α naphthoflavone α nf

Fine Dust (PM₁₀-like) (ERM^®-EZ100) was purchased from the European Reference Materials (ERM) (St. Louis, MO, USA). Fine Dust particles are composed of the size range of 10 μm (x ≤10 μm). α-naphthoflavone (α-NF) which is known as an inhibitor of AhR and used for positive control, and oleanolic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

The human hepatoma cell line HepG2 (European Collection of Cell Cultures, ECACC No 85011430) was grown in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin, and 4 mM l-glutamine (cell culture media and supplementations were obtained from PAA Laboratories) maintained at 37 °C and 5% CO₂. Cells were seeded in culture medium with 10% FBS (or 10% dextran-coated charcoal treated FBS (DCC-FBS) for transfection assays) for 24 h. HepG2 cells were either placed on 60 mm-diameter plates (0.375 × 10⁶ cells/mL) for RNA extraction or on 24 well-plates (0.12 × 10⁶ cells/mL) for transfection assays and RNA extraction from transfected cells. Cells were treated with TCDD 1 nM (Promochem) and/or E2 10 nM (Sigma–Aldrich) dissolved in dimethyl sulfoxide (DMSO, max. 0.25%; Sigma–Aldrich) in complete phenol red-free DMEM with 0.5% FBS (or without FBS for transfection assays). Additionally, simultaneous treatments with the AhR antagonist α-naphthoflavone (α-NF, Sigma–Aldrich) or the pure anti-estrogen ZK 191 703 (kindly provided by Dr. Karl-Heinrich Fritzemeier, Bayer-Schering, Germany) were performed.