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Detection and Identification of Free Radicals Generated by UV and Visible Light in Ex Vivo Human Skin
Abstract
Citation: IFSCC Magazine , 11 (2008) (3) 207–215
Contrary to the skin biological end points used for determination of the sun protection factor and UVA protection factor, generation of excess free radicals in skin – mainly reactive oxygen species – is potentially the source of much skin damage and so represents a more general biophysical answer to the effects of sun exposure of different wavelengths. By applying electron spin resonance spectroscopy to human skin biopsies, we determined in previous work a free radical action spectrum covering the ultraviolet and visible light range. Convolution of the action spectrum with sunlight spectral irradiance (280–700nm) showed the importance of visible light in free radical generation. This unexpected finding led us to perform further investigations. Firstly, an existing sun simulator was modified so that its output truly mimics the sun's full spectrum, including visible light. Human skin biopsies were irradiated either by this device or a conventional UV source, confirming our previous calculations: half of the free radicals are generated in the 400–700nm visible wavelength range. Secondly, the visible spectrum of the modified sun simulator was divided into narrow‐band lights using different pairs of short‐ and long‐pass filters. Human skin biopsies impregnated with specific spin traps were exposed to the different narrow‐band lights to identify different types of free radicals. Generation of dangerous radical species like • O2‐, • OH and • CH‐R was observed in different parts of the visible range, confirming the relevance of the free radical action spectrum and its ability to predict skin protection.
Keywords: UV + VIS solar simulator, ex vivo human skin, free radicals, action spectrum, Electron Spin Resonance
... Under which conditions the mixture switches from beneficial to deleterious has remained an unanswered question of general importance. Here we pre sent evidence for the existence of a universal free radical threshold value (FRTV), which defines the borderline between the advantageous and adverse effects of free radicals in the human body [17] . ...
... ESR Spectroscopy ESR x-band spectroscopy was performed in all experiments under the conditions described in detail by Zastrow and colleagues [10,12,17] . Spin trap PBN (phenyl-N-tert-butylnitrone) and spin label PCA [3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy] allowed the quantification of the total amount of free radicals under varying irradiation conditions. ...
... Spin trap PBN (phenyl-N-tert-butylnitrone) and spin label PCA [3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy] allowed the quantification of the total amount of free radicals under varying irradiation conditions. DMPO (5,5-dimethyl-1-pyrroline-N-oxide) and DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide) spin traps were used to distinguish carbon-centered LOS from free ROS, respectively [17] . ...
Three evolutionary sources create 'primary' reactive oxygen species (ROS) and 'secondary' lipid oxygen species (LOS), forming the human body's 'free radical ground state'. We present evidence for the existence of a universal free radical threshold value (FRTV), defining the borderline between advantageous and adverse effects of free radicals observed above the free radical ground state. Based on standard vitamin D doses, the calculated amount of ∼3.5 × 10(12) rad/mg ROS/LOS tissue represents the tolerated number of free radicals in skin tissue - defined as FRTV. By means of quantitative ESR x-band spectroscopy, the FRTV was experimentally verified using ex vivo human skin irradiated with ultraviolet + visible (UV + VIS), UVB + UVA and VIS light. In addition, we investigated whether this threshold is also existent in internal organs by extending our experiment to fresh porcine liver. Based on the determination of ROS/LOS below and above the FRTV, ROS > LOS was characterized as beneficial and LOS > ROS as deleterious to the organism, respectively. Results of the experiments using porcine liver confirmed the appearance of the FRTV at radical generation ∼3.5 × 10(12) rad/mg. The relation ROS/LOS before and after the FRTV was consistent with the results determined for the skin. We conclude that the FRTV, theoretically calculated and experimentally confirmed, should be considered as a new 'universal body constant'. © 2015 S. Karger AG, Basel.
... Several research groups are actively investigating visible light (VL) effects on skin. In addition to increasing pigmentation, VL has caused melanin photosensitization, generating singlet oxygen ( 1 O2), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and cell death (Assis et al., 2021;Mahmoud et al., 2010;Zastrow et al., 2009;Setlow et al., 1993;Tonolli;Baptista;Chiarelli-Neto, 2021;Tonolli et al, 2020;Chen et al., 2019;Chen et al., 2021). ...
