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1.
Toxicol Lett ; 324: 104-110, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32092453

RESUMO

Dietary and microbial indoles can act as ligands and activators of pregnane X receptor (PXR), with implications in human intestinal health. In the current study, we examined the effects of simple mono-methylated indoles (MMIs) on the activity and function of PXR, using a series of human hepatic and intestinal cell models. Indoles 1-MMI and 2-MMI strongly induced CYP3A4 and MDR1 mRNAs in human intestinal adenocarcinoma cells LS180, but not in primary human hepatocytes. The levels of CYP3A4 mRNA were increased by 1-MMI and 2-MMI in wild type, but not in PXR-knock-out human hepatic progenitor HepaRG cells, implying the involvement of PXR in CYP3A4 induction by MMIs. Utilizing reporter gene assay, we observed dose-dependent activation of PXR by all MMIs, and their efficacies and potencies were comparable. Tested MMIs also displayed moderate antagonist effects on PXR, revealing about partial agonist effects of these compounds. As demonstrated using the Chromatin immunoprecipitation assay (ChIP),1-MMI increased PXR occupancy of the CYP3A4 promoter. Time-Resolved Fluorescence Resonance Energy Transfer revealed that MMIs are weak ligands of human PXR. Collectively, we show that MMIs are ligands and partial agonists of human PXR, which induce PXR-regulated genes in human intestinal cells.


Assuntos
Hepatócitos/efeitos dos fármacos , Indóis/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Receptor de Pregnano X/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/fisiologia , Hepatócitos/metabolismo , Humanos , Indóis/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos
2.
Toxicol Lett ; 321: 131-137, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31877331

RESUMO

Prior exposures to chemicals/agents may alter epigenome in such a way that subsequent exposure to the same or different xenobiotic would produce different responses. Understanding the mechanism for this "priming" effect is of clinical significance in avoiding adverse drug-drug interactions. Here we reported a dramatic priming effect of dimethyl sulfoxide (DMSO) on pregnane X receptor (PXR)-mediated gene regulations and analyzed the underpinning epigenetic mechanism. We showed that DMSO (1.25-2.5 %) pretreatment has a profound effect in enhancing the expression of PXR target genes. This priming effect persisted up to 48 h. Mechanistically, DMSO pretreatment reduced H4K12 acetylation and therefore enhanced the subsequent rifampicin stimulated histone H4R3 methylation on the regulatory region of PXR target gene CYP3A4. We showed that protein arginine methyltransferase 1 (PRMT1), which methylates H4R3, was important for priming by DMSO. Inhibition of methyltransferase by the pharmacological inhibitor adenosine dialehyde (AdoX), or RNAi knockdown of PRMT1, abolished the DMSO priming effects. On the other hand, Trichostation A (TSA) pretreatment, which increases histone acetylation and therefore suppresses H4R3 methylation, also abolished the DMSO priming effects. Based on the above observation, we proposed a model of sequential order of histone methylation and acetylation on the transcription "relay".


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Dimetil Sulfóxido/toxicidade , Epigênese Genética/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Histonas/metabolismo , Receptor de Pregnano X/agonistas , Acetilação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Metilação , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo
3.
Environ Pollut ; 255(Pt 2): 113329, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31600704

