International Journal of Molecular Sciences Article Effects of Aging on Hair Color, Melanosome Morphology, and Melanin Composition in Japanese Females Takashi Itou 1,* , Shosuke Ito 2 and Kazumasa Wakamatsu 2 1 Kao Corporation, R&D - Hair Care Products Research, Tokyo 131-8501, Japan 2 Department of Chemistry, Fujita Health University School of Medical Sciences, Toyoake, Aichi 470-1192, Japan * Correspondence: [email protected]; Tel.: +81-3-5630-9972 Received: 26 June 2019; Accepted: 27 July 2019; Published: 31 July 2019 Abstract: In a previous study, we showed that the size of melanosomes isolated from Japanese female hairs enlarges with age, and this affects the hair color. In this study, we analyzed the age-dependent changes in hair melanin in order to further explore the factors related to hair color changing by aging. A significant positive correlation with age was found in the total melanin amount (TM) and the mol% of 5,6-dihydroxyindole (DHI) units, while no correlation was found in pheomelanin mol%. TM showed significant correlations with hair color parameters and the melanosome volume, suggesting that hair color darkening by aging is caused by the increase in TM due to the enlargement of the size of melanosome. From the measurement of absorbance spectra on synthetic eumelanins with different ratios of DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), we found that the increase in DHI mol% also contributes to the darkening of hair color by aging. In addition, the level of pyrrole-2,3-dicarboxylic acid (PDCA), a marker of DHI melanin, showed a significant negative correlation with the aspect ratio of melanosome, suggesting a contribution of DHI melanin to the change in melanosome morphology by aging. Keywords: hair; aging; melanosome; morphology; melanin; 5,6-dihydroxyindole; 5,6-dihydroxyindole-2-carboxylic acid 1. Introduction Human scalp hair contains melanins, which have important roles such as the protection of the head from harmful ultraviolet (UV) light, the elimination of toxic heavy metals and chemicals [1], and the attractive appearance. Melanins are produced in melanocytes in hair follicles. Graying is the most drastic change by aging in human hair, and its causes have been well studied regarding a decrease in the number of melanocytes and a depletion of melanocyte stem cells in hair follicles [2,3]. Research on the causes of hair color is important in forensic science, and the relationship between genes and hair color has recently been studied [4–6]. For blonde hair, it is known that the darkening of hair color occurs as a child grows up; however, the reason for this has not been clarified [7]. There are not many studies related to hair color changing with age apart from hair graying. Hair color mainly depends on the quantity, the quality, and the distribution of the pigment melanin [8]. There are two types of melanin, black to brown eumelanin and yellow to reddish-brown pheomelanin, and their biosynthesis pathways are well known [8]. Eumelanin consists of two kinds of moiety; one is from 5,6-dihydroxyindole (DHI), and the other is from 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHICA melanin exhibits a potent hydroxyl radical-scavenging activity, whereas DHI melanin does not [9]. It was proposed that the formation of weak aggregates accounts for a greater accessibility of free radicals to DHICA compared to the compact π-stacked DHI melanin [10]. Int. J. Mol. Sci. 2019, 20, 3739; doi:10.3390/ijms20153739 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3739 2 of 15 Commo et al. [11] found that the hair tone of East Asian (Chinese) and Caucasian hair became darker with age, and that their total melanin amount (TM) increased with age. They also analyzed contents of pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and found that the ratio of PDCA/PTCA was significantly dependent on age for Caucasian and African American hairs, meaning that an eumelanin component, DHICA mol%, changed by aging, while the correlation was not significant for East Asian hairs. Ito et al. [12] proposed the method to quantify pheomelanin content more accurately by treating hairs with hot HCl to remove protein components from hair prior to an alkaline hydrogen peroxide (H2O2) oxidation. They found a constant level of pheomelanin in human black to brown hairs. The color of Japanese female hair is generally thought to be black, but it was found that the color changes with age [13]. A significant age dependence on the morphology of hair melanosomes was also found. The minor axis of isolated melanosomes significantly increased, and as a consequence, the aspect ratio (major/minor axes) significantly decreased with age. These morphological parameters showed significant correlations with the hair color, although the subject number used for the correlation analysis was only 10. In addition to that, the hair melanin, which directly relates to the hair color, was not analyzed in the previous study. In this study, we analyzed the hair melanin of Japanese females (n = 33) of a wide age range in order to explore the relationship among age, hair color, melanosome morphology, and melanin composition. 