RESUMO
Repeated stress led to antipodal directions in immune system and cytochrome P450 activities of normotensive and hypertensive rats. Enhancement of the Reaction of Delayed Hypersensitivity, suppression of cytochrome P450-mediated monooxigenase activities were observed in Wistar rats. On the contrary, in the NISAG decrease of the Reaction Delayed Hypersensitivity, elevation of cytochrome P450-mediated monooxigenase activities were observed, as comparison with Wistar rats.
Assuntos
Hipersensibilidade Tardia/imunologia , Hipertensão/imunologia , Oxigenases de Função Mista/imunologia , Estresse Fisiológico/imunologia , Animais , Hidrocarboneto de Aril Hidroxilases/imunologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP1A1/imunologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/imunologia , Citocromo P-450 CYP2B1/metabolismo , Hipersensibilidade Tardia/enzimologia , Hipertensão/enzimologia , Oxigenases de Função Mista/metabolismo , Ratos , Ratos Wistar , Esteroide Hidroxilases/imunologia , Esteroide Hidroxilases/metabolismo , Estresse Fisiológico/enzimologiaRESUMO
Cytochrome P4502B is an isoform of cytochrome P450 (P450) that is induced by the anticonvulsant drug phenobarbital. Here, we demonstrate the constitutive expression and predominant localization of CYP2B in neurons of rat brain. Administration of phenobarbital to rats resulted in selective induction of P450 levels in cortex and midbrain, while other regions were unaffected. Immunohistochemical localization of P4502B in brains of phenobarbital treated rats revealed localization of P4502B in neuronal cells, most predominantly the reticular neurons in midbrain. The anticancer agent 9-methoxy-N(2)-methylellipticinium acetate (MMEA) has been shown to exhibit preferential neuronal toxicity in vitro. Pretreatment of rats with phenobarbital potentiated the toxicity of intrathecally administered MMEA in vivo, as seen by the degeneration of reticular neurons. Thus, induction of P450 in selective regions of brain by phenobarbital would profoundly influence xenobiotic metabolism in these regions, especially in clinical situations where phenobarbital is coadministered with other psychoactive drugs/xenobiotics.
Assuntos
Anticonvulsivantes/farmacologia , Hidrocarboneto de Aril Hidroxilases , Encéfalo/enzimologia , Sistema Enzimático do Citocromo P-450/biossíntese , Síndromes Neurotóxicas/enzimologia , Fenobarbital/farmacologia , Oxirredutases do Álcool , Animais , Antineoplásicos/toxicidade , Encéfalo/efeitos dos fármacos , Citocromo P-450 CYP2B1/biossíntese , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/imunologia , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450 , Elipticinas/toxicidade , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Mesencéfalo/patologia , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/patologia , Oxirredutases N-Desmetilantes/biossíntese , Oxirredutases N-Desmetilantes/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Ativação TranscricionalRESUMO
1. The study aimed to ascertain the enzyme kinetic basis for breed differences in the biotransformation of propofol in dog and to identify the responsible canine cytochrome P450 (CYP) isoenzymes. 2. The NADPH-dependent formation of 4-hydroxypropofol (the rate-limiting biotransformation in dog) was assayed using hepatic microsomes from the male greyhound and beagle, and from both sexes in mixed-breed dogs (five of each). 3. Enzyme kinetic analysis revealed that whereas there were no significant differences in Km, Vmax averaged > 3-fold lower in greyhound compared with beagle (p = 0.032). Although average Vmax was > 3-fold higher in the male compared with female mixed-breed dogs, this difference did not achieve statistical significance (p = 0.095), probably because of the high variability of data from mixed-breed dogs. 4. Chloramphenicol (a specific CYP2B11 inhibitor) and diethyldithiocarbamate (a non-specific CYP2 inhibitor) inhibited propofol hydroxylation in all microsomes. Quinine (a CYP2D15 inhibitor) was also inhibitory, but only in one-half of the microsomes examined. Immuno-inhibition by anti-CYP2B1 sera resulted in > 50% reduction in metabolite formation in all dogs except mixed-breed females, which showed a 30% reduction. Differences in propofol hydroxylase activity between microsomal preparations were primarily attributed to a component that was sensitive to inhibition by chloramphenicol and anti-CYP2B1 sera. 5. The results indicate that propofol hydroxylation in dog is primarily mediated by CYP2B11 and that breed (and possibly gender) differences in propofol metabolism may result from differences in the liver content of this CYP.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/metabolismo , Propofol/metabolismo , Esteroide Hidroxilases , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Biotransformação/efeitos dos fármacos , Cruzamento , Cloranfenicol/farmacologia , Citocromo P-450 CYP2B1/imunologia , Inibidores das Enzimas do Citocromo P-450 , Família 2 do Citocromo P450 , Ditiocarb/farmacologia , Cães , Feminino , Hidroxilação , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , NADP/metabolismo , Propofol/farmacocinética , Quinina/farmacologia , Caracteres Sexuais , Especificidade da EspécieRESUMO
We have previously produced 12 monoclonal antibodies (MAbs) against rat cytochrome P450(CYP)2B1, and 8 of these inhibit CYP2B1 catalytic activity to varying extents. Using competitive binding studies we showed that this collection of 12 MAbs recognize at least 6 spatially distinct epitopes. The complete coding sequence of CYP2B1 DNA in plasmid pSR-P450 was inserted into a glutathione S-transferase (GST) expression vector pGEX-1lambdaT so that it was in frame with the GST gene. Expression of GST-CYP2B1 was detected with most of the MAbs in Western blots except those which were conformation-specific. Fourteen different constructs were then made using PCR with oligonucleotide primers having EcoRI sites at their ends and were introduced into the GST expression vector at the EcoRI site. Each fusion construct was expressed in Escherichia coli, subjected to SDS-PAGE, blotted, and probed individually with each MAb. MAbs, which inhibited catalytic activity and were mutually competitive in binding to CYP2B1 (viz. BEA33, BE44, BE45, and BE28), recognized several fusion constructs and by deduction recognized amino acids 250-261 in CYP2B1. Other antibodies inhibiting catalytic activity recognized amino acids 262-272 (BEF29) and 306-491 (BE46, B50, and BE49) on CYP2B1. Non-inhibitory MAbs BE26 and BE32 were mapped to region 380-398 in CYP2B1. It was interesting to note that MAbs BEA33 and BE26, which also recognize spatially distinct epitopes on human CYP2E1 but not rat CYP2E1, had corresponding regions of high homology in human CYP2E1 but not rat CYP2E1. Identifying the epitopes recognized by this collection of MAbs will add to our understanding the sequences that may be important for producing inhibitory and specific antibodies to closely related antigens.
Assuntos
Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/imunologia , Mapeamento de Epitopos , Glutationa Transferase/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Western Blotting , Catálise , Citocromo P-450 CYP2B1/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Citocromo P-450 CYP2B1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Metilcolantreno/farmacologia , Fenobarbital/farmacologia , Esteroide Hidroxilases/metabolismo , Animais , Northern Blotting , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B1/análise , Citocromo P-450 CYP2B1/imunologia , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/imunologia , Citoplasma/química , Técnicas Imunoenzimáticas , Hibridização In Situ , Fígado/efeitos dos fármacos , Masculino , Microssomos Hepáticos/imunologia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/análise , Esteroide Hidroxilases/imunologiaRESUMO
Previous studies have suggested that degradation of the two stereoisomers of chlorpheniramine in the liver might be catalysed by different types of cytochrome P450. Stereoselective N-demethylation of chlorpheniramine and the involvement of cytochrome P450 (CYP) isozymes have, therefore, been investigated in the liver microsomes of eight-week-old male rats. Incubation of racemic chlorpheniramine with liver microsomes from the male rat resulted in the formation of both enantiomers of monodesmethylchlorpheniramine (DMChp). Further metabolism of DMChp to didesmethylchlorpheniramine (DDMChp) did not, however, occur. The S/R enantiomeric ratio for intrinsic clearance (Vmax/Km) was approximately 2.0, suggesting that the N-demethylation was stereoselective for S-(+)-chlorpheniramine. On the other hand, although the Vmax/Km value for the formation of S-(+)- and R-(-)-DMChp increased with phenobarbitone-inducible rat-liver microsomes, there was no difference between the rates of N-demethylation