RESUMO

Transcription factors including pregnane X receptor (Pxr) and nuclear factor-erythroid 2-related factor-2 (Nrf2) are important modulators of Adenosine triphosphate-binding cassette (ABC) transporters in mammalian cells. However, whether such modulation is conserved in zebrafish embryos remains largely unknown. In this manuscript, pxr- and nrf2-deficient models were constructed with CRISPR/Cas9 system, to evaluate the individual function of Pxr and Nrf2 in the regulation of ABC transporters and detoxification of heavy metal ions like Cd2+ and Ag+. As a result, both Cd2+ and Ag+ conferred extensive interactions with ABC transporters in wild type (WT) embryos: their accumulation and toxicity were affected by the activity of ABC transporters, and they significantly induced the mRNA expressions of ABC transporters. These induction effects were reduced by the mutation of pxr and nrf2, but elevations in the basal expression of ABC transporters compensated for the loss of their inducibility. This could be an explanation for remaining transporter function in both mutant models as well as the unaltered toxicity of metal ions in pxr-deficient embryos. However, mutation of nrf2 disrupted the production of glutathione (GSH), resulting in the enhanced toxicity of Cd2+/Ag+ in zebrafish embryos. In addition, elevated expressions of other transcription factors like aryl hydrocarbon receptor (ahr) 1b, peroxisome proliferator-activated receptor (ppar)-ß, and nrf2 were found in pxr-deficient models without any treatment, while enhanced induction of ahr1b, ppar-ß and pxr could only be seen in nrf2-deficient embryos after the treatment of metal ions, indicating different compensation phenomena for the absence of transcription factors. After all, pxr-deficient and nrf2-deficient zebrafish embryos are useful tools in the functional investigation of Pxr and Nrf2 in the early life stages of aquatic organisms. However, the compensatory mechanisms should be taken into consideration when interpreting the results and need in-depth investigations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais Pesados/toxicidade , Fator 2 Relacionado a NF-E2/genética , Receptor de Pregnano X/genética , Peixe-Zebra/embriologia , Animais , Glutationa/metabolismo , Inativação Metabólica , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Esteroides/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo
4.
J Biochem Mol Toxicol ; 33(12): e22408, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617658

RESUMO

In this study, gene expression alterations of phase I to III enzymes, transcription factors, and microRNA (miRNA) in embryonic zebrafish fibroblasts (ZF4) cells after the treatment of Pb(NO3 )2 and AgNO3 were investigated, to illustrate the possible detoxification pathway of heavy metal ions. It was observed that both metals caused concentration-dependent death and moderate elevation of oxidative stress in ZF4 cells. In response to such toxicity, upregulation of multidrug resistance protein (mdr)4 and multiresistance-associated protein (mrp)1 were found. However, enhanced expression of glutathione S-transferase (gst) and cytochrome P450 (cyp)1a could only be detected during the exposure of Pb2+ . In addition, both metals induced extensive upregulation of pregnane X receptor (pxr), but only moderate elevation of E2-related factor (nrf2), while they suppressed the expression of miR-122 and miR-126. In conclusion, Pb2+ and Ag+ shared the same detoxification mechanism including ABC transporters, Pxr, and miRNA in ZF4 cells, which needs further investigation.


Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Chumbo/toxicidade , Nitratos/toxicidade , Nitrato de Prata/toxicidade , Peixe-Zebra/embriologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Food Chem Toxicol ; 133: 110792, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31472229

RESUMO

Parabens are widely used as preservatives in personal care products, medicines and foods, resulting in substantial human exposures, even though some harmful effects, such as endocrine-disrupting activity, have been reported. Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are members of the nuclear receptor superfamily, regulate the metabolism of endogenous substrates including hormones. Therefore, we hypothesized that parabens may alter hormone-metabolizing activities by acting on these receptors, and such changes could contribute to the endocrine-disrupting activity. To test this idea, we systematically examined the effects of 17 parabens on these receptors using reporter gene assays. Nine parabens significantly activated human and rat PXR. Parabens with C2-C5 (linear and branched) side chains were most active. Butylparaben and isobutylparaben also significantly activated rat CAR. We found that long-side-chain (C7-C12) parabens showed up to 2-fold activation of PPARα at 10 µM. Furthermore, pentylparaben and hexylparaben showed rat PXR antagonistic activity and rat CAR inverse agonistic activity. The activity of butylparaben towards PXR and CAR was lost after carboxylesterase-mediated metabolism. These findings confirm that parabens influence the activities of PXR, CAR and PPARα, and thus have the potential to contribute to endocrine disruption by altering hormone metabolism.