Melanoma is the most aggressive type of skin cancer due to its metastatic potential, and its incidence has increased considerably in recent decades. Sun exposure to ultraviolet A and B irradiation remains the main risk factor for the development of this type of cancer, however, other factors involved are family history, artificial tanning, melanocytic nevi, and low phototype and feomelanin photosensitization by visible light (VL). Through early diagnostic detection of the malignant lesion, it is possible to initiate therapy in an early and curative way, increasing survival with a subsequent decrease in the overall mortality rate. Advances in immunotherapy and targeted therapy have changed treatment paradigms and improved the prognosis of late-stage disease. Due to the high prevalence of the disease worldwide, the purpose of this review was to characterize the cutaneous melanoma (epidemiology, etiology, pathophysiology, clinic, diagnosis, treatment, prognosis, prevention and psychosocial aspects) such as prevention of the melanocytes against Visible Light (VL).
... This work showed that depletion of the ascorbate pool in the antioxidant defense made the skin more vulnerable to UV damage. In addition, the generation of superoxide anion radical (O 2 2 ), hydroxyl radical ( • OH), and • CH-R were detected in human skin biopsies irradiated with visible light [29]. According to this study, visible light is responsible for half of the free radicals produced in the skin due to sun exposure. ...
Recent advances in the research into skin protection from damage caused by the sun, clearly illustrates the need to adjust photoprotection policies. The old idea of sunbathing for short periods of time (without using sunscreen) and avoiding overexposure, even with filter application, seems to be the best option available. Comprehensive photoprotection necessarily requires new technologies and we hypothesize that nanoscience will play important roles in this regard. We separate possible fields of development into sun blocking, antioxidant, and excited-state suppressors and membrane protection. Examples of each of the developments in these fields involving nanoscience are described and analyzed in this chapter.
... [14][15][16] Like UVA, HEVIS appears to exert its effects mainly through the generation of ROS, accounting for a substantial part of the amount generated by natural midday sunlight in human skin. 17,18 Blue light photon ROS production efficacy corresponds to 25% of UVA in human keratinocyte mitochondria. 19 ROS detoxification in skin is achieved by low molecular weight antioxidants, such as Vitamins C and E, and carotenoids like β-carotene, as well as by enzymes and antioxidant proteins, many under the control of nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of cellular redox signaling and antioxidant defenses. ...
Background:
Solar radiation causes skin damage through the generation of reactive oxygen species (ROS). While UV filters effectively reduce UV-induced ROS, they cannot prevent VIS-induced (400 - 760 nm) oxidative stress. Therefore, potent antioxidants are needed as additives to sunscreen products.
Methods:
We investigated VIS-induced ROS formation and the photoprotective effects of the Nrf2-inducer Licochalcone A (LicA).
Results:
VIS of 400-500 nm dose-dependently induced ROS in cultured human fibroblasts at doses equivalent to 1 hour of sunshine on a sunny summer day (150 J/cm2 ). A pretreatment for 24 hours with 1 µM LicA reduced ROS formation to the level of unirradiated cells while UV filters alone were ineffective, even at SPF50+. In vivo, topical treatment with a LicA-containing SPF50+ formulation significantly prevented the depletion of intradermal carotenoids by VIS irradiation while SPF50+ control did not protect.
Conclusion:
LicA may be a useful additive antioxidant for sunscreens.
... In the near future, PBN or other suitable spin traps (along with other actives/antioxidants) may also need to be included in cosmetic products to reduce or stop ROS and other free radicals generated by high frequency-high energy (HFHE) visible light (λ = 400-450 nm) [97,98], near-infrared light (NIR) (λ = 760-3000 nm) and blue light (λ = 450-495 nm). This is important because the free radicals generated by the HFHE visible light can potentially affect 40 skin related genes [98] that can attenuate the healing process leading to weak barrier functions, inflammation and un-even pigmentation to the skin. ...
