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1.
J Hazard Mater ; 469: 133930, 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38452673

RESUMO

Dinotefuran, a neonicotinoid insecticide, may impact nontarget organisms such as Decapoda P. vannamei shrimp with nervous systems similar to insects. Exposing shrimp to low dinotefuran concentrations (6, 60, and 600 µg/L) for 21 days affected growth, hepatosomatic index, and survival. Biomarkers erythromycin-N-demethylase, alanine aminotransferase, and catalase increased in all exposed groups, while glutathione S-transferase is the opposite; aminopyrin-N-demethylase, malondialdehyde, and aspartate aminotransferase increased at 60 and 600 µg/L. Concentration-dependent effects on gut microbiota altered the abundance of bacterial groups, increased potentially pathogenic and oxidative stress-resistant phenotypes, and decreased biofilm formation. Gram-positive/negative microbiota changed significantly. Metabolite differences between the exposed and control groups were identified using mass spectrometry and KEGG pathway enrichment. N-acetylcystathionine showed potential as a reliable dinotefuran metabolic marker. Weighted correlation network analysis (WGCNA) results indicated high connectivity of cruecdysone in the metabolite network and significant enrichment at 600 µg/L dinotefuran. The WGCNA results revealed a highly significant negative correlation between two key metabolites, caldine and indican, and the gut microbiota within co-expression modules. Overall, the risk of dinotefuran exposure to non-target organisms in aquatic environments still requires further attention.


Assuntos
Microbioma Gastrointestinal , Guanidinas , Nitrocompostos , Penaeidae , Animais , Penaeidae/genética , Penaeidae/metabolismo , Penaeidae/microbiologia , Neonicotinoides/toxicidade , Neonicotinoides/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/farmacologia
2.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723058

RESUMO

Pseudomonas aeruginosa is an opportunistic human pathogen that develops difficult-to-treat biofilms in immunocompromised individuals, cystic fibrosis patients, and in chronic wounds. P. aeruginosa has an arsenal of physiological attributes that enable it to evade standard antibiotic treatments, particularly in the context of biofilms where it grows slowly and becomes tolerant to many drugs. One of its survival strategies involves the production of the redox-active phenazine, pyocyanin, which promotes biofilm development. We previously identified an enzyme, PodA, that demethylated pyocyanin and disrupted P. aeruginosa biofilm development in vitro. Here, we asked if this protein could be used as a potential therapeutic for P. aeruginosa infections together with tobramycin, an antibiotic typically used in the clinic. A major roadblock to answering this question was the poor yield and stability of wild-type PodA purified from standard Escherichia coli overexpression systems. We hypothesized that the insufficient yields were due to poor packing within PodA's obligatory homotrimeric interfaces. We therefore applied the protein design algorithm, AffiLib, to optimize the symmetric core of this interface, resulting in a design that incorporated five mutations leading to a 20-fold increase in protein yield from heterologous expression and purification and a substantial increase in stability to environmental conditions. The addition of the designed PodA with tobramycin led to increased killing of P. aeruginosa cultures under oxic and hypoxic conditions in both the planktonic and biofilm states. This study highlights the potential for targeting extracellular metabolites to assist the control of P. aeruginosa biofilms that tolerate conventional antibiotic treatment.


Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Oxirredutases N-Desmetilantes/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Tobramicina/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Humanos , Oxirredutases N-Desmetilantes/farmacologia , Engenharia de Proteínas , Pseudomonas aeruginosa/fisiologia
3.
Science ; 355(6321): 170-173, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-27940577

RESUMO

The opportunistic pathogen Pseudomonas aeruginosa produces colorful redox-active metabolites called phenazines, which underpin biofilm development, virulence, and clinical outcomes. Although phenazines exist in many forms, the best studied is pyocyanin. Here, we describe pyocyanin demethylase (PodA), a hitherto uncharacterized protein that oxidizes the pyocyanin methyl group to formaldehyde and reduces the pyrazine ring via an unusual tautomerizing demethylation reaction. Treatment with PodA disrupts P. aeruginosa biofilm formation similarly to DNase, suggesting interference with the pyocyanin-dependent release of extracellular DNA into the matrix. PodA-dependent pyocyanin demethylation also restricts established biofilm aggregate populations experiencing anoxic conditions. Together, these results show that modulating extracellular redox-active metabolites can influence the fitness of a biofilm-forming microorganism.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Biofilmes/efeitos dos fármacos , Mycobacterium fortuitum/enzimologia , Oxirredutases N-Desmetilantes/química , Oxirredutases N-Desmetilantes/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/química , Cristalografia por Raios X , DNA/química , Metilação , Oxirredução , Pseudomonas aeruginosa/fisiologia
4.
J Pharm Biomed Anal ; 48(1): 92-9, 2008 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-18584988

RESUMO

The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.


