Materials. α-TP (an all-racemic mixture of 8 isomers or dl-α-tocopherol phosphate, purity: 92.7%, cosmetic grade) was supplied by Shawa Denko (Tokyo, Japan). Natural (+) α-T (type VI, purity: 70%, 695 mg d-α-tocopherol per g, 1036 IU/g), neutral red, and Corning® cell culture flasks were sourced from Sigma-Aldrich (Dorset, UK). Dulbecco modified Eagle’s medium (DMEM, high glucose, no pyruvate, no glutamine), foetal bovine serum (FBS), L-glutamine, penicillin-streptomycin, phosphate-buffered saline (PBS) pH 7.4, TrypLE™ Express (1X, phenol red-free) were sourced from Thermofisher Scientific (Paisley, UK). Alamar blue® was purchased from Fisher Scientific (Leicestershire, UK). Triton-X was supplied from Promega (Southampton, UK). Plastic bottom black walled (to prevent UVR scatter) 96 well plates were purchased from PerkinElmer (Beaconsfield, UK). The 2’,7 dichloro-dihydro fluorescein diacetate (H2DCFDA) cellular ROS detection assay kit was sourced from Abcam (Cambridge, UK). The oxygen radical absorbance capacity (ORAC) antioxidant assay kit was supplied by Zenbio (North Carolina, USA). The 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay was sourced from Alfa Aesar (Lancashire, UK). In all the studies, correction of natural α-T to account for its 70% purity was not performed because the biological activity of natural 70% pure (+) α-T (1036 IU/g) was equivalent to the pure synthetic α-T (1100 IU/g) and this allowed easy comparison. It is accepted that some impurities are present in the natural α-T and that these may have photoprotection activity, but this was thought to be unlikely.
Radiation sources. The UVA1 source was a Loctite LED flood system with peak outputs at 385 nm (Loctite, Henkel Ltd, Hemel Hempstead, UK). The array has an irradiation surface of 97 mm x 96 mm consisting of 144 LEDs. A 300W- 16S xenon arc solar UVR simulator (Solar Light, Glenside, PA, USA) with a full solar spectrum UVR setting, complying with ISO Standard 24444 and COLIPA (now Cosmetics Europe) 2006, was the source of solar-simulated radiation (SSR). The spectral irradiances of the sources were measured as previously described 4,16 and are shown in Figure S1 and outputs are described in Table S1 in the supporting information. The 385 nm spectrum was measured at a distance of 40 cm and the SSR spectrum was measured at a distance of 0 cm. Irradiation distances were based on the output of the source being measured and were selected to be within the dynamic range of the spectroradiometer. Irradiances were routinely measured with hand-held radiometers. This was a Loctite UVA/Vis radiometer (Loctite, Henkel Ltd, UK) for the 385nm source. A typical irradiance of 88.5 mW/cm2 with irradiation time of 11 min gave a dose of 56.5 J/cm2. The solar simulator spectrum was measured using a Solar® Light PMA 2100 radiometer (Solar® Light, Glenside, Pennsylvania). The solar simulator was calibrated using spectroradiometric readings such that an irradiance of 1100 μW/cm2 for 18 s gave 2-3 standard erythema dose (SED).
Absorption spectra of photoprotective compounds. α-TP and α-T were prepared at a concentration of 400 µM in methanol. Their UVR absorbance spectra were determined with a Perkin Elmer Lambda 2 UV / VIS Spectrometer (Perkin Elmer & Co GmbH, Oberlingen, Germany) between wavelengths 250-420 nm, to assess for possible sunscreening effects.
Photostability. SSR was used to test photodegradation of 1 mM (0.05% w/v) α-TP dissolved in Tris buffer pH 7.4 and an equivalent molar concentration of α-T dissolved in ethanol using increasing doses of SSR (10-50 SED or 17.8-88.9 J/cm2). Protected samples were used as dark controls (wrapped with aluminium foil). Absorbance of the dark controls A0 and samples (AT) was measured between 280-400 nm (n=3) after each exposure using a FLUOstar-Omega microplate reader (BMG LABTECH Offenburg, Germany). Percent degradation was calculated from the ratio of the change in absorbances (AT-A0) after exposure and the absorbance of dark control (A0) at 288 nm for α-TP and 291 nm for α-T, after normalising by subtracting the blank absorbance.
