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.     

SKIN DAMAGE IN ADULT AMPHIBIANS AFTER CHRONIC EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The effects of repeated UV exposure on the skin of the European crested newt, Triturus cristatus carnifex , have been investigated. The animals were irradiated 3 times per week with a Westing-house FS40T12 fluorescent sun lamp (wavelength spectrum 275–350 nm). Two groups of animals received the same total fluence of 1.3 × 10⁵ J/m² in single fluences of either 1570 J/m² (group A) or 9430 J/m² (group C), and one group received a total fluence of 2.6 × 10⁵ J/m² in single fluences of 4710 J/m² (group B). All the animals were killed 7 months after the first UV exposure, but at different intervals after the last exposure. Striking epidermal hyperplasia was found in the newts irradiated at the lower fluence rate (group A). In the animals given the higher total fluence (group B), the most prominent skin changes were dermal fibrosis and irregular thinning and thickening of the epidermis. No significant skin changes were found in group C., in which if there had been UV lesions, they had been repaired during the 5 month interval between the last irradiation and the killing of the animals. No skin tumors developed in any experimental group.     

Protective Effect of Magnesium-L-Ascorbyl-2 Phosphate Against Skin Damage Induced by UVB Irradiation

The protective effect of magnesium-L-ascorbyl-2-phosphate (MAP) on cutaneous photodamage such as lipid peroxidation and inflammation induced by ultraviolet B (UVB) exposure (290–320nm, max. 312 nm) was investigated using hairless mice. When MAP was administered intraperitoneally to mice at a dose of 100 mg of ascorbic acid (AS) per kg body weight base immediately before irradiation (15 kJ/m²), the expected increases in thiobarbituric acid reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, were significantly reduced. However, the expected decrease in the level of cutaneous AS was unchanged. Similar results were observed for animals given 100 mg of AS-Na per kg body weight before UVB irradiation. When MAP was administered intracutaneously immediately before irradiation, the expected UVB-induced increases in TBARS and sialic acid were again significantly prevented. Ascorbic acid-Na had a less protective effect than intracutaneous MAP administration. The cutaneous AS level was significantly higher in the MAP-treated mice than in the controls, and the UVB-induced decrease in tissue AS was prevented by intracutaneous MAP administration. These results suggest that MAP protects against UVB irradiation-induced lipid peroxidation and inflammation in cutaneous tissue, regardless of the drug administration route. We found, in an in vitro experiment, that MAP was converted to AS as it crossed the epidermis, but that AS-Na did not pass through the epidermis. Furthermore, MAP was also converted to AS in serum. These results suggest that the protective effect of MAP on UVB-induced cutaneous damage is due to conversion of MAP to AS.     

THE EFFECT OF CHRONIC LOW-DOSE UVB RADIATION ON LANGERHANS CELLS, SUNBURN CELLS, UROCANIC ACID ISOMERS, CONTACT HYPERSENSITIVITY and SERUM IMMUNOGLOBULINS IN MICE

Abstract— C3H mice were irradiated three times a week for up to 6 weeks with either 500 J/m² or 1000 J/m² broadband UVB (270–350 nm) or 3000 J/m² narrowband UVB (311–312 nm; TL01 source). Each dose was suberythemal to the mouse strain used. The number of Langerhans cells (LC) in the epidermis was reduced by over 50% after 2 weeks of irradiation with the UVB source and by 20% following TL01 irradiation. Continued irradiation for up to 6 weeks resulted in no further decrease in LC numbers in the case of the UVB source but a steady decline to 40% in the case of the TL01 source. Sunburn cells were detected following irradiation with both sources but the numbers were very low in comparison with acute exposure. Ultraviolet-B exposure resulted in doubling of the thickness of the epidermis throughout the 6 weeks of irradiation while TL01 exposure did not alter epidermal thickness. Conversion of trans- to ew-urocanic acid (UCA) was observed with both UVB and TL01 sources. The percentage of cis -UCA started to return to normal after 4 weeks of TL01 exposure despite continued irradiation. As observed following a single exposure, the contact hypersensitivity (CH) response was significantly reduced following 6 weeks of UVB irradiation but was unaffected by TL01 exposure, indicating no correlation between cis -UCA levels and CH response. Total serum immunoglobulin levels remained unchanged throughout the 6 weeks of UVB or TL01 irradiation but IgE titers significantly increased in all cases in the first 2 weeks of irradiation, indicating a possible shift to a T_H2 cytokine profile. The IgE levels started to return to normal at later times. Thus chronic broadband UVB exposure induces a number of cutaneous and systemic responses that are likely to be dose dependent, while chronic TL0I exposure induces only some of the these responses.     

