Effect of Ambient Humidity on UV ⁄ Visible Photodegradation of MelaninThin Films

Anup Sharma*

Department of Physics, Alabama A&M University, Normal, AL

Received 31 January 2010, accepted 16 April 2010, DOI: 10.1111 ⁄ j.1751-1097.2010.00753.x

ABSTRACT

Photodegradation of spin-coated thin films of melanin is

investigated using UVA and visible light from light-emitting

diodes. The gradual increase in transmitted light through the film

is measured as melanin photodegrades over the course of several

hours. The photodegradation rate is measured for wavelengths in

the 365–600 nm range. It is found that the increase in ambient

humidity significantly accelerates the photodegradation process.

Implications of these observations for evolution of melanism in

biological systems are discussed.

INTRODUCTION

Melanin is a ubiquitous pigment present in the skin and hair of

mammals, feathers of birds and in the cuticles of many insects.Due to its strong light-absorption property over a wide bandof UV ⁄ visible wavelengths, it is believed to play a crucial

photoprotective role for human skin (1). In other warm-blooded (endothermic) species melanin is also responsible forfunctions such as thermoregulation (2), camouflage (3) andantibacterial protection in bird wings (4). The interaction of

melanin with light has been widely investigated (5–8). Most ofthese studies like photobleaching ⁄ photodegradation of iso-lated melanin involve samples in solution form. Recently,

several studies have investigated the photobleaching of mela-nosomes from the retinal pigment epithelium (9,10). We reporthere the first investigation involving the photodegradation of

melanin thin films over the UV ⁄ visible region between 365 and600 nm as well as the effect of ambient humidity on thin filmphotodegradation. Thin films are better suited for the objectiveof this study: to quantitatively measure the effect of ambient

humidity in the photodegradation of melanin. This study isimportant to understand the evolution of melanism as it isknown (Gloger’s rule) that the extent of dark pigmentation

(eumelanin) in endothermic species increases with the humidityof the habitat (11). Gloger’s rule has been observed with fewexceptions in species including mammals, birds and insects

including one recently studied example of the gradual dark-ening of plumage of the song sparrow (Melospiza melodia)from the arid regions of southeastern Arizona to the more

humid northwestern Washington (4). We show that ambienthumidity significantly accelerates the photodegradation ofmelanin. Gloger’s rule could in part be an evolutionary

response to this dependence of photodegradation on ambienthumidity.

MATERIALS AND METHODS

Spin-coating technique was used to make optical-quality thin films ofsynthetic eumelanin (12) (Sigma Aldrich) from its solution(50 mg mL)1) in aqueous ammonia (14.8 N). Glass slides (1 mmthick) were used for film deposition. An atomic force microscope aswell as optical attenuation in these films was used to measure the filmthickness and was found to be 25 ± 3 nm. Due to the high absorptioncoefficient of UV ⁄ visible light, the thin film nature of the melaninsample allows investigation of the effect of ambient humidity onphotodegradation by the simple technique of measuring opticaltransmission. This would not be possible with a bulk sample orsample in solution form. Optical transmission was recorded for 365 nm(UVA) and for the visible wavelengths of 460, 500 and 595 nm as themelanin film is photodegraded over an extended period of severalhours. Different light-emitting diode (LED) modules were used for theUV and visible wavelengths. The LEDs (Prizmatix) were coupled to a1 m long plastic fiber of 1.5 mm core diameter. The fiber has goodtransmission for 360 nm and longer wavelengths. A power of 7.5 mWat the exit end of the fiber was incident on a melanin-coated slideplaced inside a humidity chamber, 1 mm from the fiber end. Themeasured linewidth (FWHM) for 365 nm, 460 nm, 500 nm and595 nm LEDs is 10, 15, 20 and 20 nm, respectively. Temperaturewas maintained constant at 20�C. Light transmitting through themelanin film is intensity-modulated with an optical chopper and isdetected using a lockin-amplifier.

RESULTS AND DISCUSSION

For the four wavelengths (365, 460, 500 and 595 nm) used inthis work, the calculated light absorption by 25 nm melanin

film is 24%, 15%, 12% and 8%, respectively (12). Measuredabsorption agrees with these numbers. The magnitude of thesenumbers makes it easy to monitor the percentage changeof transmission (DT%) through the film as it is irradiated

with light over a period of 24 h (Fig. 1). Photodegradationgradually results in a hole in the film and DT% increases toreach a final constant value. Lower saturation of DT% for

increasing wavelengths corresponds to decreasing absorptioncoefficients. Figure 1a is for photodegradation inside anaqueous medium. For this measurement, a small water-filled

cell (25 mm · 15 mm · 3 mm) is used in place of the humiditychamber. It is possible to observe photodegradation of thinfilm in water as melanin has very low solubility. With 365 nmUVA light, the melanin film is totally photodegraded in less

than 5 h. This time increases for longer visible wavelengths.Figure 1b is for photodegradation of melanin in air at arelative humidity of 75%, other conditions being the same as

