Effect of UVA or UVB Irradiation on Cutaneous Lipids in Films or in Solution

The barrier function of the skin is largely due to the stratum corneum which is essentially composed of lipids. Different external factors, such as UV irradiation, affect this skin layer and are responsible for a destabilization of the supramolecular organization of its constituted lipids. In this work, mass spectrometry and infrared spectroscopy are combined to study the correlation between the formation of oxidative compounds by UV irradiation and the lipid organization. Experiments were carried out on unsaturated lipids in film or solution form, exposed to UVA or UVB irradiation. UV exposure leads to the formation of oxygenated entities in the case of lipids with an unsaturated fatty acid moiety, resulting in a decrease in their packing which is greater when the lipids are in solution. The packing decrease is even greater following UVB irradiation.     

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S). To investigate whether UVB susceptibility in humans is related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S. Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+). The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVBR and the UVB-S group. To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation. However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells. To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation. Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (1FN)-γ and not interleukin (IL)-4. In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization. Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.     

Fatty acids influence "solid" phase formation in models of stratum corneum intercellular membranes

Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skin's barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cer's small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.     

The role of cholesterol in UV light B-induced apoptosis

Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon UVB light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas-receptor (Fas) aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB irradiation. The subsequent events of death-inducing signaling complex formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Fas death domain-associated protein (Daxx) from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased nonapoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis.     

Penetration and growth of DPPC/DHPC bicelles inside the stratum corneum of the skin

The effect of dipalmitoyl phosphatidylcholine (DPPC)/dihexanoyl phosphatidylcholine (DHPC) bicelles on the microstructure of pig stratum corneum (SC) in vitro was evaluated. The physicochemical characterization of these nanoaggregates revealed small disks with diameters around 15 nm and a thickness of 5.4 nm. Upon dilution, the bicelles grow and transform into vesicles. Cryogenic scanning electron microscopy (cryo-SEM) images of the SC pieces treated with this system showed vesicles of about 200 nm and lamellar-like structures in the intercellular lipid areas. These vesicles probably resulted from the growth and molecular rearrangement of the DPPC/DHPC bicelles after penetrating the SC. The presence of lamellar-like structures is ascribed to the interaction of the lipids from bicelles with the SC lipids. The bicellar system used is suitable to penetrate the skin SC and to reinforce the intercellular lipid areas, constituting a promising tool for skin applications.     

Dendrobium nobile Lindl Polysaccharides Attenuate UVB-induced Photodamage by Regulating Oxidative Stress,Inflammation and MMPs Expression in Mice Model

Acute ultraviolet B (UVB) irradiation predominantly leads to various skin disorders caused by photodamage. The major causes of UVB-induced photodamage include oxidative stress, inflammatory infiltration and collagen degradation. The aim of the study was to elucidate whether DNP had protective effect on the skin of KM mice when exposed to UVB irradiation. The DNP protective properties to skin appearance and histopathological alterations in KM mice were evaluated by hematoxylin–eosin staining, toluidine blue staining, Gomori staining and Masson's trichrome staining and mast cell staining. In this study, DNP pretreatment promoted the activities of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase, while decreased malondialdehyde level in UVB-irradiated skin, along with downregulation of proteins expression of matrix metalloproteinases and reduction in the level of the proinflammatory cytokines. Based on these findings, we demonstrated that DNP displayed strong ameliorative effects on UVB-induced acute photodamage for the first time, indicating that it would be a promoting ingredient candidate that could be used in antiphotodamage.     

