The Photoprotective Properties of α-Tocopherol Phosphate Against Long-Wave UVA1 (385 nm) Radiation in Keratinocytes in Vitro

doi:10.21203/rs.3.rs-275304/v1

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Research Article

The Photoprotective Properties of α-Tocopherol Phosphate Against Long-Wave UVA1 (385 nm) Radiation in Keratinocytes in Vitro

https://doi.org/10.21203/rs.3.rs-275304/v1

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published 17 Nov, 2021

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Long-wave UVA1 radiation (375 nm – 400 nm) is often ignored when assessing sun protection due to its low sunburning potential, but it generates reactive oxygen species (ROS) and is poorly attenuated by sunscreens. This study aimed to investigate if α-tocopherol phosphate, (α-TP) a promising new antioxidant, could protect against long-wave UVA1 induced cell death and scavenge UVA1 induced ROS in a skin cell model.

HaCaT keratinocytes cell viability (24 h) was assessed with Alamar blue^® and neutral red assays. Radical scavenging efficacy of α-T and α-TP in vitro was monitored by the dichlorodihydrofluorescein (H₂DCFDA) ROS detection assay over 2 h. The mechanism of α-TP ROS scavenging was determined using chemical DPPH and ORAC assays.

In HaCaT keratinocytes, irradiated with 226 J/cm² UVA1 in low-serum (2%, starved) cell culture medium, pretreatment with 80 µM α-TP significantly enhanced cell survival (88%, Alamar blue) compared to control, whereas α-T pre-treatment had no effect survival (70%, Alamar blue). Pre-treatment of cells with 100 μM α-TP or 100 μM α-T before 57 J/cm² UVA1 also significantly reduced ROS generation over 2 h (24.1% and 23.9% respectively) compared to the control. However, α-TP displayed weak antioxidant activity in cell free assays suggesting its photoprotection was due to bioconversion to α-T by cellular phosphatases.

Treatment with α-TP prevented long-wave UVA1 induced cell death and scavenged UVA1 induced ROS in skin cells when added to the starved cell culture medium before UVA1 exposure by bioconversion into α-T.

Clinical Pharmacology

Nanoscience

Dermatology

α-tocopherol

UVA1

keratinocytes

radiation

The impact of terrestrial ultraviolet radiation (UVR) on the human skin, especially shortwave UVB (~295–315 nm), is well established. However, the effects of long-wave radiation, i.e., UVA (315-400 nm) are less well understood. Long-wave UVA penetrates deeper into the skin compared to UVB, reaching down into the dermis, and causing photodamage via the generation of reactive oxygen species (ROS) and cyclobutane pyrimidine dimers (CPD) ^1–3. Long-wave UVA1, near the UV-visible light interface (380 to 400 nm), is emerging as a significant risk to humans because its effects are not visible as ‘sunburn’, which is the main endpoint for sunscreen efficacy ⁴. This UVA1 range (380 to 400 nm) has also previously been shown to induce DNA damage and gene expression of matrix metalloproteinases (MMP1 and MMP9), which are thought to have an important role in photoageing through oxidative reactions⁴ and it has a role in inducing skin hyperpigmentation when the skin is not protected by sunscreens⁵.

Topically applied vitamin E (alpha-tocopherol, α-T) can inhibit UVR-induced DNA damage in human keratinocytes in vitro both pre and post-UVA exposure ⁶. However, α-T has several limitations including; (i) poor chemical and photostability ⁷, (ii) chromanoxyl radical formation, which leads to deleterious free radical reactions ^8,9, (iii) poor topical formulation solubility due its high hydrophobicity ¹⁰, and (iv) retention in the unviable outer layer of the epidermis, the stratum corneum (SC) ¹¹. One approach to overcome the limitations of α-T is to use structural derivatives, such as provitamins, that are metabolised by cutaneous phosphatases or esterases to generate α-T. Provitamins can be designed to be chemically stable and less hydrophobic and thus penetrate the SC and pass into the viable skin layers where they can act as antioxidants. α-tocopherol phosphate (α-TP) is a naturally occurring structural analogue of α-T that is water-soluble, chemically stable and, when formulated as nanoaggregates, can effectively penetrate the SC¹². Pre-treatment of an ex-vivo cultured mouse skin model with 50 μL of 9 mM α-TP has been shown to protect against UVR (63% UVB and 37% UVA) induced DNA damage and reduce the number of epidermal apoptotic sunburn cells (SBC) ¹³. However, to the best of our knowledge, no studies to date have investigated the protection of α-TP against pure long-wave UVA-induced skin damage.

