The Effect of UVB Irradiation and Oxidative Stress on the Skin Barrier-A New Method to Evaluate Sun Protection Factor Based on Electrical Impedance Spectroscopy

Abstract

Sunlight is vital for several biochemical processes of the skin organ. However, acute or chronic exposure to ultraviolet radiation (UVR) has several harmful effects on the skin structure and function, especially in the case of the failing function of antioxidative enzymes, which may lead to substantial tissue damage due to the increased presence of reactive oxygen species (ROS). The aim of this work was to investigate the combined effect of ultraviolet B (UVB) irradiation and oxidative stress on the skin barrier integrity. For this, we employed electrical impedance spectroscopy (EIS) to characterize changes of the electrical properties of excised pig skin membranes after various exposure conditions of UVB irradiation, oxidative stress, and the inhibition of antioxidative enzymatic processes. The oxidative stress was regulated by adding hydrogen peroxide (H₂O₂) as a source of ROS, while sodium azide (NaN₃) was used as an inhibitor of the antioxidative enzyme catalase, which is naturally present throughout the epidermis. By screening for the combined effect of UVB and oxidative stress on the skin membrane electrical properties, we developed a new protocol for evaluating these parameters in a simple in vitro setup. Strikingly, the results show that exposure to extreme UVB irradiation does not affect the skin membrane resistance, implying that the skin barrier remains macroscopically intact. Likewise, exposure to only oxidative stress conditions, without UVB irradiation, does not affect the skin membrane resistance. In contrast to these observations, the combination of UVB irradiation and oxidative stress conditions results in a drastic decrease of the skin membrane resistance, indicating that the integrity of the skin barrier is compromised. Further, the skin membrane effective capacitance remained more or less unaffected by UVB exposure, irrespective of simultaneous exposure of oxidative stress. The EIS results were concluded to be associated with clear signs of macroscopic tissue damage of the epidermis as visualized with microscopy after exposure to UVB irradiation under oxidative stress conditions. Finally, the novel methodology was tested by performing an assessment of cosmetic sunscreen formulations with varying sun protection factor (SPF), with an overall successful outcome, showing good correlation between SPF value and protection capacity in terms of skin resistance change. The results from this study allow for the development of new skin sensors based on EIS for the detection of skin tissue damage from exposure to UVB irradiation and oxidative stress and provide a new, more comprehensive methodology, taking into account both the influence of UVB irradiation and oxidative stress, for in vitro determination of SPF in cosmetic formulations.

Keywords: UVB irradiation; azide; catalase; cosmetic sunscreen; epidermis; hydrogen peroxide; oxidative stress; stratum corneum; sun protection factor.

Figure 1

(A) Schematic representation of the 4-electrode EIS setup, equivalent circuit, and definitions of $\mathsf{\Delta }{R}_{mem}$ and $\mathsf{\Delta }{C}_{eff}$. Two platinum wires served as working and counter electrodes and two Ag/AgCl/3M KCl electrodes were used as sensing and reference electrodes. $R_{sol}$ is the resistance of the donor and receptor solution, $R_{mem}$ is the membrane resistance, and $CPE$ is a constant-phase element used to derive the effective capacitance of the membrane, $C_{eff}$. (B) Representative data (average values ± SD, n = 3) from reference experiments with no oxidative stress parameters (i.e., neat PBS) and with oxidative stress conditions (in this case PBS containing 10 mM NaN₃ and 1 mM H₂O₂). The impedance properties of the membranes were examined for 3h without UVB irradiation, followed by 4 h of UVB irradiation. The experimental procedures used to generate the data in (B) are specified in (C) and (D).

Figure 2

Summary of $\mathsf{\Delta }{R}_{mem}$ (%) after 3 h without UVB irradiation and 4 h of total UVB irradiation (corresponding to 144 J/cm²) in combination with different stress parameters present in both the donor and receptor media. Data show average values (n = 3) with error bars showing either +SD (without UVB) or −SD (with UVB); n = 6 for A and n = 2 for F and G.

Figure 3

(A) Summary of $\mathsf{\Delta }{R}_{mem}$ (%) after 6 h UVB irradiation (216 J/cm²) and protection from topically applied sunscreen formulations. PBS with 10 mM NaN₃ and 1 mM H₂O₂ was included as control, without and with UVB irradiation. Data show average values (n = 3) with error bars showing either +SD (without UVB) or −SD (with UVB). (B) $\mathsf{\Delta }{R}_{mem}$ as a function of SPF value without and with UVB irradiation. The coefficient of determination for the regression line corresponding to the $\mathsf{\Delta }{R}_{mem}$ after UVB irradiation was r² = 0.87. (C) Schematic illustration of the experimental setup with presence of 10 mM NaN₃ and 1 mM H₂O₂ in the donor and receptor media.

Figure 4

(A) Summary of $\mathsf{\Delta }{C}_{eff}$ (%) after 6 h UVB irradiation (216 J/cm²) and protection from topically applied sunscreen formulations. PBS with 10 mM NaN₃ and 1 mM H₂O₂ was included as control, without and with UVB irradiation. Data show average values (n = 3) with error bars showing either +SD (without UVB) or −SD (with UVB). (B) $\mathsf{\Delta }{C}_{eff}$ as a function of SPF value without and with UVB irradiation. (C) Schematic illustration of the experimental setup with presence of 10 mM NaN₃ and 1 mM H₂O₂ in the donor and receptor media.

Figure 5

Excised pig skin membrane soaked in 10 mM NaN₃ and 1 mM H₂O₂ for 5 h without (A) and with (B) exposure to UVB irradiation (dosage corresponding to 180 J/cm²).