2016 Arte Poster Competition First Place Winner: Circadian Rhythm and UV-Induced Skin Damage: An In Vivo Study

Furthermore, pinpointing when maximal erythema occurs in a circadian cycle can shed light on skin protection against UV damage. From the observations of our study, it may be important to change the timing of outdoor activities from the AM to the PM in order to avoid excessive UV-induced skin damage.

All subjects were recruited through the Skin Study Center at University Hospitals Case Medical Center. Seven healthy, non-shift work, FST19 II-IV adults were enrolled after informed consent was obtained.

MED testing was performed at 8:00 h and 16:00 h using an 8-holed template and exposing 8, 1-cm² circles of buttock skin to increasing doses of SSR, a full spectrum light source that most closely resembles natural sunlight. This test was done on the right buttock for the AM MED exposure and the left buttock for the PM MED exposure. A 1000 W xenon arc solar simulator model 6271 (Oriel Instruments, Stratford, CT), with a dichroic mirror and 81017bis filter (WG320/1.5 mm), producing a spectrum of 290–400 nm was used for the irradiation at increasing length of time depending on the patient’s FST. Those with lower FST were exposed to a lower starting dose. The setup for the SSR exposure is listed in previous literature.¹⁷ The spectrum and integrated irradiance were measured with a calibrated Bentham DM 150 double monochromator spectroradiometer. Irradiance was measured routinely using an IL1700 radiometer (International Light, Newburyport, MA) equipped with a sensor for UVA (SED 033, UVA filter 19672) and UVB (SED 240, UVB filter 15541) positioned 10 inches from the light source.

After 24 hours, the areas were visually graded based on the degree of erythema to determine the visual MED. Areas that showed no redness were graded “0”, incomplete circles of pink skin were graded “Trace”, complete pink circles were graded “1”, and complete dark pink to red circles were graded “2”. The erythematous skin (full pink circle) that was exposed for the shortest duration is the visual MED. Calculating the MED is performed by measuring the amount of erythema on each exposed area as well as an adjacent non-exposed skin area, using the CR300 chromameter from Konica Minolta (Tokyo, Japan).

Linear regression was applied and 1 MED was calculated using Microsoft Excel program according to COLIPA recommendations as the dose of UV producing an increase in the redness parameter ( a) of +2.5.18

6-mm punch biopsies were obtained from both SSR-irradiated skin and non-irradiated skin approximately 24 hours post SSR. The biopsies were obtained in the areas that received the highest dose of SSR and adjacent non-irradiated skin. Samples were then analyzed using Western blot and immunohistochemistry (IHC). Western blot for XPA was performed according to protocols previously listed in literature.²⁰ Actin was used as a control. For IHC, the fixed biopsies were embedded in paraffin and serially cut into 5-μm sections. After deparaffinization and dehydration, the skin sections were heated in epitope retrieval buffer at 95–97°C for 20 minutes then cooled for 30 minutes. They were then blocked in a dilution buffer containing 5% normal goat serum (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) and 0.5% saponin (Sigma, St. Louis, MO) in 1x phosphate buffered saline (PBS) and incubated 1 hour at room temperature with dilution buffer containing polyclonal anti-XPA antibody (ThermoFisher Scientific, Eugene, OR) or with polyclonal anti-CPD antibody (CosmoBio, Tokyo, Japan). After washing in PBS, Alexa Fluor 488- or 594-conjugated goat anti-rabbit secondary antibody (Invitrogen) was used to detect primary antibody and Vectashield Mounting Medium for Fluorescence with DAPI (Vector, Burlingame, CA) was used as a nuclear marker. To exclude nonspecific antibody staining, proper isotype controls were performed in every experiment. All images were acquired using an UltraVIEW VoX spinning disk confocal system (PerkinElmer, Waltham, MA) mounted on a Leica DMI6000B microscope (Leica Microsystems, Inc., Bannockburn, IL) equipped with a HC PLAN APO 20×/0.7 objective. Confocal images of Alexa 488 or 594-conjugated anti-rabbit secondary antibody and DAPI were collected using solidstate diode lasers emitting 488-nm or 561-nm and 405-nm excitation light, respectively, and with appropriate emission filters. Images were then exported and quantitatively analyzed using MetaMorph Premier Software (Molecular Devices Corporation, Sunnyvale, CA). Quantification of XPA and CPD expression levels was performed by first tracing the nuclei of DAPI stained image and translocating the nuclei location to the corresponding antibody fluorescent image. The average pixel intensity of the circled areas were then measured and recorded via MetaMorph. Data was analyzed using Microsoft Excel.

MED between skin that was irradiated in the morning versus afternoon were analyzed via T-testing and a difference of <0.05 was considered significant. Tissue data were quantified using Metamorph software and descriptive statistics were applied.

This study has been supported in part by the National Institutes of Health Grant (5P30AR039750) via the Skin Diseases Research Center (SDRC) and the Ohio Department of Development – Center for Innovative Immunosuppressive Therapeutics (TECH 09-023). We thank Ms. A’ja Patterson and the Skin Study Center Staff for their technical assistance.