RESUMO
Aldehyde oxidases (AOXs) are a group of metabolic enzymes that play critical roles in the degradation of xenobiotics and chemicals. However, the physiological function of this enzyme in insects remains poorly understood. In this study, three TcAOX genes (TcAOX1, TcAOX2, TcAOX3) were identified and characterized from Tribolium castaneum genome. Spatiotemporal expression profiling showed that TcAOX1 expression was most highly expressed at the early pupal stage and was predominantly expressed in the antennae of adults, indicating that TcAOX1 was involved in the degradation of chemical signals; TcAOX2 expression was most highly expressed at the late pupal stage and was mainly expressed in the fat body, epidermis of larvae and adults, respectively; and TcAOX3 expression was in all stages and was primarily expressed in the head of adults. Moreover, the transcripts of TcAOX2 and TcAOX3 were significantly induced after exposure to plant oil, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to this plant toxicant, suggesting that these two genes are associated with plant toxicant detoxification. Intriguingly, knockdown of the TcAOX1 led to reductions in female egg-laying but unchanged the hatchability and the development of genital organs, suggesting that this gene may mediate fecundity by effecting the inactivation of chemical signals in T. castaneum. Overall, these results shed new light on the function of AOX genes in insects, and could facilitate the development of research on pest control management.
Assuntos
Besouros , Tribolium , Animais , Tribolium/genética , Besouros/metabolismo , Aldeído Oxidase/genética , Aldeído Oxidase/metabolismo , Interferência de RNA , Fertilidade/genética , Aldeídos/metabolismoRESUMO
The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.
Assuntos
Aldeído Oxidase/metabolismo , Coenzimas/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Aldeído Oxidase/genética , Animais , Dantroleno/farmacocinética , Avaliação Pré-Clínica de Medicamentos/métodos , Guanina/análogos & derivados , Guanina/farmacocinética , Macaca fascicularis/genética , Cofatores de Molibdênio , Mutagênese Sítio-Dirigida , Oxirredução , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato/genéticaRESUMO
Carbazeran is a potent phosphodiesterase inhibitor with species-dependent metabolic profiles in rats, dogs, and humans. In this study, we investigated the aldehyde oxidase (AOX)-mediated oxidation of carbazeran to 4-oxo derivatives in chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene with humanized livers (humanized-liver mice). Liver cytosolic fractions from humanized-liver mouse effectively catalyzed carbazeran 4-oxidation with high affinity for the substrate, similar to those of the human liver cytosolic fractions and recombinant human AOX1 protein. Furthermore, hepatocytes prepared from humanized-liver mice and humans also exhibited substantial metabolism via carbazeran 4-oxidation. After a single oral administration of carbazeran (10 mg/kg), plasma levels of 4-oxo-carbazeran, N-desethyl-4-oxo-carbazeran, and 6,7-dimethoxy-1-[4-(hydroxy)-piperidino]-4-phthalazinone (three human metabolites formed via 4-oxidation) were higher in humanized-liver mice than in the control mice. In contrast, plasma levels of O-desmethylcarbazeran (a major metabolite in dogs) in control mice were higher than those in the humanized-liver mice. Relative excreted amounts of the three 4-oxidation-derived human-specific metabolites in the urine and feces were greater for humanized-liver mice than control mice, whereas the relative excreted amounts of O-desmethylcarbazeran were predominant in the urine and feces of control mice. Thus, the production of carbazeran 4-oxo derivatives was elevated in humanized-liver mice compared with control mice, in agreement with our in vitro enzyme-mediated oxidation data. These results suggest that hepatic human AOX1 functions in humanized-liver mice at the in vivo level and that humanized-liver mice may be useful for predicting drug metabolism in humans, at least with regard to human AOX1-dependent metabolism. SIGNIFICANCE STATEMENT: We found that the production of carbazeran 4-oxo derivatives was higher in humanized-liver mice than in control mice. These results were supported by the fact that carbazeran was rapidly metabolized to 4-oxo-carbazeran in humanized-liver mouse hepatocytes expressing human aldehyde oxidase 1. These results suggest that human aldehyde oxidase 1 is functional in humanized-liver mice in vivo and that chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene transplanted with human hepatocytes may be a suitable model animal for predicting aldehyde oxidase-dependent biotransformation of drugs in humans.