Effect of Fish Bone Bioactive Peptides on Oxidative,Inflammatory and Pigmentation Processes Triggered by UVB Irradiation in Skin Cells

In the present study, we evaluated for the first time the photoprotective effect of fish bone bioactive peptides (FBBP) preparation isolated from silver carp (Hypophthalmichthys molitrix) discarded tissue using in vitro experimental models of skin cells exposed to ultraviolet B (UVB) irradiation and stressing agents. FBBP preparation was obtained by papain treatment of minced bones and centrifugal ultrafiltration, and the molecular weight (MW) distribution was characterized by size exclusion and reversed-phase high performance liquid chromatography (RP-HPLC). In vitro assessment of the effect of FBBP pretreatment in UVB-irradiated L929 fibroblasts and HaCaT keratinocytes revealed their cytoprotective activity. Their capacity to efficiently reduce reactive oxygen species (ROS) production and lipid peroxidation varied in a dose-dependent manner, and it was greater in fibroblasts. A decrease of proinflammatory cytokines secretion, in particular of tumor necrosis factor alpha (TNF-α), was found after FBBP pretreatment of THP-1-derived inflamed macrophages. Melanin production and tyrosinase activity investigated in UVB-irradiated Mel-Juso cells were lowered in direct relation to FBBP concentrations. FBBP fractions with high radical scavenging activity were separated by ion exchange chromatography, and two collagenic sequences were identified. All these results offer new scientific data on aquaculture fish bone-derived peptides confirming their ability to control the antioxidant, anti-inflammatory and pigmentation processes developed during UV irradiation of skin cells and recommend their use as valuable natural ingredients of photoprotective cosmeceutical products.     

Influence of the level of cholesteryl sulfate in the solubilization of stratum corneum lipid liposomes by sodium dodecyl sulfate

 The role played by cholesteryl sulfate (Chol-sulf) in the solubilization of liposomes modeling the stratum corneum (SC) lipids by sodium dodecyl sulfate (SDS) was studied. We determined the surfactant-to-lipid molar ratios and the bilayer/aqueous phase surfactant partition coefficients of this interaction by varying the proportion of Chol-sulf, the relative proportions of the others lipids remaining constant. These parameters were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free surfactant concentration was always similar to its critical micelle concentration indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The SDS ability to saturate and solubilize SC liposomes decreased as the proportion of Chol-sulf in the bilayers increased until a minimum was reached for a Chol-sulf proportion of about 15%. Inversely, the SDS partitioning into liposomes (or affinity with these bilayers) increased as the proportion of Chol-sulf increased until a maximum was reached at similar Chol-sulf proportions (10–15%). Hence, in these Chol-sulf proportions (similar to that existing in the intercellular lipids, which was 10%) the ability of SDS molecules to interact with liposomes exhibits a minimum despite their enhanced partitioning into liposomes. These effects may be related to the reported dependencies of the level of Chol-sulf on the abnormalities in the skin barrier function and on the SC intercellular cohesion. Received: 12 October 1999 Accepted: 20 January 2000     

Development and Validation of Analytical Methods for the Detection and Quantification of a Novel Dimeric Ceramide in Stratum Corneum and Other Layers of the Skin

Ceramides (CERs) are integral parts of the intercellular lipid lamellae of the stratum corneum (SC), which is responsible for the barrier function of the skin. Many skin diseases such as atopic dermatitis and psoriasis are associated with depletion or disturbance of the level of CERs in the SC. Administration of an exogenous novel dimeric ceramide (dCER) deep into the SC may help to stabilize the SC barrier substantially and to treat some skin disease conditions and with the help of the existing technology it might be possible to formulate various pharmaceutical dosage forms that can facilitate penetration of dCER into the SC. However, assessment of the rate and extent of permeability of the exogenous dCER involves appropriate analytical techniques which can discriminately quantify the exogenous dCER in the SC and in the other skin layers. Therefore, an attempt was made to develop an AMD-HPTLC and an HPLC/APCI-MS method for the detection and quantification of exogenous dCER in the SC as well as other skin layers. The method involved synthesis of the dCER and development of appropriate HPTLC and LC/ESI-MS methods for the separation and quantification of the dCER in the SC and deeper layers of the skin. The methods developed were optimized for quantification of a novel dCER. In comparison to the AMD-HPTLC method, the HPLC/MS method offers a higher sensitivity. Both methods could be used for the quantification of dCER in presence of a complex matrix (e.g., skin extract). The developed methods are complementary and could be used for the quantification of dCER in any further stage of substance research and industrial application. The methods developed are robust, linear and sensitive with a low limit of detection (LOD) and low limit of quantification (LOQ).

