RESUMO
Despite the utility of cyclooxygenase (COX) inhibition as an antiinflammatory strategy, prostaglandin (PG) products of COX-1 and -2 provide important regulatory functions in some pathophysiological states. Scattered reports suggest that COX inhibition may also promote adverse drug events. Here we demonstrate a protective role for endogenous COX-derived products in a murine model of acetaminophen (APAP)-induced acute liver injury. A single hepatotoxic dose caused the selective induction of COX-2 mRNA and increased PGD2 and PGE2 levels within the livers of COX(+/+) male mice suggesting a role for COX-2 in this model of liver injury. APAP-induced hepatotoxicity and lethality were markedly greater in COX-2(-/-) and (-/+) mice in which normal PG responsiveness is altered. The significantly increased toxicity linked to COX-2 deficiency could be mimicked using the selective COX-2 inhibitory drug, celecoxib, in COX(+/+) mice and was not due to alterations in drug-protein adduct formation, a surrogate for bioactivation and toxicity. Microarray analyses indicated that increased injury associated with COX-2 deficiency coincided, most notably, with a profoundly impaired induction of heat shock proteins in COX-2(-/+) mice suggesting that PGs may act as critical endogenous stress signals following drug insult. These findings suggest that COX-2-derived mediators serve an important hepato-protective function and that COX inhibition may contribute to the risk of drug-induced liver injury, possibly through both nonimmunological and immunological pathways.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Acetaminofen/toxicidade , Animais , Celecoxib , Doença Hepática Induzida por Substâncias e Drogas/mortalidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Primers do DNA/química , Dinoprostona/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Immunoblotting , Isoenzimas/antagonistas & inibidores , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Análise de Sequência com Séries de Oligonucleotídeos , Prostaglandina D2/biossíntese , Pirazóis , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Taxa de SobrevidaRESUMO
Although current studies suggest that most drug-induced allergic reactions (DIARS) are caused by immunogenic conjugates formed from the reaction of a reactive metabolite of a drug with cellular proteins, it is not clear why these reactions are relatively rare. One possible pathway that may explain the low incidence of DIARS in many cases is oral tolerance, an antigen-specific immunological hyporesponsiveness induced by oral administration of antigens. The mechanism of oral tolerance, however, is not clearly understood and is difficult to study directly with drugs, because animal models of DIARS have been elusive. We chose 2,4-dinitrochlorobenzene (DNCB) as a model compound to circumvent this problem because animal models of allergic reactions have been established for this compound. DNCB forms immunogenic 2,4-dinitrophenylated (DNP) protein conjugates that can induce immune reactions and it causes oral tolerance when it is fed to animals prior to sensitization. We hypothesized that DNP-protein conjugates may have a role in oral tolerance. To test this idea, we have begun to identify cells bearing these conjugates after the oral administration of DNCB. Female C57BL/6J mice were fed DNCB and tissues were examined after 6 and 24 h. Immunohistochemical analysis indicated the presence of DNP-protein conjugates in enterocytes of the small intestine, in macrophages and lymphocytes of the mesenteric lymph nodes, in dendritic cells and lymphocytes of the spleen, and in Kupffer cells and other sinusoidal cells of the liver. It was found that Kupffer cell depletion did not affect oral tolerance to DNCB. The findings suggest that the cells bearing DNP-protein conjugates, other than Kupffer cells, in the liver and other tissues may be important in the induction of oral tolerance against DNCB. Protein adducts of drugs administered orally may also be present in these cells, and they may have a role in the downregulation of DIARS in many individuals.
