SACRAN PROTECTS SKIN AGAINST POLLUTANTS 21 aqueous solution by aspiration with a vacuum pump, and then dried at 50°C (102 μg/cm2 of polysaccharides were deposited on each fi lter). PBS(-) was reacted with 25 μM NBD-hydra- zine in the presence of 0.05% trifl uoroacetic acid for 30 min in the dark to determine the amount of ACs. ACs were quantifi ed by measuring fl uorescence intensity (FI) (Ex 470 nm, Em 550 nm) using a microplate reader with a calibration curve prepared with acrolein as a standard substance. BaP in the PBS(-) was quantifi ed by measuring FI (Ex 360 nm, Em 450 nm) using a calibration curve prepared with BaP. Trapping effi ciency is expressed as a percent- age versus the value of the nontreated membrane fi lter. CELL CULTURE AND EXPOSURE TO TOBACCO SMOKE HaCaT keratinocytes were cultured in DMEM with 5% FBS at 37°C in a humidifi ed atmosphere containing 5% CO2. HaCaT keratinocytes were inoculated at a density of 3.5 × 104 cells per well in 96-well plates. Cells were cultured in DMEM containing PBS diffused with tobacco smoke in the presence or in the absence of polysaccharides for 24 h. mRNA EXPRESSION OF CYP1A1 The expression level of CYP1A1 mRNA in HaCaT keratinocytes, after culturing for 24 h in DMEM containing PBS diffused with smoke obtained from burning one piece of tobacco by aspiration with a vacuum pump, was evaluated using real-time quantitative PCR analysis. After removal of the medium from wells, washed adherent cells were processed for PCR analyses by the direct addition of 350 μL RLT buffer (Qiagen) containing 3.5 μL 2-ME to each well. Total RNA from the lysed cells was extracted using an RNeasy Mini Kit, according to the manufacturer’s instructions. First-strand cDNA was then synthesized using a PrimeScript RT Master Mix and T100 Thermal cycler (Bio-Rad Laboratories, Hercules, CA). Real-time PCR analysis was performed using an Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher Scientifi c Inc., Kanagawa, Japan) with 1 μL cDNA for each sample. SYBR Green Real-Time PCR Master Mix was used to detect products, and 10 μM concentra- tions of the primers were used: human CYP1A1 and glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) obtained from Takara Bio Inc. (Shiga, Japan) (i.e., NM_002046.5 and NR_045089.1). The relative amount of cDNA in each sample was normalized using GAPDH, and the melting curve was used to verify specifi city. ADVERSE EFFECTS OF TOBACCO SMOKE ON HaCaT KERATINOCYTES The infl uence of smoke obtained from burning one piece of tobacco was measured for the following parameters: cell damage, cell viability, intracellular ROS, and intracellular CPs. Cell damage was examined by measuring cell viability using the neutral red assay. Cells were cultured with DMEM containing 5% FBS and neutral red at a concentration of 33 μg/mL for 2 h. After washing with PBS, neutral red incorporated into the living cells was extracted with 30% MeOH aqueous solution with agitation. The absorbance at 550 nm of the resulting solution was measured using a microplate reader.

JOURNAL OF COSMETIC SCIENCE 22 Intracellular ROS levels were measured using H2DCFDA, a fl uorescent probe for H2O2. Cells were treated with 20 μM H2DCFDA in HBSS for 30 min. After lysing the cells with 0.1% Triton X-100 in PBS, the fl uorescence (Ex 485 nm, Em 530 nm) was measured using a fl uorescence microplate reader (Spectra Max Gemini Molecular Devices, San Jose, CA). Intracellular ROS levels are calculated as FI per μg protein and are expressed as a fold change of the value of control cells. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher). Intracellular levels of CPs were determined by fl uorescence labeling of the aldehyde group with FTSC. After fi xation with cold MeOH for 10 min, cells were incubated with FTSC in 0.1 M MES-Na buffer (pH 5.5) and 2 μM Hoechst 33342 for 1 h. CPs and nuclei were quantifi ed by image analysis using corneocytometry software (Ciel, Tokyo, Japan) after obtaining fl uorescence images with a fl uorescence microscope (Floid Cell Imaging Station Thermo Fisher Scientifi c Inc). BARRIER FUNCTION OF POLYSACCHARIDES AGAINST TOBACCO SMOKE Ex vivo study. The barrier function of polysaccharides was characterized by measuring CP levels in corneocytes following exposure to tobacco smoke. Corneocytes were obtained from the upper inner arm of fi ve human volunteers who were nonsmokers by the tape-stripping method using cellophane tape (Nichiban Co., Ltd., Tokyo, Japan) and were transferred to glass slides after dividing each piece of tape into four pieces. One piece of the corneocytes from each volunteer was used for the evaluation of one sample. The corneocytes were treated with 10 μL polysaccharide aqueous solution for 10 min at room temperature. As the control, H2O, which was the solvent for the polysaccharide aqueous solution, was ap- plied on corneocytes. After rinsing with running water and then drying, the glass slides with corneocytes were placed in a box fi lled with tobacco smoke for 2 h at room tem- perature. After a further incubation for 24 h at 37°C, CP levels of corneocytes were mea- sured by fl uorescence labeling as follows: Corneocytes on glass slides were immersed in 0.1 M MES-Na solution (pH 5.5) containing 20 μM FTSC for 1 h at 25°C in the dark. After rinsing, images were obtained using a fl uorescence microscope (Floid Cell Imaging Station, Thermo Fisher). CP levels were quantifi ed from the fl uorescence images using corneocytometry software (Ciel). Informed consent was obtained from each volunteer af- ter explaining the method of collecting corneocytes and the aim of the test. The ex vivo study was approved by the Ethical Committee of the Daito Kasei Kogyo Co., Ltd. and was performed three separate times using the same fi ve volunteers. Statistical analysis. All study data are expressed as means ± standard deviation (SD). Sig- nifi cant differences between experimental values were determined using the Wilcoxon rank-sum test, and p-values less than 0.05 are considered statistically signifi cant. RESULTS PENETRATION OF POLYSACCHARIDES INTO RHEEs In the histological study, it was observed that sacran as well as HA applied topically on RHEEs remained in the stratum corneum (Figure 2). In addition, the amount of sacran