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
The Development and Applications of Integrated In Vitro and Cell-Based Bioassays project is directed toward the development, optimization, validation and application of mechanistically-based cell- and cell-free bioassay methods for rapid and inexpensive detection of selected chemicals and classes of chemicals present in complex mixtures of hazardous substances. Dr. Denison and his research team have continued to develop and utilize their recombinant cell-based CALUX bioassays for screening of chemicals and extracts in order to detect and identify natural and synthetic activator/inhibitors of the Ah (dioxin) receptor (AhR) or estrogen receptor (ER). While screening analysis has identified a variety of natural products that can interact with and activate the AhR these compounds affect the AhR signaling pathway only transiently and thus are unlikely to be toxic. In addition, the researchers found substantial species differences in the ability of chemicals to bind to and activate the AhR and this provides insights into the reported species differences in response to AhR ligands. In additional studies, analysis of extracts of more than 38 different commercial children's plastic sippy cups and plates revealed the presence of both activators/inhibitors of the ER. While ethanol (100%) extracts of many products had substantial levels of estrogenic activity, water extracts of some products contained even greater estrogenic. While the identity of the responsible chemicals and their toxicological significance remain to be determined, these endocrine active chemicals have the potential to lead into children's food and drink. The researchers have continued to develop and utilize epithelial and epidermal cell culture models for analysis of response to cellular oxidative stress with applications to biomarkers of effect. Their studies have led to the development of a method to culture epithelial and other cell lines from tilapia fish (Oreochromis mossambicus) that have proven useful in studying the responses of this model fish species to environmental and chemical stress. In addition, the research team found that cultured human epidermal cells can activate the widely encountered personal care product ingredient triclocarban into reactive metabolites that can adduct protein sulfhydryl groups. The researchers' findings with human epidermal and mouse hair protein analysis demonstrate the utility of shotgun proteomics for characterization of normal conditions and aberrant manifestations due to disease or chemical exposures. These studies provide a foundation for examining (i) responses in fish to pollutants at a cellular level, (ii) the ability of human epidermal cells to biotransform prevalent chemicals to give macromolecular adducts and (iii) the ability of chemical exposure to perturb protein expression. The researchers have continued to evaluate the influence of Superfund chemicals on the production of regulatory lipids that influence cardiovascular diseases, inflammation and pain. Their studies have illustrated the effect of Superfund chemicals on health by their ability to change the balance of regulatory lipids toward an unhealthy setting. By this mechanism, the researchers observed that xenobiotics exposure could not only alter our risks of cancers, but also could have adverse effects on fibrotic diseases, chronic pain and inflammation, hypertension, and organ damage. Additionally, the results of their studies have also indicated that supplementation of food with omega-3 lipids, such as DHA rich oil, could help reverse some of these effects. The researchers also observed chirality-dependent effects of NDL-PCBs (such as PCB136) on RyRs and found that (-)-PCB136 which potently sensitizes RyRs, also enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. Similarly, (−)-PCB 136, but not (+)-PCB 136, increases the activity of hippocampal neurons plated on microelectrode arrays. These results support the hypothesis that such chiral isomer effects on RyR activity translate into effects of PCB 136 isomers on neuronal connectivity, and suggest that the variable enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure. Overall, the researchers' studies have led to significant improvements in and/or application of their bioassay systems, identification of new toxicants and characterization of the toxic and biological events and mechanisms occurring at these targets.