Protective effect of the isoflavonoid equol against hairless mouse skin carcinogenesis induced by UV radiation alone or with a chemical cocarcinogen

Isoflavones derived from many edible plants, such as genistein from the soybean, have well-documented antioxidant and estrogenic activity but may also be anticarcinogenic. In this study, we examined the potential of the isoflavone equol [(S)-4',7-dihydroxyisoflavane] to protect from skin carcinogenesis in the hairless mouse. Daily topical applications of equol lotions significantly protected against skin carcinogenesis induced by chronic exposure to solar-simulated UV radiation (SSUV) or by topical treatment with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or by the combined cocarcinogenic treatment of DMBA followed by chronic SSUV. Monitoring of tumor development for 40 weeks showed significantly delayed tumor appearance and reduced tumor multiplicity in all equol-treated groups. In mice treated with either SSUV or DMBA + SSUV, equol significantly reduced the proportion of tumors progressing from benign papillomas to malignant squamous cell carcinoma (SCC) by 33-58% and reduced the average diameter of SCC by 71-82%. In a short-term study, equol dose dependently inhibited the SSUV induction of the tumor promotion biomarker enzyme, ornithine decarboxylase, in the skin, suggesting the anticarcinogenic activity of equol may be attributed to its inhibition of the tumor promotion phase of carcinogenesis.     

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.     

Protective effects of an antioxidant derived from serine and vitamin B6 on skin photoaging in hairless mice

The generation of reactive oxygen species (ROS) by ultraviolet radiation (UVR) accelerates skin aging, which is known as photoaging. Because cutaneous iron catalyzes ROS generation, sequestering iron by chelating agents is thought to be an effective approach toward preventing photoaging. Previously, N-(4-pyridoxylmethylene)-l-serine (PYSer) was designed as an antioxidant to suppress iron-catalyzed ROS generation by its iron-sequestering activity. In this study, PYSer showed protective effects against skin damage in hairless mice irradiated with ultraviolet B (UV-B). Topical application of PYSer to the skin significantly delayed and/or decreased the visible wrinkle formation induced by chronic UV-B irradiation. A histological study indicated that UV-B-induced epidermal hypertrophy and lymphocytic infiltration were suppressed by PYSer. Moreover, PYSer showed suppressive activity against the UV-B-induced increase in glycosaminoglycans (GAG). These results indicate that PYSer is a promising antioxidant for the prevention of chronic skin photoaging by its iron-sequestering activity.     

Topical delivery of retinoids counteracts the UVB-induced epidermal vitamin A depletion in hairless mouse

UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.     

Alterations of hairless mouse skin exposed to chronic UV irradiation and its prevention by hydrocortisone

Ultraviolet-induced alterations of skin were investigated in a murine animal model. Groups of hairless mice were exposed to UV (black light, lambda max 352 nm; UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) for 20 weeks at a dose of 16.3 J/cm2 five times weekly on weekdays. At the end of 20 weeks irradiation, the dorsal skins were biochemically and histologically examined. Ultraviolet caused remarkable increases in amounts of hyaluronan, chondroitin sulfates and dermatan sulfates in skin (microgram/cm2). Interestingly, a significant change in a collagen content (hydroxyproline, microgram/g of dry powder) caused by UV irradiation was not observed, whereas the amount of collagen (hydroxyproline, microgram/cm2) increased remarkably. Histologically, no distinguishable thickening was observed in both upper dermis and lower dermis, but thickening of the epidermis was observed. Furthermore, the histological study indicated that UV irradiation caused a disappearance of crowds of adipocytes, alternative appearance of numerous fibroblasts and accumulation of collagen bundles and hyaluronan in lower dermis. Hydrocortisone, an anti-inflammatory agent, prevented both the fibrosis of lower dermis and the accumulation of the extracellular matrix components. Based on these results, it seems reasonable that UV penetrates into the lower dermis and causes fibrosis there, resulting from the inflammatory responses.     

