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Original Paper Skin Pharmacol Physiol 2012;25:78–85 DOI: 10.1159/000335259 Received: April 5, 2011 Accepted after revision: November 17, 2011 Published online: January 12, 2012 Differences in Susceptibility to Oxidative Stress in the Skin of Japanese and French Subjects and Physiological Characteristics of Their Skin Y. Yamashita a Y. Okano b T. Ngo c P. Buche c A. Sirvent c F. Girard c H. Masaki b a Nikkol Group, Nikoderm Research Inc., Osaka, and b Tokyo University of Technology School of Bioscience and Biotechnology, Tokyo, Japan; c Laboratoire Dermscan, Villeurbanne, France Key Words Catalase ⴢ Protein carbonylation ⴢ Oxidative stress ⴢ Ethnic groups Abstract Background: Many researchers have studied differences in conditions of ethnic skin using biophysical measurements. However, few studies to date have focused on the antioxidative capacity of the skin. Methods: We measured two parameters of oxidative stress in the stratum corneum, catalase activity and protein carbonylation of the stratum corneum (SCCP), in two ethnic groups, Japanese and French subjects, to characterize the susceptibility to oxidative stress. We also measured several physiological parameters at three different skin sites, two sun-exposed sites (cheek and dorsal aspect of the hand) and a sun-protected site (inner upper arm), in both ethnic groups. Results: Transepidermal water loss (TEWL), the size of corneocytes and skin color showed differences between sun-exposed and sun-protected sites regardless of ethnicity. Regarding ethnic differences, catalase activities and parameters of skin hydration and barrier function of Japanese subjects were higher than those of French subjects. However, SCCP values showed a trend contrary to catalase activity. The difference in the b* value indicated that the melanin content of Japanese skin was higher than that © 2012 S. Karger AG, Basel 1660–5527/12/0252–0078$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com Accessible online at: www.karger.com/spp of French skin. Pearson’s correlation analyses showed that catalase activity and SCCP values had weak relationships with water content, TEWL and skin color in both ethnic groups. Conclusion: Differences in susceptibility to oxidative stress, namely melanin content and catalase activity in the skin, induce the better skin condition of Japanese compared with French subjects. Copyright © 2012 S. Karger AG, Basel Introduction Ethnic differences in skin conditions are obvious and their characteristics have been investigated from various angles. Parameters of skin physiology and morphology, such as surface hydration, barrier function, surface lipids, surface textures, wrinkles and pores as well as skin color have been studied for various ethnic groups, and differences in some factors have been found, such as variations with age and reactive characteristics to environmental stress [1–5]. In addition, it has been shown that differences between Caucasian and African skin affect the functions of the epidermis and dermis and their cellular interactions [6]. Recently, it has also been reported that races which have differently pigmented skin types show differences in epidermal barrier function which can be Yuki Yamashita Nikoderm Research Inc. 1-6-14, Azuchimachi Chuoku, Osaka 541-0052 (Japan) Tel. +81 6 6125 3501, E-Mail yamashita @ nikoderm.com attributed to the pH of the stratum corneum based on the melanin content [7]. As UV light, ozone and pollution affect the skin [8], it can be easily predicted that there are differences in the susceptibility of the skin to oxidative stress among ethnic groups. However, few studies to date have focused on the anti-oxidative capacity of the skin according to ethnic background. Therefore, we conducted biophysical measurements (water content of the skin surface and transepidermal water loss, TEWL) and determined the size of superficial corneocytes and the integrity/cohesion of the stratum corneum in addition to the susceptibility to oxidative stress in the skin of Japanese (Asians living in Japan) and of French (Caucasians living in France) subjects. The size of corneocytes is closely related to the turnover time of the stratum corneum [1], so the condition of cornification of the epidermis can be judged from this parameter. It has