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1.
Pestic Biochem Physiol ; 175: 104829, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33993977

RESUMO

Plant essential oils (EOs) are secondary metabolites derived from aromatic plants that are composed of complex mixtures of chemical constituents. EOs have been proposed as one of the alternative methods for bed bug (Cimex lectularius L.) control. In insecticide resistant mosquitoes and tobacco cutworm, EOs synergize pyrethroid toxicity by inhibiting detoxification enzymes. However, whether EOs and their constituents enhance pyrethroid toxicity in C. lectularius has remained unknown. Therefore, this study was designed to (i) determine the effects of binary mixtures of deltamethrin (a pyrethroid insecticide) with EOs or EO constituents or EcoRaider® (an EO-based product) on mortality of insecticide resistant and susceptible bed bugs, and (ii) evaluate the effects of EO constituent pre-treatment on detoxification enzyme activities of resistant and susceptible populations. Topical bioassays with binary mixtures of deltamethrin and individual EOs (e.g., thyme, oregano, clove, geranium or coriander oils) or their major constituents (e.g., thymol, carvacrol, eugenol, geraniol or linalool) or EcoRaider® at doses that kill approximately 25% of bed bugs caused significant increases in mortality of resistant bed bugs. However, in the susceptible population, only coriander oil, EcoRaider®, thymol, and carvacrol significantly increased the toxicity of deltamethrin. Detoxification enzyme assays with protein extracts from bed bugs pre-treated with EO constituents suggested selective inhibition of cytochrome P450 activity in the resistant population, but no impacts were observed on esterase and glutathione transferase activities in either population. Inhibition of P450 activity by EO constituents thus appears to be one of the mechanisms of deltamethrin toxicity enhancement in resistant bed bugs.


Assuntos
Percevejos-de-Cama , Inseticidas , Óleos Voláteis , Piretrinas , Animais , Sistema Enzimático do Citocromo P-450 , Resistência a Inseticidas , Inseticidas/toxicidade , Nitrilos , Óleos Voláteis/toxicidade , Piretrinas/toxicidade
2.
Nat Commun ; 12(1): 2680, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976191

RESUMO

Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.


Assuntos
Fenômenos Biológicos , Diagnóstico por Imagem/métodos , Medições Luminescentes/métodos , Modelos Animais , Animais , Animais Geneticamente Modificados , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Cães , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/instrumentação , Estrutura Molecular , Reprodutibilidade dos Testes
3.
Appl Microbiol Biotechnol ; 105(10): 4177-4187, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33944982

RESUMO

Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 ∆saverm3882 ∆saverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. KEY POINTS: • Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq. • Selection validity was evaluated by an engineered S. avermitilis expression system. • Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.


Assuntos
Ferredoxinas , Streptomyces , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Japão , Streptomyces/genética , Streptomyces/metabolismo
4.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947016

RESUMO

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name "B. belcheri EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.


Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Anfioxos/enzimologia , Alcadienos/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxido de Hidrogênio/metabolismo , Cinética , Anfioxos/genética , Oxilipinas/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por Substrato
5.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33921959

RESUMO

Chloroxylenol (PCMX) is applied as a preservative and disinfectant in personal care products, currently recommended for use to inactivate the SARS-CoV-2 virus. Its intensive application leads to the release of PCMX into the environment, which can have a harmful impact on aquatic and soil biotas. The aim of this study was to assess the mechanism of chloroxylenol biodegradation by the fungal strains Cunninghamella elegans IM 1785/21GP and Trametes versicolor IM 373, and investigate the ecotoxicity of emerging by-products. The residues of PCMX and formed metabolites were analysed using GC-MS. The elimination of PCMX in the cultures of tested microorganisms was above 70%. Five fungal by-products were detected for the first time. Identified intermediates were performed by dechlorination, hydroxylation, and oxidation reactions catalysed by cytochrome P450 enzymes and laccase. A real-time quantitative PCR analysis confirmed an increase in CYP450 genes expression in C. elegans cells. In the case of T. versicolor, spectrophotometric measurement of the oxidation of 2,20-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) showed a significant rise in laccase activity during PCMX elimination. Furthermore, with the use of bioindicators from different ecosystems (Daphtoxkit F and Phytotoxkit), it was revealed that the biodegradation process of PCMX had a detoxifying nature.


