SACRAN PROTECTS SKIN AGAINST POLLUTANTS 23 in the medium of RHEEs topically treated with sacran was approximately 1%, and more than 99% of the sacran was recovered from the RHEE homogenates (Figure 2). HA also showed similar results. These results indicate that polysaccharides topically applied on RHEEs stay in the stratum corneum and do not penetrate well to the living cell layers of the epidermis. TRAPPING EFFECT OF POLYSACCHARIDES ON ACs AND BaP To examine the trapping effects of polysaccharides, the amounts of ACs and BaP in PBS(-) diffused with tobacco smoke passed through membrane fi lters treated with or without polysaccharides were quantifi ed. Sacran-treated membrane fi lters showed excellent trap- ping effects for both ACs and BaP (Figure 3). More than 90% of ACs and BaP were trapped in the sacran-treated membrane fi lter compared with the control (nontreated membrane fi lter). In the case of the HA-treated membrane fi lter, although it failed to signifi cantly trap ACs, it showed a signifi cantly high trapping effect for BaP compared with the con- trol (Figure 3). Thus, although both sacran and HA have trapping effects on ACs and BaP, sacran showed a superior effect compared with HA. SUPPRESSIVE EFFECTS OF POLYSACCHARIDES ON mRNA EXPRESSION OF CYP1A1 INDUCED BY TOBACCO SMOKE HaCaT keratinocytes cultured in DMEM containing PBS diffused with tobacco smoke through a nontreated membrane fi lter showed a predominant upregulation of CYP1A1 mRNA levels. Cells cultured in DMEM containing PBS diffused with tobacco smoke Figure 2. Penetration of biotin-conjugated polysaccharides into RHEEs. RHEEs were treated topically with a biotin-conjugated polysaccharide aqueous solution (sacran or HA) as noted and then were cultured for 24 h at 37°C. The culture media were collected to quantity biotin-conjugated polysaccharides that penetrated through the RHEEs. The histology of biotin-conjugated polysaccharides is shown as representative images (scale bars, 100 μm). The penetration or retention of biotin-conjugated polysaccharides in RHEEs is expressed as a percentage of the amount of biotin-conjugated polysaccharides applied. PBS(-) was used as a control. Each value represents the mean ± SD of three experiments.

JOURNAL OF COSMETIC SCIENCE 24 through sacran or HA-treated membrane fi lters had signifi cantly reduced CYP1A1 mRNA expression levels (Figure 4). On the other hand, when comparing sacran and HA, sacran showed a more signifi cant downregulation of CYP1A1 mRNA expression levels than HA (Figure 4). AMELIORATION OF ADVERSE EFFECTS INDUCED BY TOBACCO SMOKE ON HaCaT KERATINOCYTES Treatment of HaCaT keratinocytes with tobacco smoke showed high cytotoxicity, but sacran or HA signifi cantly reduced the cytotoxicity of tobacco smoke. Compared with the Figure 3. Trapping effects of polysaccharide-treated fi lters on ACs and BaP in tobacco smoke. ACs and BaP in PBS(-) diffused with tobacco smoke passed through fi lters treated with or without polysaccharides were quantifi ed as follows: ACs were estimated by FI (Ex 470 nm, Em 550 nm) with NBD-hydrazine in the pres- ence of trifl uoroacetic acid. BaP was quantifi ed by FI (Ex 360 nm, Em 450 nm). Calibration curves were prepared, and their concentrations were quantifi ed. Each value represents the mean ± SD of three experi- ments. Wilcoxon rank-sum test, ***p 0.001. Trapping effi ciency is expressed as a percentage versus the value of the H2O-treated membrane fi lter diffused with tobacco smoke (Control).