V9001

The effect of fentanyl on CYP3A4 activity in insect membranes (V4820, Promega Corporation, Madison, WI), human hepatic microsomes and (HC-0015, Lifeline Cell Technology, Suite Z–Frederick, MD) and mice hepatic microsomes (M1000, XenoTech. LLC, Lenexa, KS) ectopically expressing human CYP3A4 was determined using 96-well microtiter plates according to the Promega protocol. The activity of CYP3A4 was examined using the P450-GloTM CYP3A4 Assay (Luciferin-IPA) according to the protocol (V9001, Promega, USA).

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.