Jasmone Is a Ligand-Selective Allosteric Antagonist of Aryl Hydrocarbon Receptor (AhR).

Herbal extracts represent a wide spectrum of biologically active ingredients with potential medical applications. By screening minor constituents of jasmine essential oil towards aryl hydrocarbon receptor (AhR) activity using a gene reporter assay (GRA), we found the antagonist effects of jasmone (3-methyl-2-[(2Z)-pent-2-en-1-yl]cyclopent-2-en-1-one). It inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-, benzo[a]pyrene (BaP)-, and 6-formylindolo[3,2-b]carbazole (FICZ)-triggered AhR-dependent luciferase activity in a concentration-dependent manner. However, the inhibition differed markedly between TCDD, BaP, and FICZ, with the latter being significantly less inhibited. The dose-response analysis confirmed an allosteric type of AhR antagonism. Furthermore, jasmone efficiently inhibited AhR activation by AhR agonists and microbial catabolites of tryptophan (MICTs). TCDD- and FICZ-inducible CYP1A1 expression in primary human hepatocytes was inhibited by jasmone, whereas in the human HepG2 and LS180 cells, jasmone antagonized only TCDD-activated AhR. Jasmone only partially displaced radiolabeled TCDD from its binding to mouse Ahr, suggesting it is not a typical orthosteric ligand of AhR. TCDD-elicited AhR nuclear translocation was not affected by jasmone, whereas downstream signaling events, including the formation of the AhR:ARNT complex and enrichment of the CYP1A1 promoter, were inhibited by jasmone. In conclusion, we show that jasmone is a potent allosteric antagonist of AhR. Such discovery may help to find and/or clarify the use of jasmone in pharmaco- and phytotherapy for conditions where AhR plays a key role.

Gene Expression Profiling of 1α,25(OH)2 D3 Treatment in 2D/3D Human Hepatocyte Models Reveals CYP3A4 Induction but Minor Changes in Other Xenobiotic-Metabolizing Genes.

SCOPE: CYP3A4 is the most important drug-metabolizing enzyme regulated via the vitamin D receptor (VDR) in the intestine. However, less is known about VDR in the regulation of CYP3A4 and other drug-metabolizing enzymes in the liver. METHODS AND RESULTS: This study investigates whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) regulates major cytochrome P450 enzymes, selected phase I and II enzymes, and transporters involved in xenobiotic and steroidal endobiotic metabolism in 2D and 3D cultures of human hepatocytes. The authors found that 1α,25(OH)2 D3 increases hepatic CYP3A4 expression and midazolam 1'-hydroxylation activity in 2D hepatocytes. The results are confirmed in 3D spheroids, where 1α,25(OH)2 D3 has comparable effect on CYP3A4 mRNA expression as 1α-hydroxyvitamin D3 , an active vitamin D metabolite. Other regulated genes such as CYP1A2, AKR1C4, SLC10A1, and SLCO4A1 display only mild changes in mRNA levels after 1α,25(OH)2 D3 treatment in 2D hepatocytes. Expression of other cytochrome P450, phase I and phase II enzyme, or transporter genes are not significantly influenced by 1α,25(OH)2 D3 . Additionally, the effect of VDR activation on CYP3A4 mRNA expression is abolished by natural dietary compound sulforaphane, a common suppressor of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). CONCLUSION: This study proposes that VDR or vitamin D supplementation is unlikely to significantly influence liver detoxification enzymes apart from CYP3A4.

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect.

BACKGROUND: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. METHODS: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. RESULTS: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. CONCLUSION: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms.

Essential oils of culinary herbs and spices activate PXR and induce CYP3A4 in human intestinal and hepatic in vitro models.

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.

Assessment of endocrine disruption potential of essential oils of culinary herbs and spices involving glucocorticoid, androgen and vitamin D receptors.

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.

Essential oils of culinary herbs and spices display agonist and antagonist activities at human aryl hydrocarbon receptor AhR.

