RESUMEN
Novel methylindoles were identified as endobiotic and xenobiotic ligands of the human aryl hydrocarbon receptor (AhR). We examined the effects of 22 methylated and methoxylated indoles on the transcriptional activity of AhRs. Employing reporter gene assays in AZ-AHR transgenic cells, we determined full agonist, partial agonist, or antagonist activities of tested compounds, having substantially variable EC50, IC50, and relative efficacies. The most effective agonists (EMAX relative to 5 nM dioxin) of the AhR were 4-Me-indole (134%), 6-Me-indole (91%), and 7-MeO-indole (80%), respectively. The most effective antagonists of the AhR included 3-Me-indole (IC50; 19 µM), 2,3-diMe-indole (IC50; 11 µM), and 2,3,7-triMe-indole (IC50; 12 µM). Reverse transcription polymerase chain reaction analyses of CYP1A1 mRNA in LS180 cells confirmed the data from gene reporter assays. The compound leads, 4-Me-indole and 7-MeO-indole, induced substantial nuclear translocation of the AhR and enriched binding of the AhR to the CYP1A1 promoter, as observed using fluorescent immunohistochemistry and chromatin immunoprecipitation assays, respectively. Molecular modeling and docking studies suggest the agonists and antagonists likely share the same binding pocket but have unique binding modes that code for their affinity. Binding pocket analysis further revealed that 4-methylindole and 7-methoxyindole can simultaneously bind to the pocket and produce synergistic interactions. Together, these data show a dependence on subtle and specific chemical indole structures as AhR modulators and furthermore underscore the importance of complete evaluation of indole compounds as nuclear receptor ligands.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Indoles/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Línea Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Genes Reporteros/efectos de los fármacos , Células Hep G2 , Humanos , Ligandos , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
Variety of xenobiotics, including therapeutically used vitamin D analogues or environmental and alimentary endocrine disruptors, may interfere with vitamin D receptor (VDR) signaling, with serious physiological or pathophysiological consequences. Therefore, it is of topical interest to have reliable and efficient in vitro screening tools for the identification of agonists and activators of human VDR. We present here two novel stably transfected human reporter cell lines allowing rapid, high-throughput, and selective identification of VDR agonists and activators. Human colon adenocarcinoma cells LS180 were stably transfected with reporter plasmids CYP24_minP-pNL2.1[Nluc/Hygro] (IZ-CYP24 cells contain the -326/-46 sequence from the human CYP24A1 promoter) or VDREI3_SV40-pNL2.1[Nluc/Hygro] (IZ-VDRE cells contain three copies of vitamin D response elements VDRE-I from the human CYP24A1 promoter). Both cell lines remained fully functional for over two months in the culture and also after cryopreservation. Luciferase inductions ranged from 10-fold to 25-fold (RLU 10(6)-10(7)) and from 30-fold to 80-fold (RLU 10(3)-10(4)) in IZ-VDRE and IZ-CYP24 cells, respectively. Time-course analyses revealed that detection of VDR activators is possible as soon as after 8 h of incubation. Cell lines were highly selective toward VDR agonists, displaying no cross-activation by retinoids, thyroids, and steroids. As a proof of concept, we used IZ-VDRE and IZ-CYP24 cells for profiling analogues of vitamin D, and intermediates in vitamin D2 and vitamin D3 metabolic pathways against VDR transcriptional activity. The data obtained revealed significant activation of VDR not only by obligatory ligands calcitriol and ergocalcitriol but also by their precursors and degradation products.
Asunto(s)
Familia 24 del Citocromo P450/genética , Genes Reporteros , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
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.
Asunto(s)
Hepatocitos/efectos de los fármacos , Indoles/farmacología , Mucosa Intestinal/efectos de los fármacos , Receptor X de Pregnano/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Línea Celular Tumoral , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/fisiología , Hepatocitos/metabolismo , Humanos , Indoles/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Receptor X de Pregnano/genética , Receptor X de Pregnano/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal/efectos de los fármacosRESUMEN
The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first-in-class non-cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR-specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.
