There is increasing concern that perinatal exposure to organophosphate acetylcholinesterase (AChE) inhibitors (OPs) may cause cognitive problems in children. This concern is based on recent reports of widespread exposure of the public to OPs, and on animal studies indicating that the developing nervous system is more sensitive than the mature nervous system to the neurotoxic effects of OPs. It is reported that OPs cause developmental neurotoxicity at doses significantly below those that inhibit the catalytic activity of AChE. These data have been widely interpreted to mean that OPs target molecules other than AChE. However, recent evidence demonstrating that AChE functions to promote axonal growth in the developing but not mature neurons suggest an alternative explanation: OPs target AChE, but the mechanism of developmental neurotoxicity involves disruption of the morphogenic function of AChE not inhibition of its catalytic activity. Interference with the axon- promoting activity of AChE represents a biologically plausible mechanism for explaining the functional deficits observed in animals exposed perinatally to OPs. The goal of this proposal is to test the hypothesis that OPs disrupt axonal growth by interfering with the morphogenic activity of AChE.
The specific aims are to: (1) Validate dorsal root ganglion (DRG) neurons as a model system for mechanistic studies of OP-induced axonal dysmor- phogenesis; (2) Determine if AChE mediates OP effects on axonal growth; and (3) Determine if OPs alter axonal and dendritic morphogenesis in primary cultures of hippocampal and cortical neurons. Neurons cultured from AChE wildtype (+/+) and AChE null (-/-) mice will be exposed to varying levels of chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and 3,5,6-trichloro-2-pyridnol (TCP). The number, length, and branching patterns of axons and dendrites will be quantified using Metamorph imaging software following immunocytochemical localization of axon- or dendrite- specific antigens. AChE enzymatic activity will be measured using a standard colorimetric assay; cell viability, by assessing protein and DNA synthesis and uptake of vital dyes. Direct interactions between OPs and AChE will be assessed by determining if OPs inhibit adhesion of AChE coated microspheres to tissue sections of developing cerebral cortex from and by autoradiography of proteins immunoprecipitated by AChE antibody from cultured DRG neurons exposed to radiolabeled CPF. These data will not only provide mechanistic insight into the developmental neurotoxicity of OPs, but also suggest a biological basis for the increased vulnerability of the developing nervous system. Impacts of these studies include the reduction of uncertainty in risk assessment, the development and evaluation of biomarkers for OP toxicity in children, and the design of in vitro assays for screening the developmental neurotoxicity of other OPs of concern.