Assuntos
Dinitroclorobenzeno/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Irritantes/efeitos adversos , Células de Kupffer/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Administração Oral , Animais , Dinitroclorobenzeno/imunologia , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Irritantes/imunologia , Células de Kupffer/fisiologia , Fígado/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/químicaAssuntos
Anestésicos Inalatórios/efeitos adversos , Hidrocarboneto de Aril Hidroxilases , Autoanticorpos/sangue , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sistema Enzimático do Citocromo P-450/imunologia , Isoflurano/efeitos adversos , Oxigenases de Função Mista/imunologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Citocromo P-450 CYP2A6 , Ensaio de Imunoadsorção Enzimática , Evolução Fatal , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Halothane causes a mild form of liver injury in guinea pigs that appears to model the hepatotoxicity seen in approximately 20% of patients treated with this drug. In previous studies, it was concluded that the increased susceptibility of some outbred guinea pigs to halothane-induced liver injury is not caused by their inherent ability to metabolize halothane to form toxic levels of trifluoroacetylated protein adducts in the liver. In this study, we reevaluated the role of trifluoroacetylated protein adducts in halothane-induced liver injury in guinea pigs. Male outbred Hartley guinea pigs were treated with halothane intraperitoneally. On the basis of serum alanine aminotransferase levels and liver histology, treated animals were designated as being susceptible, mildly susceptible, or resistant to halothane. Immunoblot studies with the use of anti-trifluoroacetylated antibodies showed that susceptible guinea pigs for the most part had higher levels of trifluoroacetylated protein adducts in the liver 48 h after treatment with halothane than did less susceptible animals. In support of this finding, the level of trifluoroacetylated protein adducts detected immunochemically in the sera of treated guinea pigs correlated with sera levels of alanine aminotransferase activity. In addition, the levels of cytochrome P450 2A-related protein but not those of other cytochrome P450 isoforms, measured by immunoblot analysis with isoform-specific antibodies, correlated with the amount of trifluoroacetylated protein adducts detected in the livers of guinea pigs 8 h after halothane administration. The results of this study indicate that the susceptibility of outbred guinea pigs to halothane-induced liver injury is related to an enhanced ability to metabolize halothane in the liver to form relatively high levels of trifluoroacetylated protein adducts. They also suggest that cytochrome P450 2A-related protein might have a major role in catalyzing the formation of trifluoroacetylated protein adducts in the liver of susceptible guinea pigs. Similar mechanisms may be important in humans.
Assuntos
Anestésicos Inalatórios/toxicidade , Halotano/toxicidade , Proteínas/metabolismo , Ácido Trifluoracético/química , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Cobaias , Humanos , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Drug-induced hepatotoxicity causes significant morbidity and mortality and is a major concern in drug development. This is due, in large part, to insufficient knowledge of the mechanism(s) of drug-induced liver injury. In order to address this problem, we have evaluated the modulation of gene expression within the livers of mice treated with a hepatotoxic dose of acetaminophen (APAP) using high-density oligonucleotide microarrays capable of determining the expression profile of >11,000 genes and expressed sequence tags (ESTs). Significant alterations in gene expression, both positive and negative, were noted within the livers of APAP-treated mice. APAP-induced toxicity affected numerous aspects of liver physiology causing, for instance, >twofold increased expression of genes that encode for growth arrest and cell cycle regulatory proteins, stress-induced proteins, the transcription factor LRG-21, suppressor of cytokine signaling (SOCS)-2-protein, and plasminogen activator inhibitor-1 (PAI-1). A number of these and other genes and ESTs were detectable within the liver only after APAP treatment suggesting their potential importance in propagating or preventing further toxicity. These data provide new directions for mechanistic studies that may lead to a better understanding of the molecular basis of drug-induced liver injury and, ultimately, to a more rational design of safer drugs.