Asunto(s)
Imitación Molecular , Receptor X de Pregnano/química , Animales , Células Cultivadas , Citocinas , Humanos , Inflamación , Intestinos , Ligandos , Ratones , OrganoidesRESUMEN
Mono-methylindoles (MMI) were described as agonists and/or antagonists of the human aryl hydrocarbon receptor (AhR). Here, we investigated the effects of MMI on AhR-CYP1A pathway in human hepatocytes and HepaRG cells derived from human progenitor hepatic cells. All MMI, except of 2-methylindole, strongly induced CYP1A1 and CYP1A2 mRNAs in HepaRG cells. Induction of CYP1A genes was absent in AhR-knock-out HepaRG cells. Consistently, CYP1A1 and CYP1A2 mRNAs and proteins were induced by all MMIs (except 2-methylindole), in human hepatocytes. The enzyme activity of CYP1A1 was inhibited by MMIs in human hepatocytes and LS180 colon cancer cells in a concentration-dependent manner (IC50 values from 1.2 µM to 23.8 µM and from 3.4 µM to 11.4 µM, respectively). Inhibition of CYP1A1 activity by MMI in human liver microsomes was much weaker as compared to that in intact cells. Incubation of parental MMI with human hepatocytes either diminished (4-methylindole, 6-methylindole) or enhanced (7-methylindole) their agonist effects on AhR in AZ-AHR reporter cells. In conclusion, overall effects of MMI on AhR-CYP1A pathway in human cells comprise the induction of CYP1A genes through AhR, the inhibition of CYP1A catalytic activity and possibly the metabolic transformation causing loss or gain of AhR agonist activity of parental compounds.
Asunto(s)
Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1A1/biosíntesis , Inductores de las Enzimas del Citocromo P-450/farmacología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Hepatocitos/efectos de los fármacos , Indoles/farmacología , Anciano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Citocromo P-450 CYP1A1/genética , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Femenino , Hepatocitos/enzimología , Humanos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Persona de Mediana Edad , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Adulto JovenRESUMEN
Essential oils (EOs) are extensively used in food industry, gastronomy and alternative medicine. They are multicomponent mixtures of bioactive compounds; hence, their potential for food-drug interactions is substantial. In this study, we investigated the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of pregnane X receptor (PXR) and expression of cytochrome P450 3A4 (CYP3A4), using human intestinal and hepatic in vitro models. All tested EOs activated PXR in intestinal LS180 cells transiently transfected with PXR, as revealed by a reporter gene assay. Consistently, all EOs induced CYP3A4 mRNA expression in PXR-transfected LS180 cells, primary human hepatocytes and wild-type hepatic progenitor HepaRG cells. EO-mediated induction of CYP3A4 mRNA expression was nullified in PXR-knock out HepaRG cells, suggesting the involvement of PXR in these effects. Collectively, we showed that EOs of culinary herbs and spices might be common activators of PXR and inducers of CYP3A4 at doses present in foods, thereby, they might have a potential for food-drug interactions. Follow-up studies are warranted to identify the bioactive constituents in the tested EOs.
Asunto(s)
Citocromo P-450 CYP3A/biosíntesis , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Aceites Volátiles/farmacología , Receptores de Esteroides/metabolismo , Especias/análisis , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Línea Celular , Citocromo P-450 CYP2B6/biosíntesis , Citocromo P-450 CYP2B6/genética , Inducción Enzimática/efectos de los fármacos , Genes Reporteros , Hepatocitos/efectos de los fármacos , Humanos , Intestinos/efectos de los fármacos , Hígado/efectos de los fármacos , Receptor X de Pregnano , Cultivo Primario de Células , Receptores de Esteroides/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
Essential oils (EOs) of culinary herbs and spices are used to flavor, color and preserve foods and drinks. Dietary intake of EOs is significant, deserving an attention of toxicologists. We examined the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of human aryl hydrocarbon receptor (AhR), which is a pivotal xenobiotic sensor, having also multiple roles in human physiology. Tested EOs were sorted out into AhR-inactive ones (14 EOs) and AhR-active ones, including full agonists (cumin, jasmine, vanilla, bay leaf), partial agonists (cloves, dill, thyme, nutmeg, oregano) and antagonists (tarragon, caraway, turmeric, lovage, fennel, spearmint, star anise, anise). Major constituents (>10%) of AhR-active EOs were studied in more detail. We identified AhR partial agonists (carvacrol, ligustilide, eugenol, eugenyl acetate, thymol, ar-turmerone) and antagonists (trans-anethole, butylidine phtalide, R/S-carvones, p-cymene), which account for AhR-mediated activities of EOs of fennel, anise, star anise, caraway, spearmint, tarragon, cloves, dill, turmeric, lovage, thyme and oregano. We also show that AhR-mediated effects of some individual constituents of EOs differ from those manifested in mixtures. In conclusion, EOs of culinary herbs and spices are agonists and antagonists of human AhR, implying a potential for food-drug interactions and interference with endocrine pathways.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Aceites Volátiles/farmacología , Aceites de Plantas/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Especias/análisis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Culinaria , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Interacciones Alimento-Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Laurus , Myristica , Origanum , ARN Mensajero/genética , ARN Mensajero/metabolismo , Thymus (Planta)RESUMEN
Essential oils (EOs) of culinary herbs and spices are consumed on a daily basis. They are multicomponent mixtures of compounds with already demonstrated biological activities. Taking into account regular dietary intake and the chemical composition of EOs, they may be considered as candidates for endocrine-disrupting entities. Therefore, we examined the effects of 31 EOs of culinary herbs and spices on transcriptional activities of glucocorticoid receptor (GR), androgen receptor (AR) and vitamin D receptor (VDR). Using reporter gene assays in stably transfected cell lines, weak anti-androgen and anti-glucocorticoid activity was observed for EO of vanilla and nutmeg, respectively. Moderate augmentation of calcitriol-dependent VDR activity was caused by EOs of ginger, thyme, coriander and lemongrass. Mixed anti-glucocorticoid and VDR-stimulatory activities were displayed by EOs of turmeric, oregano, dill, caraway, verveine and spearmint. The remaining 19 EOs were inactive against all receptors under investigation. Analyses of GR, AR and VDR target genes by means of RT-PCR confirmed the VDR-stimulatory effects, but could not confirm the anti-glucocorticoid and anti-androgen effects of EOs. In conclusion, although we observed minor effects of several EOs on transcriptional activities of GR, AR and VDR, the toxicological significance of these effects is very low. Hence, 31 EOs of culinary herbs and spices may be considered safe, in terms of endocrine disruption involving receptors GR, AR and VDR.
Asunto(s)
Disruptores Endocrinos/efectos adversos , Aceites Volátiles/efectos adversos , Plantas Comestibles/química , Receptores Androgénicos/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Glucocorticoides/metabolismo , Especias , Antagonistas de Andrógenos/efectos adversos , Andrógenos/efectos adversos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , República Checa , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Humanos , Ligandos , Plantas Medicinales/química , Receptores Androgénicos/química , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/antagonistas & inhibidores , Receptores de Calcitriol/genética , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Activación Transcripcional/efectos de los fármacosRESUMEN
The role of vitamin D receptor (VDR) in immune responses has been broadly studied and it has been shown that activated VDR alters the levels of some interleukins (ILs). In this study, we studied the opposite, i.e. whether 13 selected pro-inflammatory and anti-inflammatory ILs influence the transcriptional activity of human VDR. The experimental models of choice were two human stably transfected gene reporter cell lines IZ-VDRE and IZ-CYP24, which were designed to evaluate the transcriptional activity of VDR. The gene reporter assays revealed inhibition of calcitriol-induced luciferase activity by IL-4 and IL-13, when 1 ng/mL of these two compounds decreased the effect of calcitriol down to 60% of the control value. Consistently, calcitriol-induced expression of CYP24A1 mRNA was also significantly decreased by IL-4 and IL-13. The expression of VDR and CYP27B1 mRNAs was not influenced by any of the 13 tested ILs. These data suggest possible cross-talk between the VDR signalling pathway and IL-4- and IL-13-mediated cell signalling.
