Abstract
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/cm2 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 (H2O2) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H2O2 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 H2O2 after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN3, 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 H2O2 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 H2O2 during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H2O2 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 H2O2 during UVA exposure.
Highlights
Deleterious damage of the skin such as photo-aging and cancers that are caused by chronic long-term exposure to solar ultraviolet (UV) radiation have been extensively documented by a large number of basic and clinical studies [1,2,3,4]
The cell viability of fibroblasts cultured in Dulbecco’s modified Eagle’s medium (DMEM) exposed to ultraviolet A (UVA) decreased significantly, but remained at the same level as non-irradiated fibroblasts cultured in DMEM when the DMEM was replaced with fresh DMEM immediately after the UVA exposure (Figure 1b)
We previously reported that H2O2 generated by UVA exposure of DMEM induces the senescence of fibroblasts [19]
Summary
Deleterious damage of the skin such as photo-aging and cancers that are caused by chronic long-term exposure to solar ultraviolet (UV) radiation have been extensively documented by a large number of basic and clinical studies [1,2,3,4]. UVB has been shown to be the main cause of skin responses to acute inflammation and for chronic solar radiation-induced photo-aging and skin cancer formation [12,13]. The mechanism of UVA-induced chronic skin damage has been reported to be caused by reactive oxygen species (ROS) produced by the transfer of photo-activated electrons and energy of chemicals, such as flavins, tryptophan-derived 6-formylindolo[3,2-b]carbazole (FICZ), porphyrins, and melanin [15,16,17]. We recently reported that chronic low dose UVA radiation induces the cellular senescence of fibroblasts in vitro at a total dose of 36 J/cm within two weeks, and suggested a pivotal role for hydrogen peroxide (H2O2) in the induction of cell senescence, which is produced by UVA absorption and possibly by riboflavin, known as a potent UVA chromophore in the medium [19]
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