Long-lasting molecular changes in human skin after repetitive in situ UV irradiation

It is known that UV modulates the expression of paracrine factors that regulate melanocyte function in the skin. We investigated the consequences of repetitive UV exposure of human skin in biopsies of 10 subjects with phototypes 2-3.5 taken 1-4 years later. The expression of melanogenic factors (TYR, MART1, MITF), growth factors/receptors (SCF/KIT, bFGF/FGFR1, ET1/EDNRB, HGF, GM-CSF), adhesion molecules (beta-catenin, E-cadherin, N-cadherin), cell cycle proteins (PCNA, cyclins D1, E2) as well as Bcl-2, DKK1, and DKK3, were analyzed by immunohistochemistry. Most of those markers showed no detectable changes at > or = 1 year after the repetitive UV irradiation. Although increased expression of EDNRB protein was detected in 3 of 10 UV-irradiated subjects, there was no detectable change in the expression of ET1 protein or in EDNRB mRNA levels. In summary, only the expression of TYR, MART1, and/or EDNRB, and only in some subjects, was elevated at > or = 1 year after UV irradiation. Thus the long-term effects of repetitive UV irradiation on human skin did not lead to significant changes in skin morphology and there is considerable subject-to-subject variation in responses. The possibility that changes in the expression and function of EDNRB triggers downstream activation of abnormal melanocyte proliferation and differentiation deserves further investigation.     

The expression of melanogenic proteins in Korean skin after ultraviolet irradiation

For proper melanin production, several specific enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase are required. Their expressions are increased after exposure to UVB. However, it is not known how long tyrosinase and TRP-1 activities continue after UV irradiation in vivo. The purpose of this study is to measure the changes in expressions of tyrosinase, TRP1, and MITF after exposure to UV on skin in a Korean population. We established an immunohistochemical staining protocol for specimens which were obtained from UV-irradiated skin in five healthy Korean males on the 2nd, 5th, 7th, 28th, and 56th days after UV irradiation. Tyrosinase, TRP-1, and MITF expressions increased until 7 days after UV irradiation and then dropped to the basal constitutive level 4 and 8 weeks later. Interestingly, tyrosinase increased prior to TRP-1. This study reveals the time-sequence of melanin-synthesized enzymes and provides important information for the clinical evaluation of the effectiveness of whitening agents.     

Quantitative and morphologic changes of Langerhans' cells after ultraviolet A irradiation of human epidermis

Three different surface markers (OKT6, HLA-DR, and adenosinetriphosphatase) were compared to identify Langerhans' cells, and the changes in number and morphology of these cells were studied at different intervals after irradiation of human skin by a 2.5-fold minimal erythema dose of ultraviolet A. Morphologic alteration and decreased surface-marker reactivity became evident on day 2 and were most pronounced on day 3 or day 4 (injury phase). The recovery phase started between day 4 and 1 week and was complete by 3 weeks. HLA-DR+/OKT6- (DR+T6-) cells were present at all time intervals. The ratio of these cells to the sum of DR+T6- and DR+T6+ cells was 0.3% before irradiation, reached a peak of 65.4% at day 4, and decreased to 0.6% by 3 weeks.     

Resveratrate protects human skin from damage due to repetitive ultraviolet irradiation

Background Ultraviolet (UV) exposure results in the production reactive oxygen species. Resveratrol has attracted considerable attentions owing to its natural abundance and multiple biological effects. Objective To investigate the protective effects of resveratrate against damage to human skin induced by repetitive solar simulator ultraviolet radiation (ssUVR). Materials and methods Fifteen healthy volunteers were enrolled, and six sites on the non‐exposed dorsal skin of each volunteer were marked for study. Sites 1–4 were exposed to ssUVR at a dosage of 1.5 minimal erythema dose for consecutive 4 days. Immediately after each exposure, one test material (resveratrate + antioxidant, antioxidant, resveratrate, vehicle) was applied to one of the four sites. Site 5 and site 6 were marked as positive control site (UVR only) and baseline control site (no treatment, no UVR). L*a*b values were assessed preprocedure and postprocedure. Skin biopsies were taken 24 h after the last irradiation. The specimens were stained to determine the number of sunburn cells and melanin content melanin. Results On resveratrate treated sites, erythema was barely seen with only slight decrease of L value and insignificant increase of *a value. Furthermore, resveratrate significantly inhibited sunburn cell formation, and decreased Fontana‐Masson staining in skin samples. Conclusions Resveratrate exerts protective effects against repetitive ssUVR‐induced sunburn and suntan.     

