Skin Chromophores Research Articles

Endogenous UVA-photosensitizers: mediators of skin photodamage and novel targets for skin photoprotection

Endogenous chromophores in human skin serve as photosensitizers involved in skin photocarcinogenesis and photoaging. Absorption of solar photons, particularly in the UVA region, induces the formation of photoexcited states of skin photosensitizers with subsequent generation of reactive oxygen species (ROS), organic free radicals and other toxic photoproducts that mediate skin photooxidative stress. The complexity of endogenous skin photosensitizers with regard to molecular structure, pathways of formation, mechanisms of action, and the diversity of relevant skin targets has hampered progress in this area of photobiology and most likely contributed to an underestimation of the importance of endogenous sensitizers in skin photodamage. Recently, UVA-fluorophores in extracellular matrix proteins formed posttranslationally as a consequence of enzymatic maturation or spontaneous chemical damage during chronological and actinic aging have been identified as an abundant source of light-driven ROS formation in skin upstream of photooxidative cellular stress. Importantly, sensitized skin cell photodamage by this bystander mechanism occurs after photoexcitation of sensitizers contained in skin structural proteins without direct cellular photon absorption thereby enhancing the potency and range of phototoxic UVA action in deeper layers of skin. The causative role of photoexcited states in skin photodamage suggests that direct molecular antagonism of photosensitization reactions using physical quenchers of photoexcited states offers a novel chemopreventive opportunity for skin photoprotection.

Noninvasive reflection spectra provide quantitative information about the spatial distribution of skin chromophores

In this work, a new method of analyzing noninvasive reflection spectra is presented. The approach explicitly models the inhomogeneity of chromophore distributions in living tissues and thus extracts not only apparent chromophore concentrations but also relative chromophore distributions in tissues. Furthermore, it works with spectra obtained with short source-detector separations where the diffusion theory of light transport through turbid media is not valid, and formerly presented methods thus fail. The effect of inhomogeneously distributed chromophores in a multicompartment model of tissues on measured reflection spectra is explained and an algorithm to deconvolute tissue spectra based on this model is presented. It is evaluated using simulated spectra and measurements on phantoms, which are made up of partially printed pieces of paper to simulate inhomogeneous dye distributions. Its applicability to real tissue is proven using reflection spectra obtained with 130 microm source-detector separation from a hemoperfusion stop experiment. The proposed model accurately determines apparent chromophore concentrations and corresponding distributions in simulated spectra and phantoms. Regarding real tissue spectra, the results correspond to former publications and the spectral reconstruction yields only minimal residuals, indicating a complete and accurate spectral deconvolution. In conclusion, the presented approach is a suitable extension and amendment to existing models of light transport through inhomogeneous samples.

Determination of optimal view angles for quantitative facial image analysis

In quantitative evaluation of facial skin chromophore content using color imaging, several factors such as view angle and facial curvature affect the accuracy of measured values. To determine the influence of view angle and facial curvature on the accuracy of quantitative image analysis, we acquire cross-polarized diffuse reflectance color images of a white-patched mannequin head model and human subjects while varying the angular position of the head with respect to the image acquisition system. With the mannequin head model, the coefficient of variance (CV) is determined to specify an optimal view angle resulting in a relatively uniform light distribution on the region of interest (ROI). Our results indicate that view angle and facial curvature influence the accuracy of the recorded color information and quantitative image analysis. Moreover, there exists an optimal view angle that minimizes the artifacts in color determination resulting from facial curvature. In a specific ROI, the CV is less in smaller regions than in larger regions, and in relatively flat regions. In clinical application, our results suggest that view angle affects the quantitative assessment of port wine stain (PWS) skin erythema, emphasizing the importance of using the optimal view angle to minimize artifacts caused by nonuniform light distribution on the ROI. From these results, we propose that optimal view angles can be identified using the mannequin head model to image specific regions of interest on the face of human subjects.

Identification of quenchers of photoexcited States as novel agents for skin photoprotection.

