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

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


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 [4][5][6], 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 [7][8][9].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.

MATERIALS AND METHODS
Sepia melanin, [13], (MelanInk @ ) 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 = 0-90 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.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 ex = 495 nm, λ em = 515 nm ).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].
T-tests were conducted to determine the statistical significance of the results.The determined P-values assume normal distributions for each group.

NO measurement as nitrite and nitrate
Pigment melanin rapidly sequesters nitric oxide (see Figures 3(a − 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.Mean ± S.D. of 3 determinations, P < .02.

Fluorescence measurements
The fluorescence intensities (i.e., DAF-2T formation) of both control and test samples increased to a steady-state value at t ∼ 20 minutes (see Figure 4).Melanin competes successfully with DAF for NO as evidenced by a steady-state fluorescence scavenging ratio = 0.706 ± 0.103 (n = 4; P < .0054).These results were qualitatively the same whether dialysis systems or melanin suspensions were used.).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 NO 2 , N 2 O 3 , O 2 •− , and ONOO − , 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].

Figure 1 :Figure 2 :
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.