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1.
Molecules ; 24(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366067

RESUMO

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Desmetilação/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Microssomos Hepáticos/enzimologia , Animais , Biotransformação/efeitos dos fármacos , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães , Corantes Fluorescentes/química , Humanos , Isoquinolinas/química , Cinética , Macaca fascicularis , Camundongos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura , Teofilina/análogos & derivados , Teofilina/farmacologia
2.
Eur J Pharm Sci ; 131: 177-194, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776468

RESUMO

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in 'suspension' for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.


Assuntos
Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1B1 , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Quinazolinonas , Antineoplásicos/farmacologia , Benzo(a)pireno/toxicidade , Bioensaio , Linhagem Celular , Cisplatino/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inibidores , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Quinazolinonas/química , Quinazolinonas/farmacologia
3.
Bioorg Med Chem ; 27(2): 285-304, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553624

RESUMO

Selective cytochrome P450 (CYP) 1B1 inhibition has potential as an anticancer strategy that is unrepresented in the current clinical arena. For development of a selective inhibitor, we focused on the complexity caused by sp3-hybridized carbons and synthesized a series of benzo[h]chromone derivatives linked to a non-aromatic B-ring using α-naphthoflavone (ANF) as the lead compound. Ring structure comparison suggested compound 37 as a suitable cyclohexyl-core with improved solubility. Structural evolution of 37 produced the azide-containing cis-49a, which had good properties in three important respects: (1) selectivity for CYP1B1 over CYP1A1 and CYP1A2 (120-times and 150-times, respectively), (2) greater inhibitory potency of >2 times that of ANF, and (3) improved solubility. The corresponding aromatic B-ring compound 59a showed low selectivity and poor solubility. To elucidate the binding mode, we performed X-ray crystal structure analysis, which revealed the interaction mode and explained the subtype selectivity of cis-49a.


Assuntos
Benzoflavonas/química , Inibidores do Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1B1/antagonistas & inibidores , Benzoflavonas/síntese química , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Inibidores do Citocromo P-450 CYP1A2/síntese química , Citocromo P-450 CYP1B1/química , Desenho de Drogas , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Solubilidade , Relação Estrutura-Atividade
4.
J Biol Chem ; 293(50): 19211-19212, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30552114

RESUMO

The cytochromes P450 (CYPs) oxidatively transform a huge number of substrates in both prokaryotic and eukaryotic organisms, but the mechanisms by which they accommodate these diverse molecules remain unclear. A new study by Bart and Scott reports two co-crystal structures of CYP1A1 that reveal structural rearrangements and flexible interaction networks that explain how the active site cavity shapes itself around new ligands. These data open the door to an increased understanding of fundamental enzyme behavior and improved searches for anti-cancer compounds.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Inibidores Enzimáticos/metabolismo , Cloridrato de Erlotinib/metabolismo , Furocumarinas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP1A1/química , Inibidores Enzimáticos/química , Cloridrato de Erlotinib/química , Furocumarinas/química , Humanos , Ligantes , Ligação Proteica , Especificidade por Substrato
5.
J Biol Chem ; 293(50): 19201-19210, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30254074

RESUMO

Human cytochrome P450 1A1 (CYP1A1) is an extrahepatic enzyme involved in the monooxygenation of structurally diverse compounds ranging from natural products to drugs and protoxins. Because CYP1A1 has a role in human carcinogenesis, inhibiting its activity may potentially aid in cancer chemoprevention, whereas utilizing CYP1A1's oxidative activity could help selectively activate anticancer prodrugs. Such potential therapeutic purposes require detailed knowledge of CYP1A1's interactions with potential ligands. Known CYP1A1 ligands also vary substantially in size, and it has not been apparent from a single existing CYP1A1 structure how larger, structurally diverse ligands are accommodated within the enclosed active site. Here, two new X-ray structures with the natural product furanocoumarin bergamottin (at 2.85 Å resolution) and the lung cancer drug erlotinib (3.0 Å) revealed binding orientations consistent with the formation of innocuous metabolites and of toxic metabolites, respectively. They also disclosed local changes in the roof of the active site that enlarge the active site and ultimately form a channel to the protein exterior. Although further structural modifications would be required to accommodate the largest CYP1A1 ligands, knowing which components of the active site are malleable provides powerful information for those attempting to use computational approaches to predict compound binding and substrate metabolism by this clinically relevant monooxygenase.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Inibidores Enzimáticos/metabolismo , Cloridrato de Erlotinib/metabolismo , Furocumarinas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Cloridrato de Erlotinib/química , Furocumarinas/química , Humanos , Ligantes , Ligação Proteica , Especificidade por Substrato
6.
Artigo em Inglês | MEDLINE | ID: mdl-30012402