Photooxidative stress is a key mechanism in UVA-induced skin photodamage. Photoexcited states of endogenous UVA chromophores such as porphyrins, melanin precursors, and cross-link-fluorophores of skin collagen exert skin photodamage by direct reaction with substrate molecules (type I photosensitization) or molecular oxygen (type II), leading to formation of reactive oxygen species. Based on our previous research on the role of photoexcited states of endogenous skin chromophores as sensitizers of photooxidative stress, we describe here the identification of a novel class of chemopreventive agents for topical skin photoprotection: quenchers of photoexcited states (QPES). QPES compounds antagonize the harmful excited state chemistry of endogenous sensitizers by physical quenching, facilitating the harmless return of the sensitizer excited state to the electronic ground state by energy dissipation. To identify QPES compounds suitable for development, we designed a primary screening assay based on QPES suppression of photosensitized plasmid cleavage using conditions that exclude antioxidants. This screen is followed with a screen to test for nonsacrificial quenching of dye-sensitized singlet oxygen ((1)O(2)) formation by electron paramagnetic resonance detection of 2,2,6,6-tetramethyl-piperidine-1-oxyl, a stable free radical indicative of (1)O(2) formation. These initial screens identified a pyrrolidine pharmacophore with pronounced QPES activity, and l-proline and other noncytotoxic proline derivatives containing this pharmacophore were then screened for efficacy in cellular models of sensitized photodamage. These compounds showed QPES protection against dye-sensitized and psoralen-UVA-induced apoptosis and suppression of proliferation in cultured human skin keratinocytes and fibroblasts. Furthermore, QPES photoprotection of reconstructed full thickness human skin exposed to solar simulated light has been demonstrated.

3-Hydroxypyridine Chromophores Are Endogenous Sensitizers of Photooxidative Stress in Human Skin Cells

Photocarcinogenesis and photoaging are established consequences of chronic exposure of human skin to solar irradiation. Accumulating evidence supports a causative involvement of UVA irradiation in skin photo-damage. UVA photodamage has been attributed to photosensitization by endogenous skin chromophores leading to the formation of reactive oxygen species and organic free radicals as key mediators of cellular photooxidative stress. In this study, 3-hydroxypyridine derivatives contained in human skin have been identified as a novel class of potential endogenous photosensitizers. A structure-activity relationship study of skin cell photosensitization by endogenous pyridinium derivatives (pyridinoline, desmosine, pyridoxine, pyridoxamine, pyridoxal, pyridoxal-5'-phosphate) and various synthetic hydroxypyridine isomers identified 3-hydroxypyridine and N-alkyl-3-hydroxypyridinium cation as minimum phototoxic chromophores sufficient to effect skin cell sensitization toward UVB and UVA, respectively. Photosensitization of cultured human skin keratinocytes (HaCaT) and fibroblasts (CF3) by endogenous and synthetic 3-hydroxypyridine derivatives led to a dose-dependent inhibition of proliferation, cell cycle arrest in G2/M, and induction of apoptosis, all of which were reversible by thiol antioxidant intervention. Enhancement of UVA-induced intracellular peroxide formation and p38 mitogen-activated protein kinase-dependent stress signaling suggest a photooxidative mechanism of skin cell photosensitization by 3-hydroxypyridine derivatives. 3-hydroxypyridine derivatives were potent photosensitizers of macromolecular damage, effecting protein (RNase A) photocross-linking and peptide (melittin) photooxidation with incorporation of molecular oxygen. Based on these results, we conclude that 3-hydroxypyridine derivatives comprising a wide range of skin biomolecules, such as enzymatic collagen cross-links, B6 vitamers, and probably advanced glycation end products in chronologically aged skin constitute a novel class of UVA photosensitizers, capable of skin photooxidative damage.

Application of Image-Based Skin Chromophore Analysis to Cosmetics

Application of Image-Based Skin Chromophore Analysis to Cosmetics

Analysis of pulsed photoacoustic signal: theoretical study of the diffusion of chromophores in the human skin

Pulsed photoacoustic spectroscopy (PPAS) provides a convenient means for in vivo and in situ monitoring of human skin properties and surface concentrations of locally applied substances, such as drugs and cosmetics. In this article, we applied the one-dimensional theoretical model of Mandelis and Royce (J. Appl. Phys. 50, 6 (1979)) to simulate the photoacoustic response of human skin and diffusion phenomena through it, for various optical absorption coefficients and sample thicknesses. This approach is analyzed through the temperature and pressure variations in the photoacoustic cell.

Some photophysical studies of cis- and trans-urocanic acid.

