RESUMO
Acetaminophen (APAP) is a well-known analgesic and antipyretic drug. It is considered to be safe when administered within its therapeutic range, but in cases of acute intoxication, hepatotoxicity can occur. APAP overdose is the leading cause of acute liver failure in the northern hemisphere. Historically, studies on APAP toxicity have been focused on liver, with alterations in brain function attributed to secondary effects of acute liver failure. However, in the last decade the pharmacological mechanism of APAP as a cannabinoid system modulator has been documented and some articles have reported "in situ" toxicity by APAP in brain tissue at high doses. Paradoxically, low doses of APAP have been reported to produce the opposite, neuroprotective effects. In this paper we present a comprehensive, up-to-date overview of hepatic toxicity as well as a thorough review of both toxic and beneficial effects of APAP in brain.
Assuntos
Acetaminofen/farmacologia , Acetaminofen/toxicidade , Encéfalo/efeitos dos fármacos , Fígado/efeitos dos fármacos , Acetilcisteína/uso terapêutico , Animais , Encéfalo/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Humanos , Fígado/metabolismoRESUMO
The gastrointestinal epithelium functions as a selective barrier to absorb nutrients, electrolytes and water, but at the same time restricts the passage into the systemic circulation of intraluminal potentially toxic compounds. This epithelium maintains its selective barrier function through the presence of very selective and complex intercellular junctions and the ability of the absorptive cells to reject those compounds. Accordingly, the enterocytes metabolize orally incorporated xenobiotics and secrete the hydrophilic metabolites back into the intestinal lumen through specific transporters localized apically. In the recent decades, there has been increasing recognition of the existence of the intestinal cellular barrier. In the present review we focus on the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) in the apical membrane of the enterocytes, as an important component of this intestinal barrier, as well as on its regulation. We provide a detailed compilation of significant contributions demonstrating that MRP2 expression and function vary under relevant physiological and pathophysiological conditions. Because MRP2 activity modulates the availability and pharmacokinetics of many therapeutic drugs administered orally, their therapeutic efficacy and safety may vary as well.
Assuntos
Intestinos/fisiologia , Intestinos/fisiopatologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Animais , Humanos , Proteína 2 Associada à Farmacorresistência MúltiplaRESUMO
Bilirubin is an endogenous antioxidant with cytoprotective properties, and several studies highlight its potential in the treatment of pro-oxidant diseases. We demonstrated that oxidative stress (OS), a key feature in most hepatopathies, induces cholestasis by actin cytoskeleton disarrangement and further endocytic internalization of key canalicular transporters, such as the bile salt export pump (Bsep) and the multidrug resistance-associated protein 2 (Mrp2) . Here, we evaluated the capability of physiological concentrations of unconjugated bilirubin (UB) to limit OS and the impairment in biliary secretory function induced by the model pro-oxidant agent, tert-butylhydroperoxide (tBuOOH). UB fully prevented the formation of reactive oxygen species and membrane lipid peroxidation induced by tBuOOH in isolated rat hepatocytes. In the isolated rat hepatocyte couplet model, UB (17.1 µM) prevented the endocytic internalization of Bsep and Mrp2 and the impairment in their secretory function induced by tBuOOH. UB also prevented actin disarrangement, as evaluated by both plasma membrane bleb formation and actin fluorescent staining. Finally, UB prevented tBuOOH-induced cPKC activation. Experiments in isolated perfused rat livers showed that UB prevents the increase in oxidized glutathione biliary excretion and the drop in bile flow and the biliary excretion of specific Bsep and Mrp2 substrates. We conclude that physiological concentrations of UB are sufficient to prevent the biliary secretory failure induced by OS, by counteracting actin disarrangement and the consequent internalization of canalicular transporters relevant to normal bile formation. This reveals an important role for UB in preserving biliary secretory function under OS conditions.
