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Extracellular cystine, the oxidized form of cysteine (Cys), is taken up by cells via the cystine transporter xCT. xCT is not expressed in the liver but is induced in primary hepatocytes under conventional cultured conditions. However, compared to wild-type hepatocytes those from the xCT-knockout mouse showed no evidence of an abnormality and the levels of both Cys and glutathione (GSH) remained unchanged. The levels of ophthalmic acid (OPT), which is produced as an alternative compound by the GSH-synthesizing pathway, became increased during the culturing of hepatocytes. It therefore appears that, in primary hepatocytes, Cys is provided by systems other than xCT, most likely via the transsulfuration pathway, but the levels that are produced are not sufficient. We also employed mouse hepatoma-derived Hepa1-6 cells, which constitutively express xCT. When Hepa 1-6 cells were cultivated in Cys-free media, the levels of intracellular Cys and GSH were decreased, compared to cells cultured in conventional media, leading to cell death accompanied by an increase in the levels of reactive oxygen species and lipid peroxidation products with characteristics similar to ferroptosis. While OPT levels were increased by only to a limited extent in Hepa 1-6 cells, primary hepatocytes cultured in Cys- and Met-free media showed a marked elevation in OPT, reaching levels nearly equivalent to the GSH levels when the cells were cultured in conventional media. Thus, OPT may become a marker for Cys insufficiency and might be used to predict pathological conditions of cells with elevated oxidative stress.Differential cysteine-supply systems between the primary mouse hepatocytes and Hepa 1-6 cells under conventional or Cys/Met-free culture. Met; Methionine, Hcy; Homocysteine, Cst; Cystathionine, Cys; Cysteine, Thr; Threonine, Glu; Glutamate, 2OB; 2-Oxo-butyric acid, 2AB; 2-Amino-butyric acid, GSH; Glutathione, OPT; Ophthalmic acid, γGCS; γ-Glutamylcysteine synthetase, GS; Glutathione synthetase; NAAT; neutral amino acid transporterxCT is induced in primary mouse hepatocytes under culture conditions.xCT-KO hepatocytes contained normal levels of cysteine and glutathione (GSH).Primary hepatocytes were viable in cysteine- and methionine-free media.Ophthalmic acid levels were equivalent to those of GSH in hepatocytes.