The histological analysis,colorimetric evaluation,and chemical quantification of melanin content in ‘suntanned’ fish

Human melanocytes respond to UV irradiation by increasing the synthesis of melanin. While much is now understood of the pathways governing this process and the nature of the melanin synthesized, little is known of melanins produced by lower vertebrates and their capacity to respond to UV. Here we report that a fish, red seabream, can undergo ‘suntanning’. Histological, colorimetric and chemical assays were performed for suntanned red seabream fish bred in net cages to analyse the melanins and compared with shaded or wild red seabream fish. For color evaluation, the L* values of suntanned fish were dramatically lower than those in the other two groups. Pyrrole‐2,3,5‐tricarboxylic acid (PTCA), an indicator of eumelanin, was detected in suntanned fish at five times higher levels than in shaded or wild fish while 4‐amino‐3‐hydroxyphenyl‐alanine (4‐AHP), a marker for pheomelanin, could not be detected in any of the samples. Histological analysis showed that melanocytes in the suntanned skin enlarged and increased in number to form a monolayer at the surface of the skin. Analysis of L* values and PTCA levels showed quite a high correlation coefficient (r = ?0.843). When comparing shaded and wild red seabream fish, the scores were closer but some significant differences were still found in some body areas. These results indicate that eumelanin accumulates in suntanned fish during the increase in skin color, which is induced by sunlight, presumably by ultraviolet radiation.     

Quantitative Analysis of Eumelanin and Pheomelanin in Humans,Mice, and Other Animals: a Comparative Review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole‐2,3,5‐tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

Structural and functional analyses of a glycoside hydrolase family 5 enzyme with an unexpected β-fucosidase activity

We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B(1)4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a β-D-fucosidase. We also present the 2.2 ? resolution structure of PbFucA and use structure-based mutational analysis to confirm the role of catalytically essential residues. A comparison of the active sites of PbFucA with those of family 5 and 51 glycosidases reveals that while the essential catalytic framework is identical between these enzymes, the steric contours of the respective active site clefts are distinct and likely account for substrate discrimination. Our results show that members of this cluster of orthologous group (COG) 5520 have β-D-fucosidase activities, despite showing an overall sequence and structural similarity to GH-5 xylanases.     

The role of metals in site-specific DNA damage with reference to carcinogenesis

We reviewed the mechanism of oxidative DNA damage with reference to metal carcinogenesis and metal-mediated chemical carcinogenesis. On the basis of the finding that chromium (VI) induced oxidative DNA damage in the presence of hydrogen peroxide (H2O2), we proposed the hypothesis that endogenous reactive oxygen species play a role in metal carcinogenesis. Since then, we have reported that various metal compounds, such as cobalt, nickel, and ferric nitrilotriacetate, directly cause site-specific DNA damage in the presence of H2O2. We also found that carcinogenic metals could cause DNA damage through indirect mechanisms. Certain nickel compounds induced oxidative DNA damage in rat lungs through inflammation. Endogenous metals, copper and iron, catalyzed ROS generation from various organic carcinogens, resulting in oxidative DNA damage. Polynuclear compounds, such as 4-aminobiphenyl and heterocyclic amines, appear to induce cancer mainly through DNA adduct formation, although their N-hydroxy and nitroso metabolites can also cause oxidative DNA damage. On the other hand, mononuclear compounds, such as benzene metabolites, caffeic acid, and o-toluidine, should express their carcionogenicity through oxidative DNA damage. Metabolites of certain carcinogens efficiently caused oxidative DNA damage by forming NADH-dependent redox cycles. These findings suggest that metal-mediated oxidative DNA damage plays important roles in chemical carcinogenesis.     

Human tyrosinase is able to oxidize both enantiomers of rhododendrol

Racemic RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a topical skin‐whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD‐quinone, which is quickly converted to RD‐cyclic quinone and RD‐hydroxy‐p‐quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(?)‐RD and S(+)‐RD enantiomers. In the presence of a catalytic amount of l ‐dopa, human tyrosinase, which can oxidize l ‐tyrosine but not d ‐tyrosine, was found to oxidize both R(?)‐ and S(+)‐RD to give RD‐catechol and its oxidation products. S(+)‐RD was more effectively oxidized than l ‐tyrosine, while R(?)‐RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase‐catalyzed conversion to toxic quinones and the concomitant production of reactive oxygen species.     

