Research Grants 2008-2009
Summary of Research Grants
- Joseph Bressler, Kennedy Krieger Research Institute
Development of a computer model predicting toxicity of mixtures of metals
- Julie Dalziel, Ag Research
The Mini-Mouse Toxin Detection Assay
- Frank Gerberick, The Procter and Gamble Company
Development of a High Throughput Method to Screen for Gene Expression Changes for the Prediction of the Skin Sensitization Potential of Chemicals
- Diane Griffin, Johns Hopkins Bloomberg School of Public Health
In Vivo Imaging with Bioflurescent Viruses
- Robert Halliwell, University of the Pacific
Characterization and Evaluation of Human Stem-Cell Derived Neurons for Developmental Neurotoxicity Studies
- Lisa Krugner-Higby, University of Wisconsin
A Closed-Head Recording Device for Neurologic Studies in Laboratory Animals
- Glenn Walker, North Carolina State University
Microfluidic Systems for Reducing Animal Use in High Throughput Toxicity Assays
- Aaron Wheeler, University of Toronto
An Invertebrate Model for Animal Behavioral Screening
- Pak Wong, University of Arizona
A Rapid Microfluidic Assay for High Thoughput Screening of Chemopreventive Compounds
- Xiaozhong Yu, University of Washington
3-D sertoli cell/genocyte co-culture system: in vitro model for developmental testicular toxicity
The “mini-mouse” toxin detection assay
Dr JE Dalziel, PhD
The project will use the voltage-gated sodium ion channel (VGSC) from human skeletal muscle to test the safety of potentially contaminated shellfish and drinking water for human consumption. When the algal toxins brevetoxin and saxitoxin bind to VGSCs they disrupt normal nerve and muscle function and can produce poisoning neurological effects that range from irritated nasal passages and coughing, to ataxia and paralysis. Shellfish biotoxins require routine monitoring worldwide to protect public health. Currently the main internationally validated biological based method to measure the toxicity of these compounds is the mouse bioassay. This has been deemed outdated by the World Health Organisation for both scientific and ethical reasons. The aim of this research is to develop the “mini-mouse” assay as a method to reduce the numbers of laboratory mice required to carry out LD50s to test the neurotoxicity of algal toxins in contaminated shellfish. The electrophysiological assay that we have developed consists of a bilayer lipid membrane supported by porous Teflon that is preloaded with VGSC protein incorporated into liposomes. We will investigate the ability of this assay to determine the concentration of brevetoxins (activators) and saxitoxins (inhibitors). By using a natural biological target for saxitoxin, this in vitro assay will recognise this dangerous toxin and other isomers that might arise. The proposed mini-mouse assay has the potential to contain a suite of different ion channel receptors against which a range of potentially harmful shellfish toxins are active.
Development of a High Throughput Method to Screen for Gene Expression Changes for the Prediction of the Skin Sensitization Potential of Chemicals
The Procter and Gamble Company
Allergic contact dermatitis is a frequent occupational health problem and is the most common type of chemical allergy in humans. Therefore, there is a need to identify those chemicals that can cause skin sensitization. For this purpose guinea pig, and more recently mouse test methods have been used. However, there is a need to eliminate completely the use of animals in skin sensitization safety assessments. In recent years, our understanding of the cellular mechanisms involved in allergic contact dermatitis has increased substantially. It is known that dendritic cells (DC) residing in the skin play a key role in the development of allergic contact dermatitis. Given the importance of these cells in the initiation of skin sensitization, it seems appropriate to explore whether there are opportunities to develop alternative approaches to hazard identification based upon chemical-induced changes in the gene expression profile of these cells. This project will explore the use of Luminex® xMAP® bead-based technologies for the development of a high throughput screening method to rapidly measure gene expression changes in allergen-treated DC-like cell lines. The observed effects on gene expression in these cell lines will be compared to data derived from exposing human peripheral blood derived-DC to the same chemical allergens. The goal of this project is to provide a quick, easy and more cost-effective in vitro method to evaluate a large number of chemicals in order to identify genes that when measured in vitro will aid in the prediction of the skin sensitization potential of chemicals in vivo.
