About this Journal Submit a Manuscript Table of Contents
Research Letters in Physical Chemistry
Volume 2008 (2008), Article ID 210616, 4 pages
http://dx.doi.org/10.1155/2008/210616
Research Letter

Pigment Melanin Scavenges Nitric Oxide In Vitro: Possible Relevance to Keloid Formation

1Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495, USA
2Department of Pharmacology and Toxicology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495, USA
3Department of Internal Medicine, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495, USA
4Clinical Research Center, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310-1495, USA

Received 9 May 2008; Accepted 2 August 2008

Academic Editor: Werner Nau

Copyright © 2008 Julian M. Menter et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Recently, nitric oxide (NO) has been implicated in the formation of keloids, preferentially formed in dark-skinned persons, and we suspected that pigment melanin itself may play a direct role by adsorbing NO. We tested the ability of cuttlefish sepia melanin to scavenge (adsorb) NO, generated in situ by 2-(N.N Diethylamino) diazeneolate-2-oxide (DEA/NO), through a dialysis membrane. NO was measured as N O 2 _ and N O 3 _ by the Griess method and as N 2 O 3 by trapping experiments with the fluorogenic substrate 4,5-diaminofluorescein (DAF-2). Initial N O 2 _ and N O 3 _ concentrations were significantly lower in the test dialyzates than in controls. Scavenging of NO was rapid enough to compete with DAF adduct formation. Both analytical methods gave comparable results. Adsorbed NO and/or its oxidized products may undergo interactions with melanin, adsorbed O 2 , and/or dermal material that may lead to keloid formation.

1. Introduction

Melanin pigments are responsible for epidermal skin coloring in humans. Melanin’s broad optical absorption and stable free radical properties and binding capability make it an effective in vivo antioxidant [1, 2] photoprotective agent [1, 3], electron transfer agent [46], with semiconductor properties [6]. People of color are particularly susceptible to keloids, a recalcitrant consequence of aberrant wound healing, characterized by excessive collagen deposition that extends beyond the original wound [79]. Recently, nitric oxide (NO) has been implicated as a key player in the formation of keloids and hypertrophic scars [10] by stimulating over-expression of inducible nitric oxide synthase (iNOS) [11] which is in proximity to melanocytes and dermal collagen [11]. That NO can diffuse through biological membranes [12] suggests that these reactive species can reach the melanosomes within nearby melanocytes, so that NO adsorption to melanin and its sequelae may play a role in keloid formation, particularly in dark-skinned individuals.

2. Materials and Methods

Sepia melanin, [13], ( M e l a n I n k @ ) was predialyzed through a Spectropore membrane (MW cutoff 6–8 kD) into 100 mL 0.1 M phosphate buffer, pH 7.4/0.1 M EDTA, followed by two changes of 0.1 M buffer alone. As a source of exogenous NO, we used DEA/NO (see Figure 1) (Sigma Chemical Co., Mo, USA). 200 mL of freshly made 0.9 mM DEA/NO stock solution (0.18 μmole) was placed into each of two stirred 25 mL graduated cylinders containing dialysis bags (Spectrum Laboratories, Inc., Calif, USA, MW cutoff 6–8 Kd) filled with (a) 3 mL of a 90 mg melanin buffer suspension (“melanin bag”) or (b) 3 mL buffer alone “control bag.” We measured DEA/NO generated—NO in 200 μL aliquots (1.44 nmole) of dialyzate test and control samples 𝑡 = 0 - 9 0  minutes as nitrite and nitrate by the Griess method with a SOFT maxPRO NO—measuring kit (Molecular Devices). Control experiments confirmed that no melanin escaped into the dialyzate, and there was no significant contamination by nitrite or nitrate prior to addition of DEA/NO.

210616.fig.001
Figure 1: Structure of DEA/NO. The sodium salt ( 𝑅 = 1 ) was used in the Griess analysis experiments. The diethylammonium salt ( 𝑅 = 2 ) was used for the trapping of NO with the fluorogenic DAF-2 to form the highly fluorescent DAF-2T.

NO was also detected by its ability of its oxidation product, N 2 O 3 , to form highly fluorescent triazoles (DAF-2T) from 4, 5-diaminofluorescein (DAF-2) in the presence of molecular O 2 [14] (see Figure 2). Initially, 10.0 nmole of DAF-2 in buffer solution was mixed in disposable fluorescence cuvettes with the appropriate amount of added buffer to make a total volume of 2.0 mL. After further addition of 1.0 mL of DEA/NO (diethylammonium salt), the fluorescence intensity of melanin and control dialyzates was monitored as functions of time on a Perkin-Elmer 650–40 fluorescence spectrophotometer ( 𝜆 e x = 4 9 5 n m , 𝜆 e m = 5 1 5 n m ). The fluorescence scavenging ratio was the ratio of fluorescence intensities of melanin and control bag dialyzates under steady-state conditions. In some experiments, a 20 mM melanin suspension in the absence of a dialysis membrane was analyzed as before. These latter results were corrected for the absorption and emission of melanin [15].