Pollution from air and sunlight has adverse effects on human health, particularly skin health. It creates oxidative stress, which results in skin diseases, including skin cancer and aging. Different types of antioxidants are used as preventative actives in skin-care products. However, they have some limitations as they also scavenge oxygen. Recently, spin traps are being explored to trap free radicals before these radicals generating more free radicals (cascading effect) and not the oxygen molecules. However, not all spin traps can be used in the topical cosmetic skin-care products due to their toxicity and regulatory issues. The present review focuses on the different pathways of reactive oxygen species (ROS) generation due to pollution and the potential use of spin traps in anti-pollution cosmetics to control ROS.
... Sunlight (especially UVA-Visible penetrating deeper into the skin [1]) has a profound effect on the skin causing skin ageing, skin cancer, and a host of skin changes. Many skin changes that were commonly believed to be due to ageing, such as easy bruising, are actually a result of prolonged exposure to sun radiation. ...
The aim of this study is to develop a new kind of R&D methodology through a VHDL-AMS functional virtual prototype (FVP) applied to cosmetic industries. Cosmetic firms are in the process of improving R&D methodologies with more formal methods. These studies are mostly based on functional models and less laboratory testing. Hence, in cooperation with Lancaster firm, we have built for the first time a unique dynamic model of biophysical and chemical phenomenons linked to human skin and sun spectrum. Human health can be strongly influenced by exposure to solar radiation. Relevant interactions regarding to health take place mainly in the skin. So, the optics of human skin is of the utmost importance with reflection and absorption properties. By combining a sun emission light model and a bio-optical human skin model, we can compute the effects of the sun radiations on various species including free radicals generation.
Melanoma is the most aggressive type of skin cancer due to its metastatic potential, and its incidence has increased considerably in recent decades. Sun exposure to ultraviolet A and B rays remains the main risk factor for the development of this type of cancer, however, other factors involved are family history, artificial tanning, melanocytic nevi, low phototype and melanin photosensitization by visible light (VL). Through early diagnostic detection of the malignant lesion, it is possible to initiate therapy in an early and curative way, increasing survival with a subsequent decrease in the overall mortality rate. Advances in immunotherapy and targeted therapy have changed treatment paradigms and improved the prognosis of late-stage disease. Due to the high prevalence of the disease worldwide, the objective was to review the main aspects described in the literature regarding the etiology, pathophysiology, clinic, diagnosis, and management of the pathology, with special emphasis on cutaneous melanoma upon VL exposure.
Photoexcitation of endogenous photosensitizers (ePS) in the human skin by sunlight culminates with the formation of electronic excited states, such as triplet excited states, singlet oxygen, and a variety of reactive oxygen and nitrogen species, either free radicals or two-electron oxidants. Considering the endogenous skin photosensitizers, melanin is the most abundant and is involved in photoprotection mechanisms, while lipofuscin is an unintentionally pigment accumulated during cell aging or oxidative stress. Both pigments severely increase the phototoxicity of visible light (VL) to skin cells. In the presence of these pigments, VL induces significant oxidative damage in nucleic acids, lipids and proteins, triggering regulated and unregulated cell death mechanisms. Besides, there is accumulation of premutagenic DNA lesions, indicated by the formamidopyrimidine glycosylase (Fpg)- and endonuclease III (Endo III)-sensitive sites in the nuclear DNA. As consequence of the photoinduced oxidative damage, skin cells, under the stimulus of VL release pro-inflammatory cytokines and metalloproteinases, prompting further cell death and skin aging. In this review we describe the photochemical properties of both melanin and lipofuscin pigments and discuss the consequences of their accumulation in terms of the phototoxicity of VL to the human skin.
