Jean-Philippe Belaidi

The human melanocyte as a particular target for UVA radiation and an endpoint for photoprotection assessment.

Abstract— The induction of DNA breaks by UVA (320–400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin‐related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.

Molecular Responses to Stress Induced in Normal Human Caucasian Melanocytes in Culture by Exposure to Simulated Solar UV

Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV‐induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV‐induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300–400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m2 UV‐B and 110 kJ/m2 UV‐A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase‐3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2‐M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two‐fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV‐A radiation only (320–400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight. They provide new data related to gene expression and suggest that melanin in light skin could contribute to sunlight‐induced genotoxicity and maybe to melanocyte transformation.

TRP-2 specifically decreases WM35 cell sensitivity to oxidative stress.

TRP-2 (dopachrome tautomerase) is a melanogenic enzyme whose expression was recently reported to modulate melanocyte response to different cytotoxic events. Here we studied a possible role of TRP-2 in the oxidative stress response in the amelanotic WM35 melanoma cell line. Cell viability assays showed that TRP-2 overexpression in WM35 cells reduced their sensitivity to oxidative stress. Comet assays linked TRP-2 expression to DNA damage protection, and high-performance liquid chromotography-tandem mass spectrometry experiments showed an increase in intracellular glutathione in TRP-2-overexpressing cells. These effects were specifically reversed when TRP-2 was silenced by RNA interference. Nevertheless, these properties appeared to depend on a particular cell environment because expression of TRP-2 failed to rescue HEK epithelial cells exposed to similar treatments.

Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity in vitro assessment.

Todays lifestyle is often associated with frequent exposure to sunlight, but some xenobiotics used in drugs, cosmetics or food chemicals can produce adverse biological effects when irradiated. In particular, they can increase the risk of photogenotoxicity already due to UV radiation itself. There is thus a need to design appropriate approaches in order to obtain relevant data at the molecular and cellular level in this field. For ethical and practical reasons, in vitro models can be very convenient at least for first evaluation tests. Here, we propose a strategy based on complementary experiments to study the photogenotoxic potential of a compound. The fluoroquinolones BAYy3118 and lomefloxacin were used as standards to demonstrate the performance of each test: photoinduced interaction with supercoiled circular DNA, photomutagenicity in the yeast Saccharomyces cerevisae, induction of DNA photodamage in cultured human skin cells as revealed by comet assay, and finally induction of specific phototoxic stress responses such as p53 activation or melanogenesis stimulation. Such a strategy should help to ensure the safety of products likely to undergo environmental sunlight exposure.

Comet assay combined with p53 detection as a sensitive approach for DNA photoprotection assessment in vitro

Abstract: A simple in vitro approach where sun formulations are spread on a quartz slide and placed over human skin cells in culture is proposed as a convenient test for photoprotection assessment at the DNA level. Using the comet assay, DNA strand breaks and oxidative DNA damage were detected. Then, accumulation of p53 protein was studied as a marker for UV‐induced genotoxic stress. Such a method was used to compare formulations with different photostability. Spectroradiometry showed that a photo‐unstable formulation lost its effectiveness in UVA screening when pre‐irradiated by simulated sunlight. As a consequence, such a formulation was not as protective as a photostable one at the genomic level.

Photo-pollution stress in skin: Traces of pollutants (PAH and particulate matter) impair redox homeostasis in keratinocytes exposed to UVA1

BACKGROUND It is likely that skin is exposed to low concentrations of pollutants such as Polycyclic Aromatic Hydrocarbons (PAH) either through topical penetration by ultrafine particles or by systemic distribution. No precise estimation of pollutants in living skin is available, but literature has reported contamination of blood by PAH at concentrations in the nanomolar range. Some pollutants (PAH for example) are photo-reactive and phototoxic: sunlight and pollution might thus synergistically compromise skin health. OBJECTIVE Here, the biological effects of particulate matter, PM extract and various PAH were compared in normal human epidermal keratinocytes (NHEK) and reconstructed skin model exposed to either daily UV (d-UV 300-400nm) or UVA1 (350-400nm). Impact of pollutants (PM, PAH or PM extract) combined to UV was studied on NHEK by measuring toxicity, redox homeostasis and GSH metabolism in NHEK. METHODS NHEK were exposed to UV from solar simulator (either d-UV or UVA1) combined with pollutants. Viability, clonogenic efficiency, redox homeostasis and GSH metabolism were assessed. RESULTS Pollutants (PAH, PM or PM extract) ±UVA1 irradiation was associated with a significant phototoxic effect that was equal to or greater than that produced by d-UV. This result is interesting considering that UVA1 represents around 80% of daily UV and reaches the dermal-epidermal junction with ease. Moreover, among PAH studied, benzo[a]pyrene and indeno[1,2,3-cd]pyrene were phototoxic at very low concentrations (nanomolar range) on cultured cells or in reconstructed epidermis and also impaired keratinocyte clonogenic potential at sub-toxic doses. ROS generation within cells and in the inner mitochondrial compartment, mitochondrial membrane depolarization and/or reduced ATP production were also noted. Meanwhile, intracellular glutathione concentrations transiently decreased several hours post-treatment and reduction of its synthesis by buthionine sulfoximine potentiated PAH phototoxicity. Consequently, expression of GSH neo-synthesis genes such as SLC7A11 or GCLc was upregulated several hours post-treatment. CONCLUSION These results obtained using PAH concentrations in the range of those reported in blood of pollution-exposed people suggest that exposure to such a photo-pollution stress, particularly if chronic, may impair cutaneous homeostasis and aggravate sunlight-induced skin damage.