Research Grants 2009-2010
Summary of Research Grants
Organotypic Culture of Cerebellar Slice for Studies of Developmental Neurotoxicity of Methylmercury
Michigan State University
Methylmercury (MeHg) is extremely toxic and among the most common forms of mercury found in the environment. MeHg causes prominent neurotoxicity. The developing brain is especially sensitive to MeHg. Our goal is to understand the mechanisms responsible for this selective vulnerability of certain cells in the brain to MeHg. During development, neurons migrate from one region of the brain to another. This process, can be studied using cultures of brain slices of neonatal rat. Migration of these neurons involves the complex interaction between chemical messengers known as neurotransmitters which either excite, or inhibit electrical activity in the neurons. We propose to develop this system, in our lab for studies of developmental neurotoxicity with MeHg. This preparation can be used for studies of acute and semichronic exposure to MeHg, thus reducing the need for semichronic injections of neonatal animals with MeHg. Furthermore, more than one preparation can be made from a single rat brain, animal, reducing the overall number of animals needed for a series of studies. Because this preparation maintains the normal intact and developing synaptic connections, it offers a greater advantage over cell culture systems in which the normal connections between neurons (known as circuits) are lost. Movement of cerebellar neurons (in particular, a group called the “granule cells”) and concurrent measures of changes in calcium within the cell will be studied using confocal laser scanning microscopic measures in response to acute and semichronic exposure to MeHg. These studies will be an important contribution to the understanding of developmental neurotoxicity with MeHg.
The Mini-Mouse Toxin Detection Assay
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.
In Vivo Imaging with Bioflurescent Viruses
Johns Hopkins Bloomberg School of Public Health Viruses are an important cause of acute and chronic disease in humans and other animals. Identification of treatments requires studies in live animals and often involve large numbers of animals in order to observe changes over time. We have shown that use of a virus expressing a light-generating compound can greatly diminish the numbers of animals needed for study of virus infection and treatment. It is similarly possible to reduce the numbers of animals needed to study the host response to infection. We will determine whether these mice can be used for monitoring the response in the brain to treatment. We will use as a model system the study of mice infected with Sindbis virus, a virus that causes encephalitis
Characterization and Evaluation of Human Stem-Cell Derived Neurons for Developmental Neurotoxicity Studies
University of the Pacific
Human stem cell-derived neurons (hSCNs) may provide simple alternative in vitro models for neurotoxicological studies.This lab has recently begun investigating the physiological and pharmacological properties of neurons derived from a human stem cell line. With support from CAAT, we have established that such hSCNs develop several of the properties of mature human neurons and that they are exquisitely sensitive to the neurotoxin, methylmercury (MeHg). We have also demonstrated that the effects of MeHg depend on the stage of cell differentiation and that cells apparently unharmed by short-term exposure to low levels of MeHg, display abnormal electrophysiological properties 7-14 days later. These data support the notion of silent neurotoxicity. Our next series of experiments will determine the impact of exposure to ethanol, phenobarbitone and valproic acid on the electrophysiological properties of neurons derived from stem cells. Secondly, we will develop human glial cell - neuron co-cultures for the longer term (? 9 months) use of stem cell derived neurons in neurotoxicological studies; and thirdly, we aim to evaluate the impact of glia-neuron co cultures on neurotoxicity in vitro. Our study will utilize single nerve cell recording techniques and some histochemical methods. Success in these experiments will provide new data on the validity of human stem cell derived neurons as simple and reliable models for neurotoxicological studies and may provide strong support for a reduction in the need for laboratory animals in Developmental toxicity studies
A Closed-Head Recording Device for Neurologic Studies in Laboratory Animals
University of Wisconsin
Open Chambered Cranial Implants (OCCI) can interfere with the animal welfare. Our laboratory has developed and tested polyamide film microelectrode arrays for chronic EEG recordings that can replace OCCI for most neuroscience studies. The previous two years of CAAT support have focused on fabricating and testing prototypes both on the bench and in animals that were under anesthesia. These experiments have been done successfully in rats, cats and primates. Other funding has also been obtained, and survival experiments have been conducted in awake, behaving rats and primates using minimally invasive instrumentation connected to tether systems. Basic design and fabrication work has been done for a wireless prototype, but tests in animals have not yet been performed. These tests have been postponed until a wireless prototype for commercial production is made. The third and last year of support will be used for: 1) producing and testing a wireless prototype implant that can be commercially manufactured, and: 2) testing our electrode arrays, that lie on the surface of the brain, against the standard penetrating electrode system. The purpose of this second aim will be to further demonstrate that the flexible, polyamide film electrode array has comparable, and perhaps even superior capabilities compared to the penetrating electrodes that have been the standard in the field for over 40 years. This data will be necessary to market the commercial wireless recording device to investigators.
