Superfund Research Program
Zebrafish as a Detector and Discriminator of Organophosphate Exposure
The project researchers' Superfund work strongly suggests that very early embryonic exposure to chlorpyrifos (Dursban) results in an inhibition of acetylcholinesterase and that this early inhibition results in adult fish with learning deficiencies (this behavioral work was done in collaboration with the Levin laboratory). The researchers have followed the logic that if inhibition of acetylcholinesterase is the cause of the later effects that chlorpyrifos is acting as an indirect agonist of the acetylcholine receptors. This past year the project researchers have explored whether this might be occurring through the nicotinic acetylcholine receptors, the muscarinic acetylcholine receptors or both. Using the Noldus-Ethovision video tracking-software device project researchers have preliminary data that the muscarinic receptors could be the target of this effect. Adult data from the Levin laboratory supports this.
While these experiments were being conducted, project researchers have started using two different technologies to investigate mechanism: 1) Agilent zebrafish 22k microarrays and 2) an embryo dissociation technique with transgenic embryos that are driven by a gap43 promoter so that only neurons are fluorescent. This technique has allowed the researchers to fluorescently cell sort neurons and then run microarrays on the neurons.
Both of these new approaches have directed project researchers towards designing their own, focused microarray of approximately 500 genes. The researchers have found that a substantial number of neurotransmitter pathway genes are missing from the Agilent 22k zebrafish array so the researchers have selected genes specific to neurotransmitter pathways, genes that are enriched in the flourescent sorted neurons, and genes known to place a role in zebrafish neuronal development and are having Operon design and synthesize 70 mer oligos so that the researchers can make and use a more specific microarray system. The balance of using both the Agilent 22k array and this more specific array should allow the researchers to acquire valuable information about the transcriptional events occurring during organophosphate exposure.
At the present time project researchers are just beginning to evaluate a 40 microarray chlorpyrifos experiment and in January they will begin collecting samples from diazinon treated embryos because they have some data from the Levin laboratory from adult fish that they exposed to diazinon as embryos that indicates a behavioral effect.