DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

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This project focuses on biologically-based detection of hazardous agents by methods exploiting their specific mechanisms of action. First, sensitive and rapid bioassays will be developed for several classes of environmentally important toxicants. In one approach, the ability of certain agents to stimulate gene expression by binding to and activating chemical-specific DNA transcription factors will be exploited in bioassays for certain classes of chemicals. For this purpose, cells will he stably transfected with an expression vector containing a luciferase reporter gene inducibly regulated by the high affinity interaction of one of several ligand-dependent receptors (including those for estrogens, dioxin and related polycyclic aromatic hydrocarbons) with transcriptional enhancer elements inserted upstream of the reporter gene. In a complementary approach, competitive in vitro binding microplate assays for the detection of these chemicals will be developed utilizing purified receptors which have been produced in large quantities by a baculovirus expression system. Initial efforts will be focused on xenobiotics which interact with the Ah and estrogen receptors and will be ex:tended to include other chemicals (such as metals and peroxisome proliferators) that also alter gene expression by chemical-dependent receptors. Second, the ability of hazardous agents to perturb biochemical processes important for cellular growth and differentiation will be examined using human epidermal keratinocytes as targets. The direct toxicity of samples will be measured as well as the ability of samples to sensitize cultures to toxicity from agents activated by metabolism such as polycyclic aromatic hydrocarbons. In addition, based on our original findings with arsenic, the phosphorylation state and levels of select kinase and phosphatase activities will be examined. Protein kinase C isozymes, critical for proper keratinocyte function, and known (keratinocyte transglutaminase) or suspected protein substrates will be of special interest. Moreover, protein tyrosine phosphate levels and effects on specific DNA transcription factors (AP2, steroid hormone receptors) and differentiation markers (involucrin) will be monitored as appropriate. This information will help in tracing signal transduction pathways with which toxicants interfere. Finally, heat shock protein isoform expression and subcellular localization will be examined as a measure of cell stress. Effects of chemical treatment will be compared to actual isoform perturbation in human skin tumors and arsenical keratoses. Validation of effects seen in cultured keratinocytes will be performed using human skin in organ culture. For each assay above, applicability to environmental samples provided by other Superfund components will be tested using purified and complex mixtures of model agents as well as extracts of combustion residues from plastics, metals and other constituents present at hazardous waste sites.