Abstract
A keratinocyte culture derived from rat sublingual epithelium has been developed and characterised both morphologically and enzymically. Morphologically the culture closely resembles that found in vivo. The culture has been studied comprehensively using light and electron microscopy (scanning and transmission). The culture procedure has been optimised such that minimal amounts of time and materials are required. The culture, although heteroploid, shows a high degree of homogeneity with minimal fibroblastic contamination. Total adhered protein, total DNA content, 3H-thymidine incorporation, ornithine decarboxylase activity, acid phosphatase activity, prolinase and malate dehydrogenase activity have been studied at various phases in the cultures growth cycle. An initial study using 3,3', 4,4' tetrachlorobiphenyl indicated that acid phosphatase, prolinase and total protein may be the best parameters for studying toxic insult in these cells, together with morphological examination by electron microscopy and light microscopy using acridine orange as a stain. Subsequently, comparisons of the 3,3', 4,4' and 2,2', 4,4' tetrachlorobiphenyl isomers, benzo(a)pyrene and 20-methylcholanthrene were made. Acid phosphatase and prolinase appeared to be useful markers of the toxic effects of these compounds showing alterations consistent with some in vivo findings. These changes were observed at concentrations of the compounds that did not produce significant cytotoxicity. Morphological examination showed changes with some similarities to some carcinomas in vivo. The effects of these compounds on keratin production within the cells has also been undertaken. A comparison has also been made of the relative toxicities of several compounds in the keratinocyte culture and in the human embryonic lung fibroblast line (BCL-Dl); the keratinocyte system appeared to be more sensitive to irritant compounds. Studies of the metabolic capabilities of the culture using benzo(a)pyrene as substrate have also been undertaken. Although the capacity of these cells to metabolise foreign compounds was low, they show hydroxylation, glucuronidation and sulphation activity. In longer term tests (14-21 day), the system shows enhanced sensitivity, the point of commitment of the culture (i.e., differentiation and stratification) being a critical time. The methods used showed good reproducibility and were easily performed. The system may, therefore, provide a useful sensitive screening test for detection of compounds affecting differentiation (e.g. irritants and carcinogens).