An action spectrum for the elicitation of erythema in skin persistently sensitized by photobound 8-methoxypsoralen.

Human skin can be rendered persistently photosensitive by topical application of aqueous 8-methoxypsoralen (8-MOP) and exposure to a suberythemogenic dose of more than 380 nm radiation. We report an action spectrum for the elicitation of phototoxic erythema induced by a second exposure of skin pretreated in this way. After correction for unsensitized skin erythema this action spectrum resembles the absorption spectrum of the 4',5'-monoadduct of 8-MOP to DNA. This suggests that the monoadduct is the chromophore for erythema elicited by the second irradiation, and supports the DNA crosslink as the crucial photoproduct causing phototoxic erythema due to 8-MOP in human skin.

Photoaugmentation in the hairless mouse: a study using ornithine decarboxylase activity and alteration of DNA synthesis as markers of epidermal response.

Photoaugmentation is the potentiation of UVB-induced cutaneous erythema by UV irradiation. We have examined other cutaneous responses to UVB irradiation-the 4 hr depression of DNA synthesis, the 48 hr stimulation of DNA synthesis, and the induction of ornithine decarboxylase (ODC), to determine whether these were also susceptible to augmentation by UVA, which does not cause these responses when administered alone. No photoaugmentation of DNA synthesis, stimulation or ODC induction occurred. The early depression of DNA synthesis was slightly augmented for this did not consistently reach significance.

Recovery of skin from a single suberythemal dose of ultraviolet radiation.

Previous studies of exposure of normal skin to ultraviolet radiation have demonstrated a cumulative effect lasting greater than 24 h when repeated suberythemal exposures are given. However, the time course of recovery from a single suberythemal dose of ultraviolet A (UVA) or ultraviolet B (UVB) has not been determined. We show here for the first time that the period required for recovery of normal skin (as measured by delayed erythema) following a single suberythemal dose of UVA is between 30 and 48 h, and for UVB is between 24 and 30 h. Photoprotection was noted for both UVA and UVB from the fifth through the ninth day after the single suberythemal exposure, but was only statistically significant on the fourth day after UVB exposure. The curve depicting recovery from a single suberythemal dose of UVA or UVB from the first irradiation time through the fourth day after irradiation may be described as an exponential decay curve. Formulas are given for both UVA and UVB which describe the exponential nature of this curve. These formulas may be used to predict the exact difference in erythema threshold between preirradiated and normal skin. From the fourth day after exposure to the ninth day, the curve is nearly constant. The nature of the recovery curve in the first 4 days after exposure suggests that an exponential decay process occurs in UVA or UVB damage, consistent with unstable photoproduct decay, DNA repair, or constitutive enzymatic processes.

UVA-induced erythema, pigmentation, and skin surface temperature changes are irradiance dependent.

Human cutaneous erythemogenic and melanogenic responses to long-wave (UVA) ultraviolet radiation were investigated using irradiances ranging from 5-50 mW/cm2. Skin surface temperature changes resulting from the different irradiances were also compared. In general, threshold doses for erythema and pigmentation were higher when UVA was administered at the lowest irradiance (5 mW/cm2) than at the highest (50 mW/cm2). Erythema was maximal immediately after exposure to UVA. The most intense responses (erythema with edema, or intense pigmentation) were induced more frequently by the highest irradiance. Components of both the erythema and the pigment response to UVA are therefore irradiance-dependent. The greatest increase in skin surface temperature was observed after exposure to the highest irradiance.

Lack of photoprotection against UVB-induced erythema by immediate pigmentation induced by 382 nm radiation.

Immediate pigment darkening (IPD) was induced on the backs of 11 human volunteers of skin types III and IV by exposing the skin to UVA radiation (382 nm). The minimum erythema dose (MED) of UVB radiation was also determined by exposing sites to graduated doses of 304 nm radiation. The order of exposure of distinct anatomic areas was as follow: UVB followed by IPD induction; IPD induction followed by UVB; IPD induction followed 3 h later by UVB; and UVB only. Erythema responses induced by UVB were graded by inspection 24 h later and the MEDs in the 4 areas were compared. The induction of IPD before UVB exposure caused no significant change in the MED compared to sites receiving UVB only, or receiving UVA radiation after UVB, confirming that the IPD reaction does not protect against UVB-induced erythema. There was also no evidence of photorecovery, i.e., an increase in the MED of UVB resulting from exposure to longer wavelength, UV or visible radiation following UVB exposure.

Effect of glutathione depletion on sunburn cell formation in the hairless mouse.