Assuntos
PPAR alfa/metabolismo , Parabenos/farmacologia , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Agonismo Inverso de Drogas , Humanos , Masculino , Microssomos Hepáticos/metabolismo , PPAR alfa/agonistas , PPAR alfa/genética , Parabenos/metabolismo , Receptor de Pregnano X/antagonistas & inibidores , Receptor de Pregnano X/genética , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética
6.
Int J Mol Sci ; 20(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374856

RESUMO

The pregnane X receptor (PXR) is the main nuclear receptor regulating the expression of xenobiotic-metabolizing enzymes and is highly expressed in the liver and intestine. Recent studies have highlighted its additional role in lipid homeostasis, however, the mechanisms of these regulations are not fully elucidated. We investigated the transcriptomic signature of PXR activation in the liver of adult wild-type vs. Pxr-/- C57Bl6/J male mice treated with the rodent specific ligand pregnenolone 16α-carbonitrile (PCN). PXR activation increased liver triglyceride accumulation and significantly regulated the expression of 1215 genes, mostly xenobiotic-metabolizing enzymes. Among the down-regulated genes, we identified a strong peroxisome proliferator-activated receptor α (PPARα) signature. Comparison of this signature with a list of fasting-induced PPARα target genes confirmed that PXR activation decreased the expression of more than 25 PPARα target genes, among which was the hepatokine fibroblast growth factor 21 (Fgf21). PXR activation abolished plasmatic levels of FGF21. We provide a comprehensive signature of PXR activation in the liver and identify new PXR target genes that might be involved in the steatogenic effect of PXR. Moreover, we show that PXR activation down-regulates hepatic PPARα activity and FGF21 circulation, which could participate in the pleiotropic role of PXR in energy homeostasis.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/metabolismo , PPAR alfa/metabolismo , Receptor de Pregnano X/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/genética , Deleção de Genes , Perfilação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X/genética , Ativação Transcricional , Transcriptoma
7.
Mol Pharmacol ; 96(3): 345-354, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31436536

RESUMO

Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (KD = 1.42 × 10-05). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.


Assuntos
Fenobarbital/farmacologia , Receptor de Pregnano X/química , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Células Cultivadas , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Receptor de Pregnano X/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Triptofano/metabolismo
8.
J Ethnopharmacol ; 245: 112103, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31336134

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cholestasis is a clinical syndrome caused by toxic bile acid retention that will lead to serious liver diseases. Ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) are the only two FDA-approved drugs for its treatment. Thus, there is a clear need to develop new therapeutic approaches for cholestasis. Here, anti-cholestasis effects of the lignans from a traditional Chinese herbal medicine, Schisandra sphenanthera, were investigated as well as the involved mechanisms. MATERIALS AND METHODS: Adult male C57BL/6J mice were randomly divided into 9 groups including the control group, LCA group, LCA with specific lignan treatment of Schisandrin A (SinA), Schisandrin B (SinB), Schisandrin C (SinC), Schisandrol A (SolA), Schisandrol B (SolB), Schisantherin A (StnA) and Schisantherin B (StnB), respectively. Mice were treated with each drug (qd) for 7 days, while the administration of lithocholic acid (LCA) (bid) was launched from the 4th day. Twelve hours after the last LCA injection, mice were sacrificed and samples were collected. Serum biochemical measurement and histological analysis were conducted. Metabolomics analysis of serum, liver, intestine and feces were performed to study the metabolic profile of bile acids. RT-qPCR and Western blot analysis were conducted to determine the hepatic expression of genes and proteins related to bile acid homeostasis. Dual-luciferase reporter gene assay was performed to investigate the transactivation effect of lignans on human pregnane X receptor (hPXR). RT-qPCR analysis was used to detect induction effects of lignans on hPXR-targeted genes in HepG2 cells. RESULTS: Lignans including SinA, SinB, SinC, SolA, SolB, StnA, StnB were found to significantly protect against LCA-induced intrahepatic cholestasis, as evidenced by significant decrease in liver necrosis, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activity. More importantly, serum total bile acids (TBA) and total bilirubin (Tbili) were also significantly reduced. Metabolomics analysis revealed these lignans accelerated the metabolism of bile acids and increased the bile acid efflux from liver into the intestine or feces. Gene analysis revealed these lignans induced the hepatic expressions of PXR-target genes such as Cyp3a11 and Ugt1a1. Luciferase reporter gene assays illustrated that these bioactive lignans can activate hPXR. Additionally, they can all upregulate hPXR-regulate genes such as CYP3A4, UGT1A1 and OATP2. CONCLUSION: These results clearly demonstrated the lignans from Schisandra sphenanthera exert hepatoprotective effects against LCA-induced cholestasis by activation of PXR. These lignans may provide an effective approach for the prevention and treatment of cholestatic liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colestase/tratamento farmacológico , Lignanas/uso terapêutico , Receptor de Pregnano X/genética , Substâncias Protetoras/uso terapêutico , Schisandra , Animais , Ácidos e Sais Biliares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/induzido quimicamente , Colestase/metabolismo , Colestase/patologia , Fezes/química , Células HEK293 , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Lignanas/farmacologia , Ácido Litocólico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Substâncias Protetoras/farmacologia
9.
Toxicol In Vitro ; 60: 347-358, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31233785

