Abstract
The RTE cell line is a keratinocyte line derived from explant cultures of stratified squamous epithelium (Rat Tongue Epithelium). The culture procedure for initiating new cell lines was improved, and the cells were proposed as an in vitro model for skin, since they were found to proliferate, stratify and differentiate in a simplified culture system in the absence of specific substrate requirements (collagen, feeder layers, etc) and in a simple culture medium (without the need for growth factors, high serum levels, etc. ). The morphology of RTE cells grown in various ways (monolayers and stratified cultures grown on plastic, on collagen, and raised to the air interface) was investigated. This was compared with the parent tissue (rat tongue) and with human skin, using both light and electron microscopy, where close similarities could be found. Prolinase activity (cleavage of Pro-X iminodipeptides) was measured in RTE cells during long term culture (one month). This enzyme activity was found to change around the time of confluence when cells began stratifying, and was thought to be a marker for cell differentiation from previous work (Hopley 1986). Although these changes were found to be reproducible, their significance was unclear, and attempts to relate them to collagen synthesis (basement membrane turnover in particular) were unsuccessful. Attempts were made to validate the cell line as a model for cyotoxicity testing using a range of diverse compounds with known in vivo toxicity (skin, eye, oral LD50 and oral 90 days) and using cell proliferation as an endpoint. However, it was difficult to correlate in vitro IC50 values with in vivo classifications of toxicity. A smaller range of related compounds was then selected, and structure-activity relationships were investigated using surfactants (anionic, cationic and non-ionic) as model compounds. Acid phosphatase activity was measured after 4 hours exposure, and neutral red and kenacid blue staining after 3 days exposure as a measure of cell proliferation. All three endpoints gave the same toxicity ranking order as found in vivo, i. e. cationic > anionic > non-ionic. In addition, acid phosphatase activity was found to be a specific early marker for damage in keratinocytes. Surfactant treatment produced a peak of measured activity in these cells which was not reproduced in a 3T3- fibroblast cell line. Lastly, RTE cells were investigated as a model for studying DNA-damaging agents using the unscheduled DNA synthesis (UDS) assay of Williams (1976). Work with the directly acting alkylating agent, MNNG (N-methyl-N1-nitro-N-nitrosoguanidine) showed that RTE cells could be used. Metabolism plays an important role in the toxic effects of compounds, since these may subsequently be activated to more toxic species or deactivated to relatively harmless ones. The capacity of RTE cells for metabolism was therefore investigated using the UDS assay and treating with benzo (a) pyrene (BP) , which is inert as the parent compound and requires metabolism to exert its genotoxicity. RTE cells were found not to metabolise BP to any biologically significant degree. Therefore, it appears that RTE cells can be useful as a model for the study of agents having a direct action on skin, although their use may be limited for compounds requiring metabolic activation.