Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

<div><p>Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1), which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE)-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones.</p> </div

Effect of visnagin and khellin exposure on CYP1A1 expression and activity in HepG2 cells.

<div><p>A) HepG2 cells were treated with 10 µM visnagin, 10 µM khellin, and/or DMSO for 4 h, 8 h, 16 h, and 24 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. * - value is significantly different from DMSO-treated cells (<i>p</i> < 0.05).</p> <p>B) HepG2 cells were pre-treated with 20 µM MNF for 1 h and then exposed to 10 µM visnagin or 10 µM khellin for additional 16 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. * - value is significantly different from DMSO-treated cells (<i>p</i> < 0.05). # - value is significantly reduced in comparison to cells treated with VIS and KHEL, respectively; (<i>p</i> < 0.05).</p> <p>C) HepG2 cells were treated with visnagin (VIS; 1 µM-20 µM), khellin (KHEL; 1 µM-20 µM), 1 µM 3MC, 5 nM TCDD, and/or vehicle (DMSO; 0.1% v/v) for 48 h. Thereafter, western blotting analyses for detection of CYP1A1 and actin were performed as described in Materials and Methods section. The representative western blot analysis of two independent experiments (passages) is presented.</p> <p>D) HepG2 cells were treated with visnagin (VIS; 1 µM-20 µM), khellin (KHEL; 1 µM-20 µM), 1 µM 3MC, 5 nM TCDD, and/or vehicle (DMSO; 0.1% v/v) for either 16 h (upper panel) or 48 h (lower panel). EROD activity was determined as described in Materials and Method section. Analyses were performed in three independent experiments and are shown as fold induction over untreated cells. * - value is significantly different from DMSO-treated cells (<i>p</i> < 0.05).</p> <p>E) HepG2 cells were treated with TCDD (5 nM) for 48 h. Thereafter, substrate mixture was supplemented with increasing doses of visnagin (VIS 1 nM -20 µM) or khellin (KHEL; 1 nM -20 µM) and EROD activity was determined as described in Materials and Methods section. Data are mean from three independent experiments and are expressed as percentage (%) of TCDD-mediated induction (i.e. induction by TCDD = 100%). * - value is significantly different from TCDD-treated cells (<i>p</i> < 0.005).</p></div

Visnagin and khellin stimulate XRE-driven luciferase activity in AZ-AHR cells.

<p>Stably transfected AZ-AHR cells were treated for 24 h with visnagin (VIS; 0.001 µM-20 µM), khellin (KHEL, 0.001 µM-20 µM), 5 µM 3MC and/or vehicle (DMSO; 0.1% v/v). Analyses were performed in four independent experiments and are expressed as fold induction over untreated cells. * - Value is significantly different from DMSO-treated cells (<i>p</i> < 0.05).</p

Visnagin and khellin modulate the expression of several AHR target genes in HepG2 cells.

<p>HepG2 cells were pre-treated for 1 h with 20 µM MNF or 0.1% (v/v) DMSO and were subsequently exposed to 10 µM visnagin (VIS) or 10 µM khellin (KHEL). After 16 h, RNA was isolated and reverse transcribed and the expression of CYP1B1, AhRR, PAI-2 and VEGF was measured by qRT-PCR. The data are mean from three independent experiments and were normalized to beta-actin expression. * - value is significantly increased compared to DMSO-treated cells; § - value is significantly reduced in comparison to cells treated with VIS and KHEL, respectively; (<i>p</i> < 0.05).</p