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AIM 1. Identify functional changes in human or rat astroglia-like and neuron-like cell lines exposed to neurotoxic agricultural pesticides. The hypothesis to be tested is that each toxicant induces unique spatial and temporal perturbations of cellular homeostasis mechanisms that can be observed with non-invasive imaging tools and that these perturbations are indicative of chemical-induced tissue and organ damage in vivo. <P>AIM 2. Compare the functional responses of cell lines to agricultural pesticides with those of primary cell cultures. The working hypothesis is that a subset of responses will be similar between cell lines and primaries cultures, thus identifying endpoints useful for future validation in more complex systems.
Non-Technical Summary: Agricultural workers and their family members are exposed many neurotoxic and potentially neurotoxic chemicals, such as insecticides, rodenticides, fungicides, heavy metals, and organic solvents. Some chemicals that were previously recognized as developmental neurotoxicants are now suspected factors in the onset of neurodegenerative diseases in aging. Many potentially neurotoxic chemicals or their mixtures have not been tested for neurotoxicity because of costs and the number of animals needed for testing under current guidelines. Therefore, toxicologists are interested in improved methods for screening potential neurotoxicants and their mixtures. Reliable cell culture systems might greatly accelerate the screening of agricultural chemicals for neurotoxicity and the rate at which mechanistic and therapeutic data are generated. What is lacking is a battery of valid, common core endpoints that can be measured in cell cultures. These endpoints must accurately detect the neurotoxic chemicals without false negatives and must detect subtle abnormalities in cell function, not just cell death. One can measure a host of diagnostic endpoints in an apparently healthy person, such as blood pressure, to determine underlying illness. Analogous endpoints are needed in cell cultures. The proposed work seeks to develop a very sensitive screening system for cellular neurotoxicity of known and novel chemicals that would provide a major new direction for their investigation. The tools developed will have applicability to high throughput organ- and tissue-level assessment of cellular responses to new or untested chemicals. <P> Approach: Common core endpoints will be sought in two astrocyte-like glial cell lines, two neuronal cell lines, and rat primary astrocyte and primary neuronal cultures tested with well-known neurotoxic agricultural chemicals, such as paraquat and parathion, as well as fluoroaceate as a positive control, and carmustine as a negative control. Two glial (rat C6 and human CCF-STTG1) cells lines and rat primary astrocytes cell lines will be tested through various stages of chemical- or hormonal-induced differentiation. Similarly two neuronal (rat PC12 and human SY5Y) cell lines will be tested through various stage of differentiation. The cells will be exposed for up to 7 day to test substances, and functional endpoints of toxicity will be measured by highly sensitive imaging assays. The positive control will be tested to demonstrate that the cell cultures are responding with sufficient sensitivity to the potential neurotoxic agent. We will employ a panel of cytotoxicity tests to establish sublethal concentrations and exposure regimens and then test the chemicals at sub-cytotoxic concentrations with a panel of much more sensitive mechanistic assays for cell injury. The mechanistic assays will comprise a comprehensive assessment of cell function in living cells with imaging tools for examining mechanisms of toxicant action at the cell level. Endpoints analyzed will be Ca2+ homeostasis, indicators of stress, and measurements of homeostatic responses to toxicant exposure.