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1.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638963

RESUMO

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the "140s" FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the "140s loop" by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the "140s loop".


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Transporte de Elétrons/genética , Elétrons , Mononucleotídeo de Flavina/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sequência de Aminoácidos , Animais , Família 2 do Citocromo P450/metabolismo , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Glicina/genética , Cinética , Microssomos/metabolismo , Mutagênese Sítio-Dirigida/métodos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Oxirredução , Ligação Proteica , Conformação Proteica , Coelhos
2.
J Biol Chem ; 296: 100645, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839156

RESUMO

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Polímeros/metabolismo , Propilaminas/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/química , Catálise , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Coelhos
3.
Vet J ; 270: 105625, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33641805

RESUMO

Cimicoxib is a selective COX-2 inhibitor (coxib) registered for the treatment of pain and inflammation in dogs. Pharmacokinetics of some coxibs have been described in dogs and cats. In cats, total body clearance values are lower and terminal half-lives of the coxibs are longer than those in dogs. The aim of this work was to evaluate if this is also the case for cimicoxib. For this purpose, blood pharmacokinetics and urinary excretion after IV administration were compared between these species. The in vitro metabolism of cimicoxib was also evaluated using canine and feline microsomes. In canine and feline microsomes, the formation rate of demethyl-cimicoxib, a phase 1 metabolite, was decreased in presence of quinidine, a specific human cytochrome P450 (CYP)2D6 inhibitor. IC50 values were 1.6 µM and 0.056 µM with canine and feline microsomes, respectively. As quinidine was about 30 times more potent in feline microsomes, the affinity of cimicoxib to the enzyme was considered to be about 30 times lower than that in canine microsomes. Total body clearance (ClB) of cimicoxib, was 0.50 L/h kg in dogs and 0.14 L/h kg in cats (P < 0.01) and terminal half-life, T½λz, was 1.92 and 5.25 h, respectively (P < 0.01). Dose eliminated in urine was 12.2% in dogs and 3.12% in cats (P < 0.01). Conjugated demethyl-cimicoxib represented 93.7% of this amount in dogs and 67.5% in cats. Thus cimicoxib, like other veterinary coxibs, was eliminated more slowly in cats. Both CYP2D15 (the canine ortholog of CYP2D6) and UDP-glucuronyltransferase enzyme systems have reduced ability to produce metabolites of cimicoxib in cats.


Assuntos
Gatos/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacocinética , Cães/metabolismo , Imidazóis/farmacocinética , Sulfonamidas/farmacocinética , Administração Intravenosa/veterinária , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/metabolismo , Gatos/urina , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Inibidores de Ciclo-Oxigenase 2/urina , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães/urina , Imidazóis/administração & dosagem , Imidazóis/urina , Microssomos Hepáticos/metabolismo , Quinidina/farmacologia , Especificidade da Espécie , Sulfonamidas/administração & dosagem , Sulfonamidas/urina
4.
FASEB J ; 35(5): e21469, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33788981

RESUMO

Mycotoxins are toxic secondary metabolites produced by food-contaminating fungi, which lead to global epigenetic changes and cause toxicity to both farm animals and humans. However, whether mycotoxins induce gene-specific epigenetic alterations associated with inducible downstream gene expression is unclear as are the underlying regulatory mechanisms. Here, we found that T-2 toxin and its deacetylated metabolites but not deoxynivalenol (DON) or other representative mycotoxins highly induced the expression of cytochrome P450 1A4 (CYP1A4) in both Leghorn male hepatoma (LMH) cells and chicken primary hepatocytes, and this effect was related to the regulation of both aryl hydrocarbon receptor (AhR) and DNA methylation. We used methylation-sensitive restriction enzyme digestion-qPCR (MSRE-qPCR) and chromatin immunoprecipitation (ChIP) assays and found that the binding of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) to highly methylated CpG island 3-2 at the enhancer of CYP1A4 was accompanied by the recruitment of the repressive histone modification marker H3K27me3, inducing a silent state. In turn, T-2 toxin stimulation enriched the binding of AhR to demethylated CpG island 3-2, which facilitated p300 and H3K9ac recruitment and ultimately generated an activated chromatin structure at the enhancer by increasing the active histone modification markers, including H3K4me3, H3K27ac, and H3K14ac. Interestingly, T-2 toxin-induced AhR activation also facilitated RNA polymerase II binding to CpG island 2, which may form a transcriptionally active chromatin structure at the promoter and ultimately transactivate CYP1A4. Our findings provide novel insights into the epigenetic regulation of T-2 toxin-induced gene expression.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas Aviárias/metabolismo , Carcinoma Hepatocelular/patologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Toxina T-2/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Proteínas Aviárias/genética , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Galinhas , Ilhas de CpG , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/genética , Transcrição Genética
5.
PLoS One ; 16(2): e0247020, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33592039