Assuntos
Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/análise , Espectrometria de Massas em Tandem/métodos , Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/farmacologia , Bioensaio , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/análise , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/metabolismo , Dextrometorfano/farmacologia , Diclofenaco/metabolismo , Diclofenaco/farmacologia , Interações Medicamentosas , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Midazolam/metabolismo , Midazolam/farmacologia , Miniaturização , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/farmacologia , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade por Substrato/efeitos dos fármacos , Tacrina/metabolismo , Tacrina/farmacologia , Fatores de Tempo
5.
Eur J Clin Pharmacol ; 63(9): 821-7, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17598095

RESUMO

OBJECTIVE: The aim of this study was to assess the drug interaction potential of psychotropic medication on methadone N-demethylation using cDNA-expressed cytochrome P450 CYP enzymes. METHODS: Methadone was incubated with various drugs (n = 10) and cDNA-expressed CYP3A4, CYP2D6, CYP2B6, CYP2C19 and CYP1A2 enzymes to screen for their inhibition potency. The nature of enzyme selective activity for inhibition was further investigated for potent inhibitors. To test for a mechanism-based component in inhibition, all substances were tested with preincubation and without. 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) concentration was determined by liquid chromatography/tandem mass spectrometry following liquid/liquid extraction. RESULTS: Formation of EDDP was catalysed by CYP3A4, CYP2D6 and CYP2C19. The N-demethylation of methadone was preferentially inhibited by amitriptyline, buprenorphine, methylenedioxymethamphetamine (MDMA) and zolpidem. Both amitriptyline and buprenorphine were strong, reversible inhibitors of CYP3A4. Similarly, amitriptyline and MDMA were identified as inhibitors of CYP2D6. Zolpidem revealed a mechanism-based inhibition of CYP3A4. CONCLUSION: Amitriptyline, MDMA and zolpidem are likely to slow down conversion of methadone and to increase its area under the curve (AUC). A consideration of the in vitro evidence of drug-methadone interactions should help to improve patient care during methadone maintenance treatment.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Metadona/metabolismo , Algoritmos , Amitriptilina/metabolismo , Amitriptilina/farmacologia , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Cloridrato de Atomoxetina , Cromatografia Líquida/métodos , Citalopram/metabolismo , Citalopram/farmacologia , Clozapina/metabolismo , Clozapina/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Metadona/farmacologia , Metilação , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/farmacologia , Propilaminas/metabolismo , Propilaminas/farmacologia , Psicotrópicos/metabolismo , Psicotrópicos/farmacologia , Piridinas/metabolismo , Piridinas/farmacologia , Pirrolidinas/antagonistas & inibidores , Pirrolidinas/metabolismo , Espectrometria de Massas em Tandem/métodos , Zolpidem
7.
J Toxicol Environ Health A ; 68(4): 299-314, 2005 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-15799453

RESUMO

The metabolism of territrem B (TRB) and territrem C (TRC) in liver microsomes of 14-wk-old male and female Wistar rats was investigated. Metabolism of TRB to 4beta-hydroxylmethyl-4beta-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4beta-hydroxylmethyl-4beta-demethylterritrem C (MC) were observed in both genders. However, the amounts of MB2, MB4, and MC formed in females were much lower than in males. To investigate which cytochrome P-450 (CYP450) isoforms were involved in each step, four CYP450 isotype-specific inhibitors (furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A1, CYP2B1, CYP2C11, or CYP3A2 were used. Formation of MB2, MB4, and MC was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. Anti-CYP3A2 antibody completely inhibited MB, MB, and MC formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 produced no marked effect. Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms (1Al, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. The amounts of MB2, MB4, and MC formed by male and female rat liver microsome preparations were related to the testosterone 6beta-hydroxylase activity and CYP3A1/2 protein content of the preparation. Immunoblotting showed that CYP3A1 was expressed in both genders, but at different levels, while CYP3A2 was only expressed in males. These results suggest that the formation of MB2, MB4, and MC in liver microsomes from 14-wk-old rats of either gender is mediated by CYP3A1 and CYP3A2.