Cutaneous protection against ultraviolet B (UVB) radiation damage by endogenous glutathione (GSH) was evaluated in the epidermis of the hairless mouse by measuring the influence of GSH depletion on sunburn cell (SBC) formation. Cellular GSH exerts antioxidant effects and recent studies have suggested a role for oxygen radicals in the production of SBC. Hairless mice (Skh/h 1) received oral treatment with buthionine S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase, to deplete cutaneous GSH; 4 d later their ears were exposed to UVB radiation. BSO treatment significantly reduced GSH levels in the epidermis to 10-15% of control levels. Twenty-four hours after UVB exposure, SBC counts in the ears of animals with and without BSO treatment were measured, and those exposed to UVB were found to have increased. Greater numbers of SBC were found in the ears of BSO-treated mice exposed to 15 or 20 mJ/cm2 UVB, than in non-BSO-treated mice exposed to the same UVB doses. At higher UVB doses, there were no statistically significant differences between the groups. The results show that endogenous GSH provides the epidermis with measurable protection against injury by low or moderate UVB doses.

Effect of cutaneous hypoxia upon erythema and pigment responses to UVA, UVB, and PUVA (8-MOP + UVA) in human skin.

The effect of oxygen deprivation upon UVA-, UVB-, and PUVA-induced pigment and erythema responses in normal human skin was examined. Before exposure, varying degrees of hypoxia in the skin of the forearm were achieved by inflating a sphygmomanometer cuff applied to the upper arm. After the transcutaneously measured pO2 had stabilized, sites on the inner forearm were exposed to UVA, UVB, or 8-MOP + UVA radiation, to determine dose thresholds for the induction of erythema and pigmentation at different cuff pressures. Inflation of the cuff to greater than systolic pressure completely inhibited immediate and delayed pigment responses (IPD, DT) to UVA doses greater than 10 times the normal pigmentation threshold dose. UVA-induced delayed erythema responses were partially inhibited by cuff inflation: 2.7 times the minimal erythema dose of UVA was necessary to cause an erythema response when exposure occurred during vascular occlusion. In contrast, erythema and pigment responses to UVB and PUVA were unaltered by cuff pressures exceeding systolic pressure during exposure. Inhibition of UVA-induced erythema and pigment responses by vascular occlusion were reversed by the transcutaneous diffusion of 100% O2. These findings indicate that the cutaneous responses to UVA and UVB occur by separate pathways differing with respect to O2 dependence. Our findings agree with those of other studies which indicate that PUVA-induced phototoxicity and melanogenesis are not O2-dependent.

Epidermal DNA synthesis: a new disc technique for evaluating incorporation of tritiated thymidine.

We present a new technique for evaluating epidermal DNA synthesis as measured by incorporation of tritiated thymidine. Standard areas of epidermis were isolated by glueing plastic discs to the skin followed by heating on a hot plate at 60 degrees C. The discs were then cut out and the dermis gently separated and removed by incubation at 60 degrees C in 2 M potassium bromide. The discs with attached epidermis were washed in 0.25% acetic acid to remove unincorporated tritiated thymidine and then counted in a liquid scintillation counter. Results were expressed as disintegrations per minute per disc. This technique showed good correlation to the hydroxylapatite column and perchloric acid methods for extracting DNA, but was considerably less tedious and subject to fewer errors. Incubation of discs in DNAse indicated radioactivity was primarily in DNA. The disc technique is simple, rapid and inexpensive and could be performed in large numbers even in unsophisticated laboratories.

Coal tar phototoxicity: kinetics and exposure parameters.

We examined two manifestations of coal tar phototoxicity: delayed erythema and skin pain (tar smarts) by quantifying the amount (dose) of UVA and exposure conditions required to induce these phenomena in normal human skin. The minimal UVA dose required to induce delayed erythema (minimal phototoxic dose or MPD) and the minimal UVA dose required to induce an immediate smarting reaction (minimal smarting dose or MSD) were recorded in 32 subjects in a variety of settings. A log-log dose-response model described the relation between the interval of time tar was left on the skin and lowering of MPD. We examined 4 different methods of tar removal and showed that several methods using more than water alone were equally effective--judging by resultant phototoxicity. The time between tar removal and UVA irradiation is important. Even 30 min was sufficient for the MPD to increase from 3.77 +/- 1.55 to 6.1 +/- 4.0 J/cm2 (p less than 0.02). The smarting reaction shows a similar dependence on the time interval between tar removal and exposure. The mean MSD was less than the mean MPD at all times tested. Both manifestations of coal tar phototoxicity, reduced delayed erythema threshold and susceptibility to the smarting reaction, persisted at least 30 h after tar removal.

