JOURNAL OF COSMETIC SCIENCE 102 concentrations. Irradiations were performed in phosphate-buffered saline (PBS) before medium renewing w/wo ingredient for 24 h. RHEs were irradiated from 300 to 500 mJ/cm2 w/wo active ingredient in PBS, then replaced in maintenance medium w/wo ingredient for 24 h at 37°C/5% CO2. Cell viability. HaCaT cells viability was determined with a specifi c MTT test, and absor- bance was measured using a spectrophotometer equipped with λ = 570 nm. Dosage of infl ammatory and immunosuppressive mediators. Supernatants collected from HaCaT cells/RHE (stored at -80°C until use) were examined for secreted interleukin (IL)-6, IL-8, TNF-alpha and Galectin-7 protein levels using commercially available enzyme- linked immunosorbent assay (ELISA) kit (Biotechne). DNA damages. Cyclobutane pyrimidine dimers (CPDs) were quantifi ed in HaCaT by enzyme immunoassay Oxiselect cellular UV-induced DNA damage ELISA kit (Cell biolabs). In brief, cells were fi rst seeded in 96-well tissue culture plates before UVB irradiation and CPD revelation. Immunofl uorescence assays were performed on RHE cryosections 24 h after UVB irradiation with a specifi c monoclonal anti-thymine dimer antibody (Sigma, ref.T1192). Anti-infl ammatory activity. The catalytic activity of the pro-infl ammatory enzyme 5-lipoxygenase (5-LOX) was investigated in tubo using the LOX inhibitor screening assay kit (Abcam), and the hydroperoxides produced were detected and measured by a purifi ed LOX. Active ingre- dient was tested from 1 to 3 g/l and nordihydroguaiaretic acid was used as a reference LOX inhibitor. MONITORING OF IMMUNOSUPPRESSION Human T-lymphocyte line Jurkat cells were cultured at 37°C/5% CO2 in suspension in RMPI 1640 medium supplemented with 10% of FCS. Cells were seeded in 24-well Figure 5. Monitoring of CPDs in UVB-irradiated HaCaT keratinocytes treated with MTPC. All experi- ments were performed in triplicates. t-test was used for statistical analysis with *p 0.05, **p 0.01, ***p 0.001, and NS: nonsignifi cant. Figure 6. Monitoring of CPDs in UVB-irradiated RHE topically treated with MTPC.

UROCANIC ACID MIMIC AS SUNSCREEN 103 plates at 150,000 cells/well 2 days before activation with ionomycin and phorbol-12 myristate-13 acetate (Sigma) and stress application: treatment with various concentra- tions of recombinant human Galectin7 (Biotechne) for 24 h. Specifi c IL-2 and interferon (IFN)-γ cytokines were further monitored by ELISA quantifi cation (Biotechne). RESULTS UVB-ABS ORPTION PROPERTIES OF MTPC Absorption spectrum and molar extinction coeffi cient. MTPC shows an important absorption in the UVB range [280–320 nm], with a maximum at 284 nm. Its molar extinction coef- fi cient at 290 nm (13,150 L/mol/cm) is comparable to some listed Category I sun fi lters. Absorption spectrum of trans UCA is quite similar to MTPC, indicating that in the skin, these compounds may compete for the absorption of similar UVB wavelengths. Limitation of UCA isomerisation. UCA is produced in the stratum granulosum from the degradation of fi laggrin, and accumulates mainly in the stratum corneum. In human epidermis, UCA levels range from 4 to 34 nM/cm2 (4). Competition for UVB absorption was fi rst examined in tubo irradiating equimolar mixtures of UCA and MTPC, a ratio that can easily be attained after topical application of MTPC considering UCA very superfi cial localization. In such conditions, UCA isomerisation into cis UCA was halved (data not shown). Trans MTPC (Entadamide A) could also limit UCA isomerisation, but was less potent (data not shown). We further confi rmed these observations in vitro by the monitor- ing of UCA isomerisation in UVB-irradiated reconstituted human epidermis (RHE) in the presence of topical MTPC (Figure 3). Figure 7. Monitoring of pro-infl ammatory cytokines IL-8 (A) and TNFα (B) release from UVB-irradiated RHE topically treated with MTPC. Figure 8. Inhibition of 5-LOX activity by Entadamide A (trans isomer of MTPC).