Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

Radiation sources providing increased UVA/UVB ratios induce photoprotection dependent on the UVA dose in hairless mice

In studies involving mice in which doses of UVA (320-400 nm) and UVB (290-320 nm) radiation were administered alone or combined sequentially, we observed a protective effect of UVA against UVB-induced erythema/edema and systemic suppression of contact hypersensitivity. The UVA immunoprotection was mediated by the induction of the stress enzyme heme oxygenase-1 (HO-1) in the skin, protection of the cutaneous Th1 cytokines interferon-gamma (IFN-gamma) and IL-12 and inhibition of the UVB-induced expression of the Th2 cytokine IL-10. In this study, we seek evidence for an immunological waveband interaction when UVA and UVB are administered concurrently to hairless mice as occurs during sunlight exposure in humans. A series of spectra providing varying ratios of UVA/UVB were developed, with the UVA ratio increased to approximately 3.5 times the UVA component in solar simulated UV (SSUV). We report that progressively increasing the UVA component of the radiation while maintaining a constant UVB dose resulted in a reduction of both the erythema/edema reaction and the degree of systemic immunosuppression, as measured as contact hypersensitivity. The UVA-enhanced immunoprotection was abrogated in mice treated with a specific HO enzyme inhibitor. UVA-enhanced radiation also upregulated the expression of cutaneous IFN-gamma and IL-12 and inhibited expression of both IL-6 and IL-10, compared with the activity of SSUV. The results were consistent with the previously characterized mechanisms of photoprotection by the UVA waveband alone and suggest that the UVA component of solar UV may have beneficial properties for humans.     

Comparative effects of UVA and UVB irradiation on the immune system of fish

Aquatic organisms can be harmed by the current levels of solar ultraviolet radiation. We have recently shown that exposure of fish to UVB irradiation alters the functioning of the fish immune system, but the effects of UVA radiation are unknown. The present study continues this work by characterizing UVA irradiation-induced immunological changes in fish. Roach, a cyprinid fish, were exposed to a single dose of either UVA (3.6 J/cm2) or UVB (0.5 J/cm2) irradiation. Both irradiations suppressed transiently mitogen-stimulated proliferation of blood lymphocytes. UVA, but not UVB, decreased hematocrit, plasma protein, and plasma immunoglobulin levels and increased the proportions of blood cells classified as unidentified leukocytes, possibly consisting of UVA-damaged lymphocytes. UVB, but not UVA, altered the functioning of head kidney and blood phagocytes, induced granulocytosis and lymphocytopenia in the blood and increased plasma cortisol concentration. These results imply that both UVA and UVB are potent modulators of the immune defence of fish.     

Modulation of base excision repair alters cellular sensitivity to UVA1 but not to UVB1

Oxidative DNA damage has been implicated in some of the biological properties of UVA but so far not in the acute photosensitivity or cellular sensitivity. In contrast to pyrimidine dimers, oxidative DNA damage is predominantly processed by base excision repair (BER). In order to further clarify the role of oxidative DNA damage and its repair in the acute cellular response to UV light, we studied UVA1 and UVB sensitivities in three different cell model systems with modified BER. 8-Oxoguanine-DNA-glycosylase 1-/- (OGG1-/-) mouse embryonal fibroblasts and human fibroblasts in which BER was inhibited by incubation with methoxyamine were hypersensitive to UVA1, in particular to low doses. This hypersensitivity could be partially corrected by reexpression of OGG1 in OGG1-/- cells. The Chinese hamster ovary (CHO) cells with upregulated AP-endonuclease 1 exhibited reduced UVA1 sensitivity. UVB sensitivity was not altered in any of the cell models. These results indicate that DNA damage, in particular oxidative DNA damage, contributes to cellular UVA1 sensitivity and underline a pivotal role of its repair in the cellular responses to UVA1.     

Evaluation of sunscreen protection in human melanocytes exposed to UVA or UVB irradiation using the alkaline comet assay

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 microL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4 degrees C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37 degrees C were quantified using the comet assay. Tail moments (TM) (tail length x %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a chi 2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.     

