2.4. Quantification of total intracellular GSH

After culturing in test media supplemented with various concentrations of test materials, cells were harvested, washed with PBS, sonicated in 0.5% Nonidet P‐40, and centrifuged at 8,000 × g for 10 min at 4°C. The supernatant was then collected. Intracellular GSH levels were determined by the DTNB‐glutathione reductase recycling assay after protein precipitation with 5% sulfosalicylic acid, as described previously (Anderson, ¹⁹⁸⁵). In brief, GSH is oxidized by DTNB to give 5‐mercapto‐2‐nitrobenzoic acid and glutathione disulfide, and the glutathione disulfide is reduced to GSH by the action of glutathione reductase [EC 1.8.1.7] and NADPH. The rate of 5‐mercapto‐2‐nitrobenzoic acid formation is recorded at 405 nm to determine the total GSH level, which is expressed as the sum of GSH and glutathione disulfide. The total intracellular GSH level was normalized to the protein concentration in each well, which was measured with a Protein Assay Bicinchoninate kit (Nacalai Tesque), using BSA F‐V as a protein standard. The phrase “GSH level” refers to the total intracellular GSH level normalized to the intracellular protein concentration.