CAROTENOID PIGMENT ADMINISTRATION and DELAY IN DEVELOPMENT OF UV-B-INDUCED TUMORS

—The results of two studies are reported. In the first study, the carotenoid pigments, β-carotene. canthaxanthin and phytoene were administered to mice after one UV-B-induced skin tumor had developed, to see if carotenoid administration would delay the development of subsequent tumors. Canthaxanthin significantly delayed the development of subsequent tumors. In the second study, the same pigments were administered starting 10 weeks before, and for 24 weeks after, exposure to a large single fluence (8 × 10⁴J/m²) of UV-B radiation, to determine the effect of the pigments on tumor development. Phytoene significantly prevented the development of tumors to this fluence of radiation.     

HYDROGEN PEROXIDE MEDIATES UV-INDUCED IMPAIRMENT OF ANTIGEN PRESENTATION IN A MURINE EPIDERMAL-DERIVED DENDRITIC CELL LINE

Abstract— Ultraviolet-B (290–320 nm) radiation is known to impair the antigen-presenting cell (APC) function of Langerhans cells (LC), skin-specific members of the dendritic cell (DC) family. We sought to address mechanisms of this effect, focusing on the role played by hydrogen peroxide. For this purpose, we used a newly established murine DC line, XS52, which resembles epidermal LC in several respects. The APC capacity of XS52 cells, using two different CD4* T cell clones as responders, was inhibited significantly (>50%) by exposure to UV radiation (unfiltered FS20 sunlamps) at relatively small fluences (50–100 J/m²). Ultraviolet radiation also inhibited growth factor-dependent proliferation of XS52 cells. On the other hand, cell surface phenotype was relatively well preserved after irradiation; expression levels of B7-1 and B7-2 were reduced slightly, while other molecules ( e.g. Ia, CD54, CD1 la and CD18) were not affected. With respect to the role played by hydrogen peroxide, pretreatment with purified catalase (900 U/mL) prevented UV-induced inhibition of APC function. Short-term exposure to 3 miM H₂0₂ or f-butyl H₂0₂ mimicked UV radiation by inhibiting APC function. Finally, intrinsic catalase activity was substantially lower in XS52 cells compared with Pam 212 keratinocytes. These results indicate that the generation of hydrogen peroxide alone is sufficient to produce some, but not all, of the deleterious effects of UV radiation on DC derived from the skin.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED SISTER CHROMATID EXCHANGES IN POTOROUS CELLS

Abstract— Exposure to visible light after UV-irradiation showed a remarkable effect on UV-induced sister chromatid exchanges (SCEs). After 6-h exposure to visible light (3 × 10⁵ J/m²), two-thirds of the UV-induced SCEs were prevented, confirming Kato's findings. Exposure to visible light before UV irradiation had no effect. This effect of visible light on UV-induced SCEs was temperature dependent, suggesting the presence of enzymatic photoreactivation.     

QUANTITATIVE ASSESSMENT OF CUMULATIVE DAMAGE FROM REPETITIVE EXPOSURES TO SUBERYTHEMOGENIC DOSES OF UVA IN HUMAN SKIN

Abstract— Daily exposures to relatively small suberythemogenic fluences of UVA (50–200 kj/m²) for 8 days resulted in cumulative morphological skin alterations indicative of early tissue injury. Histologically, irradiated skin revealed epidermal hyperplasia, inflammation and deposition of lysozyme along the dermal elastic fiber network. Sunburn cells were also present within the epidermis. These changes were quantified by image analysis and were found to be related to the cumulative UVA fluence. A long UVA waveband (UVAI, 340–400 nm) was as effective as a broad UVA band (320–400 nm), suggesting that these changes are induced by longer UVA wavelengths.     