Assuntos
Bilirrubina/farmacologia , Colestase/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Actinas/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Bilirrubina/metabolismo , Colestase/metabolismo , Glutationa/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Técnicas de Cultura de Órgãos , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Wistar , terc-Butil Hidroperóxido/farmacologiaRESUMO
Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.
Assuntos
Colestase/metabolismo , Receptor alfa de Estrogênio/agonistas , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Fígado/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Animais , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Células Cultivadas , Colestase/genética , Dactinomicina/farmacologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Fulvestranto , Glutationa/metabolismo , Fígado/metabolismo , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Regulação para CimaRESUMO
The ability of the liver, small intestine, and kidney to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), was assessed in rats treated with glucagon-like peptide 2 (GLP-2, 12 µg/100 g b.wt. s.c. every 12 h for 5 consecutive days). An in vivo perfused jejunum model with simultaneous bile and urine collection was used. A single intravenous dose of 30 µmol/kg b.wt. 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its conjugate, DNP-SG, and dinitrophenyl cysteinyl glycine (DNP-CG), resulting from the action of γ-glutamyltransferase on DNP-SG, were determined in bile, intestinal perfusate, and urine by high-performance liquid chromatography. Tissue content of DNP-SG was also assessed in liver, intestine, and kidneys. Biliary excretion of DNP-SG+DNP-CG was decreased in GLP-2 rats with respect to controls. In contrast, their intestinal excretion was substantially increased, whereas urinary elimination was not affected. Western blot and real-time polymerase chain reaction studies revealed preserved levels of Mrp2 protein and mRNA in liver and renal cortex and a significant increase in intestine in response to GLP-2 treatment. Tissue content of DNP-SG detected 5 min after CDNB administration was decreased in liver, increased in intestine, and unchanged in kidney in GLP-2 versus control group, consistent with GLP-2-induced down-regulation of expression of glutathione transferase (GST) Mu in liver and up-regulation of GST-Alpha in intestine at both protein and mRNA levels. In conclusion, GLP-2 induced selective changes in hepatic and intestinal disposition of a common GST and Mrp2 substrate administered systemically that could be of pharmacological or toxicological relevance under therapeutic treatment conditions.
Assuntos
Dinitroclorobenzeno/farmacocinética , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Jejuno/metabolismo , Rim/metabolismo , Fígado/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bile/metabolismo , Dinitrobenzenos/metabolismo , Dinitroclorobenzeno/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Jejuno/efeitos dos fármacos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacos , gama-Glutamiltransferase/metabolismoRESUMO
UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.
Assuntos
1-Fosfatidilinositol 4-Quinase/fisiologia , Colestase/induzido quimicamente , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Androstadienos/farmacologia , Animais , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/fisiologia , Sistema Biliar/metabolismo , Carbazóis/farmacologia , Colchicina/farmacologia , Endocitose/efeitos dos fármacos , Estradiol/análogos & derivados , Glutationa/metabolismo , Técnicas In Vitro , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Perfusão , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais , Ácido Taurocólico/metabolismo , WortmaninaRESUMO
The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 µg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2',3'-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Inibidores de Adenilil Ciclases , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Didesoxiadenosina/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/enzimologia , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Imunofluorescência , Peptídeo 2 Semelhante ao Glucagon/fisiologia , Glutationa Transferase/biossíntese , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/enzimologia , Jejuno/metabolismo , Jejuno/patologia , Lactação/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: MRP2 is an intestinal ABC transporter that prevents the absorption of dietary xenobiotics. The aims of this work were: (1) to evaluate whether a short-term regulation of intestinal MRP2 barrier function takes place in vivo after luminal incorporation of nutrients and (2) to explore the underlying mechanism. METHODS: MRP2 activity and localization were assessed in an in vivo rat model with preserved irrigation and innervation. Nutrients were administered into distal jejunum. After 30-minutes treatments, MRP2 activity was assessed in proximal jejunum by quantifying the transport of the model substrate 2,4-dinitrophenyl-S-glutathione. MRP2 