The In Vitro Cytotoxicity of Ascorbate Depends on the Culture Medium Used to Perform the Assay and Involves Hydrogen Peroxide

Abstract

Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H₂O₂, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 ± 39 μM H₂O₂ in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 ± 17 μM H₂O₂ is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H₂O₂ at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H₂O₂ at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.