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Resveratrol (RSV) is a stilbene phytochemical common in food and red wine. RSV inhibits cytochrome P450 CYP3A4 activity and interacts with the pregnane X receptor (PXR), the central regulator of drug/xenobiotic metabolizing enzyme expression. In this work, we comprehensively examined the effects of 13 stilbenes (trans- and cis-resveratrol, trans- and cis-piceatannol, oxyresveratrol, pterostilbene, pinostilbene, a,b-dihydroresveratrol, trans- and cis-trismethoxyresveratrol, trans-3,4,5,4'-tetramethoxystilbene, trans-2,4,3',5'-tetramethoxystilbene, trans-4-methoxystilbene), on CYP3A4 and CYP2B6 mRNA induction, and on CYP3A4/5, CYP2C8/9/19, CYP2D6, CYP2A6, CYP2E1, CYP1A2 and CYP2B6 cytochrome P450 enzyme activities. Expression experiments in five different primary human hepatocyte preparations, reporter gene assays, and ligand binding assays with pregnane X (PXR) and constitutive androstane (CAR) receptors were performed. Inhibition of cytochrome P450 enzymes was examined in human microsomes. We found that only polymethoxylated stilbenes are prone to significantly induce CYP2B6 or CYP3A4 in primary human hepatocytes via pregnane X receptor (PXR) interaction. Natural resveratrol derivatives such as trans- and cis-RSV, oxyresveratrol, pinostilbene and pterostilbene significantly inhibit CYP3A4/5 enzymatic activities; however, only trans-RSV significantly inhibits CYP3A4/5 activity (both testosterone 6β-hydroxylation and midazolam 1´-hydroxylation) in micromolar concentrations by a non-competitive mechanism, suggesting a potential risk of food-drug interactions with CYP3A4/5 substrates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bisphenol S (BPS) is heat-stable structural analog of bisphenol A (BPA), a known endocrine disruptor. Due to the effort to replace BPA with BPS, it is essential to know if BPS is suitable non-toxic replacement without reported deleterious effects of BPA. Since most of the BPA effects are ascribed to its ability to activate nuclear receptors, we screened some prominent members of this family in order to confirm or refute some recent findings. We found that BPS insignificantly activated aryl hydrocarbon receptor (AhR) in reporter gene assay and no induction of AhR target gene CYP1A1 was observed in human hepatocytes (HH). BPS was able to act like an antagonist of pregnane X receptor (PXR) in reporter gene assay, but the expression of PXR target gene CYP3A4, was only moderately affected in HH. While BPS antagonized dexamethasone-inducible glucocorticoid receptor (GR)-dependent luciferase activity in reporter gene assay (IC 50 =52μM), it was not able to antagonize dexamethasone effects on GR-target genes, including GILZ, NFKBIA and IL-6. Synergistic effect of BPS (range 0.001-100μM) and DHT (100nM) was observed at androgen receptor (AR) activity level only. In conclusion, we show that BPS had only limited effect on tested nuclear receptors. Moreover, submicromolar concentrations of BPS affected activated AR. Thus, due to the low levels of exposure for humans, BPS is probably of no regulatory concern. However, further investigation should delineate possible impact on male/female development or sexual functions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aryl hydrocarbon receptor (AhR) is a transcription factor, the activity of which is modulated by hormones including glucocorticoids and estrogens. In this study, we examined the effects of triiodothyronine (T3), a ligand and activator of thyroid hormone receptor (TR), on transcriptional activity of AhR and the expression of its target gene CYP1A1. Study was carried out in human hepatocellular carcinoma cells HepG2 and primary cultures of human hepatocytes (HH). Gene reporter assay in stably transfected AZ-AhR cells revealed that T3 dose-dependently augmented 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AhR-dependent luciferase activity. In contrast, T3 had no effect on TCDD-inducible expression of CYP1A1 mRNA, protein and catalytic activity. Incubation of human hepatocytes with T3 had modulatory and inter-individual (7 cell cultures from 7 different liver donors) effects on both basal and dioxin-inducible CYP1A1/2. Since there was no correlation between T3 effects on CYP1A expression and T3-dependent expression of Spot14 mRNA, the involvement of additional factors besides TR is supposed. Overall, the co-incubation of normal and cancer human hepatic cells with TCDD and T3 suggested transcriptional cross-talk between AhR and TR, which may have physiological and toxicological implications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Triazole antimycotic itraconazole contains in its structure three chiral centres; therefore, it forms eight stereoisomers. Commercial preparations of itraconazole are a mixture of four cis-diastereoisomers. There is much evidence that efficacy, adverse effects, and toxicity of chiral drugs may be stereospecific. Therefore, we have prepared 4 pure cis-diastereoisomers of itraconazole and investigated their effects on transcriptional activities of xenoreceptors aryl hydrocarbon receptor AhR and pregnane X receptor PXR. Gene reporter assays showed that itraconazole dose-dependently activated both AhR and PXR, and the activation of AhR but not of PXR was enantiospecific. Itraconazole diastereoisomers transformed AhR and PXR into their DNA-binding forms, as demonstrated by electromobility shift assays. Cytochrome P450 CYP1A1 mRNA and protein were induced by itraconazole diastereoisomers in human hepatoma cells HepG2, human skin cells HaCaT, and in primary human hepatocytes. The expression of CYP3A4 in human intestinal LS180 cells was not influenced by itraconazole, but we observed downregulation of CYP3A4 in human hepatocytes. Collectively, we show that itraconazole is a dual activator of AhR and PXR, with differential effects on the target genes for xenoreceptors. The enantiospecific pattern was observed only in gene reporter assays for AhR. The data presented here might be of toxicological and clinical importance., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The development of biologically active molecules based on molecular recognition is an attractive and challenging task in medicinal chemistry and the molecules that can activate/deactivate certain receptors are of great medical interest. In this contribution, selected pyrimidine/piperidine derivatives were synthesized and tested for the ability to activate/deactivate Aryl hydrocarbon receptor (AhR) and Glucocorticoid receptor (GR). Tested compounds are shown to activate the receptors but to much lesser extent than positive controls, dioxin and dexamethasone for Ahr and GR, respectively. However, some of them antagonized the positive controls action. Although further in vivo studies are needed to fully characterize the bioactivities of these compounds, the reported in vitro evidences demonstrate that they might be used as the modulators of AhR and GR activities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Environmental pollutants parathion, bisphenol A and paraquat were not systematically studied towards the effects on the expression of phase I xenobiotics-metabolizing cytochromes P450 (CYPs). We monitored their effects on the expression of selected CYPs in primary cultures of human hepatocytes. Moreover, we investigated their effects on the receptors regulating these CYPs, particularly arylhydrocarbon receptor (AhR), pregnane X receptor (PXR) and glucocorticoid receptor (GR) by gene reporter assays. We found that parathion and bisphenol A are the activators of AhR. Moreover, they are the inducers of CYP1A1 mRNA in hepatoma cells HepG2 as well as in human hepatocytes by AhR-dependent mechanism via formation of AhR-DNA-binding complex, as revealed by gel shift assay. All three compounds possessed anti-glucocorticoid action as revealed by GR-dependent gene reporter assay and a decline in tyrosine aminotransferase (TAT) gene expression in human hepatocytes. Moreover, parathion and bisphenol A are the activators of PXR and inducers of CYP3A4 mRNA and protein in the primary cultures of human hepatocytes. In conclusion, the studied compounds displayed distinct activities towards nuclear receptors involved in many biological processes and these findings may help us to better understand their adverse actions in pathological states followed after their exposure., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The JEG-3 choriocarcinoma cell line has been proposed as a model cell line of human placental trophoblast for induction studies via aryl hydrocarbon receptor (AHR). We examined whether glucocorticoid dexamethasone influences AHR-mediated induction of CYP1A1 enzyme in the JEG-3 cell line. We found that dexamethasone dose- and time-dependently suppresses CYP1A1 transactivation in gene reporter assays, CYP1A1 mRNA induction, and upregulation of 7-ethoxyresorufin-O-deethylase (EROD) activity by 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in JEG-3 cells. Co-transfection of JEG-3 cells with glucocorticoid receptor (GR) expression construct and treatment with dexamethasone abolished the effect of MC on CYP1A1 promoter construct in transient transfection gene reporter assays. RU486, a GR antagonist, suppressed the effect of dexamethasone on MC-induced transactivation of AHR responsive reporter constructs. We also found that dexamethasone stimulates both ligand-dependent and ligand-independent degradation of AHR but not of aryl hydrocarbon receptor nuclear translocator (ARNT) protein in JEG-3 cells. In experiments with proteasome inhibitors MG132 and bortezomib, we found that the degradation is not sensitive to proteasome inhibition in JEG-3. We can conclude that dexamethasone suppresses AHR-mediated CYP1A1 induction in JEG-3 cells through the unique mechanism of AHR-GR crosstalk, which involves accelerated degradation of AHR., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food-drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)-cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 μM concentration. PEL-2 and CYA-3 at 100 μM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR-CYP1A1 signaling, implying zero potential of these compounds for food-drug interactions with respect to AhR-CYP1A1 pathway., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The pharmacological dosage of dehydroepiandrosterone (DHEA) protects against chemically induced carcinogenesis. The chemoprotective activity of DHEA is attributed to its inhibitory potential for the expression of CYP1A enzymes, which are highly responsible for metabolic activation of several mutagenic and carcinogenic chemicals. The present work investigated whether the chemoprevention by DHEA was due to diminished transcriptional activation of CYP1A genes or to the post-transcriptional modulation of CYP1A expression. In primary human hepatocytes, DHEA diminished the increase in CYP1A activities (7-ethoxyresorufin O-dealkylation and phenacetin O-dealkylation) and in CYP1A2 mRNA level induced by 3-methylcholanthrene, but did not alter the amount of CYP1A1 and CYP1B1 mRNA. The androgen receptor seemed to be involved in DHEA-mediated diminishment of CYP1A2 induction, which was attenuated in the presence of bicalutamide, the androgen receptor antagonist. The potential role of the glucocorticoid receptor and estrogen receptor in DHEA-mediated decrease in CYP1A2 induction was excluded. The developed computational model of CYP1A2 induction kinetics and CYP1A2 mRNA degradation proposed that a post-transcriptional mechanism was likely to be the primary mechanism of the DHEA-mediated diminishment of CYP1A2 induction. The hypothesis was confirmed by the results of actinomycin D-chase experiments in MCF-7 and LNCaP cells, displaying that the degradation rates of CYP1A2 mRNA were significantly higher in the cells exposed to DHEA. The novel findings on DHEA-mediated modulation of CYP1A2 mRNA stability may account for the beneficial effects of DHEA by decreasing the metabolic activation of pro-carcinogenic compounds., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

We examined the effects of anthocyanidins (cyanidin, delphinidin, malvidin, peonidin, petunidin, pelargonidin) on the aryl hydrocarbon receptor (AhR)-CYP1A1 signaling pathway in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cells. AhR-dependent reporter gene expression in transfected HepG2 cells was increased by pelargonidin in a concentration-dependent manner at 24h. Similarly, pelargonidin induced the expression of CYP1A1 mRNA up to 5-fold in HepG2 and LS174T cells relative to the induction by 5 nM 2,3,7,8-tetrachlorodibenzodioxin (TCDD), the most potent activator of AhR. CYP1A1 and CYP1A2 mRNAs were also increased by pelargonidin in three primary human hepatocytes cultures (approximately 5% of TCDD potency) and the increase in CYP1A1 protein in HepG2 and LS174T cells was comparable to the increase in catalytic activity of CYP1A1 enzyme. Ligand binding analysis demonstrated that pelargonidin was a weak ligand of AhR. Enzyme kinetic analyses using human liver microsomes revealed inhibition of CYP1A1 activity by delphinidin (IC50 78 μM) and pelargonidin (IC50 33 μM). Overall, although most anthocyanidins had no effects on AhR-CYP1A1 signaling, pelargonidin can bind to and activate the AhR and AhR-dependent gene expression, and pelargonidin and delphinidin inhibit the CYP1A1 catalytic activity., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Azoles antifungals are widespread used drugs for various medicinal indications. These drugs are well known for their numerous drug-drug interactions, which are believed to occur via inhibition of CYP3A4 enzymatic activity and consequently altering pharmacokinetic of co-administered drugs. In the current communication a complex view on the molecular interactions between azoles antimycotics and CYP3A4 is presented. Beside inhibition of CYP3A4 catalytic activity, azoles influence transcriptional activity of pregnane X receptor (PXR) and consequently expression of drug-metabolizing enzymes, including CYP3A4. Interactions between azoles and PXR occur by multiple mechanisms, including modulation of ligand-dependent activation of PXR (agonism, antagonism) or affecting recruitment of PXR co-activators SRC-1 (steroid receptor co-activator 1) and HNF4α (hepatocyte nuclear factor 4α). Miconazole and ketoconazole are antagonists of glucocorticoid receptor (GR), therefore these drugs inhibit GR-mediated expression of PXR and drug metabolizing cytochromes P450. In addition, PXR and GR are key regulators of intermediary metabolism (e.g. carbohydrate, lipids or bile acids homeostasis) and many other cellular functions (e.g. immune response), hence, the interactions between azoles and PXR/GR are of broader physiological importance. In conclusion, while inhibition of CYP3A4 enzymatic activity by azoles is considered as primary cause of azoles drug-drug interactions, the effects of azoles on PXR and GR should be taken in account. Apart from CYP3A4, azoles influence the expression and activity of others drug-metabolizing cytochromes P450., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

Valproic acid (VPA) is a wide spread anticonvulsant and mood-stabilizing agent, the use of which is associated with hepatotoxicity, bone marrow suppression and osteomalacia. In the current paper we propose a possible mechanism of VPA-induced osteomalacia involving accelerated catabolism of 1α,25(OH)(2)-vitamin D3 (VD3) due to increased expression of CYP24. We demonstrate that VPA strongly potentiates CYP24 mRNA expression by VD3 in human hepatocytes (HH) and in human embryonic kidney cells (HEK293). By the method of gene reporter assay we found that VPA increases basal and VD3-inducible activity of CYP24 promoter (pCYP24-luc) in human liver adenocarcinoma (HepG2) and in HEK293 cells in dose-dependent manner. In order to delineate the role of inhibitory effects of VPA on histone deacetylase 1 (HDAC1), we compared the effects of VPA with trichostatin A (TSA) on basal and inducible levels of CYP24 mRNA and pCYP24-luc transactivation. Transactivation of CYP24 promoter by VD3 was enhanced in the presence of both TSA and VPA. In contrast, VD3-inducible expression of CYP24 mRNA was enhanced by VPA but not by TSA, implying that HDAC1 inhibition is not the major reason for VPA effects on CYP24. We examined the effects of VPA on mitogen-activated protein kinases as the important transcriptional regulators of VDR. VPA activated extracellular signal-regulated kinase (ERK) but not c-Jun-N-terminal kinase (JNK) and p38 MAPKs. In conclusion, VPA enhances transcriptional activity of VDR and increases expression of CYP24 mRNA in the presence of VD3 in physiological concentrations. The mechanism involves activation of ERK and partly the inhibition of HDAC1., (© 2010 Elsevier Ireland Ltd. All rights reserved.)

Benzodiazepines have wide-spread used in pharmacotherapy for their anxiolytic, myorelaxant, hypnotic, amnesic and anticonvulsive properties. Despite benzodiazepines are used in clinics over 50 years, they have not been surprisingly tested for capability to induce major drug-metabolizing cytochromes P450. In the current study, we have examined the potency of Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Medazepam, Midazolam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam to induce CYP1A2 and CYP3A4 in primary cultures of human hepatocytes. Benzodiazepines were tested in therapeutic concentrations and in concentrations corresponding to their plasma levels in intoxicated patients. We found weak but significant induction of CYP3A4 mRNA by Midazolam and Medazepam, while other benzodiazepines did not induce CYP3A4 expression. None of the tested compounds induced CYP1A2 mRNA in three independent human hepatocytes cultures. In addition, employing gene reporter assays with transiently transfected hepatocarcinoma cells, we found that tested benzodiazepines did not activate aryl hydrocarbon receptor (AhR), whereas Midazolam and Medazepam slightly activated pregnane X receptor (PXR). Consistently, two-hybrid mammalian assay using hybrid fusion plasmids GAL4-PXR ligand-binding domain (LBD) and VP16-SRC-1-receptor-interacting domain (RID) confirmed PXR activation by Midazolam and Medazepam. In conclusion, Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam can be considered as safe drugs in term of their inability to induce PXR- and AhR-dependent cytochrome P450 enzymes CYP1A2 and CYP3A4. Medazepam and Midazolam slightly activated pregnane X receptor and displayed weak potency to induce CYP3A4 mRNA in human hepatocytes., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

A hypnotic drug Zolpidem is used in clinical practice for more than 25 years. Surprisingly, the effects of Zolpidem on the expression of drug-metabolizing cytochromes P450 (CYPs) were not examined yet. Recently, the unexpected capacity of several "old drugs", such as valproic acid or azoles, to induce CYPs was reported. Therefore, we tested whether Zolpidem induces the expression of important CYPs in primary cultures of human hepatocytes. Cells were treated for 24h with Zolpidem in therapeutic (0.1mg/L) and toxic (1mg/L) concentrations. The levels of CYP1A1, CYP1A2, CY2C9 and CYP3A4 mRNAs were not altered by Zolpidem, whereas model inducers dioxin and rifampicin significantly induced CYP1A and CYP2/3 gene expression, respectively. Consistently, Zolpidem did not activate aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), the key regulators of cytochromes P450s, as revealed by transient transfection gene reporter assays using HepG2 cells. We conclude Zolpidem be considered a safe drug with respect to the possible interactions through AhR- and PXR-dependent induction of drug-metabolizing CYPs.

CYP1A1 and CYP1A2 genes encode members of the cytochrome P450 superfamily of enzymes primarily involved in xenobiotic and drug metabolism. In this paper we examined the effects of synthetic glucocorticoid dexamethasone (DEX) on aryl hydrocarbon receptor (AhR)-mediated regulation of CYP1A1 and CYP1A2 genes and their enzymatic activity in primary cultures of human hepatocytes obtained from 17 donors and prepared in 3 countries. Dexamethasone significantly reduced both basal and inducible CYP1A1/2 ethoxyresorufin-O-deethylase (EROD) activities by more than 75 and 50%, respectively. Glucocorticoid receptor (GR) antagonist RU486 abolished this effect suggesting the involvement of GR in the process. In contrast, dexamethasone significantly augmented transcriptional activation of CYP1A2 mRNA but not CYP1A1 gene by prototype AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC). Dexamethasone had no effect on basal and TCDD-inducible levels of CYP1As proteins; however, it reduced the levels of AhR and GRalpha mRNAs and AhR protein levels. In addition, using RT(2) Profiler PCR Array, we found the effect of dexamethasone on the expression of several co-activators of AhR and GR nuclear receptors in the primary human hepatocytes. We conclude that dexamethasone controls CYP1A1 and CYP1A2 expression and activity in human hepatocytes via multiple mechanisms, which remain to be elucidated.