Assuntos
Acetaminofen/toxicidade , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Sequência de Bases , Primers do DNA , Fígado/metabolismo , Camundongos , Dados de Sequência MolecularRESUMO
BACKGROUND & AIMS: Enteropathy is a frequent complication of diclofenac and other nonsteroidal anti-inflammatory drugs, yet little is known about the underlying mechanism. One possibility is that reactive metabolites of diclofenac form adducts with enterocyte macromolecules, as previously shown for liver. We addressed this possibility by using immunohistochemistry to detect diclofenac adducts. METHODS: Rats were treated orally with diclofenac (10-100 mg/kg) and killed after 1-24 hours, and their gastrointestinal (GI) tracts were evaluated for ulcer number and area. Adduct distribution and intensity were assessed by immunohistochemistry by using a technique to simultaneously process and stain multiple intestinal rings. RESULTS: Drug treatment led to dose-dependent formation of both adducts and ulcers only in small intestine and only in animals with intact enterohepatic circulation. Adducts formed within enterocytes by 1 hour, translocated to the brush border, preceded ulceration and vascular protein leakage, and were intense at sites of ulceration. Adducts and ulcers exhibited a parallel distribution within intestinal quintiles: 3rd > 5th >> 1st. CONCLUSIONS: Diclofenac treatment resulted in the formation of drug adducts in enterocytes. Because this molecular change occurred before ulceration, was dose dependent, and exhibited concordant distribution with extent of ulceration, the results suggest a causal role for drug adduct formation in diclofenac enteropathy.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/metabolismo , Diclofenaco/efeitos adversos , Diclofenaco/metabolismo , Enterócitos/metabolismo , Enteropatias/induzido quimicamente , Úlcera/induzido quimicamente , Animais , Bile/metabolismo , Relação Dose-Resposta a Droga , Enteropatias/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Distribuição Tecidual , Úlcera/patologiaAssuntos
Anestésicos Inalatórios/toxicidade , Liases de Carbono-Enxofre/metabolismo , Éteres/toxicidade , Hidrocarbonetos Fluorados/toxicidade , Rim/efeitos dos fármacos , Rim/enzimologia , Anestésicos Inalatórios/metabolismo , Animais , Éteres/metabolismo , Humanos , Hidrocarbonetos Fluorados/metabolismo , Rim/fisiopatologiaRESUMO
Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4'-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirus-expressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Diclofenaco/análogos & derivados , Diclofenaco/metabolismo , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/metabolismo , Esteroide 16-alfa-Hidroxilase , Animais , Anti-Inflamatórios não Esteroides/química , Citocromo P-450 CYP3A , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/metabolismoRESUMO
BACKGROUND: Compound A, a degradation product of sevoflurane, causes renal corticomedullary necrosis in rats. Although the toxicity of this compound was originally hypothesized to result from the biotransformation of its cysteine conjugates into toxic thionoacyl halide metabolites by renal cysteine conjugate beta-lyase, recent evidence suggests that alternative mechanisms may be responsible for compound A nephrotoxicity. The aim of this study was to evaluate these issues by determining whether mercapturates and glutathione conjugates of compound A could produce renal corticomedullary necrosis in rats, similar to compound A, and whether renal covalent adducts of the thionacyl halide metabolite of compound A could be detected immunochemically. METHODS: Male Wistar rats were administered, intraperitoneally, N-acetylcysteine conjugates (mercapturates) of compound A (90 or 180 micromol/kg) or glutathione conjugates of compound A (180 micromol/kg) with or without intraperitoneal pretreatments with aminooxyacetic acid (500 micromol/kg) or acivicin (250 micromol/kg). Rats were killed after 24 h, and kidney tissues were analyzed for toxicity by histologic examination or for protein adducts by immunoblotting or immunohistochemical analysis, using antisera raised against the covalently bound thionoacyl halide metabolite of compound A. RESULTS: Mercapturates and glutathione conjugates of compound A both produced renal corticomedullary necrosis similar to that caused by compound A. Aminooxyacetic acid, an inhibitor of renal cysteine conjugate beta-lyase, did not inhibit the toxicity of the mercapturates, whereas acivicin, an inhibitor of gamma-glutamyltranspeptidase, potentiated the toxicity of both classes of conjugates. No immunochemical evidence for renal protein adducts of the thionacyl halide metabolite was found in rats 24 h after the administration of the mercapturates of compound A or in the kidneys of rats, obtained from a previous study, 5 and 24 h after the administration of compound A. CONCLUSION: The results of this study are consistent with the idea that a mechanism other than the renal cysteine conjugate beta-lyase pathway of metabolic activation is responsible for the nephrotoxicity of compound A and its glutathione and mercapturate conjugates in male Wistar rats.
