Furocoumarin-induced Epidermal Melanogenesis Does Not Protect Against Skin Photocarcinogenesis in Hairless Mice

Topical 6,4,4'-trimethylangelicin (TMA) plus UVA was used to induce intense epidermal pigmentation in inbred HRA.HRII-c/+/Skh hairless pigmented mice over a 13 day period. Subsequent UVB/UVA exposure was used to assess the photoprotective properties of this tan using skin tumors as an endpoint. Comparisons were always made with sibling albino mice. The TMA/UVA treatment was shown to be not carcinogenic when treated mice were compared with untreated control mice over 25 weeks. The tan faded despite daily exposure to UVB/UVA and did not afford any protection when TMA/UVA-treated mice with subsequent UVB/UVA were compared with pigmented mice treated with UVB/UVA only. In one group, the TMA-induced tan was maintained by application of TMA three times a week prior to UVB/UVA for the duration of the experiment. This treatment was associated with a significant increase in tumor risk in both pigmented and albino mice compared to groups treated with UVB/UVA alone. Although pigmented mice had a significant photoprotective advantage, it was shown to be outweighed by the carcinogenic risks of the TMA maintenance treatment when they were compared with mice that did not have this treatment. Nonpretanned pigmented mice developed mild pigmentation during UVB/UVA treatment that was shown to have no protective effect when those mice were compared with albinos. We conclude that induced epidermal tanning with or without furocoumarin enhancement is not an effective way to prevent skin cancer in the HRA.HRII-c/+/Skh mouse model.     

Chronic ultraviolet B radiation-induced biochemical changes in the skin of hairless mice

Skh:HR-1 hairless mice were irradiated chronically with sub-erythemal doses of UVB radiation, and a number of biochemical parameters in the skin were determined after 6, 12, 18, and 24 wk of exposure. The parameters measured were water, collagen, elastin, and glycosaminoglycan content; collagenase and elastase levels; and Bz-Tyr-OEt (N-benzoyl-L-tyrosine ethyl ester) and BAPNA (alpha-N-benzoyl-DL-arginine-p-nitroanilide) hydrolyzing activities. Data for UVB radiation-exposed and chronological age-matched control mice were compared with respect to unit area and to unit mass of skin. On a unit area of skin basis, UVB radiation exposure increased the level of most parameters. The particular exceptions were collagen and collagenase which remained constant. On a mass of skin basis, though, there is an apparent decrease in collagen content because of the increase in the other skin components. This suggests that there is insufficient collagen in UVB radiation-exposed skin to support the increasing mass of the tissue.     

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.     

Photoprotective effects of bucillamine against UV-induced damage in an SKH-1 hairless mouse model

UVB exposure of skin results in various biologic responses either through direct or indirect damage to DNA and non-DNA cellular targets via the formation of free radicals, reactive oxygen species (ROS) and inflammation. Bucillamine [N-(2-mercapto-2-methylpropionyl)-l-cysteine] is a cysteine-derived compound that can replenish endogenous glutathione due to its two donatable thiol groups, and functions as an antioxidant. In this study, we investigated the effects of bucillamine on UVB-induced photodamage using the SKH-1 hairless mouse model. We have demonstrated that UVB exposure (two consecutive doses, 230 mJ cm(-2)) on the dorsal skin of SKH-1 mice induced inflammatory responses (edema, erythema, dermal infiltration of leukocytes, dilated blood vessels) and p53 activation as early as 6 h after the last UVB exposure. Bucillamine pretreatment (20 mg kg(-1) of body weight, administered subcutaneously) markedly attenuated UVB-mediated inflammatory responses and p53 activation. We have also demonstrated that the stabilization and upregulation of p53 by UVB correlated with phosphorylation of Ser-15 and Ser-20 residues of p53 protein and that bucillamine pretreatment attenuated this effect. We propose that bucillamine has potential to be effective as a photoprotective agent for the management of pathologic conditions elicited by UV exposure.     