Asunto(s)
Genes Reporteros/genética , Interleucinas/farmacología , Receptores de Calcitriol/genética , Transcripción Genética/efectos de los fármacos , Transgenes/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Línea Celular , Humanos , Transfección , Vitamina D3 24-Hidroxilasa/genéticaRESUMEN
The mechanisms contributing to toxic effects of airborne lower-chlorinated PCB congeners (LC-PCBs) remain poorly characterized. We evaluated in vitro toxicities of environmental LC-PCBs found in both indoor and outdoor air (PCB 4, 8, 11, 18, 28 and 31), and selected hydroxylated metabolites of PCB 8, 11 and 18, using reporter gene assays, as well as other functional cellular bioassays. We focused on processes linked with endocrine disruption, tumor promotion and/or regulation of transcription factors controlling metabolism of both endogenous compounds and xenobiotics. The tested LC-PCBs were found to be mostly efficient anti-androgenic (within nanomolar - micromolar range) and estrogenic (at micromolar concentrations) compounds, as well as inhibitors of gap junctional intercellular communication (GJIC) at micromolar concentrations. PCB 8, 28 and 31 were found to partially inhibit the aryl hydrocarbon receptor (AhR)-mediated activity. The tested LC-PCBs were also partial constitutive androstane receptor (CAR) and pregnane X receptor (PXR) agonists, with PCB 4, 8 and 18 being the most active compounds. They were inactive towards other nuclear receptors, such as vitamin D receptor, thyroid receptor α, glucocorticoid receptor or peroxisome proliferator-activated receptor γ. We found that only PCB 8 contributed to generation of oxidative stress, while all tested LC-PCBs induced arachidonic acid release (albeit without further modulations of arachidonic acid metabolism) in human lung epithelial cells. Importantly, estrogenic effects of hydroxylated (OH-PCB) metabolites of LC-PCBs (4-OH-PCB 8, 4-OH-PCB 11 and 4'-OH-PCB 18) were higher than those of the parent PCBs, while their other toxic effects were only slightly altered or suppressed. This suggested that metabolism may alter toxicity profiles of LC-PCBs in a receptor-specific manner. In summary, anti-androgenic and estrogenic activities, acute inhibition of GJIC and suppression of the AhR-mediated activity were found to be the most relevant modes of action of airborne LC-PCBs, although they partially affected also additional cellular targets.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Disruptores Endocrinos/toxicidad , Bifenilos Policlorados/toxicidad , Línea Celular , Receptor de Androstano Constitutivo , Disruptores Endocrinos/metabolismo , Células Epiteliales/efectos de los fármacos , Humanos , Hidroxilación , Neoplasias/metabolismo , Bifenilos Policlorados/metabolismo , Receptor X de Pregnano , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Androgen receptor plays multiple physiological and pathological roles in human organism. In the current paper, we describe construction and characterization of a novel stably transfected human reporter cell line AIZ-AR for assessment of transcriptional activity of human androgen receptor. Cell line AIZ-AR is derived from human prostate carcinoma epithelial cell line 22Rv1 that was transfected with reporter plasmid containing 3 copies of androgen response regions (ARRs) followed by a single copy of androgen response element (ARE) from the promoter region of human prostate specific antigen (PSA) gene. AIZ-AR cells remained fully functional for more than 60 days and over 25 passages in the culture and even after cryopreservation. Time-course analyses showed that AIZ-AR cells allow detection of AR ligands as soon as after 8 hours of the treatment. We performed dose-response analyses with 23 steroids in 96-well plate format. We observed activation of AR by androgens, but not by estrogens and mineralocorticoids. Some glucocorticoids and progesterone also induced luciferase, but their potencies were 2-3 orders of magnitude weaker as compared to androgens. Taken together, we have developed a rapid, sensitive, selective, high-throughput and reproducible tool for detection of human AR ligands, with potential use in pharmacological and environmental applications.
Asunto(s)
Genes Reporteros , Receptores Androgénicos/genética , Transcripción Genética , Transfección , Antagonistas de Receptores Androgénicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cinamatos/farmacología , Células Clonales , Criopreservación , Dihidrotestosterona/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Higromicina B/análogos & derivados , Higromicina B/farmacología , Luciferasas/metabolismo , Receptores Androgénicos/metabolismo , Esteroides/farmacología , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (-)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (-)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC50 1.69 µM, Ki 0.92 µM for testosterone, IC50 1.46 µM, Ki 2.52 µM for midazolam; (-)-ketoconazole IC50 0.90 µM, Ki 0.17 µM for testosterone, IC50 1.04 µM, Ki 1.51 µM for midazolam).
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Hepatocitos/enzimología , Cetoconazol/química , Cetoconazol/farmacología , Biocatálisis/efectos de los fármacos , Citocromo P-450 CYP3A/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Células Hep G2 , Hepatocitos/efectos de los fármacos , Humanos , Cinética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Receptor X de Pregnano , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Estereoisomerismo , Transcripción Genética/efectos de los fármacosRESUMEN
Proton pump inhibitors omeprazole and lansoprazole contain chiral sulfur atom and they are administered as a racemate, i.e. equimolar mixture of S- and R-enantiomers. The enantiopure drugs esomeprazole and dexlansoprazole have been developed and introduced to clinical practice due to their improved clinical and therapeutic properties. Since omeprazole and lansoprazole are activators of aryl hydrocarbon receptor (AhR) and inducers of CYP1A genes, we examined their enantiospecific effects on AhR-CYP1A pathway in human cancer cells and primary human hepatocytes. We performed gene reporter assays for transcriptional activity of AhR, RT-PCR analyses for CYP1A1/2 mRNAs, western blots for CYP1A1/2 proteins and EROD assay for CYP1A1/2 catalytic activity. Lansoprazole and omeprazole enantiomers displayed differential effects on AhR-CYP1A1/2 pathway. In general, S-enantiomers were stronger activators of AhR and inducers of CYP1A genes as compared to R-enantiomers in lower concentrations, i.e. 1-10 µM for lansoprazole and 10-100 µM for omeprazole. In contrast, R-enantiomers were stronger AhR activators and CYP1A inducers than S-enantiomers in higher concentrations, i.e. 100 µM for lansoprazole and 250 µM for omeprazole. In conclusion, we provide the first evidence of enantiospecific effects of omeprazole and lansoprazole on AhR signaling pathway.
