Functional toxicology: tools to advance the future of toxicity testing

Abstract

The increased presence of chemical contaminants in the environment is an undeniable concern to human health and ecosystems. Historically, by relying heavily upon costly and laborious animal-based toxicity assays, the field of toxicology has often neglected examinations of the cellular and molecular mechanisms of toxicity for the majority of compounds-information that, if available, would strengthen risk assessment analyses. Functional toxicology, where cells or organisms with gene deletions or depleted proteins are used to assess genetic requirements for chemical tolerance, can advance the field of toxicity testing by contributing data regarding chemical mechanisms of toxicity. Functional toxicology can be accomplished using available genetic tools in yeasts, other fungi and bacteria, and eukaryotes of increased complexity, including zebrafish, fruit flies, rodents, and human cell lines. Underscored is the value of using less complex systems such as yeasts to direct further studies in more complex systems such as human cell lines. Functional techniques can yield (1) novel insights into chemical toxicity; (2) pathways and mechanisms deserving of further study; and (3) candidate human toxicant susceptibility or resistance genes.

Keywords: functional genomics; functional profiling; functional toxicology; toxicity testing; toxicology; yeast.

Figure 1

The concept of functional toxicology in yeast. In this example, a yeast cell with the PDR5 gene is able to survive under toxicant selection, whereas a cell deleted for PDR5 experiences susceptibility to that same toxicant. Therefore, the PDR5 gene is essential for survival in that toxicant.

Figure 2

Overview of functional profiling in yeast. About 4600 deletion strains uniquely identified by DNA sequences (barcodes) are pooled and exposed to a toxicant at multiple doses and generation times (5 or 15). Barcodes are amplified from purified genomic DNA by PCR and counted by hybridization to a microarray or high-throughput sequencing methods. Subsequent analyses of individual strains can confirm susceptibility or resistance to the toxicant.

Figure 3

Integration of functional assays across organisms. One can use functional tools across a variety of organisms, depending upon the model under study and the end goal of the investigation. For example, one may start with a screen in yeast, mouse, or human cells and extend the analyses to whole organisms such as zebrafish or rodents. Alternatively, one may start with zebrafish mutants or DT40 avian deletion cells and perform follow-up experimentation in human cells or other whole organisms. The many possibilities can advance the future of toxicity testing.