Differential effects of omeprazole and lansoprazole enantiomers on aryl hydrocarbon receptor in human hepatocytes and cell lines.

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

Effect of omeprazole and lansoprazole enantiomers on transcriptional activity of aryl hydrocarbon receptor AhR in human gene reporter cell line AZ-AHR.

<p>The cells were seeded in 96-well plates and stabilized for 16 h. <b>Panels A and B:</b> Cells were incubated for 24 h with S-OME, R-OME, rac-OME, S-LAN, R-LAN and rac-LAN at concentrations ranging from 10⁻¹⁰ M to 10⁻⁴ M. The vehicle was DMSO (0.1% v/v). After the treatment, MTT test was performed and absorbance was measured at 540 nm. Treatments were performed in triplicates. The data are the mean from experiments from six different passages of cells and are expressed as a percentage of viability of control cells. The values of IC₅₀ were calculated and are indicated in a figure. <b>Panels C - F:</b> AZ-AHR cells were incubated for 24 h with S-OME, R-OME, rac-OME, S-LAN, R-LAN and rac-LAN at concentrations ranging from 10⁻¹⁰ M to 10⁻⁴ M in the absence (Panels C and D - <i>agonist mode</i>) or in the presence (Panels E and F- <i>antagonist mode</i>) of TCDD (5 nM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates in five (<i>agonist mode</i>) or four (<i>antagonist mode</i>) independent cell passages. Representative gene reporter assays are showed. Data are expressed as a fold induction of luciferase activity over control cells (Panels C and D - <i>agonist mode</i>) or as a percentage of maximal induction attained by TCDD (Panels E and F- <i>antagonist mode</i>). The values of EC₅₀ and IC₅₀ were calculated and the average values are indicated in figures.</p

Effects of omeprazole and lansoprazole enantiomers on CYP1A1 mRNA, protein and catalytic activity in human cancer cell line HepG2.

<p>HepG2 cells were seeded in 6-well plates and stabilized for 16 h. All experiments were performed in three consecutive cell passages. Cells were incubated for 24 h (mRNA and EROD analysis) or 48 h (protein analysis) with TCDD (5 nM), vehicle (DMSO; 0.1% v/v), omeprazole (S-, R-, rac-; 10 µM, 100 µM, 250 µM) and lansoprazole (S-, R-, rac-; 1 µM, 10 µM, 100 µM). <b>Panel A:</b> Representative RT-PCR analyses of CYP1A1 mRNA are shown. The data are the mean ± SD from triplicate measurements and are expressed as a fold induction over vehicle-treated cells. The data were normalized to GAPDH mRNA levels. <b>Panel B:</b> Representative western blots of CYP1A1 protein are shown. Density of bands was quantified by densitometry and the values are indicated along with respective blots. <b>Panel C:</b> CYP1A1 catalytic activity (7-ethoxyresorufin-<i>O</i>-deethylase; EROD) was measured by spectrofluorometry with 530 nm excitation and 590 nm emission filters. Treatments were performed in triplicates. Average EROD data from three independent passages are showed. Data are expressed as a fold induction over vehicle-treated cells. An asterisk (*) indicates that the value is significantly different from the activity of vehicle-treated cells.</p

Effects of omeprazole and lansoprazole enantiomers on CYP1A1 mRNA, protein and EROD activity in primary human hepatocytes.

<p><b>Panel A</b> and <b>Panel B:</b> RT-PCR analyses of CYP1A1 and CYP1A2 mRNA and western blots of CYP1A1 and CYP1A2 from two different cultures (HH52 and Hep220770) are shown. Human hepatocytes were incubated for 24 h (mRNA analysis) or 48 (protein analysis) with S-OME, R-OME, rac-OME, S-LAN, R-LAN, rac-LAN, TCDD and vehicle (DMSO; 0.1% v/v). RT-PCR data are the mean ± SD from triplicate measurements and are expressed as fold induction over vehicle-treated cells. Data were normalized to GAPDH mRNA levels. Density of bands in western blots was quantified by densitometry and the values are indicated along with respective blots. <b>Panel C:</b> EROD and cytotoxicity: Human hepatocytes (culture Hep220774) were treated for 24 h with S-OME, R-OME, rac-OME, S-LAN, R-LAN, rac-LAN, TCDD and vehicle (DMSO; 0.1% v/v). Upper bar graph: An activity of 7-ethoxyresorufin- O-deethylase (EROD) was measured by fluorescent spectrophotometry with 530 nm excitation and 590 nm emission filters. Treatments were performed in triplicates. The data are expressed as fold induction over the value from control cells. Lower bar graph: A conventional MTT test was performed and absorbance was measured at 540 nm. Treatments were performed in triplicates. The data are expressed as percentage of viability of control cells. An asterisk (*) indicates that the value is significantly different from the activity of DMSO.</p