The 3-Dimensional MAME Culture Model: An In Vitro Screen for Agents Affecting Mammary Gland Development
Wayne State University
The mammary gland is a complex organ whose development is a carefully orchestrated, chronological process. Development is very similar in humans and rodents, and susceptible to modulation by environmental agents. For example, exposure of pregnant rodents to dioxin in late gestation profoundly suppresses mammary duct development and branching in female progeny. The only assay available for the screening of agents that modulate mammary gland development entails in vivo animal treatment, followed by whole mount analyses of removed mammary tissue. We have developed and are refining an in vitro Mammary Architecture and Microenvironment Engineering (MAME) 3D co-culture model in which primary and secondary mammary ducts, with clusters of attached mammary-like alveolar structures, develop within 8-30 days of the co-culturing of single cell suspensions of human mammary epithelial and myoepithelial cells, and myofibroblasts, in a 3-dimensional CultrexTM matrix. As occurs in vivo, inclusion of dioxin in the MAME model suppresses duct development and eliminates duct branching. Acceptance of the MAME model as an in vitro screen rests on how well the culture model: a) recapitulates normal mammary gland development, and b) identifies known disrupters of gland development. The specific aims of this application are to: 1) characterize the kinetics of alveolar and ductal growth, and lumen formation, in the MAME model; 2) characterize the spatial arrangements of the myoepithelial and epithelial cells in the ducts and alveolar-like structures; and 3) to survey the effects of select in vivo disrupters of mammary gland development in the MAME culture model
Biological Pathway Analysis in Human Dendritic Cells After Exposure to Sensitizing Chemicals
From a regulatory perspective there is an urgent need to develop non-animal models for the identification of chemical skin allergens. Dendritic cells (DC) of the skin play a central role in evoking the allergic cascade that is caused by these compounds. In previous studies, an in vitro model for skin DC was developed based on stem cells isolated from human cord blood. Gene expression studies led to an expression profile characteristic for induction of DC by chemical sensitizers. The differential expression of a selected gene set resulted in a classification model that successfully classifies chemicals as sensitizers and non-sensitizers. In a literature-based network, it could be observed that the gene markers communicate with each other and other molecules involved in inflammation, such as nuclear and cytoplasmic proteins, surface molecules and cytokines. This project will explore the role of the gene markers in the biological process of skin sensitization. The expression of the genes after skin sensitizing exposure will first be confirmed at the protein level and this may point to the involvement of the markers in key processes in sensitization. In a next step, the functional role of a priority selection of the markergenes in the sensitization response will be tested by interfering with their biological pathway. By demonstrating the functional role of a set of specific molecules in the sensitization response of DC, a significant contribution towards unraveling the mode of action of chemical skin sensitizers may be obtained which is in favor of future test development.
A Microassay Analysis Software Package for Comparisons and Extrapolations Relevant to Toxicology
Sutter, Thomas; Phan, Vinhthu
University of Memphis
Understanding the relationships between chemical structures and their effects in cultured cells and living organisms continue to be major challenges in toxicological sciences. Determining how a chemical is toxic and establishing the extent of conservation of this action between rodents and humans or between cells in culture and living organisms is essential to the development of in vitro assays that are predictive of human health consequences. One promising tool for establishing such relationships is microarray, a method to detect and quantify simultaneously the levels of all of the RNAs expressed in a cell. Despite the high throughput capacity of microarray, difficulties in the analysis of these huge datasets continue to limit the interpretation of such data. We have developed a unique approach for determining the effects of chemicals by microarray and have refined the statistical analysis of such data for understanding chemical structures and their associated modes of action. Here, we propose to develop prototype software to make these methods available and easily implemented for the comparisons described above. Such software is necessary for the important mission of CAAT in the reduction, refinement and replacement of animal use in toxicology.
A Rapid Microfluidic Assay for High Thoughput Screening of Chemopreventive Compounds
University of Arizona
Recent studies suggest that the protein Nrf2 could be a key target for cancer prevention, as it plays an important role in the regulation of endogenous antioxidants and phase II detoxifying enzymes. The goal of this study is to develop a rapid screening system for identifying new compounds that regulate Nrf2 and the body’s defense mechanisms. The study will lead to an in vitro system for rapid identification of potential chemopreventive compounds, which could potentially be leap forward in the prevention of many types of cancer. With the successful development of two molecular probe biosensors, we will evaluate and optimize the kinetics of these assays to facilitate rapid screening of potential chemopreventive compounds. In addition, we will develop a multiplexed microfluidic system to serve as a platform for high-throughput drug screening using these molecular assays. If successful, novel chemopreventive agents can be identified from a chemical compound library using the in vitro screening system. The cell-based assay will provide an alternative for reducing animal usage in predicting the effects of chemopreventive compounds and elucidating their mechanisms of action. The system will also be generally applicable to a wide range of toxicology studies.
3-D Sertoli Cell/Genocyte Co-Culture System: In Vitro Model for Developmental Testicular Toxicity
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.
Use of High Throughput Screening Methods Improves Computational Models for In Vivo Acute Toxicity Tests
University of North Carolina at Chapel Hill
Acute toxicity testing, which is broadly utilized in pharmaceutical industrials and environmental protection agencies, is conducted to determine the relative health hazard of various chemicals and drug candidates. The traditional approaches for animal acute toxicity testing are costly and time-consuming. High Throughput Screening (HTS) has been used as alternative method for animal toxicity tests in the past decades. However, all the previous reports showed that the relationship between the available HTS results and any animal toxicity test for diverse compounds is obscure. For example, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) hosted a workshop to study the toxicity in certain cell lines and animal acute toxicity for more than 300 compounds but failed to find any relationship between these two types of toxicity results. However, our recent study showed that we could combine the biological information obtained from cell testing and the chemical information obtained from chemical structures to make a reliable and predictive Quantitative Structure-Activity Relationship (QSAR) model of the animal acute toxicity based on the ICCVAM dataset. In this project, we will apply similar analysis of the relationship between other experimental data in cells and the animal acute toxicity test and extend this study to new data sets. We will develop novel computational toxicity models based on combined chemical and biological information using QSAR modeling approaches. Finally, we expect to use our computational acute toxicity predictors to directly evaluate the toxicity potential of new organic compounds.