Figure 2.

NVP and I2-D₃NVP cytochrome P450-dependent metabolism in primary human and mouse hepatocytes. Cryopreserved primary human hepatocytes (A–C) and fresh primary mouse hepatocytes (D–F) were incubated with 10 μM NVP or 12-D₃NVP for 24 or 48 h. Cytochrome P450 metabolites extracted from hepatocyte culture medium were measured using uHPLC-MS/MS (Orbitrap) detection. 12-OHNVP (A, D), 2-OHNVP (B, E), and 3-OHNVP (C, F) 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). Data are representative of the mean ± standard deviation of three (human) or four (mouse) experimental replicates. Significant differences between metabolite production with NVP and 12-D₃NVP were determined using an unpaired t test generating two-tailed P values (*P < 0.05 and ***P < 0.001).