New trends in photobiology: Photoprotection by melanin

J. Photochem. Photobiol. B: Biol., 9 (1991) 135-160 135

New Trends in Photobiology (Invited Review)

Photoprotection by melanin

Nikiforos Kollias Wellm_an Laboratories of Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114(U.S.A.)

Robert M. Sayre Department of Physics, Memphis State University and Rapid Precision Laboratories, Cordova, TN 38018-1342 (U.S.A.)

Lisa Zeise and Miles R. Chedekel Mel-Co., Vaccaville, CA 95687 (U.S.A.]

(Received December 3, 1990; accepted January 5, 1991)

Keywords. Melanin, epidermal melanin pigmentation, photoprotection.

Abstract

This paper is an attempt to summarize the current state of information on melanin and epidexmal melanin pigmentation (EMP) as photoprotective agents. The chemistry and biochemistry of melanin (the particle) and its interaction, in its various forms, with W radiation are considered. Methods of attenuation of W radiation are discussed in terms of structure and chemical constituents. Photoprotection by constitutive and facultative pigmentation is reviewed with minimum erythema dose (MED) as the end point. The issue of acclimatization to W radiation is discussed in terms of UVB phototherapy for psoriasis. Finally, skin cancer is considered as an end point and the reduction of its incidence with pigment level is discussed.

It is concluded that whilst EMP provides protection, its extent depends on the end point chosen for evaluation. MED is a convenient photobiological end point but is rather insensitive, whereas skin cancer is sensitive but impractical for laboratory studies. Our current state of knowledge of melanin lacks information on its absorption and scattering coefficients and its refractive index. Methods for the quantitative measurement of EMP are also urgently required.

1. Introduction

Melanins are a ubiquitous class of biological pigments ranging in color from the yellow and red-brown pheomelanins to the brown and black eumelanins. They are found in skin, hair, eyes, feathers, insect cuticles, soil,

loll-1344/91/$3.50 0 Elsevier Sequoia/Printed in The Netherlands

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fungi, bacteria and pathological human urine [ 11. Most recently, the melanins of the inner ear and brain of humans have received a great deal of attention [2, 31. Melanins are traditionally subdivided into three groups based on the tissue and species from which they originate. The term “allomelanin” is used to classify melanins found in the plant kingdom which are generally believed to be nitrogen-free polymeric pigments produced by the polyphenol-oxidase- catalyzed oxidative polymerization of one or more phenolic substrates [4]. Eumelanin and pheomelanin refer to melanins found in the animal kingdom; Scheme 1 depicts our current understanding of their biosynthetic origin. Eumelanins are nitrogen-containing dark brown and black pigments produced by the enzymatic oxidation of tyrosine. The fundamental monomeric building block of eumelanin is believed to be 5,6-dihydroxyindole (DHI) as depicted in Scheme 1 [ 51. The closely related sulfur-containing pheomelanins differ biogenetically only in the availability of cysteine during the initial stages of tyrosine oxidation [ 61. Neuromelanins are nitrogen-containing brown polymeric pigments produced in the brain via oxidative polymerization of endogenous catecholamines [ 71. Despite biosynthetic and structural studies over the past half decade, the detailed structures of these melanins remain unknown.

“Melanin and the distribution of melanosomes in the epidermis are the single most important factors in the protection of human skin from the effects of ultraviolet light” [8]. This strong statement is based on a large body of epidemiological data correlating decreased skin cancer incidence as a function of increased epidermal melanin pigmentation. This axiom has

OH 0 DHI \ OH tqros~nas.e

P /-

HO& NH, P

0 DHICA

I

Scheme 1. Melanin biosynthesis.

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withstood several challenges of the role of epidermal melanins. For example, Blum argues that “immunity to sunburn” cannot be considered as having any survival value and therefore is of little consequence in evolutionary development [9]. However, regardless of melanin’s original evolutionary function, there is little doubt that production of melanin in the skin of present day humans is photoprotective [ 81. In their 1974 paper on “The role of natural photoprotective agents in human skin”, Pathak and Fitzpatrick concluded that other investigators have substantiated this view over the past 30 years [8]. They also proposed a variety of mechanisms to explain how melanins accomplish this photoprotection, including the filtering and attenuation of impinging radiation by scattering, absorption and subsequent dissipation (as heat), absorption accompanied by redox reactions and absorption accompanied by electron transfer processes.

Photoprotection has been assessed by the determination of the threshold dose for the generation of UV-induced erythema 24 h after irradiation. This is termed the minimum erythema dose or MED. It is a reasonable photobiological end point especially for lightly pigmented people and for those who tan poorly.

It has been realized that whilst melanin protects human skin from UVB radiation [ 10, 111, additional protection is provided by the stratum corneum and urocanic acid [8]. Experiments on humans and animals have shown that native (constitutive) melanin pigmentation protects the skin from UV-induced damage including erythema, solar elastosis [ 121 and photocarcinogenesis [ 131. The protective role of melanin against the sun has been likened to a neutral density filter. For instance, melanin shows no characteristic absorption maxima in the visible or the UV spectrum, although its absorption increases with decreasing wavelength [ 14, 151. Furthermore, melanin has a random distribution throughout the stratum corneum and in the basal layer melanocytes and keratinocytes. Its occasional appearance in “supranuclear caps” is the only example of a specific distribution. However, there is evidence that melanin is a principal chromophore for photoinduced injury [ 16-181.

The photoprotection provided by constitutive pigmentation can be aug- mented by facultative pigmentation induced by UVB and UVA. The facultative pigmentation induced by psoralens plus UVA (PUVA) is particularly photoprotective.

In this review, the chemical and structural characteristics of melanin relative to its origin and its site in the skin will be examined, as well as its functions relative to its location in the epidermis. Although some examples will be drawn from studies on experimental animals, the discussion will be limited to human studies both in viva and in vitro.

A variety of end points of photoprotection may be considered including sunburn cells, immunosuppression, erythema, melanogenesis, phototoxicity, DNA damage (assessed by unscheduled DNA synthesis (UDS) or pyrimidine dimer formation), carcinogenesis and various enzymatic or biochemical end points. The minimal erythemal response has been the most widely used end point and will be relied upon heavily in this discussion, although consideration

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will also be given to the risk of skin cancer. Other end points will only be discussed in relation to specific topics.

