S1PR1 by SEW2871 could modulate intestinal barrier function, leading to the improvement of cholestatic hepatitis in ANIT-treated mice via the "gut-liver" axis.
Assuntos
Colestase , Hepatite , Animais , Humanos , Camundongos , 1-Naftilisotiocianato/efeitos adversos , 1-Naftilisotiocianato/metabolismo , 1-Naftilisotiocianato/toxicidade , Células CACO-2 , Colestase/metabolismo , Cromatografia Líquida , Hepatite/metabolismo , Fígado/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Espectrometria de Massas em TandemRESUMO
Cholestasis is primarily caused by bile acid homeostasis dysregulation, resulting in retention, aggregation, and accumulation of the toxic cholate in the hepatocytes. Existing therapies for cholestasis are limited, demanding the urgent development of novel drugs. As a result, targeting FXR specifically promises a unique treatment strategy for cholestasis. The current study aims to evaluate the influence of 7, 8-dihydroxy-4-methyl coumarin (DMC) against alpha-naphthyl isothiocyanate (ANIT)-induced liver injury in mice. The "Computer-Aided Drug Design" (CADD) and molecular docking study anticipated that DMC would proficiently bind and activate the FXR. Accordingly, the hepatoprotective activity of DMC against ANIT-induced hepatotoxicity and cholestasis was investigated in ANIT-treated HepaRG cells and the ANIT-induced cholestatic mouse model. Outcomes indicated the protective effects of DMC against ANIT toxicity in HepaRG cells after 24 h of intervention and animals after seven days of treatment. DMC partially blocks ANIT-induced increases in serum markers of hepatocellular injury, liver and gall bladder enlargement, and hepatic necrosis. Western blotting revealed that DMC alleviates ANIT-induced hepatotoxicity and cholestasis via activating the FXR receptor and regulating CYP7A1, the enzyme responsible for bile acid synthesis. DMC exhibited protective activity against cholestasis through activating FXR, suggesting it might be a promising strategy for preventing and treating cholestatic liver disease.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Colestase , Hepatopatias , Camundongos , Animais , Simulação de Acoplamento Molecular , Receptores Citoplasmáticos e Nucleares/metabolismo , 1-Naftilisotiocianato/toxicidade , 1-Naftilisotiocianato/metabolismo , Colestase/induzido quimicamente , Colestase/tratamento farmacológico , Colestase/metabolismo , Fígado/metabolismo , Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cumarínicos/farmacologia , Cumarínicos/uso terapêuticoRESUMO
Cholestasis is characterized by bile acid (BA) circulation disorders, which is usually related to damage of hepatocyte barrier function. Currently, patients with cholestasis face several obstacles in seeking diagnosis and therapy. Da-Huang-Xiao-Shi decoction (DHXSD) is an ancient classic formula that has been used clinically for cholestasis treatment. Nevertheless, the underlying biological activities and therapeutic mechanisms remain unclear. In this study, an alpha-naphthylisothiocyanate (ANIT)-induced cholestasis rat model was established to examine the anticholestatic effects of DHXSD using histopathological and molecular analyses. Transcriptomic analysis combined with 16S rRNA gene sequencing analysis was systematically applied to study the mechanism of action of DHXSD. Simultaneously, the effect of DHXSD on gut microbiota, short-chain fatty acids (SCFAs), and intestinal barrier function were evaluated based on the ANIT-induced cholestasis model in rats. The results showed that DHXSD effectively attenuated ANIT-induced cholestasis by reducing liver function indicators (alanine transaminase [ALT], P < 0.05; alkaline phosphatase [ALP], P < 0.05; total bile acid [TBA], P < 0.01; γ-glutamyl transpeptidase [GGT], P < 0.001) and levels of hepatotoxicity-related enzymes (P < 0.05), thus improving the recovery of histopathological injuries, and regulating levels of inflammatory cytokines (P < 0.05). In addition, 16S rRNA gene sequencing analysis combined with intestinal barrier function analysis revealed that the DHXSD significantly ameliorated ANIT-induced gut microbiota dysbiosis. Significantly altered genes in the model and treatment groups were screened using transcriptomic analysis. Sixty-eight genes and four microbial genera were simultaneously altered with opposing trends in variation after ANIT and DHXSD treatments. We built a framework for predicting targets and host-microbe interaction mechanisms, as well as identifying alternative treatment for cholestasis, which should be validated further for clinical application. In conclusion, DHXSD appears to be a promising agent for protection against liver injury. IMPORTANCE Cholestasis is a serious manifestation of liver diseases resulting in liver injury, fibrosis, and liver failure with limited therapies. To date, only ursodeoxycholic acid (UDCA) has been approved by the U.S. Food and Drug Administration for the treatment of cholestasis. However, approximately one-third of patients with cholestasis are unresponsive to UDCA. Therefore, it is urgent to search for appropriate therapeutic agents for restoring stoppage status of the bile components to treat cholestasis. In this study, we investigated how the microbiome and transcriptome data sets correlated with each other to clarify the role of microbiome alterations in host metabolism. In combination, this research offers potential molecular biomarkers that should be validated for more accurate diagnosis of cholestasis and the clinical utilisation of gut microbiota as a target for treatment.
Assuntos
Colestase , Microbioma Gastrointestinal , Estados Unidos , Ratos , Animais , 1-Naftilisotiocianato/efeitos adversos , 1-Naftilisotiocianato/metabolismo , Transcriptoma , RNA Ribossômico 16S/genética , Colestase/induzido quimicamente , Colestase/tratamento farmacológico , Colestase/metabolismo , Fígado , Ácidos e Sais BiliaresRESUMO
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.
Assuntos
1-Naftilisotiocianato , Colestase , 1-Naftilisotiocianato/metabolismo , 1-Naftilisotiocianato/toxicidade , Animais , Ácidos e Sais Biliares/metabolismo , Colestase/induzido quimicamente , Colestase/tratamento farmacológico , Homeostase , Fígado/metabolismo , Luteolina/farmacologia , Estresse Oxidativo , Ratos , Resveratrol/metabolismo , Taurina/metabolismoRESUMO
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.
Assuntos
1-Naftilisotiocianato/metabolismo , Glutationa/metabolismo , Fígado/citologia , Fígado/metabolismo , 1-Naftilisotiocianato/efeitos adversos , Animais , Células Cultivadas , Cinética , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos EndogâmicosRESUMO
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.
Assuntos
1-Naftilisotiocianato/farmacologia , Bile/metabolismo , Glutationa/metabolismo , 1-Naftilisotiocianato/sangue , 1-Naftilisotiocianato/metabolismo , Animais , Glutationa/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Isocianatos/metabolismo , Isotiocianatos/metabolismo , Microssomos Hepáticos/metabolismo , 1-Naftilisotiocianato/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/metabolismo , Animais , Argônio/metabolismo , Biotransformação , Carcinógenos/metabolismo , Indução Enzimática , Fluorenos/metabolismo , Isomerismo , Microssomos Hepáticos/enzimologia , Mutagênicos/metabolismo , Oxirredução , Paraoxon/metabolismo , Ratos , Ureia/análogos & derivados , Ureia/metabolismoRESUMO
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.
Assuntos
1-Naftilisotiocianato/farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP , Arsenitos/farmacocinética , Proteínas de Transporte/metabolismo , Glutationa/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , 1-Naftilisotiocianato/metabolismo , 1-Naftilisotiocianato/toxicidade , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Arsenitos/metabolismo , Arsenitos/toxicidade , Bile/metabolismo , Linhagem Celular , Células Cultivadas , Cães , Fígado/efeitos dos fármacos , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Ratos , Ratos Wistar , Transdução GenéticaRESUMO
The role of S-oxidation in the toxic bioactivation of alpha-naphthylisothiocyanate (ANIT) was investigated. The effects of several thione compounds, inhibitors and an inducer of the cytochrome P-450-dependent mixed function oxidase systems on the in vitro metabolism of ANIT and aminopyrine were determined. Ethionamide, sodium diethyldithiocarbamate (Na-DDTC) and S-methyl diethyldithiocarbamate (Me-DDTC), three agents known to undergo metabolism by an S-oxidative pathway and diminish ANIT's toxicity, inhibited the in vitro enzymatic metabolism of ANIT by rat liver microsomes. Methimazole failed to alter either the hyperbilirubinemic response of ANIT or the in vitro metabolism of ANIT. All four thione compounds (i.e., ethionamide, Me-DDTC, Na-DDTC and methimazole) inhibited the enzymatic metabolism of aminopyrine by rat liver microsomes. Me-DDTC was the most potent, whereas methimazole was the least potent inhibitor of aminopyrine metabolism. Phenobarbital, which potentiates, and SKF-525A, which inhibits the hepatotoxicity of ANIT in vivo, correspondingly stimulated or inhibited the NADPH-dependent metabolism of ANIT and aminopyrine by liver microsomes. N-Decylimidazole (NDI), another classical inhibitor of cytochrome P-450-dependent monooxygenase system, inhibited both the in vivo toxicity and in vitro metabolism of ANIT. NDI also diminished the enzymatic metabolism of aminopyrine by liver microsomes. Thus the results of this study indicate that metabolism of ANIT is intimately related to its toxicity and that ANIT probably undergoes its toxic bioactivation via a cytochrome P-450-dependent S-oxidative pathway.
