Cytotoxicity Screening of Compounds in HepG2 Cells

HepG2 cells were plated on 384-well tissue culture treated polystyrene plates at 2000 cells in 25 μl of HepG2 maintenance medium per well. After an overnight incubation at 37°C, the cells were dosed with test compounds in dimethyl sulfoxide (DMSO) and controls over wide ranges of concentrations (Table 1) and incubated for 72 h at 37°C. DMSO was at 0.5% in the final solution. The aqueous solubility of the compounds limited the range of concentrations tested. Cell viability was measured using the Promega CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) kit by adding the Dye Solution to each well and incubating for 3 h at 37°C. After incubation, the Solubilization Solution/Stop Mix was added to each well. Plates were incubated at 37°C for 1 h, mixed on a plate shaker for 10 min and then absorbance was read at 570 nm. Cell viability was determined by comparison against control cells in the presence of DMSO only and was measured in three independent replicates for each compound concentration. The IC₅₀ was calculated with GraphPad Prism using a Sigmoidal Dose-response (variable slope) algorithm. Chlorpromazine, a drug with a well-known and characterized hepatotoxicity (Frötschl et al., 2005 (link); Gandhi et al., 2013 (link); Morgan et al., 2019 (link)), was used as the positive control.