RESUMO

CYP4B1 belongs to the mammalian CYP4 enzyme family and is predominantly expressed in the lungs of humans. It is responsible for the oxidative metabolism of a wide range of endogenous compounds and xenobiotics. In this study, using data from The Cancer Genome Atlas (TCGA) project and the Gene Expression Omnibus (GEO) database, a secondary analysis was performed to explore the expression profile of CYP4B1, as well as its prognostic value in patients with lung adenocarcinoma (LUAD). Based on the obtained results, a significantly decreased CYP4B1 expression was discovered in patients with LUAD when compared with their normal counterparts (p<0.05), and was linked to age younger than 65 years (p = 0.0041), history of pharmaceutical (p = 0.0127) and radiation (p = 0.0340) therapy, mutations in KRAS/EGFR/ALK (p = 0.0239), and living status of dead (p = 0.0026). Survival analysis indicated that the low CYP4B1 expression was an independent prognostic indicator of shorter survival in terms of overall survival (OS) and recurrence-free survival (RFS) in patients with LUAD. The copy number alterations (CNAs) and sites of cg23440155 and cg23414387 hypermethylation might contribute to the decreased CYP4B1 expression. Gene set enrichment analysis (GSEA) suggested that CYP4B1 might act as an oncogene in LUAD by preventing biological metabolism pathways of exogenous and endogenous compounds and enhancing DNA replication and cell cycle activities. In conclusion, CYP4B1 expression may serve as a valuable independent prognostic biomarker and a potential therapeutic target in patients with LUAD.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biomarcadores Tumorais/metabolismo , Terapia de Alvo Molecular , Adenocarcinoma de Pulmão/diagnóstico , Adenocarcinoma de Pulmão/genética , Idoso , Hidrocarboneto de Aril Hidroxilases/genética , Biomarcadores Tumorais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
6.
Gastroenterology ; 160(6): 2103-2118, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33465371

RESUMO

BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.


Assuntos
Canalículos Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Colestase/genética , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-2/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/patologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colagogos e Coleréticos/uso terapêutico , Ácido Cólico , Claudina-1/metabolismo , Família 2 do Citocromo P450/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais , Feminino , Fibrose , Predisposição Genética para Doença , Hepatócitos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutação , Oxazóis/uso terapêutico , Permeabilidade , Fatores de Proteção , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/metabolismo , Junções Íntimas/ultraestrutura , Ácido Ursodesoxicólico/uso terapêutico , Proteína da Zônula de Oclusão-2/deficiência
7.
Artigo em Inglês | MEDLINE | ID: mdl-33141083

