Figure 6.

EtBr/AcrO viability staining and metabolite formation in primary mouse hepatocytes treated with NVP or 12-D₃NVP. Fresh primary mouse hepatocytes were incubated with vehicle (0.2% DMSO), 400 μM NVP, or 400 μM 12-D₃NVP for 8 h. Quantitation of nuclear EtBr incorporation from EtBr/AcrO viability costaining (A) as well as representative images of EtBr/AcrO costaining (B) are shown for these incubations. Cytochrome P450 metabolites extracted from hepatocyte culture medium were measured using uHPLC-MS/MS (Orbitrap) detection. 12-OHNVP (C), 2-OHNVP (D), and 3-OHNVP (E) were monitored using MS/MS scans for the following transitions: 283.1190 → 223.1104 m/z (12-OHNVP), 285.1315 → 225.1230 m/z (12-OHD₂NVP), 283.1190 → 161.0709 m/z (2-OHNVP), 286.1378 → 161.0709 m/z (2-OHD₃NVP), 283.1190 → 242.0798 m/z (3- OHNVP), and 286.1378 → 245.0987 m/z (3-OHD₃NVP). One O-glucuronidated metabolite (F) was extracted from the cell culture medium and subjected to uHPLC-MS (Orbitrap) analysis. The following high resolution ions were observed to assay for the presence of O-glucuronidated metabolite: 459.1510 ± 5 ppm for O-GlucNVP and 462.1700 ± 5 ppm for O-GlucD₃NVP. One glutathione conjugate (G) was extracted from cell pellets and subjected to uHPLC-MS (Orbitrap) analysis. The following high resolution ions were observed to assay for the presence of this metabolite: 572.1922 ± 5 ppm for glutathione-NVP and 575.2110 ± 5 ppm for glutathione-D₃NVP. Data are representative of the mean ± standard deviation of four experimental replicates, except for glutathione detection, which has three experimental replicates. Significant differences from vehicle (A) or for the indicated comparisons (A, C, D, E, F,G) were determined using an unpaired t test generating two-tailed P values (*P < 0.05; **P < 0.01).