GD - Online C. Goebel: Aktivierung dendritischer Zellen und Hautpenetration in vitro
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Characterization of the sensitizing potential of chemicals by in vitro analysis of dendritic cell activation and skin penetration

Dr. Carsten Goebel, Wella AG, Abteilung Produktsicherheit und Toxikologie, Darmstadt


The development of in vitro models to identify sensitizing chemicals received public interest since animal testing should be avoided whenever possible. We introduced a new approach, analyzing two essential properties of sensitizing chemicals: skin penetration and dendritic cell (DC) activation (J Invest Dermatol 122:1154 –1164, 2004). Immature DCs derived from human peripheral blood monocytes were pooled from different donors to reduce interindividual variation. DC activation was evaluated by flow cytometric analysis of CD86 highly positive cells and quantitative measurement of interleukin-1ß (IL-1ß) and Aquaporin P3 gene expression. The sensitizer 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced a dose dependent response for all three parameters, whereas the irritant sodium lauryl sulfate (SDS) did not. When two related aromatic amines, p-toluylenediamine (PTD) and hydroxyethyl-p-phenylenediamine (HE-PPD), were tested, both induced a substantial DC activation indicating their potential sensitizing properties. However, the in vitro findings contrasted with the in vivo sensitizing potential: in murine local lymph node assays (LLNA) PTD, but not HE-PTD,was identified as sensitizing using acetone/aqua/olive oil (AAOO) as vehicle. Skin penetration measurement revealed that the bioavailability of PTD from AAOO was significantly higher than that of HE-PPD. An enhanced skin penetration of HE-PPD was found when dimethysulfoxide (DMSO) was used as vehicle. On retesting HE-PPD in the LLNA using DMSO as vehicle, HE-PPD induced a specific response reflecting its activating properties on DCs in vitro. We conclude that in vitro analysis of DC activation capability allows prediction of the skin sensitizing potential provided that skin penetration data demonstrate a sufficient bioavailability of the test compound.

Background

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity reaction induced by small reactive chemicals (haptens). In pharmaceutical and cosmetic industry it is mandatory to identify chemicals that are potential ACD inducers before they become part of a new product. Currently, the sensitizing potential of chemicals is usually identified on the basis of animal studies, such as the LLNA. There is, however, an increasing public concern regarding the use of animal testing for the screening of new chemicals. The development of in vitro models for predicting the sensitizing potential of new chemicals is therefore receiving widespread interest. In vitro sensitization tests are furthermore needed to identify the relevant aspects of the complex interactions of a chemical with the different compartments of the immune system.

To date, no a reliable prediction of the sensitizing potential based on DC activation in vitro was achieved because the in vitro results did not reflect the actual ability to induce contact hypersensitivity in vivo.
We assume that the lack of the skin barrier and the difficulties in obtaining relevant DC activation data are responsible for this discrepancy. Therefore, we propose to integrate in vitro skin penetration analysis using pig skin, the most suitable model of human skin, and to use pooled immature DCs obtained from different donors to reduce inter-individual variability which limit the interpretation of DC activation data. The suitability of this approach was tested with two aromatic amines, a class of chemicals comprising several known sensitizers. The in vitro findings for the two compounds were compared with their potential to induce sensitization in vivo as measured by the LLNA in mice.

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