In the Spotlight
ARDF Awards 2013 Alternatives Grants
Published:November 20, 2013
The 2013 ARDF grants, totaling $200,000, address animal use in toxicity testing (4 winners) and education (1 winner). The four grantees in the field of toxicity testing each seek to advance the National Research Council (NRC) 2007 vision for “Toxicity Testing in the 21st Century.” That vision seeks to replace in vivo testing that assesses “apical” endpoints (e.g., carcinogenicity) with in vitro testing that assesses perturbations to molecular and cellular “toxicity pathways.” The results of pathway testing are interpreted with the aid of computational/bioinformatics tools.
The winners include:
- Patrick McMullen (Hamner Institutes for Health Sciences, Research Triangle Park, NC): Characterization and Visualization of Toxicity Pathways from High-Throughput In Vitro Screening: An Example with the Aryl-hydrocarbon Receptor
Dr. McMullen and colleagues will be seeking to advance pathway testing by using the aryl-hydrocarbon receptor as a prototype pathway. This receptor is active in sensing xenobiotic chemicals; after ligand binding, it stimulates the expression of enzymes and transporters responsible for the conjugation and clearance of these chemicals. The team will conduct a literature-based survey to map the genes involved in this pathway. The mapping of this multi-dimensional network will allow it to be visualized, interpreted, and interactively searched. This will facilitate interpretation of classical issues such a dose-response and time course of experiments. The team will also establish a statistical framework for summarizing the complex relationships visualized by these tools. The resulting approaches are anticipated to be readily transferable to other toxicity pathways.
- Gary Sayler (University of Tennessee, Knoxville, TN): Expressing Bacterial Luminescence in Human Cell Lines: Engineering Autobioluminescent Reporter Cells to Screen for Oxidative Stress
Dr. Sayler and colleagues also seek to provide tools to advance the implementation of “Toxicity Testing in the 21st Century,” particularly high-throughput testing (HTS). Other researchers have shown that engineering light-emitting capability into cells can provide an easily detectible signal that a perturbation of interest has occurred. Unfortunately, existing luminescent systems require that the cellular sample be destroyed at the time of assessing light emission (precluding sampling at multiple time points) or that a light-activating substrate be added to the system (adding an additional experimental step, which is undesirable in the fast-paced world of HTS). Dr. Sayler’s system, by contrast, needs no such experiment-ending or -interrupting steps, but instead generates light-emission intrinsically (“autobioluminescence”). The system uses bioluminescent machinery from bacteria engineered into human cells. The team will test the applicability of this tool to HTS by studying changes in a well-characterized toxicity pathway, Nrf-2 (nuclear factor erythroid 2-related factor 2), a major defense mechanism against reactive oxidants, which are damaging to cells. If successful, this system could be incorporated into HTS more broadly, giving an easily quantified read-out of cellular activity of interest. Autobioluminescence could be especially applicable to studying a chemical’s “kinetics” as it is absorbed, distributed, metabolized, and excreted (ADME) from cells, as the system allows for the non-destructive sampling of the same cells over time.
- Matthew Wright (Newcastle University, Newcastle Upon Tyne, UK): An Unlimited Supply of Rat Hepatocytes In Vitro – Replacing Animals as Donors
The liver plays an especially important role in toxicology, as a key site for the metabolism of exogenous chemicals and their clearance from the body, as well as a target for toxicity itself. Consequently, modeling liver function in vitro is an important component of replacing animals in toxicology, in general, and in realizing the NRC vision, in particular. Hepatocytes are the primary functional type of liver cell. Historically, they have been derived primarily from animals killed for that purpose, with the harvested cells used in primary culture. More recently, hepatocytes have been commercially available as cell lines (not primary cultures), but these sources have been problematic in terms of cost, supplier-imposed limitations on users, and/or low functionality. Similarly, converting stem cells to hepatocytes has proven to be difficult and expensive. Matthew Wright and colleagues from Newcastle University propose to develop an inexpensive and readily derived source of hepatocytes by differentiation from a liver cell line when exposed to glucocorticoid hormone alone. The cell line (“B-13”) is freely available. The researchers will characterize the resulting hepatocytes in three-dimensional culture for their ability to metabolize and transport drugs. The team also will provide workshops at Newcastle so that other researchers can learn to produce hepatocytes by this method. If successful, tens of thousands of animals could be spared as tissue donors for primary hepatocytes, and/or from in vivo studies of drug and chemical metabolism and toxicity.
- Karen Watanabe (Oregon Health & Science University, Portland, OR): Formulation of a Computational Model for Ovarian Development
The Toxicity Testing in the 21st Century paradigm, as we have seen, calls for the broad-based development of in vitro assays and the computational tools to interpret them. A prerequisite for elaborating this paradigm is some understanding of how a system works, so that pathway-based assays can be developed that mimic those systems. Dr. Watanabe and colleagues propose to synthesize the literature on what is known about early ovarian development, and produce a mathematical model and a graphical representation of the biochemical regulatory network during ovarian development. She has chosen ovarian development in mice for her model because of the wealth of data available for this species, with ovarian development in humans being a potential subsequent challenge. The model of ovarian development will be applied to predicting the effects of chemicals on this system, as an early screening tool. Results from in vitro testing will serve as input to the model. If this approach is successful, the model could be used for more definitive testing of putative reproductive toxicants, with even greater impact on reducing animal use.
- Ray Whalen (Colorado State University, Fort Collins, CO): Virtual Feline Anatomy and Virtual Equine Anatomy Programs
Ray Whalen and colleague at Colorado State University’s veterinary school have developed alternatives to animal use in education. Specifically, these are virtual anatomy programs that can substitute for dissecting animal cadavers. The programs use photographs of actual dissection but include interactive features and embedded movies. Students can click to rotate a structure, see deeper tissues, and learn the function of an organ. Whalen’s group began with a virtual canine program (also supported by ARDF) and will now concentrate on programs for cats and horses. As with other virtual programs, these instructional materials offer a number of advantages over actual dissection, including lower cost, repeatability, self-paced learning, and avoidance of cadaver chemical preservatives. Students who would be uneasy dissecting animals can avoid such activity. The virtual programs are applicable to students in a wide array of programs, including veterinary medicine, veterinary technicians, nursing, and college and high school anatomy. The programs would be especially valuable for students in distance-learning education, such as a number of veterinary tech programs. The CSU team’s efforts will also include comparative testing of students learning via the virtual programs versus actual dissections. Informal feedback on the programs to date has been positive. The intention is to eventually make these programs freely available online.
The ARDF congratulates the winners in its 2013 grants program!