Superfund Research Program
Development and Application of Integrated In Vitro and Cell-Based Bioassays
Project Leader: Michael S. Denison
Co-Investigator: Isaac N. Pessah
Grant Number: P42ES004699
Funding Period: 1995-2015
Final Progress Reports
Hazardous waste sites contain complex mixtures of a wide variety of toxic chemicals and the overall work in this project has been directed toward development, optimization, validation and application of a variety of cell-based bioassays for identification of diverse toxicants and characterization of their biological and toxicological effects. Using recently validated recombinant and in vitro bioassays for environmental contaminants (i.e. dioxins and related chemicals) and endocrine disruptors (xenoestrogens) the investigators have demonstrated the presence of Ah receptor agonists (dioxin-like chemicals) present in extracts from common commercial paper and plastic products that are in widespread public use. In addition, the researchers have demonstrated the existence of chemicals in water-soluble extracts of these products that can bind to and activate the AhR and exert dioxin-like effects on gene expression. Although the identity and toxicological/biological significance of these novel ligands remains to be established, these studies demonstrate that humans are chronically exposed to ligands for the Ah (dioxin) receptor from a diverse range of sources in addition to environmental samples and food products as commonly assumed. The project investigators’ previous work showed that arsenic suppresses human epidermal cell differentiation and induces the enzyme heme oxygenase-1, consistent with the production of deleterious effects through generation of reactive oxygen. Three other agents reported to generate reactive oxygen (cadmium chloride, potassium chromate, sodium vanadate) also suppress differentiation, but only cadmium induces heme oxygenase-1. The researchers have now shown that arsenic preserves the ability of epidermal cells to remain in a growth state long after untreated cells ordinarily lose this ability, whereas the other three agents do not. This property may promote neoplastic changes in the skin and thus may help explain specifically how arsenic increases human skin cancer. During the past year, the researchers have used various metabolomic approaches to investigate the cellular responses. In one manifestation, metabolic profiling of over 50 regulatory lipids are measured in a single assay, allowing the direct interrogation of inflammatory and proliferative process regulated by the key enzymes involved in polyunsaturated lipid metabolism. In an alternate approach, time-of-flight mass spectrometry has being used to produce metabolic fingerprints to evaluate a broader spectrum of small molecules for changes that correlate with chemical exposure. To date, metabolic profiling studies suggest that the relative production of specific lipid metabolites change in a dose dependent manner, while metabolic fingerprinting experiments have revealed a sensitive but as yet unidentified marker of arsenic exposure. Finally using brain neuronal cells the investigators have examined the molecular mechanisms by which non-coplanar (non-dioxin-like) polychlorinated biphenyls (PCBs) exert their neurotoxic effects. Not only have they demonstrated that these PCBs interact with and regulate the intracellular calcium signaling pathways in neuronal cells, but that they result in enhancement of neuronal signaling by endogenous neurotransmitters. These studies bring investigators one step closer to understanding the mechanism by which these PCBs produce neurotoxicity. Overall, the researchers’ cell systems not only provide them with novel approaches for the detection of selected chemicals and classes environmental chemicals but also avenues for detailed examination of their molecular mechanisms of action.