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

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

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice.

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.     

Wavelength dependence of histological, physical, and visible changes in chronically UV-irradiated hairless mouse skin

Albino hairless mice (Skh: HR-1) exposed chronically to sub-erythemal doses of UV radiation display physical, visible and histological alterations. Using narrow bandwidth radiation covering the UV radiation spectrum from 280-380 nm, the wavelength dependence of these alterations was determined. The wavelength dependence spectra indicate that for all but one parameter measured (skin sagging), UV-B radiation is considerably more efficient than UV-A radiation in producing changes in the skin. However, in natural sunlight there is considerably more UV-A than UV-B radiation, providing the potential for UV-A to have a larger contribution to skin damage than UV-B. This argues in favor of using broad spectrum photoprotective agents to shield the skin adequately from UV-induced aging. The spectra were also used to develop potential associations among events by determining which events occur at similar wavelengths. There seems to be a correspondence between mouse visible skin wrinking (UV-B event) and two histological events: increase in glycosaminoglycans and alteration in collagen. There was no obvious correspondence among UV-A-induced events.     

EFFECTS OF CHRONIC EXPOSURE ULTRAVIOLET-A INCLUDING 2% ULTRAVIOLET-B ON FREE RADICAL REDUCTION SYSTEMS IN HAIRLESS MICE

Chronic ultraviolet (UV) irradiation is known to cause a variety of changes in the skin, including wrinkles, pigmented spots and carcinogenesis. To explore time dependent changes in several parameters with chronic UV irradiation, we examined the molecular changes in connective tissue, intracellular defence enzymes and free radical antioxidant substances in hairless mice skin caused by chronic exposure to UV-A including 2% UV-B. Connective tissue changes were estimated using hydroxyproline and isodesmosine assays as a measure of collagen and elastin concentrations, respectively. After 6 weeks irradiation, the insoluble collagen and elastin were both substantially elevated, as were the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Continued UV irradiation resulted in a steady decline in SOD and lipid soluble antioxidants, while the GSH-Px remained elevated, suggesting that SOD and lipid soluble antioxidants in the skin may be involved in protecting it from UV damage and deteriorate with chronic irradiation.     

Biochemical changes in hairless mouse skin collagen after chronic exposure to ultraviolet-A radiation.

Evidence is mounting that UV-B and UV-A radiation affect skin differently in responses as diverse as erythema and elastosis. We found in this study that collagen metabolism was also differentially affected. Albino hairless mice were irradiated with two UV-A sources: (1) UVASUN 3000 (340-400 nm) for cumulative exposures of 4000 and 8000 J/cm2; (2) a xenon solar simulator filtered to provide full spectrum UV-A (320-400 nm) and long wavelength UV-A (335-400 nm) for cumulative exposures of 3000 and 4000 J/cm2 respectively. Collagen was isolated from other skin proteins by acid extraction, pepsin digestion and salt precipitation. Collagen types I and III were separated by interrupted gel electrophoresis. Ultraviolet-A rendered the collagen highly resistant to pepsin digestion. In age-matched controls only 16-18% of the total collagen remained insoluble, whereas in long wavelength UV-A-irradiated skins the insoluble fraction was as high as 87%. A dose response was noted at 4000 and 8000 J/cm2 as delivered by the UVASUN. Recovery of collagen from the pepsin soluble fraction was low in all UV-A groups and the amount of type III so small that determination of ratios of type III to I collagen was unreliable. These results suggest that chronic UV-A radiation may increase cross-linking of dermal collagen.     

