Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: development of an ex vivo test method

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40-65% when applied at 2 mg/cm(2). SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO(2)-UVB filters and a novel UVA-TiO(2) filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.     

The suppression of immunity by ultraviolet radiation: UVA, nitric oxide and DNA damage.

We have examined the mechanism by which solar-simulated ultraviolet radiation (ssUV) suppresses memory immunity to nickel in allergic humans. In initial studies, we used inbred mice to determine the contribution of different wavebands to sunlight-induced immunosuppression. We found that low dose UVA can enhance memory, medium dose UVA (half the amount in one minimum erythemal dose of ssUV) is immunosuppressive, but higher doses protect from UVB. This is genetically dependent, as it is not observed in all mouse strains. UVA caused a similar dose-related change in recall immunity in humans. ssUV dose responses determined the limits of protection provided by sunscreens from immunosuppression in humans. Immune protection factors calculated from these data correlated with UVA protection, but not with sun protection factor, showing that in commercial sunscreens that provide good UVB protection, UVA protection limits prevention of immunosuppression. N(G)-monomethyl-l-arginine acetate (l-NMMA) was used to inhibit nitric oxide (NO) production and T4N5 liposomes containing T4 endonuclease V to enhance DNA repair. Sub-erythemal ssUV caused a dose-related local suppression of recall immunity to nickel in humans. l-NMMA and the liposomes protected the nickel reaction, suggesting that NO and DNA damage are mediators of UV-induced immunosuppression in humans.     

Prevention of UVA-Induced Oxidative Damage in Human Dermal Fibroblasts by New UV Filters,Assessed Using a Novel In Vitro Experimental System

Background

UVA rays present in sunlight are able to reach the dermal skin layer generating reactive oxygen species (ROS) responsible for oxidative damage, alterations in gene expression, DNA damage, leading to cell inflammation, photo-ageing/-carcinogenesis. Sunscreens contain UV filters as active ingredients that absorb/reflect/dissipate UV radiation: their efficiency depends on their spectral profile and photostability which should then be reflected in biological protection of underlying skin.

Methods

A set of new UV filters was synthesized, and the most photostable one was compared to BMDBM, a widely used UVA filter. Cultured human dermal fibroblasts were exposed to UVA radiation which was filtered by a base cream containing or not UV filters placed above cell culture wells. The endpoints measured were: cell viability (MTT assay), ROS generation (DCFH-DA assay), mitochondrial function (JC-1 assay), DNA integrity (Comet assay) and gene expression (MMP-1, COL1A1) by RT-qPCR.

Results

The new UV filter resulted more efficient than BMDBM in preserving cell viability, mitochondrial functionality and oxidative DNA damage, despite similar inhibition levels of intracellular ROS. Moreover, expression of genes involved in dermal photoageing were positively affected by the filtering action of the tested molecules.

Conclusions

The experimental model proposed was able to validate the efficacy of the new UV filter, taking into account important cellular events related to UV-induced intracellular oxidative stress, often underestimated in the assessments of these compounds.

General Significance

The model may be used to compare the actual biological protection of commercial sunscreens and suncare products aside from their SPF and UVA-PF values.     

Sunscreens with an absorption maximum of > or =360 nm provide optimal protection against UVA1-induced expression of matrix metalloproteinase-1, interleukin-1, and interleukin-6 in human dermal fibroblasts.