Urocanic acid, an important human skin chromophore, undergoes a variety of photochemical transformations when exposed to the near-UV portion of sunlight and natural daylight, the principal reaction being the transformation from the stable trans- or (E)-form of the chromophore (trans-UA) to the biologically active cis- or (Z)-form (cis-UA), which is claimed to induce immunosuppression linked to the onset of skin cancer. This study is concerned with the comparative photophysical behaviour of the two urocanic acid isomers in aqueous solution using both continuous irradiation and pulsed irradiation techniques. The UV absorption maximum for both isomers occurs in the region of 270 nm with the absorption shape varying characteristically with pH, the cis-isomer showing a lower overall molar absorptivity. Both isomers exhibit weak fluorescence (quantum yields estimated to be less than 10(-4)) with each isomer showing small differences in the way in which pH and excitation wavelength influence the fluorescence emission characteristics. Pulsed nanosecond laser irradiation at 266 nm of aqueous solutions at pH 7 shows that both isomers undergo photo-ionisation with a quantum yield of 0.02 for the hydrated electron production, a quantum yield value comparable with that for photoisomerisation at this wavelength. Laser flash studies also show that the photo-ionised species reacts efficiently with oxygen (quenching rate kQ = 1.3 x 10(9) M(-1) s(-1)), while some preliminary experiments indicate that both cis- and trans-urocanic acids react with the semiquinone radical of L-3,4-dihydroxyphenylalanine (L-DOPA) with a fast reaction rate constant of approximately 5 x 10(7) M(-1) s(-1). The photophysical characteristics of trans-UA and cis-UA reported here are discussed in the context of other recent pulsed irradiation studies on urocanic acid over nanosecond and picosecond time scales, in an attempt to clarify the complex photo-behaviour of this interesting biomolecule.

Non-Invasive Method for Quantitative Evaluation of Exogenous Compound Deposition on Skin

Topical application of active compounds on skin is common to both pharmaceutical and cosmetic industries. Quantification of the concentration of a compound deposited on the skin is important in determining the optimum formulation to deliver the pharmaceutical or cosmetic benefit. The most commonly used techniques to date are either invasive or not easily reproducible. In this study, we have developed a noninvasive alternative to these techniques based on spectrofluorimetry. A mathematical model based on diffusion approximation theory is utilized to correct fluorescence measurements for the attenuation caused by endogenous skin chromophore absorption. The limitation is that the compound of interest has to be either fluorescent itself or fluorescently labeled. We used the method to detect topically applied salicylic acid. Based on the mathematical model a calibration curve was constructed that is independent of endogenous chromophore concentration. We utilized the method to localize salicylic acid in epidermis and to follow its dynamics over a period of 3 d.

Mechanisms of photodamage of the skin and its functional consequences for skin ageing

Chronic photodamage of the skin manifests itself as extrinsic skin ageing (photoageing) and photocarcinogenesis. DNA photodamage and UV-generated reactive oxygen species are the initial molecular events that lead to most of the typical histological and clinical manifestations of chronic photodamage of the skin. Knowledge of the UV-absorbing chromophores in the skin and of the molecular mechanisms leading to the unwanted effects of sun exposure provide a basis for the development of novel strategies for the prevention and repair of photoageing. This review provides an overview of the photochemistry of the major skin chromophores and their relationship to chronic photodamage.

Aging and Effects of Ultraviolet A Exposure may be Quantified by Fluorescence Excitation Spectroscopy In Vivo

The fluorescence properties of skin chromophores such as tryptophan and collagen cross-links might be useful markers of aging and photoaging. As the fluorescence of pepsin-digestible collagen cross-links was found to increase with aging and decrease with photoaging we investigated the characteristics of this dependence. In vivo fluorescence excitation spectra (emission at 380 nm) of SKH hairless mouse model skin are characterized by two bands centered near 295 nm and 335 nm due, respectively, to epidermal tryptophan moieties and pepsin-digestible collagen cross-links. Several groups of hairless mice were followed over a period of 18 mo to document changes in skin fluorescence with aging. Other groups of animals were exposed to either broad band or narrowband ultraviolet A radiation to determine the effects of ultraviolet A exposure on the fluorescence of the dermal collagen cross-links and to determine an action spectrum for the induced changes. We also found that the intensity of pepsin-digestible collagen cross-links in vivo increases linearly with age and that the fluorescence of epidermal tryptophan decreases linearly with age. We found that the fluorescence of pepsin-digestible collagen cross-links decreases immediately following exposure to ultraviolet A whereas epidermal tryptophan fluorescence increases. Both changes were dose dependent but the increase in tryptophan fluorescence occurred exclusively in young animals (2--6 mo old). We found that the ultraviolet-induced fluorescence decrease of pepsin-digestible collagen cross-links is wavelength specific. The action spectrum for the ultraviolet A effect on the in vivo fluorescence of pepsin-digestible collagen cross-links shows a distinct maximum at 335 nm that corresponds to the maximum in the fluorescence excitation spectrum due to pepsin-digestible collagen cross-links. Our results seem to indicate that in vivo fluorescence of epidermal tryptophan moieties and collagen cross-links in the dermal matrix may serve as markers for skin aging, for photoaging, and for immediate assessment of exposure to ultraviolet A radiation.