THE HAIRLESS MOUSE MODEL OF PHOTOAGING: EVALUATION OF THE RELATIONSHIP BETWEEN DERMAL ELASTIN, COLLAGEN, SKIN THICKNESS AND WRINKLES

Quantitative and qualitative changes in dermal collagen and elastin occur in response to chronic ultraviolet (UV) irradiation. These changes have been implicated in the genesis of the wrinkling seen in chronically irradiated, or photoaged skin. We examined the relationship between wrinkle formation and changes in dermal structural protein content and type. Skh-1 hairless mice were irradiated with suberythemal doses of UV-B three times a week for up to 20 wk. Visible wrinkling was present after 6-7 wk of irradiation. Dermal elastic fiber content was quantified by color image analysis of paraffin-embedded tissue. There was no significant difference in dermal elastic fiber content between irradiated and age-matched control mice after either 10 or 20 wk of irradiation. The effect of UV-B irradiation on total dermal collagen content, ratio of collagen type III-type I, and extent of glycosylation and crosslinking of collagen was no different in irradiated and age-matched control mice after 10 wk of irradiation. Increased epidermal thickness was evident in frozen sections after 6 wk of irradiation, and the thickness increased with continued irradiation. Dermal thickening was evident after 10 wk of irradiation. Sufficient UV-B irradiation will eventually cause changes in dermal elastin and collagen content; however, wrinkle formation precedes such changes. A causal relationship between wrinkle formation and dermal structural protein content changes in Skh-1 hairless mice could not be established in this study.     

Age dependent increase of elastase type protease activity in mouse skin. Effect of UV-irradiation

The effect of chronological aging and photoaging (UV-radiation) on elastase-type enzyme activity of hairless mouse skin was studied. Aging resulted in the increase of elastase type endopeptidase activity extractable from mouse skins. Both chronic UVA and UVB radiation resulted in a significant increase of elastase type activity. PBS extracted only small part of the elastase activity, UV-A produced an increase of about 90-120% according to the type of irradiation (xenon or UV-A SUN) and UV-B produced a 72% increase. Extraction by Triton X-100 suggested that most of the activity is bound to cells and fibrous structures. EDTA inhibited 80-90% of the elastase activity in chronologically aged skin extracts and also the activity induced by UVA radiation suggesting that metallo-elastase(s) are involved. About 30% of the UVB induced activity could only be inhibited by EDTA and about 50% by PMSF suggesting that irradiation by UVB increased more serine endopeptidase activity but also MMP-activity. Chronic UVA radiation produced an increase of skin elastase activity equivalent to that observed after 24 months of aging in non-irradiated animals (approximately 100 weeks) corresponding to approximately 90% of total life span of these mice. The total increase produced by UVB was less, but the strong increase of a serine elastase, presumably from PMN-s, appear to produce a much more pronounced biological activity as shown by the presence of fibronectin degradation products in skin extracts. Such degradation products were shown to exert harmful effects on tissues. These results may well have biological significance and distinguish chronological aging and photoaging.     

Noninvasive Assessment of UV-induced Skin Damage: Comparison of Optical Measurements to Histology and MMP Expression

Acute exposure to UV radiation (UVR) causes visible skin damage such as erythema and results in local and systemic immunosuppression while chronic exposure can result in photocarcinogenesis. These deleterious effects can be quantified by histology and by bioassays of key biological markers, including matrix metalloproteinases (MMPs), or tryptophan moieties. We now report our results in quantifying UV skin damage with noninvasive optical methods based on reflectance and fluorescence spectroscopy and compare these noninvasive measurements to histopathology and MMP-13 expression. A solar simulator with spectral output nearly identical to that of solar radiation was developed and used in our experiments. SKH1 hairless mice were exposed to solar-simulated UVR at a total dose of 21 MED delivered over 10 weeks. Changes in oxygenated and deoxygenated hemoglobin were measured by diffuse reflectance spectroscopy, and tryptophan changes were monitored via a fluorescence monitor. Our results show that there is an increase in erythema, skin fluorescence, sunburn cells and MMP-13 after a series of suberythemal doses of UV irradiation on a hairless mouse animal model. Increased skin fluorescence is observed with increasing UV exposure. The levels of MMP-13 increase as the cumulative UV dose increases but their increase does not correspond to noninvasively measured changes.     

TUMORIGENESIS BY A LONG WAVELENGTH UV-A SOURCE

Albino hairless mice (Skh-hr1) were exposed daily to radiation from a high-power long wavelength UV-A source (wavelengths longer than 340 nm). The irradiations lasted 2 h per day. The daily dose was 220 kJ/m2. Heavy scratching marks were observed in 13 out of 48 animals. However during the experiment 31 of the animals developed tumors of 1 mm or larger before any scratching was observed. The median induction time was 265 days for 1 mm tumors.     

