Effects of solar-simulated radiation dose fractionation on CD1a+ Langerhans cells and CD11b+ macrophages in human skin

BACKGROUND: There are few human studies investigating the immunosuppressive effects of exposure to solar-simulated radiation (SSR) and its relationship with sunburn/erythema, and few comparative data on the importance of SSR exposure regimens. OBJECTIVES: To evaluate whether SSR-induced erythema is a reliable end-point for assessing damage to antigen-presenting cells (APCs) in human skin. METHODS: We compared the relationship between SSR-induced erythema and alterations in epidermal CD1a+ Langerhans cells (LCs) and CD11b+ macrophages in human volunteers after single exposures to 0, 0.5, 1, 2 or 3 minimal erythema doses (MED). We also investigated whether SSR exposure leads to an accumulation or accommodation of the same end-points by comparing the effects of a relatively low cumulative SSR dose (3 MED) given in varying daily dose fractions (4 x 0.75 MED, 2 x 1.5 MED and 1 x 3 MED). RESULTS: Single SSR exposures induced a dose-dependent increase in erythema. CD1a+ LCs remaining in the irradiated epidermis showed a dose-dependent increase in cell size and altered morphology. Significant depletion of CD1a+ LCs and presence of CD11b+ macrophages only occurred in sites irradiated with 2 MED and 3 MED. Dose fractionation had no effect on the final erythemal response but the 4 x 0.75 MED and 1 x 3 MED protocols were better tolerated than 2 x 1.5 MED for alterations in CD1a+ LC and CD11b+ cell numbers. In contrast, dose fractionation protected against alterations in CD1a+ LC morphology or cell size. CONCLUSIONS: We found that erythema is a poor indicator of alterations in epidermal APCs and that dose fractionation is an important parameter in the immunological effects of ultraviolet radiation.     

Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA radiation

BACKGROUND: Apoptosis has been proposed to act as an important mechanism for eliminating keratinocytes that have been irreversibly damaged by ultraviolet (UV) irradiation. One way to induce apoptosis in keratinocytes is through activation of the cell surface receptor Fas (CD95), either with the ligand (FasL) or directly with UV radiation. OBJECTIVES: To investigate the regulation of Fas and FasL expression in human skin and the formation of apoptotic cells after in vivo exposure to UVB or long-wave UVA radiation. METHODS: Volunteers were irradiated with either 3 minimal erythema doses (MED) of UVB (n = 6) or 3 MED of long-wave UVA (n = 6) on buttock skin 12, 24 and 72 h before skin punch biopsies were taken. Expression of Fas and FasL was demonstrated by immunohistochemistry on cryostat sections. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labelling reaction. RESULTS: In five of six subjects, exposure to UVB radiation resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 24 and 72 h after irradiation. In all subjects, exposure to long-wave UVA resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 12 h after irradiation. In five of six subjects, exposure to UVB radiation resulted in temporarily decreased expression of FasL, but after 72 h the expression of FasL had returned to the preirradiation level. The expression of FasL on epidermal cells after exposure to long-wave UVA showed considerable variation. UVB irradiation was a stronger inducer of epidermal apoptosis than was UVA irradiation. The number of apoptotic epidermal cells did not correlate with expression of Fas or FasL. CONCLUSIONS: In human skin the expression of Fas on epidermal cells increases after in vivo exposure to UVB or long-wave UVA. Exposure to UVB causes a temporary decrease in the expression of FasL on epidermal cells.     