RESUMO

Fish can be simultaneously or sequentially exposed to various kinds of pollutants, resulting in combined effects. Polycyclic aromatic hydrocarbons induce cytochrome P450 monooxygenase 1A (CYP1A) expression, which catalyzes the conversion of the organophosphorus insecticide chlorpyrifos (CPF) into its most active derivative, CPF-oxon. CPF-oxon inhibits CYP1A and other enzymes, including carboxylesterases (CEs) and acetylcholinesterase (AChE). We studied the effects of an in vivo exposure to crude oil water accommodated fraction (WAF) followed by an ex vivo exposure of liver tissue to CPF on the expression of Cyp1a, AhR and ARNT mRNA, CYP1A protein and on the activity of biomarker enzymes in the rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed to WAF (62 µg L-1 TPH) for 48 h. Then, liver was dissected out, sliced and exposed to 20 µg L-1 CPF ex vivo for 1 h. Liver tissue was analyzed for mRNA and protein expression and for CEs, AChE, glutathione S-transferase (GST) and CYP1A (EROD) activity. WAF induced Cyp1a mRNA and CYP1A protein expression by 10-fold and 2.5-8.3-fold, respectively, with no effect of CPF. WAF induced AhR expression significantly (4-fold) in control but not in CPF treated liver tissue. ARNT mRNA expression was significantly lowered (5-fold) by WAF. CPF significantly reduced liver EROD activity, independently of WAF pre-treatment. CEs activity was significantly inhibited in an additive manner following in vivo exposure to WAF (42%) and ex vivo exposure to CPF (19%). CPF exposure inhibited AChE activity (37%) and increased GST activity (42%).


Assuntos
Clorpirifos/toxicidade , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Oncorhynchus mykiss/fisiologia , Poluição por Petróleo/efeitos adversos , Petróleo/toxicidade , Poluentes Químicos da Água/toxicidade , Acetilcolinesterase/química , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Aquicultura , Translocador Nuclear Receptor Aril Hidrocarboneto/antagonistas & inibidores , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Biomarcadores/metabolismo , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Clorpirifos/farmacologia , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/toxicidade , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Inseticidas/farmacologia , Fígado/enzimologia , Fígado/metabolismo , Resíduos de Praguicidas/farmacologia , Resíduos de Praguicidas/toxicidade , Poluentes Químicos da Água/farmacologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-29870789

RESUMO

Based on the assumed oestrogenic and apoptotic properties of soya isoflavones (genistein, daidzein), and following the current OECD test-guidelines and principle of 3Rs, we have studied the potential toxicity of phytochemicals on the zebrafish embryos test (ZFET). For this purpose, zebrafish embryos at 2-3 h post-fertilisation (hpf) were exposed to both soya isoflavones (from 1.25 mg/L to 20 mg/L) and assayed until 96 hpf. Lethal and sub-lethal endpoints (mortality, hatching rates and malformations) were estimated in the ZFET, which was expanded to potential gene expression markers, determining the lowest observed effect (and transcriptional) concentrations (LOEC, LOTEC), and the no-observable effect (and transcriptional) concentrations (NOEC, NOTEC). The results revealed that genistein is more toxic (LC50-96 hpf: 4.41 mg/L) than daidzein (over 65.15 mg/L). Both isoflavones up-regulated the oestrogen (esrrb) and death receptors (fas) and cyp1a transcript levels. Most thyroid transcript signals were up-regulated by genistein (except for thyroid peroxidase/tpo), and the hatching enzyme (he1a1) was exclusively up-regulated by daidzein (from 1.25 mg/L onwards). The ZFET proved suitable for assessing toxicant effects of both isoflavones and potential disruptions (i.e. oestrogenic, apoptotic, thyroid, enzymatic) during the embryogenesis and the endotrophic larval period.


Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genisteína/efeitos adversos , Isoflavonas/efeitos adversos , Fitoestrógenos/efeitos adversos , Glândula Tireoide/metabolismo , Animais , Apoptose , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Suplementos Nutricionais/efeitos adversos , Ectogênese , Embrião não Mamífero/enzimologia , Disruptores Endócrinos/efeitos adversos , Disruptores Endócrinos/metabolismo , Genisteína/metabolismo , Isoflavonas/metabolismo , Larva/enzimologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dose Letal Mediana , Receptores Estrogênicos/química , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Sementes/química , Transdução de Sinais , Soja/química , Glândula Tireoide/embriologia , Glândula Tireoide/enzimologia , Testes de Toxicidade Aguda , Peixe-Zebra , Receptor fas/agonistas , Receptor fas/química , Receptor fas/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-29763690

RESUMO

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1, 1A2, and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression.


Assuntos
Antineoplásicos Fitogênicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Hesperidina/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Carcinógenos Ambientais/química , Carcinógenos Ambientais/toxicidade , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inibidores , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Suplementos Nutricionais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Humanos , Células MCF-7 , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Dibenzodioxinas Policloradas/antagonistas & inibidores , Dibenzodioxinas Policloradas/química , Receptores de Hidrocarboneto Arílico/metabolismo
9.
Comput Biol Chem ; 74: 253-262, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29677600

RESUMO

2-phenyl-benzotriazole xenobiotic compounds (PBTA-4, PBTA-6, PBTA-7 and PBTA-8) that were previously isolated and identified in waters of the Yodo river, in Japan (Nukaya et al., 2001; Ohe et al., 2004; Watanabe et al., 2001) were characterized as powerful pro-mutagens. In order to predict the activation mechanism of these pro-mutagens, we designed a computational biochemistry protocol, which includes, docking experiments, molecular dynamics simulations and free energy decomposition calculations to obtain information about the interaction of 2-phenyl-benzotriazole molecules into the active center of cytochrome P450-CYP1A1 (CYP1A1). Molecular docking calculations using AutoDock Vina software shows that PBTAs are proportionally oriented in the pocket of CYP1A1, establishing π-π stacking attractive interactions between the triazole group and the Phe224, as well as, the hydrogen bonds of the terminal NH2 over the benzotriazole units with the Asn255 and Ser116 amino acids. Molecular dynamics simulations using NAMD package showed that these interactions are stable along 100.0 ns of trajectories. Into this context, free binding energy calculations employing the MM-GBSA approach, shows that some differences exists among the interaction of PBTAs with CYP1A1, regarding the solvation, electrostatic and van der Waals interaction energy components. These results suggest that PBTA molecules might be activated by CYP1A1. Thus, enhancing their mutagenicity when compared with the pro-mutagen parent species.


Assuntos
Compostos de Anilina/química , Citocromo P-450 CYP1A1/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Triazóis/química , Xenobióticos/química , Citocromo P-450 CYP1A1/metabolismo , Humanos , Estrutura Molecular , Termodinâmica
10.
Food Chem ; 258: 245-253, 2018 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-29655729

RESUMO

The physiological effects of the Stevia-derived compounds, rebaudioside A, stevioside and steviol have been the focus of several studies due to their use as sweeteners in food. Despite that, little is known about their potential food-drug interactions. In the present study, IPEC-J2 cells and primary hepatocytes were used to investigate the effect of rebaudioside A, stevioside and steviol on cytochrome p450 (CYP) mRNA expression. Moreover, hepatic microsomes were used to investigate direct interactions between the compounds and specific CYP activity. In IPEC-J2 no changes in mRNA expression of CYP1A1 or CYP3A29 were observed with the Stevia-derived compounds. In primary hepatocytes all three tested compounds induced a significant increase in CYP3A29 expression. The tested compounds had no direct effect on specific CYP activity. In conclusion, rebaudioside A, stevioside and steviol induce only minor or no changes to the CYP expression and activity, and are not likely to cause food-drug interactions.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos de Caurano/química , Diterpenos de Caurano/farmacologia , Glucosídeos/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Glucosídeos/química , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Ocludina/genética , Ocludina/metabolismo , Suínos
11.
Toxicol Lett ; 289: 54-62, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29545172