Assuntos
Cunninghamella/metabolismo , Trametes/metabolismo , Xilenos/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Daphnia/efeitos dos fármacos , Daphnia/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica , Lacase/metabolismo , Oxirredução , Testes de Toxicidade , Xilenos/análise , Xilenos/farmacologia
6.
Phytochemistry ; 187: 112747, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33823457

RESUMO

The leaf intercellular space is a site of plant-microbe interactions where pathogenic bacteria such as Pseudomonas syringae grow. In Arabidopsis thaliana, the biosynthesis of tryptophan-derived indolic metabolites is induced by P. syringae infection. Using high-resolution mass spectrometry-based profiling and biosynthetic mutants, we investigated the role of indolic compounds and other small molecules in the response of mature Arabidopsis to P. syringae. We observed dihydrocamalexic acid (DHCA), the precursor to the defense-related compound camalexin, accumulating in intercellular washing fluids (IWFs) without further conversion to camalexin. The indolic biosynthesis mutant cyp71a12/cyp71a13 was more susceptible to P. syringae compared to mature wild-type plants displaying age-related resistance (ARR). DHCA and structural analogs inhibit P. syringae growth (MIC ~ 500 µg/mL), but not at concentrations found in IWFs, and DHCA did not inhibit biofilm formation in vitro. However, infiltration of exogenous DHCA enhanced resistance in mature cyp71a12/cyp71a13. These results provide evidence that DHCA derived from CYP71A12 and CYP71A13 activity accumulates in the intercellular space and contributes to the resistance of mature Arabidopsis to P. syringae without directly inhibiting bacterial growth.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Doenças das Plantas , Folhas de Planta , Pseudomonas syringae
7.
Molecules ; 26(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799696

RESUMO

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Simulação por Computador , Cianobactérias , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/fisiologia , Evolução Molecular , Firmicutes , Genômica/métodos , Família Multigênica , Mycobacterium , Filogenia , Metabolismo Secundário/fisiologia , Streptomyces
8.
Plant Sci ; 307: 110905, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33902861

RESUMO

Contrary to animals, little is known in plants about enzymes able to produce fatty acid epoxides. In our attempt to find and characterize a new fatty acid epoxygenase in Arabidopsis thaliana, data mining brought our attention on CYP77B1. Modification of the N-terminus was necessary to get enzymatic activity after heterologous expression in yeast. The common plant fatty acid C18:2 was converted into the diol 12,13-dihydroxy-octadec-cis-9-enoic acid when incubated with microsomes of yeast expressing modified CYP77B1 and AtEH1, a soluble epoxide hydrolase. This diol originated from the hydrolysis by AtEH1 of the epoxide 12,13-epoxy-octadec-cis-9-enoic acid produced by CYP77B1. A spatio-temporal study of CYP77B1 expression performed with RT-qPCR revealed the highest level of transcripts in flower bud while, in open flower, the enzyme was mainly present in pistil. CYP77B1 promoter-driven GUS expression confirmed reporter activities in pistil and also in stamens and petals. In silico co-regulation data led us to hypothesize that CYP77B1 could be involved in cutin synthesis but when flower cutin of loss-of-function mutants cyp77b1 was analyzed, no difference was found compared to cutin of wild type plants. Phylogenetic analysis showed that CYP77B1 is strictly conserved in flowering plants, suggesting a specific function in this lineage.


Assuntos
Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Compostos de Epóxi/metabolismo , Ácidos Graxos/metabolismo , Magnoliopsida/enzimologia , Oxigenases/metabolismo
9.
Ecotoxicol Environ Saf ; 217: 112247, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33901780

RESUMO

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.