In vivo imaging with biofluorescent viruses
Diane Griffin and Ivorlyne Greene
Johns Hopkins Bloomberg School of Public Health
Animals are essential for the study of how viruses, such as alphaviruses, that infect the nervous system cause paralysis and death and for determining how such infections can be treated to prevent death and longterm sequelae of infection (e.g. paralysis, mental retardation, etc). Traditionally, these pathogenesis and treatment studies involve examining the brains and spinal cords of mice at multiple times after infection to determine where the virus is, how much virus is present and the host immune response to the virus. We have shown that viruses engineered to express bioluminescent molecules, such as firefly luciferase, can be used to follow virus replication and spread using only a small number of mice that can be followed individually over time. Recently, we have also shown that mice that express luciferase in a way that reflects the inflammatory response in the nervous system can be used to follow the host response to viral infection, again using only a small number of mice that can be followed individually over time. These studies also showed that the effects of treatment could be monitored as well. In this project we will combine the use of bioluminescent viruses and mice expressing host genes as bioluminescent proteins to examine the course of virus infection of the nervous system and the response to various treatments designed to improve the outcome from this type of potentially devastating infection.
Characterization and evaluation of human stem cell derived neurons for developmental neurotoxicity studies
Dr RF Halliwell & Dr L Coyne
University of the Pacific Human stem cell-derived neurons (hSCNs) may provide simple and powerful in vitro models of nervous system development but there is minimal data on their functional properties and even less establishing their validity and reliability for neurotoxicological studies.
With pilot funding from Johns Hopkins CAAT in 2007, we obtained data showing that hSCNs develop several of the molecular and electrical properties of mature human neurons and we have established that they are exquisitely sensitive to the prototype neurotoxin, methylmercury. We also observed that the effects of mercury are highly dependent on the stage of cell differentiation and, notably, that hSCNs apparently unharmed by short-term exposure to low levels of mercury, display impaired electrical properties 7-14 days later, supporting the notion of silent neurotoxicity.
The aims of this study are now to explore the sensitivity of human stem cell derived neurons to a broader range of putative neurotoxins, including ethanol and the anticonvulsants, phenobarbitone and valproic acid using both cellular and electrophysiological measures and to begin investigating the underling mechanisms for neurotoxicity.
These experiments will provide new data on the validity of human stem cell-derived neurons as simple, ethically acceptable and reliable models for neurotoxicological studies. Data generated in this study provides strong support for a reduction in the need for laboratory animals.
A Closed-Head Recording Device For Neurologic Studies In Laboratory Animals
Lisa Krugner-Higby DVM, Ph.D.
University of Wisconsin
The ultimate goal of this research is to produce a fully implantable, wireless, intercranial device that can replace open-chambered head caps in most neurologic research applications in animals. A fully-implantable, wireless device would mean that the implant would not be open to the environment, would be more comfortable for the animal, and the fact that it is wireless would mean that animals would not need to be restrained in order to record electrical activity from the brain. In order to achieve this ultimate goal, it is necessary to achieve many intermediate steps. In the previous year of funding, implants were designed, constructed and tested in terminal experiments in rhesus monkeys. Implants suitable for use in rats were also designed and constructed. In the second year of funding, the implant for use in rhesus will be modified for complete wireless operation. This prototype wireless device will be tested in anesthetized animals given several common pain relief drugs. The recording device for use in rats will be implanted in anesthetized rats and recordings made to test the device prior to designing a smaller wireless system that could be used in the smaller species. All of these preliminary tests will be made in anesthetized animals that will not be allowed to recover. Additional funds from other sources will be sought in order to conduct tests of the wireless implant in animals that were fully awake. Eventually, the wireless form of the device will be made available to the research community.
