Establishment of an In Vitro Photoallergy Test Using NCTC2544 Cells and IL-18 Production

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of eratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm2) by investigating the effect of UVAirradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)- treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxydibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals

ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to low molecular weight chemical allergens. The development of ACD requires the activation of innate immune cells, such as keratinocytes (KC), necessary for the maturation and the migration of dendritic cells (DC), which in turn are required for the activation of specific T cells. Human KC constitutively express several cytokines, including pro IL-1 alpha, pro IL-1 beta and pro IL-18. In vivo it has been demonstrated that IL-18 plays a key proximal role in the induction of allergic contact sensitization, favoring Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-\u3b1, IL-8 and IFN-\u3b3 (Shornick et al., 1996; Wang et al., 1999, Cumberbatch et al., 2001). Toxicologists have the responsibility of identifying and characterizing the skin and respiratory allergenic potential of chemicals, and estimating the risk they pose to human health. Growing political and practical resistance to toxicity testing in animals has driven the development of animal-free methods for screening and prioritization of toxicants, including those causing allergic hypersensitivity. The purpose of this thesis was to develop an alternative in vitro test based on the keratinocytes and IL-18 to characterize the allergenic potential of low molecular weight chemicals, and to understand the molecular mechanism(s) underlying chemical allergen-induced IL-18 production. In addition to human keratinocytes cell lines (NCTC2544, HaCaT, HPKII), commercially available reconstituted human epidermis 3D-epidermal models were also used as experimental model. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the KC and skin organotypic culture as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified. To perform these studies 22 contact allergens, 12 photoallergens/photoirritant compounds, 3 respiratory allergens and 9 irritants chemicals were used. The choice of chemicals was dictated by the SENS-IT-IV programme as relevant and representative of the \u2018universe\u2019 of irritants, respiratory and contact allergens. Phototoxic chemicals were selected based on compounds used in similar published studies and reported to cause phototoxicity. Results obtained indicate that the NCTC2544 IL-18 assay is able to discriminate contact allergens and photoallergens from irritants/photoirritants and respiratory allergens. Important factors including compound solubility, chemical reactivity and metabolic activation, which may mask the potential allergenicity of some chemicals, must be considered in the development of in vitro tests. Submerged cell culture may be unfavourable for many of the respiratory sensitizers, due to chemical instability; for this reason we have tested IL-18 production also in reconstituted human epidermis, which allows application in organic solvent, i.e. acetone: olive oil. The lack of metabolic activation may be a relevant problem in case of proaptens. However, NCTC 2544 cells posses both phase I and II metabolic activation capacity (Gelardi et al., 2001), and positive results were indeed obtained with the proaptens tested (eugenol and cinnamic alcohol). A sensitivity of 87%, specificity of 95% and an accuracy of 90% was obtained (Corsini et al., 2009; Galbiati et al., 2011). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The combination of the epidermal equivalent potency assay with the release of IL-18 lead to the development of an in vitro model able to identify contact allergens and rank them according to their potency. One other objective of this thesis was to study the signal transduction pathways involved in PPD, DNCB and citral-induced IL-18. For such purpose several inhibitors were used. To investigate the intracellular source of ROS, specific inhibitors of the three main cellular sources of ROS, namely DPI, a NADPH synthetase inhibitor; rotenone, a mitochondrial electron transport inhibitor; allopurinol, a xanthine oxidase inhibitor, were used. Z-VAD-FMK, a cell-permeant pan caspase inhibitor, that irreversible binds to the catalytic site of caspases, and a neutralizing anti-TLR4 antibody were used to investigate the role of the inflammasome and TLR4. Glycirrizic acid, a direct inhibitor of HMGB1 protein, was used to establish the role of HMGB1 as possible DAMP associated with allergen-induced IL-18. To specifically investigate the signal transduction pathway involved in PPD-induced IL-18 production selective inhibitors were used: GF109203X to inhibit PKC, PDTC and Bay 11-70-85 to inhibit NF-\u3baB, and SB203580, as p38 MAPK inhibitor. The results obtained during this three year of research activity have clearly shown that the in vitro methods based on NCTC2544 and IL-18 production are able to discriminate contact/photocontact allergens from irritants/photoirritants and respiratory allergens. Furthermore, the combined use of the epidermis in vitro model with the IL-18 production, beside the ability to identify sensitising compounds, is able to rank them according to their potency. With respect to the molecular mechanisms behind skin sensitization I could demonstrate that different intracellular sources of ROS are triggered by different contact allergens. Allergens-induced IL-18 production is dependent upon NF-\u3baB and p38 MAPK activation and TLR4 and inflammasome activation. Among the DAMPs, the evolutionarily conserved non-hystone chromatin-binding protein HMGB1 is released into the extracellular space following exposure to contact allergens, resulting in TLR4 activation and IL-18 neosynthesis. Even if more studies are necessary to elucidate the mechanisms that are involved in chemical allergens-induced oxidative stress, the signalling pathways activated and their role in contact allergy, data clearly indicated a pivotal role of ROS in chemical allergy. Consequently, the redox state of the cell becomes imbalanced, with the activation of several pathways, including MAPK, such as SAPK/JNK, ERK1/2 and p38, NF-\u3baB, Akt/ASK1 or Keap1/Nrf2 pathways, resulting in a inflammatory and cytotoxic response with the production of costimulatory molecules, cytokines, chemokines, and phase 1 detoxifying enzymes. On the basis of the results obtained, the following scenario could be imagined: chemical sensitisers can induce oxidative stress owing to their elecrophilicity, which in turn activates the inflammasome and HMGB1 release (and possible other DAMPs), which can activate TLR4. Activation of TLR4 will results in NF-\u3baB and p38 MAPK activation and in the neosynthesis of IL-18