localization was determined by quantitative confocal microscopy. Participation of extracellular mediators was evaluated using selective inhibitors and by immunoneutralization. Intracellular pathways were explored in differentiated Caco-2 cells. RESULTS: Oleic acid, administered intraluminally at dietary levels, acutely stimulated MRP2 insertion into brush border membrane. This was associated with increased efflux activity and, consequently, enhanced barrier function. Immunoneutralization of the gut hormone glucagon-like peptide-2 (GLP-2) prevented oleic acid effect on MRP2, demonstrating the participation of this trophic factor as a main mediator. Further experiments using selective inhibitors demonstrated that extracellular adenosine synthesis and its subsequent binding to enterocytic A2B adenosine receptor (A2BAR) take place downstream GLP-2. Finally, studies in intestinal Caco-2 cells revealed the participation of A2BAR/cAMP/PKA intracellular pathway, ultimately leading to increased MRP2 localization in apical domains. CONCLUSION: These findings reveal an on-demand, acute regulation of MRP2-associated barrier function, constituting a novel physiological mechanism of protection against the absorption of dietary xenobiotics in response to food intake.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Peptídeo 2 Semelhante ao Glucagon , Animais , Células CACO-2 , Humanos , Mucosa Intestinal , Nutrientes , Ratos , Ratos WistarRESUMO
UNLABELLED: The endogenous estradiol metabolite estradiol 17beta-D-glucuronide (E(2)17G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance-associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca(2+)-dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E(2)17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E(2)17G (2 mumol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [(3)H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose-response studies using IRHC, E(2)17G (3.75-800 muM) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl-lysylfluorescein with an IC50 of 54.9 +/- 7.9 muM. Gö6976 (1 muM) increased the IC50 to 178.4 +/- 23.1 muM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 coincided with complete protection against E(2)17G-induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E(2)17G also increased paracellular permeability in IRHC, which was only partially prevented by Gö6976. The cPKC isoform PKCalpha, but not the Ca(2+)-independent PKC isoform, PKCepsilon, translocated to the plasma membrane after E(2)17G administration in primary cultured rat hepatocytes; Gö6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E(2)17G, as detected via western blotting. CONCLUSION: Our findings support a central role for cPKC isoforms in E(2)17G-induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route.
Assuntos
Cálcio/metabolismo , Colestase/induzido quimicamente , Colestase/metabolismo , Estradiol/análogos & derivados , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Carbazóis/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Estradiol/efeitos adversos , Estradiol/farmacologia , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-épsilon/antagonistas & inibidores , Ratos , Ratos Sprague-DawleyRESUMO
The effect of the cholestatic estrogens ethynylestradiol (EE) and estradiol 17beta-D-glucuronide (E2-17G) on expression and activity of intestinal multidrug resistant-associated protein 2 (Mrp2, Abcc2) was studied in rats. Expression and localization of Mrp2 were evaluated by Western blotting, real-time polymerase chain reaction, and confocal immunofluorescence microscopy. Mrp2 transport activity toward dinitrophenyl-S-glutathione (DNP-SG) was assessed in vitro in intestinal sacs. EE, administered subcutaneously at a 5 mg/kg b.wt. dose, for 5 consecutive days, produced a marked decrease in Mrp2 expression at post-transcriptional level, without affecting its normal localization at the apical membrane of the enterocyte. This effect was selective because expression of other ATP-binding cassette proteins such as breast cancer resistance protein and Mrp3 were not affected and that of multidrug resistance protein 1 was only minimally impaired. Consistent with down-regulation of expression of Mrp2, a significant impairment in serosal to mucosal transport of DNP-SG and in protection against absorption of this same compound were registered. Simultaneous administration of EE with spironolactone (200 micromol/kg b.wt./day for 3 days), an Mrp2 inducer, prevented these alterations, confirming down-regulation of expression of Mrp2 by EE as a major component of functional changes. Incorporation of E2-17G (30 microM) in the serosal medium of intestinal sacs decreased serosal to mucosal transport of DNP-SG, probably because of competitive inhibition, without affecting normal Mrp2 expression or localization. Our data indicate impairment of function of intestinal Mrp2 by both cholestatic estrogens, although through a different mechanism. This finding represents an aggravation of deteriorated hepatic Mrp2 function that could further increase bioavailability of specific xenobiotics after oral exposure.
