Riboflavin Plays a Pivotal Role in the UVA-Induced Cytotoxicity of Fibroblasts as a Key Molecule in the Production of H(2)O(2) by UVA Radiation in Collaboration with Amino Acids and Vitamins

To investigate environmental factors that contribute to ultraviolet A (UVA)-induced oxidative stress, which accelerates the senescence and toxicity of skin cells, we irradiated human fibroblasts cultured in commonly used essential media with UVA and evaluated their viability and production of reactive oxygen species. The viability of fibroblasts exposed to a single dose of 3.6 J/cm(2) UVA was not reduced when cultured in Hanks balanced salt solution, but it was significantly decreased when cultured in Dulbecco’s modified Eagle’s medium (DMEM), which contains various amino acids and vitamins. Furthermore, cell viability was not reduced when fibroblasts were cultured in DMEM and treated with a hydrogen peroxide (H(2)O(2)) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H(2)O(2) was dramatically increased by UVA photosensitization when riboflavin (R) coexisted with amino acids such as tryptophan (T), and found that R with folic acid (F) produced high levels of H(2)O(2) after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN(3), which is a singlet oxygen quencher, suppressed only R and T photosensitization. Lastly, we examined the effects of antioxidants (L-ascorbic acid, trolox, L-cysteine, and L-histidine), which are singlet oxygen or superoxide or H(2)O(2) scavengers, on R and F or on R and T photosensitization, and found that 1 mM ascorbic acid, Trolox, and L-histidine were strongly photosensitized with R, and produced significant levels of H(2)O(2) during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H(2)O(2) production by UVA photosensitization. These data suggest that a low concentration of R-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H(2)O(2) during UVA exposure.