The sun protection factor (SPF) inadequately defines broad spectrum photoprotection: demonstration using skin reconstructed in vitro exposed to UVA,UVBor UV-solar simulated radiation

Wavelength specific biological damage has been previously identified in human skin reconstructed in vitro. Sunburn cell and pyrimidine dimers were found after UVB exposure, and alterations of dermal fibroblasts after UVA exposure. These damages permitted us to discriminate UVB and UVA single absorbers. The present study shows that these biological effects can be obtained simultaneously by a combined UVB + UVA exposure using ultraviolet solar simulated light (UV-SSR), which represents a relevant UV source. In addition, the protection afforded by two broad spectrum sunscreen complex formulations was assessed after topical application. These two formulations displayed the same sun protection factor but different UVA protection factors determined by the persistent pigment darkening (PPD) method. Dose response experiments of UVA or UV-SSR showed that the preparation with the highest PF-UVA provided a better protection with regard to dermal damage compared to the other formulation. Using an original UVB source to obtain the UVB portion of SSR spectrum, the preparations provided the same protection. This study strikingly illustrates the fact that the photoprotection afforded by two sunscreen formulations having similar SPF values is not equal with regard to dermal damage related to photoaging.     

Ultraviolet radiation-induced damage to human Langerhans cells in vivo is not reversed by ultraviolet A or visible light

Exposure of human skin in vivo to UVB radiation induces pyrimidine dimers in DNA and alters the morphology and function of epidermal Langerhans cells. Cells in human skin have been reported to contain a photoreactivation repair mechanism that, following exposure to UVA or visible light, repairs UVB-induced pyrimidine dimers. The purpose of this study was to determine whether exposure to photoreactivating light would also reverse the UVB-induced morphologic alterations in human Langerhans cells. The skin of eight healthy volunteers was exposed to a low dose of UVB radiation (between 0.75 and 1.5 times the minimal erythema dose), and immediately thereafter exposed to photoreactivating light from either BLB fluorescent lamps (UVA radiation) or incandescent bulbs (visible light). After exposure to UVB radiation, the number of ATPase+ epidermal Langerhans cells was reduced in all subjects to between 21% and 65% of that in unirradiated skin, and the majority of the remaining cells exhibited morphologic alterations. Exposure of the UVB-irradiated skin to photoreactivating light did not reverse or reduce these effects. We conclude that UVB-induced morphologic alterations of human Langerhans cells are not subject to photoreactivation. These results imply either that pyrimidine dimers are not involved in these effects of UVB irradiation, or that photoreactivation does not occur in human Langerhans cells in situ.     

Comparative immunotoxicology of ultraviolet B exposure I. Effects of in vitro and in situ ultraviolet B exposure on the functional activity and morphology of Langerhans cells in the skin of different species

Ultraviolet (UV) B-induced morphological and functional changes in the skin of mice, rats and humans were investigated. Changes in the morphological structure of Langerhans cells (LC), the major antigen-presenting cells in the skin, using confocal laser scanning microscopy, were found in mouse and rat skin after in situ exposure to high doses of UVB radiation (FS40) (3–9 kJ/m²). Similar UVB doses failed to induce alterations in the morphological structure of human LC. Alterations in the function of epidermal cells (especially LC) were studied, using the mixed skin lymphocyte response (MSLR). In vitro UVB exposure of epidermal cells (EC), derived from the skin of the different species, revealed that low doses of UVB radiation impaired the stimulatory capacity of these cells dose-dependently; mouse epidermal cells were most UVB-susceptible, while human cells were least UVB susceptible. For suppression of the stimulatory capacity of EC after in situ UVB exposure of skin tissue, higher doses of UVB radiation than the in vitro UVB exposure were needed in all species tested. Also in this in situ set-up mouse epidermal cells were most UVB-susceptible, and human epidermal cells were least UVB-susceptible. The magnitude of differences in susceptibility for UVB-induced changes in the stimulatory capacity of EC after in situ and after in vitro exposure experiments was similar. Firstly, it may be concluded that UVB impairs the functional activity of LC at a lower dose than that which alters the morphology of these cells. Secondly, it is clear that epidermal cells, especially LC, from the skin of rodents are more susceptible to UVB than epidermal cells derived from human skin. It is important to account for these differences in susceptibility when data on the effects of UVB radiation on the immune system in rodents are extrapolated to humans.     

