Overview of mathematical H295R steroidogenesis model. The computational model is based on an in vitro steroidogenesis experimental design with two compartments: culture medium and H295R cells ( Figure 1 ). The model consists of steroid transport and metabolic pathways. The transport pathways include cellular uptake of CHOL (steroid precursor) and MET and the import and secretion of 14 adrenal steroids (PREG, HPREG, DHEA, PROG, HPROG, DIONE, T, DCORTICO, CORTICO, ALDO, DCORT, CORT, E 1 , and E 2 ). The metabolic pathway includes conversion of CHOL into the 14 adrenal steroids and inhibition of steroidogenic enzymes by MET. Development of various aspects of the model is described in detail below.
This Test Guideline describes an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T). The human H295R adreno-carcinoma cell line, used for the assay, expresses genes that encode for all the key enzymes for steroidogenesis. After an acclimation period of 24 h in multi-well plates, cells are exposed for 48 h to seven concentrations of the test chemical in at least triplicate. Solvent and a known inhibitor and inducer of hormone production are run at a fixed concentration as negative and positive controls. At the end of the exposure period, cell viability in each well is analyzed. Concentrations of hormones in the medium can be measured using a variety of methods including commercially available hormone measurement kits and/or instrumental techniques such as liquid chromatography-mass spectrometry. Data are expressed as fold change relative to the solvent control and the Lowest-Observed-Effect-Concentration. If the assay is negative, the highest concentration tested is reported as the No-Observed-Effect-Concentration.