Toxicity of L-ascorbic acid to L929 fibroblast cultures: relevance to biocompatibility testing of materials for use in wound management.

Abstract

Fibroblast cultures are often used to evaluate materials intended for medical use, cytotoxicity being taken as an indicator of bioincompatibility. Such an approach has previously been taken with ascorbic acid in determining its value in wound healing. We have now reexamined the toxicity of L-ascorbic acid to L929 fibroblast cells in culture. Concentrations of ascorbic acid between 0.5 mM and 11 mM were tested. At concentrations above 2 mM, ascorbic acid was found to inhibit cell proliferation, with cell viability decreasing as the concentration was increased. This effect could be prevented by the addition of either superoxide dismutase or catalase to the culture medium. Assays of glutathione and glutathione disulfide were carried out on 8 day old cultures exposed for 24 h to the same concentrations of ascorbic acid. A dose-related depletion of glutathione occurred whilst glutathione disulfide levels remained essentially constant. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities were induced by ascorbic acid at all concentrations tested but the ratio of NADP to NADPH nevertheless increased as the concentration of ascorbic acid increased. Finally, ATP in cells from 8-day-old cultures became depleted in the presence of ascorbic acid at concentrations in excess of about 5 mM when assayed after 24 h incubation. These biochemical changes and the concomitant cytostatic/cytotoxic effects may be ascribed to the reactive oxygen species produced by the autoxidation of ascorbic acid in the culture medium. Ascorbic acid breakdown products appeared not to be directly involved. In addition, our results suggested that superoxide acted cooperatively with hydroxyl to elicit these effects on the fibroblasts. It is evident from this study that the microenvironment surrounding fibroblasts in culture may differ fundamentally from that surrounding fibroblasts in a healing wound, making it impossible to extrapolate directly to an in vivo situation and hence to make any recommendations from these results concerning the use of ascorbic acid in wound healing.