210616.fig.002
Figure 2: Reaction of DAF with NO to form the highly fluorescent DAF-2T.

𝑇 -tests were conducted to determine the statistical significance of the results. The determined 𝑃 -values assume normal distributions for each group.

3. Results

3.1. NO Measurement as Nitrite and Nitrate

Pigment melanin rapidly sequesters nitric oxide (see Figures 3(a) and 3(b)). At 𝑡 = 0 , the m e a n ± S . D . of 3 experiments afforded scavenging ratios of 0 . 5 0 3 ± 0 . 2 3 2 ( 𝑃 < . 0 3 ) for N O 2 and 0 . 3 2 3 ± 0 . 2 6 6 for N O 3 ( 𝑃 < . 0 2 ). Initial [ N O 3 ]/ [ N O 2 ] ratios were ~1.5.

fig3
Figure 3: (a) Determination of NO as N O 2 by Griess analysis in 0.1 M phosphate buffer, pH 7.4. Black circles: initial amount of N O = 1 . 4 4  nmole in “melanin bag” (see text). White circles: initial amount of 1.44 nmole in the absence of melanin (“blank bag”; see text). M e a n ± S . D . of 3 determinations, 𝑃 < . 0 3 . (b) Determination of NO as N O 3 by Griess analysis in 0.1 M phosphate buffer, pH 7.4 under the same conditions as in Figure 3(a). Black circles: “melanin bag.” White circles: “blank bag”; see text. M e a n ± S . D . of 3 determinations, 𝑃 < . 0 2 .
3.2. Fluorescence Measurements

The fluorescence intensities (i.e., DAF-2T formation) of both control and test samples increased to a steady-state value at 𝑡 2 0 minutes (see Figure 4). Melanin competes successfully with DAF for NO as evidenced by a steady-state fluorescence scavenging 𝑟 𝑎 𝑡 𝑖 𝑜 = 0 . 7 0 6 ± 0 . 1 0 3 ( 𝑛 = 4 ; 𝑃 < . 0 0 5 4 ). These results were qualitatively the same whether dialysis systems or melanin suspensions were used.

210616.fig.004
Figure 4: Uptake of NO by sepia melanin as determined by trapping with DAF in 0.1 M phosphate buffer, pH 7.4 (see text). Black Circles: with added melanin (20 μM in 3 mL). White circles: without added melanin M e a n ± S . D . of 4 determinations, 𝑃 < . 0 0 5 .

4. Discussion

Melanin scavenging of NO takes place rapidly (see Figures 3(a) and 3(b)). The lower steady-state fluorescence intensity in the presence of melanin (see Figure 4) confirms that scavenging competes successfully with the relatively slow [14] triazole formation, and strongly suggests that NO itself is initially adsorbed to melanin. Adsorbed NO can form a variety of active species in oxygen—containing solution including N O 2 , N 2 O 3 , O 2 , and O N O O , and could change melanin’s oxidation state [13, 16]. Any of these might stimulate keloid formation and might offer a basis for the observation that African-Americans, with higher and more robust concentrations of melanosomes, are more susceptible to keloids than are Caucasians.

Reaction of gaseous NO in solution with H 2 O 2 and/or O 2 arising from melanin autoxidation [17] is unlikely to be significant. Since melanin acts as an efficient “pseudodismutase” [17], the steady-state [ O 2 ] is low outside the melanin “cage.” Hydrogen peroxide does not react with NO [18].

Acknowledgments

The authors gratefully acknowledge the support from MBRS Grant no. GM08248 and RCMI Grant no. RR 03034. They also thank Dr. Gianluca Tosini for critically reading the manuscript. This work was presented in preliminary form at the 4th L'Oreal Symposium on Ethnic Hair and Skin, Miami, Fla, USA November 9-11, 2007.