Assuntos
Colestase/metabolismo , Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Mucosa Intestinal/metabolismo , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ratos WistarRESUMO
Complement, an important effector mechanism of the immune system, is an enzymatic cascade of approx. 30 serum proteins leading to the amplification of a specific humoral response. It can be activated through the classical or alternative pathways, or through the mannose-binding lectin pathway. The activation of the classical pathway is initiated by the binding of the C1 component to antigen-bound antibodies, known as immunocomplexes. C1 is a complex of one molecule of C1q, two molecules of C1r and two molecules of C1s. C1q contains three copies of a Y-shaped fundamental unit with globular heads included in its structure, which play a major role in the interaction with the Fc portion of immunoglobulins. Deficient or exacerbated activation of the complement system leads to diseases of variable severity, and pharmacological inhibition of the complement system is considered as a therapeutic strategy to ameliorate the inflammatory effects of exacerbated complement activation. Bilirubin is a product of haem degradation by the concerted action of haem oxygenase, which converts haem into biliverdin, and biliverdin reductase, which reduces biliverdin to UCB (unconjugated bilirubin). UCB exerts both cytoprotective and cytotoxic effects in a variety of tissues and cells, acting either as an antioxidant at low concentrations or as an oxidant at high concentrations. In the present review, we describe in detail the anti-complement properties of bilirubin, occurring at levels above the UCB concentrations found in normal human serum, as a beneficial effect of potential clinical relevance. We provide evidence that UCB interferes with the interaction between C1q and immunoglobulins, thus inhibiting the initial step in the activation of complement through the classical pathway. A molecular model is proposed for the interaction between UCB and C1q.
Assuntos
Via Clássica do Complemento/imunologia , Hiperbilirrubinemia/imunologia , Inflamação/prevenção & controle , Antioxidantes/farmacologia , Bilirrubina/farmacologia , Bilirrubina/fisiologia , Complemento C1q/metabolismo , Inativadores do Complemento/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Citoproteção/fisiologia , Humanos , Inflamação/imunologia , Estresse Oxidativo/imunologiaRESUMO
Multidrug resistance-associated protein 2 (MRP2/ABCC2), a hepatocyte canalicular transporter involved in bile secretion, is downregulated in cholestasis triggered by lipopolysaccharide. The human aquaporin-1 (hAQP1) adenovirus-mediated gene transfer to liver improves cholestasis by incompletely defined mechanisms. Here we found that hAQP1 did not affect MRP2/ABCC2 expression, but significantly increased its transport activity assessed in situ with endogenous and exogenous substrates, likely by a hAQP1-induced increase in canalicular membrane cholesterol amount. Our results suggest that hAQP1-induced MRP2/ABCC2 activation contributes to the cholestasis improvement.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aquaporina 1/fisiologia , Bile/metabolismo , Colestase/metabolismo , Hepatócitos/metabolismo , Animais , Aquaporina 1/genética , Colestase/terapia , Técnicas de Transferência de Genes , Hepatócitos/citologia , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Ratos WistarRESUMO
Hyperbilirubinemia and complement-mediated immune attack on hepatocyte membrane are common features of certain hepatic diseases. To assess whether unconjugated bilirubin (UB) counteracts complement-mediated hepatocytolysis, we first generated a rabbit polyclonal antibody (Ab) against rat hepatocyte plasma membrane (RHPM). An assay performed with isolated rat hepatocytes in the presence of the polyclonal Ab and rat serum as complement donor demonstrated that UB inhibits cell lysis, as lactate dehydrogenase release into the medium was inhibited by the pigment in a dose-dependent manner. Immunofluorescence microscopy studies showed that UB significantly attenuates the binding of C3 to the hepatocyte-Ab complex. Further enzyme immunoassay studies showed that UB interferes the binding of C1q to purified anti-RHPM IgG, also in a dose-dependent manner. A dot-blot assay showed that [14C]-UB binds to C1q and human serum albumin (HSA) to a similar extent. A differential spectrum analysis of UB in the presence of C1q further confirmed that the pigment interacts with this protein. In conclusion, we demonstrated an inhibitory action of UB on complement-mediated Ab-induced hepatocytolysis, this action being evidenced at pathophysiological pigment concentrations (171 microM and higher). A direct binding of the pigment to C1q is likely involved.