Effect on topical 5-methoxypsoralen on tumorigenesis induced in albino and pigmented hairless mouse skin by UV irradiation

A new line of the Skh:HRII hairless pigmented mouse (black juvenile coat) is described which has been selectively bred for the capacity to respond consistently to simulated solar UV radiation with a continuous and strong tan. This mouse demonstrates a degree of protection from chronic UV-induced tumorigenesis when compared with the Skh:HRI hairless albino mouse, and has been used here to study the effect of induced melanogenesis on phototumorigenesis. Mice were irradiated for 10 weeks with incremental doses of simulated solar UV radiation (UVA + B) from a fluorescent tube source which induced tumours in 100% of albino mice and 93% of black mice by 200 days (minimally oedemal), or with 60% of this dose (sub-oedemal) which induced tumours in 85% of albino mice and 65% of black mice. Mice were also exposed to the UVA component of these radiation sources, obtained by window glass filtration. The effect of topical 5-methoxypsoralen (5-MOP) was examined, at either 0.003% with minimally oedemal UVA + B or its UVA component alone, or at 0.01% with sub-oedemal UVA + B or its UVA component alone, in both albino and black mice. The 5-MOP concentrations were selected as the maximum concentration which did not increase the erythema and oedema responses after a single exposure to minimally oedemal or sub-oedemal UVA + B. At 200 days, the tumorigenic response to sub-oedemal UVA + B was significantly increased by topical 5-MOP, to 100% in albinos and 93% in black mice. In contrast, tumorigenesis in response to minimally oedemal UVA + B was unaffected by topical 5-MOP. The UVA component alone of either irradiation regime was not tumorigenic under these conditions. When combined with topical 5-MOP, the UVA of minimally oedemal UVA + B became moderately tumorigenic, and resulted in a tumour incidence of 23% in albinos and 14.5% in black mice. However, the UVA component of sub-oedemal UVA + B, when combined with topical 5-MOP, was highly tumorigenic specifically in albino mice, inducing tumours in 93% of albino mice but in only 27% of black mice. Tan intensity resulting from minimally oedemal UVA + B was not enhanced by topical 5-MOP, and its UVA component combined with 5-MOP resulted in only a minimal tan. However, the tan intensity resulting from sub-oedemal UVA + B with topical 5-MOP was strongly increased, although its UVA component combined with 5-MOP did not produce a perceptible tan.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protection of organic pigments against photocatalysis by encapsulation

A model organic pigment (β copper phthalocyanine) was encapsulated by silica using water glass as a precursor. It is shown that the thickness, porosity and uniformity of silica shells (coatings) around individual pigment particles depend significantly on pH and temperature of synthesis. It is further demonstrated that the obtained silica shells can serve as an efficient protection against the highly reactive products of photocatalysis. The degree of protection depends not only on the thickness of silica shells but also on their porosity.     

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.     

Protection of a protein against irradiation-induced degradation by additives in the solid state

The impact of ionizing radiation on a globular protein (porcine somatotropin, pST) in the solid state was studied using rate of dissolution, high-performance liquid chromatography, and Electron spin resonance (ESR) in the presence of different additives. o-Vanillin stabilized pST against irradiation-induced degradation whereas effects of trolox and isopropyl alcohol were less significant. Stabilization effect of o-vanillin has been related to the energy transfer from pST molecules to the additive which was facilitated by formation of covalent bonds between o-vanillin and pST molecules. Anticorrelation between the level of free radicals and chemical degradation (i.e. degradation increased with decrease in a free radical level) was observed in the presence of o-vanillin.     

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.     

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.     

Protection against UVB irradiation by natural filters extracted from lichens

Natural substances extracted from lichens and boldo tree were tested in vivo and in vitro as possible UV-light filters. The protection factors were compared with that found for the references: Nivea sun Spray LSF 5, octylmethoxycinnamate (OMC) and 4-tert.-butyl-4'-methoxy dibenzoylmethane (BM-DBM). The stability of the single compounds was studied through UV-Vis spectroscopy. Usnic acid resulted to be the best UVB filter, with an in vivo protection factor similar to Nivea sun Spray LSF 5. Most of the single compounds studied in vitro resulted to have higher or similar filtering power than octylmethoxycinnamate. The protection factors as well as the good UV-light absorption of their photo-products suggest that these natural substances may be useful as new filters in sun-screen preparations.     

