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Isolation, characterization and assessment of the photoprotective effect of two fungal melanins Olaizola, Carolina 2012

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ISOLATION, CHARACTERIZATION AND ASSESSMENT OF THE PHOTOPROTECTIVE EFFECT OF TWO FUNGAL MELANINS  by  Carolina Olaizola  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies  (Plant Science)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  November 2012  ®Carolina Olaizola, 2012  Abstract Melanins are a diverse group of pigmented biopolymers present in living organisms at all phylogenetic levels. Properties such as light scattering and absorption through a wide range of wavelengths, and scavenging of free radicals are common to all melanins, and render them as molecules having the capacity to protect against UV damage. Fungal melanins are more chemically diverse than animal melanin, and have radioprotective properties. In this research, I isolated melanins from the basidiomycete Agaricus bisporus and the ascomycete Grosmannia clavigera, and assessed their photoprotective effect in human skin cells. Both fungal melanins differed in physicochemical characteristics and functional groups evidenced by infrared spectral analyses. Differences in the evaluated bioactivity were also observed. Agaricus bisporus melanin behaved similar to synthetic DOPA melanin, significantly enhancing cell viability measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in human dermal fibroblasts irradiated with a high dose of ultraviolet B (UVB). Agaricus bisporus melanin also effectively reduced the amount of reactive oxygen species (ROS) induced by UVB in these cells. Measurements of light transmission at 315 nm of melanin solutions suggest that the observed photoprotective effect was likely to be due to a UV filter effect. No photoprotection was observed in cells exposed to UVB in the presence of Grosmannia clavigera melanin. Unlike synthetic DOPA melanin, both fungal melanins were potentially cytotoxic for human dermal fibroblasts. My results suggest that differences observed in the fungal melanin bioactivity are possibly linked to differences in their chemistry and optical properties.  	
    ii	
    Table of Contents Abstract  ii  Table of Contents  iii  List of Tables  v  List of Figures  vi  List of Abbreviations  viii  Acknowledgements  xi  Dedication  xii  Chapter 1: Background and Research Goals 1.1 Introduction 1.2 Review of literature 1.2.1 Melanins 1.2.2 Melanin in fungi 1.2.3 Melanin in animals 1.2.3.1 Cutaneous melanin 1.2.4 Proposed functions for melanins 1.3 Rationale and research objectives Chapter 2: Isolation and Characterization of Fungal Melanins 2.1 Introduction 2.2 Materials and methods 2.2.1 Chemicals and reagents 2.2.2 Fungal material 2.2.3 Melanin extraction 2.2.4 Melanin purification 2.2.5 Identification and analysis of melanins 2.2.5.1 Chemical tests 2.2.5.2 UV-visible and IR spectroscopy 2.2.5.3 NMR spectroscopy 2.3 Results 2.3.1 Chemical identification 2.3.2 UV-visible and IR spectra 2.3.3 NMR spectra 2.4 Discussion 2.5 Conclusions Chapter 3: In vitro Assessment of Cytotocixity and Photoprotection of Fungal Melanins to Human Skin Cells 3.1 Introduction 3.2 Materials and methods 3.2.1 Materials 3.2.2 Samples preparation 3.2.3 Cell culture  1 1 2 2 8 9 10 12 16 18 18 21 21 21 22 22 23 23 24 24 25 25 26 29 30 35  	
    36 36 39 39 39 40 iii	
    3.2.4 UVB irradiation 3.2.5 Evaluation of cytotoxicity of melanins 3.2.6 Evaluation of UVB phototoxicity 3.2.7 Evaluation of the effect of melanins on UVB-induced phototoxicity 3.2.8 Evaluation of the effect of melanins on UVB-induced ROS 3.2.9 Measurement of light transmission by solutions of melanins 3.2.10 Data analysis 3.3 Results 3.3.1 Effect of melanins on HDF proliferation 3.3.2 Effect of UVB dose on HDF viability 3.3.3 Effects of melanins in UVB-induced phototoxicity in HDF 3.3.4 Effects of melanins in ROS production by UVB-irradiated HDF 3.3.5 Transmission of light by melanin solutions in HBSS/20mM HEPES buffer 3.4 Discussion 3.5 Conclusions Chapter 4: General Conclusions and Suggestions for Future Research Bibliography Appendix I: 1H NMR spectrum of G. clavigera melanin Appendix II: 13C NMR spectrum of G. clavigera melanin Appendix III: Biosynthetic pathways of melanins  	
    41 41 42 43 44 46 46 46 46 47 49 51 52 52 57 59 63 78 85 96  iv	
    List of Tables Table	
  2.1	
    Chemical	
  tests	
  used	
  to	
  identify	
  melanins	
   	
    Table	
  3.1	
    Protocols performed with melanins and HDF irradiated with a phototoxic dose of UVB  44  Concentrations at which melanins showed significant bioactivity in HDF  57  Table	
  3.2	
    	
    	
    	
    	
    25	
    v	
    List of Figures Figure 2.1  Chemical tests used to identify melanins  26  Figure 2.2  UV-visible spectra of melanins  27  Figure 2.3  IR spectrum of A. bisporus melanin  28  Figure 2.4  IR spectrum of G. clavigera melanin  29  Figure 3.1  Effect of melanins in HDF proliferation  47  Figure 3.2  Cell viability of HDF exposed to different irradiation intensities of UVB measured after 24 and 48 h  48  Cell viability of HDF exposed to different intensities of UVB covered or uncovered with Mylar film  49  Cell viability of HDF irradiated with 800 mJ/cm2 of UVB in presence of melanins  50  Reduction of UV-induced reactive oxygen species (ROS) in HDF irradiated in buffer containing melanins  51  Figure 3.6  Transmission (%) of light at 315 nm by melanin solutions  52  Figure A-I.1  1  H NMR spectrum  78  Figure A-I.2  1  H NMR spectrum, zoom of figure A-I.1  79  Figure A-I.3  1  Figure 3.3 Figure 3.4 Figure 3.5  Figure A-I.4 Figure A-I.5 Figure A-I.6 Figure A-I.7  Figure A-II.1  	
    H NMR spectrum, frequency expansion in the region from 0.50 to 1.00 ppm  80  1  H NMR spectrum, frequency expansion in the region from 1.10 to 1.60 ppm  81  1  H NMR spectrum, frequency expansion in the region from 1.85 to 2.40 ppm  82  1  H NMR spectrum, frequency expansion in the region from 2.5 to 4.9 ppm  83  1  H NMR spectrum, frequency expansion in the region from 4.8 to 6.0 ppm 13  C NMR spectrum  84 85  vi	
    Figure A-II.2  13  C NMR spectrum, frequency expansion in the region  from 13.1 to 15.0 ppm Figure A-II.3  13  C NMR spectrum, frequency expansion in the region  from 21.2 to 23.1 ppm Figure A-II.4  13  13  13  13  13  13  13  13  93  C NMR spectrum, frequency expansion in the region  from 129.4 to 131.6 ppm Figure A-II.11  92  C NMR spectrum, frequency expansion in the region  from 126.4 to 129.2 ppm Figure A-II.10  91  C NMR spectrum, frequency expansion in the region  from 33.2 to 34.6 ppm Figure A-II.9  90  C NMR spectrum, frequency expansion in the region  from 30.6 to 32.0 ppm Figure A-II.8  89  C NMR spectrum, frequency expansion in the region  from 28.1 to 29.5 ppm Figure A-II.7  88  C NMR spectrum, frequency expansion in the region  from 25.9 to 27.6 ppm Figure A-II.6  87  C NMR spectrum, frequency expansion in the region  from 23.8 to 25.6 ppm Figure A-II.5  86  94  C NMR spectrum, frequency expansion in the region  from 173.5 to 176.5 ppm  95  Figure A-III.1 Schematic pathway of DOPA melanin biosynthesis  96  Figure A-III.2 Schematic pathway of GHB melanin biosynthesis  97	
    Figure A-III.3 Schematic pathway of DHN melanin biosynthesis  98  	
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    List of Abbreviations ANOVA  analysis of variance  ATR-FTIR  attenuated total reflectance fourier transform infrared spectroscopy  BC  British Columbia  C  carbon  CM-H2DCFDA  5-(and-6)-chloromethyl-2',7’-dichlorodihydrofluorescein diacetate, acetyl ester  13  C NMR  carbon-13 nuclear magnetic resonance  CO2  carbon dioxide  DHN  1,8-dihydroxynaphthalene  DMSO  dimethyl sulfoxide  DNA  deoxyribonucleic acid  DOPA  dihydroxyphenylalanine  EDTA  ethylenediaminetetraacetic acid  ELISA  enzyme-linked immunosorbent assay  EPR  electron paramagnetic resonance  ESCA  electron spectroscopy for chemical analysis  ESR  electron spin resonance  g  relative centrifugal force  GHB  γ-glutaminyl-4-hydroxybenzene  FeCl3  ferric chloride  FTIR  fourier transform infrared spectroscopy  	
    viii	
    H2 O2  hydrogen peroxide  HCl  hydrochloric acid  HBSS  Hanks’ Balanced Salt Solution  HDF  human dermal fibroblasts  HEPES  4-(2-hydroxy- ethyl)-1-piperazine ethane sulphonic acid  1  proton nuclear magnetic resonance  H NMR  HPLC  high-performance liquid chromatography  IC50  50% inhibitory concentration  IPA  isopropyl alcohol  IR  infrared  KOH  potassium hydroxide  LSD  laser desorption/ionization  LSGS  low serum growth supplement  M  molar  MALDI  matrix assisted laser desorption/ionization  MHz  megahertz  mJ/cm2  milijoules per square centimeter  MTT  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide  N  nitrogen  NaOCl  sodium hypochlorite  nm  nanometer  NMR  nuclear magnetic resonance  	
    ix	
    O  oxygen  OD  optical density  OH  hydroxyl  p53  tumor protein 53  PBS  phosphate buffered saline  pH  potential of hydrogen  ppm  parts-per-million  ROS  reactive oxygen species  SD  standard deviation  UBC  University of British Columbia  UK  United Kingdom  US  United States  UV  ultraviolet  UVA  ultraviolet A  UVB  ultraviolet B  XP  xeroderma pigmentosum  XPS  X-ray photoelectron spectroscopy  µM  micromolar  	
    x	
    Acknowledgements I would like to express my deepest gratitude to my supervisor, Associate Professor Dr. Eduardo Jovel, who gave me the opportunity to embark on research that has fulfilled my dreams. His financial, intellectual and emotional support were crucial to the completion of my Masters Program. I would also like to sincerely thank Dr. Tanya Wahbe for her support and helpful advice with the writing of my thesis. I appreciate the valuable friendship from both. A huge thanks to Research Associate, Zyta Abramowski, who was not only a mentor on my learning path since the day I arrived, but who also offered me her unconditional friendship and support. Thanks also to my committee members, Dr. Karen Bartlett and Dr. Colette Breuil who guided me with useful advice during committee meetings. I am really grateful to Dr. Colette Breuil and her staff, who provided me with one of the fungi. Dr. Breuil always gave her time, good advice and support whenever I asked. I thank Dr. John Kadla and Dr. Jennifer Braun for providing the IR spectra of my samples. I also feel grateful to the NMR facilities staff at the UBC Chemistry Department. A special thanks to my former chemistry professor and now dear friend, Dr. Nuri Rivero, who from Venezuela was able to guide me and get me out of ‘quagmire’ many times during my research. She also assisted with the interpretation of IR and NMR spectra. And finally, I would like to mention many other people, hoping not to forget anyone, who in one way or another offered great help during this journey: Pablo Cubeddu, Sylwester Czaplicki, Antonio Flores, Ranganayaki Nandanavanam, Anusha Samaranayaka, Agnieszka Stokloska, Greg Stopps, Selvarani Vimalanathan, Ye Wang and Xin Xin Xue.	
  	
