Sharon A. Miller

UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin

DNA damage induced by UV radiation is a critical event in skin photocarcinogenesis. However, the role of racial/ethnic origin in determining individual UV sensitivity remains unclear. In this study, we examined the relationships between melanin content and DNA damage induced by UV exposure in situ in normal human skin of different racial/ethnic groups, phototypes, and UV sensitivities. The minimal erythema dose (MED) was established for each subject exposed to UVA/UVB radiation, and skin was biopsied before as well as 7 min, 1 day, and 1 wk after UV exposure. There was great variation among individuals in the amount of DNA damage incurred and rates of its removal. The results show that after exposure to 1 MED of UV, the skin of subjects from all groups suffered significant DNA damage, and that increasing content of constitutive melanin inversely correlated with the amount of DNA damage. It is clear from these results that measured erythemal UV sensitivity of the skin (MED) is a more useful predictor of DNA photodamage than is racial/ethnic origin or skin phototype and that rates of DNA damage removal following UV radiation may be the critical determinant of the UV sensitivity (including predisposition to cancer) of the skin.

Human skin responses to UV radiation: pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis

Melanin plays an important role in protecting the skin against UV radiation, and melanomas and basal/squamous cell carcinomas occur more frequently in individuals with fair/light skin. We previously reported that levels of melanin correlate inversely with amounts of DNA damage induced by UV in normal human skin of different racial/ethnic groups. We have now separately examined DNA damage in the upper and lower epidermal layers in various types of skin before and after exposure to UV and have measured subsequent apoptosis and phosphorylation of p53. The results show that two major mechanisms underlie the increased photocarcinogenesis in fair/light skin. First, UV‐induced DNA damage in the lower epidermis (including keratinocyte stem cells and melanocytes) is more effectively prevented in darker skin, suggesting that the pigmented epidermis is an efficient UV filter. Second, UV‐induced apoptosis is significantly greater in darker skin, which suggests that UV‐damaged cells may be removed more efficiently in pigmented epidermis. The combination of decreased DNA damage and more efficient removal of UV‐damaged cells may play a critical role in the decreased photocarcinogenesis seen in individuals with darker skin.—Yamaguchi, Y., Takahashi, K., Zmudzka, B. Z., Kornhauser, A., Miller, S. A., Tadokoro, T., Berens, W., Beer, J. Z., Hearing, V. J. Human skin responses to UV radiation: pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis. FASEB J. 20, E630–E639 (2006)

Long-wavelength UVA radiation induces oxidative stress, cytoskeletal damage and hemolysis

Abstract— We investigated the ability of the different wavelength regions of UV radiation, UVA(320–400 nm), UVB(290–320 nm) and UVC(200–290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (>340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB‐ and UVC‐irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA 1‐exposed samples. The decrease in RCN was dependent on dose(0–1625 kj/m2), time(0–78 h postirradiation) and cell density (106‐107 cells/mL). Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy(200–700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.

NON‐NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non‐nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m2 for the UVASUN lamp, 75 to 390 J/m2 for the FS20 lamp and 3.8 to 17.2 J/m2 for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non‐nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non‐nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.

Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV irradiation

Abstract:  Substantial differences in DNA damage caused by a single UV irradiation were found in our previous study on skin with different levels of constitutive pigmentation. In this study, we assessed whether facultative pigmentation induced by repeated UV irradiation is photoprotective. Three sites on the backs of 21 healthy subjects with type II–III skin were irradiated at 100–600 J/m2 every 2–7 days over a 4‐ to 5‐week period. The three sites received different cumulative doses of UV (1900, 2900 or 4200 J/m2) and were biopsied 1 day after the last irradiation. Biomarkers examined included pigment content assessed by Fontana–Masson staining, melanocyte function by expression of melanocyte‐specific markers, DNA damage as cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53, apoptosis determined by TUNEL assay, and levels of p21 and Ser46‐phosphorylated p53. Increases in melanocyte function and density, and in levels of apoptosis were similar among the 3 study sites irradiated with different cumulative UV doses. Levels of CPD decreased while the number of p53‐positive cells increased as the cumulative dose of UV increased. These results suggest that pigmentation induced in skin by repeated UV irradiation protects against subsequent UV‐induced DNA damage but not as effectively as constitutive pigmentation.