Assuntos
Anestésicos Inalatórios/toxicidade , Liases de Carbono-Enxofre/toxicidade , Éteres/toxicidade , Hidrocarbonetos Fluorados/toxicidade , Anestésicos Inalatórios/química , Animais , Éteres/química , Hidrocarbonetos Fluorados/química , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Éteres Metílicos/química , Éteres Metílicos/metabolismo , Éteres Metílicos/toxicidade , Necrose , Ratos , Ratos Wistar , SevofluranoRESUMO
Idiosyncratic adverse drug reactions are unpredictable, target multiple organ systems, and often become life-threatening events. Although the causes of idiosyncratic adverse drug reactions are not known in most cases, evidence suggests that they may be mediated through immunological mechanisms. It is generally thought that for a drug to lead to an immune response, it must first become covalently bound to a carrier protein. Since most drugs are unreactive, it is usually a reactive metabolite that is expected to form covalent adducts. However, it is not clear why more people do not develop immune reactions against drug-protein adducts. One possible explanation is that orally administered drugs may lead to oral tolerance in most individuals through mechanisms similar to that found with orally administered antigens. However, very little is known regarding the interaction of drugs with gut-associated lymphoid tissue of the small intestine, where oral tolerance can develop. As an initial step to test this hypothesis, we have investigated whether diclofenac, a commonly used nonsteroidal antiinflammatory drug, can lead to protein adducts in rat small intestine. Diclofenac was administered to rats by gastric gavage. Immunoblot analysis of small intestine homogenates and isolated enterocyte subcellular fractions with drug-specific antiserum revealed 142-, 130-, 110-, and 55-kDa protein adducts of diclofenac. The 142- and 130-kDa adducts of diclofenac were identified as aminopeptidase N (CD13) and sucrase-isomaltase, respectively, by amino acid sequence analyses and by their reactions with protein-specific antibodies. The adducts were localized by immunohistochemistry and found primarily in the mid-villus and villus-tip enterocytes and also in the dome overlying Peyer's patches. Similar adducts were detected immunochemically in villus-tip enterocytes of animals treated with halothane or acetaminophen. These results show that intestinal protein adducts of drugs can be formed in gut-associated lymphoid tissue where they may lead to the down-regulation of drug-induced allergic reactions in many individuals.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/toxicidade , Intestino Delgado/efeitos dos fármacos , Tecido Linfoide/efeitos dos fármacos , Acetaminofen/metabolismo , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/imunologia , Proteínas de Transporte/metabolismo , Diclofenaco/química , Diclofenaco/imunologia , Regulação para Baixo , Hipersensibilidade a Drogas/imunologia , Feminino , Halotano/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Tecido Linfoide/metabolismo , Masculino , Camundongos , Nódulos Linfáticos Agregados/efeitos dos fármacos , Nódulos Linfáticos Agregados/metabolismo , RatosRESUMO
Sulfonamide antimicrobials cause a delayed-onset, hypersensitivity-type syndrome characterized by fever, skin rash and multiorgan toxicity occurring 7 to 14 days after initiation of therapy. The pathogenesis is believed to be immune-mediated. We investigated whether patients with delayed-onset sulfonamide hypersensitivity reactions had antibodies recognizing hapten-microsomal protein conjugates and/or native microsomal proteins. By immunoblotting using rat liver as a source of microsomal protein, 17 of 21 patients had antibodies recognizing one or more of three native endoplasmic reticulum proteins of 55 kDa (14 of 21 patients), 80 kDa (4 of 21 patients) or 96 kDa (3 of 21 patients) in size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No control subjects (n = 11) and only 1 of 18 patients with adverse events not consistent with sulfonamide hypersensitivity reactions had antibodies against these microsomal proteins under the conditions used. Only 1 patient had antibodies that recognized the sulfonamide hapten, sulfamethoxazole. The 55-kDa protein was identified as protein disulfide isomerase. The 80-kDa protein was identified as grp78. The 96-kDa protein was not identified. Delayed-onset sulfonamide hypersensitivity reactions are therefore primarily associated with antibodies recognizing specific protein epitopes and not anti-drug antibodies.