RESUMO

The antidepressant, venlafaxine (VFX), and climate change stressors, such as increased water temperature and decreased dissolved oxygen, are current threats to aquatic environments. This study aimed to determine how microRNAs (miRNAs) and predicted targeted transcripts were altered in livers of zebrafish exposed to these stressors, and livers of their un-exposed F1 and F2 offspring. Following a 21 day exposure to multiple stressors (1 µg/L VFX, +5 °C ambient, 50% O2), then a subsequent 21 day recovery, relative abundances of cyp3a65, hsp70, hsp90, and ppargc1a and miRNAs predicted to target them (miR-142a, miR-16c, miR-181c, and miR-129, respectively) were measured in the liver via quantitative PCR (RT-qPCR). There were significant decreases in miR-142a in the exposed F0 generation and the exposed F1 generation. While there were no changes detected in cyp3a65 relative abundance, there was a significant inverse relationship between cyp3a65 and miR-142a. Hsp70 expression significantly increased in the F1 generation, which persisted to the F2 generation and the relative abundance of hsp90 significantly increased in all generations. There was a significant reduction in miR-181c in the F1 generation, but there was no significant relationship between miR-181c and hsp90. Finally, there was a significant decrease in ppargc1a relative abundance in the F1 generation which was associated with an increase in miR-129. Combined, these results suggest that parental exposure to multiple, environmentally relevant stressors can confer transcriptional and epigenetic responses in the F1 and F2 generations, although identifying which stressor is a driving force becomes unclear.


Assuntos
Clima , Fígado/efeitos dos fármacos , MicroRNAs/genética , Transcrição Genética/efeitos dos fármacos , Cloridrato de Venlafaxina/toxicidade , Peixe-Zebra/genética , Animais , Antidepressivos/toxicidade , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Hipóxia/fisiopatologia , Fígado/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Estresse Fisiológico/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
8.
Gene ; 767: 145162, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32987105

RESUMO

The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2 months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4 weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/genética , Fígado/metabolismo , Esteroide Hidroxilases/genética , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Esteroide Hidroxilases/metabolismo
9.
J Ethnopharmacol ; 264: 113354, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32898626

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Geissoschizine methyl ether (GM), an indole alkaloid from Uncaria hook, is an active ingredient in the traditional Japanese Kampo medicine yokukansan, which is used to treat neurosis, insomnia, irritability, and night crying in children. AIM OF THE STUDY: Recent our pharmacokinetic studies suggested that there may be gender differences in the plasma concentrations of GM in rats, but not in humans. However, the details of this difference remain unverified. The purpose of this study was to clarify the reasons for the gender differences in rats. MATERIALS AND METHODS: GM plasma pharmacokinetics was compared in male and female rats orally administered yokukansan (4 g/kg). To confirm the involvement of cytochrome P450 (CYP) in GM liver metabolism, GM was incubated with male and female rat liver S9 fraction in the absence or presence of 1-aminobenzotriazole (a nonspecific CYP inhibitor). CYP isoforms involved in GM metabolism were estimated using recombinant rat CYP isoforms and anti-rat CYP antibodies. RESULTS: The maximum GM plasma concentrations were significantly higher in female than in male rats. When GM was incubated with rat liver S9 fractions, GM reduction was more striking in male S9 (69.3%) than that in female S9 (10.0%) and was completely blocked with nonspecific CYP inhibitor 1-aminobenzotriazole. Screening experiments using recombinant rat cytochrome P450 (CYP) isoforms showed that CYP1A1, CYP2C6, CYP2C11, CYP2D1, and CYP3A2 were involved in GM metabolism. Of these CYP isoforms, the use of anti-rat CYP antibodies indicated that male-dependent CYP2C11 and CYP3A2 were predominantly involved in the liver microsomal GM metabolism with gender differences. CONCLUSIONS: These results suggest that the cause of gender differences in plasma GM pharmacokinetics in rats is most likely because of male-dependent CYP2C11 and CYP3A2, and provide also useful information to further evaluate the pharmacological and toxicological effects in future. This study is the first to demonstrate that the gender differences in plasma GM pharmacokinetics in rats are caused by the gender-dependent metabolism of GM.


