N7505

The following reagents were obtained: ketamine (Dopalen injectable^®, Paulínia, Brazil), xylazine (Anasedan injectable^®, Paulínia, Brazil), Tween 80 (Synth^®, Diadema, SP, Brazil), hexadecyltrimethylammonium bromide (HTAB) (Sigma Aldrich^® H5882, Steinheim, Germany), o-dianisidine dihydrochloride (Sigma Aldrich^® D3252, Steinheim, Germany), dextran sulphate sodium salt (DSS)—colitis grade (36,000–50,000 MW, cod. 02160110-CF, MP Biomedicals, LLC, Solon, OH, USA), hydrogen peroxide (Synth^®), horseradish peroxidase (Sigma Aldrich^® P8250, Steinheim, Germany), trichloroacetic acid (TCA) (Sigma Aldrich^® T4885, Steinheim, Germany), reduced L-glutathione (Sigma Aldrich^® G-4251, Steinheim, Germany), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma Aldrich^® N-7505, Steinheim, Germany), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma Aldrich^® D-8130, Steinheim, Germany), and buffer RIPA (Sigma Aldrich^® R0278, Steinheim, Germany). IL-1β mouse, IL-10 mouse, CXCL-MIP-2 mouse, and TNF-α mouse were obtained from R&D Systems^®, Inc. (Minneapolis, MN, USA).

NQO1 activity was assessed by measuring the levels of NAD(P)H via absorbance at 340 nm in a Versamax plate reader (Molecular Devices; San Jose, CA, USA) at 37 °C. The reaction mixture in a final volume of 200 µl contained 50 mM ammonium bicarbonate, 0.1 mg/ml BSA, 2 mM EDTA, 200 µM NAD(P)H as electron donor (Sigma–Aldrich, N7505) and 2 µg of recombinant NQO1 (for final enzyme concentration of 0.5 µM). Positive and negative controls were included by adding 400 µM of the NQO1 known substrate menadione (Sigma–Aldrich, M5625) in the presence or absence of the NQO1 inhibitor dicoumarol at 100 µM, respectively. To assess whether the compounds were substrates of NQO1, 100 µM of the indicated compounds were added instead of menadione in the absence or presence of dicoumarol to measure specific NQO1 activity. Measurements were made every 10 sec for 20 min. For analytical high-performance liquid chromatography with diode array detection (DAD) and electrospray ionization mass spectrometry (HPLC-MS), the in vitro reactions were filtered using Amicon Ultra-0.5 centrifugal filter devices with a 30 kD molecular weight cutoff (Merck-Millipore) applying centrifugation for 10 min at 13,000 × g. 100 µl of flow-through samples were injected on an Agilent/HP 1200 system 6110 mass spectrometer with electrospray ionization (ESI+). Absorbance was monitored at 305 ± 90.

Mouse blood samples used for the EPR analyses were stored at 20–25°C in the dark until subsequent thawing for nitrite/nitrate assay. Human RBCs were collected as described above and used for NO₂^- and NO₃^- quantification immediately. Briefly, after hemoglobin precipitation with cold ethanol/chloroform (v: v– 1: 5) and vortex agitation, the mixture was centrifuged at 10,000xg for 10 minutes at 4°C. Aliquots of clear supernatants were used to detect NO₂^- and NO₃^-. For nitrite measurements, supernatants were incubated with Griess reagents according to Griess Reagent Kit instructions (G-7921, ThermoFisher). The nitrite concentrations were determined using a calibration curve (1–100 μmol/L) of sodium nitrite in deionized water. For nitrate measurements, NO₃^- was first reduced to NO₂^- in presence of NADPH 80 μmol/L (N-7505, Sigma) and nitrate reductase 50mU / 100 μl (N-7205, Sigma). The resulting total concentrations of nitrite and nitrate in samples (including products formed after NO oxidation) were determined by comparison with a calibration curve (1–100 μmol/L of sodium nitrite in deionized water). The nitrate concentrations were determined by the difference between the total NO oxidation products and nitrite concentrations from the same aliquots. Measurements of the absorbance at 548 nm were performed with a Spectramax i3 (Molecular Devices, LLS, USA).