UVB and UVC, but not UVA, potently induce the appearance of T6- DR+ antigen-presenting cells in human epidermis

Non-Langerhans cell, antigen-presenting T6- DR+ epidermal cells (EC) appear 3 days following broad band ultraviolet radiation exposure of human skin and are responsible for the increased antigen presentation capacity of EC seen 3 days after UV exposure. To determine the UV wavelengths that induce T6- DR+ EC, volar forearm skin of 10 human volunteers was irradiated in vivo with 4 minimal erythema doses (MED) each of pure UVA (mean 482 J cm-2), UVB (mean 0.390 J cm-2), and UVC (mean 0.397 J cm-2). The purity of the light sources was as follows: UVB, 98% of the emission was in the UVB range; UVC, 97% of the irradiance was in the UVC range; UVA, 100% of the energy had wavelengths longer than 340 nm. Three days after UV irradiation with 4 MED of each wavelength band, suction blister-derived EC suspensions were prepared from the UV-exposed and unirradiated sites. Percentages of T6+ DR+ Langerhans cells (LC) and T6- DR+ EC were quantitated. Relative to control EC, which contained 2.4 +/- 0.3% T6+ DR+ LC, the mean percentage (+/- SEM) of T6+ DR+ LC contained within UV-exposed EC was significantly decreased as follows: UVB, 0.5 +/- 0.2%; UVC, 0.9 +/- 0.1%; UVA, 0.5 +/- 0.2% (n = 10). T6- DR+ EC, absent in control EC, were induced both by UVB, 5.2 +/- 1.7% and UVC; 1.5 +/- 0.4%. Despite the use of more than 1200 times greater doses in J cm-2 of UVA than UVB and UVC, UVA was a poor inducer of T6- DR+ EC (0.5 +/- 0.2%) and in about half of these individuals, T6- DR+ EC were undetectable. The UV wavelengths for induction of T6- DR+ EC lies predominantly within the UVB band, but also to a lesser extent within the UVC band. These wavelengths appear to be analogous to both the wavelengths for generation of increased host susceptibility to UV-induced murine tumors and to the wavelengths for UV-induced systemic suppression of contact hypersensitivity. However, our data indicate that UV wavelengths for decreasing the number of T6+ DR+ LC in humans differs from the wavelengths for induction of systemic suppression of contact hypersensitivity in mice. Taken together, these data suggest that the appearance of T6- DR+ EC, but not the disappearance of T6+ DR+ LC, following UV exposure may be related to the induction of such antigen-specific suppressor T cells.     

Lack of photoprotection against UVB-induced erythema by immediate pigmentation induced by 382 nm radiation

Immediate pigment darkening (IPD) was induced on the backs of 11 human volunteers of skin types III and IV by exposing the skin to UVA radiation (382 nm). The minimum erythema dose (MED) of UVB radiation was also determined by exposing sites to graduated doses of 304 nm radiation. The order of exposure of distinct anatomic areas was as follow: UVB followed by IPD induction; IPD induction followed by UVB; IPD induction followed 3 h later by UVB; and UVB only. Erythema responses induced by UVB were graded by inspection 24 h later and the MEDs in the 4 areas were compared. The induction of IPD before UVB exposure caused no significant change in the MED compared to sites receiving UVB only, or receiving UVA radiation after UVB, confirming that the IPD reaction does not protect against UVB-induced erythema. There was also no evidence of photorecovery, i.e., an increase in the MED of UVB resulting from exposure to longer wavelength, UV or visible radiation following UVB exposure.     

