RESUMEN
Fat accumulation (steatosis) in ballooned hepatocytes alters the expression of membrane transporters in Zucker fatty (fa/fa) rats. The aim of the study was to quantify the functions of these transporters and their impact on hepatocyte concentrations using a clinical hepatobiliary contrast agent (Gadobenate dimeglumine, BOPTA) for liver imaging. In isolated and perfused rat livers, we quantified BOPTA accumulation and decay profiles in fa/+ (normal) and fa/fa hepatocytes by placing a gamma counter over livers. Profiles of BOPTA accumulation and decay in hepatocytes were analysed with nonlinear regressions to characterise BOPTA influx and efflux across hepatocyte transporters. At the end of the accumulation period, BOPTA hepatocyte concentrations and influx clearances were not significantly different in fa/+ and fa/fa livers. In contrast, bile clearance was significantly lower in fatty hepatocytes while efflux clearance back to sinusoids compensated the low efflux into canaliculi. The time when BOPTA cellular efflux impacts the accumulation profile of hepatocyte concentrations was slightly delayed (2 min) by steatosis, anticipating a delayed emptying of hepatocytes. The experimental model is useful for quantifying the functions of hepatocyte transporters in liver diseases.
Asunto(s)
Hígado Graso/metabolismo , Hígado Graso/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Proteínas de Transporte de Membrana/metabolismo , Obesidad/metabolismo , Obesidad/patología , Animales , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/metabolismo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Meglumina/análogos & derivados , Meglumina/farmacocinética , Meglumina/farmacología , Compuestos Organometálicos/farmacocinética , Compuestos Organometálicos/farmacología , Perfusión , Ratas , Ratas Zucker , Reología/efectos de los fármacosRESUMEN
Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Desarrollo de Medicamentos/métodos , Hepatocitos/efectos de los fármacos , Cultivo Primario de Células/métodos , Quimera por Trasplante , Animales , Canalículos Biliares/citología , Canalículos Biliares/metabolismo , Células Cultivadas , Niño , Preescolar , Ciclosporina/farmacología , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Femenino , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones SCID , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Canalículos Biliares/metabolismo , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/farmacología , Canalículos Biliares/efectos de los fármacos , Línea Celular Tumoral , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Colágeno/farmacología , Dexametasona/farmacología , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos/métodos , Interacciones Farmacológicas , Humanos , Laminina/farmacología , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteoglicanos/farmacologíaRESUMEN
Many drugs can induce liver injury, characterized by hepatocellular, cholestatic or mixed hepatocellular-cholestatic lesions. While an inflammatory stress is known to aggravate hepatocellular injury caused by some drugs much less evidence exists for cholestatic features. In this study, the influence of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α), either individually or combined, on cytotoxic and cholestatic properties of antibiotics was evaluated using differentiated HepaRG cells. Six antibiotics of various chemical structures and known to cause cholestasis and/or hepatocellular injury in clinic were investigated. Caspase-3 activity was increased with all these tested hepatotoxic drugs and except with erythromycin, was further augmented in presence of cytokines mainly when these were co-added as a mixture. TNF-α and IL-1ß aggravated cytotoxicity of TVX more than IL-6. Bile canaliculi (BC) dilatation induced by cholestatic drugs was increased by co-treatment with IL-6 and IL-1ß but not with TNF-α. Reduced accumulation of carboxy-dichlorofluorescein, a substrate of the multi-drug resistance-associated protein 2, in antibiotic-induced dilatated BC, was further extended in presence of individual or mixed cytokines. In conclusion, our data demonstrate that pro-inflammatory cytokines either individually or in mixture, can modulate cholestatic and/or cytotoxic responses to antibiotics and that the extent of these effects is dependent on the cytokine and the cholestatic antibiotic.