Effect of Laser Thermal Injury on Langerhans Cells in Mouse and Hairless Guinea Pig Epidermis

To examine the effect of laser thermal injury on Langerhans cells (LC) within the epidermis, the dorsal skin of mice and hairless guinea pigs was exposed to varying levels of laser irradiation using a thulium laser at a wavelength of 2.0 μm. At 6, 24 and 48 h post irradiation, animals were euthanized, skin samples prepared for histology and the epidermis obtained and stained by major histocompatibility complex‐II staining (mice) or ATPase assay (hairless guinea pigs) for the enumeration of LC. Mouse skin exhibited histological evidence of thermal damage at 24 h post irradiation at even the lowest dose (0.14 W) and decreases in the numbers of epidermal LC were observed at all doses and decreases were proportional to dose. In contrast, hairless guinea pig skin only showed consistent histological evidence of thermal damage at the highest dose of irradiation (0.70 W) at 24 and 48 h post irradiation and exhibited a statistically significant decrease in numbers of epidermal LC only at this dose. Thus, epidermal LC depletion occurred in the skin of both mice and hairless guinea pigs in response to laser treatment and the magnitude of depletion directly correlated with the extent of thermal damage both within and between species.     

Advantages of using hairless mice versus haired mice to test sunscreen efficacy against photoimmune suppressions

We tested the hypothesis that the strain of mice used in sunscreen protection experiments may influence immune protection. Ultraviolet (UV) dose-response curves were done in the presence or absence of a sun protection factor (SPF) 15 sunscreen using SKH1:hrBR or C3H/HeN mice. SKH1:hrBR mice showed a higher sensitivity to the suppressive effects of UV radiation (50% immune suppression equal to 5.2 kJ/m2 UVB in SKH1:hrBR mice versus 18.5 kJ/m2 in C3H mice). Immune protection factors (IPF) and an erythema protection factor (Ery-PF) for SKH1:hr mice were derived. The Ery-PF in hairless mice was 13.5, which was similar to the SPF of 15 measured in humans. When IPF were calculated as a ratio of minimal immune suppressive doses, the IPF for the SKH1:hrBR mice was 8.23 and the IFP for the C3H/HeN mice was 1.92. When IPF were estimated using the entire UV dose-response range, they were equal to 9.01 for SKH1:hrBR mice and 1.79 for the C3H/HeN mice. Because IPF and SPF can be measured directly in hairless mice, we suggest that the use of hairless mice may provide a better model to measure sunscreen efficacy, especially when the use of human volunteers is inappropriate, unethical or impossible.     

Unexpected photolysis of the sunscreen octinoxate in the presence of the sunscreen avobenzone

A major concern raised about photostability studies of sunscreen products is that the photodegradation of sunscreens does not readily translate into changes in product performance. This study examines the correlation between photochemical degradation of sunscreen agents and changes in protection provided by sunscreen films. Films of a commercial sunscreen product containing avobenzone, oxybenzone and octinoxate were irradiated using a fluorescent UV-A phototherapy lamp with additional UV-B blocking filter. Periodically, during irradiation the transmittances of the films were measured and samples collected for chemical analysis of the sunscreen agents using high-performance liquid chromatography techniques. The results show that UV-induced changes in UV transmittance of sunscreen films correlate with changes in concentration of sunscreen agents. In a parallel experiment, we also irradiated a thin film of the same product in the cavity of an electron spin resonance (ESR) spectrometer. We report the concomitant photolysis of avobenzone and octinoxate that predominates over expected E/Z photoisomerization and that irradiation of a film of this product produced free radicals detected by ESR spectroscopy that persisted even after exposure had ended.     