Protection of immobilized sulfhydryl groups against autooxidation by alterations in their microenvironment

Immobilized sulfhydryl groups were prepared by partial thiolation of NH₂-glass beads. The microenvironment of the immobilized SH groups was varied by different chemical modifications of neighboring NH₂ groups. Introduction of a strong charge in the surroundings of immobilized sulfhydryls results in their dramatic stabilization against autooxidation. This effect is due to the salting of O₂ from the surface microlayer of the thiolated beads.     

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.     

Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse

This study was conducted to assess protective effect of an antioxidant protein, sericin, on UVB-induced acute damage and tumor promotion in mouse skin. In experiment 1, HR-1 hairless mice were treated with 180 mJ/cm2 of ultraviolet B light (UVB) once daily for 1 and 7 days. The treatment for 7 days caused red sunburn lesions of the skin. The intensity of red color and area of these lesions were inhibited by the topical application of sericin at the dose of 5 mg after UVB treatment. Immunohistochemical analyses showed that the application of sericin significantly suppressed UVB-induced elevations in 4-hydroxynonenal (4-HNE), expression of cyclooxygenase-2 (COX-2) protein, and proliferating cell nuclear antigen (PCNA)-labeling index in the UVB-exposed epidermis. In experiment 2, HR-1 hairless mice were treated with 200 nmol of 7,12-dimethylbenz [alpha] anthracene (DMBA) followed 1 week later by irradiation with 180 mJ/ cm2 of UVB twice weekly for 22 weeks. The protective effect of sericin was evident in terms of significant reduction in tumor incidence and tumor multiplicity at the dose of 5 mg. The results suggest that sericin possesses photoprotective effect against UVB-induced acute damage and tumor promotion by reducing oxidative stress, COX-2 and cell proliferation in mouse skin.     

Study on the alcoholysis of isoflavone catalyzed by ionic liquids

Alcoholysis of soybean isoflavone glucosides by butanol catalyzed by acidic ionic liquids was studied. The effects of the ionic liquid catalyst type, catalyst concentration, reaction time and reaction temperature on glycoside conversions, and aglycons yields were investigated. The optimum reaction conditions are found to be as follows: 0.036 g mL⁻¹ of ionic liquid [BIM]HSO₄ as catalyst, reaction temperature at 104±1°C, reaction time of 100 min. Under these optimum reaction conditions near complete conversions of the three kinds of glycosides (daidzin, glycitin and genistin) are obtained. Furthermore, the kinetics parameters for the alcoholysis were estimated. The activation energies of alcoholysis for the three kinds of isoflavone glucosides are 124 kJ mol⁻¹, 67 kJ mol⁻¹ and 115 kJ mol⁻¹, respectively.     

Effect of l-menthol on the permeation of indomethacin, mannitol and cortisone through excised hairless mouse skin.

The effect of l-menthol on the skin permeability of mannitol, cortisone or indomethacin was examined by an in vitro penetration technique with hairless mouse skin. The donor solution was prepared with phosphate buffered saline, ethanol:buffered saline (20:80, v/v) or ethanol:buffered saline (20:80, v/v) containing 1% (w/v) l-menthol. Although ethanol showed little enhancing effect, l-menthol in an aqueous ethanol vehicle at pH 7.4 increased the permeability coefficients of mannitol and indomethacin by about 100 times that of the control (an aqueous vehicle) and increased that of cortisone by about 10 times. l-Menthol, however, scarcely enhanced the penetration of indomethacin at pH 3.0, the majority of the species being in unionized form. These results suggested that the menthol-ethanol-aqueous system enhanced skin permeability through a direct effect on the polar and/or lipid pathways, while the thermodynamic activity of the penetrant molecule in the delivery vehicle might also influence the effectiveness of the penetration enhancer.     

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.