DNA repair enzymes in sunscreens and their impact on photoageing—A systematic review

Abstract Background DNA damage is one of the main factors responsible for photoageing and is predominantly attributed to ultraviolet irradiation (UV‐R). Photoprotection by conventional sunscreens is exclusively prophylactic, and of no value, once DNA damage has occurred. As a result, the demand for DNA repair mechanisms inhibiting, reversing or delaying the pathologic events in UV‐exposed skin has sparked research on anti‐photoageing and strategies to improve the effect of conventional sunscreens. This review provides an overview of recent developments in DNA repair enzymes used in sunscreens and their impact on photoageing. Methods A systematic review of the literature, up to March 2019, was conducted using the electronic databases, PubMed and Web of Science. Quality assessment was carried out using the Newcastle‐Ottawa scale (NOS) to ensure inclusion of adequate quality studies only (NOS > 5). Results Out of the 352 publications, 52 were considered relevant to the key question and included in the present review. Two major enzymes were found to play a major role in DNA damage repair in sunscreens: photolyase and T4 endonuclease V. These enzymes are capable of identifying and removing UV‐R‐induced dimeric photoproducts. Clinical studies revealed that sunscreens with liposome‐encapsulated types of photolyase and/or T4 endonuclease V can enhance these repair mechanisms. Conclusion There is a lack of randomized controlled trials demonstrating the efficacy of DNA repair enzymes on photoageing, or a superiority of sunscreens with DNA repair enzymes compared to conventional sunscreens. Further studies are mandatory to further reveal pathogenic factors of photoageing and possible therapeutic strategies against it.


| INTRODUC TI ON
Intrinsic ageing of the skin is characterized by a slow and steady decrease of tissue function, for example tissue elasticity. 1 Additional exposure of human skin to environmental factors can lead to an overlapping damage referred to as extrinsic ageing, photoageing or premature skin ageing. 1 Photoageing describes changes in clinical, histological and functional characteristics of habitually UV-exposed skin, which develop gradually over decades. 2 Most of the unwanted skin changes during ageing such as wrinkle formation, laxity, leathery appearance or pigment changes are driven by photoageing. It also advances the loss of the skin's reparative and regenerative potential leading to higher vulnerability and an impaired wound healing response. [2][3][4] DNA damage represents one of the main causative factors of photoageing and is predominantly attributable to ultraviolet-radiation (UV-R). 2,[5][6][7] Strategies to prevent or minimize photoageing include protection against UV-R and preservation of antioxidant homoeostasis. 3 Recent studies investigated detrimental effects of UV-R at the cellular and molecular levels to identify biological targets of UV-R and the resulting cascade of impairment of cell functions and tissue degradation. 8,9 Both UV-A and UV-B radiations, including mutations of key regulator genes, have been found to cause DNA damage. 2 Despite endogenous DNA repair mechanisms, DNA damage can persist and accumulate with chronic UV exposure, cumulatively leading to photoageing and skin cancer development. 2,10 An alarming increase of UV-R due to the depletion of the stratospheric ozone layer and decrease in clouds and aerosols, especially in the northern mid-latitudes, has been observed at the Earth's surface in the last two decades. 3,11 In addition, the exposure of human skin to environmental and artificial UV-R has increased significantly due to changes in social behaviour, including an increase in average life expectancy. 3,4 As a result, there is an ever increasing demand for DNA repair treatments to inhibit, delay or reverse the process of these specific events.
Photoprotection by the application of conventional sunscreens is solely of prophylactic nature, and of no value, once DNA damage has occurred. 6,12 Whilst the existing literature has mainly addressed the impact of DNA repair enzymes on the UV-associated development of malignancies, the present review aims to discuss recent findings regarding the biological process of photoageing and the role of DNA repair enzymes. An overview of the most relevant and currently available sunscreens containing DNA repair enzymes is provided below.

