Lipid Peroxidation as the Mechanism Underlying Polycyclic Aromatic Hydrocarbons and Sunlight Synergistic Toxicity in Dermal Fibroblasts.

Light and atmospheric pollution are both independently implicated in cancer induction and premature aging. Evidence has been growing more recently on the toxic synergy between light and pollutants. Polycyclic aromatic hydrocarbons (PAHs) originate from the incomplete combustion of organic matter. Some PAHs, such as the Benzo[a]pyrene (BaP), absorb ultraviolet A (UVA) wavelengths and can act as exogenous chromophores, leading to synergistic toxicity through DNA damage and cytotoxicity concomitant to ROS formation. In this study, we shed light on the mechanism underlying the toxic synergy between PAHs and UVA. Using dermal fibroblasts co-exposed to UVA and BaP, we have demonstrated that the photosensitization reaction causes mortality, which is most likely caused by ROS accumulation. We have shown that these ROS are concentrated in the lipids, which causes an important induction of lipid peroxidation and malondialdehyde, by-products of lipid peroxidation. We have also shown the accumulation of bulky DNA damage, most likely generated by these by-products of lipid peroxidation. To our knowledge, this study represents the first one depicting the molecular effects of photo-pollution on dermal skin.

NRF2 in dermo-cosmetic: From scientific knowledge to skin care products.

The skin is the organ that is most susceptible to the impact of the exposome. Located at the interface with the external environment, it protects internal organs through the barrier function of the epidermis. It must adapt to the consequences of the harmful effects of solar radiation, the various chemical constituents of atmospheric pollution, and wounds associated with mechanical damage: oxidation, cytotoxicity, inflammation, and so forth. In this biological context, a capacity to adapt to the various stresses caused by the exposome is essential; otherwise, more or less serious conditions may develop accelerated aging, pigmentation disorders, atopy, psoriasis, and skin cancers. Nrf2-controlled pathways play a key role at this level. Nrf2 is a transcription factor that controls genes involved in oxidative stress protection and detoxification of chemicals. Its involvement in UV protection, reduction of inflammation in processes associated with healing, epidermal differentiation for barrier function, and hair regrowth, has been demonstrated. The modulation of Nrf2 in the skin may therefore constitute a skin protection or care strategy for certain dermatological stresses and disorders initiated or aggravated by the exposome. Nrf2 inducers can act through different modes of action. Keap1-dependent mechanisms include modification of the cysteine residues of Keap1 by (pro)electrophiles or prooxidants, and disruption of the Keap1-Nrf2 complex. Indirect mechanisms are suggested for numerous phytochemicals, acting on upstream pathways, or via hormesis. While developing novel and safe Nrf2 modulators for skin care may be challenging, new avenues can arise from natural compounds-based molecular modeling and emerging concepts such as epigenetic regulation.

Comprehensive characterization of naturally occurring antioxidants from the twigs of mulberry (Morus alba) using on-line high-performance liquid chromatography coupled with chemical detection and high-resolution mass spectrometry.

INTRODUCTION: The mulberry tree (Morus alba L.) is a prolific source of biologically active compounds. There is considerable growing interest in probing M. alba twigs as a source of disruptive antioxidant lead candidates for cosmetic skin care product development. OBJECTIVE: An integrated approach using high-performance liquid chromatography (HPLC) coupled with either chemical detection (CD) or high-resolution mass spectrometry (HRMS) was applied to the hydroalcoholic extract of M. alba to detect and identify lead antioxidant compounds, respectively. MATERIAL AND METHODS: The twigs were weighed, powdered and homogenized using a mill and the extract was prepared using 70% aqueous ethanol. The antioxidant metabolites were detected with HPLC coupled with CD (based on the ORAC assay) and their structural identification was carried out using a Q-Exactive Orbitrap MS instrument. RESULTS: Using this approach, 13 peaks were detected as overall contributors to the antioxidant activity of M. alba, i.e. mulberrosides (A & E), oxyresveratrol & its derivatives, moracin & its derivatives and a dihydroxy-octadecadienoic acid, which together accounted for >90% of the antioxidant activity, highlighting the effectiveness of the integrated approach based on HPLC-CD and HPLC-HRMS. Additionally, a (3,4-dimethoxyphenyl-1-O-ß-D-apiofuranosyl-(1″ → 6')-O-ß-D-glucopyranoside was also discovered for the first time from the twig extract and is presented here. CONCLUSION: To our knowledge, this is the first report from M. alba twigs using HPLC-CD and HPLC-HRMS that identifies key compounds responsible for the antioxidant property of this native Chinese medicinal plant.

Vitamin C prevents epidermal damage induced by PM-associated pollutants and UVA1 combined exposure.

Particulate matter is suspected to be substantially involved in pollution-induced health concerns. In fact, ultrafine particles (UFPs) contain polycyclic aromatic hydrocarbons (PAHs) known as mutagenic, cytotoxic and sometimes phototoxic. Since UFPs reach blood circulation from lung alveoli, deep skin is very likely contaminated by PAHs coming from either skin surface or blood. As photoreactive, benzo(a)pyrene (BaP) or indenopyrene (IcdP) is involved in the interplay between pollution and sunlight. In order to better characterize this process, experiments were carried out on reconstructed human epidermis (RHE) in a protocol mimicking realistic exposure. Concentrations of PAHs comparable to those generally reported in blood were used together with chronic irradiation to low dose UVA1. On a histological level, damaged cells mainly accumulated in a suprabasal situation, thus reducing living epidermis thickness. Stress markers such as IL1-α or MMP3 secretion increased, and surprisingly, the histological position of Transglutaminase-1 within epidermis was disturbed, whereas position of other differentiation markers (keratin-10, filaggrin, loricrin) remained unchanged. When vitamin C was added in culture medium, a very significant protection involving all markers was noticed. In conclusion, we provide here a model of interest to understand the epidermal deleterious consequences of pollution and to select efficient protective compounds.

In Vitro Methods to Simulate Pollution and Photo-Pollution Exposure in Human Skin Epidermis.

Tissue homeostasis of an individual is a finely orchestrated phenomenon that ensures integrity and steady state in health. Emerging evidence indicates that the environment, especially ambient air pollution, has a lasting impact on this equilibrium (Beelen et al., Lancet 383:785-795, 2014). Environmental pollution consists of diverse entities, namely, particulate matter (PM 2.5, PM 10), ozone, and UV rays, among others (Heroux et al., Int J Public Health 60:619-627, 2015). Understandably, skin epidermis is the first and the most exposed tissue to such a wide range of substances and bears the assault. Previous studies have established that exposure to atmospheric pollution aggravates several skin disorders as, for instance, eczema, acne, lentigines or macules, and wrinkles (Araviiskaia et al., J Eur Acad Dermatol Venereol 33:1496-1505, 2019). While pollutants can interact with skin surface, contamination of deep skin by particulate matter (either ultrafine particles or by some polycyclic aromatic hydrocarbon (PAH) moieties) is also highly probable, particularly because PAH were detected in blood and inside the cortex of hair (Guo et al., Sci Total Environ 427-428:35-40, 2012; Palazzi et al., Environ Int 121:1341-1354, 2018). Importantly, concentrations of contaminant PAH in the blood are very low, in the nanomolar range (Neal et al., Reprod Toxicol 25:100-106, 2008); thus PAH levels in the skin might be in a similar range. Furthermore, it has been shown that some PAH (e.g., benzo[a]pyrene, indenopyrene) are phototoxic under UVA irradiation through a strong production of reactive oxygen species, ultimately leading to skin cancer in mice (Burke and Wei, Toxicol Ind Health 25:219-224, 2009). Since UVA1 (340-400 nm) can reach deep dermis, it can thus be assumed that photoactivation of PAH contaminants in living skin may locally induce a significant stress. In order to study the molecular mechanisms that are affected due to this exposure, there is an increasing need to develop reliable and diverse methods that simulate pollution exposure.

Pollution and Sun Exposure: A Deleterious Synergy. Mechanisms and Opportunities for Skin Protection.

BACKGROUND: Pollutants are diverse chemical entities, including gases such as ozone and particulate matter PM. PM contains toxic chemicals such as polycyclic aromatic hydrocarbons (PAHs). Some PAHs can induce strong oxidative stress under UVA exposure. Pollution aggravates some skin diseases such as atopy or eczema, but epidemiological data also pointed to a correlation with early occurrence of (photo)-aging markers. OBJECTIVE: This paper aims at reviewing current literature dealing with dermatological effects of pollution, either on in vitro models or using in vivo approaches (including humans). It particularly focuses on the probable deleterious synergy between pollutants and sunlight. RESULTS: An exhaustive analysis of literature suggests that skin may be impacted by external stress through oxidation of some of its surface components. However, pollutants detected in plasma may also be provided to deep skin by the circulation of the blood. Oxidative stress, inflammation and metabolic impairments are among the most probable mechanisms of pollution- derived dermatological hazards. Moreover these stresses should be amplified by the deleterious synergy between pollution and sunlight. Some experiments from our lab identified few PAHs inducing a huge toxic stress, at nanomolar concentrations, when exposed to long UVA wavelengths. Prevention strategies should thus combine surface protection (long UVA sunscreens, antioxidants) and enhanced skin tissue resistance through stimulation of the natural antioxidation/detoxification pathway Nrf2. CONCLUSION: In people exposed to highly polluted environments, pollutants and sunlight may synergistically damage skin, requiring a specific protection.

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.

Melanocytes as instigators and victims of oxidative stress.

Epidermal melanocytes are particularly vulnerable to oxidative stress owing to the pro-oxidant state generated during melanin synthesis, and to the intrinsic antioxidant defenses that are compromised in pathologic conditions. Melanoma is thought to be oxidative stress driven, and melanocyte death in vitiligo is thought to be instigated by a highly pro-oxidant state in the epidermis. We review the current knowledge about melanin and the redox state of melanocytes, how paracrine factors help counteract oxidative stress, the role of oxidative stress in melanoma initiation and progression and in melanocyte death in vitiligo, and how this knowledge can be harnessed for melanoma and vitiligo treatment.

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.

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.

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.

The EpiSkin phototoxicity assay (EPA): development of an in vitro tiered strategy using 17 reference chemicals to predict phototoxic potency.

The aim of the study was to investigate the ability of human reconstructed epidermis EpiSkin(LM) to identify the phototoxic potency of topically or systemically applied chemicals (EPA: EpiSkin phototoxicity assay). Three classes, according to their available human phototoxic potential, were evaluated: systemic phototoxic compounds, topical phototoxic chemicals and non-phototoxic compounds. Non-cytotoxic concentrations of chemicals were applied topically or directly added to the underlying culture medium in order to mimic a systemic-like administration. Following treatment, tissues were exposed to non-cytotoxic dose of UVA (50 J cm(-2)). Cell viability and pro-inflammatory mediators (IL-1alpha) were investigated 22 h after UVA exposure. Our results show that the phototoxic potential of chemicals can be determined using cell viability combined with inflammatory mediator measurements (cytokine IL-1alpha) in a 3-D epidermis model. Moreover, the EPA was able to discriminate efficiently between phototoxic and non-phototoxic products. Furthermore, the EPA is sensitive to the administration route in the prediction of the phototoxic potency of the tested chemical. Differences observed between the two routes of administration (topical or systemic-like) may be linked in part to chemicals bioavailability which depends on specific penetration potential, epidermis barrier function and also on keratinocytes absorption/metabolization processes. Results were very promising and showed a very good sensitivity (92.3%) and an excellent specificity (100%) with an overall accuracy of 94.1%. The performances of the EPA showed that the EpiSkin(LM) model is an interesting tool able to integrate decision-making processes to address the question of phototoxicity linked to the application site.

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.