Assuntos
4-Butirolactona/análogos & derivados , Abelhas/fisiologia , Fungicidas Industriais/toxicidade , Inseticidas/toxicidade , Piridinas/toxicidade , 4-Butirolactona/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Imidazóis , Inseticidas/metabolismo , Neonicotinoides , Toxicogenética , Triazóis
10.
Molecules ; 26(6)2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804713

RESUMO

Nutmeg is a traditional spice and medicinal plant with a variety of pharmacological activities. However, nutmeg abuse due to its hallucinogenic characteristics and poisoning cases are frequently reported. Our previous metabolomics study proved the hepatotoxicity of nutmeg and demonstrated that high-dose nutmeg can affect the synthesis and secretion of bile acids and cause oxidative stress. In order to further investigate the hepatotoxicity of nutmeg, normal saline, 1 g/kg, 4 g/kg nutmeg were administrated to male Kunming mice by intragastrical gavage for 7 days. Histopathological investigation of liver tissue, proteomics and biochemical analysis were employed to explore the mechanism of liver damage caused by nutmeg. The results showed that a high-dose (4 g/kg) of nutmeg can cause significant increased level of CYP450s and depletion of antioxidants, resulting in obvious oxidative stress damage and lipid metabolism disorders; but this change was not observed in low-dose group (1 g/kg). In addition, the increased level of malondialdehyde and decreased level of glutathione peroxidase were found after nutmeg exposure. Therefore, the present study reasonably speculates that nutmeg exposure may lead to liver injury through oxidative stress and the degree of this damage is related to the exposure dose.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Peroxidase/metabolismo , Myristica/toxicidade , Proteômica , Sementes/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Masculino , Camundongos , Estresse Oxidativo
11.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802237

RESUMO

Breast cancer is the most frequent cancer in the female population worldwide. The role of germline genetic variability in cytochromes P450 (CYP) in breast cancer prognosis and individualized therapy awaits detailed elucidation. In the present study, we used the next-generation sequencing to assess associations of germline variants in the coding and regulatory sequences of all human CYP genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 22 prioritized variants associating with a response or survival in the above evaluation phase were then analyzed by allelic discrimination in the large confirmation set (n = 802). Associations of variants in CYP1B1, CYP4F12, CYP4X1, and TBXAS1 with the response to the neoadjuvant cytotoxic chemotherapy were replicated by the confirmation phase. However, just association of variant rs17102977 in CYP4X1 passed the correction for multiple testing and can be considered clinically and statistically validated. Replicated associations for variants in CYP4X1, CYP24A1, and CYP26B1 with disease-free survival of all patients or patients stratified to subgroups according to therapy type have not passed a false discovery rate test. Although statistically not confirmed by the present study, the role of CYP genes in breast cancer prognosis should not be ruled out. In conclusion, the present study brings replicated association of variant rs17102977 in CYP4X1 with the response of patients to the neoadjuvant cytotoxic chemotherapy and warrants further research of genetic variation CYPs in breast cancer.


Assuntos
Neoplasias da Mama , Sistema Enzimático do Citocromo P-450 , Variação Genética , Terapia Neoadjuvante , Proteínas de Neoplasias , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Taxa de Sobrevida
12.
Nat Commun ; 12(1): 2260, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859207

RESUMO

Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ligantes , Redes e Vias Metabólicas , Aromatase/metabolismo , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Humanos , Lipossomos/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Especificidade por Substrato
13.
Planta ; 253(5): 89, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33818685

RESUMO

MAIN CONCLUSION: BmG10H-1 transcript from B. monnieri was functionally active. BmG10H-1 promoter drives GUS activity in response to MeJA and wounding. BmMYB35 regulates BmG10H-1 transcript by binding to its promoter. Geraniol 10-hydroxylase (G10H) is one of the important regulatory cytochrome P450 monooxygenase, which is involved in the biosynthesis of monoterpene alkaloids. However, G10H is not characterized at the enzymatic or at the regulatory aspect in B. monnieri. In the present study, we have identified two transcripts of BmG10H (BmG10H-1and BmG10H-2) and characterized the methyl jasmonate (MeJA) and wound responsive BmG10H-1 transcript from B. monnieri. BmG10H-1 showed induced expression after 3 h of MeJA and wounding treatment in the shoot. Yeast purified recombinant BmG10H-1 protein is enzymatically active, having Vmax of 0.16 µMsec-1 µg-1 protein and catalyzes the hydroxylation of geraniol to 10-hydroxy geraniol. The BmG10H-1 promoter was isolated by using the genome walking method. BmG10H-1 promoter can drive GUS expression in transgenic Arabidopsis thaliana. GUS activity of MeJA and wound-treated Arabidopsis seedlings were found to be increased as compared to the control untreated seedlings, whereas no GUS activity was found in deleted MeJA responsive and W-box cis-elements. This shows that the BmG10H-1 promoter contains functional MeJA (TGACG) and wound responsive (TGACCT) cis-elements. Further, shoot specific and MeJA responsive recombinant BmMYB35 protein was purified, which binds with the MYB recognition cis-element (TGGTTA) present in the BmG10H-1 promoter and transcriptionally activates the reporter gene in yeast. In conclusion, the characterization of MeJA and wound responsive BmG10H-1 provides novel information about its transcriptional regulation by binding with MYB transcription factor in B. monnieri.