Lasers for the dermatologist.

This article reviews the current uses of lasers in dermatological practice. It outlines safety issues and legislation and attempts to summarize the relevant physics and light interactions. The article is divided into sections pertaining to selective photothermolysis of structures containing the major skin chromophores: haemoglobin, melanin and water. The lasers used and conditions treated are discussed with reference to a literature review.

The Soluble Eumelanin Precursor 5,6‐Dihydroxyindole‐2‐carboxylic Acid Enhances Oxidative Damage in Human Keratinocyte DNA after UVA Irradiation‡

Soluble melanin precursors are present in serum and may act as skin chromophores contributing to UVR-induced oxidative damage. Our study aimed to determine whether the soluble eumelanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) photosensitizes DNA damage in human keratinocytes exposed to UVA irradiation. The HaCaT keratinocytes were incubated with and without DHICA, before irradiation with broadband UVA (320-400 nm). The DNA photodamage was assessed using the comet assay that detects frank single-strand breaks (SSB) and specific oxidative lesions with the addition of endonuclease III. Without DHICA incubation, there was no significant increase in SSB, compared to unirradiated cells, for doses up to 48.5 J/cm2 (< 1 minimum erythemal dose). Preincubation with 0.5 microM DHICA caused an increase in SSB at every UVA dose (significant from 12.1 to 48.5 J/cm2), while varying the DHICA concentration (0.125-2 microM) showed this effect to be concentration dependent such that SSB increased and endonuclease III-sensitive sites decreased with increasing DHICA concentration. The irradiation of cells in the presence of antioxidants (catalase, mannitol and histidine) suggests that DHICA-induced photosensitization is mediated via singlet oxygen and, to a lesser extent, hydroxyl radicals. These results indicate that DHICA can induce strand breaks with UVA at clinically relevant doses via a mechanism involving reactive oxygen species.

Urocanic Acid Photochemistry and Photobiology

Urocanic acid (2-propenoic acid, 3-[ lH-imidazol-4(5)-yl], UA)? is one of the smallest molecules to have stimulated global interest among biologists, environmentalists, photochemists, photobiologists, medicinal chemists and immunologists. Its history can be traced back more than a century to when it was first found in the urine of dogs (1). Interest in the molecule remained dormant until the middle of the century but rekindled in the late 1940s when it was detected in animal skin and sweat. This led to the proposal that UA acts as a natural sunscreen, possibly as a specific photoprotecting agent for DNA because of the overlap of the UA and DNA absorption spectra (2). Since 1983 there has been an explosive growth in UA research, primarily as a consequence of the proposal by De Fabo and Noonan (3) that the cis photoisomer (cUA) of trans-UA (tUA) could be responsible for the phenomenon of photoimmunosuppression. In a recent literature survey we found that about 25 research papers have appeared every year over the last decade involving UA. Several comprehensive reviews of the molecule's photobiology, photochemistry and photophysics have appeared (4-

Pulsed photoacoustic spectroscopy applied to the diffusion of sunscreen chromophores in human skin: the weakly absorbent regime

Pulsed photoacoustic spectroscopy was used to study the penetration of sunscreen chromophores into human skin. This study focuses on basic solutions containing single typical filter molecules, as used in current sunscreens, dissolved in mineral oil. The pulsed form of the photoacoustic technique was preferred because it provides more detailed information on the filter distribution within the different layers of human skin. A new methodology provides better insight into the diffusion process through signal analysis in the time and frequency domains, allowing for global and depth-related characterization. The penetration of the chromophore influences the response signal by inducing changes in the optical and thermal properties at different depths within the medium. The light scattering effect of titanium dioxide was demonstrated by the same technique.