Evaluation of the protective effect of sunscreens on in vitro reconstructed human skin exposed to UVB or UVA irradiation

We have previously shown that skin reconstructed in vitro is a useful model to study the effects of UVB and UVA exposure. Wavelength-specific biological damage has been identified such as the formation of sunburn cells (SBC) and pyrimidine dimers after UVB irradiation and alterations of dermal fibroblasts after UVA exposure. These specific effects were selected to evaluate the protection afforded by two sunscreens after topical application on the skin surface. Simplified formulations having different absorption spectra but similar sun protection factors were used. One contained a classical UVB absorber, 2-ethylhexyl-p-methoxycinnamate. The other contained a broad-spectrum absorber called Mexoryl SX, characterized by its strong absorbing potency in the UVA range. Both filters were used at 5% in a simple water/oil vehicle. The evaluation of photoprotection on in vitro reconstructed skin revealed good efficiency for both preparations in preventing UVB-induced damage, as shown by SBC counting and pyrimidine dimer immunostaining. By contrast, only the Mexoryl SX-containing preparation was able to efficiently prevent UVA-specific damage such as dermal fibroblast disappearance. Our data further support the fact that skin reconstructed in vitro is a reliable system to evaluate the photoprotection provided by different sunscreens against specific UVB and UVA biological damage.     

Differences in Sensitivity to UVC, UVB and UVA Radiation of a Multidrug-Resistant Cell Line Overexpressing P-Glycoprotein

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best-studied mechanism involves the expression of a membrane protein that acts as an energy-dependent efflux pump, known as P-glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non-MDR) cell line.     

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.     

Comparison of The Effectiveness of Sunscreen Lotions Using A TiO2-based UVA/UVB Dosimeter

This paper describes a simple method for the evaluation of the effectiveness of a sunscreen product. It illustrates the basic principle of semiconductor (such as TiO₂) band gap, band-gap excitation, conduction-band electron trapping, and its application as an effective dosimeter in the UVB and UVA regions. The color change of the dosimeter, prepared from TiO₂ sol, can be quantitatively determined with a UV-vis spectrometer or qualitatively by visual inspection. The experiment can be used as a freshman-chemistry laboratory exercise or performed as a classroom demonstration for high school students or nonchemistry-major college students.     

Differential effects of UVA1 and UVB radiation on Langerhans cell migration in mice

The UVB (280-315 nm)- and UVA1 (340-400 nm)-induced migration of Langerhans cells (LC) from the epidermis and accumulation of dendritic cells (DC) in the lymph nodes draining the exposed skin site of C3H/HeN mice have been investigated. One minimum erythemal dose (MED) of UVB (1.5 kJ/m2) and of UVA1 (500 kJ/m2) were chosen, which have been shown previously to suppress delayed hypersensitivity (DTH). UVB irradiation resulted in a reduction in epidermal LC numbers, local to the site of the exposure, which was most apparent 12 h after exposure, but, in contrast, UVA1 had no significant effect even at 72 h after exposure. UVA1 did not exert any protection against the UVB-mediated depletion in LC numbers. The reduction in local LC following UVB exposure was prevented by systemic (intraperitoneal) treatment of mice with neutralising antibodies to either tumor necrosis factor (TNF)-alpha or interleukin (IL)-beta 2 h prior to the irradiation. It has been reported previously that UVB exposure caused an increase in the number of dendritic cells (DC) in the lymph nodes draining the irradiated skin site. In the present study we have shown that UVA1 had a similar effect. Pretreatment of the mice with neutralising antibodies to IL-1beta (by intraperitoneal injection) substantially inhibited DC accumulation induced by both UV regimens. However, anti-TNF-alpha antibodies affected only the UVB-induced increase, and did not alter the elevation in DC numbers observed following UVA1 exposure. These results indicate that UVB causes the migration of LC from the epidermis and an accumulation of DC in the draining lymph nodes by a mechanism that requires both TNF-alpha and IL-1beta. In contrast, UVAI does not cause LC migration from the epidermis and the accumulation of DC in the draining lymph nodes observed following UVA1 exposure requires IL-1beta, but not TNF-alpha. It is likely therefore that UVA1 acts through a different mechanism from UVB and may target a cutaneous antigen presenting cell other than LC, such as the dermal DC.     