Organic UV filters utilized in sunscreen have been developed targeting more efficient UV protection in their history. Sunscreens in the early days focused on prevention of sunburn mainly caused by UVB; thus organic UVB filters became the first commercial examples of synthetic organic UV filters. Among them, Ethylhexyl methoxycinnamate (EHMC) became most popular due to balanced performance and handling advantages. In the 1970s, Butyl methoxydibenzoylmethane (BMBM) was commercialized as the first organic UVA filter, reflecting increased awareness of photoaging mainly caused by UVA. Meanwhile, however, the photostability of EHMC and BMBM had been discussed more often. Therefore, photostability is considered an important characteristic in the molecular design of new-generation organic UV filters. In addition to conventional oil/water soluble organic UV filters, aqueous dispersion of particulate organic UV filers became available. Due to aqueous dispersion, these particulate organic UV fitters provide water phase protection, which efficiently prevent UV light from passing through holes originating from the water phase. These particulate organic UV filters can also provide so-called “Triple Action” efficient UV protection through the combination of absorption, reflection and multi-scattering. In addition to properties and effects, modern UV filters must satisfy broad needs of product safety and regulation from the viewpoint of sustainability. An overview of recent regulatory updates related to organic UV filters is briefly summarized here.
Zusammenfassung Es wird experimentell gezeigt, dass die Bildung von freien Radikalen – hauptsächlich reaktiven Sauerstoffspezies (ROS) – die
allgemeine photobiologische Antwort für die Haut-Sonnenlicht-Wechselwirkung darstellt. Das freie Radikal-Aktionsspektrum (Wellenlängenabhängigkeit)
für ultraviolettes und sichtbares Licht (280–700 nm) wird mittels quantitativer ESR bestimmt. Sichtbares Licht produziert
etwa 50% des totalen oxidativen Stresses, generiert durch Sonnenlicht. Wie in den anderen Bereichen können hoch reaktive •O-
2-, •OH- und •CHR-Radikale in dieser Region identifiziert werden. Die Menge der erzeugten Radikale korreliert mit der Beleuchtungsstärke
(Lux), die auf der Haut erreicht wird. Die Erzeugung freier Radikale durch nahes Infrarot (NIR, 700–1600 nm) in menschlicher
Haut wird gezeigt. Die Radikalgenerierung ist von der Bestrahlungsstärke und der durch das NIR in der Haut induzierten stationären
Temperatur abhängig. Die Temperaturabhängigkeit der Radikalbildung folgt der physiologischen Fieberkurve des Menschen. Über
das gesamte aktive Sonnenspektrum werden im menschlichen Hautgewebe freie Radikale des gleichen Typs erzeugt. Der Bereich
der schädlichen oder nützlichen Wirkung von Sonnenlicht definierter Qualität könnte künftig durch die Einführung eines Schwellenwertes
für freie Radikale in der menschlichen Haut charakterisiert werden.
The demand for evidence-based cosmetic treatments against visible signs of skin aging is growing. Nonsurgical procedures in particular are becoming more and more popular. Although until recently the primary aim in cosmetic dermatology was softening of facial lines and wrinkles, replacement of volume loss and facial contouring is now rapidly catching up. However, modern cosmetic dermatology takes into consideration all three key areas of skin aging ("3-D" approach): (1) wrinkles and lines, (2), loss of volume and contour, and (3), skin surface and texture impairment. A "holistic" approach accessing the entire face, and combining different treatment modalities, achieves best aesthetic results. This article presents minimal-invasive treatment modalities to address all three key areas of facial skin aging. In addition novel treatment modalities and combinations are discussed. Overall aesthetic results are now aimed to be more "natural" appearing, achieving a harmonious look, and should include evaluation and treatment of extra-facial, exposed skin such as the neck, décolletage, and hands.
The ultraviolet (UV) region of solar radiation is a critical factor in the initiation and development of a number of skin diseases. However, it is not only skin which is directly exposed to solar light that is affected by UV radiation, through low molecular weight mediators, generated upon irradiation, "non-skin" tissues can also be affected. The aim of this study was to examine in detail, the acute effects of UVA and UVB wavebands on hairless mice. Female SKH-1 hairless mice were exposed to a single dose of UVB (200, 800 mJ/cm(2)) or UVA (10, 20 J/cm(2)) using a solar simulator. The effects on haematological parameters, activity and/or expression of antioxidant enzymes, level of glutathione (GSH), markers of oxidative damage (lipid peroxidation and carbonylated proteins) were analysed in erythrocytes, plasma, liver and whole skin homogenates. No macroscopic changes were observed either 4 or 24 h after UVA/UVB exposure. The blood count showed a significant increase in leukocyte number and reduction of platelets 4 h following UVA and UVB irradiation, which disappeared 24 h after irradiation except for the higher UVA dose. Changes in oxidative stress-related parameters, particularly activity of catalase (CAT) and superoxide dismutase (SOD) and level of GSH and lipid peroxidation products, were found in skin, erythrocytes and liver. The expression of several enzymes (CAT, SOD, glutathione transferase (GST), nicotinamide adenine dinucleotide (phosphate) quinone oxidoreductase (NQO1) and hem oxygenase-1 (HO-1)) in skin was affected following UVA and UVB radiation. Increase in carbonylated proteins was found in plasma and skin samples.
