Photochemotherapy: identification of a metabolite of 4, 5', 8-trimethylpsoralen.

A compound, 4, 8-dimethyl, 5'-carboxypsoralen (DMeCP), has been identified in mouse urine as a major metabolite of the photoactive drug, 4, 5', 8-trimethylpsoralen (TMeP). This drug is widely used in the treatment of vitiligo and psoriasis. DMeCP is fluorescent, and nonphotosensitizing when tested on guinea pig skin. DMeCP also occurs in the urine of human patients receiving TMeP orally.

Erythropoietic protoporphyria. Photoactivation of the complement system.

The complement system was analysed in 14 asymptomatic patients with erythropoietic protoporphyria. In the majority of the sera studied the levels of complement components C1, C4, C2, and C3 were within the normal range. Upon ultraviolet light (330--460 nm) irradiation of the serum samples in vitro, a marked decrease in total hemolytic activity accompanied by reduction of C1, C4, C2, and C3 levels was observed. The loss of total hemolytic activity can be directly correlated with the levels of protoporphyrin (PP) and similar changes can be obtained in normal serum upon addition of PP followedf by ultraviolet light irradiation. It is postulated that after irradiation the excited PP develops the capacity to activate the complement sequence with the production of cleavage products, which may contribute to the skin changes observed in these patients upon sun exposure.

Dietary lutein/zeaxanthin partially reduces photoaging and photocarcinogenesis in chronically UVB-irradiated Skh-1 hairless mice.

Lutein and zeaxanthin are xanthophyll carotenoids with potent antioxidant properties protecting the skin from acute photodamage. This study extended the investigation to chronic photodamage and photocarcinogenesis. Mice received either a lutein/zeaxanthin-supplemented diet or a standard nonsupplemented diet. Dorsal skin of female Skh-1 hairless mice was exposed to UVB radiation with a cumulative dose of 16,000 mJ/cm(2) for photoaging and 30,200 mJ/cm(2) for photocarcinogenesis. Clinical evaluations were performed weekly, and the animals were sacrificed 24 h after the last UVB exposure. For photoaging experiments, skin fold thickness, suprapapillary plate thickness, mast cell counts and dermal desmosine content were evaluated. For photocarcinogenesis, samples of tumors larger than 2 mm were analyzed for histological characterization, hyperproliferation index, tumor multiplicity, total tumor volume and tumor-free survival time. Results of the photoaging experiment revealed that skin fold thickness and number of infiltrating mast cells following UVB irradiation were significantly less in lutein/zeaxanthin-treated mice when compared to irradiated animals fed the standard diet. The results of the photocarcinogenesis experiment were increased tumor-free survival time, reduced tumor multiplicity and total tumor volume in lutein/zeaxanthin-treated mice in comparison with control irradiated animals fed the standard diet. These data demonstrate that dietary lutein/zeaxanthin supplementation protects the skin against UVB-induced photoaging and photocarcinogenesis.

Molecular aspects of drug photosensitivity with special emphasis on psoralen photosensitization reaction.

Photosensitization reactions involve phototoxic reactions and photoallergic reactions, which are less common. Common drugs and chemicals that photosensitize humans are listed. Phototoxic reactions include the following: 1) direct photosensitization (type I reactions), in which the reactions of the triplet-state sensitizer are directly attributable to a component other than oxygen (e.g., DNA, proteins, and cell membranes), and 2) indirect photosensitization (type II reactions), in which the triplet state of a sensitizer reacts first with molecular oxygen, producing an "active oxygen" intermediate that subsequently reacts with the biologic system. The active oxygen intermediates are singlet oxygen (1O2)k, superoxide radical anions (O(2), and hydroxy radicals. Psoralen-induced skin photosensitization appears to involve both type I and type II reactions. The formation of monofunctional bifunctional psoralen-DNA photoadducts (a type I reaction) is probably responsible for cell damage, cell death, mutation, and even skin carcinogenesis. The erythema response appears to be the result of a type II reaction.

Role of ultraviolet radiation in the induction of melanocytic tumors in hairless mice following 7,12-dimethylbenz(a)anthracene application and ultraviolet irradiation.

We examined the role of UVR (UV radiation) (UVA, 320-400 nm; UVB, 290-320 nm; and the combination of UVA and UVB) as a promoter in the induction of cutaneous melanoma. One hundred and seventy hairless mice (Skh-hr2), 6-8 weeks old, were treated in 8 groups: group I, DMBA [7,12-dimethylbenz(a)anthracene] plus UVA; group II, DMBA plus UVA plus UVB; group III, DMBA plus UVB; group IV, DMBA; group V, UVA; group VI, UVA plus UVB; group VII, UVB; group VIII, control. DMBA (0.5% solution) was applied once to promote the formation of dermal melanocytic nevus-like lesions while UVR treatments were conducted 3 times/week for 30 weeks. The mice were examined periodically for the development of multiple pigmented lesions, papillomas, squamous cell carcinomas, melanomas, and lymphomas. Treatment with DMBA plus UVA, DMBA plus UVB, and DMBA plus UVA plus UVB stimulated the development of multiple pigmented nevus-like lesions (85-100%) in mice of groups I, II, III, and IV. Upon necroscopy, 27-33% of animals in groups I, II, and III receiving UVR treatments developed clinically and histologically characterized melanomas. Treatment with DMBA alone did not produce melanomas. DMBA-treated animals in groups I, II, and III which received UVR treatments also developed lymphomas (21-50%). Animals treated with DMBA alone or those that received UVB or the combination of UVB plus UVA (without DMBA) developed only papillomas and squamous cell carcinomas (25-47%). Skin tumors were analyzed for the presence of point mutations in the ras gene. Polymerase chain reaction amplification of DNA and selective oligonucleotide hybridization revealed mutations in the 61st codon of the N-ras gene in the precursor nevus-like lesions and melanoma samples studied. This study suggests that UVR (both UVA and UVB) plays a role as a promoter in the stimulation of melanoma and lymphoma development in hairless mice.

Production of active oxygen species (1O2 and O2-.) by psoralens and ultraviolet radiation (320-400 nm).

Furocoumarins (psoralens) are potent skin photosensitizing agents that are used in combination with long-wavelength ultraviolet radiation (320-400 nm) in the treatment of psoriasis and other skin diseases. Twelve linear and angular psoralens, capable of forming monofunctional and bifunctional adducts with DNA, were examined with a view to elucidate the role of 1O2 and O2-. in evoking skin photosensitization reactions and skin carcinogenesis. The results showed that both linear psoralens (capable of forming interstrand cross-links) and isopsoralens (angular, monofunctional type) and 3-carbethoxypsoralen (a linear and monofunctional type) produced 1O2 and O2-., although at varying degrees. Psoralen and 3-carbethoxypsoralen produced 1O2 greater than isopsoralens (angelicins). However, nonphotosensitizing angelicin, 5-methylangelicin, and 4,8-dimethyl-5'-carboxypsoralen produced 1O2 greater than 8-methoxypsoralen and 5-methoxypsoralen. The three monofunctional angelicin derivatives (isopsoralens) produced more O2-. than 8-methoxypsoralen, 5-methoxypsoralen, and 3,4'-dimethyl-8-methoxypsoralen. 3-Carbethoxypsoralen, a potent generator of 1O2 and a moderate producer of O2-., was highly photolabile. Until recently, skin photosensitization reactions (erythema, edema, damage to DNA or the membrane of cutaneous cells, the inhibition of scheduled DNA synthesis and skin carcinogenesis, etc.) were believed to involve photocyclo-addition of psoralens to DNA mediated by a type-I or anoxic reaction (a sensitizer-substrate interaction through the transfer of hydrogen atoms or electrons, but no direct involvement of molecular oxygen). Oxygen-dependent sensitized photodynamic reactions of type-II, involving the production of reactive oxygen (1O2 and O2-.), were believed not to mediate psoralen photosensitization reactions. We suggest that 1O2 and O2-. may also participate in skin photosensitization and cell membrane-damaging reactions. The fact that certain monofunctional isopsoralens produce 1O2 and O2-. at rates comparable to or better than bifunctional psoralens suggests that these reactive moieties of oxygen could play a major role in explaining their recently observed carcinogenic property and cell membrane-damaging reactions (e.g., edema or inflammation, etc.).

An electron microscopic study of the photochemical cross-linking of DNA in guinea pig epidermis by psoralen derivatives.

Albino guinea pigs were treated with psoralen derivatives plus 320--400 nm ultraviolet radiation, and DNA was extracted from their epidermis. The DNA was assayed for the presence of interstrand cross-links by standard denaturation-renaturation assays and by a new technique, electron microscopy of the DNA under totally denaturing conditions. The latter method allows individual cross-links to be directly observed and counted. When either 4,5',8-trimethylpsoralen or 8-methoxypsoralen was applied topically to the skin (8--20 microgram/cm2) or administered orally (10--12 mg/kg body weight), followed by exposure to 320--400 nm ultraviolet radiation, most of the epidermal DNA was found to contain a high frequency of cross-links. For example, oral or topical trimethylpsoralen treatment gave an average of one cross-link per 250 nucleotide pairs or about 3 . 10(5) cross-links per guinea pig chromosome. When the dose of either drug was decreased 20-fold to the level used in the clinical treatment of psoriasis, however, no cross-links coulld be detected in the epidermal DNA. The electron microscopic assay is sensitive enough that we can put an upper limit of 1 cross-link per 10(6) nucleotide pairs (80 cross-links per chromosome) for the low dose studies. The significance of these findings to the understanding of the effectiveness of psoralens in psoriasis therapy is discussed.

Characterization of superoxide dismutase from mammalian skin epidermis.

Superoxide dismutase provides a protective defense mechanism in cellular compartments against the potential cytotoxicity of superoxide anion generated by ultraviolet radiation. Little information is available about the nature of superoxide dismutase in mammalian skin. We report the isolation and characterization of superoxide dismutase from human, guinea pig, and mouse epidermis. Copper-zinc superoxide dismutase was detected in all the mammalian skin specimens examined. Manganese superoxide dismutase was detected in human and guinea pig epidermis but not in the newborn or adult albino CD1 mouse epidermis. Electrophoresis studies of the extracted and partially purified skin superoxide dismutase on polyacrylamide gel slabs in the presence of sodium dodecylsulfate showed the characteristic molecular weights for subunits of 16,500 for copper-zinc superoxide dismutase, and 23,500 for mangano superoxide dismutase. Studies under nondenaturing conditions revealed significant differences in the mobility of the enzymes, depending on the sources of superoxide dismutase. The mouse epidermal copper-zinc superoxide dismutase was found similar to the bovine liver copper-zinc superoxide dismutase used as an internal standard. The copper-zinc superoxide dismutase of human skin and guinea pig skin showed activity-stained bands characterized by a higher mobility than the same enzyme from mouse or bovine liver. Quantitative data using the beta-NADH oxidation method indicated a 5-10-fold lower content of superoxide dismutase in mammalian epidermis in comparison with other tissues examined during this study, or compared with reported values in the literature.

Studies on the nature of in vitro and in vivo photosensitization reactions by psoralens and porphyrins.

This study was directed to examine the role of type II (photodynamic) reactions involving the production of reactive oxygen species (singlet oxygen, superoxide anion, and hydroxy radicals) in in vitro and in vivo photosensitization reactions induced by skin photosensitizing chemicals. Several porphyrins and psoralens, as model compounds representing examples of endogenous and exogenous photosensitizers, were examined for their ability to (a) produce singlet oxygen and superoxide anions, (b) induce damage to membranes and associated microsomal P-450, (c) promote lipid peroxidation of microsomal lipids of liver and epidermal cells, and (d) induce skin photosensitization reactions in vivo. Dose-response study in vitro of singlet oxygen production in H2O and D2O and inhibition studies involving the production of singlet oxygen and superoxide anion by specific quenchers indicated significant production of singlet oxygen by porphyrins, about 5-20 times higher than psoralen at 10(-5) M and 10(-6) M concentration and irradiation dose of 1-5 J/cm2 of UVA (greater than 320-400 nm radiation). The comparative studies on aerobic photodegradation of microsomal P-450 of guinea pig epidermis and liver indicated a significantly greater destruction of P-450 with porphyrins than with psoralens. A membrane-lipid peroxidation study involving malondialdehyde production, using liver and epidermal microsomal fractions with and without porphyrins, psoralens, and UVA radiation, indicated 10-20 times increased production of malondialdehyde with UVA and porphyrins than with psoralens.

The nature and molecular basis of cutaneous photosensitivity reactions to psoralens and coal tar.

The basic aspects of cutaneous photosensitization reactions and the mode of therapeutic effectiveness of psoralens and coal tar, the two groups of photosensitizing agents used extensively in the photochemotherapy of psoriasis, have been reviewed. Psoralen-induced skin photosensitization and the therapeutic action of psoralens involve two distinct types of reactions, and these two reactions occur independently of each other and concurrently when the psoralen-treated skin (oral or topical) is exposed to 320 to 400 nm of radiation. The first, type I, is an oxygen-independent reaction and primarily involves photoreaction with DNA; the second, type II, is a sensitized reaction dependent on oxygen and involves the formation of singlet oxygen ((1)O(2)). The photoreactive form of psoralen is its triplet state, and the sites of reaction are (1) the cell membrane of the epidermal, dermal, and endothelial cells; (2) the cytoplasmic constituents, such as enzymes, RNA, lysosomes, etc.; (3) the cell nuclei (DNA and chromatin); and (4) the sensitized production of (1)O(2), which is responsible for cell-membrane damage and vasodilation. The major damage would be initiated by a type I reaction and would be seen in the form of nuclear damage to DNA resulting from the interaction of psoralen with DNA and to a lesser extent with RNA. The skin photosensitization response (erythema, edema, membrane damage, etc.) would result from a type II reaction involving the generation of (l)0(2). Crude coal tar (CCT), widely used in the Goeckerman therapy for psoriasis, also produces type I and type II reactions. The therapeutic and photosensitizing actions of CCT are due to (1) the photoconjugation of the photoreactive ingredients of CCT with DNA, causing inter-strand cross-links; and (2) the production of (1)O(2). CCT is an efficient producer of (1)O(2), more so than 8-methoxypso-ralen, and is responsible for cell-membrane damage and cellular edema.

The nature and molecular basis of cutaneous photosensitivity reactions to psoralens and coal tar.

The basic aspects of cutaneous photosensitization reactions and the mode of therapeutic effectiveness of psoralens and coal tar, the two groups of photosensitizing agents used extensively in the photochemotherapy of psoriasis, have been reviewed. Psoralen-induced skin photosensitization and the therapeutic action of psoralens involve two distinct types of reactions, and these two reactions occur independently of each other and concurrently when the psoralen-treated skin (oral or topical) is exposed to 320 to 400 nm of radiation. The first, type I, is an oxygen-independent reaction and primarily involves photoreaction with DNA; the second, type II, is a sensitized reaction dependent on oxygen and involves the formation of singlet oxygen (1O2). The photoreactive form of psoralen is its triplet state, and the sites of reaction are (1) the cell membrane of the epidermal, dermal, and endothelial cells; (2) the cytoplasmic constituents, such as enzymes, RNA, lysosomes, etc.; (3) the cell nuclei (DNA and chromatin); and (4) the sensitized production of 1O2, which is responsible for cell-membrane damage and vasodilation. The major damage would be initiated by a type I reaction and would be seen in the form of nuclear damage to DNA resulting from the interaction of psoralen with DNA and to a lesser extent with RNA. The skin photosensitization response (erythema, edema, membrane damage, etc.) would result from a type II reaction involving the generation of 1O2. Crude coal tar (CCT), widely used in the Goeckerman therapy for psoriasis, also produces type I and type II reactions. The therapeutic and photosensitizing actions of CCT are due to (1) the photoconjugation of the photoreactive ingredients of CCT with DNA, causing interstrand cross-links; and (2) the production of 1O2. CCT is an efficient producer of 1O2, more so than 8-methoxypsoralen, and is responsible for cell-membrane damage and cellular edema.

The role of active oxygen (1O2 and O(2)) induced by crude coal tar and its ingredients used in photochemotherapy of skin diseases.

Crude coal tar (CCT) and certain photoreactive ingredients of CCT are photosensitizing agents used in the treatment of skin diseases (psoriasis, atopic eczema, etc.). Limited information is available in elucidating the mode of action of CCT in clearing psoriasis or causing skin photosensitization reactions. The production of singlet oxygen (1O2) and superoxide radicals (O(2) or HO2), the formation of interstrand cross-links (ICL) in DNA, and the skin photosensitization reaction caused by CCT or the ingredients present in tar preparations have been examined. Both type I (oxygen-independent) and type II (sensitized reactions requiring molecular oxygen) reactions are induced by CCT. Our data show that CCT and some of the photoreactive ingredients present in CCT produce 1O2, O(2), and ICL in DNA upon exposure to UVA radiation. Based on the equivalent concentration, the efficiency of various agents to produce 1O2 was of the following order: hematoporphyrin greater than phenanthridine greater than acridine greater than methylene blue greater than CCT greater than fluoranthrene greater than anthracene greater than pyrene greater than 8-methoxypsoralen greater than anthralin greater than chloroquine greater than anthralin dimer. The O(2) formation with CCT and its ingredients was also of the same order except for anthracene which was found to be a strong producer of O(2). The therapeutic effectiveness of CCT appears to be due to: (a) its cytotoxic effects, and (b) the production of 1O2, O(2), and ICL by CCT and its photoreactive ingredients. The skin photosensitizing (smarting, edema, and erythema responses) and carcinogenic properties of CCT may also be related to the production of 1O2 and O(2) and the formation of ICL which appear to be responsible for inducing the damage to the DNA and cell membrane.

A study of the relationship between photosensitizing and therapeutic activity of 4,5',8-trimethylpsoralen, and its major metabolite 4,8-dimethyl, 5'-carboxypsoralen.

The molecular basis for the clinically observed differences in the skin photosensitizing activity and therapeutic effectiveness of the topically applied and orally administered drug trimethylpsoralen (TMP) was investigated. TMP, when tested topically, is a very potent photosensitizing and therapeutically effective furocoumarin in the treatment of psoriasis. When administered orally, however, it is significantly less photosensitizing and therapeutically a less effective drug than the commonly used furocoumarin 8-methoxypsoralen. This decreased reactivity of oral TMP is attributable to its poor solubility and rapid in vivo metabolic transformation to several inactive (nonphotosensitizing) metabolites, one of which is referred to as 4,8-dimethyl,5'-carboxypsoralen (DMeCP). The supporting evidence has been obtained by: (a) isolation of the urinary metabolite DMeCP and subsequent comparison of its properties with the synthetically prepared DMeCP and its methyl ester; (b) examining the dark and photochemical interactions of TMP, DMeCP, and DMeCP methyl ester with DNA and determining their ability to form interstrand cross-links with DNA; and (c) studying the inhibition of DNA and RNA synthesis in Ehrlich ascites tumor cells and the killing of bacteria and T2 bacteriophages. The structure-activity relationship of TMP and DMeCP also has been examined in normal human subjects and in patients with psoriasis. The order of topical therapeutic effectiveness in terms of ability to clear psoriasis plaques appeared to be: TMP greater than 8-MOP greater than DMeCP methyl ester greater than DMeCP. The data also suggest the methyl ester of DMeCP to be an interesting nonphotosensitizing furocoumarin that photoconjugates to DNA better than 8-MOP and is therapeutically effective in psoriasis.

The effect of psoralens on hepatic and cutaneous drug metabolizing enzymes and cytochrome P-450.

Psoralens are tricyclic furocoumarins with potent photosensitizing properties in the skin and are now widely used in the treatment of several dermatologic diseases. In this study the effect of 3 different psoralens 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and isopsoralen on hepatic microsomal drug-metabolizing enzymes and cytochrome P-450 has been assessed in mice and rats. 8-MOP administered orally to CD-1 mice daily for 6 days caused 2-3 fold increases in hepatic aryl hydrocarbon hydroxylase (AHH), ethylmorphine N-demethylase and cytochrome P-450. The absorbance maximum of the induced cytochrome was at 450 nm. Aniline hydroxylase activity was unchanged. Chronic administration of 8-MOP to hairless mice caused significant enhancement of hepatic ethylmorphine N-demethylase and cytochrome P-450 but had no effect on AHH; whereas chronically administered TMP had no significant effect on any of these parameters. Isopsoralen and TMP administered orally to CD-1 mice daily for 6 days had no effect on any of these liver enzymes or on hepatic P-450. 8-MOP administered daily for 6 days to rats caused a greater than 4-fold enhancement of AHH and greater than 2-fold enhancement of ethylmorphine N-demethylase and cytochrome P-450. These studies indicate that orally administered 8-MOP induces hepatic drug-metabolizing enzymes and cytochrome P-450 to a lesser extent than do the barbituates and suggest that this drug could influence the rate of biotransformation of concomitantly administered drugs in patients undergoing PUVA therapy.

Cutaneous effects of pulsed nitrogen gas laser irradiation.

The effects of pulsed nitrogen gas laser emission (337.1 nm wavelength) were studied on human skin. The laser provides high-intensity monochromatic UVA radiation and can elicit delayed erythema in an actual exposure time of about 1 msec (105,000 pulses, each lasting 10 nsec, delivered over 210 sec). The effects of nitrogen laser irradiation were compared clinically and histologically with conventional erythemogenic UVA and UVB exposures from xenon arc or mercury arc lamps and were found to be similar in many respects. The minimal erythema dose is comparable to that obtained using more conventional continuous light sources which have more than 100 times lower intensity. A phototoxicity comparison of oral and topically applied psoralens is presented, indicating that the laser may prove useful in comparing photosenitizing capacity of certain compounds.

The detection of carotenoid pigments in human skin.

Carotenoid pigments were extracted chemically from both epidermis and dermis obtained from non-carotenemic individuals at autopsy. Absorption maxima characteristic of beta-carotene were found in the extracts of specimens of epidermis following cantharidin application in volunteers made carotenemic by the ingestion of beta-carotene (180 mg/day for 10 weeks). These maxima were absent in the extracts of epidermis obtained from the volunteers before beta-carotene ingestion.

Prolonged ultraviolet light-induced erythema and the cutaneous carcinoma phenotype.

A considerable amount of evidence exists in support of the role of ultraviolet radiation as a major etiologic factor in human skin cancer, both melanoma and carcinoma types. On the basis of epidemiologic studies a phenotype has been described which helps to identify the persons who are more susceptible to skin cancer. In an attempt to further define this population, patients with cutaneous carcinoma and a normal control group were exposed to artificial ultraviolet light (UVL) and the erythema and tanning responses of each group were measured over a 21-day period. UVL-induced erythema was prolonged in a significantly higher percentage of patients with skin cancer than in control patients, lasting two to three weeks after single exposures to 6 and 8 times the patient's minimal erythema dose. The presence of prolonged erythema correlated with this history of previous skin cancer but did not correlate with other established risk factors for cutaneous carcinoma, i.e., fair skin, light hair and light eyes, easy sunburning and poor tanning, and Celtic ancestry. Prolonged erythema following UVL radiation may therefore represent an additional risk factor and help to identify the skin cancer-susceptible population.

Detection of DNA-psoralen photoadducts in mammalian skin.

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 micrograms/cm2 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 micrograms/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. There was a greater propensity for the proliferative cells to be damaged by PUVA. PUVA induced nuclear damage up to 700 micron depth in the dermis. The usefulness of the IF test in detecting DNA damage in microgram and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA.

A comparison of the melanocyte response to narrow band UVA and UVB exposure in vivo.

The visible cutaneous pigmentary response to ultraviolet-A (UVA) is immediate and, following sufficient exposure, may persist, whereas ultraviolet-B (UVB)-induced pigmentation appears after a delay of several days. We compared the in vivo response of melanocytes to single and multiple exposures of narrow band UVA and UVB irradiation which produced visibly equal increases in pigmentation. Using a xenon-mercury source matched to a monochromator, human volunteers were exposed to 304 (+/- 5) and 365 (+/- 10) nm radiation. Biopsies were performed 1, 7, and 14 days after irradiation. For each biopsy, the number of melanocytes per square millimeter of epidermis was determined using L-3,4-dihydroxyphenylalanine (dopa)- and tyrosine-incubated split epidermal preparations. Vertical sections were also examined. At days 7 and 14, after both 304 and 365 nm radiation, melanocytes were more intensely dopa-positive than in unirradiated controls, and demonstrated enlarged perikarya and a greater number of enlarged dendrites. Following both 304 and 365 nm radiation the number of dopa-positive melanocytes was increased at days 7 and 14 by 44% and 58%, respectively. Tyrosine positivity, an indicator of enhanced tyrosinase activity and increased melanin formation, was absent in controls and at day 1, and became positive in all but one sample at day 7 and day 14. Therefore, one day after UVA exposure, visible pigmentation but not tyrosinase activity was increased. At day 7, the number of tyrosine-positive melanocytes approximately equaled the number of dopa-positive melanocytes. Although UVA and UVB induce different pigmentary responses, their effects on melanocyte number and function were indistinguishable.

Dermatological and ocular examinations in rabbits chronically photosensitized with methoxsalen.

Four groups of female Dutch-belted rabbits (Oryctulagus cuniculus) were given methoxsalen (12 mg/kg) or placebo by oral intubation and 1 hr later were exposed to UVA for either 2 or 8 hr. This procedure was repeated 5 days each week for 18 mo. A fifth group received no drug and no UVA exposure. The skin of the animals given methoxsalen and UVA showed signs of acute and chronic phototoxicity. Multiple peripheral blood parameters of hepatic, renal and hematologic function were normal and were not different between groups. Complete ophthalmoscopic examinations were performed periodically. No cataracts were seen in any of the animals. This data provides the perspective that in one species the daily dose of methoxsalen and UVA required to induce chronic cutaneous photosensitization is lower than the daily dose required to induce cataracts. It is inadvisable to interpret this data as suggesting that no risk exists for patients being treated with oral methoxsalen photochemotherapy. The experimental evidence supporting photosensitization as a cause of cataracts and implicating a role of lens DNA in this cataractogenesis is reviewed. Because methoxsalen-UVA alterations of lens DNA or protein could lead to delayed onset of cataracts, and because of the serious nature and potential preventability of phototoxic lens opacification, appropriate protective eye wear is recommended for all patients receiving oral psoralen photochemotherapy.