Comparative protection efficiency of UVA- and UVB-induced tans against erythema and formation of endonuclease-sensitive sites in DNA by UVB in human skin.

UVA- and UVB-induced tans which were visually identical with each other were induced in separate sites on the lower back of 5 normal human volunteers of good tanning ability. Tanning was achieved by 4 exposures to UVA and UVB administered over an 8-day period. One week after the last exposure the protection afforded by the two types of tan against UVB-induced erythema and against UVB-induced DNA damage was measured. Protection against erythema was measured by comparison of the minimal erythema doses of UVB in tanned and untanned skin. Protection against DNA damage was assessed by comparing the numbers of endonuclease-sensitive sites in epidermal DNA extracted from biopsies taken from tanned and untanned sites exposed to the same dose of UVB. The UVB tans conferred significant protection (mean 2.98-fold) against UVB-induced erythema. UVA tans were not associated with significant protection (mean 1.4-fold). In contrast, both UVA- and UVB-induced tans were associated with a similar reduction in yield of endonuclease-sensitive sites in epidermal DNA (in UVA tan to 47% and in UVB tan to 45% of the yield in untanned skin). Protection conferred by the tans against erythema was therefore not paralleled by protection against DNA damage.

Cutaneous photosensitization by 8-methoxypsoralen: order-dependent synergism between radiation less than 380 nm and broadband UVA.

Human skin treated with topical 8-methoxypsoralen (8-MOP) was exposed sequentially to radiation at wave-lengths longer than 380 nm and to broadband UVA (320-400 nm). A striking, order-dependent synergism with respect to the induction of cutaneous phototoxicity as measured by delayed erythema was present. When exposure to greater than 380 nm radiation preceded exposure to broadband UVA, the effect was synergistic. When the order was reversed, the effect was approximately additive. This synergism is best explained by UVA-induced conversion to DNA cross-links of the monoadducts formed by prior exposure at greater than 380 nm. The direct implication is that cross-linking of DNA by psoralen is the major important event in cutaneous phototoxicity due to psoralens. Skin treated with 8-MOP and markedly suberythemogenic doses of radiation greater than 380 nm remained synergistically sensitized to small doses of UVA for at least 72 h, long after photosensitization by 8-MOP alone had disappeared in control sites. This suggests slow in vivo repair of those psoralen-DNA monoadducts capable of being subsequently converted to DNA cross-links.

Prolonged skin photosensitization induced by methoxsalen and subphototoxic UVA irradiation.

Topical 8-methoxypsoralen (8-MOP) was used to briefly provide free psoralen sufficient for marked cutaneous photosensitization, but only a small dose of UVA was delivered initially, in an effort to produce many monoadducts but few crosslinks. After ample time for clearance of the remaining free psoralen a second UVA exposure was delivered. The second exposure should not have generated any additional monoadducts in the absence of free psoralen, but the remaining monoadducts could be converted to crosslinks. The observation of a prolonged persistent UVA-photosensitive state caused by prior, very small doses of UVA given while free 8-MOP was present strongly suggests that psoralen-DNA crosslinks per se initiate much of the phototoxic effect of 8-MOP on skin, and that monoadducts induce much less acute inflammatory response. Because erythema was studied as the end point, the data say nothing about relative contributions of monoadducts vs crosslinks in causing mutagenesis, hyperpigmentation, therapeutic or other cutaneous responses. Other explanations for the induced persistent photosensitive state are also possible, but less tenable or entirely hypothetical.

A new liquid formulation of 8-methoxypsoralen: bioactivity and effect of diet.

