Daidzein dz

MAO-Glow™ Assay kit was obtained from Promega (Madison, WI, USA). Genistein (GST) (purity > 98%) and tyramine HCl were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The isozymes hMAO-A and hMAO-B derived from recombinant baculovirus and their aliquoted active units (U) were obtained from Sigma-Aldrich. Each hMAO-A and hMAO-B stock was diluted with 10 mM HEPES in cold Hank's Balanced Salt Solution (HBSS) (pH 7.4) and kept in −80°C until use. Daidzein (DZ) (purity ≥ 98%) and selective standard MAOIs including DEP and clorgyline (CLORG) and other materials were also purchased from Sigma-Aldrich (St. Louis, MO, USA).

NCI-H295R cells were exposed to seven well-characterized inhibitors of steroidogenesis, and then the concentrations of the steroids in the culture medium were measured to estimate the enzyme inhibition to evaluate the performance of the simulation model. The adrenal steroidogenic inhibitors included aminoglutethimide (AGT, Bachem AG, Bubendorf, Switzerland), o,p′-DDD (DDD, Sigma-Aldrich Inc., St. Louis, MO), spironolactone (SP, Sigma-Aldrich Inc., St. Louis, MO), metyrapone (MP, Sigma-Aldrich Inc., St. Louis, MO), ketoconazole (KC, Wako Pure Chemical Industries, Ltd., Osaka, Japan), miconazole (MC, Wako Pure Chemical Industries, Ltd., Osaka, Japan), and daidzein (DZ, Sigma-Aldrich Inc., St. Louis, MO). The cells were also exposed to four adrenal toxicants whose adrenal toxicity is not mediated through steroidogenesis inhibition. The toxicants were acrylonitrile (AN, Wako Pure Chemical Industries, Ltd., Osaka, Japan), salinomycin (SM, Sigma-Aldrich, Inc., St. Louis, MO), thioguanine (TG, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and fumaronitrile (FN, Wako Pure Chemical Industries, Ltd., Osaka, Japan). All chemicals were dissolved in DMSO (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and added to the culture medium at 1 : 1000 dilutions.