Assuntos
Alcaloides Indólicos/sangue , Microssomos Hepáticos/efeitos dos fármacos , Caracteres Sexuais , Uncaria , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP3A/metabolismo , Família 2 do Citocromo P450/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Alcaloides Indólicos/metabolismo , Alcaloides Indólicos/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos Hepáticos/enzimologia , Plasma/efeitos dos fármacos , Plasma/metabolismo , Ratos , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/metabolismo
10.
Curr Drug Metab ; 21(13): 1040-1051, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33267758

RESUMO

BACKGROUND: Retrorsine is one of the hepatotoxic pyrrolizidine alkaloids, which could be converted into a highly reactive metabolite, dehydroretrorsine, by CYP3A, and to a lesser extent by CYP2C and CYP2B. OBJECTIVE: We employed Cyp3a knockout (3AKO) mice to investigate whether the absence of CYP3A could attenuate dehydroretrorsine formation and the role of CYP2C and CYP2B in the formation. METHODS: Blood and liver samples were collected after intragastrical administration of 35 mg/kg retrorsine or saline for seven days in wild-type (WT) and 3AKO mice. Blood pyrrole-protein adducts were semi quantified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The formations of glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (GSH-DHP) and the activities of CYP3A, CYP2B and CYP2C were evaluated in the liver microsomes of WT and 3AKO mice before and after treatment. The metabolic phenotype of retrorsine was determined in human liver microsomes. The gene and protein expression of retrorsine metabolism-related CYP450s in the liver was measured by quantitative real-time PCR method and western blotting method. The serum cytokine level was detected by the ELISA method to reveal the potential mechanism of Cyp3a, Cyp2b and Cyp2c downregulation. RESULTS: After an oral administration of 35 mg/kg retrorsine for seven days, the blood exposures of DHP adducts between WT and 3AKO mice were similar, consistent with the comparable formation of GSH-DHP in their liver microsomes. The chemical inhibitor experiment in liver microsomes indicated the predominant role of CYP3A and CYP2C in GSH-DHP formation in WT and 3AKO mice, respectively. Real-time qPCR analysis showed that the expressions of Cyp2b10 and Cyp2cs increased 2.3-161-fold in 3AKO mice, which was consistent with protein changes. The increased CYP2B activity in 3AKO mice supported the potential role of CYP2B in GSH-DHP formation. After a seven-day treatment of retrorsine, the yields of GSH-DHP were lower than the untreated ones in both alleles, accompanied by the decreased mRNA of Cyp3a, Cyp2b and Cyp2c. The increased serum IL6 might mediate the retrorsine-induced downregulation of Cyp450s. CONCLUSION: These data demonstrated the increased transcription of Cyp2c and Cyp2b caused by Cyp3a ablation, which played a vital role in the metabolic activation of retrorsine, and long-term exposure of retrorsine can reduce the CYP450 activities.


Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Alcaloides de Pirrolizidina/farmacocinética , Esteroide Hidroxilases/metabolismo , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Hidrocarboneto de Aril Hidroxilases/genética , Sistema Enzimático do Citocromo P-450/genética , Família 2 do Citocromo P450/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Microssomos Hepáticos , Modelos Animais , Alcaloides de Pirrolizidina/administração & dosagem , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esteroide Hidroxilases/genética , Ativação Transcricional
11.
Int J Mol Sci ; 22(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375250

RESUMO

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Metabólica , Nicotina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Fumaça/efeitos adversos , Animais , Animais Recém-Nascidos , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Regiões Promotoras Genéticas
12.
Food Funct ; 11(11): 10058-10069, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33135718

