EFFECT OF PERMING/BLEACHING ON CUTICLES 221 distilled water contammg 0.1 % Tween 20 in a Falcon 50-ml conical tube (Becton Dickinson, Franklin Lakes, NJ) at 3 7 ° C for 20 hr. After passing through nylon mesh, the cuticle suspension was subjected to turbidity measurement at 600 nm (7). The amount of delaminated cuticle was estimated through OD600, adopting the standard curve calibrated by the preweighed cuticle suspensions (Figure la). SIZE MEASUREMENT OF CUTICLE FRAGMENTS Delaminated cuticles were centrifuged at 14,000g for 10 min, and resuspended at 0.2% in 1 % Tween 20 solution. Finally, the cuticle suspension was mixed with an equal volume ofVectorshield (Vector Laboratory, Burlingame, CA) on a slide and a cover glass put on. Utilizing an LSM510 fluorescent confocal microscope (Carl Zeiss AG, Gottin­ gen, Germany) with a 488-nm argon laser light, we observed autofluorescence from delaminated cuticle fragments under the following setting parameters: pinhole: 200 detector gain: 900 ampl offset: -0.2 and ampl gain: 1.0. From the resultant confocal images, the averaged area of cuticle fragments was computed with NewQube gradational analysis software (Nexus, Tokyo, Japan). ASSAY OF TOTAL AND Sl00A3 PROTEINS Permanent-wave-lotion-immersed hair was concentrated by ultrafiltration after alkyla­ tion of thiol groups with iodoacetamide. Protein amounts were estimated using the Bradford assay (8). Human recombinant S100A3 protein was prepared as described previously (9,10). ID-PAGE of S100A3 was performed using precast 4-12% Bis-Tris Nu-PAGE (7 .8 x 6.3 x 0.1 cm) according to the supplier's manual (Invitrogen, Carls­ bad, CA). The separation was started at 200 V for 3 5 min. S 1 00A3 protein levels were quantified by Western blot analysis (5 ). Quantification was performed, using human recombinant S100A3 as a standard, by image-analyzing software (Scion, Frederick, MD). a b C 1.0 � 0. 7 � 0. 9 Bleach * Perm * "' )OB J 0.6 0.8 I.. � 0.7 * i 0.5 (U ' � 0.6 0 0.6 � 0.4 � 0.5 □� · · D 0.4 • l\bn-treat a 03 a 0.4 •Perm ""Cl "i 0.3 (1) 1ii 0.2 +-' 0.2 A Bleach .£ -� 0.2 E 0.1 j 0.1 J!! (1) (1) 0 0 0 0 0.010 0.020 0 0.3 1.0 3.0 0 3 10 Cuticle (w/� Thioglych:llate (%) f-i:i02 (%) Figure 1. Increase of delaminated cuticles upon hair treatment chemicals. (a) Correlation between cuticle fragments and turbidity. Note the amounts of delaminated cuticle from permed (b) and bleached hair (c). Values represent the average of three independent experiments ± SD. Statistical analyses were performed by t-test of the values obtained without hair treatment chemicals. * p 0.05.

222 JOURNAL OF COSMETIC SCIENCE 2D GEL-ELECTROPHORESIS OF SlO0A3 IN DELAMINATED CUTICLES Delaminated cuticles (1 mg) from bleached hair were extracted with 100 mM dithio­ threitol containing 200 mM Tris-HCl buffer (pH 7 .6). Extracts were concentrated by precipitation with trichloroacetic acid. The precipitate was applied to modified two­ dimensional electrophoresis, as previously described (3 ). Isoelectric focusing was per­ formed according to standard protocols using IPG strips with a narrow pH gradient of 3-5 (Sigma-Aldrich, St. Louis, MO). The second dimension was performed under the same conditions described above, using the Zoom type of Nu-PAGE gel at 7 .8 x 6.3 x 0.1 cm. The mean density of spots and their proximal background zone on silver-stained 2D-P AGE gels were measured using image-analyzing software (Scion). Subtracted mean densities were used for proportion calculation. RESULTS At first, we examined whether the turbidity (OD 600 ) accurately represents the concen­ tration of the cuticles in suspension. OD 600 was linearly increased up to 1.0, depending on the amount of suspended cuticle (Figure la). No difference in OD 600 was observed among non-treated, permed, and bleached hair samples. Consistent with previous re­ ports (7), the cuticles of both permed and bleached hair were more easily delaminated compared to untreated ones (Figure 16,c). The amount of delaminated cuticles from bleached hair was increased as a higher concentration of hydrogen peroxide was applied. In the case of permed hair, however, application of an excessive concentration (3%) of thioglycolate results in reduction of the delamination. This might be attributed to the lesser rubbing of the wavy hair with the water-stirring method. Due to their very flat shape and their transparent nature, it is difficult to clearly identify delaminated cuticle fragments by conventional microscopy. In this study, we observed the autofluorescence of delaminated cuticles using confocal fluorescent microscopy (Fig­ ure 2a). Confocal images were processed using gradation analysis software. The trans­ formed gradation image revealed that the average area of each cuticle fragment, delami­ nated from permed hair, was about twice as large as that of untreated hair (Figure 26), whereas those of bleached hair were 30% smaller compared to the normal ones (Figure 2c). These results indicate that the physiological characteristics of cuticles from the bleached hair are distinct from those of the permed hair. We previously detected SlO0A3 in the permanent waving lotion (5). We examined the correlation of Sl00A3 elution by perming treatment with the enlargement of cuticle delamination. Treatment of hair fiber at the minimum thioglycolate concentration required to elute SlO0A3 into permanent waving lotion (�0.3%, Figure 3) resulted in significant enlargement of the cuticles. Although application of higher thioglycolate concentration increased the amount of eluted S 1 00A3 protein, the enlargement of delaminated cuticles reached a plateau at 1 % concentration (Figure 26). These results indicate that the loss of S 1 00A3 protein, even in a low amount, results in cuticle delamination. In contrast to permanent waving, bleaching did not result in a release of the Sl00A3 protein (data not shown). Nevertheless, cuticles of bleached hair were easily delaminated (Figure le) and fragmented into small pieces (Figure 2c). In this study, we performed 2D PAGE analysis of extracts of the cuticles delaminated from bleached hair. Although the