UVB-induced leucocyte trafficking in the epidermis of photosensitive lupus erythematosus patients: normal depletion of Langerhans cells

BACKGROUND: The pathogenic mechanisms of UV-induced skin lesions of lupus erythematosus (LE) are unknown. In a recent study of pathogenic mechanisms of polymorphic light eruption (PLE), significantly more Langerhans cells (LCs) persisted in the epidermis after UVB overexposure than in healthy individuals. Interestingly, the same phenomenon was observed in one subacute cutaneous lupus erythematosus (SCLE) patient. It could therefore be hypothesized that both photodermatoses share a common pathogenic mechanism of photosensitivity. In the present study, we tested this hypothesis by investigating leucocyte trafficking in the initial phase of cutaneous LE after intense UVB exposure. METHODS: In 22 photosensitive LE patients (12 chronic discoid lupus erythematosus, seven systemic lupus erythematosus and three SCLE) and nine age/sex-matched controls, uninvolved buttock skin was exposed to six minimal erythemal dose (MED) UVB radiation. Subsequently, biopsies were taken after 24, 48 and 72 h, and one control biopsy was taken from unirradiated skin. Skin sections were stained for the presence of LCs, neutrophils and macrophages. Areal percentages of positively stained cells within the epidermis were quantified and compared between the patients and controls. RESULTS: A gradual decrease of epidermal LCs and a gradual increase of epidermal neutrophils and macrophages at several timepoints after six MED irradiation was observed equally in both LE patients and controls. CONCLUSION: Immunohistopathology of irradiated uninvolved skin of photosensitive LE patients did not reveal the same pathologic trafficking of LCs and neutrophils as described for PLE patients. We conclude that different mechanisms are operative in the pathogenesis of PLE and photosensitive LE.     

Photoageing: the role of UVB, solar-simulated UVB, visible and psoralen UVA radiation

We have studied the morphological and histological effects of chronic exposure of two strains of haired mice (albino Balb/c and pigmented C3H-) to various types of radiation. UVB (280-320 nm) radiation from unfiltered sunlamp bulbs for 20 and 30 weeks produced marked epidermal acanthosis and dyskeratosis which was reversible in mice exposed for 20 weeks followed by a 20-week rest. In the dermis the elastic fibres were altered and in the pigmented mice these changes had almost completely reversed 29 weeks after cessation of exposure. Cellulose acetate-filtered sunlamp bulbs (greater than 289 nm) produced similar but less marked changes. Chronic exposure to UVA (320-400 nm) radiation produced minimal alterations in the dermis while the epidermis was normal. Visible (greater than 400 nm) radiation in large doses produced no degenerative changes. Methoxsalen/UVA radiation produced epidermal acanthosis, dermal sclerosis, and alteration of elastic fibres, which was prominent in both strains at 40 weeks. These findings suggest that the UVB component of sunlight is largely responsible for photoageing of the skin. Furthermore, chronic exposure to methoxsalen/UVA therapy is likely to potentiate solar-induced photoageing.     

Contrasting effects of an ultraviolet B and an ultraviolet A tanning lamp on interleukin-6, tumour necrosis factor-α and intercellular adhesion molecule-1 expression

BACKGROUND: Recent studies have demonstrated that a tanning lamp emitting predominantly ultraviolet (UV) A induces significant yields of the type of potentially mutagenic DNA damage that are associated with the onset of skin cancer (i.e. cyclobutane pyrimidine dimers). UV-induced immunosuppression is also an important event leading to skin cancer. OBJECTIVES: To the modulation of key immunological molecules following exposure to a broad-spectrum UVB lamp and a predominantly UVA-emitting tanning lamp using model in vitro systems. METHODS: We compared secretion and mRNA expression of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in normal human epidermal keratinocytes, and interferon (IFN)-gamma-induced intracellular adhesion molecule (ICAM)-1 in normal human fibroblasts irradiated in vitro with a broad-spectrum UVB lamp or with a Philips 'Performance' tanning lamp. RESULTS: With broad-spectrum UVB irradiation, upregulation of IL-6 and TNF-alpha mRNA was detected 6 h after irradiation, and a dose-dependent increase of cytokines in the supernatants of irradiated cells was found 24 h after irradiation. In contrast, there was no cytokine secretion and little evidence for mRNA upregulation following exposure to a tanning lamp. When cells were exposed first to broad-spectrum UVB, then the tanning lamp, UVB-induced cytokine secretion was inhibited, although mRNA levels were upregulated to a level close to that observed with UVB alone. By using a Schott WG 320 nm filter to attenuate the level of UVB relative to UVA emitted by the tanning lamp, the inhibition of cytokine secretion was shown to be associated with UVA exposure. Both UV sources inhibited IFN-gamma-induced ICAM-1 mRNA expression in a dose-dependent fashion. By using a Schott WG 335 nm filter, inhibition of ICAM-1 mRNA expression by the tanning lamp was shown to be associated with UVB exposure. CONCLUSIONS: These results suggest that UV sources emitting different levels of UVA and UVB have differential effects on the modulation of different immunoregulatory molecules, and indicate that there are potential interactions between these wavelengths.     