Assuntos
Aldeído Oxidase/metabolismo , Carbamatos/farmacocinética , Administração Oral , Adolescente , Adulto , Idoso , Animais , Biotransformação , Carbamatos/administração & dosagem , Células Cultivadas , Criança , Pré-Escolar , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos , Estudos de Viabilidade , Feminino , Cobaias , Hepatócitos/metabolismo , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oxirredução , Coelhos , Ratos , Proteínas Recombinantes/metabolismo , Suínos , Porco Miniatura , Quimeras de Transplante/metabolismo , Adulto JovemRESUMO
Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.
Assuntos
Aldeído Oxidase/metabolismo , Ciona intestinalis/genética , Expressão Gênica , Mitocôndrias Cardíacas/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Aldeído Oxidase/genética , Animais , Ciclo do Ácido Cítrico/genética , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Camundongos , Mitocôndrias Cardíacas/genética , Consumo de Oxigênio/genética , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismoRESUMO
Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico, in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.
Assuntos
Aldeído Oxidase/metabolismo , Preparações Farmacêuticas/metabolismo , Aldeído Oxidase/antagonistas & inibidores , Aldeído Oxidase/química , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Especificidade da EspécieRESUMO
Accurate prediction of human pharmacokinetics for drugs remains challenging, especially for non-cytochrome P450 (P450) substrates. Hepatocytes might be suitable for predicting hepatic intrinsic clearance (CLint) of new chemical entities, because they can be applied to various compounds regardless of the metabolic enzymes. However, it was reported that hepatic CLint is underestimated in hepatocytes. The purpose of the present study was to confirm the predictability of human hepatic clearance for P450 and non-P450 substrates in hepatocytes and the utility of animal scaling factors for the prediction using hepatocytes. CLint values for 30 substrates of P450, UDP-glucuronosyltransferase, flavin-containing monooxygenase, esterases, reductases, and aldehyde oxidase in human microsomes, human S9 and human, rat, and monkey hepatocytes were estimated. Hepatocytes were incubated in serum of each species. Furthermore, CLint values in human hepatocytes were corrected with empirical, monkey, and rat scaling factors. CLint values in hepatocytes for most compounds were underestimated compared to observed values regardless of the metabolic enzyme, and the predictability was improved by using the scaling factors. The prediction using human hepatocytes corrected with monkey scaling factor showed the highest predictability for both P450 and non-P450 substrates among the predictions using liver microsomes, liver S9, and hepatocytes with or without scaling factors. CLint values by this method for 80% and 90% of all compounds were within 2- and 3-fold of observed values, respectively. This method is accurate and useful for estimating new chemical entities, with no need to care about cofactors, localization of metabolic enzymes, or protein binding in plasma and incubation mixture.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fígado/enzimologia , Taxa de Depuração Metabólica , Modelos Biológicos , Aldeído Oxidase/metabolismo , Animais , Esterases/metabolismo , Glucuronosiltransferase/metabolismo , Hepatócitos , Humanos , Fígado/citologia , Macaca fascicularis , Masculino , Microssomos Hepáticos , Oxigenases/metabolismo , Ratos , Especificidade da EspécieRESUMO
1. Aldehyde oxidase (AO enzymes)-mediated oxidation predominantly occurs at a carbon atom adjacent to the nitrogen on aromatic azaheterocycles. In the current report, we identified that AO enzymes oxidation took place at both the C-2 and C-4 positions of the methylquinoline moiety of Compound A based on data from mass spectrometric analysis, AO enzymes "litmus" test, and comparison with authentic standards. 2. To assess the potential for inadequate coverage for these two AO enzyme-mediated metabolites in nonclinical safety studies, given concerns due to differences in AO enzymes expression between preclinical species and humans, the human circulating levels of the two AO enzyme-mediated metabolites were predicted prospectively using in vitro and in vivo models. Both formation clearance and elimination clearance of the two metabolites were predicted based on in vitro to in vivo correlation and comparison with in vivo data from rats. 3. The result showed that the 4-OH metabolite of Compound A would account for less than 3% of the total drug-related exposure in human plasma, while the exposure to the 2-oxo metabolite would be relatively high (â¼70%). 4. The predicted human exposure levels for the two metabolites are in similar ranges as those observed in monkeys. These data taken together support the advancement to clinical development of Compound A.