ACTIVATION OF THE HUMAN IMMUNODEFICIENCY VIRUS PROMOTER BY UVA RADIATION IN COMBINATION WITH PSORALENS OR ANGELICINS

Abstract— The effects of mono‐ and bifunctional furocoumarins plus UVA radiation (PUVA and related treatments) on the human immunodeficiency virus‐1 (HIV‐1) promoter were studied using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV‐1 promoter. The experiments were performed with three psoralens (5‐methoxypsoralen, 5‐MOP; 8‐methoxypsoralen, 8‐MOP; and 4′‐aminomethyl‐4,8,5′‐trimethyl‐psoralen, AMT) and four angelicins (angelicin; 4,5′‐diniethylangclicin, 4,5′‐DMA; 6,4′‐dimethylangelicin, 6,4′‐DMA; and 4,6,4′‐trimethylangelicin, TMA). The drugs alone and UVA radiation alone showed no erect on the HIV promoter. However, when the cells were incubated with the furocoumarins at 0.1–40 μg/mL and then irradiated. the HIV promoter was activated in distinct fluence ranges, i.e. (1) no promoter activity was discernible at low fluences (e.g. at 0.1 μg/mL of 8‐MOP up to 100 kJ/m2), (2) as the fluence was increased, the promoter activity increased to reach a maximum (10–50‐fold with respect to the unexposcd samples), and (3) as the fluence was further increased, the promoter activity decreased. Similar (although shifted on the fluence scale) pattcrns were observed with either > 340‐nm UVA radiation or with UVA radiation contaminated with a small amount of UVB radiation (typical for PUVA lamps). The effective fluences were inversely related to the drug concentration. Experiments with 5‐MOP and 8‐MOP indicated reciprocity of the drug concentration and radiation hence. The HIV promoter response patterns were similar for monofunctional angelicins and bifunctional psoralens. This indicated that the furocoumarin‐DNA crosslinks are not a prerequisite for the promoter activation and that the monoadducts suffice to elicit the HIV promoter response. The HIV promoter‐activating effectiveness of diKcrent drugs correlated with their photosensitizing potential. Thus, among psoralens the effectiveness order was AMT >. 5‐MOP >8‐MOP, and among angelicins: TMA > 6,4′‐DMA > 4,5′‐DMA > angelicin. The ektiveness did not vary substantially for 5‐MOP, 8‐MOP, 4,5′‐DMA, and 6,4′‐DMA. The combined drug and UVA radiation doses were higher than those that elicit cellular responses or those that may be received by the human white blood cells during cxtracorporeal PUVA therapy (photopheresis).

The effects of topically applied glycolic acid and salicylic acid on ultraviolet radiation-induced erythema, DNA damage and sunburn cell formation in human skin.

BACKGROUND alpha-Hydroxy acids (alphaHAs) are reported to reduce signs of aging in the skin and are widely used cosmetic ingredients. Several studies suggest that alphaHA can increase the sensitivity of skin to ultraviolet radiation. More recently, beta-hydroxy acids (betaHAs), or combinations of alphaHA and betaHA have also been incorporated into antiaging skin care products. Concerns have also arisen about increased sensitivity to ultraviolet radiation following use of skin care products containing beta-HA. OBJECTIVE To determine whether topical treatment with glycolic acid, a representative alphaHA, or with salicylic acid, a betaHA, modifies the short-term effects of solar simulated radiation (SSR) in human skin. METHODS Fourteen subjects participated in this study. Three of the four test sites on the mid-back of each subject were treated daily Monday-Friday, for a total of 3.5 weeks, with glycolic acid (10%), salicylic acid (2%), or vehicle (control). The fourth site received no treatment. After the last treatment, each site was exposed to SSR, and shave biopsies from all four sites were obtained. The endpoints evaluated in this study were erythema (assessed visually and instrumentally), DNA damage and sunburn cell formation. RESULTS Treatment with glycolic acid resulted in increased sensitivity of human skin to SSR, measured as an increase in erythema, DNA damage and sunburn cell formation. Salicylic acid did not produce significant changes in any of these biomarkers. CONCLUSIONS Short-term topical application of glycolic acid in a cosmetic formulation increased the sensitivity of human skin to SSR, while a comparable treatment with salicylic acid did not.