RESUMO

Pregnane & Xenobiotic Receptor (PXR), one of the members of nuclear receptor superfamily, acts as a 'master-regulator' of drug metabolism and disposition machinery (DMD). Activation of PXR enables detoxification and elimination of toxic xenobiotics/endobiotics, and defends our body against chemical insults. On the contrary, PXR activation also imposes a serious concern for drug-drug interactions (DDIs). Such DDIs could either decrease the efficacy or lead to accumulation of co-administered drugs at toxic level. Therefore, it is desirable that during drug development process the small drug molecules are screened on PXR-platform prior to their clinical trial and prevent late stage failures. In view of this, we have selected a group of anti-diabetic drug molecules to examine if the success and potential failure of small molecule modulators can be pre-assessed and judiciously correlated on PXR platform. For this purpose, we have examined the PXR activation potential of the selected anti-diabetic drugs. Subsequent to screening of these anti-diabetic drugs, we elaborated the study further with rosiglitazone and pioglitazone (thiazolidinediones, TZDs) which are oral anti-diabetic formulations and have been in controversy owing to their association with cardiotoxicity and bladder cancer respectively. Our study revealed that some of the selected anti-diabetic drugs possess PXR activation potential, implying that these can up-regulate the expression of CYP3A4, UGT1A1, MDR1 and thereby can be predicted to inflict undesirable consequences.


Assuntos
Hipoglicemiantes/farmacologia , Receptor de Pregnano X/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interações de Medicamentos , Glucuronosiltransferase/genética , Humanos , Pioglitazona/farmacologia , Receptor de Pregnano X/genética , RNA Interferente Pequeno/genética , Rosiglitazona/farmacologia
10.
Am J Physiol Endocrinol Metab ; 317(2): E350-E361, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31211619

RESUMO

We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr-/-mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr-/- mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr-/- or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr-/- mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr-/- mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr-/- mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr-/- mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.


Assuntos
Células Endoteliais/efeitos dos fármacos , Indóis/farmacologia , Receptor de Pregnano X/agonistas , Receptor de Pregnano X/fisiologia , Vasodilatação/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Células Cultivadas , Células Endoteliais/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Indóis/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota/efeitos dos fármacos , Microbiota/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor de Pregnano X/genética , Vasodilatação/genética
11.
Drug Metab Pharmacokinet ; 34(3): 172-180, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31101589

RESUMO

Rifamycin SV (rifamycin), is a member of the ansamycin family of antimicrobial compounds which kills bacteria commonly associated with infectious diarrhea and other enteric infections. Rifamycin has been found to be effective in experimental animal models of gut inflammation and its efficacy in these settings has been attributed partially to immunomodulatory non-bactericidal activities. This study aimed to further evaluate the anti-inflammatory activities of rifamycin by analyzing its effect on two key regulators of inflammation: PXR and NFκB. Rifamycin stimulated PXR transcriptional activity in two PXR reporter cell lines and induced expression of two genes known to be regulated by PXR and are directly involved in cellular detoxification: CYP3A4 and PgP. Moreover, CYP3A4 metabolic activity was induced by rifamycin in HepG2 cells. Rifamycin also antagonized TNFα and LPS-induced NFκB activities and inhibited IL1ß-induced synthesis of inflammatory chemokine, IL8. Although reciprocal regulation of PXR and NFkB by rifamycin was not directly addressed, the data suggest that in the absence of PXR, inhibition of NFκB by rifamycin is not dependent on PXR stimulation. Thus, rifamycin exhibits potent anti-inflammatory activities, characterized by in vitro PXR activation and concomitant CYP3A4 and PgP induction, in parallel with potent NFκB inhibition and concomitant IL8 inhibition.


