Superfund Research Program
Zebrafish as a Detector and Discriminator of Organophosphate Exposure
Dr. Linney and his research team’s Superfund work on chlorpyrifos exposure of zebrafish has progressed in several distinct and interrelated directions: They have a growing amount of circumstantial evidence that chlorpyrifos exposure is inhibiting acetylcholine esterase and this is resulting in later learning defects in adult fish (this being done in collaboration with Dr. Levin's laboratory—as such the researchers are trying to determine if the target for this effect is specific to muscarinic or nicotinic acetylcholine receptors). As an attempt to determine whether they can detect behavioral or motility effects in larvae that parallel the adult learning defects the research team has been working with the Noldus Ethovision video tracking computer interrogation system to evaluate motility differences correlating with exposures. Their preliminary results with this system suggests that certain motility changes appear to track with chlorpyrifos exposure and that exposing embryos to a muscarinic agonist shows at least one of these tracking differences. 1) The research team is now exploring whether the adult learning defects might be due to a specific effect upon muscarinic receptors. 2) Using real-time, quantitative PCR they have identified mRNA corresponding to a choline cetyltransferase gene in the first 3 days of embryonic development—using these sequences they have had a BAC library screened for large genomic clones containing this gene so that they can use its sequences to drive fluorescent reporters so that they can identify, in real-time, the location and development of neurons producing acetylcholine as a neurotransmitter—this would allow the researchers to visualize events occurring during these exposure studies.
In keeping with the renewal applications goals, Project investigators are setting up to preliminarily examine the effects of two other organophosphate pesticides upon zebrafish development. They will be exposing zebrafish with diazonin and parathion for preliminary behavioral studies by the Levin laboratory. At the present time most of their efforts continue to be focused upon chlorpyrifos exposures since they believe this is the most efficient way in which they might determine mechanism.
The focused advances that have been made are associated with: 1) the increased use of the Noldus Ethovision evaluative tool for behavioral differences imposed by pesticide exposure; 2) the increased use of the zebrafish genomic information for the examination of the genes involved in these processes and the design of a new family of transgenic reporters. In this next year the research team expects to make use of the Agilent 22k zebrafish array for examining pathways affected by these organophosphate exposures.