Prediction of the cytotoxicity of additives in cosmetics on UV-exposed human skin keratinocytes

多年来，对羟基苯甲酸甲酯（MP）一直被用作化妆品中的防腐剂。在这项研究中，我们研究了紫外线B（UVB）暴露对MP处理的人皮肤角质形成细胞的影响。将HaCaT角质形成细胞在含MP的培养基中培养24小时，暴露于UVB（15或30 mJ/cm 2）并进一步培养另外24小时。随后的细胞活力通过基于MTT的测定进行定量，细胞死亡通过荧光显微镜和流式细胞术进行鉴定。氧化应激，一氧化氮（NO）的产生和细胞脂质过氧化反应，用荧光探针测定。此外，核因子κ B和激活蛋白-1的活化通过电迁移率凝胶位移分析进行评估。MP的实际浓度（0.003%）有一点或没有影响细胞活力，氧化应激，NO的产生，脂质过氧化和激活HaCaT角质形成细胞的核转录因子。低剂量UVB对HaCaT角质形成细胞的这些参数也几乎没有影响。然而，UVB暴露显着增加MP处理的HaCaT角质形成细胞的细胞死亡，氧化应激，NO的产生，脂质过氧化和转录因子的激活。这些结果表明，MP被认为是化妆品中安全的防腐剂，当暴露在阳光下时，可能对人体皮肤产生有害影响。

For many years, methylparaben (MP) has been used as a preservative in cosmetics. In this study, we investigated the effects of ultraviolet-B(UVB)exposure on MP-treated human skin keratinocytes. HaCaT keratinocyte was cultured in MP-containing medium for 24 h, exposed to UVB (15 or 30 mJ/cm2) and further cultured for another 24 h. Subsequent cellular viability was quantified by MTT-based assay and cell death was qualified by fluorescent microscopy and flow cytometry. Oxidative stress, nitric oxide (NO) production and cellular lipid peroxidation were measured using fluorescent probes. In addition, activation of nuclear factor kappa B and activator protein-1 was assessed by electro-mobility gel-shift assay. Practical concentrations of MP (0.003%) had a little or no effect on cellular viability, oxidative stress, NO production, lipid peroxidation and activation of nuclear transcription factors in HaCaT keratinocytes. Low-dose UVB also had little or no effect on these parameters in HaCaT keratinocytes. However, UVB exposure significantly increased cell death, oxidative stress, NO production, lipid peroxidation and activation of transcription factors in MP-treated HaCaT keratinocytes. These results indicate that MP, which has been considered a safe preservative in cosmetics, may have harmful effects on human skin when exposed to sunlight.