Contact sensitizers modulate the arachidonic acid metabolism of PMA-differentiated U-937 monocytic cells activated by LPS.

For the effective induction of a hapten-specific T cell immune response toward contact sensitizers, in addition to covalent-modification of skin proteins, the redox and inflammatory statuses of activated dendritic cells are crucial. The aim of this study was to better understand how sensitizers modulate an inflammatory response through cytokines production and COX metabolism cascade. To address this purpose, we used the human monocytic-like U-937 cell line differentiated by phorbol myristate acetate (PMA) and investigated the effect of 6 contact sensitizers (DNCB, PPD, hydroquinone, propyl gallate, cinnamaldehyde and eugenol) and 3 non sensitizers (lactic acid, glycerol and tween 20) on the production of pro-inflammatory cytokines (IL-1ß and TNF-α) and on the arachidonic acid metabolic profile after bacterial lipopolysaccharide (LPS) stimulation. Our results showed that among the tested molecules, all sensitizers specifically prevent the production of PMA/LPS-induced COX-2 metabolites (PGE(2,) TxB(2) and PGD(2)), eugenol and cinnamaldehyde inhibiting also the production of IL-1ß and TNF-α. We further demonstrated that there is no unique PGE(2) inhibition mechanism: while the release of arachidonic acid (AA) from membrane phospholipids does not appear do be a target of modulation, COX-2 expression and/or COX-2 enzymatic activity are the major steps of prostaglandin synthesis that are inhibited by sensitizers. Altogether these results add a new insight into the multiple biochemical effects described for sensitizers.

Development of a mechanistic SAR model for the detection of phototoxic chemicals and use in an integrated testing strategy.

Phototoxicity is of increasing concern in dermatology, since modern lifestyle is often associated with exposure to sunlight. The most commonly reported process is via oxidative reactions. Therefore characterizing the "photo-pro-oxidant" potential of a compound early in its industrial development is of utmost interest, especially for compounds likely to undergo sunlight exposure in skin. Today there is a need for filtering compounds to be tested in the 3T3 neutral red uptake in vitro test for phototoxicity since testing requires resources. A computational model aiming at predicting the mechanisms that imply the generation of reactive oxygen species was developed using a diverse set of 56 chemicals having 3T3 NRU data. An historical mechanistic (Q)SAR model developed for polycyclic aromatic hydrocarbons was used to derive the new mechanistic model: descriptors were selected upfront to describe the modeled phenomenon. The historical parabolic relationships between phototoxicity and the energy gap (E(GAP)) between energies of the highest occupied molecular orbital and the lowest unoccupied molecular orbital was confirmed. The model predicts chemicals to be "phototoxic or photodegradable", or "non-phototoxic and non-photodegradable". A four-step testing strategy is proposed to enable the reduction of experimental testing with the in silico model implemented as a first screen.

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.

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.

In vitro tools for photobiological testing: molecular responses to simulated solar UV of keratinocytes growing as monolayers or as part of reconstructed skin.

Epidermal keratinocytes are critical targets for UV-induced genotoxicity as their transformation by sunlight overexposure can lead to skin cancer such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Therefore, assessment of photoprotection should involve early markers associated with DNA photodamage. Here, the same normal human keratinocytes either in monoculture (KC) or in full thickness reconstructed skin (RS) were compared with respect to their response to simulated solar UV (SSUV) exposure. Irradiation conditions (spectral power distribution and doses) were designed to mimic environmental zenithal UV from sunlight. At doses where survival was higher than 80%, comet assay showed more single strand breaks (SSB) and cyclobutane pyrimidine dimers (CPD) in keratinocytes in RS than in KC one hour post-exposure. The transcription factor p53 was activated in both models. While in KC p53 accumulation displayed a linear dose-dependency up to 24 h post-exposure, in RS it followed a bell-shaped profile and reverted to its basal rate. QRT-PCR demonstrated that among genes controlled by p53, P21 and MDM2 were clearly induced by SSUV in KC, whereas GADD45 expression was strongly and almost exclusively up-regulated in RS. Nrf2-dependent antioxidant genes (Ferritin light chain, NQO1) were only induced in RS, yet at low doses for NQO1. In vitro models such as KC or RS allowing the development of quantitative methodologies should be used as surrogates for in vivo tests assessing photogenotoxicity.

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.

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.

Skin DNA photodamage and its biological consequences.

It is well established that ultraviolet (UV) radiation from sunlight damages skin cells' DNA. Wavelengths in the UVB range are absorbed by DNA and can induce mutagenic lesions such as pyrimidine dimers. On the other hand, genotoxic effects of solar UVA are mainly mediated by the activation of endogenous photosensitizers resulting in the generation of a local oxidative stress. Exogenous chemicals, such as drugs like psoralens or fluoroquinolones, sometimes amplify UV-induced harmful effects. DNA damage can lead to mutations and genetic instability. This is one of the reasons why sunlight overexposure increases the risk of skin cancer. But DNA photolesions can also be involved in other skin-specific responses to UV radiation: erythema, immunosuppression, and melanogenesis are examples reported in the literature. The aim of this short review is to summarize the general knowledge in the field of UV-induced DNA damage. Besides the biological consequences of DNA photolesions, this article also deals with technologies used for their detection and shows how helpful such approaches can be to assess photoprotection provided by sunscreens.

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.

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 (gamma-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.

SFTG international collaborative study on in vitro micronucleus test V. Using L5178Y cells.

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.

Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds?

Today reconstructed skin models that simulate human skin, such as Episkin, are widely used for safety or efficacy pre-screening. Moreover, they are of growing interest for regulatory purposes in the framework of alternatives to animal testing. In order to reduce and eventually replace results of in vivo genotoxicity testing with in vitro data, there is a need to develop new complementary biological models and methods with improved ability to predict genotoxic risk. This can be achieved if these new assays do take into account exposure conditions that are more relevant than in the current test systems. In an attempt to meet this challenge, two new applications using a human reconstructed skin model for in vitro genotoxicity assessment are proposed. The skin is the target organ for dermally exposed compounds or environmental stress. Although attempts have been made to develop genotoxicity test procedures in vivo on mouse skin, human reconstructed skin models have not been used for in vitro genotoxicity testing so far, although they present clear advantages over mouse skin for human risk prediction. This paper presents the results of the development of a specific protocol allowing to perform the comet assay, a genotoxicity test procedure, on reconstructed skin. The comet assay was conducted after treatment of Episkin with UV, Lomefloxacin and UV or 4-nitroquinoline-N-oxide (4NQO). Treatment with the sunscreen Mexoryl was able to reduce the extent of comet signal. A second approach to use reconstructed epidermis in genotoxicity assays is also proposed. Indeed, the skin is a biologically active barrier driving the response to exposure to chemical agents and their possible metabolites. A specific co-culture system (Figure 1) using Episkin to perform the regular micronucleus assay is presented. Micronucleus induction in L5178Y cells cultured underneath Episkin was assessed after treatment of the reconstructed epidermis with mitomycin C, cyclophosphamide or apigenin. This second way of using human reconstructed skin for genotoxicity testing aims at improving the relevance of exposure conditions in in vitro genotoxicity assays for dermally applied compounds.

Importance of UVA photoprotection as shown by genotoxic related endpoints: DNA damage and p53 status.

In order to demonstrate the importance of photoprotection in the UVA range (320-400 nm), an in vitro approach where sun formulations are spread on a quartz slide, and placed over human keratinocytes in culture is proposed as a convenient test for photoprotection assessment at the DNA level. Using the comet assay, DNA strand breaks, oxidative DNA damage or drug-induced DNA breaks were assessed. Accumulation of p53 protein was also studied as a marker for UV-induced genotoxic stress. Such a method was used to compare two formulations with different photostability. Spectroradiometry showed that a photounstable formulation lost its effectiveness in UVA screening when pre-irradiated by simulated sunlight (UVB+UVA). As a consequence, it was also shown that this formulation was not as protective as the photostable one at the genomic level. These data demonstrate that the loss of absorbing efficiency within UVA wavelengths due to photounstability may have detrimental consequences leading to impairments implicated in genotoxic events.

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.

Molecular responses to photogenotoxic stress induced by the antibiotic lomefloxacin in human skin cells: from DNA damage to apoptosis.

Photo-unstable chemicals sometimes behave as phototoxins in skin, inducing untoward clinical side-effects when exposed to sunlight. Some drugs, such as psoralens or fluoroquinolones, can damage genomic DNA, thus increasing the risk of photocarcinogenesis. Here, lomefloxacin, an antibiotic from the fluoroquinolone family known to be involved in skin tumor development in photoexposed mice, was studied using normal human skin cells in culture: fibroblasts, keratinocytes, and Caucasian melanocytes. When treated cells were exposed to simulated solar ultraviolet A (320-400 nm), lomefloxacin induced damage such as strand breaks and pyrimidine dimers in genomic DNA. Lomefloxacin also triggered various stress responses: heme-oxygenase-1 expression in fibroblasts, changes in p53 status as shown by the accumulation of p53 and p21 proteins or the induction of MDM2 and GADD45 genes, and stimulation of melanogenesis by increasing the tyrosinase activity in melanocytes. Lomefloxacin could also lead to apoptosis in keratinocytes exposed to ultraviolet A: caspase-3 was activated and FAS-L gene was induced. Moreover, keratinocytes were shown to be the most sensitive cell type to lomefloxacin phototoxic effects, in spite of the well-established effectiveness of their antioxidant equipment. These data show that the phototoxicity of a given drug can be driven by different mechanisms and that its biologic impact varies according to cell type.

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

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.

Photogenotoxicity of mammalian cells: a review of the different assays for in vitro testing.

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.