UNDERSTANDING CHEMICAL ALLERGEN POTENCY: CONTRIBUTION OF KERATINOCYTES

Repeated exposure to chemical allergens increases the risk of becoming sensitized. Once an individual has become sensitized, any following exposure to the same chemical may result in allergic contact dermatitis (ACD). The risk to develop ACD is considered a serious health issue and the identification of potential sensitizing agents within consumer products is therefore crucial. With the enforcement of the 7th Amendment to the EU Cosmetics Directive (76/768/EEC) in March 2013, currently known as the Cosmetics Regulation (EU 1223/2009), a ban on the use of animals was introduced for identifying repeated dose toxicity endpoints of chemicals used in cosmetic ingredients and products. This ban results in an urgent need for the development of suitable non-animal methods for safety testing. The development of animal alternatives has become even more urgent due to the Registration, Evaluation, Authorisation and Restriction of CHemicals (REACH) regulation, which may demand toxicity tests for chemicals produced in quantities of over 1 ton per year. Over the last years, many in vitro models have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare, public opinions and legislation constrains. The development of in vitro, in chemico or in silico models for predicting the sensitizing potential of new chemicals is receiving widespread interest. Keratinocytes (KCs) play a key role in skin sensitization, as they provide the essential danger signals, they are involved in the protein haptenation process, and supply enzymes that are necessary for the metabolic activation of prohapten. Human KCs constitutively express several cytokines, including pro-interleukin (IL)-1\uf061, pro-IL-1\uf062 and pro-IL-18. Evidences provided from our group has shown that IL-18 production in human KCs can be used as a sensitive method to identify contact allergens, discriminating them from respiratory allergens and irritants with a sensitivity of 87%, specificity of 95% and an accuracy of 90%. IL-18 is synthesized as preform, which require proteolytic maturation by cysteine protease caspase-1, which must first be activated by the inflammasome. More recently, we demonstrated the possibility of combining the Reconstituted human Epidermis (RhE) potency assay with the assessment of IL-18 release to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (Gibbs et al., 2013). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The purpose of this thesis was to understand the role of several genes and proteins involved in contact allergen-induced NLRP3 inflammasome activation and IL-18 production, and their possible correlation with allergenic potency. Another objective of this thesis was to extend the list of chemicals tested in the RhE IL-18 potency assay, and to provide a simple method for the in vitro estimation of the expected sensitization induction level. Results obtained during these three years of research activity have shown that several proteins involved in NLRP3 inflammasome activation/regulation were modulated by contact allergens. In particular I focused my attention on the role of NLRP12 and B lymphocyte induced maturation protein-1 (Blimp-1) in IL-18 production. The expression of NLRP3, ASC and caspase-1 activation were investigated by Western blot analysis and the NLRP12 localization characterized by immunoprecipitation. Regarding potency classification the results obtained using RhE IL-18 potency assay are very promising, and further compounds should be tested to better define the applicability and limitation of RhE IL-18 potency test

