Research Grants 1997-1998

Cellular-based Assays to Detect Toxic Agents and Elucidate the Underlying Genetic and Biochemical Mechanisms of Toxicity

Michael Scott Dubow, PhD
McGill University, Montreal, Quebec

The reduction and/or replacement of whole animals in the assessment of potential dangers is an increasingly coveted goal by the public, regulatory agencies and scientists. One of the major difficulties in the achievement of this goal resides in the fact that the underlying mechanisms of toxicity (which ultimately result in observable effects) in whole animals have been difficult to dissect, and thus reproduce in vitro. The major goal of our research is to identify and characterize the mechanisms of toxicity of (selected classes of) toxic compounds at the molecular level in such a manner that we obtain cellular and molecular-based assays for toxicity measurements, as well as important information for the prediction, treatment and ultimate elimination of the effects engendered by these chemicals.

The underlying tenet of our research is that cells will augment or repress the expression of specific genes, upon exposure to bioavailable doses of a toxic agent, in order to reorient cell physiology to cope with the stress. My laboratory has taken a two-pronged approach, using both bacterial (Escherichia coli) and human (HeLa) cells in culture to identify and characterize these genetically-programmed responses to chemical exposure. In bacteria, we prepared a collection ("library") of luciferase gene fusions (using the luciferase-encoding luxA,B genes from the marine bacterium Vibrio harveyi) and have identified a number of clones whose luminescence (and thus luciferase expression) is increased upon cellular exposure to important toxic compounds.

These clones are continuing to be validated for use as luminescent "biosensors" to detect particular toxic agents and, due to the luciferase "gene tagging" used in our approach, the toxin-inducible gene(s) and encoded proteins and regulatory circuits are being identified and characterized to elucidate the fundamental mechanisms of toxicity. We are also expanding our research using human cells in culture to extrapolate our knowledge in bacterial cell responses. Our approach is to isolate and identify poly(A)+mRNAs which are increased upon cellular exposure to aluminum, tributlytin and atrazine using the creation and cloning of magnetic bead-based "subtraction libraries" and the new technology of "differential display."

We have chosen to use HeLa cells as they are a fibroblast cell line, and this cell type represents the type of cell that would be first exposed to a particular toxic agent in a whole animal. In both approaches, the toxin-affected cDNAs are being cloned, identified by DNA sequence, and will ultimately be used to develop molecular probes for the elaboration of non-whole animal assays for toxicity assessment. In addition, we are continuing to screen a Lambda gt11 cDNA expression library (prepared from HeLa cells) for clones which produce human polypeptides that bind to (radiolabelled) phenol or atrazine.

These clones are being identified by "plaque hybridization" and will furnish potential targets for the characterization of the cytotoxicity of these compounds. In this manner, we will continue to use the tools of molecular biology and biotechnology for the development of cellular and molecular-based assays to provide quantitative measurements of biologically-relevant doses of a toxic agent and a concomitant reduction in the necessity of using whole animals for safety evaluation.