Asunto(s)
Antibacterianos/efectos adversos , Canalículos Biliares/efectos de los fármacos , Colestasis/inducido químicamente , Citocinas/farmacología , Canalículos Biliares/fisiología , Proteína C-Reactiva/metabolismo , Caspasa 3/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colestasis/metabolismo , Fluoresceínas/metabolismo , HumanosRESUMEN
Cholestasis is a condition wherein bile flow is interrupted and lithocholic acid is known to play a key role in causing severe liver injury. In this study, we performed in-depth analysis of the morphological changes in bile canaliculi and the biological role of villin in cholestasis using lithocholic acid-stimulated HepG2 human hepatocarcinoma cells. We confirmed disruption of the bile canaliculi in liver sections from a liver allograft patient with cholestasis. Lithocholic acid caused strong cytotoxicity in HepG2 cells, which was associated with abnormal morphology. Lithocholic acid reduced villin expression, which recovered in the presence of nuclear receptor agonists. Furthermore, villin mRNA expression increased following small interfering RNA (siRNA)-mediated knockdown of the nuclear farnesoid X receptor and pregnane X receptor. Villin knockdown using siRNA caused cell growth arrest in HepG2 cells. The effect of villin-knockdown on whole-genome expression in HepG2 cells was analyzed by DNA microarray. Our data suggest that lithocholic acid caused cell growth arrest by suppressing villin expression via farnesoid X receptor and pregnane X receptor in HepG2 cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácido Litocólico/farmacología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Canalículos Biliares/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colestasis/patología , Células Hep G2 , HumanosRESUMEN
HepaRG is an original human hepatoma cell line, acquiring highly differentiated hepatic features when exposed to dimethylsulfoxide (DMSO). To search alternatives to DMSO, which may exert some toxicity, we have analyzed the effects of forskolin (FSK), a cAMP-generating agent known to favor differentiation of various cell types. FSK used at 50 µM for 3 days was found to promote polarization of high density-plated HepaRG cells, i.e., it markedly enhanced the formation of functional biliary canaliculi structures. It also increased expressions of various hepatic markers, including those of cytochrome P-450 (CYP) 3A4, of drug transporters like NTCP, OATP2B1 and BSEP, and of metabolism enzymes like glucose 6-phosphatase. In addition, FSK-treated HepaRG cells displayed enhanced activities of CYP3A4, NTCP and OATPs when compared to untreated cells. These polarizing/differentiating effects of FSK were next shown to reflect not only the generation of cAMP, but also the activation of the xenobiotic sensing receptors PXR and FXR by FSK. Co-treatment of HepaRG cells by the cAMP analog Sp-5,6-DCl-cBIMPS and the reference PXR agonist rifampicin reproduced the polarizing effects of FSK. Therefore, FSK may be considered as a relevant alternative to DMSO for getting polarized and differentiated HepaRG cells, notably for pharmacological and toxicological studies.
Asunto(s)
Carcinoma Hepatocelular/patología , Polaridad Celular , Colforsina/farmacología , Neoplasias Hepáticas/patología , Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/patología , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , AMP Cíclico/análogos & derivados , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/genética , Receptor X de Pregnano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rifampin/farmacología , Transducción de SeñalRESUMEN
Cholestasis represents a major subtype of drug-induced liver injury and novel preclinical models for its prediction are needed. Here we used primary human hepatocytes (PHH) from different donors in 2D-sandwich (2D-sw) and/or 3D-spheroid cultures to study inter-individual differences in the response towards cholestatic hepatotoxins after short-term (48-72 hours) and long-term repeated exposures (14 days). The cholestatic liabilities of drugs were determined by comparing cell viability upon exposure to the highest non-cytotoxic drug concentration in the presence and absence of a non-cytotoxic concentrated bile acid mixture. In 2D-sw culture, cyclosporine A and amiodarone presented clear cholestatic liabilities in all four PHH donors tested, whereas differences in the susceptibility of the various PHH donors towards the cholestatic toxicity of bosentan, chlorpromazine and troglitazone were observed. In PHH from one donor, the cholestatic liabilities of chlorpromazine and troglitazone could only be detected after long-term repeated exposures when maintained in 3D-spheroid culture, but not after short-term exposures in either 2D-sw or 3D-spheroid culture, suggesting that cholestatic hepatotoxicity may require time to develop. In conclusion, inter-individual susceptibility exists towards drug-induced cholestasis, which depends on the compound as well as the exposure time.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Variación Biológica Poblacional , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Colestasis/inducido químicamente , Hepatocitos/efectos de los fármacos , Anciano , Canalículos Biliares/metabolismo , Canalículos Biliares/patología , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colestasis/metabolismo , Colestasis/patología , Relación Dosis-Respuesta a Droga , Femenino , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Medición de Riesgo , Esferoides Celulares , Factores de TiempoRESUMEN
Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3â»6 days, were placed into two groups. The high Al group (n = 8) received PN with 63 µg/kg/day of Al, while the low Al group (n = 7) received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry were studied by transmission electron microscopy (TEM) and ImageJ software analysis. Results: The canalicular space was smaller and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow.