α-TP, when applied topically to the skin, forms nano-sized aggregates that fuse with the SC, fluidises it and allows passage into the epidermis where it can act as an antioxidant, or convert into α-T to offer UVR protection¹⁴^,¹⁵. The aim of this work was to investigate the photoprotection potential of α-TP against long-wave UVA1-induced cell viability reduction and oxidative stress in an experimental model of HaCaT human keratinocytes.

UVA1 source emission and absorption spectra of the test antioxidants. The UVR emission spectrum (385 nm Loctite UVA) was compared with the absorption spectra of the test compounds (α-TP and α-T) to exclude any possibility of UVR filtering i.e., sunscreening effect. No overlap was seen between the UVR source and either compound (Fig 1). Absorption of α-TP (MW: 554.6 g/mole) and α-T (MW: 431 g/mole) is primarily between 270-320 nm (UVC and UVB) with peaks at 287 nm and 290 nm, respectively.

Photostability. α-T showed up to 14.6% reduction in maximum absorbance at 50 SED compared to 0 SED (p = 0.01, paired t-test, comparing two conditions) confirming its photodegradation (Fig 2 and S2). The total absorbance for α-TP showed no significant change at either 50 or 0 SED (Fig 2).

Photoprotective compound cell tolerability. α-TP was well tolerated in HaCaT keratinocytes and the LD₅₀ was comparable using the Alamar blue^® and neutral red assays (1676 and 1070 µM, respectively) (Fig. 3). α-T was also well tolerated by HaCaT keratinocytes dissolved in 0.5% ethanol in the cell culture medium with no statistical reduction in cell viability after the application of test concentrations 0.61-10,000 μM (Fig. S2 in the supporting information).

UVA1 dose-response studies. Alamar blue^® and neutral red assays gave comparable results (Fig 4). For example, after 57 J/cm² the mean cell viability was 92.5% vs 89.9%; after 115 J/cm² it was 86.4% vs. 88.0%, after 140 J/cm² it was 86.0% vs. 81.6%, after 170 J/cm² was 80.3% vs 77.6% and after 226 J/cm² it was 71.4% vs. 73.0%, respectively. The UVA1 dose 226 J/cm² was selected to perform the photoprotection studies using cell viability reduction as the endpoint.

UVA1 cell survival studies. There was a dose-response (57-226 J/cm²) for UVA1 irradiation in the absence of any treatment (Fig 5). When the HaCaT keratinocytes were pre-treated with 80 µM α-T or α-TP in low-serum (2%) medium prior to UVA1 irradiation (226 J/cm²) only treatment with α-TP significantly increased the cell survival compared to cells irradiated with the control vehicle (78%) using both Alamar blue® (88%) and neutral red assays (93%) (Alamar blue®- p = 0.009, neutral red- p = 0.014, one-way ANOVA with Dunnett’s multiple comparisons test) while α-T did not (70-74%) (Fig. 5).

UVA1 ROS scavenging studies. Generation of ROS in HaCaT cells was significantly higher after UVA1 compared to the unirradiated cells (p < 0.0001, paired t-test, comparing two conditions, for the raw data see Fig. S3 in the supporting information). The positive control, i.e., the chemical generation of ROS using 250 µM TBHP, generated fewer ROS compared to UVA1 (p < 0.0001, paired t-test, comparing two conditions). Pre-treatment of HaCaT with 100 µM α-TP before irradiation with UVA1 caused a significant reduction of ROS generation (ca. 24.1% in ROS generation compared with the vehicle control, p = 0.0026, paired t-test, comparing two conditions). α-T produced a similar reduction in ROS to α-TP (ca. 23.9% reduction in ROS generation, p = 0.0034, compared to the vehicle control, paired t-test, comparing two conditions) (Fig. 6).

1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay. The α-TP exhibited poor DPPH activity, with an IC₅₀ value of 1038 µM or 575.7 μg/ mL (see Fig. S4 in the supporting information). The IC₅₀ values of L-ascorbic acid (L-AA) and α-T were 18.7 and 24 µM, respectively using curve fitting (Fig 7).