*Corresponding author email: anup.sharma@aamu.edu (Anup Sharma)� 2010 TheAuthor. Journal Compilation. The American Society of Photobiology 0031-8655/10

Photochemistry and Photobiology, 2010, 86: 852–855

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in Fig. 1a. Clearly, in Fig. 1b, the photodegradation process isslower compared with that in Fig. 1a. Likewise, Fig. 1c is forthe photodegradation of melanin in air at a relative humidity

of 40%, other conditions being the same as in Fig. 1a.Figure 1c shows photodegradation only for 365 nm (upperplot) and 460 nm (lower plot). At a low (40%) relative

humidity, photodegradation for longer wavelengths (500,595 nm) is too small to be measured.

Photodegradation rates are measured from the plots in

Fig. 1. For the purpose of comparison, we define this rate asthe inverse of the time required to change DT by half itsmaximum value. These rates are plotted in Fig. 2 as a function

of relative humidity for 365 and 460 nm wavelengths. Whilemelanin photodegradation is fastest in an aqueous medium, itis also significantly affected by relative humidity in airmedium. For 365 nm, photodegradation in air at 75%

humidity is 10-fold faster than at 40% humidity. As asurprising observation, at high humidity, visible wavelengths(e.g. 460 nm at 80% humidity) can be more effective in

photodegrading melanin than UVA wavelengths at very lowhumidity (e.g. 365 nm at 10% humidity). This has implica-tions for the photoprotective role of melanin against the

effects of solar radiation as the extent of damage caused to thestratum corneum by UV ⁄ visible wavelengths could dependstrongly on the ambient humidity. Figure 3 shows thedependence of the photodegradation rate on the intensity of

365 nm light for a relative humidity of 75%. As the lightintensity is halved, it takes twice as long for the same changeof transmission intensity, showing that photodegradation

depends linearly with light intensity. Similar to the observedlight absorption by melanin, the photodegradation is propor-tional to the intensity of light. This observation agrees with

the published results (13) showing that the absorptionspectrum of melanin over the 300–600 nm range coincideswith its action spectrum for the generation of melaninradicals. Thus Fig. 3 is consistent with the accepted role of

photoinduced radicals of melanin for its subsequent photode-gradation.

Figure 4a shows the appearance of a ‘‘hole’’ in the melanin

film as it is exposed for 24 h at different wavelengths (365, 460,500 nm) and relative humidity (50%, 75%). This correlateswith the observations in Fig. 1 that photodegradation

increases at higher humidity and shorter wavelength. Similarto the photodissociation of some organic compounds with UVlight (14,15), we believe photodegradation of melanin results in

smaller, more polar fragments. To demonstrate this, melaninfilm is exposed to UV ⁄ visible light through a microscopic maskhaving an array of opaque squares and a transparent grid.Light falling on a melanin film through the mask photode-

grades it in a grid pattern (squares being unexposed). Theresults are shown in Fig. 4b for 365 nm light. Here, patterningis done for 20 h at a relative humidity of 43%. When such a

melanin film is placed in a stream of humid air, condensation

0 10 200

10

200 10 20

0

10

20

30

400 10 20

0

10

20

(c)

Time (hours)

40% Rel. Hum.

(a)

(b) 75% Rel. Humidity

Aqueous Medium

ΔT (%

)ΔT

(%)

ΔT (%

)

Figure 1. Measurement of melanin thin film photodegradation withUVA (365 nm), and visible light at 460, 500 and 595 nm. Percentageincrease of transmission (DT%) through the melanin film is observedas it is irradiated with light over a period of 24 h. (a) Photodegradationin a water cell. Curves from top to bottom are for 365, 460, 500 and595 nm light. (b) Photodegradation in air at a relative humidity of75%. Curves from top to bottom are for 365, 460, 500 and 595 nmlight. (c) Photodegradation in air at a relative humidity of 40% for365 nm (upper curve) and 460 nm.

0 20 40 60 80 1000

0.5

1

1.5

Rat

e (h

our-1

)

Rel. Humidity (%)

365 nm

460 nm

Figure 2. Measured dependence of photodegradation rates for mela-nin thin films using UVA (365 nm) and visible (460 nm) wavelengths.

0 10 200

10

20

30

Time (hours)

ΔT (%

)

Figure 3. Dependence of melanin photodegradation on the intensity of365 nm light. Thin film sample is in air with a relative humidityof 75%. The film is irradiated over a diameter of 7 mm with a power of10 mW (lower plot) and 20 mW (upper plot).

