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Keywords:

  • melanin;
  • eumelanin;
  • pheomelanin;
  • melanogenesis;
  • melanosome;
  • casing model

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Melanin is a natural pigment produced within organelles, melanosomes, located in melanocytes. Biological functions of melanosomes are often attributed to the unique chemical properties of the melanins they contain; however, the molecular structure of melanins, the mechanism by which the pigment is produced, and how the pigment is organized within the melanosome remains to be fully understood. In this review, we examine the current understanding of the initial chemical steps in the melanogenesis. Most natural melanins are mixtures of eumelanin and pheomelanin, and so after presenting the current understanding of the individual pigments, we focus on the mixed melanin systems, with a critical eye towards understanding how studies on individual melanin do and do not provide insight in the molecular aspects of their structures. We conclude the review with a discussion of important issues that must be addressed in future research efforts to more fully understand the relationship between molecular and functional properties of this important class of natural pigments.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Pigmentation of hair, skin, and eyes in animals is mainly a manifestation of the presence of melanin. Melanin is synthesized in melanocyte cells, within membrane-bound organelles termed melanosomes. Melanocytes in mammals and birds produce two chemically distinct types of melanin, black to brown eumelanin and yellow to reddish-brown pheomelanin (Ito, 2003; Ito and Wakamatsu, 2006; Ito et al., 2000; Prota, 1992, 1995; Prota et al., 1998). Spurred in part by the observation that these two types of melanin react differently – eumelanin acts as a photoprotective anti-oxidant while pheomelanin exhibits phototoxic pro-oxidant behavior (Chedekel et al., 1978, 1980; de Leeuw et al., 2001; Samokhvalov et al., 2005; Takeuchi et al., 2004) – there is an increasing research effort to understand the molecular structure of melanins, the organization of these constituent molecules within intact melanosomes, and how these composite structures perform biological function(s). Thus it is timely to assess the connections between these efforts, which integrate across various length scales – molecule to cell to tissue – and disciplines. Review articles on melanin have appeared in recent years, covering a wide range of chemical and biological topics including the chemistry of melanogenesis (Ito, 2003), the chemical and physical properties of synthetic and natural eumelanins (Meredith and Sarna, 2006), mixed melanogenesis (Ito and Wakamatsu, 2008), the morphology of natural melanosomes (Liu and Simon, 2003), the regulation of skin pigmentation and the effects of UV exposure (Brenner and Hearing, 2008), the physiological roles of ocular melanosomes (Hu et al., 2008), the regulation of melanin synthesis in vivo (Abdel-Malek et al., 2008), the role of iron and neuromelanin in brain aging (Zecca et al., 2004), the physiological and pathogenic aspects of neuromelanin (Zucca et al., 2004), the role of neuronal pigmented autophagic vacuoles in aging and disease (Sulzer et al., 2008), and the spatial and photochemical properties of melanins and melanosomes (Simon et al., 2008). It is not our intention to rehearse these topics, but rather to offer an interdisciplinary perspective integrating across length scales on the structure and function of melanins and melanosomes. Advances in our understanding of melanogenesis and the role melanin plays in the human body require the confluence of the physical, chemical and biological sciences, and recent progress has been spurred by advancing and applying techniques developed in these different domains to a common purpose.

Our review is organized as follows. We first present a brief summary of the monomeric building blocks of the two types of melanins and then follow with a more in depth examination of the biochemistry of melanogenesis of these pigments, focusing on the pivotal role of dopaquinone in both, and recounting our current understanding of molecular transformations following monomer formation. That discussion presents the current understanding of the ‘molecular’ scale of the individual melanins. We then explore the current status of the chemical/biochemical control of mixed melanogenesis – characteristic of most natural systems – and the structural consequences of that control, as evidenced by studies of intact melanosomes. To explore melanosome function, we discuss the consequences of UV exposure and the subsequent oxidative damage to the iris stroma melanosome, produced through mixed melanogenesis, within the context of the molecular and morphological properties revealed in the earlier sections. While we focus on reviewing the literature pertinent to these topics, the discussion raises important unanswered challenges, which, like the material covered in the review itself, will require interdisciplinary approaches to address.

Molecular foundations of eumelanin and pheomelanin

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Early, yet extensive studies conducted nearly 50 years ago in Naples (for reviews see, Nicolaus, 1968; Prota, 1992; Prota et al., 1998) and New Castle (Swan, 1974; Swan and Waggott, 1970) suggested that eumelanin is a very heterogeneous polymer consisting of different oxidative states of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units, and pyrrole units derived from their peroxidative cleavage (Figure 1). Prota and his successors continued extensive biosynthetic studies to identify melanogenic intermediates beyond DHI and DHICA (up to tetramers) involved in eumelanogenesis. Although considerable advances have been made recently (d’Ischia et al., 2009; Panzella et al., 2007), these works do not change our basic views about the structure of eumelanin.

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Figure 1.  Structure of eumelanin and pheomelanin (Ito and Wakamatsu, 2008). Note that these structures of eumelanin and pheomelanin are only representative ones formulated on the basis of biosynthetic and degradative studies, as discussed in the text. The positions with (–COOH) in eumelanin structure are connected either to –H or–COOH; these positions may also be available for attachment to other units. The carbonyl group between the pyrrole and indole rings may also be a methylene group, as suggested by mass spectra of synthetic as well as natural eumelanins (Napolitano et al., 1996b; Novellino et al., 2000). The arrows indicate sites for attachment to other units. How the benzothiazine, benzothiazole and isoquinolene units are connected is a matter of conjecture. (Figure used with permission, Blackwell Publishing.)

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Most current knowledge of pheomelanin chemistry results from studies in the late 1960s conducted by Prota et al. (for reviews see, Prota, 1972; Thomson, 1974). Those early results, combined with more recent findings (e.g., Di Donato and Napolitano, 2003; Napolitano et al., 2008; Wakamatsu et al., 2009), led us to propose a representative structure (Figure 1) for pheomelanin that consists of benzothiazine moiety with contributions from benzothiazole and isoquinoline moieties. How these monomer units are connected is not established, although the connection through carbon atoms should play a major role to explain absorption throughout the visible region.

Over the past 30 years, chemical analyses have been perfected that enable the quantification of the amount of eumelanin and pheomelanin present in a naturally occurring sample of melanin (Ito and Fujita, 1985). These works collectively reveal that more often than not, natural melanin pigments consist of both eumelanin and pheomelanin in varying ratios (Ito and Wakamatsu, 2003; Naysmith et al., 2004; Thody et al., 1991). Thus, rather than focus solely on the structural and functional properties of pure eumelanin or pheomelanin, there is a need to understand how mixed melanins are made, and how their molecular and functional properties differ from the pure melanins.

Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Eumelanin and pheomelanin both are derived from the common precursor dopaquinone (DQ) formed by oxidation of the common amino acid l-tyrosine by tyrosinase (Figure 2). It had been widely believed that 3,4-dihydroxyphenylalanine (dopa) is formed first on the way to DQ. However, Cooksey et al. (1997) showed that ortho-quinones, such as DQ, are formed directly during the initial stage of melanogenesis. The chemistry of ortho-quinones is closely involved with melanogenesis as they are extremely reactive molecules (Ito and Wakamatsu, 2008). Rate constants for most of the important steps in melanogenesis have been reported based on pulse radiolysis studies (Land and Riley, 2000; Land et al., 2001, 2003).

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Figure 2.  Biosynthetic pathways leading to eumelanin and pheomelanin production (Ito and Wakamatsu, 2008). Note that the activities of tyrosinase, Tyrp1 and Tyrp2 are involved in the production of eumelanin, while only tyrosinase (and the precursor amino acid cysteine) is necessary for the production of pheomelanin. (Figure used with permission, Blackwell Publishing.)

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The first step (= 3.8/s) in eumelanogenesis is a relatively slow process involving the intramolecular addition of the amino group to produce cyclodopa (Land and Riley, 2000; Land et al., 2003). However, as cyclodopa is formed, it is rapidly oxidized to dopachrome through a redox exchange (= 5.3 × 106/M/s)(Land et al., 2003). Comparing these two rate constants reveals that the production of dopachrome is faster than the production of cyclodopa when the cyclodopa concentration is above 0.7 μM. Thus cyclodopa does not accumulate during the early phase of eumelanogenesis, but dopachrome does.

Dopachrome is an orange-red pigment and has a half-life of about 30 min (first-order rate constant of 4.0 × 10−4/s). It spontaneously decomposes by decarboxylation at neutral pH to give DHI and DHICA in a 70:1 ratio (Palumbo et al., 1987b). However, in the presence of dopachrome tautomerase (Dct), also termed tyrosinase-related protein-2 (Tyrp2), dopachrome undergoes tautomerization to preferentially produce DHICA (Palumbo et al., 1991). The ratio of DHICA to DHI in natural eumelanins is thus determined by the activity of Dct (Tsukamoto et al., 1992). Metal cations, especially Cu2+, also accelerate the dopachrome rearrangement and affect the DHICA/DHI ratio, but Dct seems to be more effective in catalyzing the tautomerization (Palumbo et al., 1987a, 1991).

Recently, the redox exchange reaction between DHI and DQ has been studied (Edge et al., 2006). Redox exchange with DHI proceeds, but not to completion, with a rate constant of = 1.4 × 106/M/s. On the other hand, the reaction with DHICA more obviously does not go to completion and is much slower (= 1.6 × 105/M/s). It was concluded that during eumelanogenesis, DHI oxidation takes place by redox exchange with DQ, although such a reaction is likely to be less efficient for DHICA. Thus, DHICA may require its oxidation to the quinone form by a direct action of tyrosinase in humans (Olivares et al., 2001) or by Tyrp1 in mice (Jiménez-Cervantes et al., 1994; Kobayashi et al., 1994). It is interesting to note that human Tyrp1 is unable to catalyze the DHICA oxidation (Boissy et al., 1998). The activities of these tyrosinase-related proteins, Tyrp1 and Tyrp2, greatly affect the quantity and quality (the ratio of DHI to DHICA and the degree of polymerization) of eumelanins produced (Lamoreux et al., 2001; Ozeki et al., 1995).

We now turn to a discussion of the formation of pheomelanin, or pheomelanogenesis. The major course of pheomelanogenesis has been clarified through the extensive studies carried out recently by the Naples group. In summary, pheomelanogenesis proceeds through several distinctive steps at the monomer levels (Figure 3): (1) addition of cysteine to DQ to produce cysteinyldopa (CD) isomers (= 3 × 107/M/s), (2) redox exchange between CD and DQ to produce CD-quinones and dopa (= 8.8 × 105/M/s), (3) cyclization of CD-quinones through dehydration to form the ortho-quinonimine (QI) with a rate constant of = 6.0/s (Napolitano et al., 1999), and (4) rearrangement (with/without decarboxylation) of QI to form the 7-(2-amino-2-carboxyethyl)-5-hydroxy-2H-1,4-benzothiazine (BT) and 7-(2-amino-2-carboxyethyl)-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (BTCA) – in a ratio of 85:15 (Napolitano et al., 1994, 2000a) with a rate constant of = 10/s (Napolitano et al., 1999).

image

Figure 3.  Early and late stages of pheomelanogenesis (Wakamatsu et al., 2009). Note that only products derived from 5SCD are shown and those from 2SCD are not shown. Products in parentheses are those with a short half time and are not isolable. (Figure used with permission, Blackwell Publishing.)

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It should be noted that in the tyrosinase oxidation in which a low concentration of QI is formed under a high concentration of CD, the above pathway is bypassed to form the 7-(2-amino-2-carboxyethyl)-5-hydroxy-3,4-dihydro-2H-1,4-benzothiazine-3-carboxylic acid (DHBTCA) through a redox exchange between QI and CD (Prota et al., 1970). DHBTCA is then oxidized back to QI through another redox reaction involving DQ (Wakamatsu et al., 2009). Thus, BT (and BTCA) is the ultimate monomer precursor of pheomelanin. BT (and BTCA) is a rather unstable molecule and decays over a few seconds (= 0.5/s; Napolitano et al., 1999). Before being incorporated into pheomelanin, a minor part of BT (and BTCA) may undergo a structural modification to form 7-(2-amino-2-carboxylethyl)-5-hydroxy-3-oxo-3,4-dihydro-2H-1,4-benzothiazine (ODHBT) and 6-(2-amino-2-carboxyethyl)-4-hydroxy-benzothiazole (BZ). The three monomeric units, BT (and BTCA), ODHBT, and BZ, are eventually polymerized to form pheomelanin. In a freshly synthesized sample of pheomelanin, the benzothiazine moieties, BT (and BTCA) and ODHBT, appear to be major structural components. However, those benzothiazine moieties are further degraded to the benzothiazole moieties in older samples of pheomelanin (Greco et al., 2009; Wakamatsu et al., 2009).

In the preceding section, DQ is shown to play pivotal roles in promoting eumelanogenesis. As mentioned above, DQ is also involved in the production of CD in the first step of pheomelanogenesis. In fact, tyrosinase oxidation of dopa in the presence of excess cysteine generates a high yield of 5-S-cysteinyldopa (5SCD) (74%) and 2-S-cysteinyldopa (2SCD) (14%) together with minor amounts of the 6-S-cysteinyldopa (6SCD) (1%) and a di-adduct, 2,5-S,S′-dicysteinyldopa (5%) (Ito and Prota, 1977). Dopaquinone also plays pivotal roles in the following two redox exchange reactions: oxidation of CD to form CD-quinone and oxidation of DHBTCA to form QI. For the latter reaction, the rate constant is not known, but a recent study showed that the reaction proceeds very rapidly (Wakamatsu et al., 2009). The process of oxidative polymerization appears to be very complex, but certainly involves oxidation of several labile (non-isolable) intermediates. It is possible that DQ also promotes such oxidation reactions through the redox exchange.

Insights from the comparison of synthetic and natural melanins

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

In 1980 (Pawelek, 1991; Pawelek et al., 1980), Pawelek’s group discovered a dopachrome conversion factor, Dct or Tyrp2 and subsequently, Aroca et al. reported DHICA as the product of the reaction involving this enzyme (Aroca et al., 1990). Until then, eumelanin was believed to be DHI rich, because the decarboxylation of dopachrome is the major pathway taken in the absence of any extrinsic factor under neutral pH. Therefore, we analyzed the contents of DHICA-derived units in various eumelanins (Ito, 1986). Two analytical methods were used to evaluate the DHICA-derived units: acid-catalyzed decarboxylation and permanganate oxidation to give pyrrole-2,3,5-tricarboxylic acid (PTCA; Figure 4). It was shown that synthetic dopa melanin contains only trace amounts of DHICA-derived units, while melanins from Sepia, B16 melanoma, and mouse black hair consist of about equal amounts of DHI- and DHICA-derived units. This study has become a milestone in the eumelanin research showing the significance of DHICA-derived units in the structure of natural eumelanins (Meredith and Sarna, 2006).

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Figure 4.  Degradation products, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), from 5,6-dihydroxyindole (DHI)-melanin and 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. Note that oxidants used are acidic potassium permanganate (Ito and Fujita, 1985) or alkaline hydrogen peroxide (Ito and Wakamatsu, 1998) for DHICA-melanin, but only alkaline hydrogen peroxide is suitable for DHI-melanin (Ito and Wakamatsu, 1998). Also, the yield of PTCA from DHI-melanin with a connection to the next monomer at the C2 position is traced (Ito and Fujita, 1985; Ito and Wakamatsu, 1998).

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The ratio of DHI to DHICA in natural and synthetic eumelanins can be estimated from the ratio of pyrrole-2,3-dicarboxylic acid (PDCA) to PTCA upon alkaline peroxide oxidation (Figure 4). PDCA and PTCA are makers of DHI- and DHICA-derived units, respectively (Ito and Wakamatsu, 1998; Napolitano et al., 1995). The PTCA/total melanin is also useful for the same purpose when total melanin is determined as an absorbance at 500 nm after solubilizing eumelanin samples in hot Soluene-350 plus water (Ozeki et al., 1996, 1997b). With this methodology, the effects of Dct (Tyrp2) activity was assessed comparing the PTCA/total melanin ratios between black and slaty mice, the latter possess the Dct activity less than one-third that of black. It was found that slaty hair contains eumelanin consisting of DHI/DHICA ratio of ∼3:1 while black hair DHI/DHICA ratio ∼1:3 (Lamoreux et al., 2001; Ozeki et al., 1995). The role of Tyrp1 in eumelanogenesis appears to control the molecular size of eumelanin produced. Our studies showed that brown mice that lack Tyrp1 activity produce eumelanin with lower molecular weights than wildtype, black mice (Ozeki et al., 1996, 1997b).

Pyrrole carboxylic acids are produced from eumelanins through peroxidative cleavage. It is therefore generally believed that the dihydroxyindole moiety is degraded to the pyrrole moiety in the polymeric structure of eumelanins (Pezzella et al., 1997). In fact, free PTCA, in addition to other pyrrole carboxylic acids, can be isolated from eumelanosomes obtained from Sepia, human black hair, and bovine eyes (Ward et al., 2008). The yields of free pyrrole carboxylic acids are surprisingly high, as much as 20% of those after hydrogen peroxide oxidation. It would be of considerable interest to determine whether the natural PTCA content can serve as a marker of (photo)aging of eumelanin in vivo.

When we study the structural features of synthetic and natural pheomelanin pigments, two different approaches are feasible. One is the biosynthetic approach starting from a mixture of dopa (or tyrosine) and cysteine or 5SCD. The use of 5SCD as a precursor makes it possible to produce less complex mixtures of pheomelanin intermediates and degradation products upon HI hydrolysis. To oxidize 5SCD to the pheomelanin polymer, various chemical oxidants such as ferricyanide and persulfate or biological oxidants such as peroxidase/H2O2 have been used (Di Donato et al., 2002; Napolitano et al., 2008). However, a major drawback of using such artificial oxidation systems is that the major pathway of pheomelangenesis bypasses the production of DHBTCA as an intermediate. On the other hand, with the use of tyrosinase, a natural oxidizing enzyme, DHBTCA becomes a major intermediate before polymerization begins to take place. With this system, it has been shown that a freshly prepared synthetic pheomelanin consists mostly of benzothiazine moiety with a minor contribution of benzothiazole moiety (Wakamatsu et al., 2009). Another possible monomer unit, isoquinoline moiety, may arise from the Pictet-Spengler type condensation between the alanyl side chains and carbonyl intermediates (Fattorusso et al., 1970; Manini et al., 2000). Modification of the alanyl side chains to form an aromatic group is consistent with biosynthetic studies (Chedekel et al., 1987; Deibel and Chedekel, 1984). A candidate for the latter is formaldehyde (see below), a putative product in the conversion of BT to BZ. In addition to BT (and BTCA), several monomeric precursors, BZ and 6-(2-amino-2-carboxyethyl)-4-hydroxy-2-methylbenzothiazole (MeBZ), ODHBT, and the isoquinoline derivatives are likely to constitute the pheomelanin polymer.

Another approach is the chemical degradation of natural pheomelanins. This approach has been quite successful not only in elucidating the structure of pheomelanin pigments (Fattorusso et al., 1969) but also in quantitatively analyzing pheomelanin in tissue samples (Ito and Wakamatsu, 2003; Wakamatsu et al., 2002). This approach is also useful in elucidating the course of pheomelanin production (Wakamatsu et al., 2009). A number of useful degradation products were isolated in a landmark study by Minale et al. in Naples (Fattorusso et al., 1969). Two types of degradation reactions are especially informative (Figure 5). One is the reductive hydrolysis with HI, which produces 4-amino-3-hydroxyphenylalanine and 3-amino-4-hydroxyphenylalanine (4-AHP and 3-AHP, respectively), BZ, MeBZ (Wakamatsu et al., 2009). The isoquinoline derivatives (Figure 6, structure 1) are another important degradation product (Fattorusso et al., 1970). Advantage of the HI hydrolysis is that the degradation products are produced in relatively high yields with retention of the original structure. The other is the oxidation either by acidic permanganate or by alkaline hydrogen peroxide. Both gave a similar set of oxidation products arising from pheomelanin. They are thiazole-4,5-dicarboxylic acid (TDCA) and thiazole-2,4,5-tricarboxylic acid (TTCA), degradation products from BZ and BZ unit connected to another monomer unit at the C2 position, respectively (MeBZ does not give rise to TTCA; Wakamatsu et al., 2009). An advantage of the oxidative degradation is that those pheomelanin degradation products are produced together with degradation products arising from eumelanin, PDCA and PTCA. This makes it possible to estimate relative ratios of pheomelanin to eumelanin in tissue samples (Ito and Wakamatsu, 2003; Wakamatsu and Ito, 2002). Pyrridine-2,3,4,6-tetracarboxylic acid (Figure 6, structure 2) is also produced by permanganate oxidation. This compound serves to support the participation of isoquinoline units in pheomelanin structure (Fattorusso et al., 1969). Since then, this useful degradation product, however, has not been studied. Another interesting degradation product is the 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BZCA) (Figure 6, structure 3; and 7-(2-amino-2-carboxyethy)-4-hydroxybenzothiazole-2-carboxylic acid (BZCA-2) from 2SCD-derived pheomelanin). An advantage of BZCA (and BZCA-2) is that it is produced in a relatively high yield by hydrogen peroxide oxidation in 1 M NaOH (Greco et al., 2009; Napolitano et al., 1996c, 2000b). Although it has a benzothiazole structure, BZCA (and BZCA-2) appears to be produced from benzothiazine moiety rather than benzothiazole moiety (Greco et al., 2009; Napolitano et al., 1996c). The conversion of benzothiazine to a benzothiazole moiety is promoted by the strongly alkaline condition of 1 M NaOH (McCapra and Razavi, 1975).

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Figure 5.  Degradation products from different types of pheomelanin moieties benzothiazine (BT), benzothiazole (BZ) and 3-oxo-dihydrobenzothiazine (ODHBT) (Wakamatsu et al., 2009). Note that only products derived from 5SCD are shown for degradation products by HI hydrolysis. (Figure used with permission, Blackwell Publishing.)

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Figure 6.  Other important degradation products from pheomelanin. Structure 1: Isoquinoline derivatives (R=H or CH3); Structure 2: Pyridine-2,3,4,6-tetracarboxylic acid; Structure 3: BZCA.

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Molecular structure of eumelanin beyond the monomers DHI and DHICA

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Extensive biosynthetic studies have been performed to illuminate the chemical processes occurring following the formation of DHI and DHICA (for reviews, see d’Ischia et al., 1996, 2005, 2009; Prota et al., 1998). Upon enzymatic or chemical oxidations, DHI affords a number of dimers, trimers, and tetramers in the early stages (d’Ischia et al., 1990; Pezzella et al., 2007). Judged from the structures of oligomers isolated, the most reactive position is the 2-positions, followed by the 4- and 7-positions. The comparative reactivity of those positions has recently been substantiated with the general purpose reactivity indicator from ab initio density-functional theory calculations (Okuda et al., 2008). The theoretical prediction indicates that the dimerization of DHI proceeds through the electron-transfer-controlled reaction between the 2-position of DHI and the 4- or 7-position of its quinine form. The mode of polymerization is strongly influenced by the presence of metal cations, such as Zn2+ or Cu2+. The polymerization at the 2-positions is much favored by the presence of Cu2+ (d’Ischia et al., 1996; Napolitano et al., 1985). It is interesting to note that oxidation of the 2,4′-dimer, a major dimer, afforded a tetramer involving a coupling at the 3-position, a position not reactive in DHI (Panzella et al., 2007).

Oxidation of DHICA, catalyzed by tyrosinase/O2 or by peroxidase/H2O2, produces mixtures of dimers and trimers in which the indole units are mostly linked through the 4- and 7-positions (Palumbo et al., 1987b; Pezzella et al., 1996). The 4-position appears to be more reactive than the 7-position. Similar to the oxidation of DHI, the presence of Cu2+ significantly influences the course of oxidation in which the 3-position becomes more reactive, thus giving minor 3,4′- and 3,7′-coupled dimers, in addition to the major 4,4′-, 4,7′- and 7,7′-dimers (Pezzella et al., 1996).

Co-oxidation of DHI and DHICA with peroxidase/H2O2 affords, in addition to homodimers, some heterodimers including the 2,4′-dimer (Napolitano et al., 1993). Beyond this point, nothing is known about the linkage between DHI and DHICA units. Addressing this issue is central to developing a complete understanding of eumelanin at the molecular scale.

Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

The process of oxidation beyond the monomer level remains to be elucidated. Several dimeric and trimetic intermediates have been identified (Costantini et al., 1990; Napolitano et al., 1996a, 2001). Trichochrome pigments are dimeric pigments with structural features similar to pheomelanin pigments, where the monomer units – BT (and BTCA) and ODHBT – are connected through a double bond at the C2. Trichochromes are the only pheomelanin-like pigments with structures that have been fully characterized as having a 2,2′-bi(2H-1,4-benzothiazine) chromophore (Ito and Wakamatsu, 2006; Prota, 1972, 1992; Thomson, 1974). The close similarity in structural features of trichochromes and pheomelanin, and their coexistence in pigmented tissues, suggests that they are formed oxidatively from the same monomer units and differ only in their mode of polymerization. Therefore, trichochromes can be regarded as a minor variety of pheomelanic pigment. The spectroscopy and photochemical properties of trichochromes has been examined and the data establish that these species are not responsible for the photo-induced oxygen activation by pheomelanin (Simon et al., 2006).

It is thus logical to consider that pheomelanin pigments arise mostly from oxidative polymerization of benzothiazines between carbon atoms at the 2-position and either the C6 or C8 position. Dimerization of BT appears to involve C–C coupling at the positions ortho and para to the hydroxyl group (Napolitano et al., 1994). Involvement of an oxygen atom in the hydroxyl group to form an ether bond in this connection is unlikely to play a major role because oligomeric compounds arising from such a connection are not expected to display the general absorption typical for pheomelanin pigments.

The view that the benzothiazine moiety is the major constituent of the pheomelanin polymer stems in part that upon hydroiodic acid (HI) reductive hydrolysis, pheomelanin forms 4- and 3-AHP isomers, which are specific degradation products of benzothiazine moieties; the yield of ∼20% of AHPs is rather high when we consider the complex nature of pheomelanin structure. The belief was recently challenged by the Naples group (Napolitano et al., 2008), mostly based on spectral comparison of synthetic and natural pheomelanins with monomeric intermediates. Our recent study (Wakamatsu et al., 2009) confirmed that in the course of late stage of pheomelanogenesis, the benzothiazine moiety is gradually converted to the benzothiazole moiety based on the following observations: DHBTCA isomers were produced following the production and consumption of CD isomers, and DHBTCA isomers were then rapidly consumed to form pheomelanin polymers with a parallel increase of BZ and MeBZ at monomer and oligomer levels (Figures 3 and 4). The continuous production of benzothiazole units was also supported by the increase of TTCA and TDCA, degradation products of the benzothiazole moiety, upon alkaline hydrogen peroxide oxidation (Wakamatsu et al., 2003). This process was paralleled by a concomitant decrease of AHP in the HI hydrolysate. Additional evidence was provided by the facts that the ratio of TTCA/4-AHP in recessive yellow mouse hair was rather close to that of synthetic pheomelanin while those in red chicken feather and in red human hair were twofold and fourfold greater, respectively. Thus, the increase of benzothiazole moiety with a compensation of benzothiazine moiety appears to depend on the duration of natural pheomelanin being exposed to environmental UV radiation. It should be noted that in this process of (photo)aging of pheomelanin, 5SCD-derived benzothiazine units are preferentially degraded compared with 2SCD-derived benzothiazine units, as evidenced by the decrease of 4-AHP/3-AHP and BZCA/BZCA-2 ratios (see later; Greco et al., 2009).

Then, how are the benzothiazine units such as BT and BTCA degraded to benzothiazole units such as BZ and MeBZ? This question has not been answered with certainty. It is known that a similar system of 6-hydroxybenzothiazole is produced from a 7-hydroxydihydrobenzothiazine in a biomimetic synthesis of firefly luciferin model compound (McCapra and Razavi, 1975). In this reaction, one carbon at the 2-position is lost, but its mechanism is not known. A more closely related compound DHBTCA is known to produce MeBZ upon UV radiation. However, this photo-reaction resulted in the retention of the C2 carbon atom of the benzothiazine ring in the form of a methyl group (Costantini et al., 1994). The ring contraction is also markedly promoted by the presence of Fe3+ ion (Di Donato et al., 2002). To produce BZ, the C2 carbon atom needs to be released in the form of either formaldehyde or carbon dioxide. The possible production of formaldehyde has to be clarified, as this leads to a great biological implication due to its carcinogenic activity.

In addition, experimental studies on the spectroscopy and photochemical properties of pheomelanins reveal that different molecular constituents are responsible for the emission, transient absorption and oxygen photoconsumption properties of the pigment (Ye et al., 2008). This suggests that a distribution of molecular species are produced, reflecting the different types of chemical processes described above.

A three step pathway for mixed melanogenesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

As mentioned above, melanogenesis in vivo produces mixtures of eumelanin and pheomelanin. Therefore, in reality melanogenesis should be considered as ‘mixed melanogenesis’. We previously proposed a three-step pathway for the mixed melanogenesis, as shown in Figure 7 (Ito, 2003; Ito and Wakamatsu, 2008; Ito et al., 2000). We will not go into the details of this pathway in this review because we recently reviewed this subject (Ito and Wakamatsu, 2008). In summary, as indicated in Figure 7, the total amount of melanin produced is proportional to DQ production, which is in turn proportional to tyrosinase activity. In fact, melanogenesis proceeds in three distinct stages. The initial stage is the production of CD isomers, which continues as long as the cysteine concentration is above 0.13 μM. The second stage is the oxidation of CDs to produce pheomelanin, which continues as long as CDs are present at concentrations above 9 μM. The last stage is the production of eumelanin, which begins only after most CDs and cysteine are depleted. Therefore, the ratio of eumelanin to pheomelanin is determined by tyrosinase activity and the availability of tyrosine and cysteine in melanosomes (Land et al., 2003). It should be noted that pheomelanogenesis is kinetically favored under more acidic environment because the cyclization of dopaquinone (the first step in eumelanogenesis) proceeds slower at lower pHs while the CD-quinone cyclization (yielding the first bicyclic intermediate in pheomelanogenesis) proceeds faster (Thompson et al., 1985).

image

Figure 7.  Three-step pathway for mixed melanogenesis (Ito and Wakamatsu, 2008). Note that the course of melanogenesis proceeds in three distinct stages: cysteinyldopa-genesis, pheomelanogenesis, followed by eumelanogenesis. (Figure used with permission, Blackwell Publishing.)

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Biochemical pathways for mixed melanogenesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Some evidence supporting the three-step pathway (which underpins the casing model discussed later in this review) of mixed melanogenesis has been obtained. Our recent studies have shown that human epidermal and uveal melanocytes in culture produce pheomelanin at rather constant levels regardless of the degree of pigmentation while they produce eumelanin at levels proportional to pigmentation (Ito and Wakamatsu, 2008; Wakamatsu et al., 2006, 2008). These results fit well to the casing model of mixed melanogenesis.

Our biosynthetic studies (Ozeki et al., 1997a; Wakamatsu et al., 2009) have shown that tyrosinase oxidation of tyrosine or dopa in the presence of cysteine proceeds in the three distinct steps: CD isomers are produced first, the production of pheomelanin follows, and eumelanin finally deposits on the preformed pheomelanin.

The proposal that tyrosinase activity plays a major role in controlling mixed melanogenesis is supported by several studies. Burchill et al. (1986) examined the effects of α-melanocyte-stimulating hormone (α-MSH) on mixed melanogenesis in viable yellow mice. When pubertal mice producing a mixed-type melanin were injected with α-MSH, tyrosinase activity increased twofold and more eumelanic hair was produced with a concomitant increase in total melanin. When these pubertal mice were injected with bromocriptine (which reduces α-MSH secretion), tyrosinase activity was reduced to less than one-tenth of the untreated control and pheomelanic hair was produced along with a decrease in total melanin. Forskolin is a direct activator of adenyl cyclase and induces a rise in cAMP level, thus resulting in an activation of melanogenesis (D’Orazio et al., 2006; Spry et al., 2009). A recent study has shown that prolonged topical application of forskolin to K14-stem cell factor transgenic mice with McIre/e background (producing mostly pheomelanin) resulted in a dramatic shift to eumelanogenesis (Spry et al., 2009). Thus, eumelanin to pheomelanin ratio in the skin was increased from 0.4 in the control mice to 4 in the treated mice. The three-step pathway for mixed melanogenesis suggests that pheomelanogenesis is more strongly influenced by changes in tyrosinase activity, as also proposed by Barsh (1996). As an example, the agouti pattern of baboon hairs were analyzed (Ito et al., 2001). Dark-colored baboons (high tyrosinase activity) produced high levels of pheomelanin in the yellow bands of their agouti hairs, whereas the corresponding bands in light-colored baboons contained little melanin. These results illustrate the important role of tyrosinase activity to control mixed melanogenesis in vivo.

The significance of tyrosinase activity in mixed melanogenesis was also studied in vitro. Hunt et al. (1995) examined the effects of a superpotent synthetic α-MSH analog on melanogenesis in human melanocytes. Treatment of melanocytes with the synthetic α-MSH resulted in an increase of eumelanin content in all of the seven cell lines examined from different ethnic origins, leading to a significant shift to more eumelanic cells. In a recent study (Le Pape et al., 2008), an effect of tyrosinase inhibitor phenylthiourea on the course of mixed melanogenesis was examined, together with the effects of α-MSH and agouti signal protein (ASP). Treatment with phenylthiourea resulted in a reduction of eumelanin content to a half with a concomitant twofold increase of pheomelanin content.

Another control point in mixed melanogenesis is the concentration of cysteine in the melanosome. How is cysteine concentration in the melanosome genetically regulated? This question has been solved in a recent study by Chintala et al. (2005). That study demonstrated that the subtle gray (sut) pigment mutation in mice arose due to a mutation in the Slc7a11 gene that encodes the plasma membrane cystine/glutamate exchanger xCT. The resulting low rate of extracellular cystine transport into sut melanocytes reduces pheomelanin production with minimal or no effect on eumelanin production. In fact, the effect of the Slc7a11 sut mutation on pheomelanin production was markedly accentuated on the Ay/a background, reducing pheomelanin levels in hair to one-sixth of the control level, with a fourfold increase in the low level of eumelanin.

The effects of varying the ratio of tyrosine to cysteine have also been examined by several groups. For example, by decreasing the extracellular concentration of cystine in cultures of human melanoma cells, del Marmol et al. (1996) demonstrated a shift to more eumelanic cells resulting from the dramatic decrease of intracellular cysteine concentration. In another study, Smit et al. (1997) found a twofold increase in melanin production in human melanocytes with a decreased ratio of pheomelanin to total melanin when cells were cultured at a higher concentration of tyrosine. In a recent study, Hida et al. (2009) have recently shown that melan-a nonagouti (a/a) mouse melanocytes produce mainly eumelanin, but ASP combined with phenylthiourea and extra cysteine could induce over 200-fold increases in the pheomelanin to eumelanin ratio. However, the pheomelanin to eumelanin ratio is not high, 0.13, and another factor(s) is still necessary to induce otherwise eumelanic melanocytes to switch to make more pheomelanin.

The role of pH in controlling melanogenesis is another important issue. This aspect of melanogenesis is now receiving much attention because it was recently found that melanosomes in melanocytes from White/fair skin are acidic while those from Black/dark skin are near neutral (Smith et al., 2004). Furthermore, the great diversity in normal human skin pigmentation appears to stem from mutations in only several genes, including P, MATP, and SLC24A5 (Lamason et al., 2005; Norton et al., 2007). Available evidence suggests that mutations in those genes may result in changing the acidic environment of melanosomes (Sturm, 2006). However, the significance of pH in controlling mixed melanogenesis has only been addressed by Ancans et al. (2001). By neutralizing intramelanosomal pH in cultured human melanocytes and melanoma cells, they found that cells with increased tyrosinase activity after neutralization had a preferential increase in the production of eumelanin, which resulted in an increase in the eumelanin to pheomelanin ratio. The effects of more acidic pH on mixed melanogenesis in melanosomes are twofold: a lower activity of tyrosinase and a slower rate of dopaquinone cyclization (but a faster rate of CD-quinone cyclization) (see above; Thompson et al., 1985). Both of those effects favor pheomelanogenesis in acidic melanosomes. There is a possibility that the addition of cysteine to DQ may also be acid-catalyzed, as is the case for the CD-quinone cyclization (Thompson et al., 1985). If this would be the case, this further favors pheomelanogenesis in acidic melanosomes.

Effects of metal ions on mixed melanogenesis are another interesting and important field of research because some metal ions are present at certain levels in melanosomes (Liu et al., 2005). In eumelanogenesis, Dct (Tyrp2) plays an important role in promoting the production of DHICA in tautomerization of dopachrome. Here Cu2+ is shown to catalyze this same reaction (see above). However, it is not known whether Cu2+ is involved in this tautomerization in vivo. In pheomelanogenesis, some metal ions have been shown to modify the course of melanogenesis at the monomer level. During oxidation of 5SCD by a chemical oxidant, the presence of Zn2+ protects the carboxyl group of QI through chelate formation to preferentially form BTCA in place of BT (Di Donato et al., 2002; Napolitano et al., 2001, 2008). Under the same conditions, Fe3+ ions appear to form chelates with intermediates to accelerate the ring contraction leading to BZ (Di Donato et al., 2002). Cu2+ ions are also involved in modification of the reaction pathway, with a greater yield of the 3-oxo-derivative ODHBT. It should be noted that pheomelanosomes isolated from red human hair contain Fe3+, Zn2+, and Cu2+ at 98, 25, and 20 μmol/g melanin, respectively, in addition to 141 μmol/g of Mg2+ (Liu et al., 2005). Those levels are high enough to affect the course of pheomelanogenesis to some extents.

The morphological consequence of our current models for the chemistry/biochemistry and time course of mixed melanogenesis is schematically represented in Figure 8, in which the assembly of the pigment involves a pheomelanin core surrounded by a eumelanin outer coat. This ‘casing model’ was originally suggested by Agrup et al. (1982) based on biochemical findings. We now turn to a more detailed discussion of evidence supporting this casing model, and then address its potential implications on proposed links between melanosome composition, oxidative stress, and epidemiological data.

image

Figure 8.  Casing model for mixed melanogenesis (Ito and Wakamatsu, 2008). Note that in the process of mixed melanogenesis, pheomelanic pigment is produced first, followed by the deposit of eumelanic pigment. In the granule with the eumelanin surface, the side was intentionally cut away to reveal the inner pheomelanin core. Eumelanin is believed to act as a photoprotective anti-oxidant while pheomelanin as a phototoxic pro-oxidant. (Figure used with permission, Blackwell Publishing.)

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Direct evidence for the casing model

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

Photoemission electron microscopy – a unique, surface-sensitive imaging technique able to spatially distinguish, with sub-100 nanometer resolution – can be applied to study the surface properties of biological biomaterials (Peles and Simon, 2009). Using this approach, the natural melanin composition on the surface of melanosomes, formed in vivo, can be determined. Such studies enable a way of testing the connection between the in vitro observations that undergrid the casing model structure that results from mixed melanogenesis and the properties of naturally occurring melanosomes that contain varying amounts of the two pigments, pheomelanin and eumelanin. Taking advantage of the fact that eumelanosomes and pheomelanosomes have different oxidation potentials (Ye et al., 2006), this methodology then offers a unique approach to determine the composition (eumelanin/pheomelanin) on the surface of intact melanosomes.

Wakamatsu et al. recently reported the eumelanin:pheomelanin ratios for uveal melanocytes isolated from eyes of varying color (Wakamatsu et al., 2008). In a collaborative effort between our groups, a variety of spatial imaging studies of the melanosomes isolated from the stroma of blue-green (hazel) and dark brown irides, which are characterized by eumelanin:pheomelanin ratios of 1.3 and 14.8, respectively, were performed (Peles et al., 2009).

Scanning spatial microscopies provide insight into the size and surface morphology of iridal stroma melanosomes (Figure 9). Electron microscopy reveals these melanosomes are, ovoid shaped with width and length dimensions of 0.26 × 0.56 μm, respectively (Peles et al., 2009). A statistical size and shape analysis of the electron images revealed morphology consistent with previously reported dimensions for these melanosomes as determined by transmission electron microscopy (Hu et al., 1992, 1993). A more detailed view of the surface of these melanosomes is obtained using atomic force microscopy (AFM). Atomic force microscopy images reveal that the surface of the iridal stroma melanosomes is comprised of smaller substructures. This result is to be expected based on previous studies of naturally occurring pigments isolated from various sources (Clancy and Simon, 2001; Liu and Simon, 2003). Force microscopy images of melanosomes generally reveal smaller substructures with lateral dimensions of a few 10s of nm. These results are additionally consistent with the view of melanogenesis proposed by Brumbaugh in the late 1960′s (Brumbaugh, 1968). In that report, Brumbaugh showed that eumelanin premelanosomes comprise zigzagging longitudinal strands with crosslinks that occur every 20 nm. Further analysis of the electron micrographs during the final stages of melanogenesis revealed melanin deposition occurring around and upon these matrices.

image

Figure 9.  Top: SEM images of dark brown iris stroma melanosomes. Bottom: AFM phase images of dark brown iridal stroma melanosomes. The small substructures of the iridal stroma melanosomes are clearly observed. (Peles et al., 2009.)

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Based on these spatial imaging studies, application of the photoemission electron microscopy would actually be probing the surface of the substructures of the melanosome that sit on its surface. The photoemission image only reveals the presence of eumelanin on the surface of the melanosome. Thus, in spite of the varying eumelanin:pheomelanin ratios for melanosomes from different colored irides, these intact melanosomes exhibit a eumelanin exterior. Using the eumelanin:pheomelanin ratios from the chemical analysis, conclusions can be drawn about the variation of the thickness of the eumelanic coats for melanosomes from different colored irides. Specifically, the eumelanin exterior is 2.48 times thicker for dark brown irides than hazel irides. If we model the substructure of the melanosomes as spheres of diameter 30 nm, the eumelanic exterior would be ∼9 nm and 3.6 nm thick for the dark brown and blue-green iridal melanosome, respectively; and the corresponding diameter of the pheomelanin cores would be ∼12 and 22.8 nm, respectively. These studies are the first to directly observe structural morphology consistent with the predictions of the three step pathway for mixed melanogenesis.

The similarity in organization of neuromelanin granules and iridal stroma melanosomes, both the products of mixed melanogenesis, suggests that the morphological consequence of mixed melanogenesis may be independent of tissue type. In a related study, the ‘casing’ model was shown to be consistent with the structure of neuromelanin granules isolated from various regions of the human brain, even though this pigment differs from those described so far (Bush et al., 2006; Ito, 2006). Specifically, neuromelanin, a dark pigment present in the substantia nigra and some other regions of the brain, consists of dopamine with some incorporation of cysteinyldopamine (Wakamatsu et al., 2003; Zecca et al., 2008; Zucca et al., 2004). Thus, during neuromelanin formation, which is another example of mixed melanogenesis, the chemistry of dopaminequinone appears to play an even more important role compared with dopaquinone because of the slower cyclization of the former. The rate of cyclization of dopaminequinone to cyclodopamine is reported to be 0.37/s (El-Ayaan et al., 1998; Jameson et al., 2004), which is 10-fold slower than the corresponding reaction of dopaquinone. Thus, mixed melanogenesis in neuromelanin formation also needs to be studied further. It should be noted that the biological origin, location and temporal scale of formation of neuromelanin are quite different from those of epidermal and ocular melanin. However, the detailed description of the differences is not within the scope of this review.

Implications of the casing model: inducing and mitigating photo-oxidative stress

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

It is of interest to consider the implications of the casing model with respect to iridal melanosomes and the epidemiology of iridal melanoma. A dark-colored iris with lower relative pheomelanin concentration (high eumelanin:pheomelanin ratio) will have a thick eumelanic exterior. Therefore, even after any oxidative damage to the surface pigment, there is still a large amount of eumelanin that can be protective against oxidative stress. However, in melanosomes from light-colored irides, oxidative damage of the eumelanic surface could expose pheomelanin, which can lead to additional oxidation stress. Such a possibility is consistent with the incidence of uveal melanoma in different races and in eyes from different colored irides. Specifically, epidemiological studies on the relationship between iridal color and incidence of uveal melanoma suggest that the light-colored eye (blue, hazel, etc.) and Caucasian is at a higher risk than the dark-colored eye and African-Asians (Hu et al., 2005; Vajdic et al., 2001). Subsequent exposure of the photoreactive and pro-oxidant pheomelanin coupled to the lower total amount of melanin in lighter-colored irides could therefore be a contributor to the observed epidemiology of uveal melanoma.

This has interesting implications in considering the connection between melanin and melanoma. Studies of eumelanin and pheomelanin content of epidermal melanocytes from different donors indicate that the eumelanin:pheomelanin ratio correlates with the color of the skin and the ethnic background of the donors. Melanocytes from dark-colored skin and African-American donors have a greater amount of eumelanin and a high ratio of eumelanin:pheomelanin as compared with lighter colored skin and Caucasian donors (Wakamatsu et al., 2006). A recent study on the eumelanin and pheomelanin content in uveal melanoma cells found that melanoma cells have a very low eumelanin content and a eumelanin:pheomelanin ratio significantly lower than that from normal melanocytes (Hu et al., 2009). These differences likely render melanoma cells more susceptible to mutagenic effects of UV radiation and oxidative stress and may enhance their proliferation thereby accelerating the progression of melanoma. Epidemiological studies indicate that the incidence of cutaneous melanoma in individuals with light-colored skin is greater than that from individuals with dark-colored skin (Cress and Holly, 1997; Tsai et al., 2005). If the melanosomes in epidermal melanocytes have a similar ‘casing’ structure, then the different incidence of cutaneous melanoma in individuals with various colored skin and the progress of uveal melanoma could be explained, at least partly, by the structure of melanosomes and the different thickness of eumelanic coats.

Future directions

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References

The above discussion reveals that recent research efforts have resulted in significant progress in the understanding of melanogenesis, from the molecular level to the organization of pigments within the intact melanosome. However, several important and challenging issues remain to be resolved. First consider the nature of post-polymerization modifications of eumelanin and pheomelanin. This issue had been addressed only sporadically (Chedekel et al., 1987; Crescenzi et al., 1993; : Ozeki et al., 1997b), and is also relevant to clarifying the biodegradation of melanin and melanosomes (Borovansky and Elleder, 2003; Sarna et al., 2003). An interesting observation related to this is the marked difference in color and intensity among natural pheomelanic pigments, such as seen in red hair color in humans and yellow coat color in mice (Ozeki et al., 1996), as pointed out in our recent review (Ito and Wakamatsu, 2008). Two recent studies (Greco et al., 2009; Wakamatsu et al., 2009) have addressed the issue of post-polymerization modification of pheomelanin and have made much progress. The difference in the color of pheomelanic pigments might be related to the difference in post-polymerization modification of pheomelanin structure and also to the presence of a large proportion of eumelanin in human pheomelanic hair (Naysmith et al., 2004; Ozeki et al., 1996).

A second and equally important unsolved problem is the nature of melanins in mixed (eumelanin/pheomelanin) melanosomes. Are the two types of melanins present in their pure form or do they co-polymerize, creating molecular building blocks that possess elements of the structures of the two pure melanin? No study to date addresses these issues.

There are also important issues around the structure of pheomelanin itself and its post-polymerization modifications. It should be mentioned that we have not unambiguously established how the isoquinoline moiety (structure 1) is produced in the late stages of pheomelanogenesis. Pyridine-2,3,4,6-tetracarboxylic acid (structure 2) serves to indicate the participation of isoquinoline units in pheomelanin structure (Fattorusso et al., 1969). Unfortunately, this useful degradation product has not been studied since this original work. When we consider the possibility that the isoquinoline moiety may be of equal importance to the benzothiazine and benzothiazole species in defining the molecular structure of pheomelanin, the lack of information on the content (or even the presence) of isoquinoline moiety is a serious drawback. Another issue that remains unsolved in the post-polymerization modifications is how the benzothiazine (BT or BTCA) moiety is converted to the benzothiazole (BZ) moiety.

Another issue that deserves more attention is the effects of acidic pH on mixed melanogenesis. While melanosomes appear to differ greatly in pH depending on the ethnic origin (Smith et al., 2004), no systemic studies have been performed on the effects of acidic pH in controlling mixed melanogenesis. A recent study has shown that the activation of the cAMP pathway by α-MSH or forskolin leads to an alkalization (neutralization) of melanosomes and a fourfold increase in melanin content (Cheli et al., 2009). Although this study links the pH of melanosomes to the cAMP pathway for the first time, mixed melanogenesis was not examined.

We have also not yet identified the molecular structural unit(s) in pheomelanin responsible for the different photophysical properties exhibited by the pigment (Ye et al., 2008). The most important of these properties is the ability of pheomelanin to activate oxygen (Chedekel et al., 1978), resulting in the formation of the superoxide radical anion. The action spectrum for oxygen activation by pheomelanin isolated from red human hair (Chedekel et al.,1980) reveals a significant onset of activity at ∼280 nm. However, the melanin sample was isolated by harsh acid/base techniques used at that time, which have subsequently been shown to significantly alter the chemical and morphology of melanins (Liu et al., 2003, 2005). Using enzymatic extraction, the action spectrum for oxygen consumption by photoexcited pheomelanin shows a clear increase between 338 and 323 nm, consistent with the measurement of the photoionization threshold of 326 nm (Ye et al., 2006). This wavelength range does not correspond to the lowest energy absorption peak exhibited by DHBTCA, ODHBT, and BZ (λmax 306, 296, and 304 nm, respectively; Wakamatsu et al., 2009; see also Napolitano et al., 2008). Further work is clearly required to identify the molecular chromophore responsible for the oxygen activation by pheomelanin.

It is clear that the biological functions of melanin pigments are closely related to their structural features. In this review, we have shown how the process of mixed melanogenesis can be interpreted in terms of chemistry. However, several important issues remain to be solved through multi-disciplinary approaches. One of those important unsolved problems is the precise biochemical mechanism of switching between eumelanogenesis and pheomelanogenesis in vivo, which appears to depend not only on tyrosinase activity but also on the availability of cysteine (or cystine) in melanosomes. A recent study suggests the presence of another factor(s) controlling pheomelanogenesis (Hida et al., 2009). In this connection, the role of the recently identified cystine transporter Slc7a11 (Chintala et al., 2005) in regulating mixed melanogenesis deserves more attention.

Finally, the assembly of the pigment within the intact melanosome remains unknown. Our insight into this important issue is likely to remain at a very qualitative level until there is a better understanding of the molecular structures of melanins.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Molecular foundations of eumelanin and pheomelanin
  5. Dopaquinone plays a pivotal role in the formation of eumelanin and pheomelanin
  6. Insights from the comparison of synthetic and natural melanins
  7. Molecular structure of eumelanin beyond the monomers DHI and DHICA
  8. Molecular structure of pheomelanin beyond the cysteinyldopa monomers: converting benzothiazines to benzothiazoles in pheomelanin
  9. A three step pathway for mixed melanogenesis
  10. Biochemical pathways for mixed melanogenesis
  11. Direct evidence for the casing model
  12. Implications of the casing model: inducing and mitigating photo-oxidative stress
  13. Future directions
  14. Acknowledgements
  15. References