UVA1-induced expression of matrix metalloproteinase-1 (MMP-1) is mediated by an autocrine mechanism involving the cytokines interleukin-1 and -6 (IL-1 and IL-6). The subsequent degradation of collagen fibers is thought to be the main cause of skin wrinkling. As it is currently not known which wavelengths within the UVA1 range are responsible for these effects, we have assessed 5 UVA1 filters (experimental filters HRH21328 and HRH22127, butyl methoxydibenzoylmethane (BMDM), diethylaminohydroxybenzylbenzoic acid hexyl ester (DHBB) and anisotriazine) with different absorption maxima for their capacity to protect against UVA1-induced MMP-1 expression. To test the efficacy of these hydrophobic filters in a cell culture system, UVA1 irradiation of primary human fibroblasts was performed through a quartz microplate filled with ethanolic solutions of the UVA filters placed on top of the cell microplate. Inhibition of UVA1-induced gene expression was detected by real time RT-PCR. The efficacy to protect against UVA1-induced MMP-1 expression was wavelength dependent: the protection by HRH22127 was best, followed by HRH21328, DHBB, BMDM, and anisotriazine. In addition, HRH22127 and HRH 21328 both significantly inhibited UVA1-induced expression of IL-1alpha and IL-6 with HRH21238 being superior to HRH22127. These studies indicate that UVA1 filters with a maximum absorption at > or =360 nm are most effective in preventing UVA1 radiation-induced MMP-1, IL-1alpha, and IL-6 expression pointing towards a critical role for effective filtering beyond > or =360 nm for protection against UVA1-induced photoaging.     

UVA and endogenous photosensitizers--the detection of singlet oxygen by its luminescence

UVA irradiation (320-400 nm) comprises about 95 percent of incident midday solar ultraviolet irradiation. It penetrates skin much deeper than UVB irradiation. The absorption of UVA irradiation in endogenous chromophores frequently leads to the generation of reactive oxygen species such as singlet oxygen ((1)O(2)). (1)O(2) is an important biochemical intermediate in multiple biological processes. Beside other procedures, the direct detection of (1)O(2) by its luminescence is a powerful tool that helps to understand the generation of (1)O(2) during UVA exposure in solution, in vitro and in vivo. This article describes the endogenous photosensitizers, their ability to generate (1)O(2) under UVA irradiation, and the detection technology to visualize the action of (1)O(2).     

Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm2 UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1alpha levels increased with LOM concentration. The release of TNF-alpha and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 micromol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM.     

Effects of in vitro exposure to power frequency magnetic fields on UV-induced DNA damage of rat lymphocytes

The mechanisms of biological effects of 50/60 Hz (power frequency) magnetic fields (MF) are still poorly understood. There are a number of studies indicating that MF affect biochemical processes in which free radicals are involved, such as the biological objects' response to ultraviolet radiation (UVA). Therefore, the present study was aimed to assess the effect of 50 Hz MFs on the oxidative deterioration of DNA in rat lymphocytes irradiated in vitro by UVA. UVA radiation (150 J/m2) was applied for 5 min for all groups and 50 Hz MF (40 microT rms) exposure was applied for some of the groups for 5 or 60 min. The level of DNA damage was assessed using the alkaline comet assay, the fluorescence microscope, and image analysis. It has been found that the 1 h exposure to MF caused an evident increase in all parameters consistent with damaged DNA. This suggest that MF affects the radical pairs generated during the oxidative or enzymatic processes of DNA repair.     

Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: protection by nitroxide radicals

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.     

Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts

During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.     

Synthesis and application of a novel sunscreen-antioxidant

Background information on the inefficacy of sunscreens to provide free radical protection in skin, despite their usefulness in preventing sunburn/erythema, prompted us to synthesize a compound which would display in the same molecule both UV-absorbing and antioxidant capacities. For this purpose, the UVB absorber, 2-ethylhexyl-4-methoxycinnamate (OMC) was combined with the piperidine nitroxide TEMPOL, which has antioxidant properties. The spectral properties of the new nitroxide-based sunscreen (MC-NO) as well as its efficacy to prevent photo-oxidative damage to lipids induced by UVA, natural sunlight and 4-tert-butyl-4-methoxydibenzoylmethane (BMDBM), a photo-unstable sunscreen which generates free radicals upon UV radiation, was studied. The results obtained demonstrate that MC-NO: (a) absorbs in the UVB region even after UVA irradiation; (b) acts as free radical scavenger as demonstrated by EPR experiments; (c) strongly reduces both UVA-, sunlight- and BMDBM-induced lipid peroxidation in liposomes, measured as reduced TBARS levels; and (d) has comparable antioxidant activity to that of commonly used vitamin E and BHT in skin care formulations. These results suggest that the use of the novel sunscreen-antioxidant or of other nitroxide-based sunscreens in formulations aimed at reducing photoinduced skin damage may be envisaged.     

