MP7 Toxicogenomics in Environmental Studies
Monday, 14 November 2005: 8:00 AM - 5:30 PM in Exhibit Hall

(GON-1117-838737) Microarray Analysis of 2,4,6 Trinitrotoluene Exposure Effects in the Earthworm Eisenia fetida.

Guan, X¹, Gong, Ping¹, Inouye, L², Indest, Karl², Perkins, E², ¹ Analytical Services, Inc., Vicksburg, MS, USA² US Army ERDC, Vicksburg, MS, USA

ABSTRACT- Current and historical training activities of the U.S. Army have resulted in the release of munitions such as 2,4,6-trinitrotoluene (TNT) into the environment that may adversely impact soil organisms. We have developed an Eisenia fetida cDNA microarray and used it to assess the impact of TNT contaminated soil on E.fetida gene expression. The cDNA microarray was constructed from a cDNA library enriched for genes affected by cadmium, TNT, 2,6-Dinitrotoluene, HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). 2200 randomly picked cDNAs were spotted onto glass slides. To assess the impact of different durations of TNT exposure, time course experiments were performed at 4-d, 14-d or 28-d at 0 (control) or 100 mg/kg of TNT, a previously determined lowest observable lethal effect level for E. fetida. Three individual worm replicates were analyzed at each time point and exposure concentration in a loop design experiment. Twenty nine cDNAs were identified as significantly affected by TNT exposure. A putative mitogen-activated protein kinase involved in signal transduction was induced at 4-d. At 4-d and 14-d, exposure resulted in down regulation of genes involved in cellular protein degradation (ubiquitin activating enzyme) and oxygen transport (hemoglobin linker chain, hemoglobin subunit B2, hemoglobin chain d1) in addition to several unknown genes. After 28-d of exposure, genes are induced involved in cellular protein degradation (ubiquitin activating enzyme, lumbrokinase-3), lysosomal degradation (beta-hexosaminidase), and several unknown genes. At 28-d, down regulated genes included a putative transcription factor, a non-heme-iron oxygen transport protein, and a trichohyalin-similar gene. Lasting effects on hemoglobin genes were not seen. These data suggest that TNT inhibits oxygen transport systems at short periods of exposure, with longer exposures resulting in significant damage to cellular proteins and macromolecules.

Key words: Microarray, Earthworm Eisenia fetida, Gene expression, Biomarker