When human skin is exposed to ultraviolet radiation (UVR), in particular ultraviolet-B (UVB; 290-320 nm), photolesions of the DNA occur, which promote light-induced skin ageing, mutations, cell death and the onset of carcinogenic events. Photolyase (EC 4.1.99.3) is a DNA-repairing enzyme that can reverse damage caused by UV radiation. The underlying study investigates the effects of a conventional sunscreen to which photolyase has been added and clarifies the question of whether this reduces the in vivo formation of cyclobutane pyrimidine dimers (CPDs) and UVR-induced apoptosis in the skin.

Ten study participants (skin type II on the Fitzpatrick skin scale) were exposed to the minimum erythema dose of simulated solar UV radiation for four consecutive days. 30 minutes before each exposure, the test substances (vehicle placebo, sunscreen with factor 50 and sunscreen with photolyase from Anacystis nidulans) were applied topically to three different sides. One side was left untreated and another was exposed exclusively to simulated solar UV radiation. Biopsy samples were taken 72 hours after the last irradiation. The amount of CPDs and the extent of apoptosis were measured by ELISA. The photolyase-added sunscreen was superior to the conventional sunscreen in reducing both the formation of CPDs and apoptotic cell death (in both cases with P<0.001). Furthermore, the addition of photolyase to a conventional topically applied sunscreen significantly contributed to a reduction in UVR-induced DNA damage and apoptosis. Previous studies showed that the UVB component of solar radiation triggers the formation of two major photoproducts: cis-syn cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts. Among the DNA photoproducts formed by UVB absorption of DNA, CPDs are mainly formed by the photo-(2+2) cycloaddition of the 5,6 double bond of the two adjacent pyrimidine nucleotides. CPDs disrupt the normal replication and transcription process of DNA and are thought to be a causative factor in skin cancer. There is also evidence to suggest that severely UVB-damaged cells undergo apoptosis in favor of surrounding normal cells. Accordingly, UVB-mediated apoptosis is currently considered a protective mechanism that prevents malignant transformation by eliminating cells whose DNA has been extensively damaged by UVB. Photoprotection is the primary preventive and therapeutic strategy against UVB-induced DNA damage and skin cancer. In addition to conventional behavioral measures such as wearing sun-protective clothing, reducing sunlight exposure time and using sunscreen products, an innovative approach to the urgent clinical challenge of photoprotection is the topical application of xenogenic DNA repair enzymes. Two different methods have been established for this purpose: the use of T4 endonuclease V and the application of photolyase. T4 endonuclease has been shown to be clinically useful in patients affected by nucleotide excision repair defects caused by pre-malignant and malignant skin lesions. However, the use of photolyase, which is also capable of removing UVB-induced CPDs from normal cells in vivo, has been shown to be more effective than T4 endonuclease V in repairing damage.
Photolyase (EC 4.1.99.3) is a monomeric DNA repair flavoenzyme of 50-60 kDa in size that repairs damage caused by UVR radiation. This enzyme occurs naturally in almost every organism exposed to sunlight, with the exception of placental mammals and humans.

Previous in vitro studies have shown that CPDs are effectively repaired by photolyase through a catalytic photocycle called photoreactivation, which utilizes the energy of blue light. Therefore, photoreactivation can be defined as the reversal of the harmful effects of UV radiation that occur after or during exposure of the organism to near-UV or blue light (300-500 nm; maximum action spectrum: 430-460 nm). Photolyase obtained from the cyanobacterium Anacystis nidulans contains a light-harvesting chromophore, 8-hydroxy-5-deazaflavin (8-HDF) and the flavin adenine dinucleotide (FAD), which is significantly involved in catalytic activity. In a clinical study conducted by Stege et al. it was shown that a liposome formulation applied to the skin containing photolyase derived from Anacystis nidulans protects against damage caused by UVB radiation, such as erythema and immunogenic hypersensitivity reaction. Based on this, it was hypothesized for the underlying study that the addition of photolyase to a conventional sunscreen may enhance its protective effect by reducing the formation of CPDs at the DNA level and UV-induced apoptosis.

RESULTS

The average MED for the simulated sunlight UV radiation was 59±10 mJ/cm2. The MEDs were recorded individually for each study participant and the study was started with the individually determined MEDs as initial values for the sunlight simulator. Repeated irradiation significantly increased the formation of CPDs in the positive control area irradiated only with UV radiation as well as in the vehicle and UV-irradiated area, 19 times higher in both areas compared to the initial value, P<0.001. The individually applied sunscreen partially, but not completely, suppressed the formation of CPDs by 62% (P<0.001 vs. positive control group and vehicle + UV-irradiated areas). However, the sunscreen with photolyase reduced the formation of CPDs by ~93% and was therefore clearly more effective than sunscreen without photolyase (P<0.001). Cell Death Detection ELISAPlus was used as a method to quantify DNA fragmentation based on the detection of mono- and oliginucleosomes in the cytoplasmic fractions of skin biopsies obtained under different experimental conditions. Repeated irradiation significantly increased apoptosis in the positive control area irradiated only with UV radiation as well as in the vehicle and UV-irradiated areas, 8.1 times higher in both areas compared to baseline, P<0.001. The individually applied sunscreen partially, but not completely, suppressed the formation of CPDs by 40% (P<0.001 vs. positive control group and vehicle + UV-irradiated areas). However, the sunscreen with photolyase reduced apoptosis by ~82% and was therefore clearly more effective than sunscreen without photolyase (P<0.001). A study by Stege et al. showed that topical application in vivo of photolyase-containing liposomes to UVB-irritated skin followed by exposure to photoreactivating light reduced the number of dimers induced by UVB radiation by 45-50%.

Conclusion

The study results of the underlying study confirm and extend existing findings on the usefulness of the application of photolyase for human photoprotection. A study conducted by Decome et al. has already shown that photolyase photoactivated by UVA radiation, which was contained in liposomes, efficiently repaired CPDs and reduced the level of single-strand breaks in keratinocytes by a factor of 2.6 to 3.3 after a single dose of UVB radiation.

Remarkably, the in vivo model used in the underlying study was repeatedly exposed to ssUV radiation. Since the accumulation of residual DNA damage caused by repeated exposure to ssUV radiation plays a key role in the development of skin cancer, the results of the underlying study clearly show that photolyase-containing topical preparations are superior to conventional sunscreens in reducing cancerous and pre-cancerous skin lesions and sunlight-induced skin aging. The data obtained in the underlying study demonstrate that the addition of photolyase to a conventional sunscreen significantly enhances its effect against both ssUV radiation-induced DNA damage and apoptopic cell death and is therefore well suited as a cosmetic photochemoprevention agent.

Underlying study:

Berardesca, E. et al: Reduced ultraviolet-induced DNA damage and apoptosis in human skin with topical application of a photolyase-containing DNA repair enzyme cream: Clues to skin cancer prevention; in: MOLECULAR MEDICINE REPORTS 5: 570-574, 2012; DOI: 10.3892/mmr.2011.673.