Cell Culture. The immortalised human adult low calcium temperature (HaCaT) keratinocyte cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This cell line has two p53 spontaneously transformed point mutations. HaCaT cells were cultured in DMEM supplemented with 10% FBS, 5% penicillin-streptomycin, and 5% glutamine, maintained in a humidified incubator at 37°C with 95% air and 5% CO2. Cells were cultured to around 80% confluence in 75 cm2 plastic flasks (Corning, New York, USA). Cells were plated into plastic bottom black wall 96 well plate (Perkin Elmer) and left to reach a confluence of 70-80% before being used for experiments.
Cell tolerance of photoprotective compounds. The cytotoxic potential of α-TP and α-T was evaluated using the Alamar blue®17 (Fisher Scientific, Loughborough, UK) and neutral red18,19 (Sigma-Aldrich, Dorset, UK) viability assays in HaCaT keratinocytes. Alamar blue® is a metabolic function (redox) indicator, which measures viable cells’ ability to reduce resazurin, to fluorescent resorufin. The neutral red assay assesses the ability of viable cells to incorporate the cationic dye into their lysosomes. Two assays with different mechanisms of action were employed to strengthen conclusions. The cells were treated for 24 h with α-TP (0.0001-10 mM) in prewarmed cell culture media at 37°C (cell culture media was used as a control) or α-T (0.0001- 5 mM) in 0.5 % ethanol mixed with prewarmed full media at 37°C (0.5 % ethanol in cell culture media was used as a control). The test compounds were aspirated from each well and the culture media containing 1:10 Alamar blue® or 1:100 neutral red solution was added to each well for 1.5 h or 3 h, respectively. The Alamar blue fluorescence intensity was measured directly at excitation/emission = 570/585 nm using an Infinite 200 PRO spectrofluorometer (Tecan Group Ltd, Männedorf, Switzerland). For neutral red, cells were washed three times in PBS to remove excess stain and then de-stain solution (50% v/v ethanol, 49% ddH2O, 1% glacial acetic acid (GAA)) was added. Neutral red optical density was measured at 540 nm. Cell viability for α-T or α-TP treated cells was calculated as a percentage of the control value. Each condition was tested in triplicate from three different passage numbers ranging from 16-20 passages.
UVA1 dose-response studies. HaCaT viability was determined over a range of 385 nm doses (56.5-225.9 J/cm2). This was determined after irradiation using Alamar blue® and neutral red assays with a FLUOstar-Omega microplate reader (BMG LABTECH, Offenburg, Germany). The viability was calculated as a percentage of the unirradiated value.
UVA1 cell survival studies. Cell starved low-serum (2%) media was used, as reported by Nakayama, to reduce the level of endogenous α-T 13. HaCaT cells, were added to black-walled 96-well plates (10,000 cells per well) and left overnight to adhere. They were washed twice with warmed PBS (100 μL/well) and treated with 80 µM of α-TP (1.5% tris buffer in medium was used as a control) or α-T (0.5 % ethanol in medium was used as a control), and then incubated at 37oC with 5% CO2 for 24 h. After aspiration of treatment solutions, the cells were washed twice with warmed PBS (100 μL/well), then fresh PBS (100 μL/well) was added to each well and the cells were irradiated with UVA1 without the plate lid. Each well was exposed individually for up to 44 min to obtain a maximum UVA1 dose of 226 J/cm2. Cells were kept on a cooling platform to keep them at around 37°C. Unirradiated controls (covered with foil) were kept in the same conditions as the longest exposure time to ensure any differences observed were due to the UVA1 exposure rather than any confounding factors. The cells were washed, and the cell viability was assessed using both the Alamar blue® and neutral red assays 24 h post-irradiation.