RESUMO

Kaempferol has been confirmed to be effective in improving metabolic diseases such as diabetes and obesity. However, its effect and mechanism in nonalcoholic steatohepatitis (NASH) are unclear. We aim to confirm whether kaempferol could improve NASH and find the corresponding differential genes and metabolites. Transcriptomics combined with metabolomics was used to investigate the alterations in genes and metabolites expression after kaempferol treatment in mice with high-fat-diet-induced NASH. The results showed that kaempferol reduced the level of alanine transaminase (ALT), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) in serum and triglyceride (TG), lipid droplets, and inflammatory cell infiltration in liver. Further, 277 differentially expressed genes (DEGs) were identified through liver transcriptomics and the five most obvious DEGs were found to be CYP2b9, Cyp4a12b, Mup17, Mup7, and Mup16, which revealed that HFD induced fatty acid degradation, ribosome, and glyoxylic acid and dicarboxylic acid metabolism. Nine serum metabolites (methylcysteine, l-tryptophan, adrenic acid, d-2-hydroxyglutaric acid, tartaric acid, p-cresol sulfate, l-alanine, l-tryosine, and glutaconic acid) and 3 liver differential metabolites (gallic acid, γ-lindenic acid, and l-phenylalanine) were also identified, while the pathways were mainly involved in phenylalanine, tyrosine, and tryptophan biosynthesis; and phenylalanine metabolism. Integrating transcriptomics and metabolomics analyses indicated that kaempferol possesses the ability to improve NASH associated with energy metabolism, lipid metabolism, oxidative stress, and inflammation-related pathways. This study provides a powerful means of multiomics data integration and reveals the potent therapy and biomarkers for kaempferol.


Assuntos
Quempferóis/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , LDL-Colesterol/sangue , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Dieta Hiperlipídica/efeitos adversos , Humanos , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Transcriptoma , Triglicerídeos/sangue
13.
Int J Mol Sci ; 21(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105604

RESUMO

Abnormal hepatic insulin signaling is a cause or consequence of hepatic steatosis. DPP-4 inhibitors might be protective against fatty liver. We previously reported that the systemic inhibition of insulin receptor (IR) and IGF-1 receptor (IGF1R) by the administration of OSI-906 (linsitinib), a dual IR/IGF1R inhibitor, induced glucose intolerance, hepatic steatosis, and lipoatrophy in mice. In the present study, we investigated the effects of a DPP-4 inhibitor, linagliptin, on hepatic steatosis in OSI-906-treated mice. Unlike high-fat diet-induced hepatic steatosis, OSI-906-induced hepatic steatosis is not characterized by elevations in inflammatory responses or oxidative stress levels. Linagliptin improved OSI-906-induced hepatic steatosis via an insulin-signaling-independent pathway, without altering glucose levels, free fatty acid levels, gluconeogenic gene expressions in the liver, or visceral fat atrophy. Hepatic quantitative proteomic and phosphoproteomic analyses revealed that perilipin-2 (PLIN2), major urinary protein 20 (MUP20), cytochrome P450 2b10 (CYP2B10), and nicotinamide N-methyltransferase (NNMT) are possibly involved in the process of the amelioration of hepatic steatosis by linagliptin. Thus, linagliptin improved hepatic steatosis induced by IR and IGF1R inhibition via a previously unknown mechanism that did not involve gluconeogenesis, lipogenesis, or inflammation, suggesting the non-canonical actions of DPP-4 inhibitors in the treatment of hepatic steatosis under insulin-resistant conditions.


Assuntos
Imidazóis/efeitos adversos , Linagliptina/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Pirazinas/efeitos adversos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Glicemia/metabolismo , Família 2 do Citocromo P450/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Imidazóis/farmacologia , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Perilipina-2/metabolismo , Pirazinas/farmacologia , Esteroide Hidroxilases/metabolismo , Triglicerídeos/sangue
14.
Mol Pharmacol ; 98(6): 658-668, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33055223

