β nicotinamide adenine dinucleotide phosphate β nadph

This method was carried out as described previously with modifications^{35 (link)}. The treated cells were resuspended in homogenization buffer containing 0.25 M sucrose (Sigma, St. Louis, MO), 20 mM Tris–HCl at pH 7.4 (Bio-Rad Laboratories, Hercules, CA), 1 mM EDTA (Sigma, St. Louis, MO), and 0.1% protease inhibitor cocktail (Roche, Mannheim, Germany). Reaction mixture consisting of 100 μl sample, 2 mg/ml biliverdin reductase from rat liver, 1 mM β-nicotinamide adenine dinucleotide phosphate (β-NADPH; Sigma, St. Louis, MO), 2 mM glucose-6-phosphate (Sigma, St. Louis, MO), 1 U glucose-6-phosphate dehydrogenase (G6PD; Sigma, St. Louis, MO) and 25 μM hemin (Sigma, St. Louis, MO) was prepared fresh and incubated at 37 °C for 1 h in the dark. The formed bilirubin was extracted with chloroform and the change in optical density at 464–530 nm was measured in a microplate reader (Multiskan Go; Thermo Fisher Scientific, Waltham, MA). HO activity was expressed as picomoles of bilirubin formed per milligram of protein per hour by using an extinction coefficient of 40 mM^-1/cm for bilirubin in chloroform and protein concentration of the sample.

The substances utilized for cell culture and reagent-grade chemicals include Dulbecco’s Modified Eagle’s Medium (DMEM), streptomycin, penicillin, phosphate-buffered saline (PBS), trypsin/EDTA solutions, newborn calf serum (NBCS), sodium azide (NaN₃), β–nicotinamide adenine dinucleotide phosphate (β-NADPH), GSH, GSSG, NAC, BSO, N-ethylmaleimide (NEM), o-phtaldialdehyde (OPT), t-octylphenoxypolyethoxyethanol (Triton-X100), 1-chloro-2,4-dinitrobenzene (CDNB), tris hydroxymethyl aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (MeOH) was acquired from Merck Life Science S.L. (Madrid, Spain). A Milli-Q water purification system (Millipore, Bedford, Burlington, MA, USA) was used to produce deionized water (resistivity < 18 MΩ cm). Standards of T-2 (MW: 466.52 g/mol), Neosolaniol (Neo; MW: 382.40 g/mol), T-2 triol (MW: 382.45 g/mol), and T-2 tetraol (MW: 298.33 g/mol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of the mycotoxins were made in MeOH at the proper working concentrations and kept at a constant temperature of −20 °C in the dark.