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.     

Protection from solar simulated radiation-induced DNA damage in cultured human fibroblasts by three commercially available sunscreens

Exposure to solar radiation can produce both acute and chronic changes in the skin, including sunburn, edema, immunosuppression, premature skin aging, and skin cancer. At the cellular level, solar radiation can produce adverse structural and functional changes in membrane proteins and lipids and in chromosomal and mitochondrial DNA. The increasing awareness of these adverse effects has led the public to demand better photoprotection. In this study, the alkaline comet assay was used to evaluate the photoprotective effects of three commercially available sunscreens at sun protection factors (SPF) 15 and 30. Human fibroblasts were used as target cells to conveniently study the effects of solar simulated radiation on DNA damage in the presence and absence of sunscreens. When human fibroblasts were exposed to various doses of solar simulated radiation, DNA damage, as measured in sunscreen-protected cells by the comet assay, was not significantly different from that detected in unexposed cells. At 1.0 and 1.5 minimal erythemal doses (MED), all sunscreens, at both SPF 15 and 30, provided nearly 100% photoprotection to the fibroblasts. Further studies are required to elucidate the role of UVA in the production and repair of DNA damage in cells exposed to sunlight.     

长波紫外线辐射对脊椎动物的影响

臭氧层空洞的加剧使紫外辐射不断加强,造成的环境污染已经成为广为关注的问题,但大多数都集中在中波紫外线(UVB)的生物学作用及其与两栖动物衰减关系的研究,有关长波紫外线(UVA)对生物体损伤作用的研究却甚少。本文综述了近年来UVA对脊椎动物损伤的研究概况,从细胞结构、酶活性、遗传物质、膜结构以及免疫系统等方面阐述了UVA的损伤机理,并对低剂量辐射诱导兴奋效应对紫外损伤的保护作用进行了简要概述。最后分析和总结了关于UVA损伤研究所存在的问题及两栖动物作为实验用动物模型在研究紫外线损伤中的作用。     

Photodegradation of carotenoids in human subjects

Photodegradation of vitamins in vitro is responsible for large losses of these nutrients in foods, beverages, and semisynthetic liquid formula diets. In vivo photodegradation of vitamins has been reported for riboflavin in jaundiced infants exposed to blue light and for folate in patients with chronic psoriasis given photochemotherapy. Two recent studies of normal subjects have also shown that photodegradation of carotenoids in plasma occurs with cumulative exposure of the skin to an artificial light source having maximal spectral emission in the UVA range. Females showed a larger effect of the UV light on their plasma carotenoid levels than males. These observations have identified a need for further investigation of the role of sunlight exposure as a determinant of plasma carotenoid levels and vitamin A status in human subjects.     

Modeling of SEB-induced host gene expression to correlate in vitro to in vivo responses

Detection of exposure to biological threat agents has relied on ever more sensitive methods for pathogen identification, but that usually requires pathogen proliferation to dangerous, near untreatable levels. Recent events have demonstrated that assessing exposure to a biological threat agent well in advance of onset of illness or at various stages post-exposure is invaluable among the diagnostic options. There is an urgent need for better diagnostic tools that will be sensitive, rapid, and unambiguous. Since human clinical cases of illness induced by biothreat agents are, fortunately, rare, use of animal models that closely mimic the human illness is the only in vivo option. Such studies can be very difficult and expensive; therefore, maximizing the information obtained from in vitro exposures to peripheral blood mononuclear cells (PBMCs) provide an opportunity to investigate dose/time variability in host responses. In our quest to study staphylococcal enterotoxin B (SEB) induced host gene expression patterns, we addressed two core issues using microarray analysis and predictive modeling. Our first objective was to determine gene expression patterns in human PBMCs exposed to SEB in vitro. Second, we compared the in vitro data with host responses gene expression patterns in vivo using PBMCs from an animal model of SEB intoxication that closely replicates the progression of illness in humans. We used cDNA microarrays to study global gene expression patterns in piglets intoxicated with SEB. We applied a supervised learning method for class prediction based on the k-nearest neighbor algorithm for the data obtained in piglets exposed to SEB in vivo against a training data set. This data set included gene expression profiles derived from in vitro exposures to eight different pathogens (Bacillus anthracis, Yersinia pestis, Brucella melitensis, SEB, cholera toxin, Clostridium botulinum toxin A, Venezuelan equine encephalitis, and Dengue-2) in PBMCs. We found that despite differences in gene expression profiles between in vitro and in vivo systems, there exists a subset of genes that show correlations between in vitro and in vivo exposures, which can be used as a predictor of exposure to SEB in vivo.     