Assuntos
Anti-Inflamatórios/farmacologia , NF-kappa B/antagonistas & inibidores , Receptor de Pregnano X/genética , Rifamicinas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Células CACO-2 , Técnicas de Cultura de Células , Citocromo P-450 CYP3A/genética , Células HT29 , Células Hep G2 , Humanos , NF-kappa B/genética , Transdução de Sinais
12.
Methods Mol Biol ; 1966: 163-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041746

RESUMO

Reporter assays are useful to study nuclear receptor activation and for example to evaluate the propensity of novel drug candidates to cause induction of drug-metabolizing cytochrome P450 enzymes. Here, we describe a protocol for a reverse transfection system to study the activation of human nuclear receptors constitutive androstane receptor and pregnane X receptor. The system provides long-term stability and uniformity of DNA-carrier complexes, thus avoiding the inherent variation in conventional transfection methods. Further, the system is easily adaptable for different studies. It offers reproducible and reliable results for early drug development and mechanistic studies related to nuclear receptor activation and resulting changes in gene expression.


Assuntos
Hepatócitos/metabolismo , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Transfecção/métodos , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Humanos , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo
13.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083458

RESUMO

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.


Assuntos
Elasmobrânquios/classificação , Elasmobrânquios/genética , Evolução Molecular , Redes Reguladoras de Genes , Filogenia , Receptor de Pregnano X/genética , Animais , Células COS , Genes Reporter , Inativação Metabólica/genética , Luciferases/metabolismo , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sintenia/genética , Ativação Transcricional/genética
14.
Chem Biol Interact ; 308: 185-193, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31132328

RESUMO

Cytochrome P450 3A (CYP3A) activity is inhibited, and its expression is suppressed during many diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanism is controversial. Here, we report that PXR may not take part in the downregulation of CYP3A during NAFLD. Hepatic CYP3A11 (major subtype of mouse CYP3A) mRNA and protein expression was significantly decreased in both mice fed a high-fat diet (HFD) for 8 weeks and palmitate (PA)-treated mouse primary hepatocytes. Similarly, in HepG2 cells, PA treatment significantly suppressed the CYP3A4 (major subtype of human CYP3A) mRNA level and promoter transcription activity. However, Western blotting analysis found an induction of PXR nuclear translocation during NAFLD in both in vivo and in vitro models. Moreover, immunofluorescence determination also found nuclear translocation effect of PXR by PA stimulation in HepG2 cells. In addition, the siRNA knockdown of PXR did not affect the suppressive effects of PA on the CYP3A4 promoter transcription activity and mRNA levels in HepG2 cells. Similarly, PXR knockdown also did not affect the suppressive effects of PA on CYP3A11 mRNA and protein expression levels in mouse primary hepatoctyes. Taken together, the results showed that the suppressive effect of CYP3A transcription was independent of PXR regulation.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Dieta Hiperlipídica , Hepatopatia Gordurosa não Alcoólica/patologia , Receptor de Pregnano X/metabolismo , Animais , Citocromo P-450 CYP3A/genética , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/veterinária , Palmitatos/farmacologia , Receptor de Pregnano X/antagonistas & inibidores , Receptor de Pregnano X/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transcrição Genética/efeitos dos fármacos
15.
J Agric Food Chem ; 67(17): 4967-4975, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30955332

RESUMO

Rooibos tea ( Aspalathus linearis) is a well-known South African herbal tea enjoyed worldwide. Limited reports indicate the potential of rooibos tea to alter the activity of certain cytochrome P450 (CYP450) isozymes. In this study, the phytochemical investigation of MeOH extract of A. linearis (leaves and stems) resulted in the isolation and characterization of 11 phenolic compounds. The MeOH extract exhibited significant inhibition of the major human CYP450 isozymes (CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19). The strongest inhibition was observed by the extract for CYP3A4 (IC50 1.7 ± 0.1 µg/mL) followed by CYP2C19 (IC50 4.0 ± 0.3 µg/mL). Among the tested phytochemicals, the most potent inhibitors were isovitexin on CYP3A4 (IC50 3.4 ± 0.2 µM), vitexin on CYP2C9 (IC50 8.0 ± 0.2 µM), and thermopsoside on CYP2C19 (IC50 9.5 ± 0.2 µM). The two major, structurally related compounds aspalathin and nothofagin exhibited a moderate pregnane-X receptor (PXR) activation, which was associated with increased mRNA expression of CYP3A4 and CYP1A2, respectively. These results indicate that a high intake of nutraceuticals containing rooibos extracts may pose a risk of herb-drug interactions when consumed concomitantly with clinical drugs that are substrates of CYP enzymes.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Aspalathus/química , Sistema Enzimático do Citocromo P-450/química , Preparações de Plantas/química , Receptor de Pregnano X/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aspalathus/metabolismo , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Inocuidade dos Alimentos , Humanos , Folhas de Planta/química , Preparações de Plantas/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Chás de Ervas/análise
16.
Mol Genet Genomic Med ; 7(5): e574, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30955239

