Toxicity Evaluation of Drugs on Mammalian Cells

Toxicity of drugs to mammalian cells was carried out in the human cell line HFF, using a previously described method [41 (link)], with slight modifications. Briefly, HFF cells were grown in a medium consisting of 500 mL Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), 50 mL new-born calf serum (NBCS; Gibco, Cleveland, TN, USA), 5 mL penicillin/streptomycin (Gibco), and 5 mL L-Glutamax (200 mM, Gibco), at 37 °C/5% CO₂ up to ~80% confluence in vented flasks. For the assay, 100 µL of the cell suspension (3 × 10⁵ cells/mL) was added to each well of a 96-well plate. The plate was incubated at 37 °C/5% CO₂ for 24 h to allow for cell adhesion, after which 100 µL of a serial drug dilution was added (prepared in a separate sterile plate). Phenylarsine oxide (PAO; Sigma) was used as the positive control. The cells were then incubated for a further 30 h before the addition of 10 µL of 125 mg/L resazurin solution, and underwent a final incubation for 24 h. Fluorescence measurements and data analysis were performed, as described above. The selectivity index was calculated as EC₅₀ (HFF)/EC₅₀(427-WT).