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Title: Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing

Abstract

The potential of rat epidermal keratinocyte (REK) organotypic culture (ROC) with proper stratum corneum barrier as a model for screening skin irritants was evaluated. The test chemicals were selected from ECETOC database (1995) and the observed in vitro irritation potential was compared to ECETOC in vivo primary irritation index (PII), to EU risk phrases, and to the harmonized OECD criteria. Chemicals were applied onto the stratum corneum surface of ROC for 30 min and samples were taken from the underlying medium at 4 and 8 h after exposure. Cell membrane integrity (determined by LDH assay) and pro-inflammatory effect (determined by IL-1{alpha} release) were verified at both time points and correlated to PII values. The best correlation (R {sup 2} = 0.831) was seen with LDH leakage test. Based on obtained data, chemicals were classified according to criteria defined by EU and OECD. From 12 chemicals, only two were incorrectly classified according to OECD criteria when using LDH leakage and IL-1{alpha} release as irritation markers. At the end of experiment, chemical-treated ROC cultures were fixed and histological changes were assessed. Typical signs for irritation were lightly stained cytoplasm, condensed nuclei, cellular vacuolization, eosinophilic cytoplasms, and blebbing. These irritation effects of chemicalsmore » were graded visually into four classes (A-D). The extent of morphological perturbations of the cultures mostly correlated with PII. The present results indicate the validity of the ROC model in predicting skin irritation potential of chemicals and show that the use of set of irritation markers with different mechanistic responses gives more information on irritation than if only one marker was used.« less

Authors:
 [1];  [2];  [3];  [4];  [2];  [3]
  1. Department of Pharmaceutics, University of Kuopio, PO Box 1627, 70211 Kuopio (Finland). E-mail: sari.pappinen@uku.fi
  2. Department of Anatomy, University of Kuopio, PO Box 1627, 70211 Kuopio (Finland)
  3. Department of Pharmaceutics, University of Kuopio, PO Box 1627, 70211 Kuopio (Finland)
  4. (Finland)
Publication Date:
OSTI Identifier:
20722024
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 208; Journal Issue: 3; Other Information: DOI: 10.1016/j.taap.2005.02.014; PII: S0041-008X(05)00110-9; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CELL CULTURES; CELL MEMBRANES; CHEMICAL INDUSTRY; CYTOPLASM; ECOLOGY; HEALTH HAZARDS; IN VITRO; IN VIVO; INFLAMMATION; LACTATE DEHYDROGENASE; MORPHOLOGY; RATS; SKIN; SODIUM; SULFATES

Citation Formats

Pappinen, Sari, Pasonen-Seppaenen, Sanna, Suhonen, Marjukka, Department of Pharmaceutics, Biopharmaceutical In Vitro Drug Evaluation Laboratory, University of Kuopio, PO Box 1627, 70211 Kuopio, Tammi, Raija, and Urtti, Arto. Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing. United States: N. p., 2005. Web. doi:10.1016/j.taap.2005.02.014.
Pappinen, Sari, Pasonen-Seppaenen, Sanna, Suhonen, Marjukka, Department of Pharmaceutics, Biopharmaceutical In Vitro Drug Evaluation Laboratory, University of Kuopio, PO Box 1627, 70211 Kuopio, Tammi, Raija, & Urtti, Arto. Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing. United States. doi:10.1016/j.taap.2005.02.014.
Pappinen, Sari, Pasonen-Seppaenen, Sanna, Suhonen, Marjukka, Department of Pharmaceutics, Biopharmaceutical In Vitro Drug Evaluation Laboratory, University of Kuopio, PO Box 1627, 70211 Kuopio, Tammi, Raija, and Urtti, Arto. Tue . "Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing". United States. doi:10.1016/j.taap.2005.02.014.
@article{osti_20722024,
title = {Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing},
author = {Pappinen, Sari and Pasonen-Seppaenen, Sanna and Suhonen, Marjukka and Department of Pharmaceutics, Biopharmaceutical In Vitro Drug Evaluation Laboratory, University of Kuopio, PO Box 1627, 70211 Kuopio and Tammi, Raija and Urtti, Arto},
abstractNote = {The potential of rat epidermal keratinocyte (REK) organotypic culture (ROC) with proper stratum corneum barrier as a model for screening skin irritants was evaluated. The test chemicals were selected from ECETOC database (1995) and the observed in vitro irritation potential was compared to ECETOC in vivo primary irritation index (PII), to EU risk phrases, and to the harmonized OECD criteria. Chemicals were applied onto the stratum corneum surface of ROC for 30 min and samples were taken from the underlying medium at 4 and 8 h after exposure. Cell membrane integrity (determined by LDH assay) and pro-inflammatory effect (determined by IL-1{alpha} release) were verified at both time points and correlated to PII values. The best correlation (R {sup 2} = 0.831) was seen with LDH leakage test. Based on obtained data, chemicals were classified according to criteria defined by EU and OECD. From 12 chemicals, only two were incorrectly classified according to OECD criteria when using LDH leakage and IL-1{alpha} release as irritation markers. At the end of experiment, chemical-treated ROC cultures were fixed and histological changes were assessed. Typical signs for irritation were lightly stained cytoplasm, condensed nuclei, cellular vacuolization, eosinophilic cytoplasms, and blebbing. These irritation effects of chemicals were graded visually into four classes (A-D). The extent of morphological perturbations of the cultures mostly correlated with PII. The present results indicate the validity of the ROC model in predicting skin irritation potential of chemicals and show that the use of set of irritation markers with different mechanistic responses gives more information on irritation than if only one marker was used.},
doi = {10.1016/j.taap.2005.02.014},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 208,
place = {United States},
year = {Tue Nov 01 00:00:00 EST 2005},
month = {Tue Nov 01 00:00:00 EST 2005}
}
  • The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highlymore » induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures of human keratinocytes. • Keratinocyte-expressed MMP-10 accumulates in the dermal compartment. • Keratinocyte K14 promoter-driven TIMP-1 expression ablates TCDD-induced phenotypes.« less
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