Epithelial cells, specifically hair follicle keratinocytes, for testing purposes are readily obtainable from the bulbous end of hairs mechanically plucked from the scalp or other part of the body. Experiments will be carried out on secondary (finite lifespan) hair follicle keratinocyte lines obtain from plucked hairs of Sprague Dawley rats and human volunteers. The rats will be treated topically or systematically with benzo(a)pyrene (B(a)P), hexachlorobiphenyl, methylmercury or 7,12 dimethylbenz(a) anthracene (DMBA). Initial topical doses in 1.0 ml acetone will be: DMBA-0.5 mg, B(a)P-1.0 mg, hexachlorobiphenyl-1.0 mg, methylmercury-0.5 mg. For hexachlorobiphenyl, benzo(a)pyrene and methylmercury, rats will be placed on long term test to establish the carcinogenicity of these compounds by topical exposure. The carcinogenicity of DMBA on rat skin is adequately known. Human hair follicle keratinocytes will be exposed in vitro to benzo(a)pyrene, hexachlorobiphenyl, methylmercury or 7,12 dimethylbenz (a)anthracene. The following specific assays will be performed on the rat epidermal cells or on rat or human keratinocytes as indicated: 1. Percentage mitosis completion (cell survival) as the proportion of cells that synthesize DNA and complete mitosis. These measurements will be made by pulsing the cells with HTdR, fixing 12 hrs later and counting doublets (side-by-side labeled cells) on whole mount autoradiographs (invivo) or newly subcultured dishes (in vitro). 2. The number of 3DNA single strand breaks per unit DNA as determined by alkaline elution (pH=12.3) of HTdR-labeled DNA from exposed cells or descendants of exposed cells. The analysis is performed by computerized liquid scintillation counting, profile analysis, and curve plotting. 3. the amount of keratin alteration in secondary human and rat keratinocytes as measured by the presence of keratins associated with transformation. The keratins are detected by fluoresceinated antibody probes.
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