Histologic changes associated with ultraviolet A-induced erythema in normal human skin

We have examined the effects of a standardized, moderately erythemogenic dose of long-wave ultraviolet (UVA) radiation on normal human skin, with the use of an appropriately filtered solar simulator and sequential biopsy specimens processed as 1-μm Epon-embedded sections. Histologic changes were present immediately after irradiation and evolved slowly during the 48-hour study. The epidermis manifested slight intracellular and intercellular edema and progressive loss of Langerhans cells to approximately one-fifth control values. A dermal infiltrate of neutrophilic polymorphonuclear leukocytes was present in all postirradiation specimens and peaked at 3 hours. A perivascular lymphocytic infiltrate, moderate endothelial cell enlargement, mast cell hypogranulation, occasional massive venular dilation, and sparse red blood cell extravasation were also noted. Overall, our findings expand and quantify earlier impressions that, compared to UVB, UVA has a relatively greater histologic effect on the dermis than on the epidermis, depletes epidermal Langerhans cells, and recruits neutrophils into irradiated human skin.     

Epidermal langerhans cell depletion after artificial ultraviolet B irradiation of human skin in vivo: apoptosis versus migration

Ultraviolet B radiation can suppress cellular immunity. One of the mechanisms related to this immunosuppression is the disappearance of Langerhans cells from the epidermis. The aim of this study was to establish the mechanism of ultraviolet B-induced Langerhans cell disappearance in healthy individuals. The two most likely mechanisms for Langerhans cell disappearance are apoptosis and migration. Apoptosis was assessed in vivo by exposing buttock skin of 10 healthy volunteers to six minimal erythema doses of ultraviolet B. Only very few apoptotic Langerhans cells could be observed in sections from the ultraviolet B-exposed skin. Migration of Langerhans cells cannot be established in skin sections and suction blisters were therefore raised in an attempt to trap migrating Langerhans cells in the sub-basal membrane blister fluid. Blisters were raised on the flexor side of the lower arm of 30 healthy volunteers at several time points after exposure of the skin to six minimal erythema doses of ultraviolet B. Blister fluid was collected and blister roofs were removed to check for Langerhans cell disappearance. Langerhans cells were detected in the blister fluid of the ultraviolet B-exposed skin and not of the unexposed skin. The number of Langerhans cells in the blister fluid peaked at about 18 h after ultraviolet exposure, which coincided with the largest depletion of Langerhans cells in the blister roof. A fraction (20-30%) of the Langerhans cells in the blister fluid stained positive for DNA damage (cyclobutyl pyrimidine dimers), showing that they originated from the epidermis. Ultraviolet B-induced Langerhans cell disappearance appears to be mainly attributable to migration.     

Pyrimidine dimer induction and repair in cultured human skin keratinocytes or melanocytes after irradiation with monochromatic ultraviolet radiation

We compared the susceptibilities of cultured melanocytes and keratinocytes to dimer induction in DNA by monochromatic ultraviolet (UV) radiation. Keratinocytes as well as melanocytes were derived from human foreskin, grown as a monolayer in petri dishes, covered with phosphate-buffered saline containing 0.1% glucose, and irradiated. UV irradiation was carried out at 254, 297, and 302 nm as well as with a light source emitting predominantly 312 nm. The induction of pyrmidine dimers was assessed by determination of the number of T4 endonuclease V-sensitive sites (ESS). We found a slightly higher response for dimer induction in melanocytes at 254, 297, and 302 nm; this difference was only significant at the 297-nm wavelength. Action spectra for pyrimidine dimer induction were derived from the exposure-response data obtained. The action spectra mimic to a large degree the action spectra for dimer induction in other cultured mammalian cells. The repair rate during a post-irradiation period lasting up to 24 h was substantially the same for the two cell types. The percentage of T4 endonuclease V-sensitive sites (ESS) remaining 9 and 24 h after irradiation was 45% and 30%, respectively.     

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation

Abstract The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m². The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of β₁ integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing β₁ integrin were divided into two subpopulations, denoted β₁ ^high or β₁ ^low. The proportion of β₁ ^high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of β₁ ^low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that β₁ ^high and β₁ ^low populations of basal epidermal cells in HSEs respond differently to UV irradiation. Received: 30 October 1997     

Changes in ultraviolet absorption of sunscreens after ultraviolet irradiation.

The incidence of skin cancer is increasing rapidly and sunscreens have been recommended in order to reduce damage from sunlight. In this investigation we have studied the change in the absorption spectrum of some photoactive organic species in sunscreens after ultraviolet A and ultraviolet B irradiation in a dose normally encountered during a full day in the sun. Samples of a number of photoactive compounds commonly used in sunscreens were irradiated with ultraviolet A and ultraviolet B light. A UVASUN 2000 MUTZHAS sunlamp was used for ultraviolet A irradiation and an Essh? Corona mini, equipped with two Philips TL12 20 W lamps, was used as the ultraviolet B source. The ultraviolet A dose was 100 J per cm2. The ultraviolet B dose corresponded to 20 minimal erythema doses. The absorption spectra of the compounds were recorded before and after irradiation. The absorbance of 2-ethylhexyl 4-methoxycinnamate was reduced significantly, whereas 3-(4-methylbenzyliden)camphor seemed to be rather stable. The benzophenones studied seemed to be relatively stable. In the case of 4-tert. butyl-4'-methoxy-dibenzoylmethane there was a rapid decrease in the ultraviolet A absorption leading to unsatisfactory protection in the ultraviolet A region. 4-Isopropyl-dibenzoylmethane also lost most of its ultraviolet protective capacity after irradiation with ultraviolet A. Ultraviolet B seemed to have a minor effect on all the samples. It is important for the clinician not only to know the initial absorption spectrum in the ultraviolet region for a specific sunscreen substance, but also whether it is altered during irradiation and in what way. This study including gas chromatography and mass spectrometry analysis indicates that some of the photoactive organic species commonly used today in sunscreens are unstable following ultraviolet irradiation.     

