An Ultrastructural Evaluation of the SKIN2TM ZK1200 System as an In Vitro Model of the Human Oral Mucosa

Dorthe Arenholt-Bindslev, Lis Andersen Torpet and Kaj Josephsen

At present, there are no validated experimental models for the preclinical oral mucosa irritancy screening of dental materials and oral hygiene products. Standardised in vitro models for the assessment of skin irritancy have been marketed, and have made possible the application of such models in preclinical skin irritancy testing. Some of these are currently undergoing international interlaboratory validation. The aim of the present study was to evaluate the histology of the commercially available SKIN2TM ZK1200 system as a potential in vitro model of the human oral mucosa. Histological evaluation by light microscopy and transmission electron microscopy over 6 days revealed a fibroblast-rich matrix covered by an 8–12 cell layer of stratifying epithelium, which shared a number of basic characteristics with non-keratinised human oral mucosa (for example, desmosomes, dispersed tonofilaments, glycogen deposits, membrane-coating granules, and keratohyalin granules, both the spherical and ribosome-associated types). Exposure to a methacrylate-based dental fissure sealant for 24 hours caused structural changes in the epithelium which were not reflected by a cytochemical cytotoxicity assay (MTT).
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The Detection of Differentiation-inducing Chemicals by using Green Fluorescent Protein Expression in Genetically Engineered Teratocarcinoma Cells

Sarah Adler, Martin Paparella, Cristian Pellizzer, Thomas Hartung and Susanne Bremer

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold
increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.
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