RESUMO

Benzo[a]pyrene (B[a]P), the most extensively studied carcinogen in cigarette smoke, has been regarded as a critical mediator of lung cancer. It is known that B[a]P-mediated Aryl hydrocarbon Receptor (AhR) activation stimulates the mitogen activated protein kinases (MAPK) signaling cascade in different cell models. MAPK pathway disturbances drive alterations in cellular processes, such as differentiation, proliferation, and apoptosis, and the disturbances may also modify the AhR pathway itself. However, MAPK involvement in B[a]P metabolic activation and toxicity in lung tissues is not well understood. Here, we used a non-transformed human bronchial epithelial lung cell line, BEAS-2B, to study the participation of ERK 1/2 kinases in the metabolic activation of B[a]P and in its related genotoxic effects. Our results indicate that B[a]P is not cytotoxic to BEAS-2B cells at relatively low concentrations, but it enhances CYP1A1 gene transcription and protein induction. Additionally, B[a]P promotes Src and ERK 1/2 phosphorylation. Accordingly, inhibition of both Src and ERK 1/2 phosphorylation decreases CYP1A1 protein induction, AhR nuclear translocation and production of B[a]P adducts. Together, these data suggest a crosstalk between AhR and the members of the MAPK pathway, ERK 1/2 mediated by Src kinase. This interaction is important for the adequate AhR pathway signaling that in turn induces transcription and protein induction of CYP1A1 and B[a]P-induced DNA damage in BEAS-2B cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Benzo(a)pireno/toxicidade , Carcinógenos Ambientais/toxicidade , Citocromo P-450 CYP1A1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Hidrocarboneto Arílico/agonistas , Mucosa Respiratória/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Adutos de DNA/química , Adutos de DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/química , Receptores de Hidrocarboneto Arílico/metabolismo , Mucosa Respiratória/metabolismo
12.
Comp Biochem Physiol C Toxicol Pharmacol ; 206-207: 54-64, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29555404

RESUMO

Polar cod is an abundant Arctic key species, inhabiting an ecosystem that is subjected to rapid climate change and increased petroleum related activities. Few studies have investigated biological effects of crude oil on lipid metabolism in this species, despite lipids being a crucial compound for Arctic species to adapt to the high seasonality in food abundance in their habitat. This study examines the effects of dietary crude oil exposure on transcription levels of genes related to lipid metabolism (peroxisome proliferator-activated receptors [ppar-α, ppar-γ], retinoic X receptor [rxr-ß], palmitoyl-CoA oxidase [aox1], cytochrome P4507A1 [cyp7α1]), reproduction (vitellogenin [vtg-ß], gonad aromatase [cyp19a1]) and biotransformation (cytochrome P4501A1 [cyp1a1], aryl hydrocarbon receptor [ahr2]). Exposure effects were also examined through plasma chemistry parameters. Additional fish were exposed to a PPAR-α agonist (WY-14,643) to investigate the role of PPAR-α in their lipid metabolism. The dose-dependent up-regulation of cyp1a1 reflected the activation of genes related to PAH biotransformation upon crude oil exposure. The crude oil exposure did not significantly alter the mRNA expression of genes involved in lipid homeostasis except for cyp7α1 transcription levels. Plasma levels of cholesterol and alanine transaminase showed significant alterations in fish exposed to crude oil at the end of the experiment. WY exposure induced a down-regulation of ppar-α, an effect contrary to studies performed on other fish species. In conclusion, this study showed clear effects of dietary crude oil exposure at environmentally relevant concentrations on xenobiotic biotransformation but revealed only weak alterations in the lipid metabolism of polar cod.