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.

Structural changes in amber due to uranium mineralization.

The presence of uranium, with a bulk mass fraction of about 1.5 wt% and radiolytic alterations are a feature of Cenomanian amber from Krizany, at the northeastern edge of the North Bohemian Cretaceous uranium ore district. Pores and microcracks in the amber were filled with a mineral admixture, mainly in the form of Zr-Y-REE enriched uraninite. As a result of radiolytic alterations due to the presence of uranium, structural changes were observed in the Krizany amber in comparison with a reference amber from Nové Strasecí in central Bohemia; this was of similar age and botanical origin but did not contain elevated levels of uranium. Structural changes involved an increase in aromaticity due to dehydroaromatization of aliphatic cyclic hydrocarbons, loss of oxygen functional groups, an increase in the degree of polymerization, crosslinking of CC bonds, formation of a three-dimensional hydrocarbon network in the bulk organic matrix, and carbonization of the organic matrix around the uraninite infill.

Opportunities and challenges in using human hepatocytes in cytochromes P450 induction assays.

INTRODUCTION: Identification of inducers of xenobiotic-metabolizing cytochromes P450 (CYP) is of topical interest. The issue mainly concerns three sectors: (i) preclinical testing of drug candidates and testing existing drugs and their combinations; (ii) food safety applications with regard to additives, contaminants, and adulterants; (iii) environmental applications, comprising detection and identification of endocrine disruptors. AREAS COVERED: A literature search was performed using the PubMed database, covering state-of-the-art of human hepatocyte (HH) culture use, and their exploitation for the identification of P450 inducers. A list of CYP inducers identified by HHs is provided. EXPERT OPINION: Primary cultures of HHs had long been considered as a gold standard for induction assays of xenobiotic-metabolizing enzymes. Owing to several shortcomings of HHs, alternative approaches such as immortalization of HHs, use of cell lines, generation of clonal cell lines from HHs, use of induced pluripotent stem (iPS) cells, cells from humanized animals, etc., were employed. While yielding particular advantage, overall, alternatives to HHs still remain an avenue for discrete applications or technical situations. Thus, HHs remain the most suitable model for complex CYP induction studies. The summary may be effectively expressed by strength/weakness/opportunity/threats analysis.

Effects of anthocyans on the expression of organic anion transporting polypeptides (SLCOs/OATPs) in primary human hepatocytes.

Anthocyans (anthocyanins and their aglycones anthocyanidins) are colorful pigments, naturally occurring in fruits. They exhibit many biological effects and have potent health benefits. Anthocyans are widely used as dietary supplements and the safety of products containing them is of great importance. To investigate whether anthocyans influence the expression of hepatic uptake transporters from the organic anion transporting polypeptide (SLCO gene/OATP protein) family, we carried out studies on primary cultures of human hepatocytes. The hepato-cellular accumulation of widely used drugs such as statins and some anticancer drugs is mediated by the liver-specific OATP1B1 and OATP1B3, thus any interference with expression of these particular transporters might influence therapeutic outcomes. We evaluated the effects of 21 anthocyanins and their corresponding 6 anthocyanidins on the expression levels of SLCO1B1/SLCO1B3 by RT-qPCR. Changes in OATP protein levels were confirmed by western blotting. Our data show that OATP1B1 responds differently to anthocyans compared with OATP1B3. We observed the induction of SLCO1B1 gene and OATP1B1 protein in four hepatocyte samples by the anthocyanins malvin chloride, malvidin-3-O-galactoside chloride and cyanidin-3-O-sophoroside chloride. For SLCO1B3, a reduction in the expression levels was seen with delphin chloride and the anthocyanidin pelargonidin. Although the values varied considerably between primary hepatocyte isolates from different individuals, a mean induction of SLCO1B1 (up to 60%) and reduction of SLCO1B3 (by less than 25%) were detected. We propose that the effects of anthocyans derived from high dose dietary supplements may have to be taken into account in patients undergoing a therapy with drugs transported by OATP1B1 and OATP1B3.

Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes.

Anthocyanidins and anthocyanins are pharmacologically active constituents of various berry fruits, such as blueberry and cranberry. These compounds are also contained in massively used nutritional supplements based on extracts or dry matter from berry fruits. The current study evaluated the effects of anthocyanidins and anthocyanins on the expression and catalytic activity of major drug-metabolizing enzymes CYP2C9, CYP2A6, CYP2B6, and CYP3A4 in primary cultures of human hepatocytes and human liver microsomes. Expression of mRNA was quantified by qRT-PCR. Expression of proteins was evaluated by Western blotting and immunochemiluminescence. The catalytic activity of CYP enzymes was measured by HPLC using specific enzyme substrates. Tested anthocyanidins (6) and anthocyanins (21) did not induce the expression of mRNA and protein of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 genes in human hepatocytes. Catalytic activities of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 enzymes were inhibited by all anthocyanidins to different extents (e.g., delphinidin inhibits CYP3A4 by >90% at 100 µM with IC50 = 32 µM). Of 21 anthocyanins tested, only cyanidin-3-O-rhamnoside (CYP3A4 by >75% at 100 µM with IC50 = 44 µM) and two glycosides of delphinidin significantly inhibited examined cytochromes P450. It may be concluded that in the ranges of common ingestion of either food or dietary supplement an induction or significant inhibition of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 activity is most probably not expected.

Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines.

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.

Dexamethasone controls aryl hydrocarbon receptor (AhR)-mediated CYP1A1 and CYP1A2 expression and activity in primary cultures of human hepatocytes.

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.

Primary cultures of human hepatocytes as a tool in cytotoxicity studies: cell protection against model toxins by flavonolignans obtained from Silybum marianum.

The aim of this study was to evaluate the cytoprotective effects upon primary human hepatocytes of silymarin extract and its main flavonolignans following exposure to the cytotoxic actions of model toxins. The conditions for the hepatocyte intoxication were optimised for allyl alcohol, carbon tetrachloride, D-galactosamine and paracetamol. Silymarin extract, silychristin and silydianin did not exert cytotoxicity (10-100 microM), whereas silybin and isosilybin at higher concentrations and after longer incubation periods were cytotoxic. All main flavonolignans of silymarin tested displayed concentration-dependent cytoprotection against the toxic effects of both allyl alcohol and carbon tetrachloride but neither paracetamol nor galactosamine. The best protection was achieved by silydianin and silychristin and to a lesser degree by silymarin, while silybin and isosilybin were less effective. It is concluded that these differing outcomes result from the varying abilities of the Silybum marianum substances tested to stabilize the cell membrane, exert antioxidant properties and exhibit intrinsic toxicity.

Effect of silybin and its congeners on human liver microsomal cytochrome P450 activities.

Silybin and related flavonolignans form a major part of the Silybum marianum extract, silymarin, which has been used to treat liver diseases for hundreds of years. Although regarded as safe, many of the extract constituents remain thus far untested for their possible effects on liver biotransformation enzymes. Cytochromes P450 (CYP) are very important in this regard. We tested the effect of four flavonolignans: silybin, its hemisynthetic derivative dehydrosilybin, silydianin, and silycristin on three specific CYP activities: bufuralol 1'-hydroxylation (CYP2D6), p-nitrophenol hydroxylation (CYP2E1), and nifedipine oxidation (CYP3A4). All flavonolignans displayed dose-dependent inhibition of these activities with IC(50) values in the micromolar range. The inhibition was competitive or mixed as revealed by double reciprocal plots of kinetic experiments. However, the inhibition is not considered to be relevant for therapy because physiological concentrations of the individual flavonolignans do not exceed 0.5 microM. The data support the use of the extract as a dietary supplement.