RESUMO

BACKGROUND: Genetic polymorphisms in numerous pharmacogenetics studies were regarded as the essential factors involved in the response to or metabolism of drugs. These genetic variants called very important pharmacogenetic (VIP) variants played a role in drugs metabolism, which have been summarized in the PharmGKB database. In this study, we genotyped 80 VIP variants from the PharmGKB in 100 members of Blang volunteers from Yunnan province. METHODS: Based on the PharmGKB database, we genotyped 80 VIP variants loci located in 47 genes. We used χ2 tests to evaluate the significant loci between Blang and the other populations, including ASW, CEU, CHB, CHD, GIH, JPT, LWK, MEX, MKK, TSI, and YRI. The global variation distribution of the significant variants was observed from the ALlele FREquency Database. And then, we used F-statistics (Fst), genetic structure, and phylogenetic tree analyses to ascertain the genetic affinity among 12 populations. RESULTS: Comparing the Blang with the other 11 populations from the HapMap Project, the statistical results revealed that rs3814055 (NC_000003.12:g.119781188C>T) of nuclear receptor subfamily 1 group I member 2 (NR1I2, OMIM# 603,065) was the most significant variant, followed by rs1540339 (NC_000012.12:g.47863543C>T) of vitamin D receptor (VDR, OMIM#601,769). Furthermore, we found that genotype frequency of rs3814055 in the Blang was closer to the populations distributed in Miao. And genetic structure and F-statistics indicated that the Blangs had a relatively closer affinity with CHD, CHB, and JPT populations. In addition, the Han nationality in Shaanxi was closer to it. CONCLUSIONS: Our results will complement the pharmacogenomics information of the Blang ethnic group and provide a theoretical basis for safer drug administration for Blang.


Assuntos
Variantes Farmacogenômicos , China , Frequência do Gene , Humanos , Receptor de Pregnano X/genética , Receptores de Calcitriol/genética
17.
PLoS One ; 14(3): e0214338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908543

RESUMO

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.


Assuntos
Clonagem Molecular/métodos , Citocromo P-450 CYP3A/genética , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Sítios de Ligação , Bovinos , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Transfecção
18.
Toxicology ; 416: 1-14, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30711707

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is affecting up to one-third of the general population in western countries. While the major cause of NAFLD is related to an unhealthy lifestyle, recent evidence has shown a role of chemical exposure in the induction and progression of NAFLD. Perfluorooctanoate (PFOA) is a ubiquitous environmental contaminant that exerts its hepatotoxicity mainly through the activation of peroxisome proliferator-activated receptor α (PPARα). We examined how PFOA might affect the progression of NAFLD and whether a preexisting fatty liver intensified or alleviated the effects of PFOA in the livers. As such, male C57BL/6 mice were fed with a low-fat control diet (CD) or a high fat diet (HFD) for 16 weeks to model normal or steatotic livers, respectively. Mice were then administered with PFOA (1mg/kg/d) by oral gavage for an additional 2, 8, and 16 weeks. Dietary treatment was continued throughout the whole study. We found HFD induced hepatic steatosis, lobular inflammation, and progressive fibrosis in mice. As expected, PFOA activated PPARα, constitutive androstane receptor (CAR) and pregnane X receptor (PXR), regardless of the diet. Gene expression analysis showed the interactions between HFD and PFOA on hepatic nuclear receptors were time-dependent. Hepatocytes growth as measured by DNA synthesis and cell growth genes induced by PFOA were exacerbated in the HFD group after 2 weeks, along with the enhanced activation of PPARα. In contrast, PFOA decreased the severity of hepatic steatosis. In HFD-fed mice, the hepatic triglyceride levels were reduced to 75%, 47%, and 40%, after 2, 8, and 16 weeks of PFOA treatment, respectively, compared to vehicle controls. Transcriptomic analysis showed the preexisting NAFLD enhanced PFOA related lipid oxidation pathways in mice. HFD induced hepatic fibrosis as measured by collagen staining and fibrosis gene markers were also attenuated by PFOA. Taken together, this study demonstrated that the preexisting NAFLD might impact on many biological effects induced by PFOA and thus need to be carefully considered as a factor in risk assessment.


