Dapsone

The TAS2R agonists chloroquine diphosphate, quinine hydrochloride dihydrate, saccharin sodium hydrate, denatonium benzoate, 1,10-phenanthroline hydrochloride monohydrate, ofloxacin, strychnine hemisulphate, erythromycin, dapsone, carisoprodol, and sodium cromoglycate were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and diphenidol hydrochloride was provided by TCI Europe (Zwijndrecht, Belgium). All products were solubilized and diluted in sterile water, with the exception of erythromycin, dapsone, and carisoprodol, which were solubilized in DMSO and then diluted in water. Antibiotics, DMSO, L-glutamine, trypan blue dye, heat-inactivated fetal calf serum and LPS (from E. coli serotype 0111:B4) were purchased from Sigma (St. Louis, MO, United States). RPMI medium was from Eurobio Biotechnology (Les Ulis, France).

RPMI 1640 with Glutamax, DMEM/F12 (1:1), Hanks' buffered salt solution (HBSS), fetal bovine serum (FBS), and Amplex red were purchased from Invitrogen, Paisley, UK. Pest (penicillin, streptomycin), neomycin, ionomycin, phorbolmyristateacetate (PMA), diphenyleneiodoniumchloride (DPI), dapsone, ML-171, Phox-I2, xanthine, hypoxanthine, xanthine oxidase, DMSO, DPPH (2,2-diphenyl-1-1picrylhydrazyl), Tween20, Sucrose, flavin adenine dinucleotide (FAD), Phosphatidic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), horseradish peroxidase (HRP) and NADPH were purchased from Sigma–Aldrich. HEK293 overexpressing Nox4 (CJ Nox4) cells were purchased from Redoxis, Lund, Sweden. HEK 293 cells expressing Nox5 and CHO cells expressing Nox1 were a kind gift from the Vincent Jaque Center Medical Universitaire, Geneva, Switzerland. GKT136901, a Nox1/Nox4 selective inhibitor, was a kind gift from prof. Harald HH Schmidt (Maastricht University, Netherlands).

NO levels in culture supernatants of macrophages were quantified by the accumulation of nitrite as a stable end product, according to a modified Griess method [19 (link)]. Cell culture supernatant (100 µL) was mixed with 14 mM 4,4′-diamino-diphenylsulfone (Dapsone, Sigma-Aldrich) in 2 M HCl (50 µL) and 0.1% N-1-naphthylenediamine dihydrochloride (50 µL) in deionized water. The absorbance of the tested culture supernatants at 550 nm was compared with a sodium nitrate standard (NaNO₂) curve.

Pyrrole was purchased from Merck, Germany, and then purified through distillation. Copper sulfate, sodium nitrate, sulfadiazine, acetone, sulfuric acid, hydrochloric acid, dapsone, sulfamethoxazol, dopamine, ascorbic acid, and uric acid were obtained from Sigma Aldrich, Germany. Hexacyanoferrate (II/III) was obtained from FlukaChemika.

BV2 microglial cells were seeded at 2 × 10⁴ cells/well and treated with compounds at increasing concentrations during 24 h in complete culture media. Then, treatments were removed, and BV2 cells were co-incubated with compounds and LPS (100 ng/mL) (O127:B8, SigmaAldrich, L3129) for additional 18 h in culture media supplemented with 1% FBS. Each plate included non-treated cells as basal nitrite production. Thereafter, nitrite production was determined by using the modified Griess assay. In brief, samples (150 μL) were mixed with DAPSONE (75 μL) (SigmaAldrich, A74807) and NEDA (75 μL) (SigmaAldrich, 33461), and the mixture was incubated at room temperature for 5 min. Light absorption was measured at 550 nm in a microplate reader (SPECTROstar nano, BMG Labtech). Basal nitrite production was referred to as 100% of nitrite production, and all data were normalized to the basal condition. Concentration needed to reduce nitrite production to 50% was calculated from graphical representation of percentage of nitrite production vs. compound concentration, and data were fitted by non-linear regression and interpolated to 50%.