Catherine Cohen

Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study.

The Episkin model took part in the prevalidation study on in vitro tests for acute skin irritation of chemicals, which was carried out during 1999 and 2000. This prevalidation study was co-ordinated and supported by the European Centre for the Validation of Alternative Methods (ECVAM). During Phase 1 and Phase 2 of this study, reproducibility and transferability of the method were verified. Unfortunately, the performance of the method in terms of predictive ability was considered insufficient, due to a low specificity. In order to improve the performance of the Episkin method, the existing protocol was refined. This refinement consisted in reducing the exposure time of epidermis with chemicals. Sensitivity, specificity and accuracy of the new method were 70, 80 and 75%, respectively, thus meeting the acceptance criteria as defined by the Management Team. The Episkin method is now ready to enter a validation study of in vitro tests for acute skin irritation.

Key Regulatory Role of Dermal Fibroblasts in Pigmentation as Demonstrated Using a Reconstructed Skin Model: Impact of Photo-Aging

To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

Iron chelation can modulate UVA-induced lipid peroxidation and ferritin expression in human reconstructed epidermis.

Background/Purpose: As ferritin has been identified as an important factor in antioxidant defense in cultured human skin cells, we evaluated UVA‐induced lipid hydroperoxides (LPO) production and ferritin expression in reconstructed human epidermis in vitro.