UVA1 ROS scavenging studies. HaCaT keratinocytes were seeded at a concentration of 25,000 cells per well in a black-walled, clear bottom 96-well plate and left for 24 h to adhere. They were washed in PBS and pretreated with 100 µL of a 100 µM α-TP or α-T for 24 h and then immediately washed with 1 x PBS followed by incubation with 100 µL of a 20 µM DCFDA solution in the dark for 45 min. The cells were then washed with PBS followed by UVA1 irradiation (57 J/cm2 (10 min exposure)). Tertiary butyl hydroperoxide (TBHP) was added into the assigned positive control wells at concentration 250 µM. Fluorescence intensity from cells of each well was measured using a FLUOstar-Omega microplate reader (BMG LABTECH, Offenburg, Germany) with excitation/emission = 485/520 nm. The mean (n=3) area under the curve (AUC) fluorescence intensity over 2 h was calculated for each condition. The mean difference in AUC fluorescence intensity between treatments, and the unirradiated vehicle control were calculated.
1,1-diphenyl-2-picryl-hydrazyl (DPPH) Assay. DPPH is hydrophobic dye that participates in hydrogen atom transfer (HAT) reactions, but strong hydrogen-bonding solvents such as methanol interfere with release of hydrogen atoms and thus enhance single electron transfer (SET) over HAT 20–22. A series of DPPH (6.25 - 0.0008 mM) solutions was prepared in the solvent in which the test compound was dissolved, (ascorbic acid was dissolved in DMSO, α-T was dissolved in methanol, and α-TP was dissolved in water). Aliquots of these samples were transferred to a 96-well plate, protected from light, placed on a shaker at room temperature for 30 min and their absorbances were measured at 517 nm using an Infinite 200 PRO spectrofluorometer (Tecan Group Ltd, Männedorf, Switzerland). Each condition was tested in triplicate. The average percentage inhibition of DPPH was calculated. Linear regression analysis (inhibition vs. concentration) was carried out to calculate the effective concentration for 50% inhibition (IC50) for each compound.
Oxygen radical absorbance capacity (ORAC) Assay. The ORAC assay tests a compound’s ability to inhibit peroxyl radicals (ROO●) from oxidising fluorescein. This assay was carried out with the ORAC Antioxidant Assay Kit (Zenbio, Research Triangle Park, North Carolina, USA) according to the manufacturer’s instructions. Trolox standards were prepared in the assay buffer (0-100 µM) along with serial dilutions of the test compounds. The fluorescein working solution (150 µL) was added to the wells of a 96 well plate, with 25 µL of each of the standards or test compound in duplicate, and the plate was incubated at 37°C for at least 15 min. A 2,2’-azobis-2-methyl propanimidamine dihydrochloride (APPH) working solution was then added to each well (25µL) to start the reaction. Fluorescence was measured in a preheated incubation chamber (37°C) using an Infinite 200 PRO spectrofluorometer (Tecan Group Ltd, Männedorf, Switzerland) with excitation/emission = 485/530 nm taken immediately and then every minute for 30 min. Standard curves were generated for each compound and the area under the curve (AUC) calculated. Each compound tested was then expressed as a Trolox equivalent concentration.
Statistical analysis. All data are expressed as the mean ± standard deviation (SD) where n=3 unless stated otherwise. The homogeneity of variance (Levene’s test) and the normality (Shapiro-Wilk test) of all sample group data were assessed prior to statistical analysis that was performed using the Statistical Package of Social Sciences program, SPSS version 17 (IBM Corp., USA) with a significance level of 0.05. Comparisons were performed using the student’s t-test, paired t-test comparing two conditions, ANOVA with multiple comparisons tests (Tukey’s, or Dunnett’s test), linear regression, non-linear regression. Data are presented using Prism software (GraphPad Prism, version 5.02, December 2008).
Mais M. Saleh - Investigation, Original draft preparation, Methodology, Reviewing and Editing K P Lawrence, Supervision, Methodology, Reviewing and Editing. Stuart A. Jones Conceptualization, Supervision, Reviewing and Editing. Antony R. Young, Conceptualization, Supervision, Reviewing and Editing.