Assuntos
Bilirrubina/farmacologia , Membrana Celular/efeitos dos fármacos , Complemento C1q/metabolismo , Proteínas Inativadoras do Complemento/farmacologia , Hepatócitos/efeitos dos fármacos , Animais , Anticorpos/imunologia , Bilirrubina/metabolismo , Membrana Celular/imunologia , Células Cultivadas , Complemento C1q/imunologia , Relação Dose-Resposta Imunológica , Técnicas Imunoenzimáticas , L-Lactato Desidrogenase/metabolismo , Masculino , Microscopia de Fluorescência , Ratos , Ratos WistarRESUMO
Renal and intestinal disposition of acetaminophen glucuronide (APAP-GLU), a common substrate for multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), was assessed in bile duct-ligated rats (BDL) 7 days after surgery using an in vivo perfused jejunum model with simultaneous urine collection. Doses of 150 mg/kg b.w. (i.v.) or 1 g/kg b.w. (i.p.) of acetaminophen (APAP) were administered, and its glucuronide was determined in bile (only Shams), urine, and intestinal perfusate throughout a 150-min period. Intestinal excretion of APAP-GLU was unchanged or decreased (-58%) by BDL for the 150 mg and 1 g/kg b.w. doses of APAP, respectively. In contrast, renal excretion was increased by 200 and 320%, respectively. Western studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, whereas Mrp3 was substantially increased in liver and not affected in kidney or intestine. The global synthesis of APAP-GLU, determined as the sum of cumulative excretions, was higher in BDL rats (+51 and +110%) for these same doses of APAP as a consequence of a significant increase in functional liver mass, with no changes in specific glucuronidating activity. Expression of apical breast cancer resistance protein, which also transports nontoxic metabolites of APAP, was decreased by BDL in liver and renal cortex, suggesting a minor participation of this route. We demonstrate a more efficient hepatic synthesis and basolateral excretion of APAP-GLU followed by its urinary elimination in BDL group, the latter two processes consistent with up-regulation of liver Mrp3 and renal Mrp2.
Assuntos
Acetaminofen/análogos & derivados , Acetaminofen/metabolismo , Fígado/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/administração & dosagem , Acetaminofen/urina , Animais , Ductos Biliares/cirurgia , Relação Dose-Resposta a Droga , Mucosa Intestinal/metabolismo , Rim/metabolismo , Ligadura , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ratos WistarRESUMO
Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.
Assuntos
Dapsona/farmacologia , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Feminino , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
The canalicular membrane represents the excretory pole of hepatocytes. Bile is an important route of elimination of potentially toxic endo- and xenobiotics (including drugs and toxins), mediated by the major canalicular transporters: multidrug resistance protein 1 (MDR1, ABCB1), also known as P-glycoprotein, multidrug resistance-associated protein 2 (MRP2, ABCC2), and the breast cancer resistance protein (BCRP, ABCG2). Their activities depend on regulation of expression and proper localization at the canalicular membrane, as regulated by transcriptional and post-transcriptional events, respectively. At transcriptional level, specific nuclear receptors (NR)s modulated by ligands, co-activators and co-repressors, mediate the physiological requirements of these transporters. This complex system is also responsible for alterations occurring in specific liver pathologies. We briefly describe the major Class II NRs, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), and their role in regulating expression of multidrug resistance proteins. Several therapeutic agents regulate the expression of relevant drug transporters through activation/inactivation of these NRs. We provide some representative examples of the action of therapeutic agents modulating liver drug transporters, which in addition, involve CAR or PXR as mediators.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos dos fármacos , Hepatopatias/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Acetaminofen/farmacologia , Acetaminofen/uso terapêutico , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Receptores Citoplasmáticos e Nucleares/metabolismo , Esteroides/farmacologia , Esteroides/uso terapêuticoRESUMO
Vesicle-based trafficking of hepatocellular transporters involves delivery of the newly-synthesized carriers from the rough endoplasmic reticulum to either the plasma membrane domain or to an endosomal, submembrane compartment, followed by exocytic targeting to the plasma membrane. Once delivered to the plasma membrane, the transporters usually undergo recycling between the plasma membrane and the endosomal compartment, which usually serves as a reservoir of pre-existing transporters available on demand. The balance between exocytic targeting and endocytic internalization from/to this recycling compartment is therefore a chief determinant of the overall capability of the liver epithelium to secrete bile and to detoxify endo and xenobiotics. Hence, it is a highly regulated process. Impaired regulation of this balance may lead to abnormal localization of these transporters, which results in bile secretory failure due to endocytic internalization of key transporters involved in bile formation. This occurs in several experimental models of hepatocellular cholestasis, and in most human cholestatic liver diseases. This review describes the molecular bases involved in the biology of the dynamic localization of hepatocellular transporters and its regulation, with a focus on the involvement of signaling pathways in this process. Their alterations in different experimental models of cholestasis and in human cholestatic liver disease are reviewed. In addition, the causes explaining the pathological condition (e.g. disorganization of actin or actin-transporter linkers) and the mediators involved (e.g. activation of cholestatic signaling transduction pathways) are also discussed. Finally, several experimental therapeutic approaches based upon the administration of compounds known to stimulate exocytic insertion of canalicular transporters (e.g. cAMP, tauroursodeoxycholate) are described.
Assuntos
Colestase/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Bile/metabolismo , Membrana Celular/metabolismo , Colagogos e Coleréticos/uso terapêutico , Colestase/tratamento farmacológico , Modelos Animais de Doenças , Endocitose , Retículo Endoplasmático Rugoso/metabolismo , Endossomos/metabolismo , Exocitose , Humanos , Fígado/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Transporte Proteico , Transdução de Sinais , Xenobióticos/metabolismoRESUMO
The mechanisms involved in spironolactone (SL, 200 micromol/kg body weight, 3 days i.p.)-induced choleresis were explored in vivo by evaluating bile salt export pump (Bsep)-, multidrug resistance-associated protein 2 (Mrp2)-, and anion exchanger 2 (AE2)-mediated secretory processes in rat liver. Hepatic bile salt metabolism was also analyzed. Total bile flow was significantly increased by SL, primarily due to an increase in bile salt-independent bile flow, whereas bile salt secretion was decreased. SL did not produce any choleresis in TR(-) rats. SL decreased the de novo bile salt synthesis rate in concordance with impaired microsomal cholesterol 7 alpha-hydroxylase activity, thus leading to a decrease in endogenous bile salt pool size. In contrast, the maximum secretory rate of tauroursodeoxycholate as well as expression of Bsep protein detected by Western blotting were not affected. Thus, decreased bile salt availability for canalicular transport rather than transport capability itself likely explains reduced biliary secretion of bile salts. Biliary secretion of glutathione, an endogenous substrate of Mrp2, and HCO(3)(-), the AE2 substrate, were increased by SL, as a main factor explaining enhanced bile salt-independent bile flow. Western blot studies revealed increased expression of Mrp2 in response to SL whereas AE2 content remained unchanged. Enhanced activity and expression of Mrp2 was confirmed by analyzing the excretion rate of dinitrophenyl S-glutathione, an exogenous substrate of Mrp2, in isolated hepatocytes and by immunofluorescence microscopy, respectively. We conclude that SL increased bile flow mainly by increasing the biliary secretion of glutathione species and HCO(3)(-); increased expression of Mrp2 is also involved.