Assuntos
Proteínas de Peixes/metabolismo , Gadiformes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Petróleo/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Colesterol 7-alfa-Hidroxilase/antagonistas & inibidores , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Clima Frio , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Proteínas de Peixes/agonistas , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/genética , Gadiformes/crescimento & desenvolvimento , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Noruega , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , PPAR alfa/antagonistas & inibidores , PPAR alfa/genética , PPAR alfa/metabolismo , Pirimidinas/farmacologia , Reprodutibilidade dos Testes , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
13.
Environ Sci Pollut Res Int ; 25(17): 16420-16426, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29043584

RESUMO

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.


Assuntos
Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP2C19/química , Sistema Enzimático do Citocromo P-450/química , Bifenilos Policlorados/química , Catálise , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Oxirredução , Estereoisomerismo
14.
Chemosphere ; 192: 105-112, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29100118

RESUMO

Phytochemicals are widely present in fruits, vegetables and other plants and have great health benefits owing to their antioxidant properties. They are naturally found in the aquatic environment as well as discharged from sewage treatment plants after their large consumption. Little is known about their impact on fish; particularly in light of their interactions with pharmaceuticals. Therefore, this study was designed to determine the effects of diosmin, naringenin, quercetin and idole-3-carbinol on CYP1A-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity on rainbow trout hepatic microsomes in the presence of two pharmaceuticals: clotrimazole and dexamethasone. The interactions between the phytochemicals and pharmaceuticals used in this study were determined using a combination index. Hepatic microsomes were exposed to two concentrations (1-or 50 µM) of phytochemicals and pharmaceuticals separately and in combinations. Singly, clotrimazole inhibited EROD activity 40% and 90% of control, while dexamethasone did not. Naringenin and diosmin inhibited EROD activity alone up to 90% and 55% respectively, but activities were further inhibited in the presence of either pharmaceutical. The preliminary study of combinations of clotrimazole with phytochemicals primarily showed synergistic effects. While EROD activity was not inhibited in the presence of quercetin or indole-3-carbinol, significant and synergistic inhibition was detected when either of these was combined with clotrimazole or dexamethasone.


Assuntos
Clotrimazol/química , Citocromo P-450 CYP1A1/química , Dexametasona/química , Diosmina/química , Proteínas de Peixes/química , Flavanonas/química , Indóis/química , Quercetina/química , Animais , Clotrimazol/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Dexametasona/farmacologia , Diosmina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Peixes/metabolismo , Flavanonas/farmacologia , Indóis/farmacologia , Cinética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Oncorhynchus mykiss/metabolismo , Quercetina/farmacologia
15.
Cell Biochem Biophys ; 76(1-2): 91-110, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28353142

RESUMO

Cytochrome P450 (CYP) 1A and 2B subfamily enzymes are important drug metabolizing enzymes, and are highly conserved across species in terms of sequence homology. However, there are major to minor structural and macromolecular differences which provide for species-selectivity and substrate-selectivity. Therefore, species-selectivity of CYP1A and CYP2B subfamily proteins across human, mouse and rat was analyzed using molecular modeling, docking and dynamics simulations when the chiral molecules quinine and quinidine were used as ligands. The three-dimensional structures of 17 proteins belonging to CYP1A and CYP2B subfamilies of mouse and rat were predicted by adopting homology modeling using the available structures of human CYP1A and CYP2B proteins as templates. Molecular docking and dynamics simulations of quinine and quinidine with CYP1A subfamily proteins revealed the existence of species-selectivity across the three species. On the other hand, in the case of CYP2B subfamily proteins, no role for chirality of quinine and quinidine in forming complexes with CYP2B subfamily proteins of the three species was indicated. Our findings reveal the roles of active site amino acid residues of CYP1A and CYP2B subfamily proteins and provide insights into species-selectivity of these enzymes across human, mouse, and rat.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Animais , Sítios de Ligação , Domínio Catalítico , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Humanos , Ligações de Hidrogênio , Ligantes , Camundongos , Conformação Molecular , Quinidina/química , Quinidina/metabolismo , Quinina/química , Quinina/metabolismo , Ratos , Software , Especificidade da Espécie
16.
Environ Sci Pollut Res Int ; 25(5): 3977-3984, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27613629

