Jean-Roch Meunier

The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis.

The expression of genes encoding antioxidant and/or phase 2 detoxifying enzymes can be enhanced in response to various environmental stresses. The main transcription factor involved in this response is nuclear factor erythroid 2‐related factor 2 (Nrf2). Nrf2 activity is negatively regulated by the protein Kelch‐like‐Ech‐associated‐protein 1 (Keap1). While the roles of Nrf2 and phase 2 genes in chemoprevention of carcinogenesis have been well described; only few studies have dealt with their role in skin cancer. Normal human keratinocytes (NHK) and melanocytes (NHM) were treated by chemical inducers of the Nrf2 pathway or by small interfering RNAs (siRNA) used to knock down Keap1 mRNA. The above treatments resulted in significant stimulation of NQO‐1 (NADPH‐Quinone‐Oxidoreductase 1) gene expression. GCL (γ‐Glutamyl‐cysteinyl‐ligase) gene was also induced but interestingly increased mRNA encoding the catalytic, heavy subunit GCLC was mainly stimulated in NHK, whereas the mRNA encoding the modifier, light subunit GCLM was mostly induced in NHM. HO‐1 (Heme Oxygenase 1) gene induction was relatively strong in NHM, but generally absent in NHK, except when the cells were subjected to cytotoxic doses of the above chemicals. Exposure to solar UV (UVB + UVA, 300–400 nm) or to UVA alone (320–400 nm) confirmed this trend, but interestingly, at doses where cell growth reduction was comparable, UVA was generally more efficient than solar UV in inducing phase 2 genes. When siRNAs directed against Nrf2 were used, a strong down‐regulation of NQO‐1 expression was observed in both, NHM and NHK, whereas reduction of HO‐1 expression was mainly detected in NHM. To our knowledge, this is the first study comparing phase 2 gene modulation in NHK and NHM. The results hereby presented should contribute to a better understanding of the molecular mechanisms involved in skin adaptation to environmental stress.

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.

Photogenotoxicity of Mammalian Cells: A Review of the Different Assays for In Vitro Testing¶

Abstract During the past several years, phototoxicity has been studied at the molecular level, and these studies have provided new insights in the field of DNA lesion characterization, DNA repair and cell response to ultraviolet (UV)-induced stress. The development of new antibiotics and antiinflammatory drugs has highlighted the necessity to develop the assessment of phototoxicity in the safety evaluation of new chemical compounds. This paper aims at reviewing the known molecular mechanisms of the cellular response to UV-induced stress, the in vitro methods that can be proposed and used to screen for toxicity of sunlight and the photosensitization process resulting from the activation of drugs by light. UV sources, biological systems and endpoints of interest in that particular objective are listed. Phototoxic effects span from the cytotoxic–apoptotic effect to the induction of primary DNA damage, DNA repair and a variety of stress genes acting on the cell cycle and the fate of the cell. Ultimately, it can lead to the induction of hereditary DNA modification. A variety of assays are proposed to specifically address all these particular consequences of UV-induced toxicity.

Increased Melanogenesis is a Risk Factor for Oxidative DNA Damage— Study on Cultured Melanocytes and Atypical Nevus Cells†

Melanin synthesis is an oxygen‐dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment‐forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine‐induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox‐sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single‐cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person.

Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin™ and full thickness model from Episkin™

BACKGROUND Episkin and full thickness model from Episkin (FTM) are human skin models obtained from in vitro growth of keratinocytes into the five typical layers of the epidermis. FTM is a full thickness reconstructed skin model that also contains fibroblasts seeded in a collagen matrix. OBJECTIVES To assess whether enzymes involved in chemical detoxification are expressed in Episkin and FTM and how their levels compare with the human epidermis, dermis and total skin. METHODS Quantification of the mRNA expression levels of phases 1 and 2 metabolizing enzymes in cultured Episkin and FTM and human epidermis, dermis and total skin using Realtime PCR. RESULTS The data show that the expression profiles of 61 phases 1 and 2 metabolizing enzymes in Episkin, FTM and epidermis are generally similar, with some exceptions. Cytochrome P450-dependent enzymes and flavin monooxygenases are expressed at low levels, while phase 2 metabolizing enzymes are expressed at much higher levels, especially, glutathione-S-transferase P1 (GSTP1) catechol-O-methyl transferase (COMT), steroid sulfotransferase (SULT2B1b), and N-acetyl transferase (NAT5). The present study also identifies the presence of many enzymes involved in cholesterol, arachidonic acid, leukotriene, prostaglandin, eicosatrienoic acids, and vitamin D3 metabolisms. CONCLUSION The present data strongly suggest that Episkin and FTM represent reliable and valuable in vitro human skin models for studying the function of phases 1 and 2 metabolizing enzymes in xenobiotic metabolisms. They could be used to replace invasive methods or laboratory animals for skin experiments.

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.

In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic HCE model: application to 435 substances from consumer products industry.

The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation.

Transcriptional Upregulation of Nrf2-Dependent Phase II Detoxification Genes in the Involved Epidermis of Vitiligo Vulgaris

Oxidative stress is widely believed to be a contributing factor in vitiligo pathogenesis. To explore mechanisms by which epidermis responds to mounting oxidative stress, we investigated the involvement of phase II detoxification genes in vitiligo. Phase II detoxification pathways have recently been identified as being important in the regulation of epidermal skin homeostasis. In this study we show that the key transcription factor nuclear factor E2-related factor 2 (Nrf2) and the downstream genes NAD(P)H:quinone oxidase-1 (NQO-1), γ-glutamyl cystine ligase catalytic subunit (GCLC), and γ-glutamyl cystine ligase modifying subunit (GCLM) are upregulated in the lesional epidermal skin of subjects with vitiligo vulgaris. The differences between lesional and nonlesional skin were further investigated by studying the induced expression of Nrf2-dependent transcripts in skin punch biopsies using curcumin and santalol. Surprisingly, nonlesional skin showed induction of all transcripts while a similar effect was not observed for the skin punches from the lesional skin. The use of curcumin and santalol on epidermal cells showed that keratinocytes were more susceptible to apoptosis, whereas melanocytes induced phase II genes under the same concentrations with negligible apoptosis. Our studies provide new insights into the role of phase II detoxification pathway in maintaining skin homeostasis and sustaining redox balance in vitiligo patients.

Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through induction of apoptosis

Essential oils are complex mixtures of odorous and volatile compounds derived from secondary plant metabolism. They can be isolated from many plants by mechanical pressing or hydro- and steam-distillation and are known to induce a wide range of biological effects through their antibacterial, antifungal, cytotoxic, antioxidant and antimutagenic activities. In order to explore their beneficial properties on human skin cells, we investigated the effects of an essential oil from rosewood Aniba rosaeodora (REO) on the human epidermoid carcinoma cell line A431, on immortal HaCaT cells thought to represent an early stage of skin carcinogenesis, on transformed normal HEK001 keratinocytes and on primary normal NHEK keratinocytes. In a defined range of concentrations, REO selectively killed A431 and HaCaT cells. The same treatments had only a minor cytotoxic effect on HEK001 and NHEK cells. Preferentially in A431 and HaCaT cells, REO triggered the production of reactive oxygen species, induced depolarization of the mitochondrial membrane and caused caspase-dependent cell death characterized by phosphatidylserine externalization, an early marker of apoptosis. Both intrinsic and extrinsic apoptotic pathways were implicated in REO-induced cell death. The identification of selective induction of apoptosis in precancerous and cancerous skin cells by REO highlights the potential anticancer activity of this essential oil.

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.