Assuntos
Hidrocarboneto de Aril Hidroxilases/farmacologia , Oxirredutases N-Desmetilantes/farmacologia , Piranos/metabolismo , Animais , Catálise , Citocromo P-450 CYP3A , Feminino , Masculino , Microssomos Hepáticos/enzimologia , Micotoxinas , Ratos , Ratos Wistar
8.
Cancer Gene Ther ; 12(5): 497-508, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15746946

RESUMO

Virus-directed enzyme prodrug therapy (VDEPT) is an emerging strategy against cancer. Our approach is a P450-based VDEPT that consists of using cyclophosphamide (CPA) as a prodrug and a Cytochrome P450 2B6/NADPH cytochrome P450 reductase fusion protein (CYP2B6/RED) as a prodrug-activating enzyme. Due to the heterogenous expression of proteins in tumor cells, basal reductase activity may not be sufficient to supply CYP2B6 with electrons, the fusion protein should enable the expression of both proteins at high levels in tumor cells. CYP/RED fusion proteins have never been previously expressed in mammalian cells, to enable expression the fusion protein was cloned into an adenoviral vector and subsequently several pulmonary tumor cell lines were infected. The CYP2B6/RED fusion protein was detected by Western blot, its mRNA by Northern blot, and its heme incorporation into an active form by spectral analysis. Infection with the fusion gene increased RED activity in microsomes by a factor of 3 compared to the control. After infection and treatment with CPA, in cell lines with low endogenous RED, the fusion protein mediated significantly higher CPA-induced cytotoxicity compared to cells expressing solely CYP2B6. In conclusion, the fusion protein is functional for VDEPT by providing one protein for higher levels of CPA metabolism.


Assuntos
Adenoviridae/genética , Antineoplásicos Alquilantes/uso terapêutico , Hidrocarboneto de Aril Hidroxilases/genética , Ciclofosfamida/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , NADPH-Ferri-Hemoproteína Redutase/genética , Oxirredutases N-Desmetilantes/genética , Pró-Fármacos/uso terapêutico , Antineoplásicos Alquilantes/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Ciclofosfamida/metabolismo , Citocromo P-450 CYP2B6 , Vetores Genéticos , Humanos , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/metabolismo , NADPH-Ferri-Hemoproteína Redutase/farmacologia , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
9.
Int J Clin Pharmacol Ther ; 43(2): 109-16, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15726880

RESUMO

OBJECTIVE: The present study evaluates the acute effect of a single-dose itraconazole administration on CYP3A phenotype, as measured by cortisol MR ratio in urine. METHODS: Twenty-four healthy Uruguayan subjects recruited according to strict inclusion criteria participated in an open-label, randomized, two-period, crossover study designed to evaluate the bioequivalence of an itraconazole formulation (Traconal 100 mg, Achê Labs, São Paulo, Brazil). The study comprised two treatment periods separated by a wash-out period of 14 days. In each period a series of venous blood samples were drawn over 48 hours. Three urine samples were obtained for CYP3A phenotyping: pre-dose, 24 and 48 hours after dosing. Blood and urine samples were assayed for itraconazole, beta-hydroxycortisol and cortisol using a validated chromatographic method. RESULTS: The ratio of the mean AUC0-inf. T/AUC0-inf. R was included in the bioequivalence range, however, due to high variability, the CI90% was not. It was found that the cortisol metabolic ratio (MR) showed inhibition relative to basal activity in a proportion of subjects 24 hours (68 +/- 6.1%, mean +/- CI95%) and 48 hours (80 +/- 7.3%, mean +/- CI95%) after ingestion of itraconazole. A significant correlation was found between itraconazole AUC0-inf. and normalized basal CYP3A MR for the reference (r = 0.62, t = 3.72, p = 0.001) and the test product (r = 0.74, t = 5.22, p = 0.00003). A good correlation existed between basal cortisol MR and the elimination half-life of itraconazole. CONCLUSIONS: The findings are in line with the hypothesis that the determination of the bioavailability of highly variable CYP3A substrates might be improved by simultaneous non-interfering phenotyping. If this is confirmed, a new methodological paradigm may need to be developed in order to take account of metabolic variability in bioequivalence evaluation of this group of drugs.