| ME THODS
We reviewed the medical literature in order to identify all studies investigating DNA repair enzymes in sunscreens, their ability to reduce or reverse DNA damage and their impact on photoageing.
A systematic search of the literature published until March 2019 was conducted using the electronic databases PubMed and Web of Science as well as relevant reference lists.
Titles and abstracts were screened for the following key terms (variably combined): "DNA repair enzymes," "sunscreen," "photoageing," "photoaging," "skin-ageing," "treatment," "photolyase," "endonuclease." Additionally, the reference lists of included articles were manually screened for further relevant publications. 352 publications evaluating the efficacy of DNA repair enzymes in sunscreens and their effect on photoageing were identified. If the abstract did not determine eligibility, full-text evaluation was performed. Investigation of sunscreens containing DNA repair enzymes was determined as the fundamental inclusion criterion. After elimination of duplicates (n = 89), full-text evaluation of the remaining publications was performed as shown in Figure 1. Only, the articles published in English or German language were included (exclusion of n = 2). Quality assessment was performed using the Newcastle-Ottawa scale (NOS). Studies with NOS> 5 were considered of adequate quality, whereas studies NOS < 5 were deemed substandard.
Data on study year, design and population were extracted from each study.

| RE SULTS AND D ISCUSS I ON
Various mechanisms were described to be involved in photoageing. 10 pertinent reviews were exclusively used for basic information.

| Sun-induced DNA Damage
Recent research substantially promoted the understanding of mechanisms involved in photoageing. The principal pathogenic factor for photoageing is the presence of UV-induced reactive oxygen species (ROS). Whilst low levels of ROS are continuously produced in the human body and have useful functions as important signalling molecules in physiological processes such as wound healing and repairing processes, there is evidence for a damaging effect of higher concentrations of ROS following UV-R exposure. [13][14][15] ROS activate cytoplasmic signal transduction pathways in resident dermal fibroblasts that are related to growth, differentiation, senescence and connective tissue degradation and therefore play a major role in photoageing. 4 Permanent genetic alterations in the DNA caused by ROS, such as changes in gene expression pathways related to collagen degradation and elastin accumulation, are primarily repaired by the base excision repair (BER) system. 16 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of free radical-induced oxidative lesions in the DNA and, therefore, has widely been used as a biomarker for oxidative stress and carcinogenesis. 17 Another way of DNA damage is caused by UV-R directly; it leads to formation of dimeric photoproducts between adjacent pyrimidine bases. 16 These photoproducts, namely cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), represent the most common UV-induced DNA damage. 18 In CPD, two adjacent thymine bases in the same DNA strand covalently connect to form a cyclobutane ring. In 6-4PPs, in addition to the two bases being covalently linked, a complex chemical structure is formed, where oxygen and hydrogen atoms from one base migrate to another. Both CPD and 6-4PP lead to mutagenesis, cell apoptosis and ultimately causing photoageing and skin cancer if not repaired by the body's own DNA repair mechanisms. 18,19 Several reports have shown a reduction in age-related DNA repair capacity caused by an accumulation of processes including UV-induced DNA damage, nucleotide excision repair (NER) and BER systems. This consequently contributes to the advancing of photoageing. [20][21][22] The biochemical nature and the formation of DNA alterations highly depend on the wavelength of interfering photons. 16 UV-B radiation, which is the most energetic and mutagenic component of solar radiation, is directly absorbed by DNA and induces CPD and 6-4PP development in particular. 16 Although less energetic, thus of less damaging potential than UV-B, the amount of UV-A in natural sunlight is at least 20 times higher than of UV-B. Hence, UV-A is equally involved in the development of DNA lesions. 16 The cytotoxic mechanism of UV-A is strongly oxygen-dependent and induces oxidative DNA lesions, mainly 8OHdG. 16 Sunlight exposure also induces the formation of matrix metalloproteinase 1 (MMP-1), an enzyme which is involved in the digestion of collagen 1. 23 According to Fisher et al, 24,25 exposure to UV-B not only leads to MMP-1 induction, but within hours of irradiation, leads to upregulation of the transcription factors AP-1 and NF-κB, which act as stimulatory factors for MMP-1 genes. Since the degradation of collagen is a hallmark of photoageing, MMP-1 gene modulation represents another potential working point in reducing UV-generated DNA damage. 23,24 Moreover, there is increasing evidence showing that in addition to UV-R, infrared radiation (IR) may contribute to photoageing. IR accelerates the rate of wrinkle formation by upregulating skin angiogenesis. 26 It has been demonstrated that both IR and UV-R play an important role in skin angiogenesis by causing a dysregulation between the angiogenesis inducing vascular endothelial growth factor (VEGF) and the angiogenesis inhibiting thrombospondin 2 (TSP-2). 27 It has also been suggested that the IR-induced skin angiogenesis may partially be caused by accumulation of heat in human skin. 27 IR radiation further contributes to photoageing by gene activity modulation in mitochondria in several ways, for example an increased formation of mitochondrial ROS and the induction of MMPs. 26 F I G U R E 1 Flow diagram of study inclusion. *: Included reviews were used for basic information exclusively

