Amisulpride

The following four antipsychotics drugs Olanzapine, Risperidone, Amisulpride and Aripiprazole were obtained from Sigma-aldrich (Sigma, USA). Fetal bovine serμM (Gibco BRL); RPMi-1640 medium (Gibco BRL); Dimethyl Sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, SUA); Cell counting kit-8 (CCK8) (Tongren, Tokyo, Japan) Annexin V-Fuorescein Isothiocyanate (FITC)/Propidium Iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, USA); Primary antibodies such as β-actin, BAX, BCL-2, MCL-1,Caspase-3, Cleaved-Caspase3 and β-catenin were obtained from Cell Signaling Technology (Beverly, MA, USA); Secondary antibodies for western blot analysis were obtained from Santa Cruz Biotechnology (Inc, CA, USA) and Cell Signaling Technology (Beverly, MA, USA).

The main chemicals used in this study, including FGAs (chlorpromazine, chlorprothixene, clothiapine, droperidol, flupentixol, fluphenazine, haloperidol, levomepromazine, loxapine, pimozide, prochlorperazine, sulpiride, thioridazine, and trifluoperazine) and SGAs (amisulpride, aripiprazole, brexpiprazole, clozapine, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, and zotepine) were acquired from Sigma-Aldrich (St. Louis, Missouri, USA) and were of highest purity grade. Before usage, the compounds were dissolved in dimethyl sulfoxide (DMSO) at the proper concentrations. Human hepatoma HepaRG™ cells were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). For 2 weeks, frozen cells were defrosted and kept alive in Williams’ E medium (Sigma-Aldrich, St. Louis, Missouri, USA) with 10% FetalClone™ II serum (HycloneTM, GE Healthcare, Chicago, Illinois, USA), 1 × L-glutamine, 5 μg/mL human insulin, and 50 μM hydrocortisone hemisuccinate without antibiotics as supplements. To produce differentiated hepatocyte-like features, the medium was then changed to the aforementioned medium plus 2% DMSO for an additional 2 weeks. Cells were grown at 37°C in a humidified 5% CO₂ environment. P-nitrophenylphosphate (PNPP) was used in an acid phosphatase (ACP) experiment to measure cell viability (29 (link)).

Paraoxon-ethyl was purchased from Sigma-Aldrich (St. Louis, MO, USA). Oxime K027 (purities > 98%) was synthesized at the Department of Toxicology and Military Pharmacy of the Faculty of Military Health Sciences (University of Defense, Hradec Kralove). Paraoxon-ethyl-D10, 4-nitrophenol solution (5000 μg/mL in methanol), 4-nitrophenol-2,3,5,6-D4, atropine solution (1.0 mg/mL in acetonitrile), and amisulpride were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC/MS grade acetonitrile, LC/MS grade methanol, LC grade chloroform, ammonium formate, and formic acid were purchased from Honeywell (Charlotte, NC, USA). All other drugs and chemicals of analytical grade were obtained commercially (Sigma-Aldrich) and were used without further purification. All substances were administered intramuscularly (i.m.) at a volume of 10 mL/kg b.w. to mice.

The exogenous OCT2 substrates amisulpride, cimetidine, ipratropium, lamivudine, metformin, MPP⁺, TEA and sepantronium (also known as YM155), as well as the endogenous OCT2 substrates acetylcholine, agmatine, choline, dopamine, epinephrine, serotonin and thiamine and the OCT2 inhibitor amitriptyline were provided by Sigma-Aldrich (Saint Quentin Fallavier, France). The chemical structures of the OCT2 substrates are indicated in Figure S2. The fluorescent dye DiASP, used here as a reference tracer substrate for OCT2 [59 (link),60 (link)], was purchased from Invitrogen (Carlsbad, CA, USA).