Sample preparation for DNA adduct analysis

Primary HUFs were seeded into 175-cm² flasks and exposed the next day to cytotoxic and sub-cytotoxic concentrations of glycidamide (for 24 h) or acrylamide (for 48 h) diluted in growth medium. Treatment was performed at 37 °C, 5% CO₂, and 3% O₂ (n = 4). Cells were then harvested and stored as pellets at – 20 °C until DNA was isolated using a standard phenol–chloroform extraction method as described previously (Kucab et al. 2015). DNA concentration and purity of the samples was assessed spectrophotometrically, and 20 µg DNA of each sample was dried using a SpeedVac Concentrator (SVC-100, Savant) for 1.5 h. Three biological replicates were prepared for each treatment condition. Samples were then shipped to the National Center for Toxicological Research (Jefferson, Arkansas, USA) where they were reconstituted in 100 μL water and heated at 100ºC for 15 min to release the N7-GA-Gua adduct from DNA. After cooling to room temperature, each sample was filtered through a 3 kDa filter (Centrifugal Filter Unit, Amicon Ultra–0.5 mL, Ultracel- 3 K, Merck Millipore, Ireland) that had been pre-washed twice with 400 µL water to remove residual glycerine. Filtrates (60 µL) were evaporated to dryness under vacuum and reconstituted in 60 µL acetonitrile:water:formic acid (80:20:0.1, v/v) and analysed by UPLC–ESI–MS/MS.