Oxygen radical absorbance capacity (ORAC) assay. α-T was not assessed in the ORAC assay because it is hydrophobic and insoluble in the reaction buffer. Trolox was the reference control because it is a water-soluble vitamin E derivative. The concentrations of α-TP and L-ascorbic acid equivalent to 100 µM Trolox were 7599 and 107.02 µM, respectively (Fig. 8). The order of activity was Trolox > L-AA > α-TP.

Exposure to UVA1 has been linked to DNA damage that results in formation of cyclobutane pyrimidine dimers (CPD)⁴, matrix metalloproteinases-1 (MPP-1) gene overexpression that may result in photoageing⁴, production of nitric oxide (NO) that may be beneficial for hypotension²³, and skin opsin-3 upregulation that causes pigmentation²⁴. Unlike sunscreens, topical antioxidants have been shown to reduce oxidative damage from ROS induced by UVR without the potential to interfere with vitamin D synthesis. Vitamin E (α-tocopherol, α-T) is one of the classical topical antioxidants that has been used to inhibit UVR-induced oxidative damage²⁵, it has also been shown to inhibit dark CPD in model systems^6,26 . Such CPD are formed after UVR exposure by a delayed reaction between DNA and reactive compounds, currently believed to be excited carbonyls ^26,27. Dark CPD have been reported in human skin in vivo after 385nm ⁴and SSR²⁸, but their biological significance is unknown.

Numerous limitations of α-T hinder its effectiveness in cosmeceutical products. Adding a phosphate ester to form a provitamin is a strategy that has been shown to overcome these limitations. α-TP has shown important UVA protective properties in the skin, but previous work did not establish if these benefits were due to its antioxidant properties or its ability to act as a sunscreen, as it forms liposomes (with different optical properties) when applied to skin¹⁴. Furthermore, previous work with α-TP used UVR sources with UVB content that overlapped with the absorption spectrum of α-TP (generated by the compound’s two aromatic rings)¹³. However, to the best of our knowledge no studies to date have investigated the protection of α-TP against spectrally pure UVA-induced skin damage by its antioxidant properties¹³.

Given the lack of overlap between the absorption spectra of α-TP and α-T with the UVA1 source it can be concluded that neither compound was capable of absorption/sunscreening effects. The absorbance of α-TP slightly increased with increasing SSR dose, most likely due to small changes in drug aggregation, whereas α-T absorbance significantly decreased which indicated photodegradation. The SSR dose in this study was 51 J/cm², which corresponds to 3.4 hours of UK summer sun, and is in agreement with reports that showed α-T photodegradation at a dose of 198 kJ/m² (19.8 J/cm²) using SSR (290-500 nm) and a subsequent loss of the compound’s antioxidant activity²⁹.

α-TP reduced HaCaT keratinocyte viability more than α-T (LD₅₀ was 1.1-1.8 mM vs > 4.70 mM, respectively) suggesting the phosphate group enhanced the molecule’s ability to enter the cells and cause cytotoxicity. This has also been shown in oral mucosal cells in a study that hypothesised that toxicity was caused by disruption of intracellular enzymes^30,31. Previous studies in other cells lines support selective cell toxicity by α-TP, but α-T is generally well tolerated ³⁰^,³². However, the cytotoxicity of TP strongly depends on its isomeric form with the γ -form showing better growth inhibitory activity than δ-form (IC₅₀ was 30 vs 55 µM, respectively) in human colorectal cancer cells (HCT116 and HT29 cells). In the same study, the α-form was the least inhibitory compared to other isomers (IC₅₀ > 100 µM) ³³.

Interestingly, pretreatment of human HaCaT keratinocytes with α-TP in starved low serum media (2% FBS) for 24 h exhibited superior protection against long-wave UVA1 (385nm) induced cell death compared to α-T. A similar effect has been reported by Nakayama and colleagues who found that incubation of skin cells with 2% FBS significantly depleted endogenous antioxidant α-T by 50% after 24 h incubation. They also showed that treatment with 0.5% α-TP dissolved in 2% DMEM not only inhibited depletion of endogenous α-T, but also increased its level by two-fold compared to untreated cells. In the same study, UVB irradiation was found to significantly reduce endogenous α-T in cultured skin by 50% compared to unirradiated skin. This reduction was inhibited by pre-UVB treatment with α-TP for 3 h which caused a 2-fold rise in α-T compared to irradiated cells treated with vehicle control¹³. This may be due to the release of α-T from the provitamin E within keratinocytes by their phosphatase enzymes, which prolongs the protection effect even after 24 h. Another possible reason is that the incorporation of α-TP is essential to prevent the propagation of radical species ³⁴.

