UROCANIC ACID MIMIC AS SUNSCREEN 101 (UVA) irradiation (Waldmann) and generation of intracellular ROS was visualized after incorporation of the 2’,7’-dichlorodihydrofl uorescein diacetate (CMH2-DCFDA) probe. ASSESSMENT OF PHOTOPROTECTIVE ACTIVITY Cell culture. HaCaT keratinocytes were cultured at 37°C/5% CO2 in growth media con- sisting of DMEM [1 g/l glucose, 10% fetal calf serum (FCS)] supplemented by 1% of antibiotic cocktail. All culture media were obtained from Invitrogen. RHEs were supplied from Skinethic laboratory and have been used starting at 17th day of differentiation. UVB irradiation. HaCaT cells were seeded in 6-well plates at 8000 cells/cm2 3 days before irradiation (100 mJ/cm2, Waldmann lamp) w/wo active ingredient (MTPC) at various Figure 3. In vitro competitive isomerisation assay with human reconstituted epidermis. Dosage of cis and trans UCA in UVB-irradiated RHE topically treated with the active ingredient MTPC or with a reference photoprotective cream (positive control). Figure 4. Cell viability monitoring (MTT assay) in UVB-irradiated HaCaT keratinocytes treated with MTPC. All experiments were performed in triplicates. SEM errors bars are represented in black line and t-test was used for statistical analysis with *p 0.05, **p 0.01, ***p 0.001.

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.