Jennifer Hennen

Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells.

In vitro approaches to address key steps of chemical-induced skin sensitization have been developed, but there is uncertainty how keratinocytes, which play a crucial role not only regarding xenobiotic metabolism but also skin inflammation, impact on a chemicals potential and potency to activate dendritic cells. We investigated these aspects by coculturing THP-1 cells, as surrogate dendritic cells, with HaCaT keratinocytes. We tested our HaCaT/THP-1 model with a set of 14 sensitizers, containing 7 prohaptens, and 10 non-sensitizers. Compared to exposing THP-1 alone, coculturing resulted in up to 3.1-fold enhanced maximal CD86 and/or CD54 upregulation on THP-1, and improved concentration-dependency. All 14 sensitizers were found positive for CD86 and/or CD54 upregulation based on ∆ mean fluorescence intensity (MFI) ≥ 10 for CD86 and ∆MFI ≥ 50 for CD54. Only 1 of 10 non-sensitizers was false-positive. Remarkably, coculture with HaCaT keratinocytes improved the rank correlation of the estimated minimum chemical concentrations inducing a positive response in vitro with in vivo data on sensitization potency, especially for CD54 (Spearman: r = 0.739, p = 0.006; CD86: r = 0.571, p = 0.041). These promising data suggest that the coculture model has the potential to support the prediction of sensitization potency based on in vitro data.

Assessment of skin sensitization potency of hair dye molecules in vitro

Today, the potential of cosmetic ingredients and products to induce skin sensitization must be determined without the use of animal methods. Therefore, alternative in vitro methods that are able to predict the chemicals’ relative potencies for skin sensitization are essential for quantitative risk assessment. This is of special importance for reactive molecules needed for certain applications such as hair dyeing. p-Phenylenediamine (PPD) and toluene-2,5-diamine (PTD) play a major role in hair dye-associated contact dermatitis (1), and, currently, either PPD or PTD is present in 80–99% of commercially available oxidative hair dyes (2). Hazard-based characterization classifies PPD and PTD as strong sensitizers on the basis of the murine local lymph node assay (LLNA), whereas its known trimeric PPD oxidation product, Bandrowski’s base (BB), is classified as an extreme sensitizer, showing that the increase in potency is related to the degree of PPD autoxidation (3, 4). In contrast, the recently introduced PPD derivative 2-methoxymethyl-p-phenylenediamine (ME-PPD) is classified as a moderate sensitizer, as approximately 30to 40-fold higher concentrations than with PPD or PTD were needed for the induction of sensitization in the LLNA (5). Here, we investigated whether the different sensitizing potencies determined in the LLNA for PPD, PTD, ME-PPD and BB can also be distinguished by the use of our human cell line-based coculture model. This model consists of human keratinocytes (HaCaT cells) and THP-1 cells as surrogate dendritic cells (DCs), and uses the chemical concentration that induces significant upregulation of the costimulatory molecule CD86 and/or adhesion molecule CD54 on THP-1 cells as a marker for sensitizing potency (6, 7).

Ranking skin-sensitizing hair dye molecules according to their potency by the use of human cells

The determination of the skin sensitization potential (potency) of hair dye ingredients without animal testing is crucial for their quantitative risk assessment. Besides p-phenylenediamine (PPD) and toluene-2, 5-diamine (p-toluylenediamine [PTD]), which are 2 commonly used precursors and major allergens in oxidative hair dye-associated contact dermatitis, >100 ingredients are currently used, including the precursor p-aminophenol. p-Aminophenol is used together with couplers such as 1-naphthol, m-aminophenol or resorcinol to create different shades. Animal tests have indicated that m-aminophenol, p-aminophenol, 1-naphthol and resorcinol are contact sensitizers, being classified as strong contact sensitizers by the Scientific Committee on Consumer Safety. However, whereas the precursor p-aminophenol and the couplerm-aminophenol frequently elicit patch test reactions in patch tested patients, with prevalences varying between 1.4% and 11%, patch test reactions to 1-naphthol or resorcinol are rare, although these couplers are also frequently used. Besides differences in exposure level and potential cross-reactivity patterns, differences in sensitization potency in humansmay account for such different responses in patients. We previously showed the capacity of our coculture model (COCAT) consisting of human cell lines, that is, HaCaT keratinocytes and THP-1 cells as surrogate dendritic cells, to rank PPD, PTD, and 2-methoxymethyl-p-phenylenediamine according to their potency based on the evaluation of the concentration inducing significant upregulation of the costimulatory molecule CD86 and/or adhesionmolecule CD54 on cocultured THP-1 cells. Here, we studied the sensitization potency of p-aminophenol,m-aminophenol, 1-naphthol and resorcinol in thismodel. METHODS

Respiratory sensitization: toxicological point of view on the available assays

Respiratory sensitization as a consequence of exposure to chemical products has increased over the last decades, leading to an increase of morbidity. The increased use of synthetic compounds resulted in an exponential growth of substances to which we are potentially exposed on a daily basis. Some of them are known to induce respiratory sensitization, meaning that they can trigger the development of allergies. In the past, animal studies provided useful results for the understanding of mechanisms involved in the development of respiratory allergies. However, the mechanistic understanding of the involved cellular effects is still limited. Currently, no in vitro or in vivo models are validated to identify chemical respiratory sensitizers. Nonetheless, chemical respiratory sensitizers elicit a positive response in validated assays for skin sensitization. In this review, we will discuss how these assays could be used for respiratory sensitization and if necessary, what can be learnt from these assays to develop a model to assess the respiratory sensitizing potential of chemicals. In the last decades, much work has been done to study the respiratory toxicity of inhaled compounds especially in developing in vitro assays grown at the air–liquid interface. We will discuss how possibly the tests currently used to investigate general particle toxicity could be transformed to investigate respiratory sensitization. In the present review, we describe the most known mechanism involved in the sensitization process and the experimental in vivo and alternative in vitro models, which are currently available and how to adapt and improve existing models to study respiratory sensitization.

Verbesserte Aktivierung und Detektion von Prohaptenen in einer Ko-Kultur

Chemicals can cause skin sensitization and thus induce an allergic response. Many chemicals act as sensitizers following metabolic activation, so in vitro assays — currently comprising skin cells or antigen-presenting cells — should allow for their enzymatic conversion. Both cell types are able to activate chemicals to a certain extent, our results point to an increased metabolic capacity of keratinocytes in co-culture. Thus, co-culture systems support the hazard assessment of prohaptens.