Safe cosmetics without animal testing? Contributions of the EU Project Sens-it-iv

The 7th Ammendment to the Cosmetics Directive of the European Commission (Directive 76/768/EEC2) bans the marketing of cosmetics containing animal-tested ingredients since March 2009. Excepted are only tests for repeated dose toxicity, for which the animal ban will come into effect by 2013. One major concern for cosmetics, i.e. the risk of containing skin (contact) sensitizers, has in the past been addressed almost exclusively by animal testing. It is this problem attracting the central interest of the integrated research project Sens-it-iv (Novel Testing Strategies for in vitro Assessment of Allergens, http://www.sens-it-iv.eu), funded by the EC within framework 6 since October 2005. Here, the 28 Sens-it-iv partners from 10 European States present the 5 most promising types of in vitro assays selected for further refinement. These are: (1) a human epidermal equivalent (EE) model to rank contact allergens according to their sensitizing potency, (2) identification of contact sensitizers, including pro-haptens, through intracellular production of IL-18 by the human keratinocyte cell line NCTC 2544, (3) determination of activation markers such as CD86, CD54 and most prominently CXCL8 (IL-8) on/in dendritic cell lines, (4) contact sensitizer-specific migration of MUTZ Langerhans cells towards the chemokine CXCL12, and (5) the allergen-specific activation and proliferation of na\uefve human T cells. Ongoing genomic and proteomic experiments are in the process of identifying larger sensitizer-specific biological marker signatures to be integrated into the above assays. We hope to supply the European control agencies with a basis for further validation of in vitro assays by the end of 2010

Safety evaluation of cosmetic ingredients: In vitro opportunities for the identification of contact allergens

Irritant and allergic contact dermatitis are undesired side effects in the development of drugs and cosmetics as well as after contact with environmental or industrial chemicals. Over the last decades, a great deal of progress has been made in the development of alternative In vitro test to assess these issues. Driven by the 7th Amendment to the European Cosmetic Directive, the EU policy on chemicals (the registration, evaluation, authorization and restriction of chemicals (REACH) system), the update of the European legislation on the protection of animals used in research, and emerging visions and strategies for predicting toxicity, in vitro methods are likely to play a major role in the near future. On 12 December 2013, the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM, part of the European Commission Joint Research Centre) published its Recommendation on the Direct Peptide Reactivity Assay (DPRA) for skin sensitization, capable of distinguishing sensitizers from non-sensitizers. Other assays (i.e., KeratinoSensTM assay) will follow shortly. While a number of methods are at various stages of development and use, currently it is not possible to rank chemicals for their sensitizing potency, an issue that is important for a full safety assessment. It is expected that a predictive method to totally replace animal testing will be in the form of a test battery comprising molecular, cell-based, and/or computational methods, the so-called "Integrated Approaches to Testing and Assessment". This review aims to discuss the state-of-the-art in the field of in vitro assessment of contact sensitizers

Efeitos do plastificante dibutil ftalato (DBP) em modelos in vivo e in vitro de hipersensibilidade de contato