Assuntos
1-Naftilisotiocianato/metabolismo , Imidazóis/farmacologia , Metimazol/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Tiocianatos/metabolismo , 1-Naftilisotiocianato/antagonistas & inibidores , 1-Naftilisotiocianato/toxicidade , Administração Oral , Aminopirina/metabolismo , Animais , Bilirrubina/sangue , Biotransformação , Cromatografia Gasosa , Ditiocarb/farmacologia , Etionamida/farmacologia , Hiperbilirrubinemia/induzido quimicamente , Cinética , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Oxigenases de Função Mista/metabolismo , NADP/metabolismo , Fenobarbital/farmacologia , Proadifeno/farmacologia , Ratos , Ratos EndogâmicosAssuntos
1-Naftilisotiocianato/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Microssomos Hepáticos/metabolismo , Tiocianatos/efeitos adversos , 1-Naftilisotiocianato/metabolismo , Animais , Indução Enzimática/efeitos dos fármacos , Glutationa/metabolismo , Técnicas In Vitro , Cinética , Masculino , Oxigenases de Função Mista/antagonistas & inibidores , Ligação Proteica/efeitos dos fármacos , Ratos , Tiocianatos/farmacologiaRESUMO
Bile acids (BAs) are crucial for the diagnosis, follow-up, and prognostics of liver and intestinal disorders and other diseases affecting BA metabolism. A rapid, simple, and sensitive analytical method is needed to demonstrate the full metabolic profile and simultaneously determine the individual BAs in biological samples. In our present study, an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method has been established and validated for simultaneous quantitation of 22 BAs and a metabonomic study was performed based on the chemometric analysis of the serum samples from carbon tetrachloride (CCl4)- and alpha-naphthylisothiocyanate (ANIT)-induced liver failure rats. The optimal chromatographic condition was effected by UPLC (Acquity UPLC BEH column, 1.7 microm, 2.1 mm x 100 mm) using a linear gradient elution system of methanol-5 mM ammonium acetate containing 0.01% acetic acid after a simple-step deproteinization by precipitation. The separation of the 22 BAs can be finished in less than 12 min, and the concentrations of these BAs in rat serums were simultaneously determined using a selective ions monitoring mode. The method was validated with respect to repeatability (relative standard deviation < 9.78%) and accuracy (relative errors from -13.55 to 9.58%). The range of each BA was found from not detected (nd) to 8301 ng mL(-1), respectively. Furthermore, the developed method was successfully applied to the metabonomics analysis of BAs in CCl4- and ANIT-induced liver failure rats, using principle component analysis and canonical discriminant analysis. The serum samples from the two types of rat liver injury could be distinguished from each other and from the untreated animals according to the varieties of BAs. It indicated that the level of BAs could be considered as a sensitive parameter of hepatotoxicity induced by different chemical toxins. This novel metabonomics study of BAs based on the UPLC-MS profile provides not only an accurate quantitative assay of the serum concentrations of biomarkers but also a promising methodology for evaluation of liver injury.
Assuntos
1-Naftilisotiocianato/toxicidade , Ácidos e Sais Biliares/metabolismo , Tetracloreto de Carbono/toxicidade , Falência Hepática/metabolismo , Metabolômica , 1-Naftilisotiocianato/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Tetracloreto de Carbono/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Análise Discriminante , Modelos Animais de Doenças , Fígado/efeitos dos fármacos , Fígado/metabolismo , Falência Hepática/etiologia , Masculino , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Metabonomics using high-resolution 1H-NMR spectroscopy of biofluids and pattern recognition is highly successful at distinguishing both organ- and sub-organ-specific toxicity. In the current study, this technique was investigated to distinguish the different biological effects caused by 1-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in the rat from that induced by exposure to 1-naphthylisocyanate (NI) and 1-naphthylamine (NA), two products of the metabolism of ANIT. While all three toxicants produced perturbations in similar urinary metabolites, principal components analysis of the temporal progression identified that the rapid initial glycosuria associated with ANIT toxicity was also present with NI but not NA dosing. However, longer-term perturbations in the urinary excretion of succinate, lactate and acetate were common to all three toxicants. The metabolic effects of the three compounds were also followed in blood plasma and liver tissue. Of the three toxicants, the most marked perturbations were induced by ANIT exposure, then NI, thereby indicating the effects of ANIT, NI and NA toxicity were distinct, with ANIT being the most, and NA the least, toxic of the three compounds. This indicates that metabonomics may be useful for following severity and mechanisms of toxicity in a series of related compounds during drug development.
Assuntos
1-Naftilisotiocianato/metabolismo , 1-Naftilisotiocianato/toxicidade , Líquidos Corporais/química , Doença Hepática Induzida por Substâncias e Drogas , 1-Naftilamina/análise , 1-Naftilisotiocianato/farmacologia , Animais , Análise Química do Sangue , Isocianatos/análise , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/sangue , Hepatopatias/metabolismo , Hepatopatias/urina , Espectroscopia de Ressonância Magnética , Especificidade de Órgãos , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Urina/químicaRESUMO
alpha-Naphthyl-isothiocyanate (ANIT) is a well known toxic substance which induces characteristic hepatic lesions. Its distribution in some organs and body fluids was investigated by using spectrophotometry, gas-chromatography and thin-layer chromatography. The results indicate that ANIT concentrates in the liver, kidneys and blood but not in the brain, urine and bile. Another five ANIT-like substances have also been examined, but no trace of them has been found in these organs and body fluids. The authors suppose that ANIT is metabolized to an unknown metabolite which is responsible for the toxic action of ANIT. None of the ANIT-like substances examined by us can be indentified with this metabolite.