Assuntos
Anticorpos/análise , Hipersensibilidade a Drogas/imunologia , Retículo Endoplasmático/imunologia , Proteínas de Choque Térmico , Hipersensibilidade Tardia/imunologia , Proteínas/imunologia , Sulfonamidas/efeitos adversos , Animais , Anticorpos/imunologia , Proteínas de Transporte/imunologia , Reações Cruzadas , Chaperona BiP do Retículo Endoplasmático , Humanos , Isomerases/imunologia , Masculino , Chaperonas Moleculares/imunologia , Testes de Precipitina , Isomerases de Dissulfetos de Proteínas , Ratos , Ratos Sprague-Dawley , Sulfonamidas/imunologiaRESUMO
BACKGROUND: Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease. METHODS: For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58). FINDINGS: The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers. INTERPRETATION: Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Clorofluorcarbonetos de Metano/efeitos adversos , Clorofluorcarbonetos/efeitos adversos , Exposição Ocupacional/efeitos adversos , Biomarcadores/análise , Biópsia , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Etano Clorofluorcarbonos , Citocromo P-450 CYP2E1/metabolismo , Surtos de Doenças , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Ácido Trifluoracético/metabolismoRESUMO
Diclofenac antiserum was previously developed and used to detect protein adducts of metabolites of dichlofenac in livers of mice and rats. In this study, the antibody has been used to facilitate the purification of a major 51 kDa microsomal adduct of diclofenac from the liver microsomes of male rats that were treated with diclofenac. The adduct was identified as male-specific cytochrome P4502C11 based on its N-terminal amino acid sequence, reaction with a cytochrome P4502C11 antibody, and by its absence from liver microsomes of diclofenac-treated female rats. When diclofenac was incubated with liver microsomes of control rats in the presence of NADPH, only the 51 kDa adduct was produced. The formation of the adduct was inhibited by a cytochrome P4502C11 monoclonal antibody, but not by reduced glutathione or N-alpha-acetyl-L-lysine. No adduct was detected when diclofenac was incubated with liver microsomes from female rats. Moreover, adduct formation in vivo appeared to lead to a 72% decrease in the activity of cytochrome P4502C11. The results indicate that cytochrome P4502C11 metabolizes diclofenac into a highly reactive product that covalently binds to this enzyme before it can diffuse away and react with other proteins.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/metabolismo , Diclofenaco/metabolismo , Inibidores Enzimáticos/metabolismo , Esteroide 16-alfa-Hidroxilase , Esteroide Hidroxilases/metabolismo , Animais , Inibidores das Enzimas do Citocromo P-450 , Diclofenaco/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/antagonistas & inibidores , Especificidade por SubstratoRESUMO
Protein covalent binding by xenobiotic metabolites has long been associated with target organ toxicity but mechanistic involvement of such binding has not been widely demonstrated. Modern biochemical, molecular, and immunochemical approaches have facilitated identification of specific protein targets of xenobiotic covalent binding. Such studies have revealed that protein covalent binding is not random, but rather selective with respect to the proteins targeted. Selective binding to specific cellular target proteins may better correlate with toxicity than total protein covalent binding. Current research is directed at characterizing and identifying the targeted proteins and clarifying the effect of such binding on their structure, function, and potential roles in target organ toxicity. The approaches employed to detect and identify the tartgeted proteins are described. Metabolites of acetaminophen, halothane, and 2,5-hexanedione form covalently bound adducts to recently identified protein targets. The selective binding may influence homeostatic or other cellular responses which in turn contribute to drug toxicity, hypersensitivity, or autoimmunity.
Assuntos
Especificidade de Órgãos/efeitos dos fármacos , Proteínas/metabolismo , Toxicologia/métodos , Xenobióticos/metabolismo , Xenobióticos/toxicidade , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Animais , Ligação Competitiva , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Halotano/metabolismo , Halotano/toxicidade , Hexanonas/metabolismo , Hexanonas/toxicidade , Humanos , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Ligação Proteica , Proteínas/química , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/metabolismoRESUMO
Halothane causes an idiosyncratic hepatitis that is thought to result, in part, from immune reactions against one or more lumenal endoplasmic reticulum (ER) proteins that have been covalently modified by the trifluoroacetyl chloride metabolite of halothane. In this study, we have identified a 170 kDa protein target of halothane in the liver of rats. The 170 kDa protein was first detected when proteins in lysates of hepatocytes from halothane-treated rats were immunoprecipitated with antisera against several resident ER proteins. This 170 kDa protein was found to be associated with other protein targets of halothane, including protein disulfide isomerase, a protein disulfide isomerase isoform, a 59 kDa carboxylesterase, and 78 kDa glucose-regulated protein. Immunoblotting with antiserum directed against the trifluoroacetylated hapten indicated that the 170 kDa protein was trifluoroacetylated. Based upon its subcellular localization, molecular mass, N-terminal amino acid sequence, and antigenicity, the trifluoroacetylated 170 kDa protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGGT), a lumenal ER protein that is thought to have a role in the folding of N-linked glycoproteins. Moreover, treatment of rats with halothane caused a 44% decrease in the activity of liver microsomal UGGT, and at least 36% of the change in the activity of the enzyme could be due to a decrease in the level of the protein. The results suggest that the function of UGGT in folding of N-linked glycoproteins may be affected by other resident ER proteins or xenobiotics such as halothane.
