## 4 Discussion  
In this study, we have taken advantage of the two characterizing properties of the xanthophylls lutein and astaxanthin, i.e., to be strong antioxidants and ROS scavengers, and  
Figure 5: Western blot analysis of protein extracts after blue-violet irradiation (50 J/cm\({}^{2}\)) in the absence or in presence of 100 \(\upmu\)M LUT and 100 \(\upmu\)M ASX either alone or in association. Protein expression of Nrf-2 and Kaep-1 are reported in panels (**A**,**B**) and the respective quantitative analysis in panels (**C**–**E**), in which the corresponding percentage values are indicated at the top of each bar. Each bar represents the average value \(\pm\) SD of three different experiments. SFM: Serum Free Medium. * \(p\) < 0.05 vs. CTRL; § \(p\) < 0.05 vs. SFM (irradiated cells, without any protection). One-way ANOVA, followed by [PERSON]’s test. Figure 6: Western blot analysis of protein extracts after blue-violet irradiation (50 J/cm\({}^{2}\)) in the absence or in the presence of 100 \(\upmu\)M LUT, 100 \(\upmu\)M ASX either alone or in association. Protein expression of HO-1 and AKT are reported in panels (**A**,**B**) and the respective quantitative analysis in panels (**C**,**D**), in which the corresponding percentage values are indicated at the top of each bar. Each bar represents the average value \(\pm\) SD of three different experiments. SFM: Serum Free Medium. * \(p\) < 0.05 vs. CTRL; § \(p\) < 0.05 vs. SFM (irradiated cells, without any protection). One-way ANOVA, followed by [PERSON]’s test. to shield from low wavelength irradiation, even though with a different absorption pattern). In fact, our experimental protocol was more focused on the shielding effect because we evaluated apoptosis and viability immediately after the irradiation, and cells were not allowed a recovery time, during which the antioxidant efficacy of the xanthophylls could have been more evident. Therefore, the reduction of ROS observed in Figures 2B and 3B is likely mainly contributed by this shielding effect. In turn, this might explain why lutein appears more efficient than astaxanthin, since its radiation absorbance is higher in the blue-violet light interval used in our irradiation protocol (415-420 nm). Consequently, also cell viability after irradiation was higher with the lutein rather than with the astaxanthin shield (Figures 2A and 3A), despite the superiority of astaxanthin in terms of antioxidant potential [40]. A similar pattern was also observed for apoptotic nuclei (Figure 5). Lutein and its mix with astaxanthin appeared slightly more efficient than astaxanthin alone in blunting apoptosis induction by blue-violet light irradiation. A moderate light exposure is usually not dangerous to the eye, which contains enough endogenous anti-oxidants to cope with this amount of irradiation. However, when the radiation is more intense and/or prolonged over time, such as it may happen to people in the snow, or on the sea during a clear and sunny day, it can cause temporary or even permanent blindness [41]. Furthermore, the architecture of the outer blood-retinal barrier (formed by RPE cells) can also be altered by exposure to white LED light, as shown by the disorganization of the actin cytoskeleton, the induction of endoplasmic reticulum stress response and finally the activation of apoptosis in treated rats [42]. Similar findings were reported also after exposure of mice to white LED light. Light exposure increased ROS concentration in RPE cells and concomitantly disrupted the staining patterns of tight and adheren junctions, together with the actin cytoskeleton [43]. In humans, the amount of working time under bright sunlight seems to be a risk factor for the development of age-related macular degeneration. Therefore, preventive measures, able to shield from heavy sunlight exposure, should be started early in life to prevent the development of AMD later in life [44; 45].