Asunto(s)
Hepatocitos/efectos de los fármacos , Lansoprazol/farmacología , Omeprazol/farmacología , Inhibidores de la Bomba de Protones/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Línea Celular , Hepatocitos/metabolismo , Humanos , Lansoprazol/química , Omeprazol/química , Inhibidores de la Bomba de Protones/química , EstereoisomerismoRESUMEN
In the past decade, a large number of enantiopure drugs were introduced to clinical practice, since improved therapeutic effects were demonstrated for one of the enantiomers from originally racemic drug. While the therapeutic effects and safety of enantiopure drugs were tested prior to their approval, various biological enantiospecific activities of these, often "old" drugs, remain to be elucidated. In the current paper, we examined enantiospecific effects of clinically used enantiopure drugs containing one chiral center in the structure (i.e. zopiclone, tamsulosin, tolterodine, modafinil, citalopram) towards aryl hydrocarbon (AhR), glucocorticoid (GR) and pregnane X (PXR) receptors in human reporter cell lines. The cytotoxicity (IC50), agonist (EC50) and antagonist effects (IC50) of R-form, S-form and racemic mixture for each tested drugs were determined and compared in AhR-, GR- and PXR-gene reporter cell lines. Since AhR, GR and PXR are key regulators of drug metabolism, energy metabolism, immunity and play many other physiological functions, the data presented here might be of toxicological significance.
Asunto(s)
Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/farmacología , Compuestos de Bencidrilo/química , Compuestos de Bencidrilo/farmacología , Línea Celular , Línea Celular Tumoral , Citalopram/química , Citalopram/farmacología , Cresoles/química , Cresoles/farmacología , Genes Reporteros/genética , Células Hep G2 , Humanos , Modafinilo , Fenilpropanolamina/química , Fenilpropanolamina/farmacología , Piperazinas/química , Piperazinas/farmacología , Receptor X de Pregnano , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inhibidores , Estereoisomerismo , Sulfonamidas/química , Sulfonamidas/farmacología , Tamsulosina , Tartrato de TolterodinaRESUMEN
Azole antifungal ketoconazole (KET) was demonstrated to activate aryl hydrocarbon receptor (AhR). Since clinically used KET is a racemic mixture of two cis-enantiomers (2R,4S)-(+)-KET and (2S,4R)-(-)-KET, we examined the effects of KET enantiomers on AhR signaling pathway. (+)-KET dose-dependently activated AhR in human gene reporter cell line AZ-AHR, and displayed 5-20× higher agonist activity (efficacy), as compared to (-)-KET; both enantiomers were AhR antagonists with equal potency (IC50). Consistently, (+)-KET strongly induced CYP1A1 mRNA and protein in human HepG2 cells, while (-)-KET exerted less than 10% of (+)-KET activity. In primary human hepatocytes, both enantiomers preferentially induced CYP1A2 over CYP1A1 mRNA and protein, and the potency of (+)-KET was slightly higher as compared to (-)-KET. Ligand binding assay with guinea pig liver cytosols revealed that both (+)-KET and (-)-KET are weak ligands of AhR that displaced [3H]-TCDD with comparable potency. Similarly, both enantiomers weakly transformed AhR to DNA-binding form with similar potency, as showed by EMSA, in guinea pig liver cytosolic extracts and nuclear extracts from mouse Hepa-1 cells. We also examined effects of KET on glucocorticoid receptor (GR), a regulator of AhR activity. Both KET enantiomers antagonized GR with similar potency, as revealed by gene reporter assay in AZ-GR cell line and down-regulation of tyrosine aminotransferase mRNA in human hepatocytes. Finally, we demonstrate enantiospecific antifungal activities of KET enantiomers in six Candida spp. strains. In conclusion, the significance of current study is providing the first evidence of enatiospecific effects of cis-enantiomers of ketoconazole on AhR-CYP1A pathway.