Assuntos
Caprilatos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Dieta Hiperlipídica , Poluentes Ambientais/toxicidade , Fluorcarbonetos/toxicidade , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , PPAR alfa/genética , PPAR alfa/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Medição de Risco , Transdução de Sinais , Fatores de Tempo
19.
Xenobiotica ; 49(3): 257-264, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29431552

RESUMO

Cytochrome P450, which is expressed in humans and other animals, is a superfamily of drug-metabolizing enzymes that play important roles in the metabolism of endogenous and xenobiotic substrates via oxidation, peroxidation and reduction. Of endogenous substrates, interleukin (IL)-6 is a crucial cytokine involved in inflammation in the liver. The present study aims to elucidate the mechanisms through which IL-6 modulates cytochrome P450 expression. CYP2C33 expression was found to be increased in HepLi cells and primary porcine hepatocytes treated with IL-6 in a concentration-dependent manner. IL-6 treatment also increased the expression of the transcriptional regulators, constitutive androstane receptor (CAR) and pregnane X receptor. Overexpression of CAR promoted CYP2C33 expression at the mRNA and protein levels, whereas knockdown of CAR by small interfering RNA reduced CYP2C33 expression. Luciferase assays showed that IL-6 treatment of HepLi cells and primary porcine hepatocytes increased CYP2C33 promoter activity. Co-immunoprecipitation and western blotting demonstrated that CAR and RXR could form heterodimers. IL-6 affects CYP2C33 expression through CAR/retinoid X receptor (RXR) heterodimers.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Interleucina-6/farmacologia , Receptor de Pregnano X/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Inativação Metabólica , Interleucina-6/metabolismo , Interleucina-6/fisiologia , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Suínos , Xenobióticos/metabolismo
20.
Pharmacogenomics J ; 19(2): 147-156, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29298995

RESUMO

Warfarin is primarily metabolized by cytochrome 2C9, encoded by gene CYP2C9. Here, we investigated whether variants in nuclear receptor genes which regulate the expression of CYP2C9 are associated with warfarin response. We used data from 906 warfarin users from the Quebec Warfarin Cohort (QWC) and tested the association of warfarin dose requirement at 3 months following the initiation of therapy in nine nuclear receptor genes: NR1I3, NR1I2, NR3C1, ESR1, GATA4, RXRA, VDR, CEBPA, and HNF4A. Three correlated SNPs in the VDR gene (rs4760658, rs11168292, and rs11168293) were associated with dose requirements of warfarin (P = 2.68 × 10-5, P = 5.81 × 10-4, and P = 5.94 × 10-4, respectively). Required doses of warfarin were the highest for homozygotes of the minor allele at the VDR variants (P < 0.0026). Variants in the VDR gene were associated with the variability in response to warfarin, emphasizing the possible clinical relevance of nuclear receptor gene variants on the inter-individual variability in drug metabolism.


Assuntos
Coagulação Sanguínea/genética , Estudo de Associação Genômica Ampla , Receptores de Calcitriol/genética , Varfarina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Anticoagulantes/administração & dosagem , Anticoagulantes/efeitos adversos , Anticoagulantes/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Citocromo P-450 CYP2C9/genética , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/genética , Feminino , Fator de Transcrição GATA4/genética , Genótipo , Fator 4 Nuclear de Hepatócito/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Receptor de Pregnano X/genética , Quebeque/epidemiologia , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/genética , Receptor X Retinoide alfa/genética , Vitamina K/genética , Vitamina K/metabolismo , Varfarina/administração & dosagem , Varfarina/efeitos adversos
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