Assuntos
Anestésicos Inalatórios/farmacologia , Retículo Endoplasmático/enzimologia , Glucosiltransferases/antagonistas & inibidores , Halotano/farmacologia , Chaperonas Moleculares/metabolismo , Animais , Glucosiltransferases/análise , Glucosiltransferases/química , Immunoblotting , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Testes de Precipitina , Ratos , Ratos Sprague-Dawley , Ácido TrifluoracéticoRESUMO
Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P450 2C9 and P450 1A2, respectively. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P450 2E1 and appeared to be directed against mainly conformational epitopes. In addition, because cytochrome P450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P450 2E1 may be able to bypass the immunologic tolerance that normally exists against cytochrome P450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.
Assuntos
Autoantígenos/imunologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Citocromo P-450 CYP2E1/imunologia , Halotano/toxicidade , Acetilação , Autoanticorpos/sangue , Flúor/metabolismo , HumanosRESUMO
From the studies that have been done by many laboratories over the last 2 decades, it is now clear that the toxicities produced by many drugs are due to their reactive metabolites. It is though that, in many cases, reactive metabolites cause toxicity by binding covalently to tissue proteins. However, until recently it was difficult to identify these protein targets. Due to the development of an immunochemical approach, this problem has been overcome, as is illustrated here by studies that have been conducted on the metabolic basis of the idiosyncratic hepatitis caused by the inhalation anaesthetic halothane. The major problem to solve in the future will be to determine how protein adduct formation leads to toxicity. It is possible that protein adduct formation may alter an important cellular function or may lead to immunopathology, as is thought to occur in the case of halothane hepatitis. If an allergic reaction is suspected, purified protein targets of reactive metabolites can serve as antigens for identifying sensitized individuals. This information can be used to prevent not only an allergic reaction to the drug, but possible cross-reactions to other drugs that are structurally related. Another important application of these studies is the design of safer alternative drugs that will not produce structurally similar toxic reactive metabolites.
Assuntos
Preparações Farmacêuticas/metabolismo , Farmacologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Humanos , Estrutura Molecular , Preparações Farmacêuticas/química , Relação Estrutura-AtividadeRESUMO
Diclofenac is a nonsteroidal anti-inflammatory drug that has been implicated in several cases of severe hepatotoxicity. Our previous study showed that diclofenac metabolites bound covalently and selectively to rat liver plasma membrane proteins with estimated monomeric masses of 110, 140, and 200 kDa. We report here that we have identified the 110 kDa diclofenac-labeled protein in rat liver as dipeptidyl peptidase IV, also known as CD26. In addition, we found that the activity of dipeptidyl peptidase IV in liver plasma membrane fractions was lowered after diclofenac treatment of rats. These results suggest that the hepatotoxicity associated with diclofenac might be due, in part, to the covalent modification of dipeptidyl peptidase IV.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diclofenaco/farmacologia , Dipeptidil Peptidase 4/efeitos dos fármacos , Fígado/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Diclofenaco/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-DawleyRESUMO
The function of a 58-kDa liver microsomal protein (P58) is controversial. To help clarify the physiological function of this protein, particularly in humans, a full-length human liver cDNA clone was isolated, sequenced, and expressed in milligram quantities with the use of a baculovirus expression system. The deduced amino acid sequence of the mature protein contained two thioredoxin-like active site motifs (CGHC) and in its C-terminus a nuclear localization motif (KPKKKKK), and an ER-retention/retrieval motif (QEDL). The mature form of human P58 shared 95% amino acid sequence identity with the deduced amino acid sequences of a bovine liver cDNA, 93% with a murine B lymphocyte cDNA, and 91% with a rat basophilic leukemia cell cDNA. In contrast to reports on the activities of nonhuman forms of P58, the purified expressed human P58 showed no carnitine acyltransferase or protease activities. However, it did have protein disulfide isomerase activity, indicating that the physiological activity of human liver P58 may be attributed, at least in part, to this activity.