Assuntos
Aldeído Oxidase/metabolismo , Quinolinas/química , Animais , Carbono/química , Cromatografia Líquida , Cães , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Haplorrinos , Humanos , Cinética , Masculino , Camundongos , Oxirredução , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Hydralazine has been reported as a selective mechanism-based inactivator of aldehyde oxidase (AO) and it is widely used in the pharmaceutical industry for reaction phenotyping to estimate fraction metabolized by AO and to identify AO substrates. In this study, however, hydralazine was found to inhibit CYP1A2, 2B6, 2D6, and 3A in human suspension hepatocytes under reaction phenotyping assay conditions, at concentrations that chemically knocked out most of the AO activities (≥50 µM). Furthermore, hydralazine is a time-dependent inhibitor of CYP1A2. Based on these findings, precautions need to be taken when using hydralazine as an AO inhibitor for in vitro studies because fraction metabolized by AO is likely to be overestimated and the likelihood of false positives in identifying AO substrates increases.
Assuntos
Aldeído Oxidase/antagonistas & inibidores , Citocromo P-450 CYP1A2/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Hidralazina/farmacologia , Aldeído Oxidase/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Reações Falso-Positivas , Humanos , Especificidade por SubstratoRESUMO
Inclusion of a microdose of 14C-labeled drug in the first-in-man study of new investigational drugs and subsequent analysis by accelerator mass spectrometry has become an integrated part of drug development at Lundbeck. It has been found to be highly informative with regard to investigations of the routes and rates of excretion of the drug and the human metabolite profiles according to metabolites in safety testing guidance and also when additional metabolism-related issues needed to be addressed. In the first-in-man study with the NCE Lu AF09535, contrary to anticipated, surprisingly low exposure was observed when measuring the parent compound using conventional bioanalysis. Parallel accelerator mass spectrometry analysis revealed that the low exposure was almost exclusively attributable to extensive metabolism. The metabolism observed in humans was mediated via a human specific metabolic pathway, whereas an equivalent extent of metabolism was not observed in preclinical species. In vitro, incubation studies in human liver cytosol revealed involvement of aldehyde oxidase (AO) in the biotransformation of Lu AF09535. In vivo, substantially lower plasma exposure of Lu AF09535 was observed in chimeric mice with humanized livers compared with control animals. In addition, Lu AF09535 exhibited very low oral bioavailability in monkeys despite relatively low clearance after intravenous administration in contrast to the pharmacokinetics in rats and dogs, both showing low clearance and high bioavailability. The in vitro and in vivo methods applied were proved useful for identifying and evaluating AO-dependent metabolism. Different strategies to integrate these methods for prediction of in vivo human clearance of AO substrates were evaluated.
Assuntos
Aldeído Oxidase/metabolismo , Drogas em Investigação/farmacocinética , Fígado/metabolismo , Animais , Disponibilidade Biológica , Biotransformação , Radioisótopos de Carbono , Citosol/metabolismo , Método Duplo-Cego , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/administração & dosagem , Feminino , Voluntários Saudáveis , Humanos , Técnicas In Vitro , Fígado/enzimologia , Macaca fascicularis , Masculino , Camundongos , Especificidade da EspécieRESUMO
Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.
