Dextromethorphan

The CYPs activities were assayed using 7-ethoxycoumarin (Sigma-Aldrich) as substrate of multiple CYP isoforms [62 (link)] or dextromethorphan (Sigma-Aldrich) as CYP2D6 marker substrate. The 7-hydroxycoumarin production (CYPs-dependent metabolite) was analyzed fluorometrically as previously reported [94 (link)], while the dextromethorphan metabolism was analyzed by mass spectrometry using an Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS spectrometer operated in positive electrospray mode, reported elsewhere [95 (link)]. In brief, SH-SY5Y cells were treated with the inducers for 48 h. After incubation, the medium was changed and cells were incubated for another 24 h in a culture medium containing 7-ethoxycoumarin or dextromethorphan at a final concentration of 50 µM. Then, cells and medium were separately collected and the reaction was cooled down and quenched with acetonitrile (Sigma-Aldrich, 0.5 mL). After centrifugation (3000× g, 10 mins), the supernatant was discarded and the pellet was dried and resuspended in 500 µL of distilled water. In the case of the dextromethorphan metabolism assay the pellets were dissolved in 100 µL acetonitrile (Sigma-Aldrich). The calibration curves were obtained using increasing concentrations of the analyte (0–0.1 µM) either for 7-hydroxycoumarin and dextrorphan. The enzyme activities were expressed as pmol metabolite per mg of protein in 24 h.

Kurarinone (>98%) was purchased from BioBioPha Co., Ltd. (Kunming, China). Glucose-6-phosphate, Glucose-6-phosphate dehydrogenase, NADP⁺, D-Glucose-6-phosphate, Tris-HCl, 7-hydroxycoumarin, 4-methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide, uridine 5′-diphosphoglucuronic acid (UDPGA; trisodium salt), phenacetin, diclofenac, dextromethorphan, chlorzoxazone, testosterone, (S)-mephenytoin, sulfaphenazole, quinidine, clomethiazole, furafylline, ketoconazole, and omeprazole were purchased from Sigma Aldrich (St. Louis, MO, USA). Metabolites of the probe substrates including 6-hydroxylated chlorzoxazone (2E1), O-deethylated phenacetin (CYP1A2), 4′-hydroxylated diclofenac (2C9), 4′-hydroxylated (S)-mephenytoin (2C19), O-demethylated dextromethorphan (2D6), and 6β-hydroxylated testosterone (3A4) were provided from the Research Institute for Liver Disease Co., Ltd. (Shanghai, China). Pooled liver microsomes from humans (HLMs), monkeys (MLMs), rabbits (RAMs), rats (RLMs), mouse (MOMs), dogs (DLMs), and minipigs (PLMs) were provided by the Research Institute for Liver Disease Co., Ltd. (Shanghai, China). Recombinant human supersomes (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19) were obtained from BD Gentest Corp. (Woburn, MA, USA).

Gene expression analysis by RNA sequencing (RNA-seq) and quantitative PCR is described in Supplementary Materials and methods. Albumin and urea secretion into the media was measured using a human albumin ELISA kit and the QuantiChrom™ urea assay kit (both from Bethyl Laboratories). Comparisons with fetal and adult hepatocyte data used the unpaired two-tailed Student’s t test. CYP3A activity was assessed in duplicate by incubation with P450-Glo™ CYP3A4 assay reagent (Luciferin-PFBE; Promega Ltd). For CYP analysis by mass spectrometry, cells were incubated with 1 mM testosterone or 1 mM dextromethorphan (Sigma, UK) in HCM. Conditioned medium was collected and diluted 1:1 in 0.5 μM phenacetin (Sigma) stop solution in methanol. CYP activity was calculated per min incubation. Alcohol dehydrogenase activity of cell lysates was assessed using a detection kit following the manufacturer’s instructions (Abcam, UK). Results were standardized to the amount of protein measured by Bradford assay.