References

  1. M. R. Chedekel, “Photophysics and photochemistry of melanin,” in Melanin: Its Role in Human Photoprotection, pp. 11–22, Valdenmar, Overland Park, Kan, USA, 1994.
  2. X. Zhang, C. Erb, J. Flammer, and W. M. Nau, “Absolute rate constants for the quenching of reactive excited states by melanin and related 5,6-dihydroxyindole metabolites: implications for their antioxidant activity,” Photochemistry and Photobiology, vol. 71, no. 5, pp. 524–533, 2000. View at Publisher · View at Google Scholar
  3. Y. Yamaguchi, M. Brenner, and V. J. Hearing, “The regulation of skin pigmentation,” The Journal of Biological Chemistry, vol. 282, no. 38, pp. 27557–27561, 2007. View at Publisher · View at Google Scholar · View at PubMed
  4. J. M. Menter and I. Willis, “Electron transfer and photoprotective properties of melanins in solution,” Pigment Cell Research, vol. 10, no. 4, pp. 214–217, 1997. View at Publisher · View at Google Scholar
  5. P. R. Crippa, “Oxygen adsorption and photoreduction on fractal melanin particles,” Colloids and Surfaces B, vol. 20, no. 4, pp. 315–319, 2001. View at Publisher · View at Google Scholar
  6. P. R. Crippa, V. Cristofoletti, and N. Romeo, “A band model for melanin deduced from optical absorption and photoconductivity experiments,” Biochimica et Biophysica Acta, vol. 538, no. 1, pp. 164–170, 1978.
  7. I. E. Roseborough, M. A. Grevious, and R. C. Lee, “Prevention and treatment of excessive dermal scarring,” Journal of the National Medical Association, vol. 96, no. 1, pp. 108–116, 2004.
  8. P. D. Butler, M. T. Longaker, and G. P. Yang, “Current progress in keloid research and treatment,” Journal of the American College of Surgeons, vol. 206, no. 4, pp. 731–741, 2008. View at Publisher · View at Google Scholar · View at PubMed
  9. L. Louw, “The keloid phenomenon: progress toward a solution,” Clinical Anatomy, vol. 20, no. 1, pp. 3–14, 2007. View at Publisher · View at Google Scholar · View at PubMed
  10. C. A. Cobold and J. A. Sherratt, “Mathematical modelling of nitric oxide activity in wound healing can explain keloid and hypertrophic scarring,” Journal of Theoretical Biology, vol. 204, no. 2, pp. 257–288, 2000. View at Publisher · View at Google Scholar · View at PubMed
  11. Y.-C. Hsu, M. Hsiao, L.-F. Wang, Y. W. Chien, and W.-R. Lee, “Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation,” Nitric Oxide, vol. 14, no. 4, pp. 327–334, 2006. View at Publisher · View at Google Scholar · View at PubMed
  12. M. G. Espey, K. M. Miranda, D. D. Thomas, and D. A. Wink, “Distinction between nitrosating mechanisms within human cells and aqueous solution,” Journal of Biological Chemistry, vol. 276, no. 32, pp. 30085–30091, 2001. View at Publisher · View at Google Scholar · View at PubMed
  13. L. Zeise, “Analytical methods for characterization and identification of eumelanins,” in Melanin: Its Role in Human Photoprotection, pp. 65–79, Valdenmar, Overland Park, Kan, USA, 1994.
  14. H. Kojima, N. Nakatsubo, K. Kikuchi, et al., “Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins,” Analytical Chemistry, vol. 70, no. 13, pp. 2446–2453, 1998. View at Publisher · View at Google Scholar
  15. C. A. Parker, “Apparatus and experimental methods,” in Photoluminescence of Solutions with Applications to Photochemistry and Analytical Chemistry, pp. 128–302, Elsevier, Amsterdam, The Netherlands, 1968.
  16. L. Novellino, M. D'Ischia, and G. Prota, “Nitric oxide-induced oxidation of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid under aerobic conditions: non-enzymatic route to melanin pigments of potential relevance to skin (photo)protection,” Biochimica et Biophysica Acta, vol. 1425, no. 1, pp. 27–35, 1998. View at Publisher · View at Google Scholar
  17. W. Korytowski, P. Hintz, R. C. Sealy, and B. Kalyanaraman, “Mechanism of dismutation of superoxide produced during autoxidation of melanin pigments,” Biochemical and Biophysical Research Communications, vol. 131, no. 2, pp. 659–665, 1985. View at Publisher · View at Google Scholar
  18. R. Farias-Eisner, G. Chaudhuri, E. Aeberhard, and J. M. Fukuto, “The chemistry and tumoricidal activity of nitric oxide/hydrogen peroxide and the implications to cell resistance/susceptibility,” Journal of Biological Chemistry, vol. 271, no. 11, pp. 6144–6151, 1996. View at Publisher · View at Google Scholar