Epidermal changes in human skin following irradiation with either UVB or UVA

We have demonstrated previously that following UVB irradiation to normal volunteers there is an increase in epidermal and stratum corneum thickness and an increase in the thymidine autoradiographic labeling index. These changes are coupled with alterations in epidermal glucose-6-phosphate dehydrogenase and succinic dehydrogenase activities, despite the absence of erythema clinically. The use of a sunscreen did not completely prevent these changes. In this study, we have examined the effects of repeated irradiation of human skin with either UVB or UVA alone in order to compare the changes produced in the epidermis and to ascertain whether UVA irradiation could cause these. Irradiation with either UVB or UVA alone was found to increase the mean epidermal thickness, the mean stratum corneum thickness, and mean keratinocyte height significantly. Glucose-6-phosphate dehydrogenase activity was significantly increased throughout the epidermis, and succinic dehydrogenase activity was significantly decreased. The autoradiographic labeling index was significantly increased following UVB irradiation but not following UVA irradiation. These results demonstrate that UVA alone can have a direct effect on epidermal morphology and metabolism, suggesting that protection of skin from UV radiation should include adequate protection from UVA.     

Mitochondrial dysfunction and cellular stress progression after ultraviolet B irradiation in human keratinocytes

Background: Ultraviolet (UV) radiation is the major environmental harmful factor that affects human skin. UVB radiation is known to be a potent inducer of reactive oxygen species (ROS) production and has also been associated with the generation of nitric oxide (NO), all of which have been implicated in various skin disorders. It is well known that mitochondria can also be affected by UVB, leading to alterations in their membrane structure and permeabilization with cytochrome c release, which consequently affects the cell function. However, the loss of keratinocyte mitochondrial function generated by UVB, as well as its kinetics, has not been characterized completely.
Methods: We evaluated the effect of UVB irradiation on HaCat cells' mitochondrial function, assessed by membrane potential loss and superoxide anion (O₂^•−) production, correlating with apoptosis, p53 expression, ROS levels and NO production, 0, 6, 12, 24 and 48 h post-irradiation.
Results: HaCat cells progressed toward apoptotic cell death as the time post-irradiation increased, with the highest levels found 48 h after irradiation. Increased levels of ROS were observed 6 h after irradiation while high O₂^•− levels and mitochondrial membrane depolarization were detected 12 h post-UVB. Nevertheless, NO production was not significantly increased at any of the evaluated times.
Conclusions: The kinetics of mitochondrial dysfunction after UVB irradiation in human keratinocytes progressed in a time post-irradiation-dependent manner, and they are closely related to cell death. However, there are certain levels of apoptosis, although low, in the absence of mitochondrial alterations. In addition, our data suggest that ROS play a greater role in keratinocyte UVB damage than reactive nitrogen species.     

Changes in the skin moisture contents, skin color, and skin protein conformational structures of sprague-dawley rats after ultraviolet B irradiation.

The influence of ultraviolet B (UVB) irradiation on the skin moisture contents, skin color, and protein secondary structure in the skin of alive Sprague-Dawley (SD) rats was investigated. The UVB irradiation source emitted 725 +/- 10 microW/cm(2) of UVB range with a peak at 302 nm during the exposure course of 6 h/day for 3 successive days. The moisture content of SD rat's skin first decreased with the UVB irradiation, then returned to normal level on days 1 and 2, and, finally, decreased significantly with the increase of UVB irradiation on day 3. After the 6-hour irradiation on days 1 and 2, the moisture contents of the irradiated skin of SD rat returned to normal values, suggesting repairability of the irradiated skin of SD rat during the following 18 hour period without irradiation. By increasing frequency and duration of UVB irradiation, the skin color parameters exhibited a lower L* value, a greater a* value, and a higher DeltaE value, indicating that the skin color darkened and reddened. The slight modification of protein secondary structure of the skin of SD rats after repeated UVB irradiation was evidenced. However, the infrared spectrum during the initial course of irradiation, from day 1 to day 3, exhibited a similar pattern. Thus, the alive SD rat's skin was repairable during the 18-hour period without irradiation.