Assuntos
Bile/efeitos dos fármacos , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Espironolactona/farmacologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/fisiologia , Antiporters/genética , Antiporters/fisiologia , Bile/metabolismo , Transporte Biológico , Hepatócitos/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ratos , Ratos Wistar , Proteínas SLC4ARESUMO
The ability of the liver and small intestine for secretion of dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), into bile and lumen, respectively, as well as expression of Mrp2 in both tissues, were assessed in 70-75% hepatectomized rats. An in vivo perfused intestinal model was used. A single i.v. dose of 30 micromol/kg b.w. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered and its glutathione conjugate, DNP-SG, was determined by HPLC in bile and intestinal perfusate. One and seven days after hepatectomy, biliary excretion of DNP-SG was decreased by 90 and 50% with respect to shams, respectively, when expressed per mass unit. In contrast, intestinal excretion was increased by 63% or unchanged one and seven days post-hepatectomy, respectively. Tissue content of DNP-SG 5 min after CDNB administration was substantially decreased in liver and significantly increased in intestine, one day post-hepatectomy. Western and immunofluorescence studies revealed preserved levels and localization of Mrp2 in both tissues from hepatectomized animals, irrespective of the time analyzed. In spite of preserved expression of Mrp2, the higher availability of DNP-SG in intestinal cells, likely as a consequence of increased glutathione-S-transferase-mediated conjugation of CDNB, may explain the in vivo findings. Further experiments in isolated hepatocytes suggested that decreased synthesis of DNP-SG rather than altered canalicular transport is responsible for the substantial impairment in excretion of this compound into bile. Taken together, these results indicate that the intestine may partially compensate for liver DNP-SG disposition, particularly shortly after surgery, while liver capability is recovering.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Glutationa/análogos & derivados , Glutationa/metabolismo , Hepatectomia , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Animais , Bile/metabolismo , Transporte Biológico Ativo , Biotransformação , Western Blotting , Densitometria , Dinitroclorobenzeno/metabolismo , Imunofluorescência , Hepatócitos/metabolismo , Técnicas In Vitro , Masculino , Tamanho do Órgão , Ratos , Ratos WistarRESUMO
The effects of dapsone (DDS) treatment (30 mg/kg body wt, twice a day, for 4 days) on biliary secretory function, with special emphasis on bile salt independent bile flow (BSIF), were investigated in male and in female Wistar rats. Because DDS is metabolized to its N-hydroxylated parent compound only in male rats, any gender difference in DDS effect can be causally attributed to this metabolite. The two main driving forces for BSIF, the biliary secretion of HCO(3)(-) and glutathione species, were assessed. BSIF was decreased by about 20% in male but not in female rats after DDS treatment. Basal biliary HCO(3)(-) secretion was decreased also by 20% in males. This was associated with a diminished (-37%) expression of the HCO(3)(-) canalicular transporter, anion exchanger 2 (AE2), detected by western blotting. Biliary output of reduced glutathione (GSH) was not modified by DDS irrespective of gender, whereas excretion of oxidized glutathione (GSSG) was increased by 830% in males. This latter finding confirmed a gender-dependent oxidative stress associated with formation of the N-hydroxylated metabolite of DDS. The expression of multidrug resistance-associated protein 2 (Mrp2), a putative transporter of glutathione species, was decreased by 38% as detected by western blotting, clearly dissociating from preserved or increased biliary excretion of GSH and GSSG. In conclusion, our results show an impairment of BSIF by DDS mainly due to a decreased AE2-mediated biliary excretion of HCO(3)(-), formation of the N-hydroxylated metabolite of DDS being a likely mediator. The clinical relevance of these findings is discussed.