of the enantiomers. In contrast, 3-methylcholanthrene reduced the intrinsic clearance of S-(+)-chlorpheniramine by N-demethylation and increased its value for R-(-)-chlorpheniramine, showing no stereoselectivity for the N-demethylation of chlorpheniramine. The difference between the intrinsic clearance of the two enantiomers by N-demethylation was because of differences in affinity for the catalysing enzyme. This is indicative of stereoselective involvement of the main enzyme concerned in the N-demethylation of the enantiomers, considered to be CYP 2C11. Anti-CYP 2C11 also partially inhibited the N-demethylation of racemic chlorpheniramine in rat-liver microsomes exposed to phenobarbitone and 3-methylcholanthrene. That CYP 2B1 was involved in the N-demethylation of both enantiomers was also supported by results from an experiment using phenobarbitone-inducible rat-liver microsomes. CYP1A1 did not, however, catalyse the N-demethylation of either enantiomer. These results indicate that N-demethylation of the S-(+)-enantiomer of chlorpheniramine occurs preferentially in the microsomes, demonstrating the stereoselective contribution of CYP2C11. Immunoinhibition studies suggest, moreover, that the N-demethylation of both chlorpheniramine enantiomers is catalysed by CYP2B1, but not by CYP1A1.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Clorfeniramina/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Antagonistas dos Receptores Histamínicos H1/farmacocinética , Isoenzimas/metabolismo , Microssomos Hepáticos/enzimologia , Esteroide 16-alfa-Hidroxilase , Animais , Anticorpos/imunologia , Citocromo P-450 CYP1A1/imunologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2B1/imunologia , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/imunologia , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/imunologia , Família 2 do Citocromo P450 , Técnicas In Vitro , Isoenzimas/imunologia , Masculino , Metilcolantreno/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Fenobarbital/farmacologia , Ratos , Ratos Wistar , Estereoisomerismo , Esteroide Hidroxilases/imunologia , Esteroide Hidroxilases/metabolismoRESUMO
Phencyclidine (PCP) inactivates the 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity of P4502B1 in a reconstituted system containing NADPH-cytochrome P450 (P450) reductase (reductase) and L-alpha-phosphatidylcholine, dilauroyl in a time-, concentration-, and NADPH-dependent manner. Catalytic activity of the enzyme could not be restored upon reconstitution with fresh reductase, indicating that the effect was on the P450 and not on the reductase. Although the kinetics suggested that PCP would be classified as a classical mechanism-based inactivator, protection against inactivation of P450 by PCP by the presence of an exogenous nucleophile, such as glutathione (GSH), indicated otherwise. There was no loss of spectrally detectable P450 associated with inactivation either in the presence or absence of GSH. When radiolabeled PCP was used to inactivate the enzyme and the reaction mixture analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found to be associated with P450, reductase, and catalase that had been added to protect against oxidative damage. When GSH was included in the reaction mixtures, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a marked decrease in the binding to all three proteins. Correspondingly, analysis of the components of the inactivated sample by reversed-phase HPLC demonstrated that radioactivity was associated with P450, reductase, and catalase, and that there was a marked decrease in the labeling of all three proteins in the presence of GSH. The stoichiometry of binding of radiolabeled PCP to the proteins in the incubation mixture in the absence of GSH was 4:1. In the presence of GSH, no significant amount of radioactivity was incorporated into the proteins. An anti-PCP metabolite antibody was used to detect PCP metabolite adducts bound to the inactivated enzyme by Western blot analysis. The antibody recognized adducts bound to P450, reductase, and catalase. In the presence of GSH, there was a decrease in immunoreactivity, although binding of PCP to all three proteins was still detected. Because the added nucleophile protects against inactivation and protein labeling by PCP, these data suggest that the reactive intermediate may escape from the active site and attack other sites on the P450, as well as other proteins in the milieu.