Chemicals and reagents
DQ and DQ-M were obtained from J&K Scientific Ltd. (Shanghai, China). Raloxifene, hydralazine and H218O were purchased from Aladdin reagent Co., Ltd. (Shanghai, China). The chemical structures of DQ, DQ-M, raloxifene and hydralazine are shown in Fig. 1. The purities of all standards were above 97.0% (HPLC). Male Sprague-Dawley rats were obtained from Oriental BioService Inc. (Nanjing, China). Acetonitrile, methanol, formic acid and ammonium acetate of LC–MS grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was prepared in our laboratory by ELGA LabWater system (ELGA Veolia, Bucks High Wycombe, UK). All other reagents and solvents were commercial products of analytical grade.
Preparation of rat liver cytosol
The method for preparation of rat liver cytosol has been reported in our previous study (Mao et al. 2018). Briefly, rats were sacrificed under ether anesthesia after starvation for 12h. The livers were perfused in vivo with ice-cold phosphate buffer via portal vein, and then quickly removed, rinsed and weighted. The liver tissues were pooled and minced, and then homogenized to 33% (w/v) in the ice-cold buffer (100 mM sodium phosphate, pH 7.4, 1.15% KCl, 250 mM sucrose and 1 mM EDTA). The homogenate was then centrifuged at 12,000 g for 20 min at 4°C. The supernatant was collected and centrifuged at 100,000 g for 60 min at 4°C using an Optima XPN-100 Ultracentrifuge (Beckman Coulter, U.S.). The resulting supernatant was collected as cytosol and then stored at -70°C. Protein concentrations were determined using commercial available Bradford protein assay kit.
Chemical inhibition experiments
To identify AOX catalyzing the conversion of DQ to DQ M, the AOX highly selective inhibitors were co-incubated with 1.0 µg/ml DQ (dissolved in normal saline) in rat liver cytosol (0.25 mg/ml), separately. The inhibitor concentrations were 1.0 µg/ml for raloxifene and 1.0 µg/ml for hydralazine. The chemical inhibitors raloxifene and hydralazine were the highly selective inhibitors of cytosolic AOX. Firstly, the rat liver cytosol was pre-incubated with raloxifene and hydralazine, respectively. After pre-incubation for 5 min, DQ was added to initiate the reaction, followed by incubation at 37°C for 30 min. The incubation was ended by adding ice-cold acetonitrile. Then, the incubation samples were centrifuged at 12,000 rpm for 5 min. Finally, 10 µL of clear upper layer was injected into the UPLC–MS/MS system for simultaneously determination of DQ and DQ M.
DQ oxidative metabolite oxygen source experiment
To confirm whether the oxygen atom incorporated into DQ M was derived from water or atmospheric oxygen, DQ (1.0 µg/mL) was incubated at 37°C in a shaking gas bath with rat liver cytosol (0.25 mg/ml) with H218O or H2O, respectively. Reaction was terminated after 30 min by addition of cold acetonitrile. After mixing and centrifugation, the supernatants were analyzed by a TQ-S Micro (Waters Corp., Milford, MA, USA) triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface in positive ionization mode. The samples incubated with H2O or H218O were analyzed using full scan mode, respectively.
Quantification of DQ M in rat tissues
All protocols were approved by the Animal Care and Ethical Committee of Nanjing Medical University. Three Sprague–Dawley (SD) rats (male, 200 ± 15 g, 6–8 weeks) were housed in a fixed cycle of 12 h light-dark facility with access to standard food and water. Each rat received an intragastric administration of DQ (dissolved in normal saline) at the dose of 11 mg/kg. The rats were sacrificed under ether anesthesia at 1 h after dosing, and heart, liver, lung, kidney, spleen, brain and muscle tissues were collected. All tissues (about 0.2 g per tissue) were cut into pieces and ground at 4°C with 1 mL acetonitrile. Then, the ground tissue samples were centrifuged at 12,000 rpm for 5 min. Finally, 10 µL of clear upper layer was injected into the UPLC–MS/MS system for simultaneously determination of DQ M.
Determination of metabolism of DQ in rat tissues in vitro
The rats were sacrificed under ether anesthesia, and heart, liver, lung, kidney, spleen, brain and muscle tissues were immediately collected. All tissues (about 0.1g per tissue) were cut into tiny blocks and put into six-well plate, and then added with 2 mL Dulbecco's Modified Eagle's medium (containing 1 µg/mL of DQ). The fresh tissues were cultured with DQ at 37°C for 12 h in a cell culture incubator containing 5%CO2. Then, the culture medium samples were collected and centrifuged at 12,000 rpm for 5 min. Finally, 10 µL of clear upper layer was injected into the UPLC–MS/MS system for simultaneous determination of DQ M.
The method for determination of DQ and DQ M has been developed and validated by our previous study (Mao et al. 2022). Briefly, the UPLC–MS/MS analyses were performed with a TQ-S Micro (Waters Corp., Milford, MA, USA) triple quadrupole mass spectrometer in the electrospray ionization (ESI) mode. The chromatographic separation was carried out using a CORTECS® UPLC® HILIC (100 mm × 2.1 mm, 1.6 µm) column (Waters Corp., Milford, MA, USA) at 40°C. The mobile phase composed of a mixture of 10 mM ammonium acetate with 0.5% formic acid (water phase) and acetonitrile (organic phase) was used at a flow rate of 0.35 mL/min. The step-wise elution was as follows: 70% organic phase (0-0.50 min), 30% organic phase (0.51–1.60 min), 70% organic phase (1.61–2.50 min). The source gas (nitrogen) flow was 660 L/hr, cone flow was 33 L/hr, desolvation temperature at 460°C, cone voltage was 35 V, and capillary voltage was 1.0 kV. The quantification m/z transitions of 183.1→156.6 and 199.1→155.1 and qualification m/z transitions of 183.1→129.5 and 199.1→78.3 were chosen for monitoring DQ and DQ-M, respectively
The crystal structure of AOX (PDB: 4UHW) was downloaded from the Protein Data Bank website (https://www.rcsb.org). All crystallographic water and ligands were removed from the protein using the Biovia Discovery Studio 2019 software. Then, the receptor model was optimized by protein automatic preparation function. The 3D structure of DQ was prepared via PerkinElmer Chem3D 18.0 software. The optimized structure of DQ with energy minimization was defined as docking ligand. Finally, the docking analysis of AOX with DQ was performed with the receptor and ligand interaction function via Biovia Discovery Studio 2019 software
Cell viability assay
Cell viability was assessed using the WST-1 Cell Proliferation Assay Kit (Beyotime, Shanghai, China). HCCLM3, Huh7, A549, 16HBE, HEK293T and GMC cells (100 µL) were cultured in 96-well plates (1 × 103 cells/well) for 24 h and then treated with different concentrations at 0, 50, 100 and 200 µg/mL of DQ or 0, 50, 100 and 200 µg/mL of DQ M for 24 h. Following treatment, 10 µL of the reconstituted WST-1 mixture was added to each well, mixed gently for one minute on an orbital shaker, and incubated for 2 h at 37°C in a CO2 incubator. Absorbance was measured at 450 nm using a microplate reader.