Dynamics of pigmentation induction by repeated ultraviolet exposures : dose, dose interval and ultraviolet spectrum dependence

Background  The dynamics of ultraviolet (UV)‐induced melanogenesis have been well characterized for single UV exposures. However, our knowledge of the effects of repeated UV exposures on the development of new pigmentation is limited.

MEDICAL UV EXPOSURES AND HIV ACTIVATION

This paper presents the first attempt to evaluate the potential of clinical UV exposures to induce the human immunodeficiency (HIV) promoter and, thus, to upregulate HIV growth in those skin cells that are directly affected by the exposure. Using the data for HIV promoter activation in vitro, we computed UVB and psoralen plus UVA (PUVA) doses that produce 50% of the maximal promoter activation (AD50). Then, using (a) literature data for UV transmittance in the human skin, (b) a composite action spectrum for HIV promoter and pyrimidine dimer induction by UVB and (c) an action spectrum for DNA synthesis inhibition by PUVA, we estimated the distribution of medical UVB and PUVA doses in the skin. This allowed us to estimate how deep into the skin the HIV‐activating doses might penetrate in an initial and an advanced stage of UVB or PUVA therapy. Such analysis was done for normal type II skin and for single exposures. The results allow us to predict where in the skin the HIV promoter may be induced by selected small and large therapeutic UVB or PUVA doses. To accommodate changes in skin topography due to disease and UV therapy, our considerations would require further refinements. For UVB we found that, when the incident dose on the surface of the skin is 500 J/m2 (290–320nm) (initial stage of the therapy), the dose producing 50% of the maximal HIV promoter activation (ADUVB50) is limited to the stratum corneum. However, with an incident dose of 5000 J/m2 (an advanced stage of the therapy), ADUVB50 may be delivered as far as the living cells of the epidermis and even to some parts of the upper dermis. For PUVA we found that, when the incident UVA doses are 25 or 100 kJ/m2 (320–400nm) (an initial and an advanced stage of therapy, respectively), and the 8‐methoxypsoralen concentration in the blood is 0.1 μg/mL (the desired level), the combined doses to the mid epidermis (and some areas of the upper dermis) are well below the 50% HIV promoter‐activating PUVA dose (ADPUVA50). Only under the worst scenario conditions, i. e. an exceptionally high drug concentration in the patients tissues and localization of HIV in the nearest proximity to the skin surface, would the combined PUVA dose expected during photochemotherapy exceed ADPUVA50. These results suggest that the probability of HIV activation in the epidermis by direct mechanisms is higher for UVB than for PUVA treatment. However, complexities of the UV‐inducible HIV activation and immunomodulatory phenomena are such that our results by themselves should not be taken as an indication that UVB therapy carries a higher risk than PUVA therapy when administered to HIV‐infected patients.

Differential inactivation of surrogate viruses with merocyanine 540

Bacteriophages may be useful as surrogates for animal viruses when the virucidal properties of different photosensitizing compounds are initially investigated. We studied photoinactivation of four bacteriophages, φX174, T7, PRD1, and φ6, by the dye merocyanine 540 (MC540) (15 μ g/mL). Merocyanine 540 (MC540) should be most effective with lipid‐containing viruses, since it is primarily lipophilic (but also binds to proteins). Two of the phages, PRD1 and φ6 contain lipid, with only φ6 having an external lipoprotein envelope. Filtered radiation (450–600nm) from a 750 W projector was used at16–100 W/m2. The survival curves of the different viruses clearly demonstrated different levels of sensitivity to photoinactivation by MC540, with φ6 (Do= 1.5 kJ/m2) being the most sensitive, followed by T7 (21‐fold less sensitive). While both PRD1 and (J>6 have lipid components, only φ6 was photoinactivated by MC540. Thus the internal lipid components of PRD1 were not sufficient to allow photoinactivation by this dye, at fluences up to 300 kJ/m2. For comparison, we also photoinactivated Herpes simplex virus (Do= 0.053 kJ/m2) and found it to be 28‐fold more sensitive than φ6 to photoinactivation by the same concentration of MC540. Thus φ6 may be used as a surrogate for enveloped human viruses for photoinactivation by lipophilic dyes, but the results may only be useful qualitatively.