Assuntos
Antifúngicos/farmacocinética , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/farmacologia , Itraconazol/farmacocinética , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/farmacologia , Adolescente , Adulto , Antifúngicos/farmacologia , Área Sob a Curva , Estudos Cross-Over , Citocromo P-450 CYP3A , Feminino , Humanos , Itraconazol/farmacologia , Masculino , Fenótipo , Equivalência Terapêutica
10.
J Toxicol Environ Health A ; 67(23-24): 1955-70, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15513895

RESUMO

Studies demonstrated that cocaine-induced immunosuppression is mediated by metabolites of cocaine. Although SKF 525-A inhibited cocaine N-demethylation in liver S9 fractions isolated from female B6C3F1 mice, our study showed that pretreatment of mice with SKF 525-A potentiated cocaine-induced suppression of the antibody response to sheep red blood cells. An increase in formaldehyde generation was subsequently shown following incubation of cocaine with the S9 fractions prepared from SKF 525-A-treated mice, indicating the possibility of cytochrome P-450 (CYP) induction. Therefore, the inductive effects of SKF 525-A on CYP enzyme activities and proteins were investigated in female B6C3F1 mice to elucidate the potentiation of cocaine-induced immunosuppression by SKF 525-A. When SKF 525-A was administered at 10, 20, or 40 mg/kg/d intraperitoneally for 7 consecutive days, both ethoxyresorufin O-deethylase and pentoxyresorufin O-dealkylase activities were induced dose-dependently. Furthermore, the induction of enzymatic activity was time dependent. Meanwhile, when the type of isozyme induced by SKF 525-A was analyzed by Western immunoblotting with monospecific anti-CYP 1A and anti-CYP 2B antibodies, only the CYP 2B appeared to be induced. From in vitro inhibition studies with monoclonal antibodies, it was confirmed that the induced activity of ethoxyresorufin O-deethylase by SKF 525-A was due to increased levels of CYP 2B proteins. Our present results provide an explanation for the potentiation of cocaine-induced immunosuppression by repeated exposure to SKF 525-A. Our results also indicate that ethoxyresorufin O-deethylase, a selective substrate for CYP 1A, may also be catalyzed by CYP 2B.


Assuntos
Citocromo P-450 CYP1A1/farmacologia , Citocromo P-450 CYP2B1/farmacologia , Inibidores Enzimáticos/farmacologia , Oxirredutases N-Desmetilantes/farmacologia , Proadifeno/farmacologia , Animais , Cocaína/toxicidade , Sistema Enzimático do Citocromo P-450/farmacologia , Inibidores da Captação de Dopamina/toxicidade , Relação Dose-Resposta a Droga , Indução Enzimática , Feminino , Camundongos
11.
Drug Metab Dispos ; 32(4): 447-54, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15039299

RESUMO

Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Intestinos/ultraestrutura , Microssomos Hepáticos/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Oxicodona/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/farmacologia , Biotransformação , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/genética , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Cetoconazol/metabolismo , Cetoconazol/farmacologia , Cinética , Taxa de Depuração Metabólica , Metilação/efeitos dos fármacos , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Microssomos Hepáticos/química , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Morfinanos/antagonistas & inibidores , Morfinanos/química , Morfinanos/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/farmacologia , Oxicodona/farmacologia , Oximorfona/química , Oximorfona/metabolismo , Ligação Proteica/efeitos dos fármacos , Quinidina/efeitos adversos , Quinidina/antagonistas & inibidores , Quinidina/farmacologia
12.
Environ Toxicol Chem ; 21(3): 575-83, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11883415