It is well known that ROS are significantly increased in cells during and following UVA irradiation^35,36. As UVA-related biological effects are largely mediated by ROS, their elimination is essential for protection against UVA damage. In this work, pre-irradiation treatment with either α-TP or α-T result in reduction in UVA1-induced ROS generation in HaCaTs to a similar extent (by ~24.1% and 23.9%, respectively) compared to the irradiated vehicle control treated cells. Wu et al, investigated photoprotection of HaCaT keratinocytes using UVA1 (365 nm) and monitored ROS generation. They reported that 2.9 IU/ml of α-T in serum free medium significantly reduced (80%) ROS generation compared to irradiated vehicle treated cells (742.5 vs. 3,952.2 fluorescence intensity/protein, 1/mg protein, p < 0.05) ²⁵. The difference in the degree of ROS suppression with our findings using the dichloro-dihydro-fluorescein (DCFDA) assay, may be due to the differences in UVA1 source (365 nm vs 385 nm) and UVA1 dose (8 J/cm² vs 57 J/cm²), the concentration of tested compound (2 mg/ml = 4.6 mM vs 100 µM), the concentration of H₂DCFDA (5µM vs 20 µM), and the level of supplements in growth medium (serum-free vs low-serum medium).

Studies were done with DPPH and ORAC assays without cells to determine if α-TP afforded innate antioxidant activity or was the outcome of its bioconversion to α-T. As free radical scavenging by antioxidants can proceed through single electron transfer (SET) or hydrogen atom transfer (HAT), two assays (DPPH and ORAC respectively) were used to identify the mechanisms ³⁷. α-TP exhibited poor DPPH activity compared to ascorbic acid and α-T. The α-T IC₅₀ of 19 µM was lower than previously reported at 40.6 μM, but within normal inter-laboratory variability ³⁸. The ascorbic acid IC₅₀ of 24 µM was slightly lower than previously reported, i.e., 34 µM ³⁹, 54.6 µM ⁴⁰, 49.5 µM ³⁸, but confirmed that assay worked well. This poor activity suggested that α-TP had very little antioxidant capacity per se via SET whereas α-T and ascorbic acid were potent antioxidants via this mechanism. α-TP and water-soluble ascorbic acid were compared to Trolox in the ORAC assay. Trolox activity was almost equivalent to ascorbic acid, but was 75-fold more effective than α-TP. Our findings agree well with those of Rezk et al. using the Trolox equivalent antioxidant capacity (TEAC) assay, in which they determined the TEAC for Trolox and α-TP (1.0, and 0.01, respectively) i.e., Trolox was 100-fold more potent than α-TP ⁴¹. Weak α-TP activity in two different assays indicated that bioconversion to α-T was responsible for the UVR protective in a biological model.

In conclusion, α-TP displayed weak antioxidant activity when assessed by chemical assays. In HaCaT keratinocytes it was well tolerated, and had superior photoprotection properties compared to α-T. Pre-irradiation treatment of HaCaT keratinocytes with α-TP resulted in a similar reduction in ROS generation as α-T, but α-TP penetrates more effectively into viable skin layers compared to α-T. The biological activity of α-TP is likely to be a consequence of its bioconversion to α-T by endogenous cellular phosphatases, which increase the intercellular level of α-T even after UVA1 exposure ends. Photostability studies showed α-T was photodegraded by SSR, but α-TP was very photostable. As a result of the superior photostability, its greater protection against cell viability reduction and comparable reduction in level of ROS generated by UVA1 exposure it is an excellent candidate to include in sun-protection products to reduce the impact of UVA1-induced skin damage.

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 (A_T) 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 (A_T-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 red^18,19 (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 37^oC 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).

CRediT Statement

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.

Acknowledgment

MS thanks the University of Jordan for funding her research project and Shawa Denko (Tokyo, Japan) for supplying the TPNa^® sample. We thank Professor Bernd Herzog for the loan of the UVA irradiation system.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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The Photoprotective Properties of α-Tocopherol Phosphate Against Long-Wave UVA1 (385 nm) Radiation in Keratinocytes in Vitro

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