Suppressive effect of caffeic acid and its derivatives on the generation of UVA-induced reactive oxygen species in the skin of hairless mice and pharmacokinetic analysis on organ distribution of caffeic acid in ddY mice

Caffeic acid (CA) and its analogues such as rosmarinic acid are well known as antioxidative agents. Exposure to UVA is known to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) and superoxide anion radical (*O2-) in the skin of animals, which in turn induces skin photodamage and photoaging. Because CA and its analogues quench 1O2, these compounds were topically applied to the abdominal skin of live hairless mice and were found to suppress ROS generation upon UVA exposure. Furthermore, the generation of UVA-induced ROS was also suppressed in the skin of mice that were orally given CA. In order to understand the mechanism by which CA blocks ROS production in UVA-exposed skin, the pharmacokinetics of CA upon oral administration to mice was followed and CA was found to efficiently distribute in the skin. These results suggest that skin damage by UVA-induced ROS generation is reduced by oral supplementation of CA, which has a scavenging and quenching activity against ROS.     

PROTECTION AGAINST ULTRAVIOLET B RADIATION-INDUCED EFFECTS IN THE SKIN OF SKH-1 HAIRLESS MICE BY A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA

In prior studies we and others have shown that oral feeding of a polyphenolic fraction isolated from green tea (GTP) or water extract of green tea affords protection against ultraviolet B (UVB) radiation-induced carcinogenesis in SKH-1 hairless mice (Wang et al., Carcinogenesis 12, 1527–1530, 1991). It is known that exposure of murine skin to UVB radiation results in cutaneous edema, depletion of the antioxidant-defense system and induction of ornithine decarboxylase (ODC) and cyclooxygenase activities. In this study we assessed the protective effect of GTP on these UVB radiation-caused changes in murine skin. Oral feeding of 0.2% GTP (wt/vol) as the sole source of drinking water for 30 days to SKH-1 hairless mice followed by irradiation with UVB (900 mJ/cm²) resulted in significant protection against UVB radiation-caused cutaneous edema ( P <0.0005) and depletion of the antioxidant-defense system in epidermis ( P <0.01–0.02). The oral feeding of GTP also resulted in significant protection against UVB radiation-caused induction of epidermal ODC ( P <0.005–0.01) and cyclooxygenase activities ( P <0.0001) in a time-dependent manner. Our data indicate that the inhibition of UVB radiation-caused changes in these markers of tumor promotion in murine skin by GTP may be one of the possible mechanisms of chemopreventive effects associated with green tea against UVB-induced tumorigenesis. The results of this study suggest that green tea, specifically polyphenols present therein, may be useful against inflammatory responses associated with the exposure of skin to solar radiation.     

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.     

EPR Detection of Free Radicals in UV-lrradiated Skin: Mouse Versus Human

Abstract— Ultraviolet radiation produces free radicals in Skh-1 mouse skin, contributing to photoaging and carcinogenesis. If a mouse model is a general indicator of free radical processes in human skin photobiology, then radical production observed in mouse and human skin should be directly comparative. In this work we show that UV radiation (Λ > 300 nm, 14 μW/cm² UVB; 3.5 mW/cm² UVA) increases the ascorbate free radical (Asc^.-) electron paramagnetic resonance (EPR) signal in both Skh-1 mouse skin (45%) and human facial skin biopsies (340%). Visible light (Λ > 400 nm; 0.23 mW/cm² UVA) also increased the Asc^.- signal in human skin samples (45%) but did not increase baseline mouse Asc^.-, indicating that human skin is more susceptible to free radical formation and that a chromophore for visible light may be present. Using EPR spin-trapping techniques, UV radiation produced spin adducts consistent with trapping lipid alkyl radicals in mouse skin (α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone/alkyl radical adduct; a^N= 15.56 G and a^H= 2.70 G) and lipid alkoxyl radicals in human skin (5,5-dimethylpyrroline-l-oxide/alkoxyl radical adduct; a^N= 14.54 G and a^H= 16.0 G). Topical application of the iron chelator Desferal to human skin significantly decreases these radicals (±50%), indicating a role for iron in lipid peroxidation; Desferal has previously been shown to decrease radical production in mouse skin. This work supports the use of the Skh-1 mouse as a predictive tool for free radical formation in human skin. These results provide the first direct evidence for UV radiation-induced free radical formation at near physiological temperatures in human skin and suggest that iron chelators may be useful as photoprotective agents.     

