Profiling of enantiopure drugs towards aryl hydrocarbon (AhR), glucocorticoid (GR) and pregnane X (PXR) receptors in human reporter cell lines.

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.

Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1.

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.

Pelargonidin activates the AhR and induces CYP1A1 in primary human hepatocytes and human cancer cell lines HepG2 and LS174T.

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.

Effects of flavored mineral waters on AhR-CYP1A1 signaling pathway in primary human hepatocytes and in human hepatic and intestinal cancer cells.

Food-drug interaction is an emerging phenomenon, comprising pharmacokinetic or toxicokinetic interactions between food constituents and drugs. The mechanisms include inhibition of enzymes and transporters, and induction of drug metabolizing enzymes. A prominent regulator of drug-metabolizing enzymes is an aryl hydrocarbon receptor (AhR) that transcriptionally regulates CYP1 enzymes, phase II enzymes and many other genes. In the current paper, we have examined the effects of 28 different flavored mineral waters on AhR-CYP1A1 signaling pathway in primary cultures of human hepatocytes and in human cancer cell lines HepG2 (hepatic) and LS174T (intestinal). The techniques of Western blot, RT-PCR and gene reporter assays were employed to determine the expression of CYP1A1 mRNA, protein and activation of AhR, respectively. We have identified four flavored mineral waters which activated AhR and/or induced CYP1A1. These data imply a potential of some flavored mineral waters to cause food-drug interactions. In addition, activation of AhR-CYP1A1 signaling may result in chemically-induced carcinogenesis and alteration of intermediary metabolism.