Asunto(s)
Aluminio/toxicidad , Canalículos Biliares/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatopatías/etiología , Soluciones para Nutrición Parenteral/toxicidad , Nutrición Parenteral/efectos adversos , Animales , Animales Recién Nacidos , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/metabolismo , Canalículos Biliares/ultraestructura , Hepatocitos/ultraestructura , Hepatopatías/metabolismo , Hepatopatías/patología , Microscopía Electrónica de Transmisión , Microvellosidades/efectos de los fármacos , Microvellosidades/ultraestructura , Porcinos , Porcinos Enanos , Factores de TiempoRESUMEN
Several endothelin receptor antagonists (ERAs) have been developed for the treatment of pulmonary arterial hypertension (PAH). Some of them have been related to clinical cases of hepatocellular injury (sitaxentan [SIT]) and/or cholestasis (bosentan [BOS]). We aimed to determine if ambrisentan (AMB) and macitentan (MAC), in addition to BOS and SIT, could potentially cause liver damage in man by use of human HepaRG cells. Our results showed that like BOS, MAC-induced cytotoxicity and cholestatic disorders characterized by bile canaliculi dilatation and impairment of myosin light chain kinase signaling. Macitentan also strongly inhibited taurocholic acid and carboxy-2',7'-dichlorofluorescein efflux while it had a much lower inhibitory effect on influx activity compared to BOS and SIT. Moreover, these three drugs caused decreased intracellular accumulation and parallel increased levels of total bile acids (BAs) in serum-free culture media. In addition, all drugs except AMB variably deregulated gene expression of BA transporters. In contrast, SIT was hepatotoxic without causing cholestatic damage, likely via the formation of reactive metabolites and AMB was not hepatotoxic. Together, our results show that some ERAs can be hepatotoxic and that the recently marketed MAC, structurally similar to BOS, can also cause cholestatic alterations in HepaRG cells. The absence of currently known or suspected cases of cholestasis in patients suffering from PAH treated with MAC is rationalized by the lower therapeutic doses and Cmax, and longer receptor residence time compared to BOS.
Asunto(s)
Colestasis/inducido químicamente , Antagonistas de los Receptores de Endotelina/toxicidad , Hepatocitos/efectos de los fármacos , Receptores de Endotelina/metabolismo , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/metabolismo , Canalículos Biliares/patología , Miosinas Cardíacas/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colestasis/metabolismo , Colestasis/patología , Relación Dosis-Respuesta a Droga , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Cadenas Ligeras de Miosina/metabolismoRESUMEN
In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca2+-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 µM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Colestasis/inducido químicamente , Hígado/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , terc-Butilhidroperóxido/toxicidad , Animales , Canalículos Biliares/metabolismo , Canalículos Biliares/fisiopatología , Colestasis/metabolismo , Hígado/metabolismo , Hígado/fisiopatología , Masculino , Proteína Quinasa C/metabolismo , Ratas WistarRESUMEN
Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.
Asunto(s)
Canalículos Biliares/metabolismo , Colestasis Intrahepática/metabolismo , Hígado/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasas Asociadas a rho/metabolismo , Amidas/farmacología , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Línea Celular , Colestasis Intrahepática/inducido químicamente , Humanos , Hígado/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ácido Taurocólico/metabolismoRESUMEN
Idiosyncratic drug-induced liver injury is a clinical concern with serious consequences. Although many preclinical screening methods have been proposed, it remains difficult to identify compounds associated with this rare but potentially fatal liver condition. Here, we propose a novel assay system to assess the risk of liver injury. Rat primary hepatocytes were cultured in a sandwich configuration, which enables the formation of a typical bile canalicular network. From day 2 to 3, test drugs, mostly selected from a list of cholestatic drugs, were administered, and the length of the network was semi-quantitatively measured by immunofluorescence. Liver injury risk information was collected from drug labels and was compared with in vitro measurements. Of 23 test drugs examined, 15 exhibited potent inhibition of bile canalicular network formation (<60% of control). Effects on cell viability were negligible or minimal as confirmed by lactate dehydrogenase leakage and cellular ATP content assays. For the potent 15 drugs, IC50 values were determined. Finally, maximum daily dose divided by the inhibition constant gave good separation of the highest risk of severe liver toxicity drugs such as troglitazone, benzbromarone, flutamide, and amiodarone from lower risk drugs. In conclusion, inhibitory effect on the bile canalicular network formation observed in in vitro sandwich cultured hepatocytes evaluates a new aspect of drug toxicity, particularly associated with aggravation of liver injury.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatocitos/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Miosinas Cardíacas/metabolismo , Clorpromazina/farmacología , Ciclosporina/farmacología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Microscopía Fluorescente , Miosina Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Imagen de Lapso de Tiempo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