    	
    xi	
    Dedication This thesis is especially dedicated to God, to my family and to Juan Carlos. Without all of you, I would not be here today.  	
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    Chapter 1: Background and Research Goals 1.1. Introduction Nearly all living organisms are subjected daily to the effects of ultraviolet (UV) radiation, primarily ultraviolet B (UVB) and ultraviolet A (UVA), that reaches the surface of the earth. UV radiation has a range of effects in biological systems, some of them harmful, especially at the DNA level. Skin cancer, photoaging and immunosuppression are among the main undesirable effects connected to UV exposure in humans (Matsumura & Ananthaswamy, 2004). The search for photoprotective compounds that provide sufficient UV protection and are safe for human use is an important step to help minimize skin cancer development. This is particularly important due to increased UV radiation reaching the Earth’s surface and the increase in migration of fair skinned populations to areas with high ambient UV radiation (Lucas et al., 2006). When properly applied, sunscreens have proven useful in the prevention of acute signs of sunburn (Gasparro, 2000). However, it seems that at some minimal erythema doses (the threshold dose to induce erythema 24 h after UV irradiation) they might not fully protect against DNA damage induced by UV radiation (Liardet et al., 2001; Pinnel, 2003). Another issue is that sunscreen products commonly use a combination of chemical filters, as no single compound has such a broad UV spectrum to provide enough protection, and this increases the possibility of side effects (Moloney et al., 2002). Through evolutionary adaptation, many organisms exposed to UV radiation for the majority of their lives have developed ways to diminish its negative effects. One of the most ubiquitous of these mechanisms is the synthesis of screening compounds that filter UV (Cockell & Knowland, 1999). A compound’s absorbance will depend on its structural characteristics and, in most cases, larger molecules will absorb longer wavelengths (Cockell & Knowland, 1999). In general, aromatic compounds are considered better UV screeners as their maximum absorption is 	
    1	
    usually within the UV range, and their efficacy increases with the number of substituents they possess (Cockell & Knowland, 1999). Among these compounds are melanins, which are the focus of my research. They are considered the most widespread and primitive of pigments present in living organisms, at all phylogenetic levels (Barr, 1983). In reality, the term melanin is used to encompass a group of chemically diverse pigments (Menon & Haberman, 1977) having complex polymeric structures with aromatic rings making them non-selective UVB and UVA screeners (Cockell & Knowland, 1999). Melanins have an unusual broadband absorption that increases towards the UV region of the spectrum (Riesz, 2007), and a stable free radical character that allows them to act as a sink for other free radicals (Bell & Wheeler, 1986). Although many roles have been attributed to melanic pigments, the main role in nature seems to be protection from UV radiation damage (Kollias, et al., 1991; Mosse & Marozik, 2008; Riley, 1997). In fungi, melanins are chemically more diverse than in animals and confer radioresistance against γ-radiation and X-rays (Bell & Wheeler, 1986; Dadachova et al., 2007; Dadachova & Casadevall, 2008). The possibilities for potential applications of fungal melanins in humans have been little explored. My goal in this research was to isolate and analyze two fungal melanins and evaluate their photoprotective effects against UVB in human skin cells. Through the remainder of this chapter, I review the literature concerned with melanin that might help put my research in context. At the end of this chapter, I define specific objectives I selected to achieve my research goal. 1.2  Review of literature 1.2.1 Melanins Melanins are irregular tri-dimensional macromolecules composed of phenolic or indolic  monomers that combine randomly to form a polymer (Butler & Day, 1998; Henson et al., 1999; 	
    2	
    Nicolaus, 1968). The monomeric units possibly are linked by several types of non-hydrolyzable bonds (Menon & Haberman, 1977; Prota, 1992). Melanins are heterogeneous and even when the starting substrate could be the same, the products may be different because polymerization patterns vary depending on biological or chemical conditions (Nicolaus, 1968). All have the basic structure of an aromatic ring, usually mixtures of quinone, hydroquinone and semiquinone moieties, complexed with proteins, carbohydrates and lipids (Alviano et al., 1991; Bell & Wheeler, 1986). The protein component attached is apparently a significant part of the molecule and might be essential for its biological activity (Fitzpatrick et al., 1967; Follman, 1973; Nicolaus, 1968). Melanin pigments are very stable regardless of temperature (some can resist up to 600ºC without decomposing), light, reducers and organic acids (Nicolaus, 1968; Wang et al., 2006). Their molecular weight is presumably high and there is a wide range in estimated values for pigments obtained from different sources (Aghanjanyan et al., 2005; Lea, 1947; Prota, 1992). Large amounts of water are bound to the melanin molecule (30% of its weight in some cases) and are probably important in the maintenance of its solvent-swollen state. Once completely dehydrated, the polymer becomes more aggregated and almost completely loses the capacity for physicochemical interactions (Nicolaus, 1968; Prota, 1992). In nature, they exist primarily as non-porous spherical particles (around 30 nm in diameter) that tend to join together as aggregates. These aggregates will then associate in loose clusters of melanin agglomerates (Kollias et al., 1991). Depending on the precursor and synthetic pathway, several types of melanin can be recognized:  	
    3	
    3,4-dihydroxyphenylalanine (DOPA) melanin This is the main type of melanin found in animals, but some fungi and bacteria also synthesize melanin from DOPA (Butler & Day, 1998). The precursor DOPA utilized for melanin biosynthesis can be derived directly from the hydroxylation of the amino acid L-tyrosine or from L-phenylalanine which in turn is hydroxylated to L-tyrosine and then converted to DOPA. The first steps in melanin synthesis that include the oxidation of tyrosine to form DOPA and then dopaquinone are catalyzed by the enzyme tyrosinase (Prota, 1992). Apparently some fungi can convert DOPA to melanin not only by the action of tyrosinases, but also through laccases, polyphenol-oxidases, peroxidases, catalases, or by spontaneous oxidation without any enzymatic intervention (Butler & Day, 1998; Jacobson, 2000). Catechol melanin In catechol melanin, the polymer is made up of catechol units linked by C-C and C-O-C bonds (Nicolaus, 1968). The complete synthetic pathway is not clear. This type of melanin has been found in teliospores of the fungus Ustilago maydis and the bacteria Azotobacter chroococcum (Butler & Day, 1998; Piatelli et al., 1965). Glutaminyl-4-hydroxybenzene (GHB) melanin GHB is considered the precursor of melanin in basidiospores of the mushroom Agaricus bisporus (Hegnauer et al., 1985; Rast el al., 1981), and possibly other basidiomycetes synthesize melanin from this same precursor (Bell & Wheeler, 1986; Butler & Day, 1998). In the proposed pathway, γ-glutaminyl-4-hydroxybenzene (GHB), derived from an intermediate of the shikimatechorismate pathway, is hydroxylated to γ-glutaminyl-3,4-dihydroxybenzene (GDHB). GDHB is then oxidized giving rise to γ-glutaminyl-3,4-benzoquinone (GBQ). A non-enzymatic cleavage of the γ-glutaminyl residue results in hydroxy-benzoquinone-imine, which is very reactive and  	
    4	
    prone to polymerize. Phenolase and peroxidase enzymes might be involved in its synthesis (Butler & Day, 1998; Prota, 1992; Rast et al., 1981). 1,8-dihydroxynaphthalene (DHN) melanin DHN melanin is found in ascomycetous fungi. Its synthesis starts with polyketides, which consist of simple units of acetate, propionate or butyrate that are conjoined head to tail by the enzyme polyketide synthase and then cyclized. After the polyketide cyclization, 1,3,6,8tetrahydroxynaphtalene (1,3,6,8-THN) is formed. A series of reduction and dehydration reactions produces the monomeric precursor 1,8-dihydroxynaphthalene (1,8-DHN). Its oxidation and polymerization, presumably with the enzymatic intervention of cell wall laccases or other oxidases, give rise to melanin (Butler & Day, 1998; Henson et al., 1999). DHN melanins have different colors based on differences in the polymer structure, size, crosslinking, oxidation state, cellular location and presence of other cellular components that might be attached to it (Henson et al., 1999). The term ‘synthetic melanin’ is used for melanins obtained in vitro either through: a) incubation of the monomeric melanin precursors with mushroom polyphenol oxidase, b) autoxidation by acidification of dark alkaline solutions of DOPA, or c) through persulfide or peroxide oxidation of tyrosine. Commercially available synthetic melanins are produced following the latter method (Kollias et al., 1991). Synthetic melanins are commonly used as a ‘standard’ to compare natural melanins. Although usually dark, the color of melanin varies from black to red to yellow (Nicolaus, 1968). This diversity in colors and hues results from light absorption and scattering properties and as a general rule, the smaller the size of the melanin granules the lighter is the pigment (Prota, 1992). Light absorption by melanins has no characteristic maximum and extends throughout the UV-visible and part of the infrared spectra, but it is stronger at shorter 	
    5	
    wavelengths (Anderson & Parrish, 1981; Riesz, 2007; Riley, 1997). In general, melanins are dark because they do not re-radiate the absorbed visible or invisible light, but transform the energy into rotational and vibrational activity within the molecule and then dissipate it as heat. Similarly, it is believed that these pigments may trap energy from other electronically excited molecules and convert it into heat (McGinness & Proctor, 1973). This property has been linked to a process known as photon-phonon conversion or coupling (McGinness & Proctor, 1973; Riley, 1997). This is the common mechanism explaining how melanins protect cells from biologically harmful quanta, free radicals and electronically excited molecules (Butler & Day, 1998). Additionally, melanins seem to be the only biopolymers exhibiting high concentrations of free radical centers in vitro and in vivo (Mason et al., 1960; Prota, 1992), and they can act as a ‘sponge’ for free radicals (Bell & Wheeler, 1986). Transient free radicals can be induced in melanin by UV or visible light irradiation (Blois et al., 1964; Sarna, 1992; Sarna & Plonka, 2005). An accurate definition of the structure and dimension of any melanin does not exist (Nosanchuk & Casadevall, 2003). The heterogeneity and irregularity of the molecule, and its intractability and insolubility in most solvents, are considered to be major barriers to the precise characterization of melanin using conventional biochemical and biophysical methods (Duff et al., 1988; Nicolaus, 1968). Due to these challenges, there is near agreement to use certain general chemical characteristics as diagnostic criteria to identify, at least primarily, a dark pigmented compound as melanin. These characteristics include: •  resistance to solvents, being insoluble in cold or boiling water, mineral acids and the usual organic solvents;  •  bleaching when subjected to the action of oxidizing agents, such as hydrogen peroxide (H2O2) or sodium hypochlorite (NaOCl);  	
    6	
    •  resistance to degradation by hot or cold concentrated acids;  •  capacity of directly reducing ammoniacal solutions of silver nitrate;  •  solubilization (almost always) in aqueous alkali; and  •  positive reaction for polyphenols (Butler & Day, 1998; Harki et al., 1997; Nicolaus et al., 1964; Prota, 1992). Methods employed to partially characterize melanins include: a) Electron Paramagnetic  Resonance (EPR) and Electron Spin Resonance (ESR), which show a population of stable free radicals in these molecules (Enochs et al., 1993; Prota, 1992); b) infrared (IR) spectroscopy, which provides information about the functional groups that compose the pigment and allows comparison between synthetic and natural melanins derived from the same presumed precursor (Bilińska, 1996; Pierce & Rast, 1995); c) solid-state nuclear magnetic resonance spectroscopy (NMR) (Tian et al., 2003); d) X-ray photoelectron spectroscopy (XPS), also known as electron spectroscopy for chemical analysis (ESCA), which has helped to define the chemical natures of the nitrogen and sulfur atoms (Williams-Smith & Dunne, 1976); e) X-ray diffraction, which revealed a lack of crystalline structure (Spiegel-Adolf & Henny, 1939; Thathachari & Blois, 1969); and more recently, f) mass spectrometry coupled with fast atom bombardment, laser desorption/ionization (LDI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry, which showed that the darker a melanin, the higher its molecular mass, possibly due to a higher degree of polymerization of the molecule (Bertazzo et al., 1995). Chemical or thermal degradation using drastic procedures that produce extensive breakdown of the pigment, followed by the identification of the end products (e.g., by HPLC or gas chromatography coupled with mass spectrometry) have been used to identify the monomeric units (Ito & Wakamatsu, 1998; Latocha et al., 2000; Prota, 1992; Saiz-Jimenez et al., 1995). Biosynthetic studies using the incorporation of labeled precursors sometimes in conjunction with 	
    7	
    solid-state NMR (Tian et al., 2003; Zhong et al., 2008) have revealed additional structural information. 1.2.2 Melanin in fungi The structural diversity of fungal melanins is considered to be much higher than that of animal melanins (Lüdemann et al., 1982; Rast et al., 1981). Fungi can synthesize melanin from DOPA, DHN, GHB, catechol and possibly from other phenols (Butler & Day, 1998; Rast et al., 1981). The origin of the aromatic nucleus or precursor is not always clear, but in some cases the shikimate-chorismate and polyketide pathways have been suggested (Rast et al., 1981). Enzymatic inhibitors of the presumed synthetic pathway or studies of albino mutants are employed in some cases to characterize melanin in fungi (Henson et al., 1999; Suryanarayanan et al., 2004). Melanin is considered a secondary metabolite in fungi and its presence can be a constitutive character or one that is only expressed after a trigger factor is introduced (Henson et al., 1999). The synthesis of melanin in response to diverse environmental stresses led to the belief that melanin production might be a protective response of cells (Henson et al., 1999). Sometimes melanogenesis is activated only at certain developmental stages like sporogenesis, ageing of hyphae and sclerotia formation (Hegnauer et al., 1985). Melanin may be present in fungal structures such as the appressoria or reproductive tissue, while being completely absent elsewhere (Henson et al., 1999). The spores have the highest capacity to produce and store melanin (Henson et al., 1999; Rast et al., 1981), which is believed to contribute to spore survival (Bell & Wheeler, 1986). Unlike animal melanins, which are formed intracellulary, fungal pigments are considered to be wall bound or extracellular in nature (Prota, 1992). In most melanized fungi the pigment is found primarily in the cell wall, localized in the outermost layer or embedded within the wall (as granules or layered in fibrils), or bound to cell wall chitin (Butler & Day, 1998). Observations based on electron microscopy have led some 	
    8	
    to suggest that fungal melanin exists also within subcellular compartments or intra-cytoplasmatic organelles, similar to animal melanosomes (Alviano et al., 1991; Rast et al., 1981; San-Blas et al., 1996). 1.2.3 Melanin in animals In animals, melanin can be found in the skin, hair, inner ear, pigmented epithelium of the uveal tract, the adrenal gland, leptomeninges, locus ceruleus and substantia nigra of the brain stem, bone, heart, adipose tissue, and the pulmonary and digestive tracts (Césarini, 1996; Brenner & Hearing, 2009). The main types are eumelanin (widely distributed in the animal kingdom) and pheomelanin (only found in mammals and birds) (Prota, 1992). Eumelanin is the most extensively studied of all melanins. Its color varies from brown to black and it is probably one of the most insoluble types of melanins (Slominski et al., 2004). It is a heterogeneous macromolecule composed of different ratios of two indole-quinones: 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxilic acid (DHICA) (Orlow et al., 1992; Prota, 1992; Slominski et al., 2004). Pheomelanin, on the other hand, varies in color from reddish-brown to yellow and it is more soluble in alkalis than eumelanin. It is composed of benzothiazine units and has highly variable nitrogen and sulfur content (Prota, 1992). Pheomelanins are considered more photoreactive and photolabile than eumelanins (Sarna & Plonka, 2005; Takeuchi et al., 2004). The term ‘mixed melanins’ usually refers to heteropolymerous melanins composed of a mixture of eu- and pheomelanin and having physical and analytical properties of both (Prota, 1992; Slominski et al., 2004). Pigments that might be byproducts of the synthesis of monoamine neurotransmitters (dopamine, norepinephrine and serotonine) in catecholaminergic neurons are known as neuromelanin. Its amount is higher in humans and in animals closely related to humans (Prota,  	
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    1992). Neuromelanin is brown to black in color, with mixed properties of eu- and pheomelanins (Barr, 1993; Slominski et al., 2004). In animals, melanin seems to have paradoxical effects: it scavenges free radicals but it can also produce them (Hubbard-Smith et al., 1992; Korytowski et al., 1986; Sarna & Plonka, 2005), it may have both photo protective and photosensitizing effects (Henson et al., 1999; Menon & Haberman, 1977; Takeuchi et al., 2004) and its ability to interact with a variety of other molecules such as proteins, carbohydrates, metal ions, pollutants and antibacterials could be either beneficial or detrimental (Henson et al., 1999; Sarna, 1992). Melanocytes are the archetypes of melanin-producing cells in animals. In cold-blooded vertebrates the name given to cells responsible for producing melanin is melanophores (Prota, 1992). Both melanocytes and melanophores derive from the neural crest. Cells with different ontogenic origins such as the Kupffer cells of the liver and the ink gland of cephalopods, also have been found to produce melanin (Prota, 1992). In melanocytes melanin synthesis naturally occurs within intracytoplasmatic organelles called melanosomes that also function as storage for melanin and can eventually be transferred from the melanocytes to other type of cells (Fitzpatrick et al., 1967; Jimbow et al., 1976). It has been proposed that melanogenesis takes place within melanosomes in animals because of a process involving highly reactive intermediates and free radicals that might otherwise damage cells (Slominski et al., 2004). 1.2.3.1 Cutaneous melanin The cutaneous melanocytes are considered the most metabolically active as they can proliferate and produce pigment throughout their lifespan. In contrast, extracutaneous melanocytes actively synthesize melanin only during embryonic differentiation. Thereafter its activity is typically negligible (Brenner & Hearing, 2009; Goding, 2007; Prota, 1992). This might be related to the importance that melanin pigmentation has for the skin. The skin is the 	
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    organ most exposed to the environment acting as an envelope for almost all the other organs and protecting them from external factors. Skin pigmentation by melanin might play an important role in UV protection, especially when we consider that some of the few structures of the body not covered by skin, such as the eyes and hair, also are pigmented in most cases. Melanin pigment in human skin is localized mainly in the epidermis, the most external structure of the skin, which is composed mainly of two type of cells: keratinocytes (representing ∼95% of the cell population) and melanocytes (Costin & Hearing, 2007). The cutaneous melanocytes are considered ‘incontinent’ cells as the melanin they produce is transferred to keratinocytes in the vicinity under the influence of several stimuli (Archambault et al., 1995; Quevedo, 1972). In normal human skin, eumelanin is the main pigment that cutaneous melanocytes synthetize, but certain ethnic groups (e.g., red-haired and freckled Caucasians) have small amounts of pheomelanin (Jimbow, 1995). It is the amount, size and distribution of melanin granules that accounts for the differences in skin tone we see among races as the number of melanocytes in skin varies little with genetic background (Costin & Hearing, 2007). Although the quantity and type of melanin synthesized by melanocytes is mainly under genetic control, there are factors influencing this process (e.g., age, sex, exposure to UV light) (Costin & Hearing, 2007). The amount of cutaneous melanin that an individual has, according to his/her cellular genetic programs, is known as constitutive pigmentation as it does not depend on other acquired endogenous or exogenous factors such as sun exposure (Jimbow et al., 1976). Direct skin exposure to UV light and the effect of some drugs, chemicals and endocrine changes can elicit a reversible increase in the amount of cutaneous melanin pigment, which is known as facultative or inducible pigmentation (Costin & Hearing, 2007; Jimbow et al., 1976).  	
    11	
    Melanin provides an attenuation factor for UV radiation transmission penetrating the human epidermis at an estimated 2-10 times (depending on wavelength) greater for dark skin compared to fair skin (Anderson & Parrish, 1981; Kollias et al., 1991). In fact, the incidence of skin cancer for individuals with fair skin is about 20 times higher than that of the dark skinned population (Kollias et al., 1991). However, constitutive pigmentation alone does not account for this difference, as facultative pigment production, which is induced by UV exposure and stratum corneum thickening, also has a protective role (Anderson & Parrish, 1981; Kollias et al., 1991). The facultative induced pigmentation elicited by UV radiation effects on human skin is considered to enhance the photoprotection already offered by the constitutive pigmentation (Ortonne, 2002). This induced pigmentation response is also genetically determined and is more pronounced in those having darker constitutive pigmentation (Agar & Young, 2005). The induced pigmentation is triggered, at least partially, by DNA damage and activation of the p53 tumor suppressor gene. The beneficial protective effects of induced facultative pigmentation due to a prolonged exposure to UV rays could be annulled by the associated cumulative DNA damage occurring to the cells (Eller et al., 2008; Young & Walker, 2008). Although the melanin in skin cannot completely prevent UV radiation from reaching and damaging cells, it can reduce its transmission to the cell nuclei (Kobayashi et al., 1998). 1.2.4 Proposed functions for melanins While melanin’s significance in animals and fungi might be different, many of the proposed functions are similar and will be discussed together. It is important to bear in mind however, that for fungi, the majority of research has been done in vitro with organisms being isolated from their natural ecosystem and therefore, the functions attributed to melanin might not be applicable in the natural environment (Butler & Day, 1998). In animals, because melanin is present in different tissues, its function is not well defined. Its role in skin might be different 	
    12	
    from its role in deep organs such as the brain or liver (Wood et al., 1999). It is likely that this pigment does not have a unique or main role but that it is connected with multiple functions. Some of the functions ascribed to melanins include: Protection from radiation: This role has been linked mainly with the absorption characteristics but also with antioxidant properties of melanins. In fungi the amount of melanin produced is associated with the level of resistance to radiation (Nosanchuk & Casadevall, 2003; Romero-Martinez et al., 2000). Fungi living on rocks, exposed surfaces or in extreme environments are often heavily pigmented and able to resist elevated temperatures and UV radiation (De Leo et al., 1999; Grishkan, 2011; Harutyunyan et al., 2008). In experiments with Cryptococcus neoformans, decreased susceptibility of melanized cells to the effects of UV light were reported (Wang & Casadevall, 1994). Likewise, melanin-deficient mutants of some fungi are more sensitive to UV radiation (Henson et al., 1999). It seems that fungal melanins can absorb different types of radiation and dissipate the energy by temporarily increasing their own content of free radicals, which ultimately protects the cells from free radicals (Bell & Wheeler, 1986). In animals, the role of melanin as a photoprotectant is still a subject of controversy. Nonetheless, one argument supporting melanin’s photoprotective role has been the observation of higher melanization in human skin with anthropologic origins in higher UV radiation areas, and the highest incidence of UV skin related cancer in fair skinned people who live in those areas (Kollias et al., 1991; Kricker et al., 1994; Ortonne, 2002). Hair color also has been linked to this function, as unpigmented skin in terrestrial animals is usually covered by dark fur, while hairless skin usually has pigmentation (Césarini, 1996). Antioxidant and free radical scavenger: One of the biological functions of melanin is possibly serving as a trap for free radicals, acting in this way as an antioxidant (Jacobson et al., 	
    13	
    1995; Mason et al., 1960; Sarna & Plonka, 2005). Due to its molecular structure, melanin can either receive or donate protons (Bell & Wheeler, 1986). The number of electron-exchange groups within the molecule, its redox properties and accessibility are important factors that may affect the interaction with the radicals (Rózanowska et al., 1999). Apparently, equally important is the state of aggregation of melanin, which influences its efficiency to scavenge OH radicals (Sarna, 1992). In some fungi, the antioxidant activity of melanin is connected to a higher virulence allowing them to survive some of the defensive mechanisms of the host’s immune system (Butler & Day, 1998; Jacobson & Tinnell, 1993; Romero-Martinez et al., 2000). In animals, the scavenging activity of melanin appears to be useful only for reactive species easily accessible to being quenched and thus being generated inside the melanosomes (Sarna, 1992). Binding of transition metals, toxins and drugs: Melanin is a highly efficient and fast ion exchange molecule that binds chemicals, toxins, and heavy and transition metals. This property has biological importance as it allows a melanin to chelate compounds and regulate their entry into the cells (Butler & Day, 1998; Slominski et al., 2004). In fungi, the accumulation of some metals could even represent a defense mechanism against antagonistic organisms (Henson et al., 1999). Defense mechanism: Substantial experimental research demonstrates that the presence of melanin in some fungi confers protection to the cell wall of mycelia and spores against the action of self or foreign lytic enzymes (Butler & Day, 1998; Henson et al., 1999; Jacobson, 2000). Studies of the human pathogen C. neoformans have shown that melanization in this fungus is associated with increased resistance to the action of some antifungal drugs (Butler & Day, 1998; Nosanchuk & Casadevall, 2003). Some lower vertebrates use background color matching or mimicry as a defense mechanism against predators, and it is the distribution of melanin granules and other pigments 	
    14	
    that produces those rapid color changes (Césarini, 1996; Hadley, 1972; Prota, 1992). One of the most convincing examples of melanin being used for defense is the case of cuttlefish, which produce a response known as “inking” to avoid predatory attack (Hanlon & Messenger, 1996). Thermoregulation: Melanin is capable of transforming absorbed UV and visible radiation into heat, which in animals can be dissipated through the dermal vascular network, serving in this way as a regulatory mechanism for homeostasis (Césarini, 1996). Melanin appears to make fungi more tolerant to extreme temperatures, and some fungi produce melanin only when exposed to high temperatures (Butler & Day, 1998; Nosanchuk & Casadevall, 2003). Additional roles attributed to melanin in fungi include: a) serving as water storage, helping to prevent cell desiccation (Butler & Day, 1998; Nicolaus, 1968; Rast et al., 1981), b) providing strength to structures such as appresorium of phytopathogenic fungi and the cell wall (Butler & Day, 1998; Jacobson, 2000), and c) virulence factor safeguarding pathogenic fungi from a host’s immune defense mechanisms (Alviano et al., 1991; Butler & Day, 1998; Henson et al., 1999; Nosanchuk & Casadevall, 2003). In Ophiostoma piliferum, melanin is required for developing perithecia with viable ascospores, and in other fungi may be important for sclerotial development (Henson et al., 1999). In animals, other possible functions also have been reported for melanin: a) it acts as an amorphous semiconductor (a role that seems to be of utmost importance in the inner ear, in the nervous system and possibly in the retina) (Barr, 1983; Brenner & Hearing, 2009; Goding, 2007; Prota, 1992), b) it contributes to tensile hair strength (Slominski et al., 2004), c) it is responsible for color changes associated in some species with sexual attraction clues (Césarini, 1996; Slominski et al., 2004), d) it has even been regarded by some as an “organizational molecule” that interacts with nucleic acids and controls post-translational modifications of proteins (Barr, 1983). 	
    15	
    1.3  Rationale and research objectives As already mentioned, melanic pigments have diverse origins, but the roles attributed to  them in different organisms are similar. Melanin pigmentation in human skin confers some protection against the harmful effects of UV rays, but certainly it is not enough. As a matter of fact, the maximum sun protection factor (on a scale from 2 to 100) associated with skin melanin is 10-15 for dark-skinned people and only 1-2 for Caucasians, which would make it a very weak sunscreen compound (Goding, 2007; Ortonne, 2002; Slominski et al., 2004). The amount and type of melanin in skin is genetically determined and may influence the possibility of developing skin cancer. Most literature suggests that eumelanin has antioxidant and photoprotective properties (Hoogduijn et al., 2004) whereas pheomelanin and its intermediate 5-S-cysteinyldopa are prooxidants, especially in the presence of metal ions (Jimbow, 1995; Takeuchi et al., 2004). Experiments done with pheomelanin also have shown that it might cause UVB sensitization, induce DNA damage in UVA irradiated skin cells and increase the risk for skin cancer (Agar & Young, 2005; Takeuchi et al., 2004). A large amount of research has been focused on the study of DOPA derived melanin, particularly eumelanin (Watt et al., 2009), and there is still controversy regarding whether the skin’s melanin has a photoprotective role in humans (Agar & Young, 2005; Wood et al., 1999). Meanwhile, the photoprotective activity of other types of melanin has remained underexplored. In fungi, the presence of melanin is believed to be a factor that confers survival advantage (Dadachova et al., 2007). This might be true, particularly because unlike animals that can move and avoid extreme environments, these microorganisms have to find ways to survive inhospitable conditions in the environments they inhabit (Dong & Yao, 2012). Melanized fungal species are able to colonize and thrive in areas with intense radiation (Dighton et al., 2008;  	
    16	
    Dadachova & Casadevall, 2008). In view of this, there might be a good chance that fungal melanin offers photoprotection to human skin cells. My research hypothesis was that melanins, non-derived from DOPA, might offer photoprotection to the skin cells of mammals. My research goal was to assess in vitro the photoprotective effect of two different fungal melanins to human skin cells. To reach this goal, I addressed two research objectives, which I cover in the following chapters of this thesis. In Chapter 2, I describe the methods employed to isolate melanin from the basidiomycete Agaricus bisporus and from the ascomycete Grosmannia clavigera. According to the literature (Fleet & Breuil, 2002; Rast et al., 1981), melanins in those fungi are synthesized from different precursors. I analyze some physicochemical characteristics of the isolated melanins and discuss similarities and differences I found among them. In Chapter 3, I describe my assessment of the photoprotective effects of the melanins isolated from the aforementioned fungi. I evaluate their cytotoxicity and photoprotection against UVB in human dermal fibroblasts using several in vitro bioassays. I also evaluate their capacity to filter UVB, measuring the transmission of light (315 nm) by melanin solutions. I used synthetic DOPA melanin in all experiments to compare its effect with the effect of melanins isolated from the two fungi.  	
    17	
    Chapter 2: Isolation and Characterization of Fungal Melanins 2.1  Introduction In this chapter, I address my first research objective: to extract, purify and analyze melanin  from an ascomycete and a basidiomycete. I chose to isolate melanin pigments from these two fungi because their melanins possibly exhibit different chemical structures. I chose to work with the ascomycete Grosmannia clavigera (Robinson-Jeffrey & R.W. Davidson). Previously named Ophiostoma clavigerum, it is one of the commonly known sapstain or blue-staining fungi, which produces a dark stain in the sapwood of some conifer species due to the presence of melanin (DiGuistini et al., 2007). The pathway and genes for its melanin synthesis have been studied and characterized as being derived from 1,8-dihydroxynaphthalene (DHN) (Fleet & Breuil, 2002; Wang et al., 2010). G. clavigera  belongs to the phylum  Ascomycota as its sexual spores are produced and carried in characteristic microscopic sac-like structures called asci (Holt, 2010). It is included within the ophiostomatoid fungi, a group comprising species that grow in the wood of some trees and having a symbiotic association with bark beetles. The morphological characteristics of the ophiostomatoid fungi are adapted to dispersal by arthropods. They produce slimy masses of spores at the top of long stalks that can be easily dispersed by these insects (Montoya & Wingfield, 2006; Six et al., 2003). G. clavigera has been associated with the mountain pine beetles Dendroctonus ponderosae and Dendroctonus jeffreyi, and can kill infested trees when inoculated at high densities (Lee et al., 2007; Six & Paine, 1997; Solheim & Krokene,1998). I also chose to work with the basidiomycete Agaricus bisporus (J.E. Lange) Imbach. This is a cultivated edible fungus, commonly known by diverse names such as button, white or champignon mushroom. A. bisporus is included in the phylum Basidiomycota, characterized by sexual spores that are carried externally in structures called basidia (Alexopoulos et al., 1996). In 	
    18	
    the case of A. bisporus, the basidia are non-septated and each of them bears only two spores (instead of four, as is common in other basidiomycetes) (Elliot, 1985). What is usually called a mushroom is the sexual phase of the fungus represented by the fruiting body or spore bearing structure. This fruiting body is composed of hyphae that differentiate into several tissues: the cap (or pileus), underneath which is the spore-bearing tissue or hymenium, composed of gills covered by a veil when non mature, and the stipe or stalk (Elliot, 1985). The gill tissues of mature mushrooms contain abundant melanized spores. The melanin is located mainly in the wall of the spore, where it appears either in the form of electron-dense granules or as plate-like particles of lower electron opacity (Hegnauer et al., 1985). Some specific characteristics make A. bisporus an appropriate fungus to work with – among them, the fact that it can be easily obtained from stores, it is non-pathogenic, its spore wall has a high melanin content (about one third of the cell dry weight) and its spore production is high (around 0.5 g/Kg of mushrooms) (Rast et al., 1981). The melanin in this fungus was studied and characterized (Rast et al., 1981; Stüssi & Rast, 1981). In Agaricus bisporus fruiting bodies, γ-glutaminyl-4-hydroxybenzene (GHB) is considered to be the main substrate for melanogenesis (Rast et al., 1981; Stüssi & Rast, 1981). There exist several methods for isolating melanins. Selecting an appropriate method requires important considerations such as the biological source, the type of melanin and the degree of purity desired (Prota, 1992). Because melanins are considered to be chemically inert and tightly bound to other cellular components in nature, an extraction method commonly used is the treatment of biological tissue using a combination of strong bases, acids and other harsh chemicals (Filson & Hope, 1957; Liu et al., 2003; Prota, 1992). Typically, the first step is homogenization of the tissue, followed by its treatment with a strong, hot alkaline solution to degrade most of the cellular components, and precipitation of the melanic pigment using acid (Ellis & Griffiths, 1974; Prota, 1992). As melanins are often tightly associated with proteins, 	
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    hydrolysis using boiling 6 or 7 M hydrochloric acid (HCl) from several hours to several days is commonly employed to further purify the pigment (Bell & Wheeler, 1986; Piatelli et al., 1965; Prota, 1992). Additional treatments with various organic solvents such as chloroform, ethanol, ether and/or tetrahydrofuran, also are employed after initial extraction to remove other macromolecules (such as lipids) that might remain attached (Bell & Wheeler 1986; Harki et al., 1997). Mixtures of hydrazine/absolute alcohol or thioglycolic acid/phenol have been employed as an alternative method to the alkaline/acid hydrolysis (Filson & Hope, 1957; Liu et al., 2003). Concerns exist that the aforementioned procedures, which involve drastic chemical treatments, could modify the chemical structure and properties of the melanin molecule (Kollias et al., 1991; Liu et al., 2003; Rosas et al., 2000). Other approaches using enzymatic extraction with proteolytic enzymes (Liu et al., 2003) and a mild melanin extraction protocol for cuttlefish aimed at preserving the natural composition and structure of melanin granules (Zeise et al., 1992) also have been employed. However, enzymatic extraction of melanin might yield a variable pigment with differences in protein, lipid and metal content, and therefore its results would be less reproducible (Ghiani et al., 2008). Regardless of the method employed, achieving ‘pure melanin’ seems to be a real challenge, if feasible at all (Butler & Day, 1998; Henson et al., 1999; Prota, 1992). For the extraction and purification of fungal melanins in my study I used a combination of acid/base extractions followed by treatment with the organic solvents chloroform and 1-butanol (Ellis & Griffiths, 1974; Harki et al., 1997). The methodology I used (acid/base extraction) is one of the most commonly described in the literature when dealing with melanins of fungal origin. I chose this method due to its feasibility. Chemical treatments are not believed to affect the basic structure of fungal melanins (Rast et al., 1981; Saiz-Jimenez et al., 1995).  	
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    To identify fungal pigments I used commonly employed chemical tests (Butler & Day, 1998; Rast et al., 1981; Suryanarayanan et al., 2004). To gain further knowledge of their characteristics, I analyzed the pigments using available techniques such as infrared (IR) spectroscopy and nuclear magnetic resonance (NMR). This allowed me to compare the two pigments, and to compare my results with that described in the literature. 2.2  Materials and methods 2.2.1 Chemicals and reagents For the extraction, purification and characterization of melanin in my study, I used  potassium hydroxide (KOH) flakes obtained from Sigma-Aldrich; chloroform (0.75% ethanol as preservative), hydrochloric acid (HCl), 1-butanol, hexane, ethyl acetate, acetone and hydrogen peroxide (H2O2) from Fisher Scientific; 100% ethanol from GreenField Ethanol Inc.; iron (III) chloride from Aldrich; dimethyl-d6-sulphoxide from Cambridge Isotope Laboratories, and synthetic (DOPA) melanin from Sigma. All chemicals were reagent grade. 2.2.2 Fungal material I purchased fresh Agaricus bisporus mushrooms (non organic), most with an intact veil, from a local grocery store. I scraped the gill tissue from the caps and stored a total of 110.4 g of gills in a freezer at -20°C. Grosmannia clavigera cultures were grown by a team in the Faculty of Forestry, University of British Columbia (UBC), using the following procedure: plugs of fungal specimens stored at -80°C were grown on 1% oxoid malt extract agar (33 g malt extract agar and 10 g technical agar per litre); specimens on 150 plates were covered with autoclaved cellophane. After two weeks, a scalpel was used to scrape the dark mycelia from the cellophane recovering a total of 63.5 g of fresh mycelia. The material was stored in a -20°C freezer. I freeze dried both samples resulting in 8.3 g of G. clavigera and 14.4 g of A. bisporus to be used for the extraction of melanin. 	
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    2.2.3 Melanin extraction I extracted melanin using methods reported by Ellis and Griffiths (1974) and Harki et al. (1997), with minor modifications. The procedure was employed is described as follows: homogenization of the dried samples in 1M KOH solution using a mortar and pestle, followed by reflux for 3 h at 100°C in a water bath, maintaining an atmosphere of nitrogen at all times during the reflux. I used a total of 200 ml of 1M KOH solution each time for refluxing A. bisporus material and 150 ml for G. clavigera. I filtered the solution through two layers of Whatman’s paper #1 using a vacuum pump, recovering the residue and keeping it in a sealed flask flushed with nitrogen. In order to maximize the amount of pigment obtained, I repeated the procedure using the recovered material once more in the case of A. bisporus, and ten more times for G. clavigera, using the same amount of 1M KOH solution for the reflux for every repetition. To precipitate the extracted pigment, I added concentrated HCl to the dark filtrate until pH 2, stirring the mixture with a magnetic bar. I centrifuged this mixture (IEC Clinical Centrifuge, International Equipment CO, Damon) at 5,125 g for 10 min to recover the pellet. I discarded the supernatant and washed the sediment with distilled water until the supernatant was colourless. I kept the pellet under nitrogen atmosphere in a desiccator. 2.2.4 Melanin purification I used a method described by Harki et al. (1997) for the purification of melanin. For each extraction, I added 10 ml of 7M HCl and performed acid hydrolysis in a sealed glass vial filled with nitrogen using a water bath at 100°C for 2 h. To recover the insoluble residue, I centrifuged (IEC Clinical Centrifuge, International Equipment CO, Damon) the extract at 5,125 g for 10 min. Next, I washed the pellet with a solution of 0.01M HCl and when the liquid was colourless, I began washing the pellet with distilled water until the pH of the supernatant was similar to that of distilled water. This procedure is used to remove proteins and carbohydrates (Kannan & 	
    22	
    Ganjewala, 2009) that might be attached to the melanin molecule. I stored samples under nitrogen atmosphere in a desiccator. To extract other macromolecules that might still be attached to the melanin (Harki et al., 1997), I re-dissolved the samples in 1M KOH (5 ml for each amount extracted) followed by the addition of 2 ml of chloroform and 200 µl of 1-butanol. I placed the mixture in a glass vial filled with nitrogen, and shook it in a vortex for 30 min at medium speed. I transferred the solution to small Eppendorf tubes and centrifuged (Thermo Scientific Sorvall Legend Micro 17R Centrifuge) at 6,000 g for 10 min. I discarded the chloroform phase and recovered the alkaline dark solution on the top, mixing it again with chloroform and 1-butanol in the same proportions. I repeated this procedure three times and then acidified the dark alkaline solution to pH 2, adding concentrated HCl while stirring with a magnetic bar. I recovered the precipitated pigment by centrifugation (Thermo Scientific Sorvall Legend Micro 17R Centrifuge) at 10,000 g for 10 min and then washed it several times with distilled water until the supernatant was clear and its pH was similar to the pH of distilled water. I left both samples in a desiccator filled with nitrogen to dry completely at room temperature. I stored them at all times within the desiccator protected from light and under nitrogen atmosphere. 2.2.5 Identification and analysis of melanins 2.2.5.1 Chemical tests I tested the solubility of extracted pigments from both fungi in cold and hot (90°C) water and different organic solvents using a concentration of 10 mg/ml. To test alkali solubility, I dissolved 2 mg in 5 ml of 1M KOH solution. Additional chemical tests included reaction with a 1% (w/v) aqueous solution of ferric chloride (FeCl3), reaction to an oxidizing agent (30 vol. H2O2) and precipitation by acid (7M HCl solution).  	
    23	
    2.2.5.2 UV-visible and IR spectroscopy I dissolved the extracted fungal pigments in a 1M KOH solution to a final concentration of 0.01 mg/ml and scanned the UV-visible spectra in a Shimadzu UV-Visible Spectrophotometer (Kyoto, Japan) at the Food, Nutrition and Health building, UBC. The spectra were recorded in the wavelength range of 190 to 700 nm. I also scanned a solution at the same concentration of commercial synthetic DOPA melanin in 1M KOH to compare its spectra with that of the extracted pigments and used a solution of 1M KOH as the reference blank. Using a Perkin Elmer Spectrum One FTIR spectrometer (Mod. HWS 5984) at the Faculty of Forestry, UBC, I obtained and recorded the IR spectra from 4500 to 600 cm-1 of both pigments. I powdered approximately 1 mg of each sample and analyzed them directly in the Attenuated Total Reflectance (ATR) mode using a Pike Miracle Accessory (Pike Technologies). Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) can be used for analysis of insoluble polymers in a solid state, like melanins, having the advantage of requiring minute amounts of sample and preventing solid-state transformations since the materials are not subjected to thermal or mechanical energy during sample preparation (Bugay, 2001; Pierce & Rast, 1995). 2.2.5.3 NMR spectroscopy I mixed 20.1 mg of G. clavigera and 20.5 mg of A. bisporus samples, each with 1 ml of deuterated dimethyl sulfoxide, filtered the solutions using a small plug of cotton tightly packed into a Pasteur pipette, and transferred them into a NMR tube. Proton and carbon-13 NMR spectroscopy were conducted at the Chemistry Department, UBC. Samples were analyzed in a Bruker Avance-600 with a cryoprobe operating at 600 MHz, with DMSO at 2.5 ppm for the proton and at 39.51 ppm for the carbon, using pulse program spin echo carbon.  	
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    2.3  Results The amount of pigment I obtained was 322.4 mg for A. bisporus and 382.5 mg for G.  clavigera, which means a yield of 2.2% from the total dry weight of A. bisporus gills, and 4.6% from the total dry weight of G. clavigera mycelia. Physically, both pigments can be better described as amorphous dark granules. 2.3.1 Chemical identification Both pigments extracted from A. bisporus and G. clavigera gave positive results in diagnostic chemical tests commonly used to identify melanins (Butler & Day, 1998, Harki et al. 1997) (Table 2.1 and Figure 2.1). Table 2.1 Chemical tests used to identify melanins Chemical Test  Reagent  A. bisporus pigment  G. clavigera pigment  Solubility in inorganic solvents  Water*  Insoluble  Slightly soluble  1M KOH* (2mg/5 ml)  Soluble  Soluble  Solubility in organic solvents  Ethanol* Acetone* Ethyl acetate* Chloroform* Hexane* 7 M HCl  Insoluble Insoluble Insoluble Insoluble Insoluble Positive (at pH 2)  Slightly soluble Slightly soluble Insoluble Insoluble Insoluble Positive (at pH 2)  Test for polyphenols (pigment solution in 1M KOH)  Aqueous solution of FeCl3 1% (w/v)  Positive: dark brown flocculate precipitate  Positive: dark brown flocculate precipitate  Reaction to oxidizing agent (pigment solution in 1M KOH)  H2O2 vol. 30 (10% Va)  Bleaching**  Bleaching**  Precipitation from solution in 1M KOH  *Solvents warmed up to 90°C and there was no change in solubility **Started to be evident at 1 h and completed after 24 h Va : volume of alkaline solution of melanin!  	
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    Figure 2.1 Chemical tests used to identify melanins (A) Solubility of pigments extracted from A. bisporus and G. clavigera tested in (a) cold and hot (90°C) water, (b) ethanol, (c) acetone, (d) chloroform, (e) ethyl acetate, (f) hexane and (g) 1M KOH solution. (B) Reaction of solution in 1M KOH of the pigments extracted from A. bisporus and G. clavigera with: a) H2O2 30 vol. (10% of volume of melanin solution), b) 1% FeCl3 aqueous solution (1:1 with melanin solution), c) 7M HCl until pH 2. (A)  A. bisporus  G. clavigera  (B)  !"  #"  $"  !"  #"  $"  2.3.2 UV-visible and IR spectra The UV-visible spectra for both pigments showed broadband absorption with a continuous decrease in absorbance from the UV to the visible wavelengths, with several peaks of absorption below 220 nm. Synthetic (DOPA) melanin showed a comparable absorption profile, most similar to A. bisporus melanin (Figure 2.2). The slopes of the lines that resulted after plotting the 	
    26	
    logarithm of absorbance against wavelengths 400–600 nm for A. bisporus, G. clavigera and DOPA pigments were -0.003747, -0.001891 and -0.004044, respectively.  Figure 2.2 UV-visible spectra of melanins  (#$"  A.b. melanin  ("  G.c. melanin synthetic (DOPA) melanin  '" &#$" &" %#$"  	
    absorbance  '#$"  %" !#$" !" &!!"  &$!"  '!!"  '$!"  (!!"  ($!"  $!!"  $$!"  )!!"  )$!"  *!!"  *$!"  wavelength	
  (nm)	
   The IR spectrum of A. bisporus pigment (Figure 2.3) shows a broad band centered at 3190 cm-1 attributed to stretching vibrations of C-H, N-H and/or O-H groups. The C-H could be due to the presence of aromatic rings as there is a strong band at 1621 cm-1, which corresponds to the vibration of aromatic C=C. The stretching bands at 1352 and 1436 cm-1, corresponding to bending vibrations of C-H and/or O-H, may be connected to the presence of aliphatic groups in the first case or to phenolic groups in the case of the O-H. The band at 1227 cm-1 due to C-N and C-O, would support the presence of phenols and of aromatic amines. It is difficult to say whether there is an amide group as the C=O group that complements it might be joined in the band corresponding to the aromatic C=C.  	
    27	
    	
    1227  1621  1436 1352  3190  Figure 2.3 IR spectrum of A. bisporus melanin  In the IR spectrum of the pigment from G. clavigera (Figure 2.4), stretching vibration bands from aliphatic C-H appear at 2851 and 2921 cm-1. The C-H (2921 cm-1) may be due to the presence of aromatic rings as the band at 1616 cm-1 corresponding to the vibration of aromatic C=C also appears in this case. However, it is not as strong as that seen in the A. bisporus pigment. The band at 1414 cm-1 corresponding to bending vibrations of C-H further supports the presence of aliphatic groups. The C-O bands at 1078 cm-1, and bending vibration bands at 1454 cm-1 and broad band centered at 3265 cm-1 from O-H, could be attributed to phenolic groups. The band at 1706 cm-1 attributed to the vibration of C=O is possibly due to the presence of a cetonic or acidic group. Although the presence of amides and/or amines cannot be ruled out, in the spectrum of the G. clavigera pigment there is no evidence of a stretching vibration band that corresponds to N-H groups.  	
    28	
    Figure 2.4 IR spectrum of G. clavigera melanin  1078  1171  1706 1616 1454  2921  2851  3265  !  2.3.3 NMR spectra The proton NMR (1H NMR) spectrum of G. clavigera pigment (Appendix I), shows multiplets in the region of non aromatic and phenolic protons at 5.315 ppm. Triplets at 2.173 ppm may be due to protons of carbons bonded to carbonyl groups. There are very weak signals in the region of the aromatic protons that might be coming from phenolic groups. In the carbon13 NMR (13C NMR) of the pigment extracted from G. clavigera (Appendix II), C-O signals at 174.53 ppm, which in the amplification shows an additional peak at 174.55 ppm that may be from acyl acid group carbons (C=O). There are 23 signals of saturated aliphatic carbons between 13.92 to 33.51 ppm. Signals for aromatic carbons appear from 126.96 to 131.49 ppm, with a total of 12 aromatic or heteroaromatic carbons. It was not possible to solubilise the pigment from A. bisporus in DMSO sufficiently to obtain an NMR spectrum.  	
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    2.4  Discussion The melanin extraction and purification methods I implemented yielded pigments from  both A. bisporus and G. clavigera, which generally agreed in their physico-chemical characteristics both between them and with that described in the literature. Some slight differences, however, were noticed between them. When dissolved in similar concentrations, G. clavigera pigment had a more yellowish-brown color compared with A. bisporus pigment that appeared darker. In general, the dark color of melanins is attributed to chromophoric groups such as quinones, nitroso, nitrile and azo (Alviano et al., 1991). According to Nicolaus (1968), when the oxidation of diphenols such as DOPA, 5,6-dihydroxyindole, catechol and 1,8dihydroxynaphtalene produce quinones with many active centers for polymerization, the resulting compound is generally a black pigment, whereas when the number of active centers is limited, the resulting pigment is brown, reddish-brown or yellowish-brown. Melanins with high levels of indole quinones (such as DOPA derived) appear darker because of the strong absorbance in the red portion of the spectrum (Riley, 1997). This low frequency light absorption is largely through carbonyls and/or oxygen-containing groups; melanins with fewer carbonyl groups are paler and appear more yellow or red (Babitskaya et al., 1998; Riley, 1997). The primary identification of melanins commonly relies on criteria that include general physicochemical properties (Butler & Day, 1998) as previously mentioned. This is particularly true for fungal melanins, which have not been as intensively studied as animal melanins. One chemical characteristic of melanins is their insolubility in organic solvents (Bell & Wheeler, 1986; Nicolaus, 1968). The general insolubility of melanins has been linked to its structural organization (monomeric units connected by covalent bonds and aggregated into small planar oligomers stacked in parallel aligned sheets) and the removal of the associated protein content (Riesz, 2007; Russell et al., 1980). Unlike A. bisporus, which totally fulfilled the insolubility 	
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    criteria, the pigment extracted from G. clavigera was slightly soluble in water, ethanol and acetone. It cannot be ruled out, that this partial solubilization of G. clavigera pigment is connected to structural modifications due to the acid/base treatment (Liu et al., 2003; Young, 1921) or to the presence of proteins or carbohydrates still attached to the chromophore (Bell & Wheeler, 1986). Differences in solubility of melanins have been related to origin, state of hydration or purity, and/or polymerization pattern (Claussen & Pepper, 1968). Additional solubility factors include the ionization state of the pigment’s carboxylic, phenolic and aminic groups, its polyelectrolyte nature and its amino acid content (Aghajanyan et al., 2005). The thick brown precipitate observed after the addition of an aqueous solution of FeCl3 to a solution in 1M KOH of A. bisporus and G. clavigera pigments confirms the presence of polyphenols in both fungal pigments (Kumar et al., 2011). Although pigments obtained from A. bisporus and G. clavigera were decolorised by the H2O2 solution, the reaction was slow, becoming apparent after 1 hour and completed after 24 h. The bleaching of melanin solutions by oxidizing agents such as H2O2 has been linked to the degradation of pigments (Korytowski & Sarna, 1990; Prota, 1992). This results from the preoxidation of hydroquinone groups followed by nucleophilic attack of OOH- ions, which would cause ring-opening reactions. Oxidation of hydroquinone groups by hydroxyl (OH) radicals generated in the presence of metal ions and H2O2 is also possible (Korytowski & Sarna, 1990). For bleaching to occur, solubilisation of melanin in a strong alkaline solution is necessary, as this produces ionization of its hydroxyl phenolic groups, which favours its reactivity with the oxidizing agent (Korytowski & Sarna, 1990). The solubilization of both pigments in 1M KOH and the precipitation observed when their alkaline solutions were acidified by the addition of 7M HCl can be explained by the state of aggregation of melanins being affected by the pH. At physiological pH, melanins form 	
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    negatively charged colloidal systems and are insoluble. Lowering the pH of a melanin solution causes the formation of large agglomerates and sedimentation (Prota, 1992), while an increase in the pH will rapidly produce disaggregation into clusters of smaller size from less polymerized oligomers. This behavior is connected to the presence of ionizable groups and hydrophobic interactions within the molecule (Matuszak & Wasilewska-Radwanska, 2006; Prota, 1992). The spectral properties might allow recognition of a dark pigment as melanin as in general they show an unusual broadband absorption spectrum that increases monotonically as the wavelength decreases (Das et al., 1978; Prota, 1992). The UV-visible spectra of the pigments from A. bisporus, G. clavigera and synthetic melanin is consistent with that in the literature for all melanins. However, differences in the intrinsic absorption throughout the ultraviolet and visible regions of the light spectra (Figure 2.2) are expected for different melanins (Sarna et al., 1986). The increased absorption observed toward the higher energies corresponding to UV wavelengths probably bears importance in biological systems as they may ‘capture’ the energy from the most damaging photons (Riesz, 2007). The broadband absorption of melanins is considered atypical of organic chromophores (Meredith & Sarna, 2006). Three possible explanations are mentioned for this characteristic featureless absorption spectrum of melanic pigments: a) that it represents a scattering phenomenon and not electronic absorption, b) that it is due to the amorphous semiconductor nature of the pigment, and c) that the spectrum is formed by the superposition of the spectra of many chemically different structures that compose the melanin molecule (Riesz, 2007). Consistent for melanins is the exponential nature of this absorbance in the visible range that produces linear curves with negative slopes when the logarithm of the absorption is plotted against wavelength (Baker & Andrews, 1944; Daniel, 1938; Ellis & Griffiths, 1974). The slopes obtained for the pigments extracted from A. bisporus and G. clavigera, and that obtained for 	
    32	
    synthetic DOPA melanin in my study, are in accordance with those reported in the literature for fungal melanins (from -0.0015 to -0.0040) and DOPA melanin (from -0.0018 to -0.0064) (El Bassam et al., 2002; Ellis & Griffiths, 1974; Harki et al., 1997; Suryanarayanan et al., 2004). The differences reported in the calculated slopes of melanins have been related to the state of oxidation of the molecule and not to differences among different types of melanin (Baker & Andrews, 1944; Bell & Wheeler, 1986). The IR spectrum usually provides information about the functional groups that compose the pigment and allows comparison between melanins (Bilińska, 1996; Pierce & Rast, 1995). In the pigments extracted from A. bisporus and G. clavigera, the IR spectra show an expected band close to 1600 cm-1 attributed to stretching vibrations of C=C bonds of aromatic structures. This band is considered important for the identification of melanins and typical of a conjugated quinoid structure (Babitskaya et al., 2000; Bilińska, 1996). In the IR spectra of fungal melanins, bands attributed to aromatic, hydroxylic, alcoholic, phenolic, carboxylic acid, ester, ketones, amide, amine and aliphatic groups, have all been described in the literature (Babitskaya et al., 2000; Bilińska, 1996; Russell et al., 1980). However, some of the IR studies on fungal melanins have been done with melanin that has not been ‘purified’. It can be inferred from the IR spectra the presence of aliphatic and phenolic groups in both pigments, from A. bisporus and G. clavigera. In the IR spectrum of the pigment extracted from A. bisporus, the evidence of N-H or C-N bonds confirms presence of nitrogen, as described by Rast et al. (1981). Although those same authors report that a strong band at about 1100 cm-1 is characteristic of GHB melanin, in the IR spectrum of the pigment extracted from A. bisporus, that band does not appear. This might be due to different methods for extraction, purification and storage of the pigment, and the fact that no melanin is exactly like another, even if coming from the same source. In the IR spectrum of G. clavigera, there is no clear evidence of amine or amide groups. The studies 	
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    published for the characterization of DHN melanin are few, but it has been stated that, similar to other types of melanin it has aromatic rings and hydroxyl groups, but possibly no nitrogen and carboxyl groups (Henson et al., 1999). Conventional high resolution solution-phase NMR spectroscopy, a technique commonly used to elucidate most chemical structures, has been difficult to apply in the case of melanins due to their insolubility (Duff et al., 1988). However, some studies using 13C NMR and 1H NMR spectroscopic analysis of melanins report resonances from aromatic, carboxylic and aliphatic carbons (Aime et al., 1991; Lüdemann et al., 1982). The 13C NMR and 1H NMR spectra of the G. clavigera pigment confirm the presence of aromatic, aliphatic and carbonyl groups. The presence of aliphatic carbons (olephinic) have been related to uncyclized side chains of the precursor and/or to the protein moiety attached to the pigment (Aime et al., 1991; Ghiani et al., 2008; Katritzky et al., 2002). The  13  C NMR spectrum of the G. clavigera pigment shows  preponderance in aliphatic carbon in relationship to the aromatic resonances. However, there is variability in the reported concentration, intensity and distribution of carbon resonances in the 13  C NMR spectra from different fungal species (Knicker et al., 1995; Olennikov et al., 2011).  This has been linked with the high diversity of carbon structures of fungal melanins, which is significantly different from animal melanin (Knicker et al., 1995). The melanin molecule contains mobile and rigid regions, which make the interpretation of high resolution NMR difficult (Lüdemann et al., 1982). Therefore, caution is advised in establishing a relationship between the intensity of the signals with the absolute concentration of a certain class of carbons, or to interpret the absence of a signal as the absence of a structural element (Lüdemann et al., 1982). The lower sensitivity of 1H NMR spectra for melanin analysis has been linked to the amount of quaternary carbons and the paramagnetic character of the molecule (Katritzky et al., 2002; Tian et al., 2003). 	
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    The insolubility of the pigment obtained from A. bisporus precluded its analysis by conventional NMR. Solid-state NMR, which does not require the use of a solvent, has been employed successfully to analyze some melanins (Duff et al., 1988; Knicker et al., 1995; Tian et al., 2003). However, this technique was not available to me. 2.5  Conclusions In summary, the pigments extracted from A. bisporus and G. clavigera both have  physicochemical properties common to melanins. Additionally, they show the typical and unique broadband absorption in the UV-visible spectra which monotonically decrease from lower to higher wavelengths and is characteristic of melanin pigments. The IR spectra of both pigments reveal the presence of an aromatic system, aliphatic and phenolic groups. The NMR spectra (carbon and proton) of G. clavigera pigment confirm the presence of aromatic and aliphatic carbons, phenolic and acid acyl groups. Therefore, the pigments extracted from both fungi fulfill criteria to define them as melanins. The melanins extracted from A. bisporus and G. clavigera showed some differences in color, solubility and in the IR spectra. In G. clavigera melanin, unlike A. bisporus melanin, nitrogen groups were not evidenced in the IR and NMR spectra. That both melanins have different molecular structure might be expected, not only because of their different origin, but also because of the differences observed through their analysis.  	
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    Chapter 3: In vitro Assessment of Cytotoxicity and Photoprotection of Fungal Melanins to Human Skin Cells 3.1  Introduction In this chapter, I describe an evaluation of the cytotoxicity and the photoprotection against  UVB by melanins extracted from A. bisporus and G. clavigera, using in vitro bioassays. Synthetic DOPA melanin was used for comparison of activity with fungal melanins. I used primary human dermal fibroblasts (HDF) for the experiments. Primary cells are considered a better model of an in vivo state, because unlike neoplasic or immortalized cell lines, they undergo few population doublings (‘Life Technologies Corporation’, 2011). Fibroblasts constitute the main cellular components of the dermal layer of the skin, and are primarily responsible for the synthesis and degradation of the extracellular matrix (Costin & Hearing, 2007). The dermis is a major component of the skin that provides its flexibility, elasticity and tensile strength and it also gives support to the outermost layer, the epidermis, actively interacting with its components (Chu, 2008). Although sunlight impinges the epidermis, the UV rays penetrate in a significant proportion into the dermis, most specially in caucasians (Everett et al., 1966), which are more prone to develop skin cancer (Kricker et al., 1994; Preston & Stern, 1992; Tadokoro et al., 2003). It has been estimated that approximately 1015 photons/cm2 can reach the dermis, an amount of energy that cannot be disregarded as inconsequential (Everett et al., 1966), therefore UVB radiation can induce dose-dependent DNA damage not only in the epidermis, but also in the dermal layer (Katiyar et al., 2000). To induce phototoxicity in HDF, I used UVB light (280-315 nm). The UVB photons are considered to be the most energetic reaching the Earth’s surface (Cockell & Knowland, 1999) and are absorbed by the DNA bases, mainly pyrimidines (Ravanat et al., 2001). The absorption of UVB energy by DNA leads to the formation of aberrant covalent bonds between adjacent pair 	
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    of bases producing dimeric photoproducts that have mutagenic potential (Doniger et al., 1981; Matsumura & Ananthaswamy, 2004). Chronic inflammation and immunosuppression of the skin produced by UVB also play a role in photocarcinogenesis (Halliday, 2005; Ichihashi et al., 2003). UVB radiation can initiate and promote skin cancer without the intervention of any other factor (Bowden, 2004; Claerhout et al., 2006). Typically, after an injury by UV radiation, mammalian cells activate mechanisms to repair the DNA damage through different pathways (Matsumura & Ananthaswamy, 2004), and to allow an efficient repair, they transiently enter into growth arrest (D’Errico et al., 2003). When cells have been irrepairably damaged or transformed by the effects of UVB, they are eliminated through apoptosis (Claerhout et al., 2006). The apoptotic response is a highly regulated process by means of which the cell commits ‘suicide’ based on a genetic mechanism (Majno & Joris, 1995). Cell cycle arrest and apoptosis are mechanisms possibly activated in order to avoid the replication of cells with non-repaired DNA damage (Matsumura & Ananthaswamy, 2004). To measure the cell damage induced by UVB and assess photoprotection by melanins, I used a simple assay that quantifies cell survival: the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. I also used this assay to test the cytotoxicity of melanins on HDF. MTT is considered a gold standard assay for viability/cytotoxicity (Niles et al., 2008). The method was developed by Mosmann (1983) and allows measurement of in vitro changes in cell proliferation, or in cases of cell necrosis or factor-induced cytotoxicity, a reduction in cell viability (Mosmann, 1983). It is based in a colorimetric reaction, in which the yellow tetrazole salt (MTT) is reduced to the purple compound formazan by the action of mitochondrial enzymes. This reaction only takes place in metabolically active cells (Mosmann, 1983). The insoluble purple formazan dye crystals are solubilized using a detergent, isopropyl 	
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    alcohol (IPA) or DMSO. The intensity of the purple color is directly proportional to the number of metabolically active cells and inversely proportional to the toxicity of the test material (Denizot & Lang, 1986; Mosmann, 1983). The absorbance of the colored solution is read in a spectrophotometer. UVB radiation is able to generate reactive oxygen species (ROS) that can lead to cell damage (Heck et al., 2003; Masaki & Sakurai, 1997; Peus et al., 1998). ROS generated by UVB are a consequence of redox (Heck et al., 2003) and photosensitized reactions, and they might be more important than the photons themselves to induce cell death at low doses of irradiation, producing DNA and cell membrane oxidative damage (Shindo & Hashimoto, 1998). To determine if the fungal melanins and synthetic DOPA melanin had any effect in the production of ROS induced by UVB in HDF, I performed an assay with 2’,7’dichlorodihydrofluorescein diacetate that measures generalized oxidative stress (Eruslanov & Kusmartsev, 2010; Halliwell & Whiteman, 2004). This assay can be used to screen the ability of a substance to reduce increases in intracellular ROS (Wang & Joseph, 1999). The relatively nonfluorescent compound 5-(and-6)-chloromethyl-2',7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), freely enters into cultured cells when in its esterified form. As a result of deacetylation by intracellular esterases, it becomes trapped inside (Eruslanov & Kusmartsev, 2010). ROS produced inside the cells as a consequence of an oxidative stress (e.g., UVB) react with the CM-H2DCFDA, which as a result becomes highly fluorescent. The intensity of fluorescence reflects the amount of ROS present (Kim et al., 2005). Finally, to estimate the capacity of melanins to filter UVB, I measured the transmission of light by solutions of the tested melanins.  	
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    3.2  Materials and methods 3.2.1 Materials In my experiments I used the following biochemicals and chemicals (reagent grade):  medium 106, low serum growth supplement (LSGS), antibiotic-antimycotic solution (sodium salt of penicillin G, streptomycin sulfate, and amphotericin B as Fungizone® in 0.85% saline), trypsin/EDTA, trypsin neutralizer, phenol-red free Hanks’ Balanced Salt Solution (HBSS) (containing calcium and magnesium chloride), 4-(2-hydroxy- ethyl)-1-piperazine ethane sulphonic acid (HEPES) solution, phosphate buffered saline (PBS) solution and 5-(and-6)chloromethyl-2’,7’-dichlorodihydrofluorescein  diacetate,  acetyl  ester  (CM-H2DCFDA)  purchased from Invitrogen; (+)-sodium L-ascorbate (vitamin C sodium salt), 5-fluorouracil, thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide, trypan blue and synthetic (DOPA) melanin from Sigma; triton X-100 and potassium hydroxide flakes from SigmaAldrich; 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox™) from Aldrich, isopropyl alcohol (IPA) from Fisher and 100% ethanol from GreenField Ethanol Inc. The labware I used included: 75 cm2 Falcon tissue culture flasks and clear 96-well microtest tissue culture plates with flat bottom obtained from Becton Dickinson Labware, US; Falcon black 96-well microtest assay plates with clear bottom from Becton Dickinson, UK and Mylar film (type D, 0.127 mm thick) from Precision Plastics, Delta, BC. 3.2.2 Samples preparation I prepared stock solutions of 10 mg/ml in 0.5M KOH of the melanins (from A. bisporus, G. clavigera, and synthetic DOPA), warming to 90ºC to assure complete solubilisation. From the stock solutions, I prepared different concentrations of the samples for each assay, diluting them first with 0.5M KOH and then with culture media or buffer (according to the assay), to the final working concentrations. The final concentration of KOH solution in media/buffer in all wells 	
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    was 0.01M, which was tested in pilot experiments to be safe for the cells. Compounds used for positive control wells in the assays: vitamin C and 5-fluorouracil, were dissolved in water, and Trolox™ was dissolved in 100% ethanol. Final working concentrations in media/buffer were prepared same as for melanins. The final concentration of ethanol in wells was 1%, tested in pilot experiments to not affect cells. 3.2.3 Cell culture I cultured HDF from neonatal foreskin (Cascade Biologics, Invitrogen) in medium 106 supplemented with LSGS and 1% of an antibiotic-antimycotic solution (10,000 units/ml of penicillin-base, 10,000 µg/ml of streptomycin-base, and 25 µg/ml of amphotericin B). I maintained cells to grow in an incubator at 37°C with a humidified atmosphere of 5% CO2 in air. Initially, I grew cells in 75 cm2 tissue culture flasks until the monolayers reached 80-90% confluence, and then sub-cultured them. For all experiments, I transferred cells to 96-well plates. The procedure I used included the following steps: I added 10 ml of trypsin/EDTA solution to the 75 cm2 tissue culture flask and immediately after, removed 6.5 ml and left the remaining 3.5 ml for 2 min, for cells to detach. I added 10 ml of trypsin neutralizer solution, pipetting it several times into the flask. I recovered cells by centrifugation (Sorvall Legend XTR, Thermo Fisher Scientific centrifuge) at 180 g for 5 min at 20ºC, blended the pellet in 10 ml of culture media and mixed 20 µl of this solution with 20 µl of trypan blue. I counted cells using a hemacytometer and then plated them at the desired density. For the experiments, I used cells passage 4-6. Conditions for incubation of cells remained the same (37°C with a humidified atmosphere of 5% CO2 in air) during all experiments.  	
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    3.2.4 UVB irradiation The UVB source was a wood cabinet 64 cm long, 11.5 cm wide and 11 cm deep containing two Philips UVB broadband bulbs model TL 20W/12 (Light Sources Inc., US). According to the manufacturer, the emission range of bulbs is 290-320 nm with a peak at 302 nm. I irradiated the cells in 96-well tissue culture plates covered with the lid and localized at 5 cm from the UVB source. To avoid the formation of medium derived toxic photoproducts and the absorption of energy by phenol red from the media during irradiation (Belleti, 2007; Stoien & Wang, 1974), I irradiated cells in buffer solution. To monitor the dose of UVB, I used a UV (UVA or UVB) radiometer/dosimeter (UV Minder, Chromaline Corporation, Duluth, MN, US) with the sensor placed at 5 cm from the UVB source and covered with a transparent lid from a 96-well plate. As dark controls, I used sham-irradiated cells from wells covered with black tape and aluminum foil. 3.2.5 Evaluation of cytotoxicity of melanins To measure the cytotoxicity of the melanins from A. bisporus, G. clavigera and synthetic DOPA, I used proliferating HDF. I seeded cells (1 x 104 cells per well) in a 96-well plate and incubated them overnight to allow them to attach. I then replaced the media in each well with 200 µl of new media containing tested compounds (melanin from A. bisporus, G. clavigera or synthetic DOPA), in concentrations of 200, 20, 2, 0.2, 0.02 and 0.002 µg/ml. I used similar concentrations of the cytotoxic compound 5-fluorouracil as a positive control. After 72 h of incubation, I performed the MTT assay according to the method described by Denizot and Lang (1986), with some modifications. The procedure included aspiration of the medium containing the tested compounds and addition of 200 µl of serum free medium containing MTT (0.5 mg/ml) to each well, followed by incubation for 2 h. I then removed media containing MTT and added to each well 150 µl of IPA (to solubilise purple formazan crystals), pipetting several times until 	
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    the mixture had a uniform color. I quantified the absorbance of the solutions at 544 nm using an ELISA reader (Fluostar/Polarstar Galaxy, BMG Labtechnologies, Germany). The optical density (OD) of the blank (IPA) was subtracted from the OD of samples and control, previous to calculations. I quantified cell viability using the formula: % viability = (absorbance of test sample /absorbance of control*) x 100. *Mean absorbance value for vehicle (0.01M KOH) treated cells. This value represented 100% cell viability. The % of cell inhibition is calculated with the formula: % inhibition = 100 - % of viability. 3.2.6 Evaluation of UVB phototoxicity To define the experimental system before investigating the possible photoprotection offered by the melanins, I conducted pilot experiments to measure the effect of different UVB intensities on HDF viability. I seeded cells in 96-well plates (1.6 x 104 cells per well), allowing them grow to achieve confluence. Prior to the UVB challenge (24 h) I replaced media in each well by 100 µl of media with vehicle (0.01M KOH). For the irradiation, I removed media and added 100 µl of pre-warmed HBSS/20mM HEPES buffer. I placed cells in the incubator for 15 min and then exposed each quarter of the plate to UVB doses of 100, 200, 400 or 800 mJ/cm2 respectively. Half of the wells in the plate were sham-irradiated and served as dark controls. Immediately after the irradiation, I replaced buffer with complete fresh medium and maintained cells in the incubator. To define the length of time to perform MTT after the UVB challenge, I measured cell viability by MTT at 24 and at 48 h after the UVB exposure. I performed MTT assay and calculations as previously described. Mean absorbance values for dark control cells represented 100% viability. 	
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    Irradiation times during experiments varied from approximately 20 min to reach a UVB dose of 100 mJ/cm2 to 3 ½ h for a dose of 800 mJ/cm2. To determine the contribution of UVA present in light emitted by the bulbs, I also performed pilot irradiation experiments with and without filtration of the light through 0.127 mm thick Mylar. Mylar film absorbs radiation in the UVB region (Allred & Giesy, 1985). In these experiments, I irradiated cells with different UVB doses as before, but half of the wells were covered by Mylar while the other half remained uncovered. Two rows of sham-irradiated wells served as dark controls. 3.2.7 Evaluation of the effect of melanins on UVB-induced phototocixity I tested the photoprotection offered to HDF by concentrations of 200, 150,100, 75, 50 and 25 µg/ml of the melanins from A. bisporus, G. clavigera and synthetic DOPA. I used 50 µg/ml of Sodium L-ascorbate (vitamin C), as a positive control for the assay (Lin et al., 2003; Shibayama et al., 2008). I designed six protocols (Table 3.1) adding the tested compounds at different stages of the experiment. I only repeated the experiment in which there was a positive response. The procedure I used was as follows: I seeded cells in 96-well plates (1.6 x 104 cells per well) and left them to grow until confluence. Depending on the protocol to carry out (Table 3.1), 24 h before irradiation I replaced media in wells by either 100 µl of media containing different concentrations of the tested compounds or by media alone. Before the irradiation, I replaced the media in the wells with 100 µl of pre-warmed HBSS/20mM HEPES buffer with different concentrations of the tested compounds or buffer alone and placed cells in the incubator for 15 min. I exposed cells to UVB, and immediately after I replaced buffer with 100 µl of media alone or media with the tested compounds. I always added vitamin C to positive control wells, 24 h both before and after, and during UVB treatment. I performed MTT assay and calculated cell 	
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    viability as previously described. Mean absorbance values of vehicle treated cells exposed to UVB represented 100% value for cell number. Survival rates in presence of the tested melanins represented percentages of the control value. Table 3.1 Protocols performed with melanins and HDF irradiated with a phototoxic dose of UVB Incubation of cells with melanins took place for 24 h, adding them to the culture media before or immediately after UVB exposure, or for 15 min in the buffer solution before irradiation and irradiating cells in their presence. (+) melanins present, (-) melanins absent. 24 h prior UVB  UVB  24 h post UVB  I  +  -  -  II  -  -  +  III  -  +  -  IV  +  +  -  V  -  +  +  VI  +  -  +  3.2.8 Evaluation of the effect of melanins on UVB-induced ROS I tested concentrations of 200, 100, 50 and 25 µg/ml of A. bisporus and synthetic DOPA melanins, and 200, 100, 50 µg/ml of G. clavigera melanin in this experiment. Cells treated with Trolox™ (50 µg/ml) served as positive controls (El Hindi et al., 2004; Koopman et al., 2008). For the induction of ROS, I used a UVB dose previously determined in laboratory experiments using the same cell line. I used the protocol described by Eruslanov and Kusmartsev (2010) with minor modifications: I plated cells (1.6 x 104 cells per well) in black 96-well microtiter plates with clear bottom and left them to grow until achieving confluence. The day of the assay, I prepared the fluorescent probe by dissolving 50 µg of CM-H2DCFDA in DMSO and diluting it 	
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    in HBSS/20mM HEPES buffer to 10 µM solution. I removed the medium and in half of the 96 wells, cells were loaded by adding to each well 100 µl of the solution containing the fluorescent probe. The other half remained CM-H2DCFDA free, adding to them only 100 µl of HBSS/20mM HEPES buffer. Loading took place in the incubator for 30 min. I then washed all wells with 200 µl of HBSS/20mM HEPES buffer to clear away excess of CM-H2DCFDA (Halliwell & Whiteman, 2004). This was followed by the addition of 100 µl of HBSS/20mM HEPES buffer containing the tested compounds. After 30 min of incubation, I induced extracellular oxidative stress by irradiation with 66 mJ/cm2 of UVB (at room temperature). One row exposed to UVB, containing only vehicle treated cells, represented 100% of UVB induced ROS, and one row of sham-irradiated cells containing only vehicle served as dark control. Following irradiation, I placed cells in an incubator for 1 h, and then lysed them by adding 100 µl of lysis buffer (1% triton in 2X PBS) to each well. After 10 min, I measured fluorescence in an ELISA plate reader (Fluostar/Polarstar Galaxy, BMG Labtechnologies, Germany), using standard and time-resolved fluorescence optics (excitation wavelength of 485 nm and emission wavelength of 520 nm). CM-H2DCFDA free wells containing tested compounds represented background fluorescence of those compounds. This background fluorescence was subtracted from the total fluorescence of irradiated cells loaded with CM-H2DCFDA and test sample. Therefore, each corrected fluorescent measurement was an indication of the amount of ROS formation within the cell, and not due to any background fluorescence. For the calculations I used the formula: % ROS= (fluorescence reading in sample treated exposed cells fluorescence reading in untreated exposed cells) x 100 The % of reduction in UVB-induced ROS was obtained by subtracting from 100 the calculated % of ROS. 	
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    3.2.9 Measurement of light transmission by solutions of melanins I prepared solutions of 200, 100, 75, 50, 25, 10, 1, and 0.1 µg/ml of melanins from A. bisporus, G. clavigera and synthetic DOPA in HBSS/20 mM HEPES buffer, and then measured transmission at 315 nm in an Ultrospec 1000 UV-Visible spectrophotometer (Pharmacia Biotech, Cambridge, England). 3.2.10 Data analysis I conducted each experiment at least three times, each one with triplicate measurements for all treatment and control groups. I considered the results of the three experiments that were more similar and expressed them as means + standard deviation (SD). I analyzed results with GraphPad Prism software (version 5.02). I calculated IC50 values (50% inhibitory concentration) using non-linear regression analysis for dose-inhibition response. To determine statistical significance, I used two-way ANOVA followed by Bonferroni as a post-hoc test, for the analysis of the phototoxicity produced by different UVB intensities measured after 24 and 48 h and in the presence/absence of Mylar film. For the other experiments I used one-way ANOVA followed by Tukey’s multiple comparison test. I considered differences significant at p < 0.05. 3.3  Results 3.3.1 Effect of melanins on HDF proliferation Incubation with 200 µg/ml of A. bisporus, G. clavigera and synthetic DOPA melanins  reduced HDF proliferation by 77.6 + 2.6, 68.6 + 1.9 % and 48.3 + 5.1 respectively, compared to non-treated cells (controls). At 20 µg/ml, A. bisporus and G. clavigera melanins had antiproliferative activity of 30.7 + 7.6 and 44 + 3.0 % respectively (Figure 3.1). The half maximal inhibitory concentration (IC50) in HDF for A. bisporus melanin was 25.3 + 7.8 µg/ml and 9.7 + 0.6 µg/ml for G. clavigera. For 5-fluorouracil, I calculated an IC50 of 1.1 +  	
    46	
    0.3 µg/ml. It was not possible to calculate the IC50 for DOPA synthetic melanin since with the highest dose tested (200 µg/ml) the percentage of cell inhibition did not reach 50%. The difference between the IC50 of A. bisporus and G. clavigera melanin and the IC50 of A. bisporus melanin and the cytotoxic compound 5- fluorouracil, was statistically significant. The IC50 of G. clavigera melanin was not significantly different from the IC50 of 5- fluorouracil. Figure 3.1 Effect of melanins in HDF proliferation Proliferating HDF were incubated for 72 h with different concentrations of A. bisporus (Ab), G. clavigera (Gc) and synthetic (DOPA) melanins. The cytotoxic compound 5-fluorouracil (5FU) was used as positive control. Cell viability was quantified by the MTT assay. Vehicle (0.01M KOH) treated cells were used as controls for 100% cell viability. Values represented are means + SD of three independent experiments, each one with triplicates for samples and control groups. *Significantly different from control group (p < 0.05).  3.3.2 Effect of UVB dose on HDF viability Irradiation of HDF with UVB doses of 200, 400 and 800 mJ/cm2 produced significant decreases in cell viability after 24 h (23.4 + 2.9, 46.9 + 5.9 and 64.7 + 1.8 % respectively), compared to non-irradiated cells (controls). After 48 h of irradiation, all UVB doses tested 	
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    significantly decreased cell viability. There was no significant difference in cell viability measured after 24 and 48 h for UVB exposure to doses of 200, 400 and 800 mJ/cm2 (Figure 3.2). The difference in cell viability of HDF irradiated by UVB doses of 100, 200, 400 and 800 mJ/cm2 covered by Mylar compared to cell viability of HDF irradiated not covered by Mylar was statistically significant (Figure 3.3). Figure 3.2 Cell viability of HDF exposed to different irradiation intensities of UVB measured after 24 and 48 h HDF were exposed to irradiation intensities of 100, 200, 400 and 800 mJ/cm2 of UVB. Cell viability was quantified by the MTT assay 24 and 48 h after the irradiation. Sham-irradiated cells served as dark controls, representing 100% viability. Values represented are means + SD of three independent experiments with multiple replicates for each UVB group. * Significantly different from values of same group (p < 0.05). Bars connected by a line have no statistical difference.  	
    48	
    Figure 3.3 Cell viability of HDF exposed to different intensities of UVB covered or uncovered with Mylar film HDF were exposed to irradiation intensities of 100, 200, 400 and 800 mJ/cm2 of UVB covered or uncovered by Mylar film. Cell viability was quantified by the MTT assay 24 h after the irradiation. Sham-irradiated cells served as dark controls representing 100% viability. Values represented are means + SD of three independent experiments with multiple replicates for each UVB group. * Significantly different from values of same group (p < 0.05). Bars connected by a line have no statistical difference.  3.3.3 Effect of melanins in UVB-induced phototoxicity in HDF Using 200 mJ/cm2, which in pilot experiments showed to be the minimum dose of UVB at which cell viability of HDF significantly decreased after 24 h, did not yield consistent results when photoprotection by melanins was tested. Therefore, I decided to use a dose of 800 mJ/cm2, which consistently decreased HDF viability by more than 50% in pilot experiments. Incubation of HDF with melanins for 24 h prior or after being exposed to UVB (protocols I, II and VI, in table 3.1) produced no difference in cell viability compared to the control group (data not shown). However, irradiation of HDF in buffer containing melanins did produce significant differences in cell viability in some treatment groups compared to controls (Figure 3.4). At concentrations > 50 µg/ml, A. bisporus and synthetic DOPA melanin significantly enhanced cell viability in HDF irradiated with 800 mJ/cm2 of UVB. Vitamin C (50 µg/ml) also was effective in 	
    49	
    significantly increasing cell viability. G. clavigera melanin did not offer effective photoprotection at any of the concentrations tested. The enhancement in cell viability showed a positive dose response, ranging from 38.7 + 5.6 % at 50 µg/ml to 57.7 + 3.2 % at 200 µg/ml for A. bisporus melanin, and from 43.8 + 6.1 % to 63.6 + 24.5 for similar concentrations of synthetic DOPA melanin. Vitamin C (50 µg/ml) enhanced cell viability by 24.8 + 8.5 %. Incubation of HDF with melanins for 24 h before or after UVB along with irradiation in their presence did not produce significant differences compared with irradiation of cells in the presence of melanins without incubation for 24 h (data not shown). Figure 3.4 Cell viability of HDF irradiated with 800 mJ/cm2 of UVB in presence of melanins Cells were exposed to 800 mJ/cm2 of UVB in buffer containing different concentrations of A. bisporus (Ab), G. clavigera (Gc) and synthetic (DOPA) melanins. Vitamin C (50 µg/ml) was added to positive control wells 24 h prior, 24 h after and during UVB irradiation. Cell viability was quantified by the MTT assay 24 h after irradiation. Vehicle (0.01M KOH) treated cells exposed to UVB were used as controls for 100% cell viability. Values represented are means + SD of three independent experiments, each one with triplicates for samples and control groups. *Significantly different from UVB control group (p < 0.05).  	
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    3.3.4 Effect of melanins in ROS production by UVB-irradiated HDF Irradiation of HDF in the presence of melanin from A. bisporus and synthetic DOPA produced a dose-dependent reduction in the production of ROS (Figure 3.5). The response was significant at concentrations of 200, 100 and 50 µg/ml for A. bisporus melanin, and at 200 and 100 µg/ml for synthetic DOPA melanin, compared to untreated cells (controls). G. clavigera melanin did not produce significant reduction in the production of ROS at any of the concentrations tested. The percent reduction of ROS by A. bisporus pigment at 200 µg/ml, 100 µg/ml and 50 µg/ml was 63 + 6.7, 38.9 + 6.3 and 22.9 + 10.0 respectively, whereas for synthetic DOPA melanin the percent reduction was 57.3 + 2.2 with 200 µg/ml and 28.7 + 8.4 with 100 µg/ml. Figure 3.5 Reduction of UVB-induced reactive oxygen species (ROS) in HDF irradiated in buffer containing melanins Cells were loaded with the fluorescent probe CM-H2DCFDA for 30 min (37°C) and then exposed to 66 mJ/cm2 of UVB to induce production of ROS. Cells were irradiated in buffer containing different concentrations of A. bisporus (Ab), G. clavigera (Gc) and synthetic (DOPA) melanins. Trolox™ (50 µg/ml) was present during irradiation in positive control wells. Fluorescence was quantified in a spectrophotometer with fluorescence optics, 10 min after lysing cells with 1% triton in 2X PBS. Vehicle (0.01M KOH) treated cells exposed to UVB represented 100% ROS induction. Values represented are means + SD of three independent experiments, each one with triplicates for samples and control groups. *Significantly different to UVB control group (p < 0.05).  	
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    3.3.5 Transmission of light by melanin solutions in HBSS/20mM HEPES buffer The percent UV transmitted at 315 nm by melanin solutions from A. bisporus, G. clavigera and synthetic DOPA melanin in buffer decreased as their concentration increased. At all concentrations tested, the percent of light transmitted was higher for G. clavigera pigment compared with the other two melanins. At 100 µg/ml, percent transmission of A. bisporus and synthetic DOPA melanins was almost zero, whereas for G. clavigera it was 6.2%. The transmission profile of melanin solutions from A. bisporus and synthetic DOPA almost overlap (Figure 3.6). Figure 3.6 Transmission (%) of light at 315 nm by melanin solutions Percent transmission at 315 nm of different concentrations of melanin solutions in HBSS/20 mM HEPES buffer was obtained using an Ultrospec 1000 UV-Visible spectrophotometer. 120.0  transmission (%)  100.0  80.0  synthetic (DOPA) melanin  60.0  A. b. melanin 40.0  G. c. melanin  20.0  0.0 0  50  100  150  200  250  concentration (!g/ml)  3.4  Discussion UVB radiation has been linked to different types of damage to skin tissues: from acute  sunburn to photocarcinogenesis (Claerhout et al., 2006; Setlow, 1974), and it can lead to cell death by different mechanisms (Shindo & Hashimoto, 1998). The cytotoxic dose of UVB varies across cell types and UVB wavelengths (e.g., narrow vs. broadband) (Cho et al., 2008). The UVB dose at which I found significant cytotoxicity in human dermal fibroblasts was 200 	
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    mJ/cm2, measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24 h (Figure 3.2). This dose is high compared to what is reported in the literature (Cho et al., 2008; Xu et al., 2010). When cells covered by Mylar film were irradiated, results showed that the measured phototoxicity was indeed due to UVB (Figure 3.3). However, the cytotoxic dose of UVB for a particular cell type in vitro is possibly influenced not only by the assay used to measure it, but by other factors, such as culture medium, passage and origin of the cells, and time between UV exposure and measurement of viability (Cho et al., 2008; Saguet et al., 2006; Shindo & Hashimoto, 1998). It is even possible that the decrease in cell viability I measured in the MTT assay, as a consequence of UVB, was an overestimation. MTT might mistake what is cell cycle arrest for cell death (Courdavault et al., 2004; Niles et al., 2008). Skin fibroblasts are reported to be more resistant than keratinocytes and melanocytes to the effects of UVB (Cho et al., 2008). They can preferentially go into cell cycle arrest instead of apoptosis after a UVB insult (D’Errico et al., 2003). The accumulation of vimentin, an important protein component of the cytoskeleton, in the cytoplasm of fibroblasts as a result of the UVB insult might contribute to this resistance (Xu et al., 2010). I can not fully explain why the UVB dose of 200 mJ/cm2 did not yield reproducible results when I assessed the effect of melanins on UVB-induced cytotoxicity in HDF, even though I maintained similar conditions as those used in the pilot experiments. One possible explanation for this inconsistency in the results obtained with 200 mJ/cm2, might be the fact that the cells had to spend more time to achieve the same dose of UVB on each subsequent exposure due to the wear of the UV bulbs. An increase in temperature due to UVB exposure for a longer amount of time may induce the expression of heat shock proteins, which increase cell tolerance to UVB irradiation (Schmidt-Rose et al., 1999; Simon et al., 1995). On the other hand, it has been estimated that sunbathing for one day at sea level produces a maximal dermal exposure that is equivalent to 1000 mJ/cm2 of UVB, already considering what is filtered through 	
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    the epidermis (Saguet et al., 2006). Therefore, using 800 mJ/cm2 more realistically reflected real conditions of acute UV exposure to test photoprotection. The photoprotective effect of melanins isolated from diverse sources has been previously investigated, but most of the studies have been conducted using DOPA derived melanin (Geng et al., 2008a, 2008b; Mosse et al., 2000). Few studies have investigated photoprotection by other types of melanin, especially those derived from fungi. Paramonov et al. (2002a, 2002b) investigated the effect of DHN melanin (obtained from ascomycetes) when applied to the skin of two human volunteers irradiated with UVA. They found that doses of 0.005 mg/ml or lower stimulated melanogenesis while higher doses produced photoburn reactions. In my experiments, I compared the photoprotective effects in human skin cells of three different types of melanin: GHB melanin from A. bisporus, DHN melanin from G. clavigera and synthetic melanin obtained from DOPA. Significant photoprotection against high doses of UVB on HDF was offered only by A. bisporus and synthetic DOPA melanins. A positive response was observed exclusively when the compounds were present during the irradiation at relatively high concentrations in the buffer solution. This might suggest that the pigments acted mainly as a shield, possibly linked to the color of the solutions, which prevented UV from reaching the cells. Dark-colored pigments have high-intensity light absorption (Babitskaya et al., 2000) and melanin pigments can filter and attenuate radiations through absorption and scattering (Agar & Young, 2005; Kollias et al., 1991). Measurements of melanin transmission in buffer solution further support this assumption, since melanin solutions decreased transmission at 315 nm in a concentration dependent manner. The concentration of A. bisporus and synthetic DOPA melanin solutions at which the transmission dramatically decreases coincides with the lowest concentration (50 µg/ml) at which those pigments offered the cells significant UVB protection. The reduced filter effect of UV light by G. clavigera melanin might be due to a lower capacity of 	
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    absorption by this pigment. However, it cannot be concluded that this is the only reason it did not offer photoprotection to cells. Its inefficacy to protect cells against UVB damage might have other possible explanations, perhaps linked to other aspects of its photochemistry. Concentrations of A. bisporus and synthetic DOPA melanins that offered effective photoprotection to cells in my experiments are in agreement with those reported by Geng et al. (2008a). They found that a bacterial melanin (derived from DOPA) in concentrations of 100 – 400 µg/ml significantly increased the viability of xeroderma pigmentosum (XP) fibroblasts irradiated with UVA. XP is a genetic disorder involving a defect in the repair mechanism of DNA damage induced by UV (Feller et al., 2010). In pilot experiments, I tested concentrations of melanins below 25 µg/ml; however, no photoprotective effect in the cells was observed. The fact that melanin from A. bisporus and synthetic DOPA may act as physical filters has importance as ideally, a sunscreen compound should prevent UV from penetrating the skin and reaching the DNA where it can produce damage. For instance, it has been shown that in keratinocytes, melanin granules are positioned above the nucleus acting as a shield for the impingent UV and preventing the formation of DNA photoproducts (Kobayashi et al., 1998). Due to its nature, melanin pigments may act not only as physical filters of UV but also as chemical filters behaving as antioxidants (Bustamante et al., 1993; Cunha et al., 2010) and scavengers of free radicals through redox reactions and electron transfer processes (Menon & Haberman, 1977; Mosse & Marozik, 2008; Sarna & Plonka, 2005). A. bisporus and synthetic DOPA melanins were also effective in reducing ROS produced by irradiated HDF. Generation of ROS has been linked to UVB-induced toxicity in dermal fibroblasts (Bae et al., 2009; Masaki & Sakurai, 1997). Therefore, it is possible that the reduction in ROS produced by melanins from A. bisporus and synthetic DOPA contributed to the enhancement in cell viability observed when phototoxic doses of UVB were used. In experiments to assess ROS production, cells were 	
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    irradiated in the presence of melanins. The reduction measured might be due to the filter effect of the pigments to UVB and not necessarily to antioxidant activity. However, the antioxidant activity of melanin from several fungi has been demonstrated in non-cellular assays (De Cássia & Pombeiro-Sponchiado, 2005; Dong & Yao, 2012; Kumar et al., 2011; Shcherba et al., 2000). Even though I tried two different non-cellular assays to assess the antioxidant capacity of the tested melanins, precipitation of the pigments when mixed with the reagents used in the assays impeded me from analyzing any result. Results from pilot experiments (data not shown) demonstrated that if irradiation was carried out without the compounds being in the buffer, there was no photoprotective effect for any of the melanins. This might be due either to the pigments not being uptaken by the cells during the incubation period or because the amount incorporated was not effective to offer photoprotection. Nonetheless, melanins are not inert compounds. When applied in the culture medium they can be incorporated by different type of cells (Hopwood et al., 1985; Pajak et al., 1983) and thus, may affect cell viability (Grossi et al., 1998; Mosse et al., 2000; Pajak et al., 1983). It has been speculated that when melanin is added to the cells’ growth medium, some fraction may enter into the nucleus and interact with the DNA, and this might be responsible for its cytotoxicity (Schmitz et al., 1995). Apparently, the effect on cell viability that melanin has when added to culture media depends both on the concentration and length of incubation time (Pajak et al., 1983). Fungal melanins have been shown to elicit inflammatory reaction and antibody production when injected in mice (Nosanchuk et al., 1998; Nosanchuk & Casadevall, 2003), meaning that they can have important biological effects. A study by Blinova et al. (2003) evaluated the effect in cultured human keratinocytes of DHN melanin obtained from several ascomycetes. They found that the effect on proliferation was not uniform for all the melanins tested, but at a concentration of 100 µg/ml all had inhibitory effects. The results I obtained in 	
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    proliferating cells show that A. bisporus and G. clavigera melanin are both potentially cytotoxic to HDF, especially G. clavigera melanin whose IC50 value was not significantly different to that of 5-fluorouracil. The IC50 is considered to be an accurate predictor of human toxicity (Barile et al., 1994). At the concentrations tested, synthetic DOPA melanin proved to be safe for HDF. The results for DOPA melanin are in accordance with what has been reported in the literature. Grossi et al. (1998) found that melanin obtained by autoxidation from L-DOPA in concentrations up to 250 µg/ml did not affect survival in a human mammary epithelial cell line. Likewise, a study by Östergren et al. (2005) reported that synthetic DOPA melanin at concentrations up to 0.5 mg/ml did not affect cell viability of cultured neuron like PC12 cells. Table 3.2 summarizes the bioactivity of the tested melanins in the assays done with HDF.  Table 3.2 Concentrations at which melanins showed significant bioactivity in HDF  Melanin type  A. bisporus G. clavigera Synthetic (DOPA)  3.5  Cytotoxicity Inhibition of IC50 cell proliferation 20-200 !g/ml 25.3 !g/ml 20-200 !g/ml 9.7 !g/ml 200 !g/ml > 200 !g/ml  Photoprotection from UVB phototoxicity  Reduction of UVB-induced ROS  50-200 !g/ml 50-200 !g/ml  50-200 !g/ml 100-200 !g/ml  Conclusions Melanin from A. bisporus and synthetic DOPA at concentrations from 50 µg/ml  significantly blocked transmission of UVB and offered effective photoprotection to HDF. A. bisporus and synthetic DOPA melanins were also effective to reduce the production of ROS induced by UVB in HDF. The observed decrease in the production of ROS could perhaps be linked with the observed increase in survival of cells irradiated with cytotoxic doses of UBV in the presence of those melanins. 	
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    That these melanins have antioxidant activity cannot be concluded from my experiments, but this possibility cannot be disregarded either. Concentrations at which A. bisporus melanin was photoprotective are potentially cytotoxic. G. clavigera did not have any photoprotective effect in HDF, and is potentially cytotoxic. Of the three melanins tested, synthetic DOPA melanin possibly offers the safest profile for HDF.  	
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    Chapter 4: General Conclusions and Suggestions for Future Research Through this research I analyzed two melanins extracted from different classes of fungi: from a basidiomycete and from an ascomycete. According to published studies, these melanins are synthesized from different precursors (Bell & Wheeler, 1986; Rast et al., 1981). I explored some of their bioactivity, more specifically their photoprotective effect on human skin cells. Melanins are, by no means, easy compounds to study. They are diverse, complex and somehow enigmatic molecules. Despite being a heterogeneous group, they share some characteristics that are useful to identify them: dark color; general insolubility in common organic solvents, water and aqueous acid; polyphenolic nature, relative resistance to degradation and broadband absorption spectrum in the UV-visible wavelengths (Butler & Day, 1998; Menon & Haberman, 1977; Prota, 1992). All these parameters were present in the pigments that I isolated from A. bisporus and G. clavigera, therefore I define them as melanins. Additional information obtained by the IR spectra of the melanins isolated from both fungi, is also in agreement with what is reported for melanins. Due to their unique properties, the isolation of melanic pigments requires an approach that is different to the methodology commonly used for the extraction of other chemical compounds from natural sources. The acid/base treatment I used to extract the melanins from A. bisporus and G. clavigera is the main method I found in the literature used to isolate fungal melanins. There are some concerns, however, about the possibility of this method modifying the structure of the pigment (Liu et al., 2003). The fact that in nature melanins are tightly bound to other macromolecules (Nicolaus, 1968; Prota, 1992), and mainly, the lack of a defined chemical structure (Nosanchuk & Casadevall, 2003) and standards for comparison especially for fungal melanins, impede me from ascertaining that the pigments I isolated are in a ‘pure’ state. The general insolubility of melanins made it challenging to apply techniques commonly employed for characterizing chemical compounds, such as high-resolution 	
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    liquid-phase NMR (Duff et al., 1988). This was the case for A. bisporus melanin whose insolubility in DMSO made it impossible to obtain a NMR spectrum. The NMR spectrum obtained for G. clavigera confirmed data obtained with the IR spectrum and revealed more details about the molecule. This is important, considering that there are no published reports analyzing the chemical structure of melanin from this fungus. Solid-phase NMR has started to be applied to study fungal melanins (Knicker et al., 1995) and it would have been a useful technique for me to better characterize both melanins, but still it is not widely available. Time and resource constraints prevented me from further characterizing the fungal melanins that I isolated. However, the differences found in the general physicochemical characteristics and IR spectra of the pigments from A. bisporus and G. clavigera allowed me to conclude that they are different types of melanin. The ability to absorb and scatter light through a wide range of wavelengths, and the free radical character are properties apparently common to all melanins (Bell & Wheeler, 1986; Enochs et al., 1993; Menon & Haberman, 1977), and have been linked to their role in photoprotection (Kollias et al., 1991; Menon & Haberman, 1977). Therefore, it was my hypothesis that melanins different to DOPA derived could offer effective photoprotection to human skin cells. It is known also, that eumelanin and pheomelanin absorb photons of UV and visible light and convert the energy into heat (Meredith & Sarna, 2006). The same property could apply to other types of melanins. It seems, however, that the effectiveness to protect cells from UV damage varies according to the type of melanin. For instance, pheomelanin has been linked to photosensitization reactions (Takeuchi et al., 2004) and its photoabsorbing capacity is considered to be insignificant (Ortonne, 2002). The two isolated fungal melanins also showed different responses in the bioassays I used in skin fibroblasts. A. bisporus behaved similarly to synthetic DOPA melanin, decreasing 	
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    damage and percent of ROS induced by UBV in HDF. The photoprotection offered by A. bisporus and synthetic DOPA melanins may be due to their capacity to efficiently filter UVB. In fact, at concentrations at which they offered protection to HDF against UVB, they were able to almost completely block transmission of UV. To confirm that the photoprotection offered to HDF was really due to a filter effect, experiments using same conditions to compare their effect to known UVB absorbers such as menthylanthranilate or 2-phenyl-benzimidazole-5-sulfonic acid (Pathak & Fitzpatrick, 1993) should be done. On the other hand, I did not observe any positive effect from G. clavigera melanin in the assays I used in human dermal fibroblasts to assess photoprotection. Differences in light absorption properties might be linked to this, but the possibility of photolysis, as reported with pheomelanin (Chedekel et al., 1978) or other types of photochemical reactions, cannot be ruled out. Both fungal melanins demonstrated potential cytotoxicity for skin fibroblasts. The fact that the concentrations at which A. bisporus melanin was effective to offer photoprotection also showed to affect cell proliferation is a drawback regarding the possibility of its application in human skin. However, conducting cytotoxicity tests in keratinocytes would be required to better assess potential problems of skin toxicity since these are the cells which actually are in contact with products applied onto the skin. Also, that the observed cytotoxicity could be due to ‘impurities’ that remained attached to the melanins after their isolation from the fungi, cannot be completely ruled out. There are some difficulties related to the chemical nature of melanins and their possible applications in biological systems. One of them is their aforementioned general insolubility. Hence one problem I had to address was finding a solvent system I could use for the experiments with human skin cells. On the other hand, the dark color of solutions with high concentrations of melanin makes it very difficulty to know if the pigment is completely dissolved in the solvent. 	
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    This limited the range of concentrations I could test for cytotoxicity, which prevented me from obtaining the IC50 value for synthetic DOPA melanin. However, unlike the two fungal melanins tested in this study, synthetic DOPA melanin seems to be safe to use in human skin cells. Another factor when considering possible applications of melanins to products for use in human skin is the lack of standardized procedures for the extraction and characterization of melanins extracted from natural sources, which would not guarantee uniform compounds. Nonetheless, melanins have many potential applications that warrant further exploration and study. Their antioxidant activity for instance, which I could not reach to assess properly, has been reported by several authors (De Cássia & Pombeiro-Sponchiado, 2005; Dong & Yao, 2012; Sichel et al., 1991) and appears to be important for their biological role as photoprotective molecules. From the results I obtained, I dare to say that the origin and the chemistry of a melanin, possibly have a major influence on its activity in a specific biological system. A better understanding regarding the structure of these pigments and how the differences in their chemistry might influence their properties could elucidate the scope of their biological activity.  	
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    Olennikov, D.N., Agafonova, S.V., Stolbikova, A.V., & Rokhin, A.V. (2011). Melanin of Laetiporus sulphureus (Bull.: Fr.) Murr sterile form. Applied Biochemistry and Microbiology, 47, 298-303. Orlow, S.J., Osber, M.P., & Pawelek, J.M. (1992). Synthesis and characterization of melanins from dihydroxyindole-2-carboxylic acid and dihydroxyindole. Pigment Cell Research, 5, 113121. Ortonne, J.P. (2002). Photoprotective properties of skin melanin. British Journal of Dermatology, 146 (Suppl. 61), 7-10. Östergren, A., Svensson, A.L., Gunnar, L., & Brittebo, E.B. (2005). Dopamine melanin-loaded PC12 cells: a model for studies on pigmented neurons. Pigment Cell Research, 18, 306-314. Pajak, S., Hopwood, L.E., Hyde, J.S., Felix, C.C., Sealy, R.C., Kushnaryov, V.M., & Hatchell, M.C. (1983). Melanin endocytosis by cultured mammalian cells. A model for melanin in a cellular environment. Experimental Cell Research, 149, 513-526. Paramonov, B.A., Turkovskii, I.I., Potokin, I.L., & Chebotarev, V.Y. (2002a). Photoprotective activity of melanin preparations from black yeast-like fungus during UV irradiation of human skin: dependence on previous photoexposure. Bulletin of Experimental Biology and Medicine, 134, 366-369. Paramonov, B.A., Turkovskii, I.I., Potokin, I.L., & Chebotarev, V.Y. (2002b). Photoprotective activity of melanin preparations from black yeast-like fungus during UV irradiation of human skin: dependence on the concentration. Bulletin of Experimental Biology and Medicine, 133, 377-379. Pathak, M., & Fitzpatrick, T. (1993). Preventive treatment of sunburn, dermatoheliosis, and skin cancer with sun protective agents. In: T. Fitzpatrick, A. Eisen, K. Wolff, I. Freedberg & K.F. Austen (Eds.), Dermatology in General Medicine, (4th ed.), Vol. 1 (1689-1717). New York: McGraw Hill. Peus, D., Vasa, R.A., Meves, A. Pott, M., Beyerle, A., Squillace, K., & Pittelkow, M.R. (1998). H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes. The Journal of Investigative Dermatology, 110, 966-971. Piatelli, M., Fattorusso, E., Nicolaus, R.A., & Magno, S. (1965). The structure of melanins and melanogenesis. Tetrahedron, 21, 3229-3236. Pierce, J.A., & Rast, D. M. (1995). A comparison of native and synthetic mushrooms melanins by fourier-transform infrared spectroscopy. Phytochemistry, 39, 49-55. Pinnel, S.R. (2003). Cutaneous photodamage, oxidative stress, and topical antioxidant protection. Journal of the American Academy of Dermatology, 48, 1-19.  	
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    Appendix I: 1H NMR spectrum of G. clavigera melanin Figure A-I.1 1H NMR spectrum The spectrum was adquired at 600 MHz in a Bruker Avance-600 spectrometer using a cryoprobe, and DMSO as solvent at 2.5 ppm.  Carolina Olaizola G clavigera (blue cap) 1H spectrum ref to DMSO 2.5 ppm  Current Data Parameters NAME co1557 EXPNO 1 PROCNO 1  5.340 5.325 5.315 5.305 5.292 3.458 2.763 2.736 2.724 2.713 2.500 2.185 2.173 2.161 2.011 1.999 1.987 1.974 1.466 1.291 1.242 1.222 0.928 0.916 0.903 0.853  JOB NO:1557 co1557 1 1  F2 − Acquisition Parameters Date_ 20101112 Time 5.04 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG zg30 TD 32768 SOLVENT DMSO NS 32 DS 2 SWH 8389.262 Hz FIDRES 0.256020 Hz AQ 1.9530824 sec RG 8 DW 59.600 usec DE 6.00 usec TE 298.0 K D1 1.00000000 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 1H P1 9.20 usec PL1 3.50 dB SFO1 600.1536009 MHz F2 − Processing parameters SI 65536 SF 600.1500063 MHz WDW EM SSB 0 LB 0.50 Hz GB 0 PC 1.00  6  5  4  3  2  1  0  ppm  1.31 9.38 0.53 2.12  7  1.03 1.52  8  0.85  9  92.02  10  1.00  11  1.98  12  	
   	
   	
   	
   	
   	
    	
    78	
    Figure A-I.2 1H NMR spectrum, zoom of figure A-I.1  	
   	
    	
    79	
    Figure A-I.3 1H NMR spectrum, frequency expansion in the region from 0.50 to 1.00 ppm 	
    	
    80	
    Figure A-I.4 1H NMR spectrum, frequency expansion in the region from 1.10 to 1.60 ppm 	
    Current Data Parameters NAME co1557 EXPNO 1 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.04 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG zg30 TD 32768 SOLVENT DMSO NS 32 DS 2 SWH 8389.262 Hz FIDRES 0.256020 Hz AQ 1.9530824 sec RG 8 DW 59.600 usec DE 6.00 usec TE 298.0 K D1 1.00000000 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec  1.222  1.242  1.291  Carolina Olaizola G clavigera (blue cap) 1H spectrum ref to DMSO 2.5 ppm  1.466  JOB NO:1557 co1557 1 1  ======== CHANNEL f1 ======== NUC1 1H P1 9.20 usec PL1 3.50 dB SFO1 600.1536009 MHz F2 − Processing parameters SI 65536 SF 600.1500063 MHz WDW EM SSB 0 LB 0.50 Hz GB 0 PC 1.00  1.50  1.45  1.40  1.35  1.30  1.25  1.20  1.15  1.10  ppm  9.38  1.55  1.31  1.60  	
   	
   	
    	
    81	
    Figure A-I.5 1H NMR spectrum, frequency expansion in the region from 1.85 to 2.40 ppm 	
    	
   	
   	
    	
    82	
    Figure A-I.6 1H NMR spectrum, frequency expansion in the region from 2.5 to 4.9 ppm	
   	
    	
   	
   	
   	
    	
    83	
    Figure A-I.7 1H NMR spectrum, frequency expansion in the region from 4.8 to 6.0 ppm 	
    	
    	
    84	
    Appendix II: 13C NMR spectrum of G. clavigera melanin 13  Figure A-II.1 C NMR spectrum The spectrum was adquired at 600 MHz in a Bruker Avance-600 spectrometer using a cryoprobe, and DMSO as solvent at 39.51 ppm.  	
    85	
    13  Figure A-II.2 C NMR spectrum, frequency expansion in the region from 13.1 to 15.0 ppm  	
    86	
    13  Figure A-II.3 C NMR spectrum, frequency expansion in the region from 21.2 to 23.1 ppm  21.96  Carolina Olaizola G clavigera (blue cap) 13C{1H} spectrum ref to DMSO 39.51 ppm  22.08  JOB NO:1557 co1557 2 1  Current Data Parameters NAME co1557 EXPNO 2 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.19 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG carbon_spinecho_sp TD 32768 SOLVENT MeOD NS 2048 DS 4 SWH 37593.984 Hz FIDRES 1.147277 Hz AQ 0.4358777 sec RG 23170.5 DW 13.300 usec DE 33.25 usec TE 298.0 K D1 1.00000000 sec d11 0.03000000 sec D20 0.00010000 sec d21 0.00011500 sec DELTA 0.89999998 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 13C P1 15.00 usec P8 2000.00 usec PL1 −1.40 dB SFO1 150.9229288 MHz SP13 2.18 dB SPNAM13 Crp60comp.4 SPOFF13 0.00 Hz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 100.00 usec PL2 3.50 dB PL12 24.22 dB PL13 25.00 dB SFO2 600.1530000 MHz F2 − Processing parameters SI 32768 SF 150.9079094 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40  23.1  	
    23.0  22.9  22.8  22.7  22.6  22.5  22.4  22.3  22.2  22.1  22.0  21.9  21.8  21.7  21.6  21.5  21.4  21.3  21.2  ppm  87	
    13  Figure A-II.4 C NMR spectrum, frequency expansion in the region from 23.8 to 25.6 ppm  24.46  25.10  Carolina Olaizola G clavigera (blue cap) 13C{1H} spectrum ref to DMSO 39.51 ppm  25.21  JOB NO:1557 co1557 2 1  Current Data Parameters NAME co1557 EXPNO 2 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.19 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG carbon_spinecho_sp TD 32768 SOLVENT MeOD NS 2048 DS 4 SWH 37593.984 Hz FIDRES 1.147277 Hz AQ 0.4358777 sec RG 23170.5 DW 13.300 usec DE 33.25 usec TE 298.0 K D1 1.00000000 sec d11 0.03000000 sec D20 0.00010000 sec d21 0.00011500 sec DELTA 0.89999998 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 13C P1 15.00 usec P8 2000.00 usec PL1 −1.40 dB SFO1 150.9229288 MHz SP13 2.18 dB SPNAM13 Crp60comp.4 SPOFF13 0.00 Hz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 100.00 usec PL2 3.50 dB PL12 24.22 dB PL13 25.00 dB SFO2 600.1530000 MHz F2 − Processing parameters SI 32768 SF 150.9079094 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40  25.6  	
    25.5  25.4  25.3  25.2  25.1  25.0  24.9  24.8  24.7  24.6  24.5  24.4  24.3  24.2  24.1  24.0  23.9  23.8  ppm  88	
    13  Figure A-II.5 C NMR spectrum, frequency expansion in the region from 25.9 to 27.6 ppm  	
    89	
    13  Figure A-II.6 C NMR spectrum, frequency expansion in the region from 28.1 to 29.5 ppm  28.51  28.57  28.71 28.69  28.80  28.88  29.01 28.99 28.96  Carolina Olaizola G clavigera (blue cap) 13C{1H} spectrum ref to DMSO 39.51 ppm  29.06  JOB NO:1557 co1557 2 1  Current Data Parameters NAME co1557 EXPNO 2 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.19 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG carbon_spinecho_sp TD 32768 SOLVENT MeOD NS 2048 DS 4 SWH 37593.984 Hz FIDRES 1.147277 Hz AQ 0.4358777 sec RG 23170.5 DW 13.300 usec DE 33.25 usec TE 298.0 K D1 1.00000000 sec d11 0.03000000 sec D20 0.00010000 sec d21 0.00011500 sec DELTA 0.89999998 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 13C P1 15.00 usec P8 2000.00 usec PL1 −1.40 dB SFO1 150.9229288 MHz SP13 2.18 dB SPNAM13 Crp60comp.4 SPOFF13 0.00 Hz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 100.00 usec PL2 3.50 dB PL12 24.22 dB PL13 25.00 dB SFO2 600.1530000 MHz F2 − Processing parameters SI 32768 SF 150.9079094 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40  29.5  	
    29.4  29.3  29.2  29.1  29.0  28.9  28.8  28.7  28.6  28.5  28.4  28.3  28.2  28.1  ppm  90	
    13  Figure A-II.7 C NMR spectrum, frequency expansion in the region from 30.6 to 32.0 ppm  	
    91	
    13  Figure A-II.8 C NMR spectrum, frequency expansion in the region from 33.2 to 34.6 ppm  33.64  Carolina Olaizola G clavigera (blue cap) 13C{1H} spectrum ref to DMSO 39.51 ppm  33.91  JOB NO:1557 co1557 2 1  Current Data Parameters NAME co1557 EXPNO 2 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.19 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG carbon_spinecho_sp TD 32768 SOLVENT MeOD NS 2048 DS 4 SWH 37593.984 Hz FIDRES 1.147277 Hz AQ 0.4358777 sec RG 23170.5 DW 13.300 usec DE 33.25 usec TE 298.0 K D1 1.00000000 sec d11 0.03000000 sec D20 0.00010000 sec d21 0.00011500 sec DELTA 0.89999998 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 13C P1 15.00 usec P8 2000.00 usec PL1 −1.40 dB SFO1 150.9229288 MHz SP13 2.18 dB SPNAM13 Crp60comp.4 SPOFF13 0.00 Hz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 100.00 usec PL2 3.50 dB PL12 24.22 dB PL13 25.00 dB SFO2 600.1530000 MHz F2 − Processing parameters SI 32768 SF 150.9079094 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40  34.6  	
    34.5  34.4  34.3  34.2  34.1  34.0  33.9  33.8  33.7  33.6  33.5  33.4  33.3  33.2  ppm  92	
    13  Figure A-II.9 C NMR spectrum, frequency expansion in the region from 126.4 to 129.2 ppm  126.96  127.55  127.76 127.74  Carolina Olaizola G clavigera (blue cap) 13C{1H} spectrum ref to DMSO 39.51 ppm 127.93 127.88  JOB NO:1557 co1557 2 1  Current Data Parameters NAME co1557 EXPNO 2 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.19 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG carbon_spinecho_sp TD 32768 SOLVENT MeOD NS 2048 DS 4 SWH 37593.984 Hz FIDRES 1.147277 Hz AQ 0.4358777 sec RG 23170.5 DW 13.300 usec DE 33.25 usec TE 298.0 K D1 1.00000000 sec d11 0.03000000 sec D20 0.00010000 sec d21 0.00011500 sec DELTA 0.89999998 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 13C P1 15.00 usec P8 2000.00 usec PL1 −1.40 dB SFO1 150.9229288 MHz SP13 2.18 dB SPNAM13 Crp60comp.4 SPOFF13 0.00 Hz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 100.00 usec PL2 3.50 dB PL12 24.22 dB PL13 25.00 dB SFO2 600.1530000 MHz F2 − Processing parameters SI 32768 SF 150.9079094 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40  129.2  	
    129.0  128.8  128.6  128.4  128.2  128.0  127.8  127.6  127.4  127.2  127.0  126.8  126.6  126.4  ppm  93	
    13  Figure A-II.10 C NMR spectrum, frequency expansion in the region from 129.4 to 131.6 ppm  	
    94	
    13  Figure A-II.11 C NMR spectrum, frequency expansion in the region from 173.5 to 176.5 ppm  Carolina Olaizola G clavigera (blue cap) 13C{1H} spectrum ref to DMSO 39.51 ppm 174.53  JOB NO:1557 co1557 2 1  Current Data Parameters NAME co1557 EXPNO 2 PROCNO 1 F2 − Acquisition Parameters Date_ 20101112 Time 5.19 INSTRUM av600cp PROBHD 5 mm CPTCI 1H− PULPROG carbon_spinecho_sp TD 32768 SOLVENT MeOD NS 2048 DS 4 SWH 37593.984 Hz FIDRES 1.147277 Hz AQ 0.4358777 sec RG 23170.5 DW 13.300 usec DE 33.25 usec TE 298.0 K D1 1.00000000 sec d11 0.03000000 sec D20 0.00010000 sec d21 0.00011500 sec DELTA 0.89999998 sec MCREST 0.00000000 sec MCWRK 0.01500000 sec ======== CHANNEL f1 ======== NUC1 13C P1 15.00 usec P8 2000.00 usec PL1 −1.40 dB SFO1 150.9229288 MHz SP13 2.18 dB SPNAM13 Crp60comp.4 SPOFF13 0.00 Hz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 100.00 usec PL2 3.50 dB PL12 24.22 dB PL13 25.00 dB SFO2 600.1530000 MHz F2 − Processing parameters SI 32768 SF 150.9079094 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40  176.5  	
    176.0  175.5  175.0  174.5  174.0  173.5  ppm  95	
    Appendix III: Biosynthetic pathways of melanins 	
   Figure A-III.1 Schematic pathway of DOPA melanin biosynthesis (Adapted from Bell & Wheeler, 1986)  Tyrosine Tyrosinase 3,4-dihydroxyphenylalanine (DOPA) Tyrosinase Dopaquinone  Leucodopachrome  Dopachrome  5.6-dihydroxyindole  Indole-5,6-quinone  Melanochrome  Melanin  	
    96	
    Figure A-III.2 Schematic pathway of GHB melanin biosynthesis (Adapted from Rast et al., 1981)  Intermediate from the shikimate-chorismate pathway  !-glutaminyl-4-hydroxybencene (GHB) Peroxidase/phenolase !-glutaminyl-3,4-dihydroxybencene (GDHB) Peroxidase/phenolase  !-glutaminyl-3,4-benzoquinone (GBQ) Non-enzymatic polymerization Melanin  	
    97	
    Figure A-III.3 Schematic pathway of DHN melanin biosynthesis (Adapted from Butler & Day, 1998) 	
   	
    Acetate Polyketide synthase 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8,- THN) Reductase Scytalone Dehydratase 1,3,8-trihydroxynaphthalene (1,3,8-THN) Reductase Vermelone Dehydratase 1,8-dihydroxynaphthalene (1,8-DHN) Polymerase Melanin  	
    98	
    

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