RESUMO

CYP2A enzymes metabolically inactivate nicotine and activate tobacco-derived procarcinogens [e.g., 4-[methylnitrosamino]-1-(3-pyridyl)-1-butanone]. Smoking decreases nicotine clearance, and chronic nicotine reduces hepatic CYP2A activity. However, little is known about the impact of smoking or nicotine on the expression of CYP2A in the lung. We investigated 1) the levels of human lung CYP2A mRNA in smokers versus nonsmokers and 2) the impact of daily nicotine treatment on lung CYP2A protein levels in African green monkeys (AGMs). Lung CYP2A13, CYP2A6, and CYP2A7 (and CYP1A2) mRNA levels in smokers and nonsmokers were assessed in Gene Expression Omnibus data sets (GSE30063, GSE108134, and GSE11784). The impact of chronic, twice-daily, subcutaneous nicotine at two doses (0.3 and 0.5 mg/kg) versus vehicle on lung CYP2A protein levels was assessed. The impact of ethanol self-administration was also investigated, with and without nicotine treatment. Smokers versus nonsmokers (from GSE30063 and GSE108134) had lower (1.04- to 1.12-fold) levels of lung CYP2A13, CYP2A6, and CYP2A7 (and higher CYP1A2) mRNA. Both doses of nicotine tested decreased AGM lung CYP2A protein (3- to 7-fold). Ethanol self-administration had no effect on AGM lung CYP2A protein, and there was no interaction between ethanol and nicotine. Our results suggest that smoking was associated with a reduction in human lung CYP2A13, CYP2A6, and CYP2A7 mRNA, consistent with the role of nicotine treatment in reducing AGM lung CYP2A protein. This regulation by smoking/nicotine will increase interindividual variation in lung CYP2A levels, which may impact the localized metabolism of inhaled drugs and tobacco smoke procarcinogens. SIGNIFICANCE STATEMENT: CYP2A13 and CYP2A6 are expressed in the lung and may contribute to local procarcinogen activation. Smokers had lower lung CYP2A mRNA levels compared with nonsmokers. Lung CYP2A protein expression was decreased by systemic treatment with nicotine. Decreased lung CYP2A expression may alter smoking-related lung cancer risk and tissue damage from other inhaled toxins. This novel regulatory impact of nicotine, including nicotine found in smoking-cessation nicotine-replacement therapies, may have potential benefits on smoking-related lung cancer risk.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Pulmão/patologia , Fumar/patologia , Esteroide Hidroxilases/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/genética , Chlorocebus aethiops , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Pulmão/efeitos dos fármacos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Masculino , Microssomos Hepáticos , Nicotina/toxicidade , não Fumantes , RNA Mensageiro/análise , Análise de Sequência de RNA , Fumaça/efeitos adversos , Fumantes , Fumar/efeitos adversos , Esteroide Hidroxilases/análise , Esteroide Hidroxilases/genética , Tabaco/química , Tabaco/toxicidade
15.
Drug Metab Pharmacokinet ; 35(6): 497-504, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32958406

RESUMO

Human cytochrome P450 (or CYP) inhibition rates were investigated in sera from high fat diet (HFD)-induced type 2 diabetes (T2D), T2D recovered, and asymptomatic mice models to verify whether P450 inhibition assays could be used for the detection of disease, evaluation of therapeutic effect, and early diagnosis of T2D. In T2D mice, the blood glucose levels markedly increased; while blood glucose levels of recovered mice exceeded 200 mg dL-1, these eventually returned to the levels seen in control mice. In asymptomatic mice fed with short term HFD (stHFD), no changes in blood glucose levels were observed. The inhibition rates of CYP1A2, CYP2A13, and CYP2C18 in T2D mice significantly increased. Whereas in recovered mice, these changes returned to the same levels noted in the control mice. Changes in the inhibition rates of CYP2A13 and CYP2C18 in stHFD mice were similar to those in T2D mice. A receiver operating characteristic (ROC) curve analysis showed high area under the ROC curve (AUC) values (0.879-1.000) of CYP2A13 and CYP2C18 in T2D and stHFD mice, indicating their high diagnostic accuracy. Collectively, this study validates the P450 inhibition assay as a method for the therapeutic evaluation and early diagnosis of T2D mouse models.


Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Inibidores das Enzimas do Citocromo P-450/sangue , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Doenças Assintomáticas , Biomarcadores/sangue , Citocromo P-450 CYP1A2/metabolismo , Inibidores do Citocromo P-450 CYP1A2/sangue , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica , Diagnóstico Precoce , Feminino , Humanos , Camundongos Endogâmicos C57BL , Valor Preditivo dos Testes , Proteínas Recombinantes/metabolismo
16.
J Chem Inf Model ; 60(10): 5026-5035, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-32808774

