Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol

A three-dimensional culture of human keratinoeytes exposed at the air- liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, 35S-methionine incorporation, ILl-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the epical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD50 values (40 J/cm2) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.