W3 AM Toxicogenomics in Environmental Studies
Wednesday, 16 November 2005: 8:00 AM - 11:40 AM in Ballroom 3

(RIS-1117-837421) Effects of chlorpyrifos exposures on juvenile rainbow trout gene expression and behavioral endpoint analyses.

Rise, M.¹, Rise, M.¹, Miller, S.¹, Schmoldt, A.¹, ¹ Great Lakes WATER Institute, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA

ABSTRACT- We develop tools to study biologically significant impacts of low levels of organophosphate pesticides (OPs) in water, and to resolve molecular mechanisms by which OPs can cause neurological and behavioral defects in vertebrates. OPs are widely used in U.S. and international agriculture. OPs exert their acutely toxic effects by inhibiting acetylcholinesterase, which is critical to the normal functioning of cholinergic synapses. A toxicogenomic approach was used to characterize response of the juvenile rainbow trout transcriptome to 48 hour sub-acute (1-100 ppb) waterborne chlorpyrifos exposure relative to vehicle control. A reference design microarray experiment, using 3600 gene GRASP cDNA microarrays with Genisphere Array 50 reagents and methods, identified candidate molecular biomarkers of chlorpyrifos exposure, including a novel HSP. Results were validated using quantitative RT-PCR (QPCR) on pooled samples. Subsequent QPCR of individual fish samples will provide an assessment of biological variability of biomarker expression. We created a suppression subtractive hybridization (SSH) cDNA library from chlorpyrifos-exposed and control tissues to identify novel trout genes responsive to chlorpyrifos. Rainbow trout sequences in this SSH library had significant (E-value < 1e-10) BLASTX hits against known vertebrate genes, including spinal cord-derived growth factor, annexin A5, translation factor sui1, activated protein kinase C receptor, beta crystalline B, lung cancer oncogene 7, and a novel HSP. Select SSH-identified genes will be validated by QPCR. In addition, we are developing methods for studying changes in behavior that are associated with chlorpyrifos exposure. We employ EthoVision, a video tracking system that enables automatic behavior recognition and analysis of locomotory tracks. Early results demonstrate that fish activity can be evaluated with this software. Behavioral profiling, and functional annotation of chlorpyrifos-responsive genes, will add detail to our understanding of potentially deleterious changes associated with OP exposure in fish, and provide biomarkers for assessing biological impacts of OPs on feral fish populations.

Key words: chlorpyrifos, microarray, rainbow trout, behavior