RESUMO

Dioxins and dioxin-like compounds can be analyzed by bioanalytical screening methods to evaluate their biotoxicity. In vitro bioassays, based on 7-ethoxyresorufin-O-deethylase (EROD) and the activity of cytochrome P450 1A1 and the aryl hydrogen receptor (AhR) pathway, are employed for the evaluation of bioanalytical equivalents (BEQ) of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) from a wide variety of sample matrices. Here, we present the evaluation of 11 humic soil samples derived from forest stands across Germany and a comparison of the BEQ values against toxic equivalents (TEQ, PCDD/Fs+PCBs) derived by chemical analysis. BEQ values ranged from 8.8 to 34.1 while TEQ values from 13.9 to 60.5 pg/g dry weight. Additional two subsequent mineral layers were analyzed to identify the BEQ/TEQ gradient vertically, showing a TEQ decrease of 85.1 and 93.8 % from the humic to the first and second mineral layers, respectively. For BEQ values, a decrease as well as an increase was detected. BEQ measurements were performed with and without sample clean-up. Omitting clean-up revealed about 20 times increased BEQ values presumably due to non-persistent bioactive compounds not detected by chemical analysis. The results we present suggest that the EROD assay can be used for the screening of large sample quantities for the identification of samples showing dioxin and dioxin-like contaminations even at low levels, which can then be further analyzed by chemical analysis to identify the congener composition. The study also shows that EROD results give a qualitative image of the contamination. EROD seems to be interfered with cross-contaminants specifically for soils with high biological activity as forest layers.


Assuntos
Dioxinas/análise , Poluentes do Solo/análise , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/química , Florestas , Cromatografia Gasosa-Espectrometria de Massas , Alemanha , Ratos
17.
Environ Sci Pollut Res Int ; 25(12): 11192-11204, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28144864

RESUMO

The pharmaceutical products are emerging pollutants continuously released into the environment, because they cannot be effectively removed by the wastewater treatment plants. In recent years, questions have been raised concerning the environmental risks related to these pollutants. The goal of this research was to evaluate the responses in Lemna minor after 7 days and in Corbicula fluminea after differing durations (1, 3, 7, and 19 days) of exposure to the psychoactive drug mixture (valproic acid, citalopram, carbamazepine, cyamemazine, hydroxyzine, oxazepam, norfluoxetine, lorazepam, fluoxetine, and sertraline) in different concentrations (0, 0 + ethanol, drug concentration (DC) 1 = river water concentration, DC2 = effluent concentration, and DC3 = 10× effluent concentration). In this aim, growth parameters of L. minor, gluthathione S-transferase (GSTs), catalase (CAT), ethoxyresorufin-O-deethylase (EROD) and/or gene expressions (pi-gst, cat, cytochrome P450 4 (cyp4), multidrug resistant 1 (mdr1), and superoxide dismutase (sod)) were measured. GST activities increased significantly in L. minor exposed to DC3, but no changes were found in CAT activity. In C. fluminea, EROD activity was induced significantly in both gill and digestive gland tissues after 3 days' exposure to DC3, while a GST increase was observed only in digestive gland tissues, suggesting that these pharmaceuticals induced an oxidative effect. Gene expression analysis revealed transient transcriptomic responses of cyp4, sod, and mdr1 under drug concentrations 2 or 3 and no change of expression for the other genes (cat and pi-gst) or condition (environmental drug concentration) tested. Finally, the data reported in this study represent important ecotoxicological information, confirming that this enzyme family (cyp4, sod, and mdr1) may be considered as a sensible and early indicator of exposure to drugs and emphasizing the involvement of selected genes in detoxification pathways.