Microfluidic Systems For Reducing Animal Use In High Throughput Toxicity Assays
North Carolina State University
The system developed in this project will address the growing need for alternative animal models in toxicological studies. To meet this need we propose to develop an in vitro microfluidic screening system, capable of simultaneously testing many compounds, that will minimize or eliminate animal use in irritant testing. A solvent-resistant polymer will be used to construct the microfluidic devices so that a wide range of test compounds, including organics, can be used for irritant screening. Cultures of human epidermal keratinocytes (HEK) will be maintained within the devices and used as the model system, thus eliminating animal use for these studies. We will validate the irritant potential of benzene and toluene, components in a wide variety of consumer products, by exposing them to HEK and assaying for the secreted pro-inflammatory markers IL-8 and TNFa. The microfluidic screening system will use one one-hundredth the volume of a traditional 96 well titer plate, thus reducing the amount of cells and reagents needed for irritant screening. The proposed microfluidic system will have wide-ranging impact in all areas of toxicology since it can be used with any cell type and with any soluble chemical compound.
An Invertebrate Model For Animal Behavioral Screening
University of Toronto
A new technology has recently become commercially available: automated, multiplexed behavioral analysis platforms. These systems facilitate the identification (via digital video and automated image analysis) of rat or mouse behaviors that are associated with neurological diseases. This development has led experts to predict a significant increase in the use of mammalian subjects for behavioral testing in drug discovery and other applications. A potential solution to the ethical and resource issues associated with this trend is the replacement of mammals with simple invertebrates such as Caenorhabditis elegans, a 1 mm-long, 959-cell, soil-dwelling nematode. We propose to test whether a combination of microfluidic mazes and C. elegans behavioral assays can be useful as a replacement for mammalian animal models. If successful, this could lead to a reduction in the use of mammals in applied and basic behavioral research, which would support the central aim of the Center for Alternatives to Animal Testing (CAAT).
A Rapid Microfluidic Assay For High-Throughput Screening Of Chemopreventive Compounds
Pak Kin Wong, PhD
University of Arizona
The overall goal of the project is to develop a rapid screening system for identifying chemopreventive compounds. Chemopreventive compounds often exert their effects by inducing the expression of cytoprotective enzymes to defend cells from oxidative stress and reactive carcinogenic intermediates. The expression of many cytoprotective enzymes is regulated by the NF-E2-related factor 2 via antioxidant-responsive elements. Here, we plan to develop two molecular probe designs for rapid detection of the ARE-bound Nrf2 protein and Nrf2-mediated gene expression for identifying potential chemopreventive compounds. The molecular probe biosensors will form the technological core of a high-throughput screening system. To evaluate the assays, human dermal fibroblasts are chosen as an in vitro skin-like model. The development will lead to a rapid, quantitative, sensitive, and specific screening system for rapid identification of chemopreventive compounds. The system will provide an alternative for reducing animal usage in predicting the effects of chemopreventive compounds and elucidating their mechanisms of action. The assays should also be generally applicable to a wide range of toxicology studies, as they are compatible with various in vitro models.
3-D Sertoli Cell/Genocyte Co-Culture System: In Vitro Model For Developmental Testicular Toxicity
Xiaozhong Yu, MD, PhD
University of Washington
The development of in vitro models that monitor and which can identify altered testis development would provide important alternatives to in vivo testing. Such methods would allow for the assessment of reproductive and developmental effects induced by environmental agents and thus lead to significant refinement and reduction of in vivo animal use. Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three dimensional gonocyte/Sertoli cells co-culture (GSC) model with ECM, demonstrated this culture system creates an in vivo-like niche and supports germ-line stem cell functioning within a 3-D environment, permits the formation of a testicular-like multilayered architectural bio-structure that mimic in vivo characteristics of seminiferous tubules. We feel strongly that this novel in vitro GSC system will provide investigators with a simple, efficient, and highly reproducible alternative for the assessment of reproductive toxicity and the screening of testicular developmental toxicants. Thus, the purpose of this study is to further refine and characterize our in vitro 3-D GSC culture as a simple, efficient tool for screening testicular developmental toxicant by using a range of known in vivo male reproductive and developmental toxicants. This will be a systems based analysis using genomics and GO-quant technologies. We will also apply this 3-D GSC system to develop appropriate cell-based assays and biomarkers of susceptibility for testicular toxicity through the use of real-time nanotechnology biosensor assay methods. The establishment of a simple, efficient in vitro 3-D GSC culture model has the potential to greatly reduce the number of animals used through its ability to increase and refine the type of information obtained.