Radical production and DNA damage induced by carcinogenic 4-hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus

4-Hydrazinobenzoic acid, an ingredient of mushroom Agaricus bisporus, is carcinogenic to rodents. To clarify the mechanism of carcinogenesis, we investigated DNA damage by 4-hydrazinobenzoic acid using ³²P-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes. 4-Hydrazinobenzoic acid induced Cu(II)-dependent DNA damage especially piperidine-labile formation at thymine and cytosine residues. Typical hydroxyl radical scavengers showed no inhibitory effects on Cu(II)-mediated DNA damage by 4-hydrazinobenzoic acid. Bathocuproine and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H₂O₂ in the DNA damage. These findings suggest that H₂O₂ generated by the autoxidation of 4-hydrazinobenzoic acid reacts with Cu(I) to form reactive oxygen species, capable of causing DNA damage. Interestingly, catalase did not completely inhibit DNA damage caused by a high concentration of 4-hydrazinobenzoic acid (over 50 μM) in the presence of Cu(II). 4-Hydrazinobenzoic acid induced piperidine-labile sites frequently at adenine and guanine residues in the presence of catalase. 4-Hydrazinobenzoic acid increased formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA, whereas 4-hydrazinobenzoic acid did not increase the formation of 8-oxodG in the presence of catalase. ESR spin-trapping experiments showed that the phenyl radical was formed during the reaction of 4-hydrazinobenzoic acid in the presence of Cu(II) and catalase. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF/mass) spectrometry analysis showed that phenyl radical formed adduct with adenosine and guanosine. These results suggested that 4-hydrazinobenzoic acid induced DNA damage via not only H₂O₂ production but also phenyl radical production. This study suggests that both oxidative DNA damage and DNA adduct formation play important roles in the expression of carcinogenesis of 4-hydrazinobenzoic acid.     

Decrease in the particle size of low-density lipoprotein (LDL) by oxidation

A radical reaction of low-density lipoprotein (LDL) causes fragmentation and cross-link of apolipoprotein B-100 (apoB). LDL (50 microg/ml) was subjected to the well-studied oxidation with Cu(2+) (1.67 microM). The concentration of alpha-tocopherol decreased to 10% of the initial level during the first 30 min. After this lag time, the conjugated diene content, as measured by absorption at 234 nm, started increasing and the residual apoB at 512 kDa determined by immunoblot after SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) was also decreased. The particle size of LDL determined by nondenaturing gradient gel electrophoresis decreased steadily during the initial 120 min, when residual native apoB was only 30% of the initial level. Plasma was also oxidized with Cu(2+) (400 microM). Under this condition, a clear lag time was not observed and alpha-tocopherol content, apoB, and the LDL particle size were decreased simultaneously. Based on these experiments, we propose that an oxidation reaction is involved in the formation of small dense LDL.     

Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation

Hair pigmentation is one of the most conspicuous phenotypes in humans. Melanocytes produce two distinct types of melanin pigment: brown to black, indolic eumelanin and yellow to reddish brown, sulfur‐containing pheomelanin. Biochemically, the precursor tyrosine and the key enzyme tyrosinase and the tyrosinase‐related proteins are involved in eumelanogenesis, while only the additional presence of cysteine is necessary for pheomelanogenesis. Other important proteins involved in melanogenesis include P protein, MATP protein, α‐MSH, agouti signaling protein (ASIP), MC1R (the receptor for MSH and ASIP), and SLC7A11, a cystine transporter. Many studies have examined the effects of loss‐of‐function mutations of those proteins on mouse coat color pigmentation. In contrast, much less is known regarding the effects of mutations of the corresponding proteins on human hair pigmentation except for MC1R polymorphisms that lead to pheomelanogenesis. This perspective will discuss what we have/have not learned from mouse coat color pigmentation, with special emphasis on the significant roles of pH and the level of cysteine in melanosomes in controlling melanogenesis. Based on these data, a hypothesis is proposed to explain the diversity of human hair pigmentation.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.