been reported that the strength of cohesion of the stratum corneum can be judged from the state of the stratum corneum removed by tape-stripping [9]. A thicker stratum corneum abraded by the adhesive tape means a lower integrity/cohesion of the stratum corneum and leads to reduced barrier function of the epidermis [10]. We analyzed the degree of stratum corneum abrasion by the tape-stripping method and used this parameter as an indicator of the integrity/cohesion of the stratum corneum. In addition, we examined two characteristics of the skin associated with oxidation, the antioxidative capacity of the stratum corneum, namely catalase activity, and carbonylated proteins in the stratum corneum (SCCP). Catalase is one of the foremost antioxidant defense enzymes in the stratum corneum, and it is closely related to skin conditions [11–13]. Proteins in the stratum corneum, such as keratins, are modified by oxidation or glycation reactions to produce carbonylation proteins [14–16]. Therefore, SCCP is an excellent marker to show the degree of skin damage due to oxidative stress, and it has been reported that SCCP levels increase at sun-exposed body sites [17]. Several noninvasive methods have been reported that are useful for the determination of oxidative stress and antioxidative capacity of human skin. For example, the interaction between carotenoids and free radicals can be investigated using electron paramagnetic resonance spectroscopy or resonance Raman spectroscopy [18, 19]. Chemiluminescence decay analyses are also useful tools to measure the effects of topically applied antioxidants under in vivo conditions [20]. On the other hand, reactive carbonyl compounds of lipid peroxides in skin lipids can be detected in trace levels using analytical instruments in vitro [21]. However, Oxidative Stress in the Skin of Japanese and French Subjects those methods are not adequate to study large numbers of specimens. Therefore, we measured catalase activity and SCCP levels using tape-stripped stratum corneum which is one of the simplest non-invasive measurement methods for skin antioxidative capacity. In this study, we measured these parameters on sunexposed and on sun-protected sites of the body in both ethnic groups, and considered their locational and ethnic differences in relation to the susceptibility of the skin to oxidative stress. Moreover, to clarify the effect of differences in the susceptibility to oxidative stress on skin conditions, we used Pearson’s correlation analysis between catalase activities/SCCP values and the biophysical parameters. Materials and Methods Subjects This study was conducted to determine the skin characteristics of two different ethnic groups, 92 Asian subjects living in Japan (Japanese; 43 males and 49 females, 22–64 years old) and 104 Caucasian subjects living in France (French; 52 males and 52 females, 18–64 years old). In both groups, the number of subjects in 10-year age groups and their mean ages were adjusted to be approximately the same. The mean ages of the Japanese and French subjects were 41.1 8 12.8 and 40.4 8 14.4 years, respectively. Written informed consent was obtained from each subject before the study. Biophysical Measurements The water content of the skin surface was measured using a skin hygrometer SKICON 200EX (IBS Ltd., Japan) for Japanese subjects and using a Corneometer CM825 (Courage and Khazaka, Germany) for French subjects. The values obtained using the SKICON 200EX were converted to the Corneometer CM825 values using a calibration curve reported in a previous paper [22]. TEWL was measured using an AS-CT1 (Asahi Biomed) for Japanese subjects and using an AquaFlux AF100 for French subjects. The measurement values obtained using the AquaFlux AF100 were converted to AS-CT1 values based on the results of a validation study using both instruments, as follows: y = 0.8482x – 1.4002 (y: values obtained by AS-CT1, x: values obtained by AquaFlux AF100). Skin color was measured using a CM-2600d (Konica Minolta, Japan) for Japanese subjects and using a CR300 (Konica Minolta) for French subjects. These instruments have different measuring diameters (CM-2600d: ⌽ 3 mm; CR300: ⌽ 8 mm), but L* (lightness), a* (redness) and b* (yellowness) values of the skin can be obtained using each of them. The superficial stratum corneum was removed using