Orientador : Prof. Dr. Anderson Joel Martino AndradeCo-orientador : Prof. Dr. Michel Fleith OtukiTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 29/10/2015Inclui referênciasResumo: A prevalência de doenças alérgicas tais como dermatite, asma e rinite, tem aumentado, e isso poderia ser devido à presença de substâncias químicas ambientais com potencial adjuvante, ou seja, capazes de exacerbar a resposta imune. Ésteres de ftalatos, plastificantes amplamente encontrados no ambiente, têm sido reportados como participantes no desenvolvimento de doenças alérgicas agindo como adjuvantes. A hipersensibilidade de contato (HSC) é uma reação alérgica que resulta de contatos repetidos da pele com substâncias químicas chamadas haptenos, e consiste em duas fases: sensibilização e elicitação. O plastificante dibutil ftalato (DBP) é capaz de aumentar a resposta inflamatória em HSC induzida por alérgeno quando associado em ambas as fases da HSC, mas em doses muito mais altas que aquelas potencialmente relevantes para a exposição humana. Além disso, os mecanismos moleculares envolvidos nesse efeito adjuvante não estão completamente elucidados. O presente estudo pretendeu investigar os efeitos do DBP na fase de elicitação da HSC induzida por oxazolona em modelo animal de edema de orelha, usando camundongos BALB/c e doses mais baixas de DBP. Ainda, investigamos alguns aspectos relacionados aos mecanismos envolvidos nesse efeito adjuvante. Para isso, avaliamos os efeitos do DBP isoladamente ou associado a alérgenos em dois ensaios in vitro, o ensaio de ativação de NCTC 2544 (representativo de ativação de queratinócitos) e o ensaio de ativação de THP-1 (substituto de ativação de células dendríticas). Foi também investigado se a indução de estresse oxidativo poderia ser parte do mecanismo molecular responsável pelo efeito adjuvante induzido pelo DBP. Ainda avaliamos a possível participação da ativação dos receptores de potencial transitório A1 e V1 (TRPA1 e TRPV1) no efeito adjuvante do DBP no modelo de HSC induzida por oxazolona. In vivo, os parâmetros avaliados foram edema de orelha, 24, 48 e 72 horas após a elicitação, atividade das enzimas N-acetil-?-d-glucosaminidase (NAG) e mieloperoxidase (MPO) (marcadores de infiltração de leucócitos), análise histológica (contagem de células infiltradas, espessura da derme e epiderme) e dosagem de hidroperóxidos de lipídeos (LOOH) e glutationa reduzida (GSH). Nos experimentos in vitro, a produção de interleucina 18 (IL-18) foi avaliada em células NCTC 2544, e a expressão do cluster de diferenciação 86 (CD86) e mRNA de interleucina 8 (IL-8) além da produção de IL-8 e de espécies reativas de oxigênio (EROs) foram avaliados em células THP-1. In vivo, o DBP, nas duas maiores doses (0,4 e 4 mg/orelha), foi capaz de aumentar a resposta inflamatória envolvida na HSC induzida por oxazolona, como observado por aumento no edema de orelha, hiperplasia dérmica e epidérmica, contagem de células infiltradas e atividade de NAG e MPO. Antagonistas TRPA1 e TRPV1 reverteram parcial ou completamente, respectivamente, o efeito adjuvante do DBP neste modelo animal de HSC, sugerindo que a ativação destes TRPs seja parte do mecanismo molecular envolvido no efeito adjuvante. In vitro, DBP potencializou a ativação de células THP-1, como foi demonstrado pelo aumento da expressão de CD86 e IL-8 mRNA e liberação de IL-8 após exposição a associações de DBP com Citral e imidazolidinil ureia , além do aumento da expressão de CD86 em células THP-1 tratadas com DBP associado a oxazolona. Por outro lado, não foi observado efeito adjuvante nas células NCTC 2544, já que não houve aumento na produção de IL-18 após exposição a p-Fenilenodiamina associado ao DBP, sugerindo que o efeito adjuvante do DBP esteja relacionado com maior ativação de células dendríticas e não de queratinócitos. Nossos resultados indicam que o DBP pode agir como adjuvante imunológico tanto in vivo quanto in vitro, e que esse efeito poderia estar relacionado com uma maior ativação de células dendríticas e que envolva ao menos parcialmente a ativação de TRPA1 e TRPV1. Palavras-chave: dibutil ftalato, hipersensibilidade de contato, efeito adjuvante, queratinócitos, células dendríticas, TRPA1, TRPV1, espécies reativas de oxigênio, estresse oxidativo.Abstract: The prevalence of allergic diseases, such as atopic dermatitis, asthma and rhinitis has been increasing, and this could