Assuntos
Aldeído Oxidase/metabolismo , Glycine max/metabolismo , Vírus do Mosaico/fisiologia , Nitrato Redutase/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/fisiologia , Proteínas de Soja/fisiologia , Xantina Desidrogenase/fisiologia , Agrobacterium tumefaciens , Coenzimas/biossíntese , Sequência Conservada , DNA Complementar/genética , DNA de Plantas/genética , Resistência à Doença , Vetores Genéticos , Metaloproteínas/biossíntese , Dados de Sequência Molecular , Cofatores de Molibdênio , Filogenia , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Pteridinas , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Soja/genética , Glycine max/genética , Glycine max/virologia , Regulação para Cima , Xantina Desidrogenase/genéticaRESUMO
Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 µg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.
Assuntos
Cicatrização/fisiologia , Xantina Desidrogenase/metabolismo , Aldeído Oxidase/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Proliferação de Células , Suplementos Nutricionais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Expressão Gênica , Tecido de Granulação/metabolismo , Peróxido de Hidrogênio/metabolismo , Queratinócitos/metabolismo , Masculino , Camundongos , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tungstênio/metabolismo , Tungstênio/farmacologia , Xantina Desidrogenase/antagonistas & inibidores , Xantina Desidrogenase/genéticaRESUMO
The mechanistic understanding of interactions between diet-derived substances and conventional medications in humans is nascent. Most investigations have examined cytochrome P450-mediated interactions. Interactions mediated by other phase I enzymes are understudied. Aldehyde oxidase (AO) is a phase I hydroxylase that is gaining recognition in drug design and development programs. Taken together, a panel of structurally diverse phytoconstituents (n = 24) was screened for inhibitors of the AO-mediated oxidation of the probe substrate O(6)-benzylguanine. Based on the estimated IC50 (<100 µM), 17 constituents were advanced for Ki determination. Three constituents were described best by a competitive inhibition model, whereas 14 constituents were described best by a mixed-mode model. The latter model consists of two Ki terms, Kis and Kii, which ranged from 0.26-73 and 0.80-120 µM, respectively. Molecular modeling was used to glean mechanistic insight into AO inhibition. Docking studies indicated that the tested constituents bound within the AO active site and elucidated key enzyme-inhibitor interactions. Quantitative structure-activity relationship modeling identified three structural descriptors that correlated with inhibition potency (r(2) = 0.85), providing a framework for developing in silico models to predict the AO inhibitory activity of a xenobiotic based solely on chemical structure. Finally, a simple static model was used to assess potential clinically relevant AO-mediated dietary substance-drug interactions. Epicatechin gallate and epigallocatechin gallate, prominent constituents in green tea, were predicted to have moderate to high risk. Further characterization of this uncharted type of interaction is warranted, including dynamic modeling and, potentially, clinical evaluation.
Assuntos
Aldeído Oxidase/antagonistas & inibidores , Aldeído Oxidase/metabolismo , Dieta/efeitos adversos , Interações Alimento-Droga/fisiologia , Catequina/efeitos adversos , Catequina/análogos & derivados , Catequina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Ligantes , Oxirredução , Relação Quantitativa Estrutura-Atividade , Chá/efeitos adversos , Xenobióticos/metabolismoRESUMO
1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fígado/efeitos dos fármacos , Preparações Farmacêuticas/metabolismo , Acetamidas/farmacocinética , Acetamidas/farmacologia , Aldeído Oxidase/metabolismo , Animais , Quimera , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatite/virologia , Humanos , Fígado/fisiologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Farmacocinética , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Ativador de Plasminogênio Tipo Uroquinase/genética , Varfarina/farmacocinética , Varfarina/farmacologiaRESUMO
Metabolism by aldehyde oxidase (AO) has been responsible for a number of drug failures in clinical trials. The main reason is the clearance values for drugs metabolized by AO are underestimated by allometric scaling from preclinical species. Furthermore, in vitro human data also underestimates clearance. We have developed the first in silico models to predict both in vitro and in vivo human intrinsic clearance for 8 drugs with just two chemical descriptors. These models explain a large amount of the variance in the data using two computational estimates of the electronic and steric features of the reaction. The in vivo computational models for human metabolism are better than in vitro preclinical animal testing at predicting human intrinsic clearance. Thus, it appears that AO is amenable to computational prediction of rates, which may be used to guide drug discovery, and predict pharmacokinetics for clinical trials.
Assuntos
Aldeído Oxidase/química , Desenho de Fármacos , Coenzimas/química , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Fígado/enzimologia , Metaloproteínas/química , Microssomos Hepáticos/efeitos dos fármacos , Modelos Químicos , Cofatores de Molibdênio , Oxigênio/química , Farmacocinética , Pteridinas/química , Análise de Regressão , SoftwareRESUMO
Sulfur is the sixth most abundant element in life and an important building block of proteins and cellular metabolites. Plants like bacteria can synthesize their sulfur-containing biomolecules from sulfate, where sulfite is an intermediate of the sulfur assimilation pathway. Above a certain threshold SO(2)/sulfite is cytotoxic and is rapidly metabolized to avoid damage. However, the existing data show considerable differences in basal sulfite levels both between species and apparent discrepancies in measured levels in the same species. In order to resolve this question we employed a sulfite detection method using chicken sulfite oxidase and developed an independent enzymatic assay, based on the specific detection of sulfite by sulfite reductase and compared those measurements to a modified colorimetric fuchsin-based method, specific for sulfite detection. We show here that when properly used the sulfite levels detected by the three methods can yield identical results. Furthermore, to examine the capacity of the plant to detoxify sulfite we injected sub-lethal sulfite solutions (yet, several folds higher than the basal levels) into Arabidopsis and tomato leaves and monitored the excess sulfite turnover. Within 3h of sulfite injection, more than 80% of the injected sulfite in Arabidopsis and 91% in tomato were oxidized to sulfate, demonstrating the high capacity of the sulfite oxidation mechanism/s in plants.
Assuntos
Folhas de Planta/metabolismo , Plantas/metabolismo , Sulfitos/metabolismo , Aldeído Oxidase/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Cisteína/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/metabolismo , Oxirredução/efeitos dos fármacos , Extratos Vegetais/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Plantas/efeitos dos fármacos , Padrões de Referência , Sulfatos/metabolismo , Sulfito Oxidase/metabolismo , Sulfitos/toxicidade , Nicotiana/efeitos dos fármacos , Nicotiana/metabolismoRESUMO
The upper lip and primary palate form an essential separation between the brain, nasal structures and the oral cavity. Surprisingly little is known about the development of these structures, despite the fact that abnormalities can result in various forms of orofacial clefts. We have uncovered that retinoic acid is a critical regulator of upper lip and primary palate development in Xenopus laevis. Retinoic acid synthesis enzyme, RALDH2, and retinoic acid receptor gamma (RARγ) are expressed in complementary and partially overlapping regions of the orofacial prominences that fate mapping revealed contribute to the upper lip and primary palate. Decreased RALDH2 and RARγ result in a median cleft in the upper lip and primary palate. To further understand how retinoic acid regulates upper lip and palate morphogenesis we searched for genes downregulated in response to RARγ inhibition in orofacial tissue, and uncovered homeobox genes lhx8 and msx2. These genes are both expressed in overlapping domains with RARγ, and together their loss of function also results in a median cleft in the upper lip and primary palate. Inhibition of RARγ and decreased Lhx8/Msx2 function result in decreased cell proliferation and failure of dorsal anterior cartilages to form. These results suggest a model whereby retinoic acid signaling regulates Lhx8 and Msx2, which together direct the tissue growth and differentiation necessary for the upper lip and primary palate morphogenesis. This work has the potential to better understand the complex nature of the upper lip and primary palate development which will lead to important insights into the etiology of human orofacial clefts.