an adhesive Sellotape (Nichiban, Japan) and was then stained with an aqueous solution containing 0.5% brilliant green (Aldrich Chemical Institutes Inc., Japan) and 1.0% gentian violet (Sigma-Aldrich Co., Japan) [23]. The size of corneocytes was calculated by obtaining the number of pixels using Photoshop 5.0 (Adobe) on images obtained using an optical microscope at 200-fold magnification [23]. The Skin Pharmacol Physiol 2012;25:78–85 79 Cheek a French Dorsal hand Measured site 18 16 14 12 10 8 6 4 2 0 ** 25 ** ** b ** 15 10 5 0 Cheek Upper arm ** ** 20 b* value a* value L* value Japanese 80 70 60 50 40 30 20 10 0 Dorsal hand Cheek Upper arm Measured site c Dorsal hand Upper arm Measured site Fig. 1. Differences in skin color between measurement sites and ethnic groups. The parameters of skin color (L*, a* and b* values) were measured. The cheek and the dorsal aspect of the hand were defined as sun-exposed sites, and the inner upper arm as a sun-protected site. Significant differences between Japanese and French subjects are shown at all sites for a* (b) and b* values (c), but not for L* values (a). ** p ! 0.01 (Student’s t test). thick abrasion, which is an indicator of the integrity/cohesion of the stratum corneum, is the ratio of the number of pixels of the overlapped area of corneocytes to the total number of pixels of corneocytes. All skin measurements were performed on two sun-exposed sites (the cheek and the dorsal aspect of the hand) and on a sunprotected site (inner upper arm). Measurement of Catalase Activity and SCCP Levels Catalase activity and SCCP levels were measured in tapestripped stratum corneum. Tape strips were taken sequentially using adhesive Sellotape (Nichiban). The first tape strip was discarded, the second was used to measure the SCCP level and the third was used to measure catalase activity. Catalase activity was measured based on the scavenging of hydrogen peroxide (H2O2) [13]. Briefly, each piece of tape with the stratum corneum was punched out with a 6-mm diameter punch, was placed into a BM15 1.5-ml tube (BM Equipment Co. Ltd., Japan) containing 250 nmol H2O2 (Wako, Japan) and was mixed for 120 min. After the incubation, the solution was used to quantify the remaining H2O2. H2O2 levels were determined by reaction with 4-aminoantipyrine (Wako) and phenol (Wako) in the presence of horseradish peroxidase. Reaction products were quantified by measuring the absorbance at 550 nm. Catalase activity is defined as the amount of scavenged H2O2 normalized to the total protein of the stripped stratum corneum. SCCP levels were determined using a previously reported method [17]. Briefly, the tape-stripped stratum corneum was fixed on a glass slide and was stained with 40 mM fluorescein-5thiosemicarbazide (AnaSpec Inc., USA) in 0.1 M 2-morpholinoethane sulfonic acid-Na buffer (pH 5.5; Dojindo Laboratories, Japan) and was then observed using a fluorescence microscope Eclipse-80i (Nikon, Japan). Fluorescence images were captured with a CCD camera DS-U1 (Nikon) and were analyzed using Pho- 80 Skin Pharmacol Physiol 2012;25:78–85 toshop 5.0 (Adobe). SCCP levels are defined as the average fluorescence intensity of the stratum corneum area in a test image. All measurements were performed on skin of the cheek, of the dorsal aspect of the hand and of the inner upper arm. Statistical Analysis Tukey’s test was performed to compare the parameters of the measured sites. Student’s t test was performed to compare the skin characteristics between Japanese and French subjects, and Pearson’s correlation analysis was also performed between catalase activity/SCCP values and the other skin physiological parameters using software for statistical analysis, SPSS Statistics 17.0 (SPSS Inc.). Correlation analysis was performed on both original and logarithm-converted data. Results Locational Differences At first, we compared differences between sun-exposed and sun-protected skin sites. As described in the Materials and Methods, we defined the cheek and the dorsal aspect of the hand as sun-exposed sites, and the inner upper arm as a sun-protected site considering topical sun exposure frequencies. In skin from both ethnic groups, the L* value was significantly higher in sun-protected skin of the inner upper arm compared to sun-exposed skin of the dorsal aspect of the hand (table 1). In