Assessment of erythema and skin perfusion by digital image analysis and scanner laser Doppler.

In the present study, a model for measuring the intensity of skin erythema by computer-assisted digital image analysis (DIA) was elaborated. The DIA technique was compared with laser Doppler perfusion imaging (PIM) to evaluate the relationship between erythema and skin perfusion. To produce erythema, three different types of nicotine patches (Nicotinell®, Nicorette® and Nicorette® placebo) were randomly applied ventrally on the upper arms for the time period recommended by the manufactures. After removal of the patches, colour photographs for subsequent DIA were taken and laser Doppler scanning for PIM was performed. The relationship between PIM and DIA was estimated intra- and inter-individually. A statistically significant linear correlation between PIM and DIA data was found, but the relationship was relatively weak. This may be explained by the fact that DIA and PIM are influenced by different physiological factors such as skin chromophores and velocity of moving cells. The results strengthen the theory that erythema and perfusion measurements reflect separate physiological phenomena and that the methods should be considered complements to each other in the experimental situation.

Pulsed photoacoustic study of the diffusion of chromophores in human skin.

The technique of pulsed photoacoustic spectroscopy was used to investigate the diffusion of chromophores in human skin. The kinetic of diffusion has been studied for five solutions at different concentrations in a mixture of chromophores, as used in commercial sunscreens. In addition to the classical macroscopic interpretation of the diffusion process, a new method is shown to give more detailed information on chromophore presence at different depths in skin. For the first time, results are expressed in the frequency domain by means of the Fourier transform applied to the photoacoustic signal. The spectra are discussed versus the depth in skin samples and the time of diffusion kinetics. This new method of data analysis is shown to be very useful for understanding the influence of the internal structure of a medium on the penetration rate of chromophores into skin.

Depth determination of chromophores in human skin by pulsed photothermal radiometry

We report on the application of pulsed photothermal radiometry (PPTR) to determine the depth of in-vitro and in-vivo subsurface chromophores in biological materials. Measurements provided by PPTR in combination with a nonnegative constrained conjugate-gradient algorithm are used to determine the initial temperature distribution in a biological material immediately following pulsed laser irradiation. Within the experimental error, chromophore depths (50-450 µm) in 55 in-vitro collagen phantoms determined by PPTR and optical low-coherence reflectometry are equivalent. The depths of port-wine-stain blood vessels determined by PPTR correlate very well with their locations found by computer-assisted microscopic observation of histologic sections. The mean blood-vessel depth deduced from PPTR and histologic observation is statistically indistinguishable (p > 0.94).

Imaging laser heated subsurface chromophores in biological materials: determination of lateral physical dimensions

We describe a non-contact method using infrared radiometry to determine lateral physical dimensions of laser heated subsurface chromophores in biological materials. An imaging equation is derived that relates measured radiometric temperature change to the reduced two-dimensional temperature increase of laser heated chromophores. From measured images of radiometric temperature change, the lateral physical dimensions of chromophores positioned in an in vitro model of human skin are determined by deconvolution of the derived imaging equation using a non-negative constrained conjugate gradient algorithm. Conditions for optimum spatial resolution are found by analysis of a derived radiometric transfer function and correspond to superficial chromophores and/or weak infrared absorption in a laser irradiated biological material. Analysis indicates that if the infrared attenuation coefficient is sufficiently small (i.e., less than 10 ), infrared radiometry in combination with a deconvolution algorithm allows estimation of lateral physical dimensions of laser heated subsurface chromophores in human skin.

Depth profiling of laser-heated chromophores in biological tissues by pulsed photothermal radiometry

A solution method is proposed to the inverse problem of determining the unknown initial temperature distribution in a laser-exposed test material from measurements provided by infrared radiometry. A Fredholm integral equation of the first kind is derived that relates the temporal evolution of the infrared signal amplitude to the unknown initial temperature distribution in the exposed test material. The singular-value decomposition is used to demonstrate the severely ill-posed nature of the derived inverse problem. Three inversion methods are used to estimate solutions for the initial temperature distribution. A nonnegatively constrained conjugate-gradient algorithm using early termination is found superior to unconstrained inversion methods and is applied to image the depth of laser-heated chromophores in human skin.