RESUMO

The plasticity of cytochromes P450 (P450s) is known to contribute significantly to their catalytic capacity of metabolizing various substrates. Although numerous studies have been performed, factors governing the plasticity and dynamics of P450s are still not fully understood. In this study, taking CYP2B4 as an example, we dissect the protein plasticity and dynamics in different environments. CYP2B4 is featured by a high degree of plasticity, which exhibits open, closed, and intermediate states. By analyzing the CYP2B4 crystal structures, we identified the structural features for the closed, open, and intermediate states. Interestingly, formation of the dimer structure was found in the open and intermediate states. The subsequent molecular dynamics (MD) simulations of the open structure in water confirmed the importance of the dimer form in stabilizing the open conformations. MD simulations of the closed and open structures in the membrane environment and the free energies for opening the F-G cassette obtained from the umbrella sampling calculations indicate that the membrane environment is important for stabilizing the F-G cassette. The dynamical network analysis indicates that Asp105 on the B-C loop plays an important role in transiting the structure from the open to the intermediate state. Our results thus unveil the mechanisms of dimer formation and open-to-intermediate transition for CYP2B4 in the water and membrane environments.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Simulação de Dinâmica Molecular , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Conformação Proteica
17.
Biomolecules ; 10(7)2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659980

RESUMO

Alternaria molds simultaneously produce a large variety of mycotoxins, of which several were previously reported to induce enzymes of phase I metabolism through aryl hydrocarbon receptor activation. Thus, we investigated the potential of naturally occurring Alternaria toxin mixtures to induce Cytochrome P450 (CYP) 1A1/1A2/1B1 activity. Two variants of an extract from cultured Alternaria alternata, as well as the toxins alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX-I), and altertoxin II (ATX-II), were tested singularly and in binary mixtures applying the 7-ethoxy-resorufin-O-deethylase (EROD) assay in MCF-7 breast cancer cells. Sub-cytotoxic concentrations of the two toxin mixtures, as well as ATX-I, ATX-II and AOH, exhibited dose-dependent enhancements of CYP 1 activity. ATX-I and ATX-II interacted synergistically in this respect, demonstrating the two perylene quinones as major contributors to the extract's potential. Binary mixtures between AOH and the two altertoxins respectively exhibited concentration-dependent antagonistic as well as synergistic combinatory effects. Notably, AME showed no efficacy towards EROD enzyme activity or impact on other toxins' efficacy. Hence, this study provides insights into synergistic and other combinatory effects of Alternaria toxins in natural co-occurrence scenarios in the context of AhR signalling pathway activation in breast cancer cells.


Assuntos
Alternaria/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Neoplasias da Mama/metabolismo , Micotoxinas/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzo(a)Antracenos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lactonas/farmacologia , Células MCF-7 , Perileno/análogos & derivados , Perileno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo
18.
Brain Res Bull ; 163: 57-64, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32707261

RESUMO

Cytochrome P450 (CYP) epoxygenases have been considered the main producers of epoxyeicosatrienoic acids (EETs) through the oxidation of arachidonic acid (AA). EETs display various biological properties, notably their powerful anti-inflammatory activities. In the brain, EETs have proven to be neuroprotective and to improve neuroinflammation. However, it is known that inflammation could modify CYP expression. We have previously reported that an inflammatory process in astrocytes is able to down-regulate CYP2J3 and CYP2C11 mRNA, protein levels, and activity (Navarro-Mabarak et al., 2019). In this work, we evaluated the effect of neuroinflammation in protein expression of CYP epoxygenases in the brain. Neuroinflammation was induced by the intraperitoneal administration of LPS (1 mg/kg) to male Wistar rats and was corroborated by IL-6, GFAP, and Iba-1 protein levels in the cortex over time. CYP2J3 and CYP2C11 protein levels were also evaluated in the cortex after 6, 12, 24, 48, and 72 h of LPS treatment. Our results show for the first time that neuroinflammation is able to downregulate CYP2J3 and CYP2C11 protein expression in the brain cortex.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/metabolismo , Regulação para Baixo/fisiologia , Mediadores da Inflamação/metabolismo , Esteroide 16-alfa-Hidroxilase/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Encéfalo/efeitos dos fármacos , Família 2 do Citocromo P450/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Masculino , Ratos , Ratos Wistar , Esteroide 16-alfa-Hidroxilase/antagonistas & inibidores
19.
Environ Toxicol Chem ; 39(9): 1693-1701, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32452045

