Study On Developing Evaluation Method Of UV Protection Efficacy Of Sunscreen Cosmetics In Vitro

Background:In recent years, more and more attention has been paid to the biological effects of UVA. Many studies have shown that the ultraviolet ray is one of the main environmental factors in creating a large increase of reactive oxygen species (ROS) in the cells. Mostly, UVA exerts indirect biological influence by generation of superfluous ROS. The cells in a physiological state usually produce less ROS, and ROS are rapidly removed by both enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and non-enzymatic antioxidants such as the glutathione, vitamin C, vitamin E, coenzyme Q, etc, which maintain the pro-oxidant/anti-oxidant balance resulting in cell and tissue stabilization. However, a over exposure to UVA may overwhelm the skin anti-oxidant defence mechanisms and lead to pro-oxidant/anti-oxidant disequilibrium. So the two factors above, that is, overproduction of ROS and pro-oxidant/anti-oxidant disequilibrium, eventually cause oxidative damages to the cells. By measuring the biological markers (such as SOD, CAT, GSH-Px, ROS and 8-isoprostane) of the oxidative damages to cells caused by ultraviolet radiation , this study can not only reflect the body's antioxidant capacity, but also indirectly reflect the extent of body's injury from free radical.Studies have proved that sunscreen cosmetics can play an important role in preventing human body from the damages caused by ultraviolet radiation. At the present time, there are two international methods, including in vivo and equipment measurements (UV spectroscopy and SPF method), which are generally adopted to assess the efficacy of sunscreen cosmetics. However, any reliable in vitro biological evaluation has not been reported yet. So we are trying to establish a kind of in vitro biological evaluation system, which is different from traditional evaluation methods. In this study, we adopted a method, in which the oxidative damages to cells induced by UVA radiation and is used as our theoretical basis, and the in vitro cultured HaCaT cells are took for biological materials. This method quantitatively determined the efficacy of sunscreen cosmetics to block ultraviolet rays by means of detecting a variety of biomarkers (such as ROS, 8-isoprostane, SOD, GSH-Px and CAT) of the oxidative damages of cells caused by ultraviolet radiation.OBJECTIVE:To establish an assessment system of the UV protection efficacy of the sunscreen cosmetics by detecting a variety of biomarkers (such as ROS, 8-isoprostane, SOD, CAT and GSH-Px ) of the oxidative damage caused by ultraviolet radiation on cells in vitro.METHODS:1. Cell culture: HaCaT keratinocytes, a spontaneously transformed human epithelial cell line, were routinely cultured in DMEM supplemented with 10% FBS, 100U/ml penicillin, and 100μg/ml streptomycin and maintained at 37℃in a 95% air/5% CO₂-humidified incubator. For experiments, cells were seeded at a quantitative density in 24-well plates or 60mm Petri dishes.2. MTT assay was used to detect HaCaT keratinocytes proliferation activity at the doses of UVA irradiation and to determine the UVA irradiation dose for the following experiment. HaCaT keratinocytes were pre-treated with a series of doses for chosen antioxidants and sunscreen cosmetics and then the cells were irradiated with a given dose of UVA irradiation. The changes of proliferation activity among the dosing groups provided data for choosing suitable doses of sunscreen cosmetics in the following experiment.3. Study on oxidative damages of HaCaT keratinocytes: ROS was labeled with the fluorescent probe (CM-H₂DCFDA) to detect the impact of UVA irradiation on cell-generated ROS. ELISA was used to detect the changes of UVA irradiation on 8-isoprostane formation. Determination of biochemical enzyme to detect UVA irradiation on the impact of SOD, GSH-Px and CAT enzyme activity. And two antioxidants (VitE and VitC) were chosen as a positive control to evaluate the stability and sensitivity of the five indicators used as biomarkers of oxidative damage.4. Study on the protective effect of sunscreen cosmetics in the oxidative damages induced by UVA irradiation: Respectively, using the five oxidative damage biomarkers above to evaluate the changes of the indicators after the intervention of the sunscreen cosmetics. To study whether these five biomarkers are applicable to evaluate the UV protective effect of the sunscreen cosmetics, and to select the most suitable evaluation indicator.RESULTS:1. MTT assay showed that:1.1 The proliferation activity of HaCaT keratinocytes had not been affected obviously at 8J/cm² UVA irradiation. The cell proliferation at this dose of irradiation maintains a good bioavailability, and is suitable for the following experiment.1.2 Pre-treated with VitE (25, 50 and 100μmol/L) and VitC (125, 250 and 500μmol/L) for 16h could significantly increase the proliferative activity of HaCaT keratinocytes. To determine the dose of sunscreen cosmetics in following tests is as follows: Sample 1 was 125, 250 and 500μg/ml, sample 2 was 62.5, 125 and 500μg/ml, sample 3 was 31.2, 62.5 and 125μg/ml, sample 4 was 62.5, 125 and 250μg/ml, sample 5 was 62.5, 125 and 250μg/ml , sample 6 was 125, 250 and 500μg/ml, sample 7 was 62.5, 125 and 250μg/ml, sample 8 was 62.5, 125 and 250μg/ml, sample 9 was 125, 250 and 500μg/ml.2. Results for oxidative damages of HaCaT keratinocytes: At 8J/cm² UVA irradiation, compared with control group (-UVA), the activities of SOD, CAT and GSH-Px significantly decreased and the content of ROS and 8-isoprostane significantly increased. Pre-treated with VitE (25, 50 and 100μmol/L) and VitC (125, 250 and 500μmol/L) for 16h, at 8J/cm² UVA irradiation, compared with control group (+UVA), the activities of SOD, CAT and GSH-Px increased in a dose-dependent manner and the formation of ROS and 8-isoprostane was dose-dependently reduced.3. Protective effects of sunscreen cosmetics in the UVA -induced oxidative damage. The results were shown in the table below:Note: + Representing that the cosmetic sample with a certain dose can remarkably change the value of the biomarkers after exposed to 8J/cm² UVA irradiation. It indicates that the cosmetic sample has protective effect on UVA-induced oxidative damages.- Representing that the cosmetic sample with a certain dose cannot change the value of the biomarkers after exposed to 8J/cm² UVA irradiation. It indicates that the cosmetic sample hasn't protective effect on UVA-induced oxidative damage.CONCLUSION:1. The results of the present study show that the three indicators including ROS, 8-isoprostane and SOD, compared with CAT and GSH-Px , are more sensitive and more suitable to adopted as biomarkers for the qualitative evaluation of the UV protective effects of cosmetics in the process of cell oxidative damage caused by ultraviolet radiation.2. The integrated application of the three indicators (ROS, 8-isoprostane and SOD) helps to accurately assess the UV protective role of sunscreen cosmetics.3. In vitro method for qualitative assessment of the UV protective effects of sunscreen cosmetics is feasible, and has some advantages such as simple, rapid, economical, and short-cycle.4. 8-isoprostane, as a new type of oxidative damage biomarkers, is first used in the in vitro biological evaluation system to determine the efficacy of sunscreen cosmetics, which has not been reported in a similar study at home and abroad.5. The in vitro biological evaluation system is only applied to qualitatively determine the efficacy of sunscreen cosmetics and a quantitative assessment is still expected to be explored in the further.