PMC

Intracellular AA (iAA) protects HL60 and U266 cells depleted from GSH and treated with As₂O₃. As₂O₃ toxicity in HL60 (A) and U266 (D) cells with (□) or without (▪) GSH. Cell viability (B,E) and colony formation (C,F) of HL60 and U266 cells depleted of GSH, loaded with vitamin C, and treated with 1 μMAs₂O₃. Cell viability was determined 48 hours after treatment. (A-F) Data are expressed as the mean ± SD of 3 independent experiments.

As₂O₃ is cytotoxic to HL60 and U266 cells. HL60 (A) and U266 (C) cells were treated with 1, 2, 3, 5, and 10 μMAs₂O₃ for 24 (○) and 48 (•) hours and viability was determined by trypan blue exclusion. Cloning efficiency of HL60 (B) and U266 (D) cells treated with 1 to 3 μM As₂O₃ or were left untreated. (A-D) Data are expressed as the mean ± SD of 3 independent experiments.

Transport and accumulation of vitamin C in HL60 and U266 cells. HL60 (A) and U266 (B) cells were incubated for the time indicated with 250 μM dehydroascorbic acid (DHA; ○) or ascorbic acid (AA; •) at room temperature. Intracellular accumulation of vitamin C in HL60 and U266 cells treated with DHA for 60 minutes at 37°C (A-B insets). (C) Inhibition of DHA transport in U266 cells treated with cytochalasin E (Cyt E; ○) and B (•). (A-C) Data are expressed as the mean ± SD of 3 independent experiments. iAA indicates intracellular AA.

Intracellular AA (iAA) protects HL60, U266, and RPMI-8226 cells from As₂O₃ cytotoxicity. Cell viability of HL60 (A) and U266 (D) cells loaded with vitamin C using DHA and exposed to As₂O₃ for 48 hours. Cloning efficiency of HL60 (B) and U266 (E) cells loaded with vitamin C and treated with 2 and 1 μMAs₂O₃, respectively. (C) HL60 cells were loaded with 4 mM iAA, treated with 3 μMAs₂O₃ for 24 or 48 hours, and apoptotic cells were stained using annexin V–FITC assay (left panel) or a TUNEL assay (right panel), respectively. (F) Cell viability of RPMI-8226 cells loaded with 4 mM AA and exposed to 1 μMAs₂O₃ for 48 hours. (A-F) Data are expressed as the mean ± SD of 3 independent experiments.

AA does not generate H₂O₂ in human plasma and the generation of H₂O₂ in plasma is dependent on the presence of free transition metal ions. Incubation of freshly isolated human plasma with increasing concentrations of AA. (A) The amount of H₂O₂ generated was determined by Amplex red. As a positive control, 100 μM AA was incubated in IMDM (last bar). (B) The generation of H₂O₂ was determined in human plasma incubated with 100 μM AA and increasing concentrations of CuCl₂. (A-B) Experiments were performed in triplicate and are expressed as the mean ± SD of 2 independent experiments.

AA generates H₂O₂ in a cell-free system that is dependent on the presence of free transition metal ions. (A) Different concentrations of AA (○) and DHA (•) were incubated in IMDM for 1 hour at 37°C and H₂O₂ was determined by Amplex red. (B) The generation of H₂O₂ by 1 mM AA in IMDM in the presence or absence of 500 U/mL catalase. (C) Generation of H₂O₂ by 100 μM of AA or 10 μM CuCl₂ in IMDM and increasing concentrations of DMP. (D) Generation of H₂O₂ by 50 μM of AA and increasing concentrations of As₂O₃ or 10 μM CuCl₂ in 50 mM sodium phosphate buffer (pH 7.4). (A-D) Experiments were performed in triplicate and are expressed as the mean ± SD of 2 independent experiments.

Extracellular ascorbic acid generates ROSs and augments the cytotoxicity of As₂O₃. Cell viability of HL60 (A) and U266 cells (B) treated with 1, 2, 3, and 5 μMAs₂O₃ with (▪) or without (□) 100 μM extracellular ascorbic acid for 48 hours. (C, left) Annexin V (FL1) and PI (FL3) flow cytometry analysis of apoptotic HL60 cells treated with AA or DHA for 20 hours. (Right) Percentage of apoptotic cells after treatment with either AA, DHA, As₂O₃, or the combination of DHA and As₂O₃.NT indicates cells not treated. (D) Cell viability of HL60 cells containing (•) or depleted of (○) GSH and treated with increasing concentrations of ascorbic acid in culture media. Cell viability was determined 48 hours after treatment. (E) Production of ROSs in HL60 cells loaded with vitamin C using DHA (•) and in cells where ascorbic acid was added in culture media (○). Data are expressed as the mean ± SD of 3 (A-B) and 2 (C-E) independent experiments.