A new encapsulated liquid preparation of methoxsalen (8-MOP) and a commonly used crystalline preparation (Oxsoralen) were compared in 12 subjects. Each subject ingested 0.6 mg/kg body weight of each formulation on different days. Six subjects ingested a low-fat meal before ingestion of drug, and 6 subjects ingested a high-fat meal. Photosensitivity was tested from 1/2 to 6 h after ingestion of 8-MOP by exposure to 320-400 nm radiation (UVA) from a filtered xenon are lamp. A series of graduated doses of UVA were administered at each time point to determine the minimum phototoxic dose (MPD). Ingestion of 8-MOP and grading of erythema were conducted in a double-blind manner, and bilaterally symmetrical exposure sites were used to test each preparation. The phototoxic reaction was observed at 24, 48, and 72 h by two experienced observers who were unaware which formulation had been ingested. The two test days were separated by 48 h. The encapsulated liquid preparation induced greater photosensitivity than Oxsoralen (mean MPDs +/- SD: 7.1 +/- 4.7 vs 12.9 +/- 6.7 J/cm2, respectively; n = 12; p less than 0.05). The encapsulated liquid preparation also induced photosensitivity earlier than Oxsoralen (mean hours after ingestion to achieve peak photosensitivity +/- SD: 2.1 +/- 1.2 vs 3.9 +/- 1.6, respectively; n = 9; borderline significance). On a low-fat diet the encapsulated liquid peaked 2.5 h earlier than Oxsoralen, as well as showing the shortest and the most predictable period of photosensitivity. However, overall, the degree and time of peak photosensitivity induced by either preparation were unaffected by diet. Ingestion of the encapsulated liquid induced photosensitivity in all 12 subjects; Oxsoralen failed to sensitize 3 subjects. Side effects were similar after both preparations. A new encapsulated liquid preparation of 8-MOP may thus allow lower doses of UVA to achieve therapeutic results in photochemotherapy, and a shortened waiting period following ingestion of drug.

An action spectrum for photoinduction of prolonged cutaneous photosensitivity by topical 8-MOP.

Suberythemogenic exposure of human skin treated with aqueous 8-MOP to radiation greater than 380 nm prolongs photosensitization to subsequent UVA from 6 to 24-72 h. One hypothesis for prolonged photosensitization is that greater than 380 nm irradiation forms 8-MOP-DNA monoadducts, which are removed more slowly than free 8-MOP and serve as a substrate for crosslinking by further UVA exposure. Sufficient DNA crosslinking results in erythema. We have examined this hypothesis by measuring the action spectrum for induction of prolonged photosensitization. Skin of normal volunteers was treated with aqueous 8-MOP (0.003%) and immediately received suberythemogenic monochromatic exposures between 334 and 430 nm. twenty-four hours later, the presence of prolonged sensitization was tested by small exposures of UVA. Erythema was evaluated 3 and 5 d later, and an action spectrum for prolonged sensitization was determined. The minimum phototoxic dose (MPD) was also determined at each wavelength. The action spectrum for prolonged photosensitization declined gradually between 334 and 425 nm. The action spectrum for delayed erythema induced by a single exposure of 8-MOP-treated skin declined rapidly from 334-390 nm. These findings are consistent with prolonged binding of 8-MOP in the skin by an initial exposure, possibly as 8-MOP-DNA monoadducts, allowing the second exposure to induce an erythemogenic event, possibly crosslinking of DNA.

Coal tar phototoxicity: characteristics of the smarting reaction.

We examined the properties and ultraviolet exposure parameters of tar smarts in an effort to elucidate the mechanisms involved. We showed that irradiation with 1 minimal smarting dose (MSD) of UVA immediately following tar removal lowered the MSD for 6 h, demonstrated by subsequent challenge with UVA. Following 3 MSDs this "memory" effect was demonstrable for 24 h. The smarting reaction was area dependent--smaller areas of exposure require higher doses of UVA to induce smarting. Smarting followed reciprocity over a 6-fold range of irradiances (2-12.5 mW/cm2) but higher irradiances required higher doses of UVA, perhaps due to a delay in the recognition and reporting of smarting. The smarting reaction and delayed erythema due to UVA and tar were equally blocked by sunscreen.

Higher pyrimidine dimer yields in skin of normal humans with higher UVB sensitivity.

We have measured UVB (280-320 nm)-induced DNA damage in skin of individuals with different sensitivities to UVB irradiation as measured by minimal erythema dose (MED). The DNA damage was susceptible to cleavage by Micrococcus luteus UV endonuclease, which recognizes pyrimidine dimers in DNA. An alkaline agarose gel electrophoresis method was used to quantitate the number of M. luteus UV endonuclease-sensitive sites in nonradioactive DNA from skin biopsies of 7 individuals irradiated with UVB (0-180 mJ X cm-2). The production of sites correlated well with MED (correlation coefficient = 0.78). The slope of the dose response curve for the most UVB-sensitive individual (MED = 24 mJ X cm-2) and for the least UVB-sensitive individual (MED = 146 mJ X cm-2) were 11.5 X 10(-4) and 2.6 X 10(-4) sites per 1000 bases per mJ X cm-2, respectively. The UVB-induced DNA damage was determined to be pyrimidine dimers by its susceptibility to cleavage by M. luteus UV endonuclease and its photoreactivability by Escherichia coli photoreactivating enzyme.

Light and electron microscopic analysis of tattoos treated by Q-switched ruby laser.