EFFECT OF CHRONIC UV EXPOSURE ON EPIDERMAL FORWARD SCATTERING-ABSORPTION INSK–1 HAIRLESS MOUSE SKIN

Abstract— Clinical and histological precancerous responses to UV irradiation are complicated dynamic functions of total dose, dose fractionation, fluence rate, and spectral distribution. This may be due, in large part, to the ability of UV to decrease epidermal-stratum corneum transmission by stimulation of hyperplasia. This work provides quantitative measurement of dose- and wavelength-dependent optical changes inSK–1 hairless mouse epidermis-stratum corneum occurring under irradiation with “monochromatic” UV wavebands, at 280, 290, 300, 307, and 313 nm. Mice were irradiated 5 days per week with a filtered Xenon-Hg high-intensity grating monochromator, starting with 0.9 minimal erythemal dose (MED), followed by incremental increases in the radiation dose by 20% of the original dose every tenth irradiation day, for2–8 consecutive weeks. Subsequent irradiations (for longer experiments) were followed by 30% incremental increases after the 8th week every 10th irradiation day until cessation of radiation at the end of 14 weeks. Irradiated and control full-thickness epidermis/ stratum corneum were examined histologically and by forward-scattering absorption spectroscopy. Chronic irradiation of hairless mice resulted in significant hyperplasia which was optically manifested by a general increase in forward-scattering absorbance. At moderate local doses (7.2 MED), the absorbance increase per MED was approximately the same for all excitation wavelengths, whereas at large total doses (? 100 MED) the optical increase per delivered MED progressively decreased in the order 313> 307> 300? 290> 280 nm. The increase in skin thickening, expressed as observed increase in absorption at 320 nm, correlated well with histological and clinical data. We propose that optical changes induced by UV-induced thickening can account in large part, if not entirely, for dynamic changes in action spectra for (pre) cancerous processes under chronic irradiation conditions.     

MOUSE SKIN PHOTOSENSITIVITY WITH DIHAEMATOPORPHYRIN ETHER (DHE) AND ALUMINIUM SULPHONATED PHTHALOCYANINE (AlSPc)

Skin photosensitivity of sun exposed sites is the major side effect of dihaematoporphyrin ether (DHE) photodynamic therapy (PDT). Reports of severe oedema and erythema have generally been anecdotal. We have studied aluminium sulphonated phthalocyanine (AlSPc) as a potential photosensitiser for PDT. In this paper we report our work comparing the skin photosensitivity reactions of DHE and AlSPc. We have studied: (i) the time course of the skin reactions, (ii) the effect of increasing time from administration of photosensitiser to irradiation, (iii) drug-skin reaction dose response. Groups of Skh I female hairless albino mice were given an intravenous bolus dose of either 0.9% saline solution, AlSPc or DHE (Photofrin II). Drug doses ranged from 0.5 to 50 mg/kg. At times ranging from 1 h to 1 month animals were irradiated with a range of doses of solar simulated radiation (SSR). The skin reaction was observed over a 2 week period. DHE reactions were always more severe than those with AlSPc. Peak skin reaction was seen at 3 h for DHE and 6 h for AlSPc. DHE reactions were still visible 2 weeks after irradiation whereas the AlSPc reaction disappeared by 48 h. Irradiation evoked a reaction up to 2 months after administration of DHE but only up to 2 weeks with AlSPc. The mean SSR dose at which a skin reaction was seen decreased with increasing dose of both agents. The rate of decrease was slower with AlSPc than DHE. This study suggests that in PDT, AlSPc will cause much less skin photosensitivity than DHE.     

Photoprotective effects of methoxycinnamidopropyl polysilsesquioxane

A new sunscreen ingredient, methoxycinnamidopropyl polysilsesquioxane (MCP-PSQ), which contains an UV-absorbing p-methoxycinnamoyl group, has been developed synthetically and evaluated using in vitro and in vivo approaches. Previous studies revealed that MCP-PSQ has a raising or boosting effect on the sun protection factor (SPF) of other sunscreen agents. In this study, we demonstrated that MCP-PSQ, an organic/inorganic hybrid compound, has photoprotective effects for human fibroblasts, and for hairless mouse and human skin. MCP-PSQ increases cell viability and suppresses the expression of p53 protein in fibroblasts after UV exposure. In addition, the numbers of sunburn cells and mast cells are reduced by topical application of MCP-PSQ on hairless mouse skin after UV irradiation. A 10% MCP-PSQ cream has higher and similar effects on SPF values for human skin compared to 5% titanium dioxide (TiO(2)) and 5% ethylhexyl methoxycinnamate (EHMC), respectively. The SPF value obtained using the MCP-PSQ cream did not drop after UV irradiation of the cream itself. However, higher dose of UV irradiation is required to guarantee the stability or photostability of the formulation. Further, there were no side effects such as erythema, edema, itch or tingling, suggesting that MCP-PSQ is a good sunscreen agent.     

