An immunohistochemical panel to assess ultraviolet radiation-associated oxidative skin injury

Abstract

Ultraviolet (UV) radiation results in a significant loss in years of healthy life, approximately 1.5 million disability-adjusted life years (DALYs), and is associated with greater than 60,000 deaths annually worldwide that are attributed to melanoma and other skin cancers. Currently, there are no standardized biomarkers or assay panels to assess oxidative stress skin injury patterns in human skin exposed to ionizing radiation. Using biopsy specimens from chronic solar UV-exposed and UV-protected skin, we demonstrate that UV radiation-induced oxidative skin injury can be evaluated by an immunohistochemical panel that stains 8-hydroxydeoxyguanosine (8-OH-dG) to assess DNA adducts, 4-hydroxy-2-nonenal (HNE) to assess lipid peroxidation, and advanced glycation end products (AGEs) to assess protein damage. We believe this panel contains the necessary cellular biomarkers to evaluate topical agents, such as sunscreens and anti-oxidants that are designed to prevent oxidative skin damage and may reduce UV-associated skin aging, carcinogenesis, and inflammatory skin diseases. We envision that this panel will become an important tool for researchers developing topical agents to protect against UV radiation and other oxidants and ultimately lead to reductions in lost years of healthy life, DALYs, and annual deaths associated with UV radiation.

Figure 1. Immunohistochemical characterization and quantification of 8-OH-dG in UV-protected and UV-exposed human skin biopsy specimens

A), C) UV-protected human skin sections stained with antibodies against 8-OH-dG. Strong nuclear staining is observed in approximately 15% of epidermal cells (arrows). B), D) UV-exposed human skin sections stained with antibodies against 8-OH-dG. Strong nuclear staining is observed in approximately 90% of epidermal cells (arrows). Moderate staining of intracelular material is observed equally in the epidermis of UV-protected as well as UV-exposed skin specimens. Original magnification A, B × 40 and C, D × 100. E) Analysis of staining intensity scores for 8-OH-dG. No difference was observed in 8-OH-dG staining of the stratum corneum of UV-exposed and UV-protected skin (0 ± 0 versus 0 ± 0). 8-OH-dG staining was significantly increased in the epidermis of UV-exposed versus UV-protected skin (3.0 ± 0.29 versus 1.5 ± 0.22; p = <0.01). 8-OH-dG staining was significantly increased in the dermis of UV-exposed versus UV-protected skin (1.6 ± 0.29 versus 1.3 ± 0.21; p = <0.05). *p < 0.05, ***p < 0.01. Data are shown as mean ± SEM; n=5.

Figure 2. Nuclear staining of 8-OH-dG in UV-protected and UV-exposed human skin biopsy specimens

Nuclear staining of 8-OH-dG in UV-protected and UV-exposexposed human skin biopsy specimens. UV-exposed human skin sections stained with antibodies against 8-OH-dG demonstrated strong nuclear staining in approximately 90% of epidermal cells and UV-protected demonstrated nuclear staining in approximately 15% of epidermal cells.

Figure 3. Immunohistochemical characterization and quantification of HNE in UV-protected and UV-exposed human skin biopsy specimens

A), C) UV-protected human skin sections stained with antibodies against HNE-adducts. B), D) UV-exposed human skin sections stained with antibodies against HNE-adducts. In UV-exposed skin samples there was a stronger staining for HNE in stratum corneum compared to the UV-protected skin (brown color, arrows). There is nuclear staining evenly observed in epidermis and dermis of both skin samples. Immunostaining is defined as low (A, C) and strong (B, D). Original magnification A, B × 40 and C, D × 100. E) Analysis of staining intensity scores for HNE. HNE staining of the stratum corneum was significantly increased in UV-exposed versus UV-protected skin (2.5 ± 0.22 versus 1.3 ± 0.12; p = <0.01). HNE staining was significantly increased in the epidermis of UV-exposed versus UV-protected skin (2.2 ± 0.18 versus 1.4 ± 0.17; p = <0.05). HNE staining was not significantly increased in the dermis of UV-exposed and UV-protected skin (1.9 ± 0.19 versus 1.2 ± 0.15). * p < 0.05, ***p < 0.01. Student t test. Data are shown as mean ± SEM; n=5.

Figure 4. Immunohistochemical characterization and quantification of AGEs in UV-protected and UV-exposed human skin biopsy specimens

A), C) UV-protected human skin sections stained with antibodies against AGEs. Weak staining is observed in epidermis (brown color staining, solid black arrow) and dermis (brown color staining, white arrow). Nuclear staining is demonstrated in approximately 10% epidermal cells. B), D) UV-exposed human skin sections stained with antibodies against AGEs. Moderate staining is observed in the epidermis (brown color, black arrows) and dermis (brown color, long white arrow). Strong nuclear staining is noted in approximately 70% of epidermal cells (brown color, short white arrows). The increased staining intensity in UV-exposed skin samples is determined to be 191% for stratum corneum, 196% for epidermis and 193% for dermis as compared to UV-protected skin samples. Original magnification A, B × 40 and C, D × 100. E) Analysis of staining intensity scores for AGEs. AGEs staining of the stratum corneum was significantly increased in UV-exposed versus UV-protected skin (1.6 ± 0.17 versus 0.9 ± 0.07; p = <0.05). AGEs staining was significantly increased in the epidermis of UV-exposed versus UV-protected skin (2.1 ± 0.16 versus 1.1 ± 0.13; p = <0.01). HNE staining was significantly increased in the dermis of UV-exposed and UV-protected skin (2.3 ± 0.19 versus 1.2 ± 0.16; p = <0.01). * p < 0.05, ***p < 0.01. Student t test. Data are shown as mean ± SEM; n=5.

Figure 5. Nuclear staining of AGEs in UV-protected and UV-exposed human skin biopsy specimens

UV-exposed human skin sections stained with antibodies against AGE demonstrated strong nuclear staining in approximately 70% of epidermal cells and UV-protected demonstrated strong nuclear staining in approximately 10% of epidermal cells.