Photostability of sunscreen products influences the efficiency of protection with regard to UV-induced genotoxic or photoageing-related endpoints

BACKGROUND: Acute as well as chronic sun exposure induces biologically damaging effects in skin including photoageing and cancer. Ultraviolet (UV)A radiation is involved in this process; it is therefore important that sunscreen products provide efficient and stable protection in this range of wavelengths. OBJECTIVES: This study based on in vitro approaches was performed to demonstrate that photostability is an essential requirement to protect against UVA-induced genetic and dermal alterations. METHODS: The protection afforded by two sunscreen products, differing with regard to their photostability, was studied using biological markers related to the genotoxic or photoageing impact of UVA or simulated solar UV radiation (UV-SSR). Comet assay was used to assess direct DNA breakage, photo-oxidized purines and lomefloxacin-induced DNA breaks in nuclei of normal human keratinocytes in culture. In similar conditions, detection of p53 accumulation was performed. The use of reconstructed skin in vitro allowed us to use a three-dimensional model to analyse the dermal and epidermal damage induced by UVA or UV-SSR exposure. Abnormal morphological features of the tissue as well as fibroblast alterations and matrix metalloproteinase-1 release induced by UV exposure have been studied after topical application of products on the skin surface. RESULTS: The results showed that the photostable product afforded better protection with regard to all the criteria studied, compared with the photounstable product. CONCLUSIONS: These data demonstrate that the loss of absorbing efficiency within the UVA wavelength domain due to photoinstability may have detrimental consequences on cell function and lead to impairments that have been implicated in genotoxic events as well as in the photoageing process.     

Alteration in murine epidermal Langerhans cell population by various UV irradiations: quantitative and morphologic studies on the effects of various wavelengths of monochromatic radiation on Ia-bearing cells

The present study was undertaken in order to clarify the exact mode of the Langerhans cell (LC) depleting process caused by UV irradiation. Following irradiation with a single dose of various wavelengths of monochromatic UV radiation (UVR), we studied the number of Ia-positive cells in mouse epidermal sheets quantitatively, particularly with regard to dose-response relationship, action spectrum, and time course change. In addition, we studied morphologic alterations of these cells using electron- and immunoelectron microscopy (EM and IEM). We obtained the following results after a single dose of UVB radiation (200 mJ/cm2 of 300 nm) or PUVA (1% of 8-methoxypsoralen (8-MOP) 20 microliter and 1 J/cm2 of 360 nm): (1) EM and IEM showed that while some LCs simply lost their Ia marker without any structural alterations, the majority of the LCs disappeared due to actual cell damage. (2) During an "injury phase," the initial 48 h, and a "recovery phase," lasting from 4-14 days after irradiation, enlargement of the size of remaining Ia-positive LCs occurred. The degree of enlargement was closely related to the degree of reduction in number, suggesting a process compensating for the loss of the LC population. (3) It was found that the recovery rate of LCs after irradiation damage was slower than that of keratinocytes, indicating different cell kinetics between these distinct cell populations in the epidermis, i.e., restoration of LCs after irradiation seems to be achieved at least partially through a repopulation process originating in the bone marrow. Studies with irradiation of various monochromatic wavebands, with or without topical 8-MOP, showed that the action spectrum for Ia-positive cell depletion activity lay within the spectrum shorter than 300 nm for UVR alone, and between 320-380 nm for 8-MOP plus UVR. Since the action spectra were similar to those for keratinocyte damage, i.e., sunburn cell formation, induction of unscheduled DNA synthesis, and to those for UVR-induced erythema, we conclude that common mechanisms underlie these types of tissue damage.     