2. Results All results including the previous study [13] are summarized in Tables S1 and S2. Missing data indicate that the sample amount was too small for the experiment. 2.1. Age Dependence of Hair Color Figure1 shows the age dependence of hair color parameters L*, a*, and b* from the ages 4–68 years old (n = 25). These values significantly decrease with age (Table1). The age dependence of the cross-sectional area for these hair fibers showed a maximum at around the 30–40’s (Figure S1), which was the same trend as reported previously [14] and indicated that the hair color had no correlation with the cross-sectional area of the hair fiber (Figure S2). Similar to the case of Caucasian hair [7], it is generally recognized for Japanese hair that the hair color is sometimes brown in childhood but turns darker as children grow up. However, there was no information about how the color of pigmented hair changes after maturity. Commo et al. [11] reported a significant age dependence of hair tone level—assessed using a light color scale of 10 steps—not only in Caucasian hair (R2 = 0.18, p = 0.0012) but also in East Asian (Chinese) hair (R2 = 0.097, p = 0.020). In this study, for pigmented hairs of Japanese females, we found higher correlations with age (R2 = 0.53, p = 3.3 10 5 for L*, R2 = 0.65, p = 1.1 10 6 for a*, and R2 = 0.60, p = 5.6 10 6 for b*), and it was × − × − × − clearly seen that the hair color continued to be darker with age after pubertal age. Int.Int. J.J. Mol.Mol. Sci. 2019,, 20,, 3739x FOR PEER REVIEW 33 of 1515 * * * FigureFigure 1.1. TheThe ageage dependencesdependences ofof hairhair colorcolor parametersparametersL L*,, aa*,, andand bb* valuesvalues ofof JapaneseJapanese femalefemale hairhair * * * * * bundles.bundles. Circles,Circles, LL*;; squares,squares, aa*;; triangles,triangles, bb*. Lines are thethe fittedfitted curves.curves. SolidSolid line,line, LL*;; dasheddashed line,line, aa*;; dot-and-dash line, b*. p = 3.3 10 5 (L*), 1.1 10 6 (a*), and 5.6 10 6 (b*). dot-and-dash line, b*. p = 3.3 ×× 10-5− (L*), 1.1 × 10× −6 (−a*), and 5.6 × 10×−6 (b−*). Table 1. p-values by the regression analysis between hair color parameters and various values. Table 1. p-values by the regression analysis between hair color parameters and various values. Hair Color Parameter Hair Color Parameter L* a* b* L* a* b* 5 6 6 Human Age (year) 3.3 10 −51.1 10 −6 5.6 10 −6 Human Age (year) × − 3.3 × 10 × 1.1− × 10 ×5.6 ×− 10 FiberFiber Cross sectionalCross sectional area (µ aream2) (μm2) 0.630.63 0.280.28 0.120.12 Major axis (μm) 0.72 0.26 0.37 Major axis (µm) 0.72 0.26 0.37 Minor axisMinor (µ axism) (μm) 0.00630.0063 0.00430.0043 0.00240.0024 MelanosomeMelanosome AspectAspect ratio ratio 4.1 10 5 4.1 × 10−51.2 101.25 × 10−5 1.5 1.510 × 510−5 × − × − × − VolumeVolume (µm3) (μm3) 0.0110.011 0.0660.066 0.0380.038 TotalTotal melanin melanin amount amount (μg/mg) 2.2 × 10−7 1.0 × 10−7 3.9 × 10−7 2.2 10 7 1.0 10 7 3.9 10 7 (µg/mg)A650/A500 1 × − 0.043 × −0.0054 × 0.015− 1 A650/A500DHI 2 (mol%) 0.0430.13 0.00540.0027 0.0150.0032 DHI 2 (mol%) 0.13 0.0027 0.0032 MelaninMelanin Pheomelanin (mol%) 0.59 0.31 0.29 Pheomelanin (mol%) 0.59 0.31 0.29 3PTCA 3 level (ng/mg) 6 1.2 × 10−6 9.47 × 10−7 1.7 × 610−6 PTCA level (ng/mg) 1.2 10− 9.4 10− 1.7 10− 4 × 7 × 10 × 9 PDCA PDCAlevel 4 (ng level/mg) (ng/mg) 8.6 10− 8.6 × 10−71.8 10−1.8 × 10−10 1.5 1.510 ×− 10−9 5 × × × PTeCA PTeCAlevel (ng5 level/mg) (ng/mg) 0.000870.00087 0.00940.0094 0.00570.0057 1 2 1 TheThe ratio ratio of of absorbance absorbance at the the wavele wavelengthngth of 650 nm (A650) (A650) and 500 500 nm nm (A500), (A500), 2 5,6-dihydroxyindole,5,6-dihydroxyindole, 3 pyrrole-2,3,5-tricarboxylic acid, 4 pyrrole-2,3-dicarboxylic acid, 5 pyrrole-2,3,4,5-tetracarboxylic acid. 3 pyrrole-2,3,5-tricarboxylic acid, 4 pyrrole-2,3-dicarboxylic acid, 5 pyrrole-2,3,4,5-tetracarboxylic acid.