2. Melanin

2. I. Melanin definitions We use the term epidermal melanin pigmentation (EMP) to include all

chemical and biochemical species as well as pigmented organelles and degradation products in both living and dead keratinocytes which contribute to what has been termed “melanin” and appear as a brown pigment in human skin. The word “melanin” will be reserved for the high polymer species which make up the granules seen electron microscopically in melanosomes. Thus precursors and metabolites of the melanin polymer, as well as premelanosomes, which are included in EMP should not be confused with melanin. EMP includes native (constitutive) and facultative pigmentation however induced.

2.2. Melanin structure 2.2.1. Gross features The melanin pigments are produced and packaged in melanocytes. These

specialized dendritic cells, of neural crest origin, are found in the basal cell layer between the epidermis and the dermis. Each melanocyte is normally surrounded by 36 keratinocytes and one Langerhans cell and this ensemble constitutes the epidermal melanin unit. Numerical variation occurs at different body locations. Melanin formation takes place in melanosomes. These organelles are eventually transferred through the dendritic processes of the melanocytes to the keratinocytes of the epidermal melanin unit during the pigmentary response. Melanosomes of Caucasians are ovoid in shape with a long dimension of 400 nm and are found in groups. The melanosomes of negroid subjects are also ovoid in shape, but are 800 nm in length and exist individually. The melanosomes are packaged in membrane-limited vesicles. The melanosome-containing keratinocytes progress towards the surface of the skin during differentiation and are eventually discarded by desquamation.

Proteins from the smooth and the rough endoplasmic reticula, the Golgi apparatus and the Golgi associated endoplasmic reticulum of the melanocytes are deposited into unpigmented, membrane-limited vesicles: the premelanosomes. As the melanosomes move from the perikaryon to the dendritic processes, pigment formation commences and is virtually complete by the time the melanosomes are transferred to the keratinocyte. Delayed tanning, stimulated by WB radiation, includes melanosome formation, melanin production and melanosome transfer, as well as an increase in the number of melanocytes. The details of the mechanism involved in UV-induced melanogenesis are poorly understood.

2.2.2. Tyrosinase Tyrosinase is the most important enzyme in the control of pigmentation

and a measure of its activity provides useful information on the melanogenic

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potential of the melanocyte. The reaction scheme is complicated by the highly unstable nature of the intermediates and the extremely stable insoluble nature of the final product. These combine to make the elucidation of the exact structure of the melanin polymer difficult. It is interesting that the nature of the melanin produced does not depend entirely on the enzymatic action of tyrosinase, but also on the availability of sulfhydryl-containing derivatives such as cysteine. Tyrosinase has a molecular weight of 55 000 when synthesized and 70 000 after subsequent glycosylation. The post- translational glycosylation of mammalian tyrosinase occurs as the enzyme is transferred through the Golgi complex and delivered to the premelanosome where the enzyme activity is expressed. The heterogeneity inherent in the glycosylation patterns results in a continuous spectrum of tyrosinases. In LJV-irradiated skin, the initial rapid increase in melanogenic activity results from the activation of a pre-existing pool of tyrosinase which is normally dormant in the cells. It has been found that the levels of tyrosinase existing in several different murine melanoma cell lines are quite similar, but do not necessarily reflect the visible pigmentation of these cells (191.

Tyrosinase acts to convert tyrosine into melanin, and until recently there was no compelling evidence or reason to suggest the involvement of other enzymes. However, there is now strong evidence [ZO-221 for the presence of an enzyme which catalyses the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and the involvement of catalase in the biosynthesis of eumelanin. It would appear that the regulation of melanogenesis is more complex than hitherto suspected and schemes may have to be revised in the near future.

2.2.3. Melanin metabolites Raper was the llrst to postulate that indole derivatives are formed as

intermediates in eumelanogenesis [20]. These derivatives, DHI and DHICA, arise from the oxidation of the 1,4-addition product of tyrosinase-generated dopaquinone [23] (see Scheme 1). The nucleophilic addition products of cysteine and dopaquinone, collectively known as cysteinyldopas, are formed as intermediates during pheomelanogenesis [23] (see Scheme 1). Significant quantities of the dihydroxyindole and cysteinyldopa intermediates together with their 0-methylated homologs escape the melanosomal compartment during active melanogenesis. Indeed, detection and quantitation of these excreted metabolites are used clinically to evaluate and diagnose malignant melanoma and to monitor the efficacy of various therapeutic protocols.

The indole and cysteinyldopa precursors and their related O-methyl derivatives are undoubtedly released into the epidermis during periods of melanogenic activity such as UV- or PUVA-induced tanning. The lipophilic nature of the indoles themselves and the O-methyl derivatives of both the indoles and cysteinyldopas would favor a relatively long half-life in the lipophilic dermalandepidermaltissue. The monocysteinyldopas (5-S-cysteinyldopa, 2-S-cysteinyldopa and 6-S-cysteinyldopa) exhibit A,, around 292 nm with moderate extinction coefficients (approximately 1100-l 300 M- ’ cm- ‘),

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whereas the indoles, their O-methyl derivatives and 2,5&S’-dicysteinyldopa exhibit A,, in the range 302-330 nm and extinction coefficients on the order of 5000-10 000 M-’ cm-‘. Thus, a natural consequence of active melanogenesis is the in s<tu production and accumulation of species in the epidermis ideally suited to behave as natural sunblocks.

2.3. Physicochemical properties of wwlunins The types of melanin studied include extracted “natural” melanins from

biological systems or “synthetic” melanins prepared in vitro.

2.3.1. Natural wmlunins The structure of epidermal melanin is unknown. It is generally agreed

that eumelanin is an insoluble irregular polymeric pigment which possesses a weak and relatively unreactive electron spin resonance (ESR) signal and a broad featureless UV-visible absorption spectrum [ 1, 181. However, melanin is not a black unreactive relative of Teflon nor a functionalized carbon black, and should not be expected to remain structurally unchanged following treatments with concentrated hydrochloric acid, strong alkali solutions or temperatures in excess of 100 “C [23]. Unfortunately, several of the commonly cited literature methods for isolation and handling of melanins involve such treatments [ 151. Melanins are fragile biopolymers whose structure can be irreversibly changed even by mild treatments such as vacuum drying. Thus, the use of chemical, physical, spectroscopic and photobiological parameters reported for melanins may lead to serious errors if such parameters are derived from abused melanins.

The ink sac of the cuttlefish Sepia oficinaZi.s is a rich source of eumelanin and several variations of a generic mild protocol for its isolation have been published [24]. These protocols do not involve specialized instrumentation or expertise, and when diligently followed will reproducibly afford a black melanin powder. The critical feature of these protocols is the lack of harsh chemical or thermal steps. The uniformity of eumelanin isolated from such protocols has led to the proposal that melanin from the ink sac of S. oflcinalti should be adopted as a primary eumelanin standard, and we strongly endorse this proposal.

2.3.2. Synthetic m.dunins The inherent problems involved in the isoIation and handhng of natura1

melanins have led to the use of the following melanin substitutes: (i) biosynthetic or enzymatically produced melanin which is commonly

obtained by incubation of mushroom polyphenol oxidase with one or more of the monomeric melanin precursors pictured in Scheme 1; dopa and tyrosine are the most commonly employed substrates;

(ii) autoxidative dopa melanin which is easily isolated by acidification of the dark solutions produced from alkaline solutions of dopa that have been allowed to come into contact with air;

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(iii) a “synthetic” melanin which has been commercially available for a number of years. Its preparation involves persulfate or peroxide oxidation of tyrosine.

The convenience of these preparations is obvious. However, we must realize that they are models for melanin, and are not synonymous with natural eumelanin. The validity of these models has not been established, and the advisability of their use in a given study must be evaluated on a case-by- case basis. In general, the enzymatic model melanins are considered the best and the chemically oxidized “synthetic” model melanins are the worst. Indeed, the autoxidation of L-dopa and the chemical oxidation of tyrosine have been repeatedly demonstrated to be unacceptable models for epidermal melanin [23]. Thus, physical constants and spectroscopic data derived from these model melanins may be of historic interest and useful for “order-of-magnitude” calculations. However, the use of such data to substantiate hypotheses in biological systems is inappropriate.

2.3.3. Particle properties of melanins One of the most widely cited properties of eumelanin is that it exists

in nature as a particle. The collective community of photobiologists, and we include ourselves in this group, has failed to examine critically the consequence of the “particle nature of melanin” with respect to its potential role as a sunscreen. Techniques and protocols developed for the analysis and characterization of polymer particles and solid surfaces have recently been used to evaluate the particle parameters of eumelanins [25]. The emerging picture has resulted in the classification of three levels of melanin particles.

(i) Primary particles which are defined as the smallest natural unit of melanin. Scanning electron micrographs were used to identify and characterize a spherical non-porous particle with a diameter on the order of 30 run, with an inherent specific surface area on the order of 160 m2 g- ’ (it is interesting to note that the dimensions of this particle conform to those reported by Mishima [25] for the coated vesicles).

(ii) Melanin aggregates which are composed of strongly associated primary melanin particles placed together in such a fashion that the measured specific surface area is significantly less than the sum of the specific surface areas of the primary particles of which it is composed. Such aggregates have been characterized for S. oficinalis melanin to be spherical in shape, exhibiting the parameters given in Table 1.

(iii) Melanin agglomerates (also reported in Table 1) which are loosely associated clusters of melanin aggregates that exhibit measured surface areas roughly equal to the sum of the surface areas of the melanin aggregates from which they are derived.

In order to examine the potential photobiological consequences of the particle nature of melanins, we propose the following analogous classification scheme for epidermal melanins: (a) melanosomes which may be viewed as melanin agglomerates; (b) melanin dust observed in the epidermis and stratum corneum which may be classified as melanin aggregates; (c) breakdown of

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TABLE 1

Particle parameters of the S&a omn&s melanin (K+ form)

Measured particle parameters Derived particle parameters

Particle diameter (d> 159 mn Specific volume=pore volume+ 0.638 cm3 g-l (l/powder density)

Specik surface area 29.31 m2 g-’ Bulk density (0) = (l/specific 1.566 g cmm3 volume

Specific pore volume 0.023 cm3 g-l Surface area = 6/(d ~0) 24.09 m2 g-l

Helium powder density 1.625 g cm-’ Parking area=(lOOO/dXDxC,) 23.9 R2 g-l

Acidic surface groups 168 peq g-l Charge density 0.669 C mm2

(CRJ

melanin dust to primary particles which results in the apparent absence of melanin in the stratum corneum of most individuals.

2.3.4. Abscn-ption properties Through the use of &-switched lasers at different wavelengths, threshold

doses for the disruption of melanosomes and an approximate absorption spectrum for the melanosome chromophore were estimated [ 261. This spectrum was found to be similar to that of synthetic autoxidative dopa melanin. The absolute absorption coefficient for the chromophore in the melanosomes was estimated [27] to be on the order of lo3 cm-’ at 400 run, dropping to lo2 cm- ’ by 700 run. This can only be an “order-of-magnitude” calculation because of the variation in size and composition of melanosomes. The melanin content of melanosomes can vary from 17.9% to 72.3% and the protein content from 5.4% to 61.4%, which leaves a residue that remains unaccounted for which can amount to as much as 34.3% of the total mass [28].

The absorption coefficient of synthetic melanin (prepared by autoxidation of dopa in water at pH 9) in aqueous solution at di.tTerent pH values is strongly dependent on the redox state and somewhat less on the pH (range 3 to 12) [29]. Based on the assumption that synthetic and extracted melanins are amorphous semiconductors [ 14, 30, 3 11, and that the principal process in absorption is the conversion of the photons to heat, an estimate was made of the carrier diffusion coefficient and the recombination time as well as the carrier mobility. It was found that the absorption coefficient is greater than lo4 cm-’ for the reduced forms at 488 and 514.5 run, whereas for the oxidized form it ranges from 5 x lo2 to lo4 cm-’ in the pH range 3-12. The thermal constants for synthetic melanin [29] are as follows: thermal conductivity, 1.5~10~~ cal cm-’ s-l ‘CT’; density, 1.5 g cmm3; specific heat, 0.61 cal g-’ T-l.

Optical constants were also measured for thin films of melanin ammonium salt and the real and imaginary parts of the index of refraction were calculated

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to be 1.65 and 0.18 respectively at 550 run [32]. The optical density of melanin free acid was found to behave in a similar manner to that of an amorphous semiconductor with an energy gap of 1.45 eV, which is low compared with the values reported earlier [ 141 (3.4 eV for choroid melanin). It should be noted that the two samples called “melanin” probably have a different structure and different amino acid constituents.

2.4. Photochemical properties of melanins and mtabolites The early stages of eumelanogenesis contain a complex mixture of

enzymic and non-enzymic reactions. Scheme 1 contains a number of highly reactive intermediates which have been hypothesized to be involved in melanogenesis [20, 33, 341. Advances in the instrumentation and techniques used to study ultrafast reactions have enabled researchers to produce and characterize such putative intermediates successfully. Indeed, the absorption spectra, lifetimes and selected reaction rate constants for dopaquinone, cysteinyldopaquinone, cyclodopa, dopachrome, indolechrome and related proposed intermediates of the neuromelanin pathway have been published [35-421. These studies have led to a detailed understanding of the non- enzymic steps occurring during the early stages of melanogenesis.

Irradiation of melanogenic metabolites, a class of endogenous sensitizers, gives rise to a number of reactive free radicals. The types of cellular damage promoted by these radicals depend on the rate constants of reactions with cellular biomolecules, the accessibility of the radicals to cellular biomolecules and the concentration of the radicals within a specific cellular environment. Although plasma levels of melanogenic metabolites are in the range 4-16 run01 1-l in humans, local concentrations in the skin, and particularly in actively pigmenting melanocytes, are undoubtedly much higher. Calculations [43] show concentration levels as high as 200 PM. It is interesting to speculate that the more hydrophobic methylated metabolites may localize within the lipid portion of biomembranes. Studies of light-induced reactions of melanin precursors have indicated that several critical cellular components including DNA, proteins and lipids are possible targets.

3. Photoprotection

Photoprotection implies that insufficient radiation reaches the biological target to elicit the chosen end point. In discussing photoprotection [44], we consider how electromagnetic radiation is propagated through the epidermis and how it interacts with the various components of EMP, leading to its attenuation. Biological experiments are considered which render support to the photoprotection concept.

3.1. The interaction of optical radiation with human skin: the origin of photoprotection

Electromagnetic radiation impinging on human skin can undergo a number of interactions. (i) It can be reflected by the surface of the stratum corneum

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or attenuated by the urocanic acid located there. (ii) It can enter the stratum corneum after experiencing a slight change in its direction of travel, where (iii) it may interact with “melanin dust” resulting in total or partial absorption or it may traverse the 15 or so cell layers of the stratum corneum (total thickness, 15-20 pm; density, 1.5 g cmV3) (451 experiencing a possible additional change in the direction of travel (scattering) and, in the case of UV radiation, further attenuation by the keratin of the flattened keratinocytes and by the aromatic amino acids. If it penetrates this far (the chances of this are very good except for wavelengths in the UVB (280-320 run)) it will (iv) enter the viable epidermis where it can interact with any of the various components of EMP, aromatic amino acids or other chromophores resulting in partial or total attenuation; if it traverses the eight or ten cells of the viable epidermis (total thickness, 150-200 pm; density, approximately 1 g cmp3) it will again experience changes in the direction of travel (scattering) and will arrive at the basal membrane (the dermal-epidermal junction). Once radiation reaches the dermis it experiences a great deal of scattering by collagen fibers and strong absorption by the hemoglobins and bilirubin.

EMP has different forms at different sites within the skin which interact differently with radiation. In the stratum corneum, EMP appears primarily as “melanin dust”, which is believed to be a degradation product of melanosomes. These are particles with a diameter smaller than 0.1 pm in most individuals except negroes where some undegraded melanosomes also exist [ 461. Differences in transmission through stratum corneum of black and white skin are generally smaller than a factor of two [47, 481. The theoretical photoprotective value of “melanin dust” in the stratum corneum is of interest because any radiation that is totally attenuated, without generating photochemical reactions, will not reach the living parts of the skin. Attenuated radiation, once converted to heat, could easily be accommodated by convection and perspiration. However, analysis of the existing data indicates that it is of small consequence in practice as a calculation based on the absorbance of melanosomes [ 271 indicates that, for a typical stratum corneum (12-20 pm), absorption by EMP of black individuals can be accounted for by a melanosome layer of 1 pm. It must also be borne in mind that although the evaluation of the absorption properties of extracted stratum corneum is useful and permits an estimate of the effective protection, the surface of the stratum corneum is quite rough with many folds. When stratum corneum is removed from full thickness skin, it usually attains an area 50%-100% greater than the remaining skin and, consequently, when extracted its true attenuation is underestimated.

EMP of the Malpighian layer is provided by premelanosomes and, more prominently, melanosomes within melanocytes. As mentioned above the melanosomes occur in groups and are partially melanized in white skin, whereas in black skin the melanosomes are fully melanized and appear individually. Melanin and its metabolites are packaged in melanosomes at the basal layer. These larger particles are distributed to keratinocytes throughout the Malpighian layers by the dendritic melanocytes. For intact epidermis,

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the UV transmission of black skin is considerably less than that of white skin. At 300 nm, black skin transmits approximately 2%-10% of the incident radiation, whereas in white skin 20%-509/o is transmitted [47, 481. If the amount absorbed within the stratum corneum is subtracted, the EMP in black skin provides an attenuation factor which is 5-10 times greater than that of white skin. Further analysis, using the absorption coefficient provided by Jacques and McAuliffe [ 271, indicates that the Malpighian EMP may be replaced by an optical path of 6-8 Frn through melanosomes. This, coupled with the l-2 pm equivalent in the stratum corneum of black skin, indicates that the potential protection from melanin is much greater in the Malpighian layer than in the superficial stratum corneum layers.

So far we have presented evidence which shows that brown EMP in skin produces an attenuation of the radiation that penetrates human epidermis. It is also clear that the most desirable distribution for effective EMP will be in the stratum corneum as this will protect the lower viable layers and will also readily enable the dissipation of the absorbed energy because of its small thickness (approximately 15 pm) in comparison with intact epidermis (approximately 150 pm). Although these results permit a better understanding of the location of the absorption by EMP, it should be remembered that they are based on small studies and verification is desirable, especially with black epidermis. Furthermore, no attempt has been made to quantify the chromophore content of the “black” skin samples. There appear to be significant differences in EMP content between deeply pigmented black skin and brown skin of blacks [la]. A further complication is introduced in in vitro measurements after separation of epidermis from dermis as EMP may be left on the dermis. This could equal the EMP content of the stratum corneum. The surface structure of the stratum corneum in intact skin will greatly affect the amount of scattering by the air-stratum corneum interface, possibly by a factor of 2-5. Although in vitro data may allow an objective measure of transmission, they cannot be used with any degree of certainty in in vivo estimations of protection. This problem can only be addressed by using biological end points in intact skin.

3.2. Photoprotection with minimum erythema dose (&TED) as the end point: constitutive pigmentation; skin type

Skin types have been defined by Fitzpatrick [49, 501 (Table 2) based on self-assessment from the first moderate unprotected sun exposure of the year (approximately 3 MED).

Studies investigating the protection of constitutive pigmentation have either compared MED response to skin type or MED to some instrumental measure of skin color. Although skin type might be expected to relate to constitutive pigmentation, no unambiguous correlation has been established [ 51, 521. The concept of skin type has drawn a great deal of criticism, but it remains the only practical clinical criterion to determine the initial dose for UV therapy (other than phototesting) and the risk of skin cancer (and the prescription of photoprotective agents). This classification is based on

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TABLE 2

Definition of skin type [68]

skin color Skin type Sunburn Tan

White White White White Brown Black

I II III IV V VI

Yes Yes Yes No No No

No Mh-lirnal Yes Yes Yes Yes

TABLE 3

A phototyping questionnaire [54]

1. Do you sunburn in the summer? Yes (0), No (1) 2. The color of your skin in the winter is:

white (0), olive (I); brown (2) 3. The color of your suntan is:

( )

( )

copper (0), light brown (l), dark brown (2) 4. Do you sunburn while tanning? Yes (0) no (1) 5. Do you bum after three sessions of suntanning?

Yes (0), no (1) 6. Does your sunburn last for more than three days?

Yes (0) no (1)

: :

( )

( )

the response of skin to the first 20 min exposure to summer sunlight. Some deficiencies may arise from the volunteers’ failure to remember their skin reactions accurately. A severe underestimation of the risk is quite usual in the most sensitive group who have difficulty tanning at all, although wishing they might [53]. Recently, Cesarini [54] has proposed an alternative classification scheme based on weighted answers to six questions (Table 3) in accordance with his definition of phototypes [55]. The sum of the responses is an indication of the sensitivity of the individual. The smaller the number, the more sensitive the person. A preliminary use of this questionnaire indicates that most individuals seem to arrive at a correct estimation of their risk.

In a study of eight volunteers, an impressive correlation was demonstrated between the UVB MED and the skin reflectance at 457 nm. However, color assessed in this study was not that due to EMP, but rather to the collection of skin chromophores that absorb in the visible, which includes EMP [56]. A similar study on 91 Caucasian volunteers, using a Minolta Chroma-meter, led to a much broader correlation between skin type and reflectance (Y) and between skin type and lightness (L). “A good correlation was also found between skin color and MED for unexposed skin of skin types II, III and Iv” [57].

Whilst the skin typing system serves a purpose for Caucasian subjects (despite its problems), it does not work well for darker populations. For

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example, in Japan and Kuwait it is necessary to define a local skin typing system which allows for a burning reaction to UV in the minimal to intense range and a tanning reaction in the moderate to intense range. In both groups, a reasonable correlation was established between skin color and intensity of burn [58, 591. It is unfortunate that, although clinically useful, such studies do not enable an estimation to be made of absorption as a function of EMP concentration, i.e. an estimate of photoprotection. We can only conclude that EMP provides photoprotection.

3.2.1. MED with broad band sources In principle, this type of study should allow the estimation of a pho-

toprotection factor, which would be very valuable for a source that simulates solar radiation. A limitation of such studies is that simulated solar radiation spectra vary and thus do not represent a given latitude at the same time of year and day. There is also a lack of quantitative techniques to estimate both constitutive and facultative pigments and their distribution.

Cripps (601 was able to demonstrate photoprotection by comparing the MED for solar simulated radiation (SSR) for skin type I individuals (21 volunteers) with that of black individuals (two volunteers). He found an effective protection factor of 9.68 and a correlation between skin type and SSR MED, although no quantitative estimate of EMP was obtained.

In a study designed specifically to determine the correlation between skin EMP and UVEI MED (47 psoriatic volunteers of skin type V), a very weak correlation was observed. A spectrophotometric method was used to estimate EMP which eliminated the contributions of other pigments. Moreover, it provided an estimate of the visible pigment level rather than the UV absorption of EMP. It was concluded that a measurement of the visible pigment level does not allow a prediction of the MED to LJVR and therefore an estimation of photoprotection (611.

Diffey and Farr [62] have shown that a “normal” range of skin reactions to UVA and UVB, measured by MED determinations, can be defined to allow the determination of “not normal” reactions. The normal range is a rather extended ellipse in the plane of UVA MED VS. UVE? MED. This enables a reasonable estimate to be made of an individual’s UVB MED from his UVA MED. If a single parameter were responsible for the MED response at both wave bands, a simple linear relationship would have been expected. The results indicate a more complex phenomenon, proposed by van der Leun [ 63, 641. An extension of the Diffey and Farr study to individuals of moderate to intense pigment level, together with an objective measure of the pigment level, would permit an estimate of UVA US. UVB photoprotection.

3.2.2. MED with wwnochrowzatic sources Studies with monochromatic sources allow the estimation of an action

spectrum for the biological end point and the possible estimation of the absorption spectrum of the chromophore. However, the logarithmic response of human skin limits the sensitivity of this technique to changes in the absorption coefficient greater than a factor of 5-10. Action spectra for

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24 h MED for skin types I and II and III and IV have been reported in the range 250-365 nm [65, 661. A study has also been reported on skin type V individuals at 295, 305, 315 and 365 nm (671 in which the 365 run MED was determined instrumentally because visual evaluation was precluded by the intense immediate pigment darkening (IPD) reaction. Action spectra comparisons show no relationship with the absorption spectra of synthetic or extracted melanin. Clearly observable differences would appear only if the absorption spectrum of EMP (in the wavelength range of these studies, i.e. 295-365 nm) showed changes in absorbance of more than an order of magnitude. Using in vivo spectral measurements of EMP, which indicate that in the 300-370 nm range the apparent absorbance varies by less than a factor of two, it is possible to estimate the photoprotective factor of the constitutive type V skin pigment as approximately 2.5 for the entire wavelength range. The epidermal melanin was measured on the volar side of the forearm on 483 volunteers of the same type V population and it was found to be 2.46, which is approximately 2.3 times that of an average light-skinned white subject. It should be noted that the incidence of skin cancer for individuals of skin type I is approximately 20 times higher than that of the skin type V population. Thus the increased protection against skin cancer in type V subjects cannot be accounted for in terms of constitutive pigment alone. The type V skin propensity for facultative pigment production in conjunction with stratum corneum thickening in response to solar radiation has an important protective role.

3.3. Photoprotection, with MED as the end point, by W- and PWA- induced pigrnentatim: facultative pigment

Facultative pigment [68] can be induced by single exposures of UVA (320-400 nm), UVB (280-320 nm) or PUVA (psoralen plus UVA). The pigmentary responses to UVA may be immediate [69-721. The transient nature of IPD has precluded an understanding of this phenomenon despite much effort. IPD is most easily observable on skin type IV (or darker) individuals. When induced by a low dose (lo-20 J cmm2) it will persist for a few minutes to a few hours, but with a dose greater than 20 J crnm2 the change in skin color may persist as a delayed pigment (DP). It has been reported that an immediate erythema accompanies UVA irradiation, but we have found that a slight forced air flow over the irradiation site can alleviate this response. UVELinduced pigment appears 3-5 days after irradiation and is at a maximum at 7 days. If the irradiation is intense (more than 3 MED), the stratum corneum is sloughed off with much of the pigment at 10-l 5 days post-exposure. Erythema and pigmentation may persist for weeks or months depending on the individual and the UV dose in relation to the minimum melanogenic dose. Sometimes the assessment of pigment reaction is confounded by an underlying vascular reaction which tends to give the pigment a richer appearance. This may be resolved spectroscopically and we often detect a weak erythema as well as pigment at 2 or 3 weeks post- exposure and sometimes at longer intervals.

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The distribution of WA-induced facultative pigmentation is mainly in the basal cell layer and therefore, although it might appear intense, it is not effective in providing protection [73]. In contrast, WB-induced facultative pigmentation is distributed in keratinocytes throughout the living epidermis, the activated melanocytes and the dead corneocytes of the stratum corneum 1741.

IPD is initially grey and changes to a brownish pigment within minutes to hours, but does not offer any protection against WB. In spectroscopic measurements we found that IPD shows very different spectral characteristics from constitutive EMP. Like EMP it shows an increase in apparent absorbance with decreasing wavelength from 720 to 400 nm, but thereafter there is a substantial decrease in apparent absorbance with decreasing wavelength showing a minimum at about 365 nm. Both the increase in absorbance in the visible and the decrease in the WA range are dose dependent; they increase in intensity with dose. These characteristics contrast with constitutive EMP which shows a monotonic increase in apparent absorbance with decreasing wavelength and a maximum at about 335 nm. The absorbance minimum of IPD at 365 nm becomes less apparent with time post-irradiation and within 2-5 days it disappears. The DP induced by WA has been reported to be photoprotective as it limits the acute response to further WA; however, repeated exposure to WA results in a persistent mononuclear inflammatory cell infiltrate and vessel wall thickening. In spectroscopic studies, there is a large decrease in absorbance at 365 nm after each exposure to WA [ 751.

Delayed pigment induced by WB is photoprotective against further WB exposure. In spectroscopic observations, we have found that WB-induced pigment is considerably more absorbing in the WB than constitutive EMP. It does not exhibit an apparent absorbance maximum at 335 nm as does EMP; the absorbance simply continues to increase with decreasing wavelength below 335 nm [75]. Although this may be due in part to the deposition of melanin “dust” in the stratum corneum, it is primarily due to the production of melanin precursors [76]. This specific increase in the WB absorption becomes evident after the seventh day post-irradiation and decreases sig- niflcantly when the stratum corneum is sloughed off. The pigment produced by WB appears to be distributed throughout the epidermis as well as in the stratum corneum and therefore by its distribution alone would be expected to be more photoprotective than WA-induced pigment.

The most effective pigmentation procedure, from the point of view of both quantitative production of pigment and photoprotection, is PWA [68). This pigment is rich and abundant and spectroscopically it appears the closest to native EMP. The effective protection generated by PWA in cases of polymorphous light eruption demonstrates the effectiveness of a PWA tan [ 77-791. Young et al. [SO] (using a 5-methoxypsoralen-containing sunscreen and SSR) have succeeded in generating adequate photoprotection in skin types I and II by changing their response to SSR to that more typical of skin types III and IV. It is interesting to note that SSR alone was not effective in stimulating any photoprotection in skin types I and II. This is an exciting

150

result as it suggests that the genetically determined pigmentary response to UV radiation can be modified. In this study the end point was unscheduled DNA synthesis rather than MED.

3.4. Acclimatization Acclimatization or protective adaption [81] implies tolerance of solar

radiation or a significant increase in the MED. Invariably it has been assumed that such protection is a result of increases in EMP. However, there are many other reasons for increased tolerance such as changes in the distribution of EMP and increased UVH attenuation by thickening of the stratum corneum. Such thickening could be further accompanied by increased folding which would enhance the scattering of radiation. In lightly pigmented Caucasians an increase in the number of freckles can be found with exposure which can also lead to localized protection [82]. Finally, there may be qualitative and/or quantitative changes in the production and distribution of erythema mediators.

Long-term exposure to solar radiation which can be considered to be the cause of acclimatization may also cause photoaging [83-851. It is known that in this process many changes take place in the dermis. Prominent among these are vascular changes which may lead to alterations of the erythema response. Changes in collagen structure and organization, such as cross- linking, and also in e&tin fibers may lead to alterations in skin optics and therefore the distribution of radiation. Few studies on acclimatization have been performed with human subjects. Kaidbey and Kligman [86] exposed eight volunteers to UVA twice a week for eight weeks and compared the protection, one week after the last irradiation, against UVA and SSR. Although much pigment was generated, the protection against an MED was a factor of only 2-3. It is important to note that the responses to five or ten times the MED were identical in the tanned and untanned sites. Stratum corneum was also extracted from the tanned and untanned sites and the protection obtained from the tanned stratum corneum (evaluated on four volunteers) was a factor of two. A minimal thickening of the stratum corneum was also demonstrated on the tanned sites compared with the untanned sites.

Cripps [60] reported an acclimatization equivalent to a sun protection factor (SPF) of 2.33 for 21 volunteers of skin types II (6), III (10) and IV (5). The experiment was conducted on individuals who sought the sunlight during 3.5 months of the summer, and protection was evaluated in the month of August in Wisconsin. The SPF was due to pigment formation as well as epidermal thickening. Sayre et al. [87] found that, for skin types II and III (the populations they worked with), winter-summer sensitivity, expressed in terms of the MED to SSR, changed by a factor of 1.2. They suggested that the skin types tested were less capable of acclimatization than the darker skin types IV, V and VI. Their sample population was classified into indoor and outdoor workers and they showed 10% and 20% changes in MED respectively for the winter-summer change. Using suberythemogenic doses and multiple exposures (five and ten daily exposures), it was found that the

151

MEDs to UVA and to UVI3 were reduced at the end of the experiment It was also observed that the minimum melanogenic dose decreased with exposure. This seems to indicate that damage can accumulate, the release of mediators involved in producing erythema or pigmentation is altered by prior exposure, receptors are somehow sensitized or the physical or optical properties of the skin are altered leading to differential wavelength penetration 1881.

A number of studies have also been performed in conjunction with phototherapy for psoriasis and other diseases. It is known that, for effective phototherapy, the dose of PUVA or UVB must be increased periodically if not at every treatment. Studies have been conducted to establish both the variation in sensitivity with time after the start of treatment and the time of recovery from the end of therapy to the pretreatment level. These parameters are important when a patient’s condition necessitates the initiation of treatment after remission. It was reported [89] that, for psoriatic patients receiving UVES phototherapy, the highest dose delivered was 18.7 + 12.0 times the original MED with a final dose of 15.2 +9.7 MED for skin types I and II (2 1 volunteers). For skin types III and IV (16 volunteers), the highest dose was 15.1+ 11.6 MED and the final dose 11.2 + 7.9 MED. It should be noted that these patients did not show a significant pigmentation increase from their UVES exposure. In an unpublished study [go], again on psoriatic patients of skin type V, MED determinations were conducted throughout therapy on uninvolved skin sites of 90 volunteers. The average MED increased with treatment up to 8 weeks when it reached 7.5 times the original MED. Thereafter the MED remained constant but showed a slight decrease with treatment for up to 20 weeks. The patients appeared to experience an increase in their EMP with therapy (three times a week), but within the first 6 weeks they returned to their original visual pigment level. The highest dose values were similar to those reported [ 90-931 for aggressive UVB regimens, although one study was on skin type III and the other on skin type V.

Although the above studies relate to psoriatic patients, the MED as- sessments were conducted on univolved skin and there is no indication that it does not behave as normal skin in terms of UVB-induced facultative pigmentation. The tolerance to UVB is enhanced greatly, although the pigment level in the skin does not increase substantially. Part of this tolerance can be ascribed to epidermal thickening [94-961. The most perplexing observation was in the skin type V patients where, after approximately 80% clearance and at constant dose (three times a week), some patients started to burn and the UVB dose had to be continuously reduced until clearance was complete. The protection achieved in the above cases is most probably not connected with EMP, but involves long-term acclimatization. This does not happen with sunlight, where facultative pigmentation becomes very apparent with long-term exposure. However, there is probably a non-EMP-related component to this acclimatization. While the photoprotection attained in the phototherapy studies was substantial, the decay time to 2 MED tolerance from the peak of 15.2 occurred 42 days after the termination of treatment.

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3.5. Other end points 3.5.1. Non-melanoma Skin cancer It has been established that the incidence of non-melanoma skin cancer

in the U.S.A. shows an inverse dependence on latitude as well as on pigment level. This means that skin cancer incidence increases with decreasing latitude for white subjects, thus showing a dependence on the annual total solar UV insolation. Conversely, it would be expected that skin cancer incidence should decrease with increasing pigmentation. This has also been confirmed [ 97, 981. In New Mexico, the annual age-adjusted rates for “Angles” was found to be ten times higher than that for “Hispanics” [97]. In Texas, it was found that the incidence rate for white males was 120 per 100 000, for Spanish surname males 20 per 100 000 and or non-white males 4.5 per 100 000 [99]. The age-adjusted rate for skin cancer incidence in the U.S.A. was found to be 232.6 per 100 000 for whites and 3.4 per 100 000 for blacks, whereas for all other cancers the incidence rate for whites was 318.9 and for blacks 347.3 per 100 000 [97]. These differences in rates can be understood in terms of UV transmission of the epidermis; recent studies have shown that sunscreens with a protection factor of two, which are of no practical value in preventing erythema, significantly inhibit some biochemical and histological changes in the dermis associated with photoaging [ 1001.

A mathematical relation (linear regression) has been found between the logarithm of the skin cancer incidence rate (per 100 000) and the total annual UV radiation for various sites in continental U.S.A. The equation in question has the form

log(incidence rate) = m X (annual UV dose) + b

where m and b are arbitrary constants, representing the slope and intercept of the straight line, and whose values are decided by fitting the data. An increase in the pigment level of the population to that of moderate pigmentation would amount to an attenuation of the incident radiation by a factor of four, Le. the incident radiation would be reduced; this can be incorporated in the equation

log(incidence rate) = m X (annual UV dose) X T+ b

where T (in this case 0.25) is the transmittance of the epidermis of the pigmented population compared with the “whites”. In effect, what this factor does is to reduce the annual total solar UV radiation which arrives at the basal layer. For a darkly pigmented person this factor (T) would have a value of 0.1, or the radiation arriving at the basal layer would be reduced by a factor of ten. We can thus define an effective slope whose value is m’ = m x T. This type of analysis can predict reductions in incidence rates as shown in Table 4 and Fig. 1 for squamous cell cancers in females.

The values given are based on a model developed by R. M. Sayre for calculating reduction in skin cancer through the use of sunscreens. The answers provide reasonable estimates of risk.

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TABLE 4

Predicted reduction in incidence rate of squamous ceII cancer in females

T

(%) m b Incidence rate

100 0.025 2.135 I

25 0.025 0.527 0.042z 10 0.025 0.214 0.0241

0.0 0.5 1 .o 1.5 2.0

ANNUAL UV COUNT/l 00000

Fig. 1. Predicted risk reduction. Age-adjusted squamous ceil carcinoma for females. Riik based. on &in transmission estimates for 25% transmittance ( -----) (moderately pigmented) and 10% transmittance (... . .) (darkIy pigmented). The data are from ref. 98.

Another calculation which allows an estimate to be made of the skin cancer incidence is based on the measurement of EMP in viva [ 1011. If we make the assumption that the remitted intensity from white Caucasian skin is of the order of 20% of the incident intensity, then we can write

where 0.8 corresponds to the pigment level of white caucasians, and the factor of two appears in the exponential because the light traverses the pigmented epidermis twice before it is remitted. Beer’s law can be considered to be almost valid for intact epidermis [ 951. This allows the calculation of the factor A, which is the product of the thickness of the absorber and the factor that relates the pigmentation index with the absorption coefficient tunes the concentration. The value obtained for A is 2.2. In experiments carried out in Kuwait on white and black skin we obtained pigment levels in the range O-10. A typical white has a pigment level of 0.8, a typical

154

Kuwaiti (similar to a Hispanic of New Mexico) has a level of 2.3 and a deep brown skin a level of 4. On this scale of pigment level, the range 3.5-10 corresponds to what we would broadly call “black” skin. Finally, a typical dark black skin has a pigment level of 7. Using these constants, the following intensities at the basal layer were obtained: whites, 0.441,; Hispanics, 0.0891,; brown-black, O.OlU,; black, 0.000451,. These numbers correspond very closely to the findings in the Texas survey. When we take the ratios of the intensities of whites to Hispanics, whites to brown black and whites to black we obtain 4.9, 37 and 980. The most significant factor about these findings is that the attenuation in intensity corresponds well to the reduction in skin cancer incidence, whereas the reduction of the MED between blacks and whites is only a factor of ten. Therefore skin cancer incidence appears to be related to the number of photons which are delivered to the basal cell layers and the MED is related to the absorbance of the epidermis.

It is important to note that unlike the MED which cannot be predicted, on an individual basis, knowing the pigment level, the skin cancer risk of a population may be accurately estimated by measuring the pigment level.

3.5.2. Sunburn cells and other end points There are no human studies where sunburn cell production has been

measured for different skin pigment levels. In a study using guinea pigs [102] it was shown that a bergapten-induced tan appeared to reduce the number of sunburn cells compared with a non-sensitized tan and normal skin; the extent of protection cannot be compared with pigment enhancement as no quantitative measurements were given.

A study has also been reported [ 1031 on black and white guinea pigs; no protection was detected in terms of unscheduled DNA synthesis when both pigmented and unpigmented areas were challenged with UVC or UVl3 radiation. In a study on human volunteers with UVB-induced pigment, it was found that the protection afforded was very dependent on wavelength [ 1041. In considering these studies it should be remembered that EMP is very likely to show photochemical changes especially during UVC irradiation and so the observation of no photoprotection or photoprotection varying with wavelength is not surprising.

4. Conclusion

Although much has been learned about the chemistry and photochemistry of melanins, and methodologies have been developed to determine quantitatively eumelanin and pheomelanin through their degradation products, questions still remain. In order to proceed with meaningful calculations of photoprotection, we need to determine the following: (i) the absorption and scattering coefficients of eumelanin, (ii) the refractive index of eumelanin, and (iii) the exact changes and distribution of eumelanin as it traverses the epidermis to the stratum corneum.

155

Although evidence has been presented which indicates that eumelanin and pheomelanin are present in human skin, their existence needs to be confirmed and quantitatively assessed as a function of pigment level in the skin and reactivity to UV radiation. The suggestion that pheomelanin is a potential phototoxin needs to be addressed.

The extent of photoprotection by melanin, or rather EMP, needs to be assessed quantitatively for various pigment levels in the skin. The use of the MED a threshold reaction with a logarithmic response, does not provide sufficient sensitivity. Other non-invasive end points need to be considered and investigated.

The protection offered by the native pigment is undeniable; the extent of this protection is variable depending on the biological end point chosen. In terms of MED, the protection reaches a maximum value of lo-15 for very black individuals, whereas for Hispanics or Kuwaitis or dark Mediter- raneans it reaches a value of 2.5. However, in terms of skin cancer the protection is substantial: a factor of 5-10 for Hispanics and a factor of 500-1000 for dark blacks. What is urgently needed in future photobiological studies on photoprotection is a conserted effort to measure non-invasively and quantitatively the EMP in conjunction with other measurements; this is starting to occur slowly [ 57, 1051. Finally, in evaluating photoprotection the interaction of pigment with other photoprotective agents needs to be considered.

Acknowledgments

We wish to take this opportunity to thank the individuals who provided us with advice and suggestions in the preparation of this paper and copies of papers to be published; these include F. Urbach, I. H. Blank, R. W. Gange, K. Kaidbey, S. L. Jacques, A. J. Thody, C. R. Taylor, P. R. Crippa, A. R. Young and L. Zeise. These individuals are not responsible for any omissions or for the content of this text.

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New trends in photobiology: Photoprotection by melanin