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K⁺-stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m²). ATPase activity measured in the absence of K⁺ and K⁺-stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m².     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

GERMICIDAL ULTRAVIOLET LIGHT INDUCES ORNITHINE DECARBOXYLASE IN MOUSE EPIDERMAL CELLS AND MODIFIES THE INDUCTION CAUSED BY PHORBOL ESTER TUMOR PROMOTERS

Germicidal ultraviolet light (UVC. 8–10 J/m²) induces ornithine decarboxylase (ODC) in mouse epidermal cells in vitro in a biphasic manner with maxima of 2–3 fold induction at 4–6 h and of 10–20 fold induction at 15–18 h after irradiation. At this dose of UVC overall protein synthesis is inhibited by 10–30% and RNA synthesis by 40–50%. Induction of both ODC peaks is prevented by actinomycin D or cycloheximide. Similar culture factors appear to influence the extent of ODC induction by UVC and by the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), since the ratio of peak activities is approximately constant at 2, whereas absolute values vary considerably between experiments. If cells are irradiated with UVC and then exposed to TPA, the effects are additive at 10 J/m², less than additive at higher and enhanced at lower doses of UVC.     

PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

Abstract The preparations of chicken erythrocyte linear double-stranded DNA and superhelical plasmid pBR322 DNA were irradiated by continuous low-intensity UV radiation (I = 25-50 W/m², λ= 254 nm) as well as by highintensity picosecond laser UV radiation (I = 10¹¹-10¹³ W/m², λ= 266 nm). The effect of DNA secondary structure alterations on the formation of liquid-crystalline dispersions from UV-irradiated DNA preparations was studied. It was shown that in the case of linear DNA, watching the disappearance of abnormal optical activity characteristic for cholesteric liquid crystal we managed to detect the presence of photochemical alterations in DNA irradiated by low-intensity UV radiation at an absorbed energy of more than 20 quanta per nucleotide. In the case of superhelical DNA using enzyme treatment of liquid-crystalline dispersions and monitoring the appearance of abnormal optical activity, we detected the presence of photochemical alterations in DNA molecules after low-intensity UV irradiation at an absorbed energy of less than 4 quanta per nucleotide. Under the latter approach using picosecond UV laser irradiation at three different light intensities we were able to distinguish the different mechanisms of fine alterations in DNA secondary structure at an absorbed energy value of about 3 quanta per nucleotide.     

Protection of UV-induced Suppression of Skin Contact Hypersensitivity: A Common Feature of Flavonoids after Oral Administration?

In this study we investigated the effect of the dietary ingredients fruit and vegetable, green tea phenol extract (GTP) and the specific flavonoid components quercetin and chrysin on the UV-induced suppression of the con-tact hypersensitivity (CHS) response to picryl chloride (PCl). The SKH-1 mice were fed with test diet from 2 or 4 weeks before and during the UV irradiation (daily, 95 mJ/cm²) and tested for the CHS ear-swelling response 10 weeks after the onset of the irradiation. For the CHS, mice were immunized with PCl by epicutaneous application on nonirradiated sites. Four days after sensitization all mice were challenged on both sides of each ear by topical application of one drop PCl. In addition, from mice fed with the fruit and vegetable mixture the number of Langerhans cells (LC) were scored in the skin and from mice fed with quercetin, quercetin levels in plasma were measured at week 11 after the start of UV irradiation. It was found that fruit and vegetable (19% in the diet), GTP (0.1% and 0.01% in the drinking water), quercetin (1% in the diet) and chrysin (1% and 0.1% in the diet), prevented statistically significantly the UV-induced suppression of CHS to PCl. In the skin of mice fed with fruit and vegetables combined with UV irradiation the number of LC were comparable to the control mice, whereas the number of LC were significantly diminished in mice treated with UV only. This protective effect on the presence of LC in the epidermis after UV irradiation, which was also observed in a previous study with quercetin, may play a role in the prevention of UV-induced immunosuppression by the flavonoids tested. In conclusion, we found protection of flavonoids against UV-induced effects on CHS, which may be a common feature of most flavonoids.     

