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 overall goals of this project are directed toward the development of validated mechanistically-based bioassay systems for inexpensive detection of chemicals and classes of chemicals in complex environmental mixtures. Modifications of the cell-based bioassay for detection of dioxin and dioxin-like chemicals have led to the development of a third generation (G3) systems with dramatically greater sensitivity of detection and magnitude of response. The G3 bioassay system is currently being evaluated by a major research laboratory in Germany for selection as the dioxin screening method of choice for governmental regulatory laboratories in the European Union member states. In addition, the estrogenic cell bioassay detection system is also currently being evaluated by the European Commission on Validation of Alternative Methods for acceptance as a validated method for chemical screening in the European Union.
During the past year, researchers have found that arsenic alters the signaling ability of certain transcription proteins in human epidermal cells, likely in concert with changes in the chromatin where they bind. In addition, researchers have found that the accumulations of certain proteins in fingernail are suppressed by exposure to high arsenic levels in the drinking water. Moreover, researchers have found that the sensitivity of cultured human epidermal cells to copper-induced oxidative stress is increased by ascorbate, an effect that also appears germane to arsenic exposure. These results highlight the usefulness of keratinocyte cultures for investigation of toxic mechanisms and biomarker development.
During the last year researchers have found that a key enzyme, epoxide hydrolase, not only is important in the regulation of blood pressure but that the enzyme is a key protein in many regulatory processes. It seems to act by altering the same pathway as common pharmaceuticals such as aspirin and Advil. Surprisingly it was found to reduce blood glucose in rodent models of diabetes and reduce pain – particularly neuropathic pain – in rodent models of diabetic pain. On the positive side these data indicate that inhibitors of the enzyme could be used as pharmaceuticals to treat major human diseases. On the negative side the data caution that environmental chemicals which alter the amount of the enzyme or alter its activity could lead to unanticipated biological effects in people exposed to these chemicals. Environmental compounds known to alter the enzyme level include plasticizers, pesticides, drugs and personal care products.
Using a receptor based screening method developed in this project, researchers also identified environmentally relevant compounds that altered the function of the ryanodine receptor isoforms 1 (RyR1) and 2 (RyR2). Collaborative studies within this project and resulted in positive hits that included triclosan, that they reported previously (Ahn et al. 2008), and twelve chemicals that produced the most significant effect, including chloranil, dichlone chlorpyriphos, o,p’-DDE and deltamethrin (Morisseau, et al. 2009).
There are over 80,000 industrial chemicals used in the U.S. at levels high enough to be on government lists, however, for the vast majority of these compounds little or nothing is known. During the last year researchers demonstrated that that the bioassay systems can be collected and run in a high throughput mode using very low technology robotics. The resulting data do not indicate the safety of a compound but rather flag certain industrial chemicals for more detailed studies. One impact of this screen has been to discover several poorly studied but common industrial compounds that at a biochemical level offers some toxic risk. A broader impact has been to demonstrate that a high throughput screen for prioritizing toxic risk is a cost effective outcome of the nation’s Superfund research effort.