Assuntos
Acetatos/metabolismo , Bacopa/genética , Bacopa/metabolismo , Ciclopentanos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Genes de Plantas/genética , Oxilipinas/metabolismo , Bacopa/enzimologia , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética
14.
Pestic Biochem Physiol ; 174: 104826, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33838719

RESUMO

Liriomyza trifolii is an invasive leafminer fly that inflicts damage on many horticultural and vegetable crops. In this study, the effects of elevated temperatures on L. trifolii tolerance to insecticides abamectin (AB), monosultap (MO) and a mixture of abamectin and monosultap (AM) were firstly investigated, then five CYP450 genes (LtCYPs) were cloned, and expression patterns and NADPH cytochrome C reductase (NCR) activity in L. trifolii were compared in response to high temperature stress and insecticide exposure. Results showed elevated temperatures induced expression of LtCYP450s, the expression level of LtCYP4g1, LtCYP4g15 and LtCYP301A1 after exposed to different high temperature were significantly up-regulated compared with the control (25 °C), while there was no significant difference in LtCYP4E21 and LtCYP18A1. Under the joint high temperature and insecticide stress, the expression of LtCYP4g15, LtCYP18A1 and LtCYP301A1 was significantly higher under elevated temperatures than that of only under AB exposure. For MO and AM exposure, only 40 °C could induce the expression of LtCYP4g15, LtCYP18A1 and LtCYP301A1. In general, the LtCYPs expression pattern was correlated with increased NCR activity and decreased mortality in response to insecticide exposure under elevated temperatures. These all demonstrated that insecticide tolerance in L. trifolii could be mediated by high temperature. This study improves our understanding of L. trifolii physiology and offers a theoretical context for improved control that ultimately reduces the abuse of insecticides and decreases exposure to non-target organisms.


Assuntos
Dípteros , Inseticidas , Animais , Produtos Agrícolas , Sistema Enzimático do Citocromo P-450/genética , Inseticidas/toxicidade , Temperatura
15.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926097

RESUMO

Ovarian hormone deficiency leads to increased body weight, visceral adiposity, fatty liver and disorders associated with menopausal metabolic syndrome. To better understand the underlying mechanisms of these disorders in their early phases of development, we investigated the effect of ovariectomy on lipid and glucose metabolism. Compared to sham-operated controls, ovariectomized Wistar female rats markedly increased whole body and visceral adipose tissue weight (p ˂ 0.05) and exhibited insulin resistance in peripheral tissues. Severe hepatic triglyceride accumulation (p ˂ 0.001) after ovariectomy preceded changes in both serum lipids and glucose intolerance, reflecting alterations in some CYP proteins. Increased CYP2E1 (p ˂ 0.05) and decreased CYP4A (p ˂ 0.001) after ovariectomy reduced fatty acid oxidation and induced hepatic steatosis. Decreased triglyceride metabolism and secretion from the liver contributed to hepatic triglyceride accumulation in response to ovariectomy. In addition, interscapular brown adipose tissue of ovariectomized rats exhibited decreased fatty acid oxidation (p ˂ 0.01), lipogenesis (p ˂ 0.05) and lipolysis (p ˂ 0.05) despite an increase in tissue weight. The results provide evidence that impaired hepatic triglycerides and dysregulation of some CYP450 proteins may have been involved in the development of hepatic steatosis. The low metabolic activity of brown adipose tissue may have contributed to visceral adiposity as well as triglyceride accumulation during the postmenopausal period.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Transtornos do Metabolismo dos Lipídeos/etiologia , Metabolismo dos Lipídeos/fisiologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/fisiologia , Dieta Hiperlipídica , Dislipidemias/metabolismo , Fígado Gorduroso/metabolismo , Feminino , Glucose/metabolismo , Insulina/metabolismo , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Transtornos do Metabolismo dos Lipídeos/fisiopatologia , Lipídeos/sangue , Lipogênese/efeitos dos fármacos , Lipólise , Fígado/metabolismo , Menopausa/metabolismo , Menopausa/fisiologia , Obesidade/metabolismo , Ovariectomia/efeitos adversos , Pós-Menopausa/metabolismo , Pós-Menopausa/fisiologia , Ratos , Ratos Wistar , Triglicerídeos/metabolismo , Ganho de Peso
16.
Xenobiotica ; 51(6): 668-679, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33879032