RESUMO

Planktonic copepods (Calanus glacialis and C. hyperboreus; n = 37) and water (n = 19) were collected to examine the spatial distribution and bioaccumulation of organochlorine contaminants (OCs) in the Alaskan and Canadian Arctic. The rank order of total OC (sigma OC) group concentrations in Calanus samples was toxaphene > or = sigma polychlorinated biphenyls (PCBs) > sigma hexachlorcyclohexane (HCH) > sigma DDT > sigma chlordane-related compounds (CHLOR) > sigma chlorobenzenes (ClBz). The dominant analyte was alpha-HCH in all water and zooplankton samples. The most abundant toxaphene congener in water and zooplankton samples was the hexachlorobornane B6-923. Organochlorine contaminant group concentrations in Alaskan zooplankton and water samples were lower than those in samples collected from sites in the eastern Canadian Arctic. Comparison of PCB and toxaphene congener profiles in zooplankton and water samples suggests that biotransformation by cytochrome P-4502B isozymes is low in Calanus, and limited phase I metabolism may occur. The log relationship of bioaccumulation factor (log BAF) versus octanol-water partition coefficient (log Kow) relationship was near 1:1 for OCs within the log Kow range of 3 to 6. A curvilinear model provided a better relationship between these two variables when OC compounds with log Kow > 6 were included. These results suggest that hydrophobic OCs (log Kow 3-6) in Calanus species are at equilibrium with the water concentrations and that physical partitioning, rather than biotransformation, is the major factor governing OC profiles in marine zooplankton.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Crustáceos/fisiologia , Monitoramento Ambiental , Poluentes Ambientais/farmacocinética , Inseticidas/farmacocinética , Bifenilos Policlorados/farmacocinética , Poluentes Químicos da Água/farmacocinética , Alaska , Animais , Regiões Árticas , Biotransformação , Canadá , Citocromo P-450 CYP2B6 , Sistema Enzimático do Citocromo P-450/farmacologia , Poluentes Ambientais/análise , Poluentes Ambientais/metabolismo , Inseticidas/análise , Inseticidas/metabolismo , Oxirredutases N-Desmetilantes/farmacologia , Bifenilos Policlorados/análise , Bifenilos Policlorados/metabolismo , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/metabolismo
13.
J Biol Chem ; 275(40): 30781-6, 2000 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-10859304

RESUMO

Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme. With native enzyme, electron transfer was observed within the radiolytically generated one-electron reduced enzyme but only at low pH (6.0). The kinetics and thermodynamics of this electron transfer in one-electron reduced enzyme may be compared with that studied previously in the two-electron reduced enzyme. In contrast to previous studies with two-electron reduced enzyme in which a pK(a) of approximately 8 was determined for the flavin semiquinone, in the one-electron reduced enzyme the semiquinone was not substantially protonated even at pH 6. 0. These results indicate that reduction of the iron-sulfur center of the enzyme significantly decreases the pK(a) of the flavin semiquinone of the active site. This provides further evidence, in conjunction with the strong magnetic interaction known to exist between the centers in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another in the active site of the enzyme.


Assuntos
Flavinas/química , Oxirredutases N-Desmetilantes/química , Catálise , Transporte de Elétrons , Compostos Ferrosos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Methylophilus methylotrophus/enzimologia , Oxirredução , Oxirredutases N-Desmetilantes/farmacologia , Fenil-Hidrazinas/farmacologia , Radiólise de Impulso , Temperatura , Termodinâmica , Raios Ultravioleta
14.
J Am Acad Dermatol ; 37(5 Pt 1): 765-71, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9366824

RESUMO

Recent case reports and studies suggest that interactions involving the cytochrome P-450 mixed function oxidase system are important causes of medication toxicity and decreased efficacy during combination drug therapy. The cytochrome P-450 3A3/4 isoenzyme is involved in many significant drug interactions. New and familiar drugs continue to be implicated as having potentially serious interactions with this group of enzymes. An understanding of the basic principles of these interactions may have a major impact on patient outcome.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/farmacologia , Fármacos Dermatológicos/farmacologia , Oxirredutases N-Desmetilantes/farmacologia , Dermatopatias/tratamento farmacológico , Antibacterianos/farmacologia , Anticonvulsivantes/farmacologia , Antidepressivos/farmacologia , Antifúngicos/farmacologia , Antituberculosos/farmacologia , Azóis/farmacologia , Ligação Competitiva , Citrus , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Macrolídeos , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Especificidade por Substrato
15.
Toxicol Appl Pharmacol ; 135(1): 1-8, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7482528