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.     

Dietary,but not topical,alpha-linolenic acid suppresses UVB-induced skin injury in hairless mice when compared with linoleic acids

Peroxidizability of fatty acids in the air is roughly proportional to the number of double bonds, but in vivo peroxidation proceeds in a more complex manner. Here, we compared the effects of dietary and topically applied oils enriched with linoleic acid (LA, 18:2n-6) or alpha-linolenic acid (ALA, 18:3n-3) on UV-induced skin injury in a strain of hairless mice. The UVB-induced erythema score was significantly lower in mice with topically applied creams containing LA and ALA than in mice with the basal cream; no significant increase in the score was detected in the ALA group compared with the LA group. However, dietary ALA inhibited the increase in erythema score after UVB irradiation compared with LA. The peroxidizability index of the skin total lipids was significantly higher, but UVB-induced prostaglandin E2 (PGE2) production was significantly lower in the group fed an ALA-rich diet compared with the group fed an LA-rich diet. The levels of thiobarbituric acid-reactive substances, estimated in the presence of butylated hydroxytoluene in the assay mixture, were not affected by UVB treatment or by the dietary fatty acids, but the severity of the skin lesion was associated with PGE2 levels. These results indicate that the type of fatty acids, n-6 or n-3, is critical for the suppression of UVB-induced skin lesion when the skin fatty acids are modified by dietary manipulation. Anti-inflammatory activity of dietary flaxseed oil with relatively high ALA and low LA contents was demonstrated in UVB-irradiated hairless mice.     

Natural Killer Cell Activity during UVR-induced Skin Tumor Formation in the Skh Hairless Mouse

Abstract— We analyzed natural killer (NK) cell activity in the hairless albino Skh/HR1 mouse, to study whether the NK cell activity plays a role during UV radiation (UVR)-induced carcinogenesis. In 4 h ^5lCr-release assays, spleen lymphocytes of specific pathogen-free (spf) Skh/HR1 mice displayed 5–10% spontaneous NK cell activity. This was comparable to NK ceil activity in C57BI/6, C3H and athymic NMRI nu/nu mice, which were also kept under spf conditions. In all strains investigated, the low spontaneous NK cell activity could be increased up to 20–30% by intraperitoneal administration of polyinosinic: polycytidylic acid (polyI:C), a standardized in vivo NK cell induction method. The polyI:C potentiation of NK cells in Skh/HR1 mice was similar to that in C57Bl/6 and NMRI, but significantly less than in C3H mice. Chronic daily UV irradiation according to a protocol that was also used for induction of carcinogenesis (11–12 weeks, 95 mJ/cm² of UV exposure from FS40 sunlamps) did not decrease NK cell activity on a cell for cell basis. Neither was the inducibility of NK spleen cell activity with poly I: C in Skh/HR1 mice during UV exposure reduced. Based on total organ basis, the pooled lymph node cells (axillary, mandibulary and inguinal lymph node) showed a doubling of NK cell activity ( P < 0.001), mainly due to an almost 100% increase in the number of lymph node cells. In conclusion, UVR does not suppress the normal or inducible NK cell activity at the time of clinical appearance of skin tumors. This suggests that such suppression of NK cell activity is not likely to contribute to UVR-induced carcinogenesis in the Skh/HRl strain.     