RESUMO

Trazpiroben (TAK-906), a peripherally selective dopamine D2/D3 receptor antagonist, is being developed for the treatment of patients with gastroparesis. The potential of trazpiroben to act as a perpetrator or a victim for cytochrome P450 (CYP)- or transporter- mediated drug-drug interactions (DDIs) was evaluated following the latest regulatory guidelines.In vitro studies revealed that trazpiroben is metabolised mainly through a non-CYP pathway (56.7%) by multiple cytosolic, NADPH-dependent reductase, such as aldo-keto reductase and short-chain dehydrogenase/reductase including carbonyl reductases. Remaining metabolism occurs through CYP3A4 and CYP2C8 (43.3%). Trazpiroben is neither an inhibitor nor an inducer of major CYP enzymes at a clinically relevant dose. It is a substrate of P-glycoprotein (P-gp) and organic anion transporting polypeptide (OATP) 1B1/1B3, but is not an inhibitor of transporters listed in the DDI guidelines at a clinically relevant dose. This is consistent with findings from CYP3A and P-gp-based clinical assessment showing no substantial change (≤2-fold) in trazpiroben exposure when co-administered with itraconazole.Collectively, trazpiroben has low potential of enzyme-mediated DDIs and is unlikely to act as a perpetrator of transporter-mediated DDIs but there may be a potential to act as a victim of OATP1B1/1B3 DDI that will be evaluated clinically.


Assuntos
Gastroparesia , Transportadores de Ânions Orgânicos , Preparações Farmacêuticas , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Proteínas de Membrana Transportadoras
17.
Food Chem ; 356: 129550, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33819785

RESUMO

Glucosinolates (GSLs) are well known for plant defense and human nutrition. In this study, broccoli seedlings were illuminated under different LED light, including white, red, blue, and 75% red + 25% blue (200 mmol·m-2·s-1) for 4 weeks to investigate the effects of LED light on GSLs and sulforaphane biosynthesis. Results showed that red light promoted GSL biosynthesis and sulforaphane accumulation because red light could induce SOT18 expression to advance aliphatic GSLs biosynthesis, whereas the high tryptophan content and the upregulation of CYP79B2, CYP79B3, and CYP83B1 were attributed to indole GSL biosynthesis. Low-level methionine content and downregulated SOT18 were the main factors inhibiting GSLs and sulforaphane accumulation under blue LED illumination. BoHY5 gene expression was induced significantly and the yeast one-hybrid assay demonstrated BoHY5 could bind to SOT18 promoter. Consequently, BoHY5 inhibited SOT18 expression, and played a negative role in the GSL biosynthetic network.