Assuntos
Araceae/metabolismo , Carbamazepina/análise , Catalase/metabolismo , Corbicula/efeitos dos fármacos , Citocromo P-450 CYP1A1/metabolismo , Água Doce/análise , Brânquias/metabolismo , Glutationa Transferase/metabolismo , Psicotrópicos/farmacologia , Superóxido Dismutase/metabolismo , Águas Residuárias/análise , Animais , Araceae/química , Citocromo P-450 CYP1A1/química , Ecossistema , Glutationa Transferase/química , Oxirredução , Superóxido Dismutase/química , Poluentes Químicos da Água/análise
18.
Environ Sci Pollut Res Int ; 25(17): 16455-16463, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28639016

RESUMO

In what has become known as the Yusho incident, thousands of people in western Japan were poisoned by the accidental ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds. In this study, we investigated the accumulation patterns of 69 PCB congeners in the blood of Yusho patients in comparison with those of non-exposed controls. The blood samples were collected at medical check-ups in 2004 and 2005. To compare the patterns of PCB congeners, we calculated the concentration ratio of each congener relative to the 2,2',4,4',5,5'-hexaCB (CB153) concentration. The concentration ratios of tetra- and penta-chlorinated congeners in the blood of Yusho patients were significantly lower than those of controls. To examine the cytochrome P450 (CYP)-dependent metabolic potential of the 2,3',4,4'5-pentaCB (CB118), CB153, and 2,3,3',4,4'5-hexaCB (CB156) congeners, we conducted PCB-CYP (CYP1A1, CYP1A2, CYP2A6, and CYP2B6) docking simulation by in silico analysis. The docking models showed that human CYP1A1, CYP2A6, and CYP2B6 isozymes have the potential to metabolize CB118 and CB153. On the other hand, it was inferred that CB156 is difficult to be metabolized by these four CYP isozymes. These results indicate that CYP1 and CYP2 isozymes may be involved in the characteristic accumulation patterns of PCB congeners in the blood of Yusho patients.


Assuntos
Citocromo P-450 CYP1A2/química , Sistema Enzimático do Citocromo P-450/química , Dioxinas/química , Bifenilos Policlorados/química , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Isoenzimas , Japão , Masculino , Bifenilos Policlorados/sangue
19.
J Agric Food Chem ; 65(34): 7440-7446, 2017 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-28782952

RESUMO

Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer, and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multistep chemical approaches. Here, we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7-trihydroxyflavone (baicalein). Molecular modeling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake-flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds.


Assuntos
Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , Flavanonas/metabolismo , Flavonoides/metabolismo , Saccharomyces cerevisiae/genética , Biocatálise , Biotransformação , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/isolamento & purificação , Expressão Gênica , Humanos , Hidroxilação , Saccharomyces cerevisiae/metabolismo
20.
Biochim Biophys Acta Gen Subj ; 1861(11 Pt A): 2852-2860, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28780123

RESUMO

BACKGROUND: Cytochromes P450 are major drug-metabolizing enzymes involved in the biotransformation of diverse xenobiotics and endogenous chemicals. Persistent organic pollutants (POPs) are toxic hydrophobic compounds that cause serious environmental problems because of their poor degradability. This calls for rational design of enzymes capable of catalyzing their biotransformation. Cytochrome P450 1A1 isoforms catalyze the biotransformation of some POPs, and constitute good starting points for the design of biocatalysts with tailored substrate specificity. METHODS: We rationalized the activities of wild type and mutant forms of rat cytochrome P450 1A1 towards 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and 3,3',4,4'-tetrachlorobiphenyl (PCB77) using experiments and molecular dynamics simulations. RESULTS: We showed that the enhanced activity of the CYP1A1 mutant towards TCDD was due to more efficient binding of the substrate in the active site even though the mutated site was over 2.5nm away from the catalytic center. Moreover, this mutation reduced activity towards PCB77. GENERAL SIGNIFICANCE: Amino acids that affect substrate access channels can be viable targets for rational enzyme design even if they are located far from the catalytic site.


Assuntos
Catálise , Citocromo P-450 CYP1A1/genética , Poluentes Ambientais/toxicidade , Inativação Metabólica/genética , Animais , Biotransformação/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Citocromo P-450 CYP1A1/química , Adutos de DNA/efeitos dos fármacos , Poluentes Ambientais/química , Humanos , Inativação Metabólica/efeitos dos fármacos , Mutação , Bifenilos Policlorados/química , Bifenilos Policlorados/toxicidade , Dibenzodioxinas Policloradas/química , Dibenzodioxinas Policloradas/toxicidade , Ratos , Especificidade por Substrato
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