Pharmacokinetics and metabolism in early drug discovery.

The need for high-throughput approaches in absorption, distribution, metabolism and excretion studies is driven by the impact of high-speed chemistry and pharmacological screening. Perhaps an even greater impact is that these studies will, in the future, provide large data sets that can be used to predict biological events related to absorption, bioavailability and metabolism of drugs. Through linking of in silico and in vitro methods, considerable progress has recently been made towards this future perspective. Despite this progress, these approaches do not yet replace in vivo methods.     

A Highlights from MBoC Selection: Cega: a single particle segmentation algorithm to identify moving particles in a noisy system

Improvements to particle tracking algorithms are required to effectively analyze the motility of biological molecules in complex or noisy systems. A typical single particle tracking (SPT) algorithm detects particle coordinates for trajectory assembly. However, particle detection filters fail for data sets with low signal-to-noise levels. When tracking molecular motors in complex systems, standard techniques often fail to separate the fluorescent signatures of moving particles from background signal. We developed an approach to analyze the motility of kinesin motor proteins moving along the microtubule cytoskeleton of extracted neurons using the Kullback-Leibler divergence to identify regions where there are significant differences between models of moving particles and background signal. We tested our software on both simulated and experimental data and found a noticeable improvement in SPT capability and a higher identification rate of motors as compared with current methods. This algorithm, called Cega, for “find the object,” produces data amenable to conventional blob detection techniques that can then be used to obtain coordinates for downstream SPT processing. We anticipate that this algorithm will be useful for those interested in tracking moving particles in complex in vitro or in vivo environments.     

Liquid chromatographic assay for common sunscreen agents: application to in vivo assessment of skin penetration and systemic absorption in human volunteers

The purpose of the present study was to develop a reverse-phase high-performance liquid chromatographic (HPLC) assay for quantifying four common sunscreen agents, namely 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 2-ethylhexylsalicylate (octylsalicylate) and salicylic acid 3,3,5-trimethcyclohexyl ester (homosalate) in a range of biological matrices. This assay was further applied to study the skin penetration and systemic absorption of sunscreen filters after topical application to human volunteers. Separation was achieved utilizing a Symmetry C(18) column with methanol-water as the mobile phase. The assay permits analysis of the sunscreen agents in biological fluids, including bovine serum albumin (BSA) solution, plasma and urine, and in human epidermis. The assay was linear (r2 > 0.99) with minimum detectable limits of 0.8 ng for oxybenzone, 0.3 ng for octylmethoxycinnamate, and 2 ng for homosalate and octylsalicylate. The inter- and intra-day variation for the four sunscreens was less than 3% at the upper end of the linear range and less than 6% at the lower end. Recoveries of sunscreens from plasma, 4% (w/v) BSA solution and epidermal membranes were within the range of 91-104%. Recoveries from urine of the four sunscreens, and oxybenzone with its metabolites were more than 86%. Up to approximately 1% of the applied dose of oxybenzone and its metabolites was detected in the urine. Appreciable amounts were also detected in the stratum corneum through tape stripping. The HPLC assay and extraction procedures developed are sensitive, simple, rapid, accurate and reproducible. Results from the preliminary clinical study demonstrate significant penetration of all sunscreen agents into the skin, and oxybenzone and metabolites across the skin.