Madhu A. Pathak

Melasma: A clinical, light microscopic, ultrastructural, and immunofluorescence study

Melasma is an acquired brown hypermelanosis of the face. Although it is thought that melasma is associated with multiple etiologic factors (pregnancy, gastric, racial, and endocrine), one of the primary causes of its exacerbation appears to be exposure to sunlight. Three patterns of melasma are recognized clinically: (1) a centrofacial pattern, (2) a malar pattern, and (3) a mandibular pattern. Examination of patients with Woods light (320--400 nm) is useful in classifying the specific type of melasma in correlation with the localization of pigment granules (melanosomes) in the epidermis and dermis. Four types of melasma are described on the basis of Woods light examination: (1) an epidermal type, (2) a dermal type, (3) a mixed type, and (4) a fourth type, described in patients of dark complexion, in which the lesions, for lack of contrast, are not discernible on Woods light examination, perhaps due to the increased number of melanosomes in the normal skin of black individuals. Light, histochemical, and electron microscopic studies revealed an increase in number and activity of type-specific melanocytes which appeared to be engaged in increased formation, melanization, and transfer of pigment granules (melanosomes) to the epidermis as well as to the dermis. The melanocyte seems to undergo a functional alteration brought about by a combination of multiple factors, including persistent sun exposure, hormonal factors, and genetic predisposition.

Skin photosensitizing agents and the role of reactive oxygen species in photoaging

In this paper, the role of reactive oxygen species in photoaging is presented. Many photosensitizing agents are known to generate reactive oxygen species (singlet oxygen (1O2), superoxide anion (O2.-) and .OH radicals). Although photoaging (dermatoheliosis) of human skin is caused by UVB and UVA radiation, the hypothesis tested here in the pathogenesis of photoaging of human skin is the free radical theory involving the generation of reactive oxygen species by UVA (320-400 nm) radiation and their damaging oxidative effects on cutaneous collagen and other model proteins. The UVA-generated reactive oxygen species cause cross-linking of proteins (e.g. collagen), oxidation of sulfydryl groups causing disulfide cross-links, oxidative inactivation of certain enzymes causing functional impairment of cells (fibroblasts, keratinocytes, melanocytes, Langerhans cells) and liberation of proteases, collagenase and elastase. The skin-damaging effects of UVA appear to result from type II, oxygen-mediated photodynamic reactions in which UVA or near-UV radiation in the presence of certain photosensitizing chromophores (e.g., riboflavin, porphyrins, nicotinamide adenine dinucleotide phosphate (NADPH), etc.) leads to the formation of reactive oxygen species (1O2, O2.-, .OH). Four specific observations are presented to illustrate the concept: (1) the production of 1O2 and O2.- by UVB, UVA and UVA plus photosensitizing agents (such as riboflavin, porphyrin and 3-carbethoxypsoralens) as a function of UV exposure dose, the sensitizer concentration and the pH of the irradiated solution; (2) the formation of protein cross-links in collagen, catalase and superoxide dismutase by 1O2 and O2.- (.OH) and the resulting denaturation of proteins and enzyme activities as a function of UVA exposure dose; (3) the protective role of selective quenchers of 1O2 and O2.- (e.g. alpha-tocopherol acetate, beta-carotene, sodium azide, ascorbic acid, etc.) against the photoinactivation of enzymes and the prevention of the protein cross-linking reaction; (4) the possible usefulness of certain antioxidants or quenchers that interact with the UVA-induced generation of reactive oxygen species in the amelioration of the process of photoaging.

Sunscreens: Topical and systemic approaches for protection of human skin against harmful effects of solar radiation

This review deals with topical and systemic approaches for protection of human skin against the harmful effects of solar radiation. Two concerns about the deleterious effects of sun exposure involve: (1) acute effects (e.g., sunburn and drug-induced phototoxicity) and (2) potential long-term risks of repeated sun exposures leading to development of solar elastosis, keratoses, induction of both nonmelanoma and melanoma skin cancer, and alteration of immune responses and functions. Action spectra of normal and abnormal reactions of human skin to acute and chronic effects of solar radiation are presented with a view to helping the physician prescribe the appropriate sunscreens. Factors that influence acute effects of sunburn are reviewed. Various artificial methods effective in minimizing or preventing harmful effects of solar radiation, both in normal individuals and in patients with photosensitivity-related problems, are discussed. Emphasis is placed on the commercially available chemical sunscreens and their properties. Sun protection factor (SPF) values of several brand-name formulations determined with a solar simulator under indoor conditions (laboratory) and with solar radiation under natural, field conditions are presented. Factors responsible for variations of SPF values observed under indoor and outdoor conditions are reviewed. Systemic photoprotective agents and their limitations are outlined. The photobiology of melanin pigmentation (the tanning reaction) is briefly discussed, with emphasis on the dangers of using quick-tanning lotions for stimulation of the tanning reaction.

Photosensitization of skin in vivo by furocoumarins (psoralens)

Abstract The molecular basis of cutaneous photosensitivity due to psoralens remains unknown. To elucidate the nature of cutaneous photosensitization in vivo, guinea-pigs kin was photosensitized with 365 nm radiation after application of 3H-labeled 4,5′,8-trimethylpsoralen. Epidermal DNA, RNA and soluble protein were isolated before and after cutaneous photosensitization. DNA of mammalian skin was characterized and its purity established. In addition, the nuclear, mitochondrial, microsomal and soluble protein fractions were also isolated after cutaneous photosensitization by 4,5′,8-trimethylpsoralen. A covalent linking of 4,5′,8-trimethylpsoralen with DNA and, to a lesser extent, with RNA was observed only when cutaneous photosensitization occurred. In none of the control experiments (five different types), was 4,5′,8-trimethylpsoralen observed to bind with epidermal DNA or RNA or to protein. Of the macromolecules studied, the DNA showed the highest reactivity in vivo with 4,5′,8-trimethylpsoralen. There was an aggregation of 4,5′,8-trimethylpsoralen photoproducts (dimers) to the 60 000 × g supernatant soluble protein by noncovalent (Van der Waals) forces. The radioactivity of 4,5′,8-trimethylpsoralen bound to DNA (the photoadduct of DNA-[3H]4,5′,8-trimethylpsoralen) could not be separated from DNA after Sephadex gel filtration, CsCl density gradient centrifugation and repeated chloroform extraction. The fact that 4,5′,8-trimethylpsoralen alone and no other photoproduct or metabolite of 4,5′,8-trimethylpsoralen was reacting with DNA was ascertained by recovering the [3H]4,5′,8-trimethylpsoralen bound to DNA after photolysis of DNA-[3H]4,5′,8-trimethylpsoralen photoadduct with 250-nm radiation and identifying it by four different chromatographic systems. Thus, during cutaneous photosensitization in vivo, 4,5′,8-trimethylpsoralen undergoes photoaddition with DNA and RNA. The formation of a C4-cycloaddition product of 4,5′,8-trimethylpsoralen with DNA and RNA appears to be responsible for epidermal-cell damage.

DNA synthesis in normal and xeroderma pigmentosum fibroblasts following treatment with 8-methoxypsoralen and long wave ultraviolet light.

Abstract Furocoumarins are known to photosensitize biological systems to 360 nm irradiation and an important mechanism is coupling of the compounds to thymine of DNA. In order to study the effect on DNA synthesis, incorporation of [ 3 H]thymidine into DNA of cultured fibroblasts was studied following 8-methoxypsoralen treatment and 360 nm irradiation. Normal and xeroderma pigmentosum cells showed identical inhibition of scheduled DNA synthesis but the latter were more sensitive to killing than the former. Repair synthesis of 8-methoxypsoralen and ultraviolet-damaged DNA could be domonstrated in normal cells but not in those from xeroderma pigmentosum. The amount of repair DNA synthesis relative to the degree of inhibition of scheduled DNA synthesis was much less for 8-methoxypsoralen and long wave ultraviolet-treated cells then those exposed to 254 nm radiation. Studies of guinea pig epidermal DNA following treatment of the skin with [ 3 H]trimethylpsoralen and long wave ultraviolet light indicated there is a progressive elimination of the photoadduct.

Usefulness of retinoic acid in the treatment of melasma

Melasma is a circumscribed brown macular hypermelanosis of the areas of the face and neck that are exposed to light. Clinical trials with various depigmenting formulations containing hydroquinone were conducted to determine the ideal concentration of hydroquinone, retinoic acid, and corticosteroids for the treatment of melasma. The compounds were tested with and without the concomitant use of topical sunscreen preparations. Based on the results of the trials and our earlier clinical experience, we conclude that treatment of melasma should involve the following: avoidance of sun exposure, constant use of broad-spectrum sunscreens, and topical application of a cream or lotion containing 2% hydroquinone and 0.05% to 0.1% retinoic acid (tretinoin). Patients should suspend use of oral contraceptives and other agents that promote skin pigmentation. The monobenzyl ether of hydroquinone should never be used in melasma therapy.

Production of singlet oxygen and superoxide radicals by psoralens and their biological significance

We have investigated a series of linear and angular furocoumarins, capable of forming either the monofunctional adducts (single strand) or bifunctional adducts (interstrand cross-links) with DNA with a view to examine the relationship of their skin photosensitizing potency, their ability to produce singlet oxygen (1O2) or superoxide radicals (O-.2 or HO.2), and their carcinogenic activity. The significance of photochemical interactions of psoralens and DNA is well known in skin photosensitization and skin carcinogenesis. Our data suggest that both monofunctional and bifunctional psoralens produce 1O2 and O-.2, and these reactive forms of oxygen may contribute to the development of skin cancer and membrane-damaging effects of these furocoumarins.

Photobiology of melanin pigmentation: Dose/response of skin to sunlight and its contents

A randomized double-blind clinical trial involving twenty-two volunteers was conducted in two locations (Orlando, FL, and St. Paul, MN) to test the efficacy of the newly designed ultraviolet monitor badges (Sun Timers), described in another paper by us in this issue of the Journal, 1 and to establish the relationship between spectral band exposure dose and the biologic responses of erythema and pigmentation. Individuals with skin types II, III, and IV, exhibiting differences in reactivity to solar radiation, were exposed to varying doses of full-spectrum sunlight through templates mounted on the lower portion of the back. Simultaneously, on the upper portion of the back, the same volunteers were exposed through two different types of polyester filters that transmitted ultraviolet A (UVA) and visible radiation. Using templates with windows, exposures to full-spectrum sunlight, UVA, and visible radiation were carried out to 1, 2, 3, 6, and 9 sunburn units (approximately 30-270 millijoules/cm2 between 10:30 A.M. and 3:30 P.M. daylight time in mid summer), measured with the aid of a Robertson-Berger meter and an IL700 International Light radiometer. Erythema and pigmentation resulting from these exposures were graded (double-blind) immediately after exposure, at 24 hours, and after 5 days. Numerical skin response ratings at each exposure dose for different spectral bands were then averaged and plotted. It was found that the UVB monitor response was predictive of a 24-hour erythema response and 5-day pigmentation response within 30% of the biologic average for skin types II, III, and IV. UVA radiation stimulated melanogenesis. The minimal melanogenic dose (MMD) for skin type II was the same as the minimal erythemogenic dose (MED). The MMD for individuals of skin types III and IV was distinctly less than their MED. Thus, melanogenesis can be stimulated with a suberythemal dose of UVB or UVA radiation. The sun protection factor values of melanin for melanized skin have been estimated to vary from 1.0 (skin type II) to 4.3 (skin types V and VI).

Sunlight and Melanin Pigmentation

When viewed from the perspective of photobiology, melanin pigmentation of human skin can be described in two categories: the first, constitutive or intrinsic skin color, and the second, facultative or inducible skin color (Quevedo et al., 1974). Constitutive skin color designates the genetically determined levels of cutaneous melanin pigmentation in accordance with the genetic programs of the cells in the absence of direct or indirect influences (e.g., solar radiation, hormones, or other environmental factors). Facultative skin color characterizes the increase in melanin pigmentation above the constitutive level and arises from the complex interplay of solar radiation and hormones upon the genetically endowed melanogenesis of the individual. The facultative skin color change brought about by solar radiation is commonly referred to as “suntan.”

Ultraviolet radiation and the development of non-melanoma and melanoma skin cancer: clinical and experimental evidence.

Clinical and experimental evidence explaining and supporting the role of UV radiation as a causal factor for the induction and promotion of nonmelanoma and malignant melanoma skin cancer are presented. While there is excellent animal experimental data and human epidemiologic evidence supporting the causal relationship of UVR (UVB, as well as UVA radiation) for basal and squamous cell carcinomas, the data establishing a direct causal relationship between melanoma and exposure to sunlight appear to be complex. They do, however, suggest a definite promotional role of sunlight in the causation of melanoma. Using a hairless pigmented mouse strain (Skh-hr2), experiments were initiated to examine the role of UVR in the induction of melanoma. A single application of DMBA as an initiator and subsequent thrice-weekly exposures to either UVB (290-320 nm) or UVA (320-400 nm) or the combined exposures of UVA and UVB resulted in the formation of blue nevus-like lesions. Repeated UVR exposures for over 30 weeks resulted in the development of melanoma (38%), as well as lymphoma and squamous cell carcinoma only in those mice that were pretreated with DMBA and had developed nevi. Mice receiving UVB, UVA, or the combination treatments of UVB plus UVA without DMBA pretreatment developed papillomas and squamous cell carcinoma but no melanoma. These studies indicate that some initiation event is essential to transform melanocytes to blue nevus-like lesions before UVR (UVB + UVA) can act as a promoter and accelerate the development of malignant melanoma, as as well as lymphoma.