A1010

After determining the optimal number, 20,000 cells were seeded onto 96-well plates. After overnight incubation, cells were treated with decreasing concentrations of arsenic trioxide (A1010, Sigma-Aldrich, St. Louis, MO, USA) or cisplatin (cis-diammineplatinum(II)dichloride, P4394, Sigma-Aldrich, St. Louis, MO, USA) in order to calculate the half-maximal inhibitory concentration (IC50). After 48 h, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma-Aldrich, M2128-10G) reagent was added. After 3 h of incubation at 37 °C, cells were lysed for about 20 h with MTT lysis buffer (50% N-N dimethylformamide, 20% sodium dodecyl sulfate, 2.5% glacial acetic acid, 2.1% 1 N HCl, at pH 4.7), and absorbance was read at 560 nm with an optical spectrometer. IC50 was determined with GraphPad Prism (Version 5 for Windows, GraphPad Software, La Jolla, CA, USA).

Teratogens, except for cigarette smoke extract, were purchased from Sigma Aldrich (St. Louis, MO, USA): tributyltin-chloride (0.1 µM, T50202), cadmium-chloride (0.5 µM, C2554), benzo-α-pyrene (0.5 µM, B1760), nicotine (0.5 µM, N-3876), bisphenol-A (10 µM, 239658), diethylstilbestrol (10 µM, D4628), arsenic(III) oxide (0.5 µM, A1010), triclosan (0.1 µM PHR1338), fenthion (0.1, 1 µM, 36552). Cigarette smoke extract (0.1 µM) was purchased from Murty Pharmaceuticals (0.1 µM, Lexington, KY, USA). Stock solutions of teratogens were made in DMSO or water, and final concentrations were adjusted to contain 0.01% DMSO (ACS grade, Fisher Scientific, USA). Sub-lethal doses were chosen based on preliminary studies (unpublished McIntyre, Killeen & Marín de Evsikova).