The effect of clinical UVA/B exposures on urinary urocanic acid isomer levels in individuals with caucasian type (II/III) skin types

Terrestrial ultraviolet radiation (UVR) exposure, consisting of ultraviolet A (320-40 nm) and B (290-320 nm), results in the photoisomerizion of epidermal trans-urocanic acid (trans-UCA) to cis-urocanic acid (cis-UCA), a potential suppressor of local and systemic immune responses. This study examines urinary UCA isomers as biomarkers of UVA/B exposure. It presents results measuring both cis- and trans-UCA in human urine samples collected from a group of study subjects (skin types II/III) that underwent controlled UVA/B exposures similar to those administered in commercial suntanning parlors. The UCA isomers were purified from urine using C18 solid-phase extraction columns followed by high-performance liquid chromatography (HPLC) with UV absorbance (268 nm) detection. The UCA biomarker was expressed as the ratio of cis-UCA to trans-UCA (UCA ratio), or as cis-UCA concentration corrected for urine volume using creatinine (cis-UCA-Cr). The UCA ratio increased over baseline in the urine of individuals exposed to UVA/B. A single exposure to approximately 70 percent minimal erythema dose (MED) of UVR (95 % UVA/5 % UVB to approximately 90 % of skin area) produced a 4.75-fold increase in the UCA ratio (p< 0.001) relative to baseline. Repeated daily UV exposures of similar doses produced a minimal increase in UCA ratio above that of the single UV exposure. These findings indicate that UCA cis-trans ratio holds promise as a biomarker for recent solar UV exposure.     

Do OKT6 positive Langerhans cells play a role in the suppression of ultraviolet-induced carcinogenesis?

Langerhans cells (LC) in epidermis are antigen presenting cells. LC may play a role in immune surveillance system and are considered to suppress development of ultraviolet (UV) induced skin cancers. We studied effect of UVB irradiation to LC of xeroderma pigmentosum (XP) and normal subjects by using OKT6 monoclonal antibody. When 3 minimal erythema dose (MED) of UVB were irradiated, density of OKT6 positive LC of XP began to decrease 6 hours after irradiation, and showed the least numbers on day 2 and returned completely to the pre-irradiation level on day 14. Further, after 3 MED irradiation, LCs of both normal subjects became the least on day 3 and returned to the pre-irradiation level on day 14. In XP variant and normal subjects, the number of LC in chronic sun-exposed skin decreased significantly in a similar way comparing to that of non-exposed skin. These results suggest that epidermal LC may not play an essential role in prevention of UV-induced tumor development.     

Oxygen intermediates are involved in ultraviolet radiation-induced damage of Langerhans cells

Experiments were conducted to determine whether ultraviolet (UV) radiation exerts its effect through the generation of oxygen intermediates on Langerhans cells (LC). Guinea pigs were exposed to one single dose of UVB (0.9-2.7J/cm2), and biopsy specimens were taken 5 days after the irradiation. The population of LC was evaluated using ATPase-stained epidermal sheets. These exposures reduced the number of LC to 20-25% of the original density. On the other hand, superoxide dismutase (SOD) (0.02-0.2 mg), a scavenger of superoxide anion, which had been injected intradermally just before UV radiation, significantly prevented the depletion of LC, although not completely (37-40% of the original density). The injection immediately after the exposure was still significantly effective, but less so. Other scavengers of oxygen intermediates including catalase, D-mannitol, and L-histidine revealed no detectable effect. A single exposure of UVB at doses of 0.3-0.6 J/cm2 did not deplete the ATPase-positive LC. However, the same dose of UVB reduced the number of LC to 70%, when exposed after the injection of an SOD inactivator, diethyldithiocarbamate, possibly due to inactivation of physiologically existing SOD. These observations indicate that oxygen intermediates such as superoxide anion or its subsequent species are generated by UV radiation exposure and damage the epidermal LC.     

Photocarcinogenesis: UVA vs. UVB radiation

Recent research is revealing combinations of disturbed oncogenic and tumor-suppressive signaling pathways by altered or missing genes in skin cancers: mutated PTCH (in the mitogenic Sonic Hedgehog pathway) and mutated p53 tumor suppressor gene in basal cell carcinomas (BCC), possibly an activated mitogenic RAS pathway and mutated p53 in squamous cell carcinomas (SCC), and possibly an activated MET/RAS pathway and inactive p16(INK4a) tumor suppressor in cutaneous melanomas. UV radiation damages DNA and can give rise to genomic alterations, varying from point mutations to crude chromosomal dislocations. UVB radiation (wavelength band 280-315 nm) is more carcinogenic than UVA radiation (315-400 nm) in experimental induction of SCC. The impact of UVB radiation can be clearly inferred from the characteristic point mutations in p53 found in human SCC and BCC. In contrast to UVB radiation, much of the mutagenic and carcinogenic action of UVA radiation appears to be mediated through reactive oxygen species (ROS). Experiments have shown that UVA1 (340-400 nm) exposure induces SCC largely without the characteristic point mutations in p53. Both UVB and UVA radiation can give rise to ROS-related point mutations (e.g. G to T) and crude genomic alterations (e.g. deletions) which may not be recognized as caused by UV radiation.     

Effect of ultraviolet (UV) A, UVB or ionizing radiation on the cell cycle of human melanoma cells

BACKGROUND: One important component of the cellular response to irradiation is the activation of cell cycle checkpoints. It is known that both ultraviolet (UV) radiation and ionizing radiation (IR) can activate checkpoints at transitions from G(1) to S phase, from G(2) phase to mitosis and during DNA replication. OBJECTIVES: To evaluate the effects of irradiation with different wavelengths on cell cycle alterations. METHODS: p53-deficient IPC-298 melanoma cells were irradiated with 10 J cm(-2) UVA, 40 mJ cm(-2) UVB, or with 7.5 Gy IR. Cell cycle effects were then determined by DNA/5-bromodeoxyuridine dual-parameter flow cytometry. RESULTS: IPC-298 cells irradiated in G(1) with UVA were not arrested at the G(1)/S transition, but at the G(2)/M transition. Despite p53 deficiency, the cells showed a G(1) arrest after UVB exposure. Furthermore, IR did not affect G(1) or S phase, but induced G(2) phase arrest. Hence, the effects of UVA, but not of UVB, on the cell cycle in p53-deficient melanoma cells are comparable with those of IR. CONCLUSIONS: UVA and IR induce radical-mediated strand breaks and DNA lesions, and UVB essentially induces thymine dimers that lead to excision repair-related strand breaks. Different cell cycle effects may be a consequence of different types of DNA damage. The results showed that UVB-irradiated p53-deficient cells are arrested in G(1). Irradiation with the solar radiation component UVB can therefore result in a beneficial retardation of tumour promotion in human skin carrying p53-mutated cell clones.     

Quantitative assessment of UV-induced changes in microcirculatory flow by laser Doppler velocimetry

Objective measurements of blood flow changes following UV irradiation in the skin of human volunteers have been made with the noninvasive technique of laser Doppler velocimetry (LDV). This optical procedure allowed perfusion (number of red cells X velocity) alterations in the cutaneous microcirculation to be monitored after exposure of the skin to UVA and UVB + UVC radiation. Response curves were obtained in 6 subjects following irradiation at 4 times the minimal UVB + UVC erythema dose (MED). Measurements were made on control (untreated) skin and on skin pretreated with a sunscreen lotion. It was found that: (1) the lotion vehicle had no protective effect, (2) the active sunscreen constituent (2-ethylhexylcinnamate, 5%) was significantly protective, and (3) the presence of bergapten (5-methoxypsoralen, 30 ppm) did not enhance or diminish the cinnamate protective effect. LDV measurements in 5 subjects were also taken during and subsequent to 5 daily exposures to 1 MED of UVB + UVC radiation. Control and pretreated skin sites were again studied and similar protective effects were observed. However, on subsequent reexposure of these sites to 4 MED of UVB + UVC, 14 days after the first of the 5 single MED doses, no significant change in skin blood perfusion was detected at either control or pretreated sites. In a separate series of experiments, LDV data were collected after UVA radiation exposures up to 15 J/cm2. No changes in microcirculation perfusion were detected in any of the situations considered. All LDV measurements were made with 2 instruments of slightly different design and were compared to subjective assessments of erythema performed by a single observer. The results suggest that LDV has significant potential as a means to quantify (1) UV exposures in excess of the MED and (2) the inhibition of UV-induced changes in microcirculatory flow by chemical protectants.     

Epidermal stratification reduces the effects of UVB (but not UVA) on keratinocyte cytokine production and cytotoxicity

Ultraviolet (UV) radiation induces cytokine release from cultured keratinocytes as well as from epidermis in vivo. The purpose of this study was to determine whether differentiation of cultured keratinocytes into stratified epithelium decreases the effects of UVA and UVB radiation on cytokine release. Interleukin-1 (IL-1)α, IL-1β and tumor necrosis factor (TNF)-α release from human keratinocytes and reconstituted human epidermis was measured after exposure to UVA or UVB radiation. Release of IL-1α, IL-1β, and TNF-α was induced by both UVA and UVB radiation from both keratinocytes and reconstituted epidermis. Release of these cytokines was correlated with cytotoxicity. Keratinocyte cultures were far more sensitive to UVB radiation than reconstituted epidermis, in terms of both cytotoxicity and cytokine release. In contrast, epidermal stratification/differentiation had much less effect on the sensitivity to UVA radiation. We conclude that epidermal stratification and the formation of a stratum corneum provide protection against UVB radiation but have limited barrier effect against UVA radiation.     

Impact of ultraviolet radiation on the expression of marker proteins of gap and adhesion junctions in human epidermis

Aims/background: We aimed to investigate the impact of ultraviolet B (UVB) as well as UVA1 on the epidermal expression of specific markers of gap and adhesion junctions. Methods: Twelve healthy subjects were enrolled in the study. The back of the subjects was irradiated with three MED‐UVB as well as three MED‐UVA1. Twenty‐four hours later, punch biopsies were taken from irradiated and non‐irradiated skin. Immunohistochemical procedures were used for the detection of connexin 43, E‐cadherin, involucrin, Ki‐67 using specific antibodies. Results: Staining intensity of connexin 43 in UVB‐exposed skin was significantly increased when compared with non‐exposed and UVA1‐exposed sites. By contrast, staining intensity of E‐cadherin in UVB‐exposed skin was significantly decreased when compared with non‐exposed and UVA1‐exposed sites. Involucrin and Ki‐67 staining of keratinocytes was significantly increased in UVB‐exposed sites as compared with non‐exposed and UVA1‐irradiated sites. Conclusions: UVB significantly alters the epidermal expression of gap and adhesion junction proteins possibly indicating a role of these proteins in the regulation of UV‐induced inflammation and development and progression of skin cancer.