     

Interactions between a skin penetration enhancer and the main components of human stratum corneum lipids

The study employed isothermal titration calorimetry (ITC) technique to investigate the interactions between a skin penetration enhancer, laurocapram, and three representatives of the human stratum corneum (SC) intercellular lipids, i.e., ceramide-3, cholesterol and behenic acid. Experiments were performed using a TAM2277 (Thermometric AB, Sweden) calorimeter. Results showed that, firstly, two laurocapram molecules were bound to one ceramide-3 molecule in propylene glycol solution. The binding was enthalpy-driven and system entropy decreased after binding. Secondly, the binding ratio between cholesterol and laurocapram molecule was one to one. It was mainly an entropy-driven process, suggesting the ordered cholesterol molecules in SC lamella were disturbed by the introduction of laurocapram. Hydrogen bonding might be the main force of the binding. The three different interaction modes showed that the chemical enhancers selectively interacted with the components of SC lipids so that the SC can still retain its barrier function.     

Daphnia pulex Fed UVB-lrradiated Chlamydomonas reinhardtii Show Decreased Survival and Fecundity†

Abstract The ability to avoid or tolerate UVB radiation (290–320 nm) probably reduces the quality of phytoplankton as food for zooplankton. Ultraviolet avoidance forces motile algae lower in the water column, reducing net primary productivity. Production of UV-absorbing compounds as a tolerance mechanism takes carbon and energy away from cell growth; these compounds are also less useful as food for herbivores. We assessed the food quality of UVB-irradiated Chlamydomonas reinhardtii Dang, by analysis of cohort life tables for the herbivore Daphnia pulex Leydig. Algal cultures were grown under fluorescent lights interspersed with UV bulbs (20°C, 16 h light: 8 h dark) and covered with either cellulose acetate (+UVB) or Mylar D (-UVB). After a 7 day UVB exposure, carotenoid levels of C. reinhardtii were significantly greater than in unexposed cultures; there were no significant differences in chlorophyll, lipid, protein or nonstructural carbohydrate levels. After an initial decrease in growth rate, UVB-exposed algae recovered and final growth rates of both cultures were similar after 7 days. Daphnia neonates were raised under optimal conditions (20°C, 16 h light:8 h dark) and fed either UVB-irradiated or control algae. Daphnia fed UVB-irradiated algae had significantly increased mortality, decreased growth rate at the time of first clutch production, delayed release of the first clutch and decreased overall fecundity (P < 0.05). Daphnia were also less efficient at clearing UVB-irradiated algae from the water column (17% removal in 18 h) when compared with Daphnia that grazed upon nonirradiated algae (85% removal in 18 h). These data suggest that even though UVB-irradiated algal populations can recover from their initial decline, their predators may not be able to utilize them as an effective food source. Therefore, the impacts of UVB radiation within     

The effect of internal lipids on the water sorption kinetics of keratinised tissues

Water has a large influence on the properties of keratinised tissues. The water diffusion properties of keratinised tissues are known to be governed by the cell membrane complex, which is mainly composed of internal lipids. The main aim of this work was to characterise the differences in the water sorption and desorption behaviour of human hair and stratum corneum (SC) both with and without internal lipids. Absorption and desorption curves were obtained using a thermogravimetric balance equipped with a controlled humidity chamber. The results demonstrate that the role of the intercellular lipids in the SC is more marked than in hair, which is likely due to the greater amount of lipids present in its structure. Therefore, lipid structures in the SC are essential both to prevent changes in the water-holding capacity of the skin and to maintain the water permeability of the SC.     

Platelet-activating factor does not mediate UVB-induced local immune suppression

The lipid mediator Platelet-activating factor (PAF) and oxidized glycerophosphocholine PAF agonists produced by UVB have been demonstrated to play a pivotal role in UVB-mediated systemic immunosuppression. Importantly, employing the ability of distant UVB irradiation to inhibit contact hypersensitivity (CHS) responses to the chemical antigen dinitrofluorobenzene (DNFB) to an area of unirradiated murine skin, we and others have demonstrated that UVB-mediated systemic immunosuppression was only observed in PAF-R expressing wild type (WT) mice and not in PAF-R-knockout (Pafr-/-) mice. As it is not known if PAF is involved in UVB-mediated local immunosuppression, these studies compared local UVB on CHS responses in WT versus Pafr-/- mice. We demonstrate that the application of DNFB onto UVB-exposed (locally) area of mouse skin resulted in a similar significant inhibition of subsequent CHS responses in both WT and Pafr-/- mice compared to sham-irradiated control mice. Furthermore, the expression of langerin, a marker for the presence of Langerhans cells was substantially reduced equally in the epidermal ears of UVB-irradiated WT and Pafr-/- mice compared to their respective sham control groups. These findings indicate that the PAF-R is not involved UVB-induced local immunosuppression.     

An ex vivo methodology to assess the lipid peroxidation in stratum corneum

Environmental risks, particularly UV radiation, provide a challenge to the function of the skin barrier. Protective measures such as the use of antioxidant products represent a possible method of providing protection to the skin.This paper reports the development of a non-invasive ex vivo method using tape strips of the outermost layers of stratum corneum (SC) from human volunteers in order to determine the effectiveness of an antioxidant emulsion topically applied to prevent lipid peroxidation (LPO) in the horny layer after an UV irradiation exposure. Two different formulations were used: formulation (A), containing Vitamin A, E and C, and formulation (B) containing fish extract. Both formulations were topically applied in vivo on volunteer forearms; then, a tape stripping of the SC of each volunteer was carried out. The lipid peroxidation was measured ex vivo after an UV irradiation of the SC samples. The amount of SC stripped to evaluate differences in lipid peroxidation, the UV irradiation intensity to form lipid peroxides and the accuracy of lipid peroxide analysis were optimized in this methodology using formulation (A). After an exposure application of seven days, a group of three strips of the outermost layers of SC of volunteers was irradiated with an intensity of 182.7 J/cm² to quantify the LPO inhibition.The percentage of LPO inhibition obtained after topical application of both formulations was in the range of 40–58% demonstrating the effectiveness of the formulations topically applied against lipid peroxidation on human SC. This methodology may be used as a quality control tool to determine ex vivo the percentage of the LPO inhibition on human SC for a variety of antioxidants topically applied.     

Retaining Skin Barrier Function Properties of the Stratum Corneum with Components of the Natural Moisturizing Factor—A Randomized,Placebo-Controlled Double-Blind In Vivo Study

The influence of a topically applied formulation containing components of natural moisturizing factor (NMF) on barrier-related parameters of the stratum corneum (SC) was investigated in vivo using confocal Raman microspectroscopy in a randomized, placebo-controlled double-blind study on 12 volunteers for 14 days. This method allowed for the elucidation of subtle differences between the verum and the placebo even though the components of the verum naturally occur in the SC. This differentiation is not possible non-invasively by conventional methods. In this study, we found that the applied verum and placebo formulations disrupted the equilibrium of water, NMF and lipids in the SC. The adverse effects of the formulation could be mitigated by incorporating it into a simplified supplementation of NMF molecules. As a long-term effect, the amount of strongly bound water increases at 30–40% SC depth (p < 0.05) and the amount of weakly bound water decreases at 30–40% SC depth (p < 0.05) for the verum. This supplement was also unexpectedly able to prevent intercellular lipids (ICL) disorganization in selected depths. In the long term, the verum treatment limited the lateral disorganization of the ICL to the upper 20% SC depth. Further research is required to elucidate the interplay of these factors in the SC, to better understand their contribution to the equilibrium and barrier function of the skin. This understanding of the interaction of these naturally occurring components could help in the future to develop and optimize topical treatments for diseases like psoriasis, atopic dermatitis, ichthyosis where the skin barrier is disrupted.     