The Cu(+) pump ATP7B plays an irreplaceable role in the elimination of excess Cu(+) by the hepatocyte into the bile. The trafficking and site of action of ATP7B are subjects of controversy. One current proposal is that an increase in intracellular Cu(+) results in the translocation of ATP7B to the lysosomes and excretion of excess Cu(+) through lysosomal-mediated exocytosis at the bile canaliculus. Here, we show that ATP7B is transported from the trans-Golgi network (TGN) to the bile canaliculus by basolateral sorting and endocytosis, and microtubule-mediated transcytosis through the subapical compartment. Trafficking ATP7B is not incorporated into lysosomes, and addition of Cu(+) does not cause relocalization of lysosomes and the appearance of lysosome markers in the bile canaliculus. Our data reveal the pathway of the Cu(+)-mediated transport of ATP7B from the TGN to the bile canaliculus and indicates that the bile canaliculus is the primary site of ATP7B action in the elimination of excess Cu(.)
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Canalículos Biliares/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/farmacología , Transcitosis/efectos de los fármacos , Animales , Canalículos Biliares/efectos de los fármacos , Brefeldino A/farmacología , Compartimento Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , ATPasas Transportadoras de Cobre , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Hep G2 , Humanos , Hidrazonas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Macrólidos/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/metabolismoRESUMEN
Estradiol-17ß-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Receptores ErbB/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrógeno/metabolismo , Hepatocitos/metabolismo , Animales , Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/metabolismo , Canalículos Biliares/fisiopatología , Células Cultivadas , Colestasis/inducido químicamente , Colestasis/metabolismo , Receptores ErbB/genética , Estradiol/metabolismo , Estradiol/farmacología , Antagonistas del Receptor de Estrógeno/farmacología , Femenino , Fulvestrant , Técnicas de Silenciamiento del Gen , Hepatocitos/efectos de los fármacos , Quinazolinas/farmacología , Ratas , Ratas Wistar , Tirfostinos/farmacología , Familia-src Quinasas/metabolismoRESUMEN
This study investigated the effects of nanoencapsulated curcumin (NEC) and praziquantel (PZQ) treatment on the resolution of periductal fibrosis (PDF) and bile canalicular (BC) abnormalities in Opisthorchis viverrini infected hamsters. Chronic O. viverrini infection (OV) was initially treated with either PZQ (OP) and subsequently treated with NEC (OP+NEC), curcumin (OP+Cur) or unloaded carriers (OP+carrier) daily for one month. OP+NEC treatment reduced the PDF by suppression of fibrotic markers (hydroxyproline content, α-SMA, CTGF, fibronectin, collagen I and III), cytokines (TGF-ß and TNF-α) and TIMP-1, 2, 3 expression and upregulation of MMP-7, 13 genes. Higher activity of NEC in reducing fibrosis compared to curcumin was also demonstrated in in vitro studies. Moreover, OP+NEC also prevented BC abnormalities and upregulated several genes involved in bile acid metabolism. These results demonstrate that NEC and PZQ treatment reduces PDF and attenuates BC defect in experimental opisthorchiasis. From the Clinical Editor: Infection by Opisthorchis viverrini leads to liver fibrosis and affects population in SE Asia. Currently, praziquantel (PZQ) is the drug of choice but this drug has significant side effects. In this study, the authors combined curcumin (NEC) and praziquantel in a nanocarrier to test the anti-oxidative effect of curcumin in an animal model. The encouraging results may pave a way for better treatment in the future.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Canalículos Biliares/patología , Curcumina/administración & dosificación , Nanocápsulas/química , Opistorquiasis/tratamiento farmacológico , Praziquantel/administración & dosificación , Animales , Antihelmínticos/administración & dosificación , Antihelmínticos/química , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/química , Canalículos Biliares/anomalías , Cricetinae , Curcumina/química , Difusión , Combinación de Medicamentos , Fibrosis/patología , Fibrosis/prevención & control , Nanocápsulas/administración & dosificación , Nanocápsulas/ultraestructura , Opistorquiasis/patología , Praziquantel/química , Resultado del TratamientoRESUMEN
