Ageing of the skin involves complex processes in which both genetic (intrinsic) and environmental (extrinsic) factors play essential roles.
Photoageing, also called extrinsic, premature, or accelerated ageing, describes the superposition of environmental effects, especially of ultraviolet (UV) radiation, on chronological skin changes. These changes result from cumulative exposure to sunlight and lead to distinct morphological changes in exposed skin.1 Clinically, photodamaged skin is characterised by the presence of abnormal elastic fibres in the dermis with a dystrophic structure as a consequence of UV, and by a dramatic decrease of distinct collagen types. Photoaged skin is coarsely wrinkled and associated with skin thickening (both epidermal and dermal compartments), marked dryness and scaliness, uneven pigmentation and lentigines.2 UV radiation is responsible for a wide variety of different acute and chronic effects on the skin. Acute UV damage may cause sunburn and cumulative long-term UV damage is implicated in photoageing and skin cancer due in part to DNA alterations.3 UVB is able to induce important photolesions in DNA with potentially mutagenic properties. The nucleotide excision repair (NER) is one of the essential systems for correcting UVinduced DNA damage. In the ageing process, the various DNA repair systems including NER, BER (base excision repair), double strand break repair and mismatch repair, decrease their abilities. Therefore, DNA damage is easy to accumulate gradually with intrinsic ageing, which is related to the progress of the phenotype of ageing. Focusing on photoageing, an excessive UV exposure to skin beyond the DNA repair capacity in skin is considered to result in an accumulation of photoproducts, which may be associated with the progression of photodamaged skin (Fig. 1).4 Hence, repair of this damage is central to maintaining cell and organ stability and viability over the lifetime of the organism. The contribution of photoageing in DNA damage and the decrease in DNA repair capacity results in the appearance of photodamaged skin. Environmental insults such as UV rays from the sun, cigarette smoke exposure and pollutants, and the natural process of ageing contribute to the generation of highly reactive species. Enhanced free radical generation during UV irradiation in skin causes premature ageing of the skin and accelerates photoageing. Aged and photoaged skin has reduced levels of the natural enzymatic and nonenzymatic antioxidant defence mechanisms. Depleted antioxidant enzyme expression in photodamaged skin is associated with higher levels of protein oxidation. In cases of increased reactive oxygen species (ROS) generation (like photoaging), antioxidant enzymes may be overwhelmed, resulting in oxidative stress and oxidative protein damage. Proteins are known to be important targets for oxidative modifications. Direct oxidation of proteins by ROS yields highly reactive carbonyl derivatives.5 Carbonylation of proteins is irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. A large number of neurodegenerative diseases are directly associated with the accumulation of proteolysis-resistant aggregates of carbonylated proteins in tissues.6 Therefore, protein carbonyl groups provide a reasonable marker for free radical-induced protein oxidation. Carbonyl groups are introduced into proteins by a variety of oxidative pathways, for instance by reactions with aldehydes (4-hydroxynonenal, malondialdehyde) produced during lipid peroxidation via Michael additions and Schiff base adducts. Lipid peroxidation represents a degradative process, which is the consequence of the production and the propagation of free radical reactions and has been implicated in the pathogenesis of numerous diseases and ageing.7 ?, ?-unsaturated aldehydes produced during the peroxidation of polyunsaturated fatty acids react with protein thiol groups to form a stable covalent thiol ether adduct carrying a carbonyl group, as do aldehydes present in cigarette smoke.8 These aldehydes are part of the family of Reactive Carbonyl Species (RCS). RCS are a heterogeneous group of small molecular weight carbonyls activated by ?, ?-unsaturation as in 4-hydroxynonenal and acrolein, ?-oxo-substitution as in as glyoxal, and ?