An animal model of solar-aged skin: histological, physical, and visible changes in UV-irradiated hairless mouse skin

Albino hairless mice (Skh:HR-l) exposed to sub-erythemal doses of UVB or UVA radiation display physical, visible, and histological alterations. Skin surface replicas, transepidermal water loss, and skin fold thickness were found to change with irradiation. Visibly, the skin wrinkled with UVB and sagged with UVA exposure. These changes were graded on 3-point scales. Histological alterations included tissue thickening, loss of elastic fibers, elastosis, loss of collagen, and increases in muco-substances. The UVB alterations occur to a much lesser extent with an SPF-15 (7% PABA and 3% oxybenzone) sunscreen product. This sunscreen product had little effect on development of UVA-induced changes. However, an efficient UVA sunscreen (Parsol 1789) did reduce the UVA-induced changes. Many of the UVB-induced alterations regressed after UVB irradiation was stopped. No regression in UVA-induced alterations was observed when UVA irradiation was stopped. Qualitatively, the effects with UVA irradiation were like those observed in mouse chronological aging. These models and the convenient physical and visible grading methods described can be used to determine the effectiveness of topical treatments, such as sunscreens.     

Thermal dependence of Raman descriptors of ceramides. Part II: effect of chains lengths and head group structures

The outermost layer of the mammalian skin, the stratum corneum (SC), represents the main skin barrier. The SC lipids have a very exceptional composition, as the main lipid classes are ceramides (CER), long-chain fatty acids and cholesterol. Information on the function of each CER subclass and on the relation between CER lipid organisation and composition is of great importance to unravel the mechanism controlling the skin barrier function. Raman spectroscopy has been increasingly used for the study of intra- and inter-molecular structures of long-chain lipid compounds. In this study, we employed Raman spectroscopy to evaluate the effect of (1) the chain length and (2) the polar head architecture on the conformational order and organisational behaviour of CERs. The relation between the structure and the stability of the organisation was studied by monitoring the thermotropic response of each CER in the temperature range between 25 and 95 °C. This work enabled the determination of a correlation between the gauche/trans ratio in the νCC region and the state of the lateral packing. Moreover, it was shown that –OH groups in the α position of the fatty acids reduce the stability while long alkyl chains reinforces the intra- and inter-chains order.     

cis-UROCANIC ACID FAILED TO AFFECT in vitro HUMAN LANGERHANS CELL ALLOSTIMULATORY FUNCTION

Abstract— -Urocanic acid (UCA) represents the major ultraviolet B (UVB, 290–320 nm)-absorbing component of the skin. Trans-UCA is naturally produced in the stratum corneum and converts to the cis isomer upon UVB irradiation. In this study, we examined the effect of purified cis -UCA (about 99% of cis isomer) on the human Langerhans cell (LC) allostimulatory function by using the mixed epidermal cell-lymphocyte reaction (MELR). We found that addition of increasing amounts (6.5–400 μg/mL) of purified cis-UCA or (rara-UCA did not modify the T-cell response supported by enriched LC (eLC: 8–25% LC) as well as purified LC (pLC: 70–90% LC) suspensions. Because cis-UCA had no effect on the allostimulatory function of untreated LC, we investigated whether this compound could modify T-cell proliferation induced by UVB-irradiated LC. The UVB exposure of eLC or pLC to 100 J/m² significantly inhibited the capacity of both suspensions to mount a T-cell response. However, addition of cis- UCA did not potentiate this UVB-induced immunosuppression. The eLC or pLC were then incubated with cis-UCA for 18 h at 37°C and washed before adding to allogeneic T cells. The obtained proliferative response was similar to that induced by control LC incubated in medium alone, demonstrating that pretreatment with cis -UCA did not alter human LC function. In conclusion, these results strongly suggest that cis-UCA has no direct effect on human LC antigen-presenting function.