Many substances are hepatotoxic due to their ability to cause intrahepatic cholestasis. Therefore, there is a high demand for in vitro systems for the identification of cholestatic properties of new compounds. Primary hepatocytes cultivated in collagen sandwich cultures are known to establish bile canaliculi which enclose secreted biliary components. Cholestatic compounds are mainly known to inhibit bile excretion dynamics, but may also alter canalicular volume, or hepatocellular morphology. So far, techniques to assess time-resolved morphological changes of bile canaliculi in sandwich cultures are not available. In this study, we developed an automated system that quantifies dynamics of bile canaliculi recorded in conventional time-lapse image sequences. We validated the hepatocyte sandwich culture system as an appropriate model to study bile canaliculi in vitro by showing structural similarity measured as bile canaliculi length per hepatocyte to that observed in vivo. Moreover, bile canalicular excretion kinetics of CMFDA (5-chloromethylfluorescein diacetate) in sandwich cultures resembled closely the kinetics observed in vivo. The developed quantification technique enabled the quantification of dynamic changes in individual bile canaliculi. With this technique, we were able to clearly distinguish between sandwich cultures supplemented with dexamethasone and insulin from control cultures. In conclusion, the automated quantification system offers the possibility to systematically study the causal relationship between disturbed bile canalicular dynamics and cholestasis.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Técnicas de Cultivo de Célula , Colágeno/química , Hepatocitos/efectos de los fármacos , Animales , Canalículos Biliares/metabolismo , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Colestasis Intrahepática/inducido químicamente , Dexametasona/administración & dosificación , Fluoresceínas/farmacocinética , Hepatocitos/metabolismo , Insulina/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.
Asunto(s)
Canalículos Biliares/efectos de los fármacos , Ácidos Cólicos/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Drogas en Investigación/farmacología , Colorantes Fluorescentes/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Absorción Fisiológica/efectos de los fármacos , Animales , Canalículos Biliares/citología , Canalículos Biliares/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Drogas en Investigación/efectos adversos , Fluoresceínas , Hepatocitos/citología , Hepatocitos/metabolismo , Cinética , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Ratas Wistar , Reproducibilidad de los Resultados , Ácido Taurocólico/metabolismoRESUMEN
Polarized hepatocytes contain tight junctions (TJs), which are among the most important junctions for sealing the bile canalicular lumen from the sinusoidal space. Alterations in TJs are implicated in chronic cholestatic liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis, which have lipid peroxidation marker elevations or antioxidant vitamin decreases. However, the effect of oxidative stress on hepatocyte polarity or liver morphology is unknown. We found that carbon tetrachloride (CCl4)-induced oxidative stress resulted in disassembly of TJs. Ultrastructural analysis revealed disruption in TJs, Golgi morphology, and expansion of the bile canalicular lumen size in CCl4-treated hepatocytes. The Par complex [Par-3-atypical protein kinase C (aPKC) and Par-6 ternary complex] regulates TJs and lumen formation, and the Par-3-aPKC complex formation was inhibited by CCl4 treatment. Moreover, the antioxidant compound vitamin E prohibited a CCl4-induced disturbance in TJs and Par-3-aPKC complex formation. aPKC phosphorylates Par-3 and down-regulates its own affinity with Par-3. Importantly, aPKC kinase activity and Par-3 phosphorylation were significantly increased in CCl4-treated rat livers. These results indicate that the Par-3-aPKC complex plays a crucial role in the maintenance of hepatocyte polarity and sealing of the bile canalicular lumen. Our findings suggest that bile canalicular lumen expansion might explain the presence of cholestasis in patients with primary biliary cirrhosis and primary sclerosing cholangitis.
Asunto(s)
Canalículos Biliares/enzimología , Canalículos Biliares/patología , Tetracloruro de Carbono/toxicidad , Polaridad Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteína Quinasa C/metabolismo , Animales , Canalículos Biliares/efectos de los fármacos , Proteínas Portadoras/metabolismo , Activación Enzimática/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/ultraestructura , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Hígado/ultraestructura , Masculino , Modelos Biológicos , Proteínas del Tejido Nervioso , Ratas Wistar , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Vitamina E/farmacologíaRESUMEN
At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17ß-D-glucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 µM) or TLC (2.5 µM) for 30 min, with or without pre-incubation with DBcAMP (10 µM) for 15 min. Then, the increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.