Assuntos
Brassica/metabolismo , Glucosinolatos/metabolismo , Isotiocianatos/metabolismo , Plântula/metabolismo , Sulfóxidos/metabolismo , Brassica/efeitos da radiação , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Iluminação , Plântula/efeitos da radiação
18.
BMC Plant Biol ; 21(1): 191, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879061

RESUMO

BACKGROUND: Cytochrome P450s (P450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. RESULTS: A comparative species leaf metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea P450s were investigated from leaf and root tissue. A total of 103 and 123 full-length P450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of P450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Representatives within the CYP71D and CYP726A were identified in Fontainea that are excellent candidates for diterpenoid synthesis, of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species and were significantly more highly expressed in root tissue compared to leaf tissue. CONCLUSION: This study presents a comprehensive overview of the P450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Diterpenos/metabolismo , Euphorbiaceae/genética , Genes de Plantas , Proteínas de Plantas/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Euphorbiaceae/enzimologia , Euphorbiaceae/metabolismo , Família Multigênica , Proteínas de Plantas/metabolismo
19.
J Hazard Mater ; 414: 125457, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33652222

RESUMO

Predicting the detoxifying mechanism and potential toxic derivatives of xenobiotic substances is significant for risk assessment. The present study delineated the detoxifying mechanism of 1-chloro-4-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene (DDT) metabolized by human P450 enzymes using a combination of molecular dynamic (MD), quantum mechanics/molecular mechanics (QM/MM) and density functional theory (DFT). This study highlights that DDT can be metabolized by P450 enzymes through the hydrogen abstraction and electrophilic addition mechanism, and the main derivatives are epoxides (2,3-oxide-DDT and 3,4-oxide-DDT), DDE and dicofol. The epoxides are unstable and the C-O bond cleavage easily occurs by the reaction with hydronium ion or hydroxyl radicals, yielding endocrine disruptor hydroxylated DDT. The eco-toxicity evaluation indicates that the derivatives of DDT are less toxic than DDT, and the solubility increase of the derivatives can accelerate their excretion from the body. The study can provide an understanding of the biotransformation of DDT by the P450 enzymes in human livers.


Assuntos
Sistema Enzimático do Citocromo P-450 , DDT , Biotransformação , Sistema Enzimático do Citocromo P-450/metabolismo , DDT/toxicidade , Humanos , Simulação de Dinâmica Molecular
20.
Med Sci Monit ; 27: e930591, 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33723203

RESUMO

BACKGROUND Cytochrome P450 (CYP) genes are necessary for the production or metabolism of fetal sex hormones during pregnancy. The second-to-fourth digit ratio (2D: 4D) is formed in the early stage of human fetal development and considered an indicator reflecting prenatal sex steroids levels. We explored the association between 2D: 4D and single-nucleotide polymorphisms (SNPs) of CYP. MATERIAL AND METHODS Correlation analysis between 2D: 4D and 8 SNPs, rs2687133 (CPY3A7), rs7173655 (CYP11A1), rs1004467, rs17115149, and rs2486758 (CYP17A1), and rs4646, rs2255192, rs4275794 (CYP19A1), was performed using data from 426 female and 412 male Chinese university students. SNP genotyping was conducted using PCR. Digit lengths were photographed and measured by image processing software. RESULTS rs2486758 (CYP17A1) correlated with left hand 2D: 4D in men (P=0.026), and rs1004467 (CYP17A1) correlated with right hand 2D: 4D in men (P=0.008) and the whole population (P=0.032). In men, allele G rs1004467 decreased right hand 2D: 4D, while allele C of rs2486758 increased left hand 2D: 4D. In women, left hand 2D: 4D was higher in genotypes with allele A of SNP rs4646 (CYP19A1) under the dominant genetic model; female DR-L was higher in genotypes with allele T of rs17115149 (CYP11A1). SNPs rs2687133 (CYP3A7) and rs1004467 (CYP17A1) were significantly correlated with right hand 2D: 4D (P=0.0107). CONCLUSIONS SNPs rs1004467 and rs2486758 of CYP17A1 are significant in the relationship between 2D: 4D and CYP gene polymorphisms under different conditions. SNP interactions between CYP genes probably impact 2D: 4D. The correlation between 2D: 4D and some sex hormone-related diseases may be due to the effect of CYP variants on the 2 phenotypes.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Dedos/anatomia & histologia , Alelos , Aromatase/genética , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Citocromo P-450 CYP3A/genética , Feminino , Frequência do Gene/genética , Estudos de Associação Genética/métodos , Genótipo , Humanos , Masculino , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Esteroide 17-alfa-Hidroxilase/genética , Estudantes , Universidades , Adulto Jovem
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