-oxo-substitution as in as malondialdehyde.9 RCS are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation.10 Intracellular RCS, it is suggested, play an important role in oxidative stress-related mutagenesis and carcinogenesis through their inhibitory effect on DNA repair mechanisms as well as on induction of DNA damage.11 Trans-4-hydroxy-2-nonenal (4-HNE) is among the most abundant and cytotoxic of the RCS. 4-HNE is a major electrophilic product of lipid peroxidation caused by oxidative stress, which is formed by radical-initiated degradation of ?-6-polyunsaturated fatty acids such as linoleic and arachidonic acids, two relatively abundant fatty acids in human cells.12 4-HNE added exogenously to or generated endogenously in cells is able to bind to various kinds of proteins, and impairs their function.13 It has been demonstrated that 4-HNE can greatly inhibit DNA repair capacity in human cells through its direct interaction with repair proteins. 4-HNE-protein adducts have been detected in various tissues in a number of human diseases, such as atherosclerosis, neurodegenerative diseases, and cancers, providing evidence for the important role that the interaction of 4-HNE with proteins may play in human diseases, including carcinogenesis. 4-HNE can inhibit NER capacity through its direct interaction with proteins involved in DNA repair.13 It is very likely that many proteins involved in DNA repair may be adducted by 4-HNE, which may result in detrimental effects on cellular DNA repair capacity, and consequently in an increase in DNA damage. A new peptide has been developed to protect skin against UV damage. Preventhelia (INCI name: Diaminopropionoyl Tripeptide-33) is a tetrapeptide obtained from a focused peptide library, specifically identified to fight the detrimental effects of UV radiation in human skin. The new peptide is born from preventive cosmetics: it acts by scavenging RCS and preventing skin from photoageing. The efficacy of Preventhelia in protecting skin from the effects of UV exposure was proved by several in vitro tests. The product was able to inhibit the formation of carbonyl proteins as a result of oxidative stress by scavenging RCS, and demonstrated photoprotective properties in different skin cell types. Finally, the activity of Preventhelia was tested regarding its ability in DNA protection and repair.
Materials and methods
Quenching ability towards 4-hydroxynonenal
A 20-fold excess solution of Preventhelia in respect to 4-hydroxynonenal was prepared. The mixture was warmed at 37°C and the quenching efficacy was monitored by means of RP-HPLC, detecting the disappearance of 4-hydroxynonenal (4-HNE) at different time intervals. Carnosine was used as a positive control.
Photoprotection test
The in vitro NRU photoprotection test is based on the determination of the protective effect of a chemical when tested in the presence of a cytotoxic dose of simulated solar light. The photoprotective effect is expressed as an increase of the uptake of the vital dye Neutral Red (NR), when measured 24 hours after treatment of human epidermal keratinocytes (HEKa) or human dermal fibroblasts (HDFa) with the test chemical and irradiation. NR is a weak cationic dye that readily penetrates cell membranes by non-diffusion, accumulating intracellularly in lysosomes. Alterations of the cell surface of the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought by the action of solar light result in a decreased uptake and binding of NR. Cell viability is determined by Neutral Red Uptake, measuring the optical density of the NR extract at 540 nm in a spectrophotometer. Cell viability of HEKa or HDFa after treatment with 1 mg/mL Preventhelia, was compared with the cell viability of irradiated (CTR+UV) and non-irradiated (CTR-UV) control cells.
DNA protection
Melanocytes (105 cells/plate) were incubated with three different concentrations of Preventhelia (0.01 ?g/mL, 0.1 ?g/mL and 1 ?g/mL) for two hours at 37°C. After this contact period, cells were irradiated with UVA (1.0 J/cm2) for no more than 3 min at 4°C. Negative controls included non-irradiated untreated cells and non-irradiated cells treated with 0.01 ?g/mL, 0.1 ?g/mL and 1 ?g/mL Preventhelia (the new peptide). UVA irradiated cells without the new peptide were used as positive controls. Finally, UVA-induced DNA breaks were analysed by the alkaline comet assay.
DNA repair
The aim of this study was to evaluate the effects of the new peptide in cellular DNA repair systems on skin cells and to further determine whether it might promote the normal DNA repair capacity. The DNA damage and the repair process were quantified by the comet assay. During the DNA repair process the breaks are rejoined and the extent of DNA migration from the treated nuclei in the assay decreases. Human dermal fibroblasts cultures were irradiated with UVB light (0.04 J/cm2) for 30 seconds in order to cause DNA damage. Immediately after irradiation, cells were exposed to different concentrations of the new peptide (0.25 ?g/mL and 0.5 ?g/mL). Then, the DNA damage was analysed by the comet assay at 15 min (control of maximum DNA damage) and 3 h after irradiation to determine the DNA reparation course. Negative controls included non-irradiated untreated cells and non-irradiated cells treated with the new peptide for 3 h. UV-irradiated cells analysed 15 minutes after irradiation were used as controls of maximum DNA damage.
Results and discussion
Quenching ability towards 4-hydroxynonenal
The new peptide was able to quench 4-HNE by 84.16% after 3 hours, and 95.3% after 6 hours of treatment (Fig. 3). These results were clearly superior to the quenching ability of carnosine with the same experimental conditions, and show that the product has an excellent quenching profile against RCS.
Photoprotection test
The new peptide proved to have a significant photoprotective effect on human epidermal keratinocytes and human dermal fibroblasts cell cultures at the tested concentration. 1 mg/mL of the new peptide induced a 92% increase in cell viability of HEKa respect to irradiated control cells. HDFa treated with the new peptide reached a 76.9% cell viability respect to a 100% cell viability in non-irradiated control cells. When compared to irradiated control cells, Preventhelia was able to increase cell viability by more than 13,000% (Fig. 4 and Fig.5).
DNA protection
Results show that no increase in DNA damage could be detected in the melanocytes treated with three different concentrations of the new peptide when compared to the control cells (Fig. 6). Pre-treatment with 3 different concentrations of tested product was able to decrease UVA-induced DNA lesions (Table 1). The product was shown to have an internal photoprotection capacity against UVA radiation with a doseresponse relationship (p<0.001).
DNA repair
As expected, 15 minutes after UVB irradiation (0 h post-irradiation), a significant percentage of DNA tail was induced in the control cells (non-treated cells), which decreased after 3 hours by the action of the normal DNA repair systems indicating that the assay was correct. There were no significant differences between Preventhelia treated and untreated cells at 15 minutes after irradiation. However, 3 hours after irradiation, the percentage of DNA tail generated was significantly reduced on new peptide treated cells compared to the control cells (Fig. 7). Results show that 3 hours after irradiation, the percentage of DNA damage decreased (Fig. 8), and consequently the DNA repair capacity increased significantly (p=0.002) with Preventhelia treatment. It can be therefore concluded that after UV irradiation, the new peptide is able to promote 1.7-fold (69%) the normal DNA repair system capacity.
Conclusions
The effects of chronological ageing are accelerated by extrinsic factors such as acute UV exposure. Preventhelia is a new peptide for cosmetic applications that has proved to present a photoprotective effect on two types of skin cells, as shown by the increased cell viability after treatment with the tested peptide. The new peptide is able to quench the most cytotoxic product of lipid peroxidation, 4-hydroxynonenal, therefore inhibiting the formation of carbonylated proteins and preventing DNA damage by the inhibition of 4-HNE-protein adducts which could inhibit NER capacity through its direct interaction with proteins involved in DNA repair. In vitro tests with different concentrations of the new peptide support the product’s efficacy: it protects skin cells from UVA-induced DNA damage and it is able to promote the DNA repair system capacity. The overall result is a complete skin protection to minimise the effects of intrinsic and extrinsic ageing. The new peptide is seen as a photoprotectant active for daily use, able to reverse and prevent the damage caused directly or indirectly by UV irradiation to DNA and proteins.
References
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