Inactivation of Vaccinia Virus by Natural Sunlight and by Artificial UVB Radiation

This study determined the sensitivity of vaccinia virus, an orthopox virus commonly used as a surrogate for variola virus (etiological agent of smallpox), exposed to UVB radiation emitted by a solar simulator, or to direct natural sunlight. The data obtained indicate that: (1) the virucidal effect of natural sunlight can be mimicked adequately by an artificial light source with similar spectral characteristics in the UVB, (2) viral sensitivity to UVB or to solar radiation can be correlated with experimental data previously obtained with UVC, (3) the correlation factor between virus inactivation by solar radiation (measured at 300 ± 5 nm) and by UVC (254 nm) is between 33 and 60, and (4) the sensitivity of viruses either dry on glass surfaces or in liquid suspension is similar when in the presence of similar amounts of cellular debris and growth media. The findings reported in this study should assist in estimating the threat posed by the persistence of virus during epidemics or after an accidental or intentional release.     

Influence of UVB,UVA and UVA1 irradiation on histamine release from human basophils and mast cells in vitro in the presence and absence of antioxidants

UV irradiation is widely used for the treatment of atopic eczema. In recent years, UVA1 phototherapy has gained increasing attention. This study analyzed the influence of different UV wavelengths--especially UVA1--on histamine release from human basophils and mast cells. The modulation of this parameter might be responsible for some of the therapeutic effects of UV irradiation. Enriched human basophils and human mast cells (HMC1 cell line) were irradiated with increasing doses of UVB, UVA and UVA1 in vitro. After irradiation, different stimulants were added to induce histamine release. In additional experiments, basophils were preincubated with superoxide dismutase, ascorbate or trolox to study the role of antioxidants in the modulation of histamine release after UV irradiation. UVA and UVA1 significantly inhibited histamine release from basophils and mast cells. UVB only had an inhibitory effect on mast cells. Preincubation with superoxide dismutase and ascorbate did not influence the inhibitory effect of UVA1 on basophil histamine release, whereas trolox decreased significantly the histamine release from nonirradiated basophils.     

N-acetyl-L-cysteine prevents DNA damage induced by UVA, UVB and visible radiation in human fibroblasts

The thiol N-acetyl-L-cysteine (NAC) is a source of cysteine for the synthesis of the endogenous antioxidant glutathione (GSH) which is depleted by ultraviolet radiation. It is also associated with the scavenging of reactive oxygen species (ROS). In this study the effects of NAC were examined in cultured human fibroblasts during prolonged exposure to ultraviolet B (UVB), ultraviolet A (UVA) and visible irradiation (280-700 nm), delivered by a 150 W xenon-arc lamp. The alkaline comet assay was used to assess the DNA damage in individual cells. It was found that incubating skin and lung fibroblasts at 37 degrees C for 1 h with an optimal 6 mM NAC supplement prior to light exposure, significantly reduced the level of DNA damage in both cell types, however, the skin fibroblasts were less sensitive to xenon-arc lamp irradiation than lung fibroblasts. NAC incubation resulted in an initial delay in DNA damage when the cells were irradiated. There was also a significant reduction in the overall levels of DNA damage observed with continued irradiation. NAC significantly reduced the DNA damage produced in lung fibroblasts depleted of normal GSH protection by the glutamylcysteinyl synthetase inhibitor, L-buthionine-[S,R]-sulfoximine. Although the specific mechanism of NAC protection has not yet been elucidated, these results support the hypothesis that NAC may protect the cells directly, by scavenging ROS induced by UVA and visible radiation, and indirectly by donating cysteine for GSH synthesis.     

Photobiological effects of UVA and UVB light in zebrafish embryos: evidence for a competent photorepair system

The consequences of UVB and UVA irradiation on hatch rate, mortality, and malformation were studied in embryonic zebrafish (Danio rerio). The use of zebrafish embryos has expanded from traditional developmental models to diverse studies, including many techniques utilizing light exposure. To characterize useful indicators of photodamage, the responses and threshold limits of UV radiation as a function of embryonic stage and fish source were evaluated. Significant differences in UVB susceptibility were observed in embryos at 3, 6-7, 12, and 24h post-fertilization (hpf), with the 1000-cell stage (3 hpf) having greatest tolerance to UVB. Embryos derived from zebrafish raised in outdoor ponds were more tolerant to UVB than were embryos from laboratory-raised fish. Combinations of UVB and UVA exposure were used to confirm the presence of a competent photorepair system in zebrafish that could return otherwise malformed embryos to a normal phenotype. Overall, embryonic zebrafish had large tolerances (LD(50) of 850 J/cm(2)) to UVA, confirming their suitability for photoactivation and photorepair studies.