INHIBITION OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED TUMOR PROMOTION IN MURINE SKIN BY SYSTEMIC EFFECTS OF ULTRAVIOLET IRRADIATION

Systemic effects of UVB irradiation (280-320 nm) have been shown to prevent subsequent chemical tumorigenesis induced by an initiation-promotion protocol. The present investigation was designed to determine whether initiation or promotion is prevented by UV irradiation. Groups of 25 B6D2F1/J mice received 12 weeks of intermittent dorsal UVB radiation treatments administered before, or 3 weeks after, initiation with a single application of 7,12-dimethylbenz[a]anthracene on the ventral skin. All mice were promoted ventrally with 5 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) applied three times weekly throughout the experiment. UV irradiation consisted of five 30-min exposures per week to a bank of 6 Westinghouse FS40 sunlamps. UV irradiation applied before or after initiation resulted in a decrease of 18-16 tumors per group of 25 mice, for a reduction of 61 and 50%, respectively, at 24 weeks after the first TPA treatment. Thus, prevention of tumor development was similar whether the UV influence was present or not during initiation. This finding suggests that the UV prevention of promotion could account for UV inhibition of skin tumors induced by an initiation-promotion regimen. Consistent with this concept, pretreatment of mice with dorsal UVB radiation was found to reduce DNA synthesis after exposure to TPA by 46%, although it did not decrease tritiated benzo[a]pyrene binding to DNA, in ventral epidermis. Thus, UVB irradiation systemically reduced TPA-induced tumor promotion in murine skin.     

Quantitative measurements of oxidative stress in mouse skin induced by X-ray irradiation

To find efficient methods to evaluate oxidative stress in mouse skin caused by X-ray irradiation, several markers and methodologies were examined. Hairless mice were irradiated with 50 Gy X-rays and skin homogenates or skin strips were prepared. Lipid peroxidation was measured using the skin homogenate as the level of thiobarbituric acid reactive substances. The level of lipid peroxidation increased with time after irradiation and was twice that of the control at 78 h. ESR spectra of skin strips showed a clear signal for the ascorbyl radical, which increased with time after irradiation in a manner similar to that of lipid peroxidation. To measure levels of glutathione (GSH) and its oxidized forms (GSSG) simultaneously, two HPLC methods, sample derivatization with 1-fluoro-2,4-dinitrobenzene and detection with a UV detector (method A) and no derivatization and detection with an electrochemical detector (method B), were compared and the latter was found to be better. No significant change was observed within 24 h after irradiation in the levels of GSH and GSSG measured by method B. The GSH/GSSG ratio may be a less sensitive parameter for the evaluation of acute oxidative stress caused by X-ray irradiation in the skin. Monitoring the ascorbyl radical seems to be a good way to evaluate oxidative stress in skin in vivo.     

PROTECTION AGAINST ULTRAVIOLET-B RADIATION-INDUCED LOCAL and SYSTEMIC SUPPRESSION OF CONTACT HYPERSENSITIVITY and EDEMA RESPONSES IN C3H/HeN MICE BY GREEN TEA POLYPHENOLS

Abstract— Exposure of skin to UV radiation can cause diverse biological effects, including induction of inflammation, alteration in cutaneous immune cells and impairment of contact hypersensitivity (CHS) responses. Our laboratory has demonstrated that oral feeding as well as topical application of a poly-phenolic fraction isolated from green tea (GTP) affords protection against the carcinogenic effects of UVB (280–320 nm) radiation. In this study, we investigated whether GTP could protect against UVB-induced immunosuppression and cutaneous inflammatory responses in C3H mice. Immunosuppression was assessed by contact sensitization with 2,4-dinitrofluorobenzene applied to UVB-irradiated skin (local suppression) or to a distant site (systemic suppression), while double skin-fold swelling was used as the measure of UVB-induced inflammation. Topical application of GTP (1–6 mg/animal), 30 min prior to or 30 min after exposure to a single dose of UVB (2 kj/m²) resulted in significant protection against local (25–90%) and systemic suppression (23–95%) of CHS and inflammation in mouse dorsal skin (70–80%). These protective effects were dependent on the dose of GTP employed; increasing the dose (1–6 mg/animal) resulted in an increased protective effect (25–93%). The protective effects were also dependent on the dose of UVB (2–32 kJ/m²). Among the four major epicatechin derivatives present in GTP, (‐)-epigallocatechin-3-gallate, the major constituent in GTP, was found to be the most effective in affording protection against UVB-caused CHS and inflammatory responses. Our study suggests that green tea, specifically polyphenols present therein, may be useful against inflammatory dermatoses and immunosuppression caused by solar radiation.     

