R3 AM Ecotoxicogenomics of Emerging Chemical Issues
Thursday, 17 November 2005: 8:00 AM - 11:40 AM in Ballroom 3

(POY-1117-845548) The Explosion of Ecotoxicogenomics: Effects of ORCs on Gene Expression in Daphnia magna.

Poynton, H¹, Kennedy, A², Guan, X², Steevens, J³, Varshavsky, J¹, Holman, P¹, Loguinov, A¹, Perkins, E³, Vulpe, C¹, ¹ UC Berkeley, Berkeley, CA, USA² Analytical Services, Inc., Vicksburg, MS, USA³ US Army Corps of Engineers, Vicksburg, MS, USA

ABSTRACT- Ongoing and historical use of ordnance by the Army has resulted in the unintentional release of ordnance related compounds (ORCs) in soil, surface water, and ground water. Current methods of on-site monitoring of ORC contamination are limited by high detection limits, low specificity, and the lack of quantitative measurements. Gene expression profiling may overcome these limitations by providing enhanced sensitivity and specificity. To illustrate the potential of gene expression profiling for detecting ORCs in the environment, we exposed Daphnia magna to 2,4,6-trinitrotoluene (1.19 mg/L) its degradation products, 2-amino dinitrotoluene (0.60 mg/L) and 4-amino dinitrotoluene (0.89 mgl/L), and cyclotrimethylenetrinitramine (RDX) (30 mg/L). Exposure concentrations were selected at 1/10 the LC50 determined by literature searches and preliminary acute toxicity assays. RNA extracted following 24-h exposures was hybridized to a 5000 Daphnia magna cDNA microarray. Gene expression data suggests that RDX affected oxidative stress related genes (glutamine synthetase-like gene), defense response (cuticle proteins, collagenase, chitinase), protein synthesis (elongation factor alpha), proteases (chymotrypsin, carboxypeptidase), and fatty acid metabolism (acyl-Coenzyme A dehydrogenase). These responses were generally distinct from expression patterns for copper, cadmium, and zinc. Zinc and RDX expression patterns overlapped in up regulation of the proteases chymotrypsin and carboxypeptidase. However, as a chemical class, the metals caused a down regulation of digestive enzymes while RDX impacts a broader array of functions. Several genes confirmed by real-time PCR to be affected by metal exposure (glutathione-S-transferase, metallothionein, and ferritin) were not affected by exposure to RDX by microarray analysis. We will discuss the differences and similarities in gene expression following exposure to the different ORCs, and compare them with the expression profiles for copper, cadmium, and zinc.

Key words: gene expression profiling, cDNA microarray, TNT, RDX