| Mechanisms of sun-induced photoageing
A central role in photoageing can be assumed for telomere-based signalling. 28 Telomere length is a molecular marker of a cell's age.
Genomic instability due to telomere shortening has been linked to several ageing-related conditions such as benign and malignant neoplasms and photo-aged skin. [29][30][31][32][33] Age-related telomere shortening can be slowed down to some degree by telomerase, a ribonucleoprotein that adds DNA sequence repetitions in the telomere region. 34,35 However, most mammalian somatic cells do not express telomerase, which leads to the progressive and cumulative loss of chromosomeprotective sequences from chromosome ends. 36 In vitro studies have linked accelerated telomere shortening to DNA-damaging agents such as ROS. 37,38 In combination with UV-induced damage to critical regulatory genes, this leads to the typical appearance of photoageing. 39 Such mechanisms have been investigated in mouse models only. 32,40,41 To understand the role of telomere shortening in human skin ageing, a respective model is required, since human telomeres are up to ten times longer and telomerase is less active as compared to mice. 32,40,41

| Current concepts of DNA repair enzymes in sunscreens
Blocking the transmission of UV-R with sunscreens is a basic element for preventing photoageing. 1 Several studies have demonstrated a protective effect of regular use of conventional sunscreens against the development of skin cancers. [42][43][44] However, they do not have a repairing effect on skin cells, which have already been damaged by sun exposure. 6 Conversely, "active photoprotection" is provided by sunscreens containing DNA repair enzymes and antioxidants in addition to the sun protection factor (SPF). 6,45 These compounds may overcome the current lack of solar radiation harm management by the dual mechanism of prevention and repair. 6,45 Previous insights into the molecular basis of photoageing have led to the development of certain enzymes that are capable of identifying and removing damaged cell DNA fragments. The two main representatives namely photolyase and T4 endonuclease V are discussed below.

| Photolyase
Photolyase, a flavoenzyme containing the flavin adenine dinucleotide molecule, acts as a catalytic cofactor and repairs UV-induced DNA damage of CPD and 6-4PPs. 19 Two different kinds of photolyases specifically repair CPD and 6-4PP and thus are usually classified as CPD photolyases or 6-4 photolyases, respectively, corresponding to their different substrates. The catalytic cofactor is conserved in the whole protein superfamily of photolyases and adopts a unique folded configuration at the active site that plays a critical functional role in DNA repair. 19 Photolyase recognizes damaged thymine dimers and restores these lesions through direct absorption of blue light by the flavin adenine dinucleotide molecule or through energy transfer from an excited second chromophore, the antenna chromophore, 19 which finally splits into individual pyrimidines and returns the electron back to the enzyme redox cofactor. 46,47 Application of photolyase is also associated with a reduction of MMP-1 in epidermal and dermal compartments of the skin. 23 Overexpression of MMP-1 in human skin cells results in the destruction of collagen that plays a key role in photoageing. 23 In addition, photolyase supports in-cell regeneration that inhibits UV-induced apoptosis and reduces skin inflammation caused by sunlight exposure by inhibition of the pro-inflammatory cytokine interleukin 6 (IL-6). 48 Due to the extremely strong mutagenic potential of CPD (that exceeds the one of 6-4PPs or other lesions, and their responsibility for the great majority of UV-induced mutations), recent studies focussed primarily on CPD photolyase as a repair enzyme. 47 18,50 All human studies reported the use of sunscreens containing chemical UV filters combined with liposome-encapsulated CPD photolyase. 12,49,51 The liposome encapsulation provides a shuttle mechanism for the enzymes across the human stratum corneum and introduces biologically active proteins into the living epidermis. 52 This mechanism may provide a new pathway for photoprotection against some forms of UV-induced skin damage. 51 Clinical trials in 2000 12 and 2011 5 found that the addition of CPD photolyase to conventional sunscreens contributes significantly to the reduction of UV-R-induced DNA damage and apoptosis when applied topically to human skin. The number of UV-B radiation-induced CPD decreased by 40%-45%, which demonstrates the ability of photolyase to actively repair damage. 12 However, these studies were conducted with small sample sizes, without a control group and mainly addressed carcinogenesis. Therefore, they are considered as "proof of concept" studies.

| T4-bacteriophage endonuclease V (T4 endonuclease V)
T4 endonuclease V is an enzyme, which was initially isolated from Escherichia coli infected with T4 bacteriophage. It initiates DNA repair at the site of UV-induced CPD, which contributes to mutations that result in non-melanoma skin cancers (NMSC) including the AK, if not repaired. 53 The enzyme binds nontarget DNA in a salt-dependent manner and screens the DNA by facilitated diffusion to locate its target site. 54 Once UV-damaged DNA is detected, cleavage occurs by two combined activities, the pyrimidine dimer-DNA glycosylase activity and the apurinic-apyrimidinic endonuclease activity. 55  efficacy and pace of naturally occurring DNA repair is enhanced approximately fourfold by the influence of T4 endonuclease V. 56 In addition, the enzyme stimulates skin regeneration as well as skin reconstruction and prevents the destruction of extracellular matrix components, which contributes to the prevention of photoageing. 57 For instance, MMP-1 induction in human skin cells, by treatment with T4 endonuclease V, is reduced in the same way as by photolyase treatment, which results in the reduction of collagen-destruction. 23 Encapsulation of T4 endonuclease V into liposomes as delivery vehicles, termed "T4N5," is required for an adequate penetration through the stratum corneum. Findings of a mouse study suggested that application of T4N5 to the skin may be a useful adjunct to sunscreens for prevention and reduction of deleterious local effects of UV-R such as sunburn cell formation. 58

| Comparison of sunscreens alone vs sunscreens containing DNA repair enzymes
Advances in the understanding of skin biology have led to the development of a diversity of treatments aimed at preventing ageing and enabling skin rejuvenation. An ideal sunscreen should comprise different features: 1) protection against UV-B radiation and longwavelength UV-A radiation; 2) stability and safety of the filters; 3) ROS scavenging capability; and ideally 4) inclusion of enzymes contributing to cellular DNA repair. 6 Recent irradiation studies indicated that the addition of DNA repair enzymes (CPD photolyase and T4 endonuclease V) to conventional sunscreens may reduce UV-R-induced molecular damage to exposed skin to a greater extent than conventional sunscreens alone. 59  indicating their efficacy in reducing CPD formation. 59 In another double-blind study by Emanuele et al, 7 the efficacy to reduce CPD formation of SPF 50 sunscreen with or without antioxidants (carnosine, atrazine, ergothioneine) and/or CPD photolyase was evaluated in biopsies of human skin obtained after irradiations.
It was shown that the combination of topical antioxidants and CPD photolyase resulted in the highest reduction of CPD and free radi-

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing was not applicable to this article as no data sets were generated or analysed during the current study.