Attenuation of DNA damage in the dermis and epidermis of the albino hairless mouse by chronic exposure to ultraviolet-A and -B radiation

Mammalian skin is vulnerable to the photocarcinogenic and photoaging effects of solar UV radiation and defends itself using a variety of photoprotective responses including epidermal thickening, tanning and the induction of repair and antiradical systems. We treated Skh-1 albino hairless mice for 60 days with ultraviolet-A (UVA) or ultraviolet-B (UVB) radiation and measured the frequency of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by a single acute sunburn dose of UVB at different stages of the chronic treatment. We found that both UVA and UVB exposure produced a photoprotective response in the dermis and epidermis and that the degree of photoproduct attenuation was dependent on dose, wavelength and the type of damage induced. Although epidermal thickening was important, our data suggest that UV protective compounds other than melanin may be involved in mitigating the damaging effects of sunlight in the skin.     

French Maritime Pine Bark (Pinus maritima Lam.) Extract (Flavangenol®) Prevents Chronic UVB Radiation-induced Skin Damage and Carcinogenesis in Melanin-possessing Hairless Mice

A French maritime pine bark extract, Flavangenol^®, is widely used as a nutritional supplement for protection against atherosclerosis, hypertension, diabetes, etc. Chronic exposure to solar UV radiation damages skin, increasing cutaneous thickness, wrinkling and pigmentation, as well as reducing elasticity, and causes skin cancer. The aim of this study was to examine the effects of flavangenol on skin damage and the incidence of skin tumors caused by long-term UVB irradiation in melanin-possessing hairless mice. The oral administration of flavangenol (60, 200 or 600 mg kg⁻¹, twice daily) significantly inhibited increases in skin thickness, and the formation of wrinkles and melanin granules, as well as increases in the diameter and length of skin blood vessels. Furthermore, it prevented increases in numbers of apoptotic, Ki-67-positive and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells, and the expression of skin vascular endothelial growth factor (VEGF) induced by chronic UVB irradiation. The effect on these biomarkers was associated with a reduction in the incidence of tumors in mice. The antiphotoaging and anticarcinogenetic activities of flavangenol may be due to inhibition of the expression of Ki-67, 8-OHdG and VEGF through a scavenging effect on reactive oxygen species.     

Protective Effect of Curcumin Against Acute Ultraviolet B Irradiation‐induced Photo‐damage

Ultraviolet B (UVB) irradiation is one of the most dangerous insults for skin and causes sunburn, erythema, photoaging and photocarcinogenesis. Curcumin (diferuloylmethane), a yellow spice derived from dried rhizomes of Curcuma longa, has been shown to possess significant anti‐inflammatory, antioxidant, anticarcinogenic, antimutagenic, anticoagulant and anti‐infective effects. However, the protective effects of curcumin against acute photo‐damage are poorly understood. In this study, we investigated the photoprotective effects of curcumin against UVB‐induced acute photo‐damage in hairless mice and immortalized human keratinocytes (HaCaT). Topical application of curcumin significantly inhibited acute UVB (540 mJ cm^?2, for 3 successive days)‐induced inflammatory cells, collagen accrementition derangement and lipid peroxidation, and effectively induced NF‐E2‐related factor 2 (Nrf2) nuclear accumulation in uncovered (Uncv) hairless mice skin. Treatment of HaCaT cells with curcumin significantly attenuated acute UVB (300 mJ cm^?2)‐induced lactate dehydrogenase release, intracellular reactive oxygen species production and DNA damage, activated the expression of the phase II detoxifying enzymes and promoted DNA repair activity. The photoprotective effect provided by curcumin was potential associated with modulation of Nrf2‐dependent antioxidant response. Our study suggested that curcumin is a potential agent for preventing and/or treating UV radiation‐induced acute inflammation and photoaging.     