Fractionated melanin (Mel-HEV), a bleached version of natural melanin, offers protection against the high energy visible (HEV/UVA) and ultraviolet (specifically UVA) irradiation making it a potential compound to be added to skin care and sunscreen formulations and other cosmetic and personal care products. Chlorpromazine (CPZ) has been shown to exhibit photosensitivity and phototoxicity reaction in vitro and in vivo. Comparative evaluation of chemotoxicity and phototoxicity using Mel-HEV and CPZ (as positive control) was performed on mouse fibroblast cell line 'Balb/c 3T3'. This is the recommended method for evaluating the phototoxic potential of compounds under the European Center of Validation of Alternative Methods (ECVAM) guidelines (OECD, 2004). This study was expanded from a mouse cell line - Balb 3T3/c to two human cell lines - HDF and HEKn for two reasons: to compare the difference between the sensitivity and behavior of two fibroblast cell lines (Balb/c 3T3 vs. HDF) and to compare the differences between two fibroblast cell lines with the keratinocyte cell line (HDF & Balb/c 3T3 vs. HEKn). It was found that Balb/c 3T3 and HEKn were both sensitive to the phototoxic potential of CPZ. However, HDF showed insensitivity to phototoxic evaluation. The test compound, Mel-HEV, was found to be non-phototoxic. The mean toxic concentration (MTC) for CPZ during HEV and UVA exposure conditions was found to be similar using Balb/c 3T3 (36.25 μg/ml) and HEKn (39.99 μg/ml) showing that cells exhibit similar responses at HEV/UVA- conditions. However, Balb/c 3T3 showed more sensitivity to CPZ at HEV/UVA+ condition (MTC=0.87 μg/ml; mean PIF=55.33; MPE=0.395) than HEKn (MTC=5.35 μg/ml; PIF=7.61; MPE=0.276) making it the preferred cell line for phototoxicity evaluations.
The proposed rules of the Food and Drug Administration (FDA) for the UVA in vitro testing procedure were applied to the evaluation of various sunscreen formulations and the following technical challenges were observed: when proposed roughened quartz substrates were used, the required coefficient-of-variation criteria were not met, and the dynamic ranges of the available transmittance analyzers were exceeded for sunscreens with high SPF values. In the proposed rules, the FDA requested comment regarding the suitability of other possible substrates. In this research, two modifications to the FDA's proposed rules were evaluated: (a) the use of an alternative substrate, Vitro Skin N-19 (IMS, Inc.) instead of roughened quartz substrate and (b) an increase in application time from 10 seconds to 30 seconds to ensure a uniform distribution of sunscreen product over the application area of the substrate. These two modifications allowed meeting the required coefficient-of-variation criteria without exceeding the dynamic ranges of the available transmittance analyzers. The modified test conditions were utilized for the evaluation of six commercial sunscreens, which fulfilled criteria of "medium" or "high" categories-based on their UVAI/UV ratios. These findings were in agreement with the statement in the proposed rules that the FDA is aware of the difficulty for current sunscreen formulations to meet the "highest" category and believes that allowing such a category will foster additional research and development in this area. To determine if it was possible to achieve a UVA rating greater than 0.95, two experimental sunscreen prototypes with bisoctrizole (USAN), bemotrizinol (USAN), avobenzone, and octocrylene were tested under the modified test conditions and attained the "highest" category. It should be noted that bisoctrizole and bemotrizinol are being evaluated by the FDA under TEA and are not permitted in the US at this time, but they are approved for use in the rest of the world.
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