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 and by the Griess method and as by trapping experiments with the fluorogenic substrate 4,5-diaminofluorescein (DAF-2). Initial and 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 , 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 [4–6], 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 [7–9]. 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.
2. Materials and Methods
Sepia melanin, [13], () 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 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, , to form highly fluorescent triazoles (DAF-2T) from 4, 5-diaminofluorescein (DAF-2) in the presence of molecular [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 (). 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].
-tests were conducted to determine the statistical significance of the results. The determined -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 , the . of 3 experiments afforded scavenging ratios of () for and for (). Initial []/ [] ratios were ~1.5.
(a)
(b)
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 minutes (see Figure 4). Melanin competes successfully with DAF for NO as evidenced by a steady-state fluorescence scavenging (; ). These results were qualitatively the same whether dialysis systems or melanin suspensions were used.
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 , , , and , 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 and/or arising from melanin autoxidation [17] is unlikely to be significant. Since melanin acts as an efficient “pseudodismutase” [17], the steady-state [] 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.