RESUMO

Acrylonitrile (VCN) is known to cause extensive gastrointestinal damage and tumors in rats. In this study the metabolism of VCN to cyanide (CN-) was characterized in the small intestinal mucosa. The majority of the metabolic reactivity was localized in the microsomal fraction and required reduced nicotinamide adenine dinucleotide phosphate for maximal activity. The intestinal metabolism of VCN to CN- was characterized with respect to VCN concentration, time, pH, and microsomal protein concentration. VCN metabolism to CN- was enhanced significantly by the addition of sulfhydryl compounds such as glutathione, cysteine, and D-penicillamine (10 mM) to 142, 161, and 189% of control, respectively. The intestinal bioactivation of VCN to CN- was enhanced by microsomes obtained from intestinal mucosa of phenobarbital (455% of control), beta-naphthoflavone (375% of control), 4-methylpyrazole (305% of control), or ethanol (165% of control)-treated rats. Addition of ethanol (80 mM) to incubation mixtures containing control or ethanol-induced microsomes resulted in significant inhibition of microsomal metabolism of VCN to CN- to 20 and 34% of control, respectively. Addition of dimethyl sulfoxide induced a similar inhibitory effect on VCN metabolism by control or ethanol-induced microsomes (8 and 26% of control, respectively). Furthermore, antibody to cytochrome P450 2E1, but not antibody to cyt P450 2B1, significantly inhibited VCN metabolism by ethanol-induced intestinal microsomes to about 25% of control. Mild inhibition (80-85% of control) of VCN metabolism was detected when antibody to cyt P450 2B1 or 2E1 was added to incubation mixtures containing Pb-induced intestinal microsomes. These findings indicate that extrahepatic tissues such as the intestinal mucosa are capable of metabolizing VCN to CN- and establish a major role of intestinal cyt P450, particularly cyt P450 2E1, in the intestinal metabolism of VCN to CN-.


Assuntos
Acrilonitrila/farmacocinética , Acrilonitrila/toxicidade , Sistema Enzimático do Citocromo P-450/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Oxirredutases N-Desmetilantes/farmacologia , Acrilonitrila/antagonistas & inibidores , Animais , Biotransformação/efeitos dos fármacos , Cianetos/metabolismo , Citocromo P-450 CYP2E1 , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/farmacologia
16.
Crit Rev Toxicol ; 23(1): 1-20, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8471158

RESUMO

Numerous halogenated hydrocarbons of the alkane, alkene, and alkyne classes are metabolized by P450 enzymes to products that elicit cytotoxic and/or carcinogenic effects. Such halogenated hydrocarbons include anesthetics (e.g., halothane and enflurane) and industrial solvents (e.g., carbon tetrachloride, chloroform, and vinylidine chloride). Formation of reaction intermediates from these compounds occurs via P450-promoted dehalogenation, reduction, or reductive oxygenation, with certain hydrocarbons undergoing all three reaction types. Of the multiple forms of P450 present in liver microsomes, P4502E1 has been identified as the primary catalyst of hydrocarbon bioactivation in animals and, most likely, in humans as well. As hepatic concentrations of this P450 enzyme are highly inducible by ethanol and similar agents, prior exposure to 2E1-inducing compounds can play a pivotal role in halogenated hydrocarbon toxicity. Considering that metabolism governs the cytotoxicity and carcinogenicity of halogenated hydrocarbons, an understanding of the mechanism(s) underlying 2E1 induction in man becomes all the more important.


Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Hidrocarbonetos Halogenados/metabolismo , Oxirredutases N-Desmetilantes/fisiologia , Anestésicos/metabolismo , Animais , Biotransformação , Citocromo P-450 CYP2E1 , Sistema Enzimático do Citocromo P-450/farmacologia , Humanos , Hidrocarbonetos Halogenados/efeitos adversos , Hidrocarbonetos Halogenados/farmacocinética , Hidrocarbonetos Halogenados/toxicidade , Oxirredutases N-Desmetilantes/farmacologia , Solventes/metabolismo , Xenobióticos/metabolismo
17.
J Pharmacobiodyn ; 7(11): 872-82, 1984 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6530649

RESUMO

Renal microsomal N-nitrosodimethylamine (NDMA) N-demethylation was measured in several species by a sensitive radiometric method using [14C]NDMA as a substrate and 2,4-dinitrophenylhydrazine (DNPH) as a trapping agent of the [14C]formaldehyde formed. The activities were the highest in mice and lowest in hamsters. The activities in rats could not be detected. Among several strains of mice studied the DDY strain was the highest in its activities. Although nicotinamide adenine dinucleotide supported NDMA demethylation by about 32% of nicotinamide adenine dinucleotide phosphate in kidney and only 16% in liver, other properties (pH profiles, km values, and effects of inhibitors) exhibited almost similar results in liver as compared to kidney.


Assuntos
Rim/enzimologia , Microssomos/enzimologia , Oxirredutases N-Desmetilantes/análise , Animais , Radioisótopos de Carbono , Cromatografia em Camada Fina , Cricetinae , Citocromo P-450 CYP2E1 , Formaldeído/biossíntese , Técnicas In Vitro , Cinética , Fígado/enzimologia , Masculino , Mesocricetus , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxirredutases N-Desmetilantes/farmacologia , Fenil-Hidrazinas , Ratos , Ratos Endogâmicos , Especificidade da Espécie
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