be related to the presence of environmental chemicals with immune adjuvancy potential, which could exacerbate the immune response. Phthalate esters, plasticizers widely found in the environment, have been reported to participate in the development of allergic diseases acting as adjuvants. Contact hypersensitivity (CHS) is an allergic reaction resulting from repeated contact of the skin with chemicals called haptens and consisting in two phases: sensitization and elicitation. The plasticizer dibutyl phthalate (DBP) has been shown to enhance the inflammatory response in allergen-induced CHS when associated both in sensitization and elicitation phases, but in much higher doses than those potentially relevant for human exposure. Besides, the molecular mechanisms involved in this adjuvant effect are not fully understood. The present study aimed to investigate the effects of DBP in the elicitation phase of oxazolone induced CHS in an animal model of ear edema, using BALB/c mice and lower doses of DBP. Additionally, we intended to investigate some aspects related to the mechanisms involved in this adjuvant effect. For that, we evaluated the effects of DBP alone or in association with allergens in two in vitro assays, NCTC 2544 activation assay (representative of keratinocyte activation) and THP-1 activation assay (surrogate for dendritic cell activation). It was also investigated if the induction of oxidative stress could be part of the molecular mechanisms underlying the adjuvant effect induced by DBP. Moreover, we evaluated the possible participation of transient receptor potential cation channel (TRP) A1 (TRPA1) and V1 (TRPV1) activation by DBP in the animal model of CHS induced by oxazolone. In vivo, the parameters evaluated were ear edema 24, 48 and 72 hours after elicitation, activity of the enzymes N-acetyl-?-d-glucosaminidase (NAG) and myeloperoxidase (MPO) (markers of leukocyte infiltration), histological analysis (infiltrated cell count, dermal and epidermal thickness) and lipid hydroperoxide (LOOH) and reduced glutathione (GSH) levels. For in vitro experiments, interleukin 18 (IL-18) production was assessed in NCTC 2544 cells and the expression of cluster of differentiation 86 (CD86) and interleukin-8 mRNA besides the release of IL-8 and reactive oxygen species (ROS) production were evaluated in THP-1 cells. In vivo, DBP in the two highest doses (0.4 and 4 mg/ear) was able to increase the inflammatory response involved in oxazolone-induced CHS, as observed by increase in ear edema, epidermal and dermal hyperplasia, infiltrated cell count and NAG and MPO activity. TRPA1 and TRPV1 antagonists reversed partially or completely, respectively, the adjuvant effect of DBP in this animal model of CHS, suggesting that the activation of these TRPs are part of the molecular mechanism involved in the adjuvant effect. In vitro, DBP potentiated THP-1 cell activation since there was increased expression of CD86 and IL-8 mRNA besides IL-8 release in THP-1 cells exposed to combinations of citral or imidazolidinyl urea with DBP, and increased upregulation of CD86 expression after exposure to DBP associated with oxazolone. On the contrary, no adjuvant effect was seen in the human keratinocyte cell line NCTC 2544, as indicated by the lack of increase in IL-18 production after exposure to p-Phenylenediamine in association with DBP, suggesting that the adjuvant effect of DBP relies on dendritic cells rather than on keratinocytes. In conclusion, our results indicate that DBP can act as an immune adjuvant both in vivo and in vitro, and this effect could be mainly related to increased dendritic cell activation and at least partially achieved by the activation of TRPA1 and TRPV1. Key words: dibutyl phthalate, contact hypersensitivity,adjuvant effect, keratinocytes, dendritic cells, TRPA1, TRPV1, reactive oxygen species, oxidative stress

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitisation potency prediction. The results of the first phase - systematic evaluation of 16 test methods - are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data, potential for throughput, transferability and accessibility in cooperation with the test method developers. A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy - combined with bioavailability and skin metabolism data and exposure consideration - is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients