Glutathione Isopropyl Ester Reduces UVB-lnduced Skin Damage in Hairless Mice

Abstract— The protective effect of administration of glutathione (GSH) isopropyl ester on photodamage, such as lipid peroxidation, inflammation and tumorigenesis induced by UV exposure (290–400 nm, max. 312 nm), was investigated using hairless mice. Pretreatment with 20 mg/kg GSH isopropyl ester prevented the increases of thiobarbituric acid-reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, which were caused by a single dose (15 kj/m²) of UV irradiation. The level of epidermal GSH in skins of the GSH ester-treated mice was maintained within normal limits. When mice were exposed to UV at a dose of 2 kj/m², three times weekly, skin tumors developed in all of them after 25 weeks. The formation of skin tumors was significantly inhibited by administration of 10 mg/kg GSH ester prior to each UV irradiation for 25 weeks. Moreover, the increases of cutaneous TBARS and serum sialic acid in the tumor-bearing mice were also prevented by continuous pretreatment with GSH ester. Even after 24 weeks, the epidermal GSH content of the pretreated mice was mostly retained compared to nonirradiated mice. However, administration of GSH prior to acute or chronic UV irradiation had no effect on the UV-induced damage. The present results suggest that the protection from photo-damage afforded by pretreatment with GSH ester is due to maintenance of a normal GSH level.     

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.     

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.     

Protective Effect of Dermal Brimonidine Applications Against UV Radiation‐induced Skin Tumors,Epidermal Hyperplasia and Cell Proliferation in the Skin of Hairless Mice

Brimonidine at 0.18%, 1% and 2% concentrations applied topically in hairless mice significantly decreased tumor burden and incidences of erythema, flaking, wrinkling and skin thickening induced by UVR. The unbiased median week to tumor ≥1 mm was increased by the 1% and 2% concentrations. The tumor yield was reduced by all concentrations at week 40 for all tumor sizes but the ≥4 mm tumors with the 0.18% concentration. At week 52, the tumor yield was reduced for all tumor sizes and all brimonidine concentrations. The tumor incidence was reduced by all concentrations at week 40 for all tumor sizes, but the ≥4 mm tumor with the 0.18% concentration and at week 52 for all tumor sizes with the 1% and 2% concentrations and with the 0.18% concentration only for the ≥4 mm tumors. Reductions in ≥4 mm tumor incidences compared to the vehicle control group were 54%, 91% and 86% by week 52 for the 0.18%, 1% and 2% concentrations, respectively. Brimonidine at 2% applied 1 h before or just after UVB irradiation on hairless mice decreased epidermal hyperplasia by 23% and 32% and epithelial cell proliferation by 59% and 64%, respectively, similar to an epidermal growth factor receptor (EGFR) inhibitor.     

Wikstroemiaganpi Extract Improved Atopic Dermatitis-Like Skin Lesions via Suppression of Interleukin-4 in 2,4-Dinitrochlorobenzene-Induced SKH-1 Hairless Mice

Plants of the genus Wikstroemia are used in Chinese traditional medicine to treat inflammatory diseases, such as arthritis, bronchitis, and pneumonia. The present study was designed to determine whether Wikstroemia ganpi (Siebold and Zucc.) Maxim. offers a potential means of treating 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) in mice. Symptoms such as redness, edema, and keratinization in AD mice induced by DNCB were alleviated by the co-application of an ethanolic extract of W. ganpi for 2 weeks. The severity of skin barrier function damage was evaluated by measuring TEWL (transepidermal water loss). TEWLs of DNCB sensitized mouse dorsal skin were reduced by the application of a W. ganpi ethanolic extract, and skin hydration was increased. In addition, the infiltration of inflammatory cells into the dermis was significantly reduced, as were blood levels of IgE and IL-4, which play an important role in the expression of AD. The results of this experiment suggest that W. ganpi is a potential therapeutic agent for AD.     

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.     

A New Kind of Biomaterials——Bullfrog Skin Collagen

Pepsin-soluble collagen was prepared from bullfrog skin and partially characterized. This study revealed interesting differences, such as molecular weight, amino acid composition,denaturation temperature (Ta), in the frog skin collagen when compared to the known vertebrate collagens. This study gives hints that bullfrog skin can be a potential, safe alternative source of collagen from cattle for use in various fields.     

Photodynamic Therapy with Topical dL-aminolevulinic Acid Delays UV Photocarcinogenesis in Hairless Mice

Abstract— Photodynamic therapy (PDT) with topical application of 8-aminolevulinic acid (ALA) followed by irradiation with visible light (ALA-PDT) is a relatively new and promising experimental treatment of superficial premalignant and malignant skin neoplasms. The purpose of this study was to determine whether ALA-PDT can prevent photocarcinogenesis in hairless mice exposed to solar UV. A total of 140 mice was divided into seven groups of 20 mice each. Group 1: solar-UV exposure. Group 2: solar UV and a cream base + visible light once a week. Group 3: solar UV and ALA-PDT once a week. Group 4: solar UV and ALA-PDT once every second week. Group 5: solar UV and ALA-PDT every fourth week. Group 6: ALA-PDT once a week. Group 7: no treatment. The time to first and to second tumor 1 mm was registered. Predefined endpoints, such as one tumor a 4 mm or an area of small confluent tumors on the back of the mice were criteria for withdrawal from the experiment. The time to first and to second tumor was significantly longer in the ALA-PDT-treated mice than in mice only exposed to solar UV and solar-UV/cream base-visible light (P < 0.005). However, we observed an increased death and accident rate in the ALA-PDT-treated groups compared to the groups not treated with ALA-PDT (chi-square test, P = 0.0250). Significantly more ALA-PDT-treated mice were withdrawn because of a tumor 4 mm ( P = 0.0005). The UV unexposed mice developed no tumors. Repetitive treatments with ALA-PDT delay photoinduced carcinogenesis in mice.     

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.     

Inhibitory Effects of Plant Tannins on Ultraviolet Light-Induced Epidermal DNA Synthesis in Hairless Mice

Naturally occurring hydrolyzable (HT) and condensed (CT) tannins and their monomeric units were tested for their ability to inhibit the stimulation of DNA synthesis by UVB radiation. Hairless mice were irradiated with either single (200 mJ/cm²) or multiple (150 mJ/cm²) doses of UVB applied at 24 h intervals and epidermal DNA synthesis was measured at different times after the last of these treatments. The peak of DNA synthesis that is observed 48–56 h after a single UVB irradiation shifts to an earlier time of 16–24 h after multiple UVB treatments. Interestingly, the early inhibitory period of DNA synthesis observed 8 h after a single UVB treatment is not detected following multiple UVB treatments. Rather, DNA synthesis is stimulated six-fold 24 h after multiple UVB treatment, a response that is higher than the peak occurring 48–56 h after a single UVB irradiation. The disappearance of the early period of inhibition when the peak of DNA synthesis shifts to an earlier time may be linked to reactive oxygen species brought to the epidermis by infiltrating leukocytes, which, in turn, act as second messengers to stimulate growth signals in cells. Topical applications of HT or CT remarkably inhibit the DNA responses to single and multiple UVB treatments, an effect that is dependent on the dose and time of administration. Indeed, the peak stimulation of DNA synthesis is maximally inhibited when 17 mg of Tarapod tannic acid (TA), an HT, are applied topically 20 min before a single UVB treatment. The polymeric tannins inhibited DNA synthesis to a greater degree than equal doses of their monomeric units, gallic acid and catechin. These results suggest that various oligomeric HT and CT may be useful against tumor-promoting responses associated with the exposure of skin to physical carcinogens.     

p53 Mutations in Hairless SKH-hr1 Mouse Skin Tumors Induced by a Solar Simulator

In this study, we investigated whether the spectrum of p53 mutations in skin tumors induced in hairless SKH-hr1 mice by a solar simulator (290–400 nm) are similar to those found in skin tumors induced in C3H mice by UV radiation from unfiltered (250–400 nm) and Kodacelfiltered (290–400 nm) FS40 sunlamps. Analysis of tumor DNA for p53 mutations revealed that 14 of 16 (87.5%) SkH-hr1 skin tumors induced by the solar simulator contained mutations. Single C → T transitions at dipyrimidine sequences located on the nontranscribed DNA strand were the most predominant type of p53 mutation. Remarkably, 52% of all p53 mutations in solar simulator-induced SKH-hr1 skin tumors occurred at codon 270, which is also a hotspot in C3H skin tumors induced by unfiltered and Kodacel-filtered FS40 sunlamps. However, T → G transversions, which are hallmarks of UVA-induced mutations, were not detected in any of the solar simulator-induced skin tumors analyzed. These results demonstrate that the p53 mutation spectra seen in solar simulator-induced SKH-hr1 skin tumors are similar to those present in unfiltered and Kodacel-filtered FS40 sunlamp-induced C3H skin tumors. In addition, our data indicate that the UVA present in solar simulator radiation does not play a role in the induction of p53 mutations that contribute to skin cancer development.     

添加剂对活性人工皮肤胶原海绵支架材料的影响

研究了壳聚糖、硫酸软骨素和肝素对胶原海绵理化性质和生物相容性的影响。结果表明：添加壳聚糖后胶原海绵的孔隙率和暖水率下降,添加硫酸软骨素后上升,而添加肝素后无明显变化。三种添加剂均可减少基质收缩,增强材料的抗降解性能,但种问差异不明显。与纯胶原海绵相比,复合海绵可进一步促进细胞的吸附和增殖,其中添加壳聚糖和肝素的效果相当,优于硫酸软骨素,有望应用于构建组织工程化人工皮肤。     

A Collagen Hydrolysate Containing Tripeptides Ameliorates Sarcopenia in Middle-Aged Mice

Collagen peptide (CP) and collagen tripeptide (CTP) are supplementary health foods that exhibit several biological effects. However, the effects of collagen on age-associated sarcopenia and its underlying mechanisms are unclear. C57BL/6J mice (n = 24, 12 months old) were divided into three dietary groups and administered AIN93G (aging control, AC; JA BIO, Suwon, Korea), AIN93G plus 0.2% CP, and AING93G plus 0.2% CTP supplement for 12 weeks. The results indicated that the CP and CTP supplements significantly increased the weight of the quadriceps tibialis anterior and gastrocnemius muscles and reduced body fat. A morphological analysis revealed that the spaces within the muscle cells were tight with attenuated fibrosis following CP and CTP supplementation. Immunohistochemistry was applied and a Western blot analysis was performed to determine the underlying mechanisms. The CTP supplement increased the expression of IGF-1, PI3K/AKT, and mTOR, whereas the CP supplement increased the expression of IGF-1 and AMPK in the gastrocnemius of aging mice. CP and CTP ameliorate age-associated sarcopenia through different mechanisms.     

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30 mJ cm⁻²) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100 µm ). Consistent with these results, bucillamine pretreatment in mice (20 mg kg⁻¹) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6–12 and 24 h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.     

Photoprotective Effects of a Formulation Containing Tannase-Converted Green Tea Extract Against UVB-Induced Oxidative Stress in Hairless Mice

Ultraviolet B (UVB) irradiation may induce the acceleration of skin aging. The purpose of this study was to develop an effective formulation containing tannase-converted green tea extract (FTGE) to inhibit UVB-induced oxidative damage. Significant (p < 0.05) prevention of the reduced form of glutathione (GSH) depletion was observed in mice treated with FTGE. The hydrogen peroxide levels of mice treated with FTGE were similar to those of UVB non-irradiated mice. No significant difference was observed between No UVB control and FTGE mice. Also, mice treated with FTGE had significant (p < 0.05) decreases in thiobarbituric acid-reactive substance levels by lipid peroxidation compared with No UVB control mice. Our data suggest that this formulation may be effective in protecting skin from UVB photodamage.     

Endogenous Retinoic Acid Required to Maintain the Epidermis Following Ultraviolet Light Exposure in SKH‐1 Hairless Mice

Ultraviolet light B (UVB) exposure induces cutaneous squamous cell carcinoma (cSCC), one of the most prevalent human cancers. Reoccurrence of cSCC in high‐risk patients is prevented by oral retinoids. But oral retinoid treatment causes significant side effects; and patients develop retinoid resistance. Exactly how retinoids prevent UVB‐induced cSCC is currently not well understood. Retinoid resistance blocks mechanistic studies in the leading mouse model of cSCC, the UVB‐exposed SKH‐1 hairless mouse. To begin to understand the role of retinoids in UVB‐induced cSCC we first examined the localization pattern of key retinoid metabolism proteins by immunohistochemistry 48 h after UVB treatment of female SKH‐1 mice. We next inhibited retinoic acid (RA) synthesis immediately after UVB exposure. Acute UVB increased RA synthesis, signaling and degradation proteins in the stratum granulosum. Some of these proteins changed their localization; while other proteins just increased in intensity. In contrast, acute UVB reduced the retinoid storage protein lectin:retinol acyltransferase (LRAT) in the epidermis. Inhibiting RA synthesis disrupted the epidermis and impaired differentiation. These data suggest that repair of the epidermis after acute UVB exposure requires endogenous RA synthesis.     

UVR reflections at the surface of the eye

Sunlight plays an etiological role in the formation of skin cancers [Phys. Med. Biol. 24 (1979) 931]. Non-melanoma skin cancers commonly arise in sun-exposed parts of the body, especially on the head and neck regions [Int. J. Dermatol. 34 (6) (1995) 398] although the amount of sun exposure that is required for the formation of skin cancers is still unknown. It is known that the larger the dose of UVR, and in particular the erythemal action spectrum, the more likely it is to form the non-melanoma skin cancers, basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). A number of regions on the face exhibit a seemingly high rate of occurrence of BCCs in relation to apparent direct exposure. One of these regions is the inner canthus located next to the eye on the bridge of the nose which has an occurrence rate of 7.1% of all BCCs that occur on the head and neck [Gen. Surg. 51 (6) (1981) 576, Aust. NZ J. Surg. 60 (1990) 855, Malignant Skin Tumours, Longman Singapore Publishers, Singapore, 1991]. The inner canthus seems to be well protected from large direct doses of ultraviolet radiation (UVR) and to explain the higher incidence of BCCs on the inner canthus it is proposed that a significant proportion of the incident UVR on the eye and surrounding areas is reflected onto the inner canthus. This paper presents a preliminary investigation of the contribution of UVR reflected to the inner canthus from the tear film covering the eye using a two-dimensional theoretical model on the horizontal plane (0 degrees elevation angle). Calculations show that up to 30% of the total radiation that is received on the inner canthus on a cellular level in this plane is reflected from the eye. A three-dimensional computer-generated ray tracing model of the eye, surrounding facial features and the inner canthus is being created to investigate the effect that these reflections have on the total dose of UVR.