contrast, the a* and b* values were lower on the inner upper arm than on the dorsal aspect of the hand (fig. 1, table 1). These results Yamashita /Okano /Ngo /Buche /Sirvent / Girard /Masaki 80 70 60 50 40 30 20 10 0 ** 20 ** 15 10 Upper arm Cheek b 1,400 SC thick abrasion (%) Size of corneocytes (mm2) Dorsal hand Measured site 1,000 25 5 a 1,200 ** 30 0 ** 800 600 400 200 0 Cheek c suggest that the skin color is darker in sun-exposed skin than in sun-protected skin in both ethnic groups. The differences of skin physiological parameters between sun-exposed and sun-protected sites in both ethnic groups are summarized in table 1. In the French subject group, significant differences were shown between the dorsal aspect of the hand and the inner upper arm in all physiological parameters measured (fig. 2) and catalase activity and SCCP levels. The same tendencies were found in the Japanese group except for water content. However, in both ethnic groups, differences in skin physiological parameters between the cheek and the inner upper arm were smaller than those between the dorsal aspect of the hand and the inner upper arm. Catalase activity was significantly higher on the sun-protected inner upper arm than on the sun-exposed dorsal aspect of the hand in both ethnic groups. The SCCP level was lower on the inner upper arm than on the dorsal aspect of the hand (table 1, fig. 3). However, similar to some of the biophysical parameters, the SCCP levels in French subjects and both of the oxidative stress markers in Japanese subjects showed no differences between the cheek and the inner upper arm. Oxidative Stress in the Skin of Japanese and French Subjects 35 ** ** Cheek Fig. 2. Locational and ethnic differences in physiological parameters of the skin. Several skin physiological parameters – water content (a), TEWL (b), size of corneocytes (c) and thick abrasion of the stratum corneum (SC, d) – were measured. The cheek and the dorsal aspect of the hand were defined as sun-exposed sites, and the inner upper arm as a sun-protected site. Significant differences between Japanese and French subjects are shown at all sites for water content and thick abrasion of the stratum corneum, at the dorsal aspect of the hand, the inner upper arm for TEWL and at the cheek for size of corneocytes. ** p ! 0.01 (Student’s t test). French TEWL (g/m2/h) Water content (AU) Japanese Dorsal hand Upper arm Measured site 50 45 40 35 30 25 20 15 10 5 0 Upper arm Measured site Dorsal hand ** ** ** Cheek Dorsal hand Upper arm d Measured site Table 1. Locational difference of skin physiological parameters Parameters Water content TEWL Size of corneocyte SC thick abrasion L* a* b* CAT SCCP Japanese French cheek vs. arm hand vs. arm cheek vs. arm hand vs. arm 0.000** 0.000** 0.000** 0.008** 0.000** 0.000** 0.000** 0.921 0.190 0.293 0.000** 0.000** 0.001** 0.000** 0.000** 0.000** 0.000** 0.000** 0.957 0.000** 0.000** 0.980 0.000** 0.000** 0.000** 0.000** 0.157 0.000** 0.000** 0.000** 0.028* 0.000** 0.000** 0.000** 0.000** 0.000** Tukey’s tests were carried out between the cheek and the inner upper arm (cheek vs. arm), and between the dorsal aspect of the hand and the inner upper arm (hand vs. arm). SC = Stratum corneum; CAT = catalase. * p < 0.05, ** p < 0.01: significant difference between the two parameters. Skin Pharmacol Physiol 2012;25:78–85 81 Japanese oxidative stress markers of the skin. Two parameters of oxidative stress, catalase activity (a) and SCCP level (b), were measured. The cheek and the dorsal aspect of the hand were defined as sun-exposed sites, and the inner upper arm as a sunprotected site. Significant differences between Japanese and French subjects are shown at all sites for both catalase activity and SCCP level. ** p ! 0.01 (Student’s t test). ** 120 ** 25 100 ** 20 SCCP level Catalase activity (nmol/μg protein) Fig. 3. Locational and ethnic differences in French ** 30 15 10 5 ** ** 80 60 40 20 0 0 Dorsal hand Cheek a Upper arm Dorsal hand Cheek b Measured site Upper arm Measured site Table 2. Correlation analysis between catalase activity and skin physiological parameters Water content TEWL Size of corneocytes SC thick abrasion SCCP L* a* b* Cheek Japanese French 0.517** –0.071 –0.221* 0.177 0.162 –0.136 –0.228* –0.124 –0.137 0.048 0.326** 0.252* –0.258* –0.194 0.114 0.148 Hand Japanese French 0.013 0.047 –0.044 –0.018 –0.057 0.043 –0.174 0.174 –0.105 –0.189 0.332** 0.278** –0.269** –0.162 0.009 –0.244* Arm Japanese French –0.06 0.038 –0.238* 0.082 –0.278** –0.040 –0.262* –0.156 –0.206* –0.255** 0.130 0.117 –0.249* –0.082 –0.084 –0.064 Each number shows the correlation coefficient of Pearson’s correlation analysis. SC = Stratum corneum. * p < 0.05, ** p < 0.01: significant correlation between the two parameters. Ethnic Differences The a* value (which indicates redness in the skin color) was significantly higher in French subjects compared to Japanese subjects, but the b* value (which indicates yellowness in the skin color) was lower (fig. 1). On the other hand, the L* value (which indicates brightness) was almost the same in the French and in the Japanese subjects (fig. 1). Contrary to expectations, the difference in skin color between the two ethnic groups was not reflected in the L* value but rather in the a* and b* values. Japanese subjects also showed a higher water content on the skin surface, a lower TEWL, and a lower abrasion of the stratum corneum compared with French subjects. Those results suggest that Japanese subjects have higher levels of skin hydration and barrier functions than French subjects (fig. 2). 82 Skin Pharmacol Physiol 2012;25:78–85 Regarding the parameters related to susceptibility of the skin to oxidative stress, the catalase activity of Japanese subjects was higher than that of the French subjects (fig. 3). In contrast, SCCP levels of Japanese subjects were higher than those of the French subjects, although higher catalase values seemed to lead to lower SCCP levels. Pearson’s Correlation between Physiological Parameters and Catalase Activity/SCCP Levels Relationships between catalase activity or SCCP level and skin physiological parameters are summarized in tables 2 and 3. Correlation coefficients in logarithm-converted data were almost the same to those in the original data; therefore, we show only the linear regression results. In the cheek skin of Japanese subjects, catalase activities had a positive correlation with water content and L* value but were negatively correlated with TEWL, thick abraYamashita /Okano /Ngo /Buche /Sirvent / Girard /Masaki Table 3. Correlation analysis between SCCP level and skin physiological parameters Water content Cheek Japanese French –0.228* –0.229* Hand Japanese French –0.289** –0.226* Arm Japanese French –0.049 –0.085 TEWL Size of SC thick corneocytes abrasion Catalase L* a* b* 0.040 –0.352** 0.320** 0.169 –0.137 0.048 –0.251* –0.187 –0.020 0.191 0.260* –0.185 –0.194 0.063 –0.134 –0.159 0.237* 0.112 –0.105 –0.189 –0.371** –0.242* 0.151 0.136 0.05 0.103 0.176 0.000 0.144 –0.016 0.316** 0.224* –0.206* –0.255** –0.177 0.056 0.148 –0.016 0.278** –0.292** 0.228* 0.310** Each number shows the correlation coefficient of Pearson’s correlation analysis. SC = Stratum corneum. * p < 0.05, ** p < 0.01: significant correlation between the two parameters. sion of the stratum corneum and the a* value (table 2). On the other hand, SCCP levels showed a negative correlation with water content and L* value, and a positive correlation with TEWL, thick abrasion of the stratum corneum and the b* value (table 3). However, in the cheek of French people, catalase activity was only related to the L* value. The relationships between physiological parameters and catalase activity/SCCP levels changed depending on the ethnicity and the body site, and they were not definitive. Correlation coefficients were not higher in general except for the relationship of catalase activity and water content in the cheek skin of Japanese subjects. Discussion Physiological parameters of the skin (water content, TEWL, size of corneocytes, thick abrasion of the stratum corneum and skin color) and parameters of oxidative stress (catalase activity and SCCP level) were initially compared between sun-exposed and sun-protected areas of the skin in relation to ethnic differences (Japanese and French subjects). TEWL, the size of corneocytes, catalase activity and SCCP levels of sun-protected skin (inner upper arm) were significantly different from sun-exposed skin (dorsal aspect of the hand), which suggests that the skin condition is better at sun-protected sites than at sunexposed sites in both ethnic groups. However, the behaviors of those parameters on the cheek, which is also a sunexposed site, were slightly changed from the dorsal aspect of the hand. Judging from the difference between cheek versus inner upper arm and dorsal aspect of the hand versus inner upper arm, it was considered that the dorsal Oxidative Stress in the Skin of Japanese and French Subjects aspect of the hand is more damaged than the cheek in both ethnic groups (table 1). This probably reflects the fact that the cheek is protected from external stimulations by sebaceous lipids (including antioxidants) and/or that it receives more skin care. Next, we compared each parameter between Japanese and French subjects. The comparison of skin color, which clearly indicates the ethnic difference, shows that Japanese subjects have lower a* values and higher b* values than French subjects. On the other hand, the L* values of both ethnic groups showed no significant difference. Skin color is affected by various factors such as melanin, hemoglobin, carotenoid, amyloid and age. In CIE L*a*b* colors of skin, a* values reflect skin redness according to the blood flow, while b* values reflect skin darkness or yellowness according to levels of epidermal melanin and carotenoid, and L* values reflect skin brightness [3, 24– 26]. L*a*b* values vary with ethnicity, and especially epidermal melanin reflects ethnic differences in skin color [27]. As the b* value is higher in Japanese than in French subjects, Japanese subjects seem to have a higher melanin content in the skin. When skin physiological parameters were compared between the two ethnic groups, Japanese subjects had a higher water content in the stratum corneum, a lower TEWL and a lower thick abrasion of the stratum corneum. From these results, we can estimate that the skin hydration and barrier functions of the skin of Japanese subjects are greater than in the skin of French subjects. Races with darker skin types have greater numbers of melanosomes and a more acidic pH of the stratum corneum, which leads to higher stratum corneum integrity and increased skin barrier function [7]. Melanin in the epidermis is also known to protect against UV damage. Judging from these considerations, it is speculated Skin Pharmacol Physiol 2012;25:78–85 83 that the more efficient barrier function against UV and external insults in the skin of Japanese subjects compared with French individuals originates from the higher melanin content. In addition, Japanese subjects have a higher catalase activity than French subjects and are considered to have a greater UV-protective capacity. High catalase activity was also observed at the inner upper arm which shows a good skin condition. We previously reported that catalase activity at sun-protected sites of atopic dermatitis patients decreased depending on the degree of severity, which indicates that catalase activities are also affected by endogenous oxidative stress [28]. It is also known that the skin hydration and barrier functions of the skin of atopic dermatitis patients are inferior to those of normal subjects [29]. Our results show that catalase activities correlate positively with water content in the stratum corneum, negatively with TEWL and positively with the L* value on the cheek of Japanese subjects. From these results, we conclude that the higher catalase activity helps maintain a healthy skin condition. On the other hand, SCCP levels, which are another marker of oxidative stress, were higher in the skin of Japanese than in that of French subjects. This is not easily explained because SCCP levels and catalase activity seem to behave in opposite manners from each other. In our results of locational differences, SCCP levels and catalase activity showed an opposite trend in both ethnic groups (fig. 3). In addition, Pearson’s correlation analysis also showed a negative correlation between catalase activity and SCCP levels, although the correlation coefficient was low (tables 2, 3). The reason for this discrepancy is due to the multiple factors that affect the production of SCCP. SCCP exists as an oxidative product of stratum corneum proteins, which are influenced by other factors in addition to UV. For example, skin dryness causes an increase in SCCP [30–32]. Sebum is also a candidate which affects the production of SCCP [33]. 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