Photochemistry and Photobiology, 2010, 86 853

of larger water droplets takes place only on the regions of thefilm that were exposed to light. Hydrophilicity of the exposed

areas of film clearly demonstrates photodegradation of mel-anin. Photobleaching of melanin in solution and the role ofdissolved oxygen in this process have been investigated (5–7).

This is known to generate hydrogen peroxide and hydroxylions. While the precise chemistry involved in photobleachingof melanin is still poorly understood, a plausible explanation(5) based on the known mechanisms involves reversible and

irreversible oxidation of melanin units. The fully reduced ⁄oxidized monomers of hydroquinones (QH2) and quinones (Q)are known to exist (16) in comproportionation equilibrium

(QH2 + Q M 2SQ + 2H+) with semioxidized melanin radi-cals (SQ). Photoexcitation of melanin as well as its hydrationcan shift this equilibrium by increased generation of radicals.

Aerobic photo-oxidation of melanin in solution is known toresult in superoxide anion which in a dismutation reaction withwater produces hydrogen peroxide and hydroxyl radicals(6,17). In one plausible scheme, hydroquinone monomers in

melanin are oxidized into semiquinones by hydroxyl radicalsfollowed by a disproportionation reaction to produce quinon-es. Final degradation is produced by a ring-opening reaction

in quinones (18). In this model involving hydroxyl radicals,the rate of photodegradation will be proportional to thehydration of melanin thin film due to adsorption of ambient

humidity. Measured variation of photodegradation rates withambient humidity in Fig. 2 is thus simply indicative ofhumidity adsorption isotherm for melanin substrate. Such a

supralinear variation of adsorbed water with ambient relativehumidity is commonly observed on hydrophilic substrates (19)like melanin.

The results described above could be applied to the

photodegradation of melanin in biological systems. Therelevance of this work is for photoprotection by melanin inthe outermost layer (stratum corneum) of epidermis but

probably not by melanosomes in the deeper layers. However,as several studies have shown, while most of the UV lightfiltration may take place in the deeper malpighian layer,

photoprotection by the stratum corneum is also important.Depending on the skin type and wavelength of light, thestratum corneum can filter out between 30% and 80% ofincident light (20–22). Water diffusion characteristics (23)

through the stratum corneum and water sorption (24) by ithave been investigated. It is known that the water content in

the stratum corneum can increase by more than an order ofmagnitude (25) as the relative humidity changes from 20% to

80%. This observation together with our results suggests thatthe photodegradation of melanin inside the skin and featherscould be strongly affected by the humidity of the external

environment. This hypothesis is supported by several studiesdealing with the effect of humidity on photobleaching of hair(26,27). While the light intensities used in this work (0.1–10 mW mm)2) are 100–1000 times greater than the terrestrial

solar radiation flux in visible and UV spectral ranges (28), theyare several orders of magnitude smaller than those usedfor laser ablation of organic films (29). As seen in Fig. 3, the

photodegradation rate varies linearly with light intensitiesused in this work and effects recorded over a course of 24 hwould take several months under terrestrial solar radiation

intensities.Gloger’s rule in evolutionary biology relates to enhanced

melanogenesis in endothermic species living in habitats withgreater levels of humidity. As an example, for birds living in

regions of higher humidity this is documented by heaviermelanin pigmentation in feathers (4). Several possible evolu-tionary factors have been suggested to explain this including:

(1) Humid regions have greater vegetation and darker pig-mentation provides better camouflage in shade (3); (2) Melaninprovides a defense against feather-eating bacteria which grow

more under humid conditions (4). Additionally, melanin infeathers provides mechanical strength and resistance againstwear. As can be inferred from our results, a higher level of

melanogenesis could also be an evolutionary response tothe observation that ambient humidity accelerates melaninphotodegradation.

Acknowledgements—This work was funded by the Center for Biopho-

tonics for Science and Technology (CBST) and Research Infrastruc-

ture for Science and Engineering (RISE) awards from NSF.

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(a) (b) Figure 4. (a) Appearance of a ‘‘hole’’ in the melanin film as it is exposed for 24 h at different wavelengths (365, 460, 500 nm) and relative humidity(50, 75%). This correlates with the observations in Fig. 1 that photodegradation increases at higher humidity and shorter wavelength. (b) Melaninfilm is exposed through a mask of transparent grid and opaque squares to 3 mW of 365 nm for 20 h at a relative humidity of 43%.Photodegradation results in polar fragments and a grid-patterned hydrophilic surface. This hydrophilicity is made visible in a stream of humid airby condensation of water droplets largely over the irradiated areas.

854 Anup Sharma

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