RESUMO

In the present study, we investigated whether the immortalized chicken hepatocellular carcinoma cell line, leghorn male hepatoma (LMH), had a comparable aryl hydrocarbon receptor (AhR) response to primary chicken embryonic hepatocytes (CEHs) when used in a well-established assay for chemical screening and prioritization. The LMH cells were grown as 2-dimensional (2D) confluent cells and 3D spheroids to determine the optimal cell culture states for chemical screening. Cytochrome P450 1A4 and 1A5 (CYP1A) activity and gene expression were compared between CEHs and LMH cells grown in 2 culture states following exposure to the dioxin-like compound 3,3',4,4',5-pentachlorobiphenyl (PCB-126). The CYP1A activity was measured using the ethoxyresorufin-O-deethylase (EROD) assay, and changes in mRNA expression associated with the AhR pathway were determined using a custom-designed polymerase chain reaction array. Among LMH cell culture states (i.e., 2D vs 3D), EROD induction was observed only in 3D LMH spheroids. Similarly, 3D spheroids had the greatest number of changes in AhR-related genes compared with confluent cells. Overall, these results suggest that LMH cells grown as 3D spheroids have a metabolic and gene expression profile that is comparable to that of CEH, and may represent a suitable animal-free alternative for in vitro screening of chemicals. Environ Toxicol Chem 2020;39:1693-1701. © 2020 SETAC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Esferoides Celulares/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Galinhas/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Bifenilos Policlorados/metabolismo , Bifenilos Policlorados/toxicidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esferoides Celulares/patologia
20.
Med Sci Monit ; 26: e922149, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32284524

RESUMO

BACKGROUND Leonurine is an active component of the traditional Chinese medicine Leonurus japonicus. This study aimed to investigate the effects of overexpressed CYP450s on the metabolic activity of leonurine. MATERIAL AND METHODS BEAS-2B cells stably expressing CYP1A1, 1A2, 2A13, 2B6, and 3A4 were constructed. CYP450s expression was identified using reverse-transcription PCR and Western blot assay. CCK-8 assay was used to evaluate the effect of leonurine on cell activity. Leonurine was incubated in vitro with CYP1A1, 1A2, 2A13, 2B6, and 3A4 metabolic enzymes to evaluate the clearance rate of CYP450 enzymes for leonurine. UPLC-MS was used to detect changes of drug concentration and discover the main metabolic enzymes affecting leonurine. RESULTS BEAS-2B cells stably expressing CYP1A1, 1A2, 2A13, 2B6, and 3A4 were successfully constructed. According to primary mass spectra and secondary mass spectra of leonurine, the main metabolic enzymes were 312.1550 [H+] and 181.0484. Compared to the control group, residue of leonurine in CYP2A13 group was significantly reduced (F=5.307, p=0.024). Compared to the 0-min group, the clearance rate of leonurine in the CYP2A13-treated group was significantly decreased at 120 min after treatment (F=7.273, p=0.007). CCK-8 results also showed that activity of BEAS-2B cells that overexpress CYP2A13 gradually decreased with increased concentration of leonurine. Although CYP2A13 demonstrated good metabolic activity for leonurine, we found that CYP1A1, 1A2, 2B6, and 3A4 had no metabolic effects on leonurine. CONCLUSIONS Leonurine can be effectively activated through CYP2A13 enzyme metabolism, and further inhibits activity of human lung epithelial cells (BEAS-2B). Therefore, CYP2A13 is a main metabolic enzyme for leonurine in BEAS-2B cells.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Ácido Gálico/análogos & derivados , Brônquios/citologia , Linhagem Celular , Ácido Gálico/farmacologia , Humanos , Inativação Metabólica
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