Postadministration Protective Effect of Magnesium-L-ascorbyl-phosphate on the Development of UVB-induced Cutaneous Damage in Mice

The effects of stable vitamin C, magnesium-L-ascorbyl-2-phosphate (MAP), administered after acute and chronic exposure to UVB irradiation were investigated using hairless mice. Intraperitoneal administration of 100 mg/kg of MAP immediately after acute exposure to 15 kJ/m² of UVB significantly prevented increases of UVB-induced lipid peroxidation in skin and sialic acid in serum, an inflammation marker. Administration of 50 mg/kg of MAP immediately after each exposure significantly delayed skin tumor formation and hyperplasia induced by chronic exposure to 2 kJ/m² of UVB. Intraperitoneal administration of 200 mg/kg of MAP produced an increase in ascorbic acid (As) levels in the serum, liver and skin within 15 min. Serum As levels quickly returned to normal, but hepatic and cutaneous levels remained elevated before returning to normal after 24 h, suggesting that MAP was converted to As in the serum and in those tissues. Ultraviolet B-induced hydroxyl radical generation in murine skin homogenates was scavenged by As-Na addition, which was directly detected by electron spin resonance (ESR). These results suggest that postadministration of MAP delays progression of skin damage induced by UVB irradiation. It is presumed that MAP, once converted to As, exhibits such inhibitory effects by scavenging hydroxyl and lipid radicals generated as a direct or indirect result of UVB exposure.     

Effects of Sphingomyelin-Containing Milk Phospholipids on Skin Hydration in UVB-Exposed Hairless Mice

Reactive oxygen species (ROS) generated by ultraviolet (UV) exposure cause skin barrier dysfunction, which leads to dry skin. In this study, the skin moisturizing effect of sphingomyelin-containing milk phospholipids in UV-induced hairless mice was evaluated. Hairless mice were irradiated with UVB for eight weeks, and milk phospholipids (50, 100, and 150 mg/kg) were administered daily. Milk phospholipids suppressed UV-induced increase in erythema and skin thickness, decreased transepidermal water loss, and increased skin moisture. Milk phospholipids increased the expression of filaggrin, involucrin, and aquaporin3 (AQP3), which are skin moisture-related factors. Additionally, hyaluronic acid (HA) content in the skin tissue was maintained by regulating the expression of HA synthesis- and degradation-related enzymes. Milk phospholipids alleviated UV-induced decrease in the expression of the antioxidant enzymes superoxidase dismutase1 and 2, catalase, and glutathione peroxidase1. Moreover, ROS levels were reduced by regulating heme oxygenase-1 (HO-1), an ROS regulator, through milk phospholipid-mediated activation of nuclear factor erythroid-2-related factor 2 (Nrf2). Collectively, sphingomyelin-containing milk phospholipids contributed to moisturizing the skin by maintaining HA content and reducing ROS levels in UVB-irradiated hairless mice, thereby, minimizing damage to the skin barrier caused by photoaging.     

Depletion of cutaneous glutathione by ultraviolet radiation

Supplemental antioxidants may have a role in ameliorating or preventing the actinic damage that can lead to cutaneous disorders such as skin cancer, hyperpigmentation, and premature aging. Glutathione is an important endogenous antioxidant and fulfills various protective functions in the skin. Irradiation of hairless mice with short (UVB) or long (UVA) wavelength ultraviolet radiation or with UVA combined with a photosensitizing psoralen (PUVA) can deplete skin glutathione levels. Ultraviolet B irradiation causes rapid transient fluctuations in the epidermal glutathione level and the relative amount present as the oxidized form. Ultraviolet A irradiation can deplete epidermal and dermal glutathione for several hours but requires much higher doses than UVB. PUVA treatments may lead to extensive and prolonged depletions of epidermal and dermal glutathione, the severity of which is dependent on the psoralen dose and may last for several days. These transient depletions, oxidations, and sometimes rapid recoveries of cutaneous glutathione levels are compatible with a role for glutathione as an endogenous photoprotective agent in the skin. Experimental evidence supports such a role: for example severe skin edema develops in mice only after about 50% of the glutathione has been depleted by PUVA treatment. Although different mechanisms are involved in each case, glutathione depletion may contribute to the production of phototoxicity by UVB, UVA, and by PUVA. Understanding the depletion mechanisms may allow the development of strategies aimed at preventing loss of cutaneous glutathione, and at reinforcing the natural protective functions of this critical cell component.