Assuntos
Genes Homeobox , Tretinoína/metabolismo , Xenopus laevis/embriologia , Família Aldeído Desidrogenase 1 , Aldeído Oxidase/metabolismo , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Larva/metabolismo , Morfogênese , Palato/anormalidades , Palato/embriologia , Receptores do Ácido Retinoico/metabolismo , Retinal Desidrogenase , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus laevis/anormalidades , Xenopus laevis/metabolismo , Receptor gama de Ácido RetinoicoRESUMO
OBJECTIVES: To investigate the metabolism of cryptolepine and some cryptolepine analogues by aldehyde oxidase, and to assess the implications of the results on the potential of cryptolepine analogues as antimalarial agents. METHODS: The products resulting from the oxidation of cryptolepine and 2-fluorocryptolepine by a rabbit liver preparation of aldehyde oxidase were isolated and identified using chromatographic and spectroscopic techniques. The antiplasmodial activity of cryptolepine-11-one was assessed against Plasmodium falciparum using the parasite lactate dehydrogenase assay. KEY FINDINGS: Cryptolepine was oxidized by aldehyde oxidase give cryptolepine-11-one. Although 2-fluorocryptolepine was found to have less affinity for the enzyme than cryptolepine, it was a better substrate for aldehyde oxidase than the parent compound. In contrast, quindoline, the 11-chloro- , 2,7-dibromo- and 2-methoxy analogues of cryptolepine were not readily oxidized. Cryptolepine-11-one was found to be inactive against P. falciparum in vitro raising the possibility that the effectiveness of cryptolepine as an antimalarial, may be compromised by metabolism to an inactive metabolite by liver aldehyde oxidase. CONCLUSIONS: Cryptolepine and 2-fluorocryptolepine are substrates for aldehyde oxidase. This may have implications for the design and development of cryptolepine analogues as antimalarial agents.
Assuntos
Aldeído Oxidase/metabolismo , Antimaláricos/metabolismo , Alcaloides Indólicos/metabolismo , Fígado/enzimologia , Malária Falciparum/tratamento farmacológico , Quinolinas/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Cryptolepis , Feminino , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oxirredução , Raízes de Plantas/química , Plantas Medicinais/química , Plasmodium falciparum , CoelhosRESUMO
Effects of vitamin E and selenium supplementation on aldehyde oxidase (AO) and xanthine oxidase (XO) activities and antioxidant status in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats were examined. AO and XO activities increased significantly after induction of diabetes in rats. Following oral vitamin E (300 mg/kg) and sodium selenite (0.5 mg/kg) intake once a day for 4 weeks, XO activity decreased significantly. AO activity decreased significantly in liver, but remained unchanged in kidney and heart of vitamin E- and selenium-treated rats compared to the diabetic rats. Total antioxidants status, paraoxonase-1 (PON1) and erythrocyte superoxide dismutase activities significantly decreased in the diabetic rats compared to the controls, while a higher fasting plasma glucose level was observed in the diabetic animals. The glutathione peroxidase activity remained statistically unchanged. Malondialdehyde and oxidized low-density lipoprotein levels were higher in the diabetic animals; however, these values were significantly reduced following vitamin E and selenium supplementation. In summary, both AO and XO activities increase in STZ-induced diabetic rats, and vitamin E and selenium supplementation can reduce these activities. The results also indicate that administration of vitamin E and selenium has hypolipidemic, hypoglycemic, and antioxidative effects. It decreases tissue damages in diabetic rats, too.
Assuntos
Aldeído Oxidase/metabolismo , Diabetes Mellitus Experimental/metabolismo , Rim/metabolismo , Fígado/metabolismo , Xantina Oxidase/metabolismo , Animais , Antioxidantes/metabolismo , Arildialquilfosfatase/sangue , Arildialquilfosfatase/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Suplementos Nutricionais , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Glutationa Peroxidase/sangue , Glutationa Peroxidase/metabolismo , Rim/efeitos dos fármacos , Lipídeos/sangue , Fígado/efeitos dos fármacos , Malondialdeído/sangue , Ratos , Ratos Sprague-Dawley , Selenito de Sódio/administração & dosagem , Selenito de Sódio/farmacologia , Estreptozocina , Superóxido Dismutase/sangue , Superóxido Dismutase/metabolismo , Vitamina E/administração & dosagem , Vitamina E/farmacologia , Vitaminas/administração & dosagem , Vitaminas/farmacologiaRESUMO
OBJECTIVE: To study the role of Quercetin in streptozotocin-induced diabetes in rats. METHODS: Wistar male rat (n=40) were allocated into three groups, control group (n=10) and Quercetin (QR) group received 15 mg/kg (IP) QR, (n=10), and diabetic group that received 55 mg/kg (IP) streptozotocin (STZ) (n=20) which was subdivided to two groups of 10; STZ group and treatment group. Treatment group received 55 mg/kg (IP) STZ plus 15 mg/kg QR, daily for 4 weeks, respectively; however, the control group just received an equal volume of distilled water daily (IP). Diabetes was induced by a single (IP) injection of streptozotocin (55 mg/kg). Animals were kept in standard condition. Twenty-eight days after inducing diabetic, 5 mL blood were collected for TAC, MDA and Ox-LDL levels and liver tissues of rat in whole groups were removed then prepared for apoptosis analysis by Tunel method. RESULTS: Apoptotic cells significantly decreased in group that has received 15 mg/kg (IP) Quercetin (P<0.05) in comparison to experimental groups (P<0.05). CONCLUSIONS: Since in our study 15 mg/kg (IP) Quercetin have significantly Preventive effect on liver cells damages by reducing number of apoptotic cells in Liver, so it seems that using it can be effective for treatment in diabetic rat.
Assuntos
Apoptose , Diabetes Mellitus Experimental/tratamento farmacológico , Hepatócitos/fisiologia , Lipoproteínas LDL/sangue , Fígado/patologia , Cebolas/química , Quercetina/uso terapêutico , Aldeído Oxidase/antagonistas & inibidores , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/uso terapêutico , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/uso terapêutico , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Quercetina/isolamento & purificação , Ratos Wistar , EstreptozocinaRESUMO
Intake of anthocyanin-rich foods has been associated with a reduced risk of cardiovascular diseases. We recently reported that a nutritional supplementation with a bilberry anthocyanin-rich extract (BE) attenuates atherosclerotic lesion development in apolipoprotein E-deficient (apoEâ»/â») mice. However, the mechanism(s) of their preventive action are not completely understood. Anthocyanins may alter mRNA levels of genes related to atherosclerosis in cultured macrophages and endothelial cells, but in vivo studies remain scarce. The aim of the present study was to explore the in vivo mechanisms of action of the same bilberry extract, administered by supplementation at a nutritional level, in the aorta of apo Eâ»/â» mice using a global transcriptomic approach. This study focused on the early stage of atherosclerosis development for better assessment of BE action on initiation mechanisms of this pathology. After a two week period, plasma lipid and antioxidant capacity were evaluated and the global genomic analysis was carried out using pangenomic microarrays. BE supplementation significantly improved hypercholesterolemia whereas the plasmatic antioxidant status remained unchanged. Nutrigenomic analysis identified 1261 genes which expression was modulated by BE in the aorta. Bioinformatic analysis revealed that these genes are implicated in different cellular processes such as oxidative stress, inflammation, transendothelial migration and angiogenesis, processes associated with atherosclerosis development/protection. Some of the most significantly down-regulated genes included genes coding for AOX1, CYP2E1 or TXNIP implicated in the regulation of oxidative stress, JAM-A coding for adhesion molecules or VEGFR2 implicate in regulation of angiogenesis. Other genes were up-regulated, such as CRB3, CLDN14 or CDH4 potentially associated with increased cell-cell adhesion and decreased paracellular permeability. These results provide a global integrated view of the mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis.