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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(10): 865-871, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31814561

RESUMO

Objective To construct a double transfected Flp-InTM CHO cell line stably expressing both cytochrome P450 family 2 subfamily A member 13(CYP2A13) and multidrug resistance-associated protein 2(MRP2). Methods We constructed the recombinant plasmids of pCMV6-NEO-CYP2A13 and pcDNA5-MRP2. The pCMV6-NEO-CYP2A13 recombinant plasmid was first transfected into Flp-InTM CHO cells, and CYP2A13-Flp-InTM CHO cells with higher CYP2A13 activity were screened using limiting dilution method and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) cytotoxicity assay. Thereafter, pcDNA5-MRP2 was transfected into CYP2A13-Flp-InTM CHO cells. The expression levels and activities of CYP2A13 and MRP2 in the double transfected cells and normal cells were detected by real-time quantitative PCR, Western blot analysis and NNK cytotoxicity assay in order to screen Flp-InTM CHO cells with stable expression of CYP2A13 and MRP2. Results Compared with non-transfected cells, the expression of CYP2A13 and the sensitivity of NNK toxicity in CYP2A13-Flp-InTM CHO cells increased. The expression of CYP2A13 and MRP2 in CYP2A13/MRP2-Flp-InTM CHO cells also increased significantly. Compared with CYP2A13-Flp-InTM CHO cells, CYP2A13/MRP2-Flp-InTM CHO cells showed no significant difference in CYP2A13 expression; the expression of MRP2 increased while the sensitivity of NNK toxicity decreased significantly. Conclusion The double transfected cell model of CYP2A13 and MRP2 has been successfully established, which lays the foundation for the study of in situ activation of respiratory carcinogens.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Células CHO , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Animais , Carcinógenos/toxicidade , Cricetinae , Cricetulus , Nitrosaminas/toxicidade , Plasmídeos , Transfecção
2.
Res Vet Sci ; 126: 178-183, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31539794

RESUMO

After oral route of administration, drug absorption is unpredictable and is governed by various factors such as multi drug resistance-1 (MDR1) an efflux transporter and drug metabolizing enzymes (like CYP3A4, CYP3A37, CYP2D6) at intestine and liver. Naturally available phyto chemicals like piperine and quercetin as well as some floroquinolones are known to inhibit MDR1 and CYP3A37 activity and increases bioavailability of co-administered drugs. This study was carried out to investigate the effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in liver and intestine of broiler chicken. After oral administration of piperine and quercetin for 3 consecutive days followed by with or without oral administration of marbofloxacin for 5 days, CYP3A37 and MDR1 mRNA expression levels were determined using quantitative real-time PCR. Total of 36 broiler chickens in seven individual groups were treated with different regimen and the mRNA expression levels at duodenum and liver were analyzed with apt statistical tools. After piperine and quercetin combined treatment with marbofloxacin, CYP3A37 mRNA expression levels were significantly down regulated by 20.57 (p = .034) and 25.95 (p = .003) folds; and MDR1 mRNA expression levels were also significantly down regulated by 11.33 (p = .012) and 33.59 (p = .006) folds in liver and duodenum, respectively. Down regulation of CYP3A37 and MDR1 mRNA in liver and duodenum indicate the combined pretreatment of piperine and quercetin may be useful for improving the pharmacokinetics of orally administered drugs which are substrates for CYP3A37 and MDR1.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Alcaloides/farmacologia , Hidrocarboneto de Aril Hidroxilases/genética , Benzodioxóis/farmacologia , Galinhas/fisiologia , Família 3 do Citocromo P450/genética , Fluoroquinolonas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Quercetina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas/genética , Inibidores das Enzimas do Citocromo P-450/farmacologia , Família 3 do Citocromo P450/metabolismo , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória
3.
Toxicol Lett ; 313: 1-10, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170421

RESUMO

The constitutive androstane receptor(CAR) activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. CAR activators, including phenobarbital, are considered rodent non-genotoxic carcinogens. Recently, trans-3,4,5,4´-tetramethoxystilbene(TMS), a potential anticancer drug (DMU-212), have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. We studied whether TMS inhibits mouse Car to protect from the PB-induced tumorigenesis. Unexpectedly, we identified TMS as a murine CAR agonist in reporter gene experiments, in mouse hepatocytes, and in C57BL/6 mice in vivo. TMS up-regulated Car target genes Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis. TMS did not change or down-regulate genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, or Mdm2, which were up-regulated by another Car ligand TCPOBOP. TMS did not increase liver weight and had no significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In murine hepatic AML12 cells, we confirmed a Car-independent proapoptotic effect of TMS. We conclude that TMS is a Car ligand with limited effects on hepatocyte proliferation, likely due to promoting apoptosis in mouse hepatic cells, while controlling Car target genes involved in xenobiotic and endobiotic metabolism.


Assuntos
Anticarcinógenos/farmacologia , Proliferação de Células/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Estilbenos/farmacologia , Animais , Anticarcinógenos/metabolismo , Apoptose/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Ligação Proteica , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Estilbenos/metabolismo
4.
J Vet Med Sci ; 81(7): 983-985, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31118352

RESUMO

Knowledge of genetic polymorphisms of cytochrome P450 (CYP), the most important xenobiotic metabolizing enzyme, is very limited in cats. Preliminarily, we investigated genetic polymorphisms in CYP2A13, one of the major CYP isoforms in the liver and lung. Four synonymous and three non-synonymous polymorphic variants were identified in feline CYP2A13 in domestic cats in Japan, without an obvious major type. Metabolic parameters, Km and Vmax, of coumarin hydroxylation of CYP2A13 were shown to range within two times for the identified non-synonymous polymorphic variants by using heterologous coexpression system in Escherichia coli. The results confirmed the polymorphic nature of CYP2A13 as a basis for effective application of medicines and prevention of adverse reactions in treatment of domestic cats.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Gatos/genética , Variação Genética , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Animais , Gatos/metabolismo , Cumarínicos/metabolismo , Feminino , Hidroxilação , Fígado/enzimologia , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA
5.
Artigo em Inglês | MEDLINE | ID: mdl-31048018

RESUMO

Enrofloxacin (ENR) is the most commonly used antibiotic in crustacean farming in China. Diet supplementation with lactic acid (LA) may, however, affect the efficacy and safety of ENR-based drugs. The aims of this study were to investigate the effects of LA on drug residues and elimination of oral ENR in Chinese mitten crab (Eriocheir sinensis) and to determine ENR and gene expression levels of drug-metabolizing enzymes in the hepatopancreas. To this end, ENR was orally administered to the crabs at a dose of 10.0 mg kg-1 body weight on the eighth day after feeding diets supplemented with 0.3%LA. The results showed that ENR levels in the hepatopancreas were significantly different at 1 and 12 h between the ENR and ENR + 0.3% LA groups (P < 0.05). Lactic acid did not significantly affect the expression of CYP2A (phase I). However, the expressions of CYP3 (phase I) and GST (phase II) were significantly up-regulated by LA during the elimination process of ENR (6-24 h). At Tmax (1 h), the expression of phosphoenolpyruvate carboxykinase (PEPCK) was induced and expression of succinate dehydrogenase (SDH) was inhibited by LA. Both of these enzymes were significantly inhibited during the elimination process of ENR. The results suggest that LA contributes to the elimination of ENR, and thus, enhances hepatopancreas biotransformation and anti-injury capacity in E. sinensis.


Assuntos
Braquiúros/efeitos dos fármacos , Enrofloxacina/farmacocinética , Inativação Metabólica/efeitos dos fármacos , Ácido Láctico/farmacologia , Administração Oral , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Aquicultura , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Braquiúros/enzimologia , Família 3 do Citocromo P450/genética , Família 3 do Citocromo P450/metabolismo , Suplementos Nutricionais , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Enrofloxacina/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/metabolismo , Inativação Metabólica/genética , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo
6.
Pharmacol Res Perspect ; 7(3): e00475, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31049204

RESUMO

Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy-bupropion, and bupropion were quantified by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. Level of mRNA and protein were determined by RT-PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (V max) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.


Assuntos
Bupropiona/farmacocinética , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Regulação para Baixo , Insuficiência Renal Crônica/induzido quimicamente , Adenina/efeitos adversos , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Masculino , Microssomos Hepáticos/metabolismo , Ratos , Ratos Wistar , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo
7.
Genes (Basel) ; 10(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939847

RESUMO

There is a special interest in the implementation of pharmacogenetics in clinical practice, although there are some barriers that are preventing this integration. A large part of these pharmacogenetic tests are focused on drugs used in oncology and psychiatry fields and for antiviral drugs. However, the scientific evidence is also high for other drugs used in other medical areas, for example, in cardiology. In this article, we discuss the evidence and guidelines currently available on pharmacogenetics for clopidogrel, warfarin, acenocoumarol, and simvastatin and its implementation in daily clinical practice.


Assuntos
Anticoagulantes/efeitos adversos , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Farmacogenética , Acenocumarol/efeitos adversos , Acenocumarol/uso terapêutico , Anticoagulantes/uso terapêutico , Hidrocarboneto de Aril Hidroxilases/genética , Doenças Cardiovasculares/epidemiologia , Clopidogrel/efeitos adversos , Clopidogrel/uso terapêutico , Citocromo P-450 CYP2C9/genética , Guias como Assunto , Humanos , Medicina de Precisão , Sinvastatina/efeitos adversos , Sinvastatina/uso terapêutico , Varfarina/efeitos adversos , Varfarina/uso terapêutico
8.
Biochem Biophys Res Commun ; 512(1): 119-124, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30876690

RESUMO

CYP2A5 is a major enzyme responsible for nicotine and cotinine metabolism in mice. Nicotine and cotinine enhance alcoholic fatty liver in wild type (WT) mice but not in CYP2A5 knockout (KO) mice, and reactive oxygen species (ROS) generated during the CYP2A5-mediated metabolism contributes to the enhancing effect. In combination with ethanol, nicotine and cotinine increased lipid peroxidation end product 4-hydroxynonenal (HNE) in WT mice but not in KO mice. In ethanol-fed KO mice, only 5 and 10 genes were regulated by nicotine and cotinine, respectively. However, in ethanol-fed WT mice, 59 and 104 genes were regulated by nicotine and cotinine, respectively, and 7 genes were up-regulated by both nicotine and cotinine. Plin 2 and Cdkn1a are among the 7 genes. Plin2 encodes adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, which was confirmed to be increased by nicotine and cotinine in WT mice but not in KO mice. Cdkn1a encodes P21 and elevated P21 in nuclei was also confirmed. HNE can increase P21 and P21 inhibit cell proliferation. Consistently, hepatocyte proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 were decreased in WT mice but not in KO mice by nicotine/ethanol and cotinine/ethanol, respectively. These results suggest that inhibition of liver proliferation via a ROS-HNE-P21 pathway is involved in nicotine- and cotinine-enhanced alcoholic fatty liver.


Assuntos
Aldeídos/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Animais , Hidrocarboneto de Aril Hidroxilases/deficiência , Hidrocarboneto de Aril Hidroxilases/genética , Proliferação de Células/efeitos dos fármacos , Cotinina/administração & dosagem , Inibidor de Quinase Dependente de Ciclina p21/genética , Família 2 do Citocromo P450/deficiência , Família 2 do Citocromo P450/genética , Modelos Animais de Doenças , Fígado Gorduroso Alcoólico/genética , Feminino , Hepatócitos/metabolismo , Hepatócitos/patologia , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/genética , Camundongos , Camundongos Knockout , Nicotina/administração & dosagem , Perilipina-2/genética , Perilipina-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos
9.
Environ Pollut ; 247: 524-533, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30708314

RESUMO

Prothioconazole (PTC) is a widely used triazolinthione fungicide with low toxicity and short residual period. However, its desulfurization metabolite, prothioconazole-desthio (PTC-d), is more persistent and has higher toxicity in terrestrial animals. In this study, the toxicokinetics (TK) and tissue distribution of PTC and PTC-d in Chinese lizards (Eremias argus) were measured following single oral dose (100 mg kg-1 body weight) treatments. TK parameters indicated that PTC was more rapidly absorbed than PTC-d, as indicated by its shorter time to reach peak concentrations in most tissues. Furthermore, the relative bioavailability of PTC in lizards was lower than that of PTC-d. Compared with PTC, PTC-d preferentially accumulated in lizards, as reflected by longer half-life of PTC-d. During the distribution process, PTC-d generated in vivo was transported from other tissues and was deposited in the skin and tail, where PTC-d may be excreted by exuviation or tail detachment. Preferential enrichment of S-enantiomer of both PTC and PTC-d were observed in all tissues. Hepatic cytochrome P450 gene expression measurement revealed that cyp1a5 and cyp3a28 exhibited the strongest responses in both treatment groups. In addition, the opposite responses of cyp2k4 in different treatment groups may indicate that this enzyme caused differences in the rates of metabolism of the two chemicals. This study compared the TK profile of PTC and its desulfurization metabolite PTC-d in lizards and demonstrated that the desulfurization of PTC could increase its ecological risk due to the higher bioavailability and persistence of PTC-d.


Assuntos
Fungicidas Industriais/toxicidade , Lagartos/metabolismo , Triazóis/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Fungicidas Industriais/metabolismo , Fígado/metabolismo , Lagartos/genética , Estereoisomerismo , Distribuição Tecidual , Toxicocinética , Triazóis/metabolismo
10.
Pest Manag Sci ; 75(10): 2744-2755, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30788896

RESUMO

BACKGROUND: Many insect cytochrome P450 proteins (CYPs) are involved in the metabolic detoxification of exogenous compounds such as plant toxins and insecticides. Tribolium castaneum, the red flour beetle, is a major agricultural pest that damages stored grains and cereal products. With the completion of the sequencing of its genome, two T. castaneum species-specific CYP genes, CYP4BN6, and CYP6BQ11, were identified. However, it is unknown whether the functions of most CYPs are shared by TcCYP4BN6 and TcCYP6BQ11, and the upstream regulatory mechanism of these two CYPs remains elusive. RESULTS: QRT-PCR analysis indicated that TcCYP4BN6 and TcCYP6BQ11 were both most highly expressed at the late pupal stage and were mainly observed in the head and gut, respectively, of adults. Moreover, the transcripts of these two CYPs were significantly induced by dichlorvos and carbofuran, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to these two insecticides. Intriguingly, knockdown of the latrophilin (lph) gene, which has been reported to be related to the insecticide susceptibility, reduced the expression of TcCYP4BN6 and TcCYP6BQ11 after insecticide treatment, suggesting that these two CYP genes are regulated by lph to participate in insecticide susceptibility in T. castaneum. CONCLUSION: These results shed new light on the function and mechanism of CYP genes associated with insecticide susceptibility and could facilitate research on appropriate and sustainable pest control management. © 2019 Society of Chemical Industry.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Receptores de Peptídeos/metabolismo , Tribolium/genética , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Óvulo/efeitos dos fármacos , Pupa/efeitos dos fármacos , Pupa/metabolismo , Tribolium/efeitos dos fármacos , Tribolium/crescimento & desenvolvimento , Tribolium/metabolismo
11.
Chem Res Toxicol ; 32(3): 484-492, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30701961

RESUMO

Human cytochrome P450 (P450) family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g., 4-ipomeanol. These and other drug-metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with H2O2 and recovery with mild reducing agents [Albertolle, M. W., et al. (2017) J. Biol. Chem. 292, 11230-11242]. The inhibition is due to sulfenylation of the heme-thiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications for chemical toxicity and observed disease-drug interactions, in which the decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza and autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (Protein Data Bank entry 5T6Q ). In simulating either the thiolate (Cys-S-) or the sulfenic acid (Cys-SOH) at the heme ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and religation of the thiolate to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Glutamina/farmacologia , Heme/metabolismo , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Coelhos
12.
Environ Toxicol Chem ; 38(3): 660-670, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30615215

RESUMO

Exposure to dioxin-like compounds is consistently associated with concentration-dependent induction of cytochrome P4501A (CYP1A) enzymes in primary cultures of avian hepatocytes. We have previously demonstrated that the median effective concentration (EC50) for induction of this response is predictive of in vivo sensitivity to dioxin-like compounds in birds. We investigated sources of interindividual variation in the CYP1A response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild herring gulls and considered how this variation may complicate dioxin sensitivity estimates based on the CYP1A bioassay. Concentration-dependent effects of TCDD on CYP1A mRNA expression were characterized in 55 hepatocyte cultures prepared from individual herring gull embryos. A large degree of variability was observed among the hepatocyte culture preparations. For example, 1) basal CYP1A4 and CYP1A5 mRNA expression varied by 20- and 126-fold, respectively, among individuals, and 2) exposure to TCDD induced CYP1A4 mRNA expression by 57-fold in the most responsive sample but did not significantly induce CYP1A4 mRNA expression above baseline values in 42% of hepatocyte culture preparations. Environmental and genetic factors contributing to the observed variability are discussed. Despite the large amount of interindividual variation, we conclude that reproducible EC50-based estimates of species sensitivity can be obtained from the CYP1A cell culture bioassay when samples are collected from relatively uncontaminated colonies. Environ Toxicol Chem 2019;38:660-670. © 2019 SETAC.


Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Proteínas Aviárias/biossíntese , Bioensaio , Charadriiformes/metabolismo , Poluentes Ambientais/toxicidade , Hepatócitos/enzimologia , Dibenzodioxinas Policloradas/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Proteínas Aviárias/genética , Variação Biológica da População , Células Cultivadas , Charadriiformes/embriologia , Charadriiformes/genética , Indução Enzimática , Hepatócitos/efeitos dos fármacos , RNA Mensageiro/biossíntese
13.
Toxicol Sci ; 167(1): 172-189, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30203046

RESUMO

Exposure to environmentally relevant chemicals that activate the xenobiotic receptors aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor alpha (PPARα) in rodent test systems often leads to increases in oxidative stress (OS) that contributes to liver cancer induction. We hypothesized that activation of the oxidant-induced transcription factor Nrf2 could be used as a surrogate endpoint for increases in OS. We examined the relationships between activation of xenobiotic receptors and Nrf2 using previously characterized gene expression biomarkers that accurately predict modulation. Using a correlation approach (Running Fisher Test), the biomarkers were compared with microarray profiles in a mouse liver gene expression compendium. Out of the 163 chemicals examined, 47% from 53 studies activated Nrf2. We found consistent coupling between CAR and Nrf2 activation. Out of the 41 chemicals from 32 studies that activated CAR, 90% also activated Nrf2. CAR was activated earlier and at lower doses than Nrf2, indicating CAR activation preceded Nrf2 activation. Nrf2 activation by 2 CAR activators was abolished in CAR-null mice. We hypothesized that Nrf2 is activated by reactive oxygen species from the increased activity of enzymes encoded by Cyp2b family members. However, Nrf2 was similarly activated in the livers of both TCPOBOP-treated wild-type and Cyp2b9/10/13-null mice. This study provides evidence that Nrf2 activation (1) often occurs after exposure to xenobiotic chemicals, (2) is tightly linked to activation of CAR, and (3) does not require induction of 3 Cyp2b genes secondary to CAR activation.


Assuntos
Microssomos Hepáticos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenobarbital/toxicidade , Receptores Citoplasmáticos e Nucleares/metabolismo , Xenobióticos/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biomarcadores/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Indução Enzimática , Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Fator 2 Relacionado a NF-E2/genética , PPAR alfa/genética , PPAR alfa/metabolismo , Fenobarbital/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Xenobióticos/metabolismo
14.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30563925

RESUMO

The effect of Cereus jamacaru ethanolic extract in rats was analyzed using genotoxicity assays and liver ABCB1 and CYP2D4 gene expression. The lyophilized extract of C. jamacaru cladodes was analyzed with LC-MS/MS. Male Wistar rats (n=36) were equally distributed into six groups that did (+) or did not (-) receive cyclophosphamide treatments: Control (-); Control (+); EXP 1 (-), and EXP 1 (+), both treated with 210 mg/kg of ethanolic extract; and EXP 2 (-) and EXP 2 (+), both treated with 420 mg/kg of ethanolic extract. After 30 d of treatment, body weight and food and water intake were monitored. Right femur of the rats and spinal canal fluid were harvested and used for genotoxicity assays, and the liver samples were used for gene expression studies. The phytochemical analysis identified novel compounds. Animals treated with C. jamacaru showed lower body weight and food ingestion compared to controls (P<0.05). The genotoxicity assay showed an absence of ethanolic extract cytotoxicity. CYP2D4 expression was higher in EXP 2 groups compared with EXP 1 (-) group (P<0.05). ABCB1A expression was higher in negative groups compared with the positive groups. These results indicated a new phytochemical characterization of C. jamacaru and its effect on food ingestion and body weight gain. Moreover, the genotoxicity assay suggested that C. jamacaru ethanolic extract treatment presents significant intrinsic genotoxic potential and the enhanced expression of ABCB1 and CYP2D4 on C. jamacaru extract treatment suggests a role of the efflux transporter and microsomal enzyme, respectively, in C. jamacaru pharmacokinetics.


Assuntos
Cactaceae/química , Extratos Vegetais/farmacocinética , Extratos Vegetais/toxicidade , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Peso Corporal/efeitos dos fármacos , Ciclofosfamida/toxicidade , Etanol/química , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Testes de Mutagenicidade , Extratos Vegetais/química , Plantas Medicinais/química , Ratos Wistar , Espectrometria de Massas em Tandem
15.
Int J Mol Sci ; 19(10)2018 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-30297679

RESUMO

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina's HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.


Assuntos
Calcitriol/farmacologia , Di-Hidroxicolecalciferóis/farmacologia , Queratinócitos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Células Cultivadas , Humanos , Queratinócitos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Hidrocarboneto Arílico/química
16.
J Pharmacol Sci ; 138(1): 46-53, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30245287

RESUMO

Hepatic steatosis is the early stage of alcoholic liver disease (ALD), may progress to steatohepatitis, fibrosis even cirrhosis. Polydatin, the primary active component of Polygonum cuspidatum Sieb. et Zucc, has been recognized to possess hepatoprotective and anti-inflammatory properties. To investigate whether polydatin alleviates ethanol induced liver injury and to elucidate the underlying molecular mechanisms, zebrafish larvae at 4 days post-fertilization (dpf) were exposed to 350 mmol/L of ethanol for 32 h, then treated with polydatin for 48 h. Oil red O, Nile Red and H&E staining were used to analyze the pathological changes in liver. The mRNA levels were measured by quantitative PCR and the antioxidant capacity was detected using H2O2-specific fluorescent probe. Here, polydatin strongly alleviated hepatic steatosis and decreased the expression levels of alcohol and lipid metabolism-related genes, including CYP2Y3, CYP3A65, HMGCRa, HMGCRb and FASN. Additionally, polydatin inhibited oxidative stress in the liver according to fluorescent probe. Moreover, significantly up-regulated expression of DNA damage-related genes (CHOP, GADD45αa) revealed that polydatin attenuated hepatic apoptosis in larvae. In conclusion, polydatin may improve the liver function of zebrafish with acute alcoholic liver injury through attenuating hepatic fat accumulation, ameliorating lipid and ethanol metabolism and reducing oxidative stress and DNA damage.


Assuntos
Anti-Inflamatórios , Antioxidantes , Glucosídeos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Estilbenos/farmacologia , Peixe-Zebra , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Família 3 do Citocromo P450/genética , Família 3 do Citocromo P450/metabolismo , Dano ao DNA/genética , Fallopia japonica/química , Expressão Gênica/efeitos dos fármacos , Glucosídeos/isolamento & purificação , Glucosídeos/uso terapêutico , Metabolismo dos Lipídeos/genética , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Estilbenos/isolamento & purificação , Estilbenos/uso terapêutico , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Arch Biochem Biophys ; 657: 65-73, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30222954

RESUMO

Tobacco and alcohol are often co-abused. Nicotine can enhance alcoholic fatty liver, and CYP2A6 (CYP2A5 in mice), a major metabolism enzyme for nicotine, can be induced by alcohol. CYP2A5 knockout (cyp2a5-/-) mice and their littermates (cyp2a5+/+) were used to test whether CYP2A5 has an effect on nicotine-enhanced alcoholic fatty liver. The results showed that alcoholic fatty liver was enhanced by nicotine in cyp2a5+/+ mice but not in the cyp2a5-/- mice. Combination of ethanol and nicotine increased serum triglyceride in cyp2a5+/+ mice but not in the cyp2a5-/- mice. Cotinine, a major metabolite of nicotine, also enhanced alcoholic fatty liver, which was also observed in cyp2a5+/+ mice but not in the cyp2a5-/- mice. Nitrotyrosine and malondialdehyde (MDA), markers of oxidative/nitrosative stress, were induced by alcohol and were further increased by nicotine and cotinine in cyp2a5+/+ mice but not in the cyp2a5-/- mice. Reactive oxygen species (ROS) production during microsomal metabolism of nicotine and cotinine was increased in microsomes from cyp2a5+/+ mice but not in microsomes from cyp2a5-/- mice. These results suggest that nicotine enhances alcoholic fatty liver in a CYP2A5-dependent manner, which is related to ROS produced during the process of CYP2A5-dependent nicotine metabolism.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Fígado Gorduroso Alcoólico/etiologia , Nicotina/efeitos adversos , Nicotina/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cotinina/efeitos adversos , Cotinina/sangue , Cotinina/metabolismo , Cotinina/urina , Família 2 do Citocromo P450/genética , Etanol/toxicidade , Fígado Gorduroso Alcoólico/metabolismo , Feminino , Técnicas de Inativação de Genes , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
18.
J Vet Pharmacol Ther ; 41(6): 815-824, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30113702

RESUMO

We previously showed that (+)-tramadol is metabolized in dog liver to (+)-M1 exclusively by CYP2D15 and to (+)-M2 by multiple CYPs, but primarily CYP2B11. However, (+)-M1 and (+)-M2 are further metabolized in dogs to (+)-M5, which is the major metabolite found in dog plasma and urine. In this study, we identified canine CYPs involved in metabolizing (+)-M1 and (+)-M2 using recombinant enzymes, untreated dog liver microsomes (DLMs), inhibitor-treated DLMs, and DLMs from CYP inducer-treated dogs. A canine P-glycoprotein expressing cell line was also used to evaluate whether (+)-tramadol, (+)-M1, (+)-M2, or (+)-M5 are substrates of canine P-glycoprotein, thereby limiting their distribution into the central nervous system. (+)-M5 was largely formed from (+)-M1 by recombinant CYP2C21 with minor contributions from CYP2C41 and CYP2B11. (+)-M5 formation in DLMs from (+)-M1 was potently inhibited by sulfaphenazole (CYP2C inhibitor) and chloramphenicol (CYP2B11 inhibitor) and was greatly increased in DLMs from phenobarbital-treated dogs. (+)-M5 was formed from (+)-M2 predominantly by CYP2D15. (+)-M5 formation from (+)-M1 in DLMs was potently inhibited by quinidine (CYP2D inhibitor) but had only a minor impact from all CYP inducers tested. Intrinsic clearance estimates showed over 50 times higher values for (+)-M5 formation from (+)-M2 compared with (+)-M1 in DLMs. This was largely attributed to the higher enzyme affinity (lower Km) for (+)-M2 compared with (+)-M1 as substrate. (+)-tramadol, (+)-M1, (+)-M2, or (+)-M5 were not p-glycoprotein substrates. This study provides a clearer picture of the role of individual CYPs in the complex metabolism of tramadol in dogs.


Assuntos
Analgésicos Opioides/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Cães/metabolismo , Microssomos Hepáticos/metabolismo , Esteroide Hidroxilases/metabolismo , Tramadol/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Gatos/metabolismo , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/genética , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Especificidade da Espécie , Esteroide Hidroxilases/antagonistas & inibidores , Esteroide Hidroxilases/genética
19.
Drug Metab Dispos ; 46(11): 1538-1545, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30154104

RESUMO

Human CYP2A6 (Cyp2a5 in mice) plays an important role in metabolism and detoxification of various drugs and chemicals. Here, we investigated a potential role of peroxisome proliferator-activated receptor γ (Ppar-γ) in circadian regulation of the Cyp2a5 enzyme. We first showed that Cyp2a5 mRNA and protein in mouse liver displayed robust circadian oscillations. Consistent with a circadian protein pattern, Cyp2a5-mediated 7-hydroxylation of coumarin was circadian time-dependent. Formation of 7-hydroxycoumarin was more extensive at a dosing time of Zeitgeber time 2 (ZT2) than that at ZT14. Interestingly, the nuclear receptor Ppar-γ was also a circadian gene. Circadian Ppar-γ protein level was strongly correlated with the Cyp2a5 mRNA level (r = 0.989). Furthermore, Ppar-γ activation (by a selective agonist, rosiglitazone) upregulated Cyp2a5 expression in Hepa-1c1c7 cells, whereas Ppar-γ knockdown downregulated Cyp2a5 expression. Also, Ppar-γ knockdown blunted the rhythmicity of Cyp2a5 mRNA in serum-shocked Hepa-1c1c7 cells. In addition, a combination of promoter truncation analysis, mobility shift, and chromatin immunoprecipitation assays revealed that Ppar-γ directly bound to a PPAR response element (i.e., the -1418- to -1396-bp region) within Cyp2a5 promoter and activated the gene transcription. Taken together, Ppar-γ was a transcriptional activator of Cyp2a5, and its rhythmic expression contributed to circadian expression of Cyp2a5.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Relógios Circadianos/genética , Família 2 do Citocromo P450/genética , Hepatócitos/fisiologia , Fígado/fisiologia , PPAR gama/genética , Animais , Linhagem Celular , Regulação para Baixo/genética , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
20.
Aquat Toxicol ; 202: 6-15, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29966910

RESUMO

The early life stages of Pacific salmon are at risk of environmental exposure to diluted bitumen (dilbit) as Canada's oil sands industry continues to expand. The toxicity and latent effects of dilbit exposure were assessed in sockeye salmon (Oncorhynchus nerka) exposed to water-soluble fractions of dilbit (WSFd) from fertilization to the swim-up stage, and then reared in clean water for 8 months. Mortality was significantly higher in WSFd-exposed embryos, with cumulative mortality up to 4.6-fold higher in exposed relative to unexposed embryos. The sublethal effects of WSFd exposure included transcriptional up-regulation of cyp1a, a concentration-dependent delay in the onset and progression of hatching, as well as increased prevalence of developmental deformities at total polycyclic aromatic hydrocarbon (TPAH) concentrations ≥35 µg L-1. Growth and body composition were negatively affected by WSFd exposure, including a concentration-specific decrease in soluble protein concentration and increases in total body lipid and triglyceride concentrations. Mortality continued during the first 2 months after transferring fish to clean water, reaching 53% in fish exposed to 100 µg L-1 TPAH; but there was no latent impact on swimming performance, heart mass, or heart morphology in surviving fish after 8 months. A latent effect of WSFd exposure on brain morphology was observed, with fish exposed to 4 µg L-1 TPAH having significantly larger brains compared to other treatment groups after 8 months in clean water. This study provides comprehensive data on the acute, sub-chronic, and latent impacts of dilbit exposure in early life stage sockeye, information that is critical for a proper risk analysis of the impact of a dilbit spill on this socioeconomically important fish species.


Assuntos
Comportamento Animal/efeitos dos fármacos , Hidrocarbonetos/toxicidade , Salmão/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Embrião não Mamífero/efeitos dos fármacos , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Campos de Petróleo e Gás , Salmão/metabolismo , Natação , Regulação para Cima/efeitos dos fármacos
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