Short-pulse laser exposures can be used to alter pigmented structures in tissue by selective photothermolysis. Potential mechanisms of human tattoo pigment lightening with Q-switched ruby laser were explored by light and electron microscopy. Significant variation existed between and within tattoos. Electron microscopy of untreated tattoos revealed membrane-bound pigment granules, predominantly within fibroblasts and macrophages, and occasionally in mast cells. These granules contained pigment particles ranging from 2-in diameter. Immediately after exposure, dose-related injury was observed in cells containing pigment. Some pigment particles were smaller and lamellated. At fluences greater than or equal to 3 J/cm2, dermal vacuoles and homogenization of collagen bundles immediately adjacent to extracellular pigment were occasionally observed. A brisk neutrophilic infiltrate was apparent by 24 h. Eleven days later, the pigment was again intracellular. Half of the biopsies at 150 d revealed a mild persistent lymphocytic infiltrate. There was no fibrosis except for one case of clinical scarring. These findings confirm that short-pulse radiation can be used to selectively disrupt cells containing tattoo pigments. The physial alteration of pigment granules, redistribution, and elimination appear to account for clinical lightening of the tattoos.

Effects of systemic indomethacin, meclizine, and BW755C on chronic ultraviolet B-induced effects in hairless mouse skin.

Chronic exposure of hairless mice to ultraviolet B (UVB) radiation is associated with inflammation as well as an altered macromolecular composition of the dermis. This study was designed to determine whether or not various systemic anti-inflammatory agents inhibit chronic UVB-induced changes in the macromolecular content of the dermis and, if so, whether each agent had the same or different effects. The agents and doses were chosen for their ability to inhibit the changes induced by a single exposure to UVB radiation (increased vasopermeability, neutrophil accumulation, and skin-fold thickness). Indomethacin, a cyclooxygenase inhibitor, and meclizine, an H1 histamine receptor antagonist, were administered from slow-release pellets. BW755C, a combined cyclooxygenase and lipoxygenase inhibitor, was administered intraperitoneally 30 min prior to UVB exposure. Animals were exposed to UVB three times per week for 20-26 weeks or were unirradiated. The elastin, glycosaminoglycan and collagen content of the skin were determined by measuring the desmosine, uronic acid, and hydroxyproline levels, respectively. The amount of each macromolecule per area of skin increased after chronic UVB exposure. The increase in desmosine was inhibited by indomethacin; the increase in hydroxyproline was inhibited by meclizine and BW755C. None of the agents inhibited the uronic acid increase. These results suggest that chronic inflammation contributes to the dermal changes seen in chronically UVB-exposed skin and that different inflammatory mediators are involved in the increases observed in elastin, glycosaminoglycans, and collagen.

Production of pyrimidine dimers in DNA of human skin exposed in situ to UVA radiation.

Cyclobutyl pyrimidine dimers, measured as sites recognized by the dimer-specific ultraviolet (UV) endonuclease from Micrococcus luteus, were produced in DNA of human skin exposed in situ to UVA (320-400 nm) radiation. The dimer yields produced by a broadband UVA source, by broadband UVA filtered to remove all light of wavelength less than 340 nm, and by narrow band radiation centered at 365 nm were similar, indicating that UVA radiation, and not stray shorter wavelength radiation, was responsible for dimer production. The identity of the UVA-induced DNA lesions was confirmed as pyrimidine dimers by photoreactivation of approximately 100% of the endonuclease-sensitive sites in vitro with the 40,000 dalton Escherichia coli photoreactivating enzyme.

Longwave ultraviolet radiation (UVA, 320-400 nm)-induced tan protects human skin against further UVA injury.

The protective effect of a UVA (320-400 nm) induced tan against cutaneous injury by further UVA-irradiation was studied by evaluating the histopathologic changes in tanned and untanned normal human buttock skin 24 h after exposure to 2 and 4 minimal erythema doses of UVA. In each subject there were fewer polymorphonuclear leukocytes and less endothelial cell prominence and vessel wall necrosis in the UVA tanned skin than in the untanned UVA-irradiated skin. In the tanned control and tanned UVA-irradiated skin there was a prominent mononuclear cell inflammatory infiltrate that was much greater than in untanned skin. In immunoperoxidase stained tissue sections, the mononuclear cells were predominantly T cells, and in all of the specimens the number of phenotypic helper/inducer cells exceeded the phenotypic cytotoxic/suppressor cells. This demonstrates that a UVA tan provides photoprotection against acute UVA exposure. In addition, tanning, with or without further UVA-irradiation, was associated with a mononuclear cell inflammatory infiltrate.