TOPICAL UROCANIC ACID ENHANCES UV-INDUCED TUMOUR YIELD AND MALIGNANCY IN THE HAIRLESS MOUSE

Epidermal urocanic acid has been postulated to be the mediator of the specific state of immunosuppression induced by UV irradiation, by which UV-initiated tumour cells are able to evade normal recognition and can survive to grow progressively into malignant tumours. These experiments demonstrate that topical application of UV-irradiated urocanic acid systemically suppresses the contact type hypersensitivity response to oxazolone in hairless mice. In addition, topically applied urocanic acid markedly increases the overt tumour yield and the degree of malignancy in hairless mice exposed chronically to daily minimally erythemal doses of simulated solar UV light. Topical urocanic acid also increases the number of latent UV-initiated tumours, detectable by croton oil promotion. Therefore UV photoproducts of urocanic acid can both systemically suppress contact hypersensitivity in the epidermis, and also enhance early survival of UV-initiated tumour cells resulting in augmentation of UV photocarcinogenesis.     

Spectral properties of topical retinoids prevent DNA damage and apoptosis after acute UV-B exposure in hairless mice

We showed in a recent study that topical retinyl palmitate prevented UV-B-induced DNA damage and erythema in humans. Given that retinyl palmitate is a precursor of retinoic acid, the biological form of vitamin A that acts through nuclear receptors, we wondered whether these protective effects toward UV-B exposure were either receptor dependent or linked to other properties of the retinoid molecule such as its spectral properties. We determined the epidermal retinoid profile induced by topical retinoic acid in hairless mice and analyzed its effect on markers of DNA photodamage (thymine dimers) and apoptosis following acute UV-B exposure; we compared these effects to those induced by other natural topical retinoids (retinaldehyde, retinol and retinyl palmitate) which do not directly activate the retinoid receptors. We then analyzed the direct action of these retinoids on UV-B-induced DNA damage and apoptosis in cultured A431 keratinocytes. Topical retinoic acid significantly decreased (approximately 50%) the number of apoptotic cells, as well as the formation of thymine dimers in the epidermis of mice exposed to acute UV-B. Interestingly, the other topical retinoids decreased apoptosis and DNA damage in a similar way. On the other hand, neither retinoic acid nor the other retinoids interfered with the apoptotic process in A431 keratinocytes exposed to UV-B, whereas DNA photodamage was slightly decreased. We conclude that the decrease of apoptotic cells in hairless mouse epidermis following topical retinoids and UV-B irradiation reflects a protection of the primary targets of UV-B (DNA) by a mechanism independent of the activation of retinoid nuclear receptors, rather than a direct inhibition of apoptosis.     

Time-dependent decrease in sunscreen protection against chronic photodamage in UVB-irradiated hairless mouse skin

To determine the time dependence of sunscreen protection against chronic photodamage in hairless mice, the time was varied (0-8 h) between topical sunscreen treatment and UVB radiation exposure. Sunscreen products with labeled sun protection factor (SPF) values of 2, 4 and 8 were evaluated; these values were verified in a guinea pig model for SPF determinations. When applied immediately prior to UVB radiation exposure, these sunscreen products were very effective in prevention of skin wrinkling and tumor formation. Onset of photodamage was delayed, the delay being greater with higher SPF values. However, the sunscreen actives were rapidly lost from the skin surface, and their protective effect diminished strikingly as the time between treatment and irradiation increased. For daily protection against chronic photodamage, this suggests a need for photoprotectants with greater substantivity to achieve a high level of protection throughout the day.