Second Round (4 Awards)

(Abstracts provided by applicants)

Screening Live Organisms for Mutagenicity
Elwood Linney, Principal Investigator Duke University, Durham, NC	R21 CA 97440-01
Transgenic mouse, rat and fish models have been constructed which allow the investigation of the effects of chemicals upon producing mutations. In all these organisms the process involves challenge with the chemical followed by isolation of the DNA and screening for mutations in transgenes representing selectable bacterial markers. While this allows for some degree of quantitativeness, the results uncovered are limited by the processing time and the selection of tissue from which DNA is obtained. In this proposal we plan to develop an imaging assay in transgenic embryos and larvae which will allow for a live, whole animal screen for mutations produced by potential cancer therapeutics. This approach will take advantage of the small size of zebrafish, allowing for microscopical observation throughout and beyond embryonic development. By evaluating live individual organs as they develop, the potential toxicity of new cancer therapeutics can be easily evaluated as to dose, and whether biochemical processing by the organism might create a by-product that is localized by the organ that does the processing. This technology will be developed by using transgenic technologies in combination with fluorescent reporter targets which, in unmutated form, will localize fluorescence to specific regions of the cells of the organism and, if mutated, a signal change would occur allowing one to scan the whole embryo for somatic mutations. The specific aims are: to develop fluorescent indicator reporter genes that will, if mutated in a target region, cause fluorescence to re-locate to another region of the cell to construct, identify and characterize germ-line transgenic zebrafish with these indicator genes using DNA microinjection technology and pseudotyped retroviral vector infection technology to evaluate the transgenic lines with a characterized mutagen(ENU) to test the proof of principle

In Vitro Systems: Predict Drug-induced Pulmonary Injury
Charles Tyson, Principal Investigator SRI International, Menlo Park, CA	R21 CA 97438-01
The National Cancer Institute has a need for development, standardization, and validation of assays that can quickly and cheaply determine or predict organ-specific toxicities of potential cancer therapeutic agents. The objective of the proposed project is to develop an in vitro lung cell assay to determine or predict potential dose-limiting pulmonary injury by anticancer agents in humans. Research in the R21 phase will focus on the development, evaluation, and optimization of one or more primary multicell systems that can meet that objective, in particular precision-cut lung slices and co-cultures of lung endothelial and epithelial cells. The assay to be developed will ultimately be able to discriminate between the major types of injury induced in lung by anticancer agents, differences in toxic potency, and interspecies differences in sensitivity. In this work, known and novel indicators will be investigated for assessing the degree and nature of the injury produced by the agents in vitro for correspondence with in vivo response, using the rat as a model and clinical chemistry and advanced proteomic and laser-MS (Jet-REMPI) techniques. Reference compounds used to develop and validate the assay will be anticancer agents with specific, well- defined, representative toxicities in animals and humans. The R33 phase will focus on developing the full capabilities of the assay as an important and critical part of the in vitro toxicity test battery needed by the NCI to screen for target organ toxicities and to aid in predicting safe dose levels and regimens for clinical trials.

P75 Expression for Assessing Neurotoxicity
William Valentine, Principal Investigator Vanderbilt University, Nashville, TN	R21 CA 97453-01
Neurotoxicity is a common dose limiting or significant side effect of many chemotherapeutic agents emphasizing the importance of assessing neurotoxicity in the screening of novel chemotherapeutic agents. Current methods of screening for neurotoxicity typically utilize morphologic endpoints. The cost and resource requirements of these methods limits the number of chemicals that can be evaluated and the utility of these assessments is diminished by their inherent sensitivity and difficulty in obtaining quantitative data. Previous work has demonstrated up regulation of several genes following toxic insults and physical injury to the peripheral nervous system. The low affinity nerve growth factor receptor (p75) has been most extensively characterized and has exhibited the greatest increases and longest duration of elevated expression after injury. The ability to detect elevations in mRNA prior to the onset of structural changes together with the ability to assess changes along the entire axon suggest that up regulation of p75 may provide a more sensitive index of neurotoxicity than morphological endpoints. The proposed project is based upon the hypothesis that elevated p75 expression in the peripheral nervous system is a general response to toxic insult that is quantifiable and precedes the onset of morphological changes. This hypothesis will be tested through exposing rats to neurotoxic agents that selectively target the neuron, axon or Schwann cell and then determining if elevated p75 transcription precedes the onset of structural changes at the light and electron microscope level. The quantity of p75 mRNA will be determined in peripheral nerve using real time RT-PCR and the levels of p75 protein in nerve evaluated using immunohistochemistry. The utility of measuring truncated p75 in plasma and urine to I detect neurotoxtcity will also he determined by ELISA and plasmon resonance. Morphologic assessments will he used to determine the temporal relationship of p75 expression to the onset of structural changes. The significance of these investigations lies in the development of more efficient and sensitive methods for detecting neurotoxicity of new therapeutic agents, monitoring neurotoxicity in patients during therapy, defining bioactivation pathways of neurotoxic agents and assessing the neurotoxicity of environmental compounds. The benefits of these methods can be realized through facilitating development of new therapeutic agents and recognizing neurotoxic chemicals before the occurrence of human neurotoxicity.

Tissue Chips for Toxicology Evaluation
Jay Gandolfi, Principal Investigator University of Arizona, Tucson, AZ	R33 CA 97449-01
While chemotherapeutic agents can be screened for their efficacy by specific cellular and molecular analyses, the toxicity it can produce in an animal (man) can occur in multiple targeted organs. Thus, while screening of a library of potential chemicals as chemotherapeutic agents can be expedited, toxicological profiling is time-consuming and expensive. The goal of this proposal is to use precision-cut tissue chips from multiple organs from mice to simultaneously examine the in vitro toxicities of a chemotherapeutic agent. This rapid examination of toxic effects in multiple target tissues will expedite profiling of the toxic potential of a new chemo-therapeutic agents. Tissue chips have the advantage that the cellular architecture of the organ is retained so site-specific toxic effects can be detected. The Aims of this Proposal are: Aim 1. Develop the procedures for preparing tissue chips from precision-cut tissue slices. In our earlier attempts to use tissue chips, we "stamped" the tissue chips from our precision-cut tissue slices. We plan to further explore this method or examine other techniques. Aim 2. Develop incubation procedures for maintaining the viability of tissue chips for incubation periods required for toxicity testing. We have incubated smaller organ sections on a rotating shaker using multiwell plates with a bead at the bottom to increase the mixing. This approach can be readily adapted to the tissue chips. Aim 3. Adapt toxicity assays to the small biomass associated with the tissue chip. We have previously adapted assays for cytotoxicity (necrosis), apoptosis, altered mitochondrial function, etc for our tissue slices from different organs. Since the chemotherapeutic agents have different mechanisms of toxicity, we will need to have a battery of analyses to characterize the toxicity. These need to be scaled down or new, more sensitive assays applied. Development of rapid analyses to expedite profiling of the toxicity will be emphasized. Aim 4. Validate the toxicity of existing chemotherapeutic agents with the tissue chips. These studies will demonstrated the tissue specificity of the chemotherapeutic agents and validate the selectivity of the tissue chips. Aim 5. Profile the toxicity of chemotherapeutic agents under development. We will coordinate with our cancer chemotherapeutic drug discovery group to obtain lead series of compounds and will profile their toxicities with the tissue chips. Our results will be compared to independent toxicity profiles obtained from contract laboratories also examining these compounds. These studies should validate if we have developed an innovative approach to rapidly profile the toxicity of potential chemotherapeutic agents.