V8751

CYP1A1, CYP2B6, CYP2C9 and CYP3A4 enzyme activities were evaluated directly in all cultured cells (HepaRG, HLCs and primary human hepatocytes) attaching to the collagen type IV-coated 6-cm dish at a density of 10⁶ cell. All cultured cells were treated with a cocktail of 20 μM rifampicin, 50 μM omeprazole, 1 mM phenobarbital and 88 μM ethanol. All treated cells were incubated for 72 h with daily medium change. CYP450 activities were assessed based on luciferase activity using the P450-glo 1A1, 2B6, 2C9 and 3A4 assays (V8751, V8321, V8791, V9001; Promega, WI). After 3-d incubation period, cells were incubated with Williams’ Media E supplemented with 100 μM Luciferin-CEE, Luciferin-2B6, Luciferin-H for 3–4 h or 3 μM Luciferin-IPA for 30–60 min. An aliquot (50 μL) of the medium was transferred to 96-well opaque white luminometer plate (Nunc, Denmark) and luciferin detection reagent was added to each well. After sitting at room temperature for 20 min, luminescence was measured with a SpectraMax M5 spectrofluorometer.

EROD assay: HT-29 cells (15,000 cells/well) treated with vehicle, UroA and UAS03 (24 h), were rinsed with HBSS buffer, and then fresh HBSS buffer was added along with 5 μM of 7-ethoxyresorufin. Cells were further incubated at 37 °C for 1 h. After the incubation time, fluorescence (Exc. 530 nm, Em. 580 nm) was measured and product (resorufin) formed was calculated from calibration standard and normalized with protein concentration.
P450-Glo Cyp1A1 luminiscence assay: HT29 cells (25,000 cells/well) were plated in 48 well plate. Cell were then treated with UroA (0.1, 1, 10, 25, and 50 µM) or UAS03 (0.1, 1, 10, 25, and 50 µM) or FICZ (0.1, 1, 10, 25, and 50 nM) for 24 h. After treatment, cells were washed to remove any residual drugs, and fresh medium containing Cyp1A1 substrate (as per protocol provided with kit Cat.# V8751; Promega) for 3 h. After incubation, 25 µl of culture medium was removed from each well and transferred to a 96-well white opaque plate and 25 µl of luciferin detection reagent was added to initiate the luminescence reaction and plate was incubated at room temperature for 20 min. After incubation, luminescence was recorded in luminometer. The data reported as fold change over vehicle treatment.