EFFECT OF PHOTOREACTIVATING LIGHT ON LETHAL AND PRE-MUTATIONAL UV LESIONS IN ESCHERICHIA COLI WP2s CARRYING THE R46 MUTATOR PLASMID

Abstract—An excision-deficient E. coli strain carrying the R46 mutator plasmid showed a different response towards photo-reactivation after UV irradiation than the same strain without plasmid. While the photoreactivation of lethal lesions was comparable in both strains, the number of UV-induced mutants per 10⁶ survivors was slightly reduced for the plasmid bearing strain by photoreactivating light at UV fluences below 60 mJ/m² but increased at higher fluences. To explain this it is proposed that some UV photoproduct(s) of DNA other than cyclobutane dipyrimidine dimers are pre-mutational lesions for error-prone DNA repair by the plasmid, P-repair, but not for SOS-repair.     

ULTRAVIOLET LIGHT-INDUCED INHIBITION OF CELL DIVISION AND DNA SYNTHESIS IN AXENICALLY GROWN REPAIR MUTANTS OF DICTYOSTELIUM DISCOIDEUM

Cell division and DNA synthesis were studied during axenic growth following 254 nm ultraviolet light (UV) irradiation of a repair-proficient parental strain ( rad⁺ , D₁₀ colony formation = 195 J/m²) and two repair mutants ( rad C. D₁₀= 50 J/m²; rad B. D₁₀= 5 J/m²) of Dictyostelium discoideum. Isopycnic CsCI gradients were used to distinguish uptake of labeled precursors into nuclear (n) and mitochondrial (m) DNA, using Netropsin to enhance the density resolution. In all strains, m-DNA synthesis was inhibited to a lesser extent than was n-DNA synthesis. For rad C, which has been shown in other experiments to be slow in incision and dimer removal, the UV-induced lags in division and n-DNA synthesis were longer than for rad⁺. However, rad B showed a more complex response. Although brief division lags were observed for < 10 J/m², little immediate division lag was detected at greater fluences. Instead, a brief period of cell multiplication of up to but not exceeding two-fold occurred, followed by a cessation of division, and then by lysis. Fluences that yielded extensive lags in n-DNA synthesis in rad^- and rad C resulted in little detectable immediate postirradiation lag in n-DNA synthesis in rad B. However, later in the postirradiation period, when DNA synthesis had resumed in rad⁺ and rad C. it gradually declined to near zero in rad B. We conclude: (1) that the more extended lag in division and n-DNA synthesis in rad C is consistent with its slower rate of excision repair, and (2) that rad B contains a defect resulting in less initial blockage of DNA replication by UV lesions.     

SUNSCREENS WITH BROAD-SPECTRUM ABSORPTION DECREASE THE trans TO cis PHOTOISOMERIZATION OF UROCANIC ACID IN THE HUMAN stratum corneum AFTER MULTIPLE UV LIGHT EXPOSURES

Abstract The trans to cis photoisomerization of urocanic acid (UCA) in skin is considered to play an important role in the mechanism of immunosuppression. We have investigated the effects of skin type and various sunscreens with low sun protection factor (SPF) on the UV-induced cis -UCA formation in human skin after exposure to artificial IJV light. The rate of cis -UCA formation depends little on the skin type and is reduced by topical application of sunscreens. The rate of cis -UCA formation decreases with increasing SPF and only broad-spectrum, highly protective sunscreens offer protection against the UV-induced formation of cis -UCA, which accumulates in the stratum corneum after multiple UV exposures. A theoretical approach to estimate the distribution of cis -UCA after irradiation indicates that this compound may diffuse into the deeper layers of the epidermis with D ∼ 10⁻¹⁷ m²/s, and that its elimination from the stratum corneum is mainly due to desquamation.