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DIFFERENTIAL DISPLAY OF TRENBOLONE AND DEHP INDUCED GENE TRANSCRIPT PATTERNS IN FATHEAD MINNOW LIVER
Citation:
Reddy, T V., A L. Miracle, R Flick, J M. Lazorchak, D L. Lattier, G P. Toth, AND M E. Smith. DIFFERENTIAL DISPLAY OF TRENBOLONE AND DEHP INDUCED GENE TRANSCRIPT PATTERNS IN FATHEAD MINNOW LIVER. Presented at Society of Environmental Toxicology and Chemistry, Salt Lake City, UT, November 18-22, 2002.
Impact/Purpose:
The indeterminate condition of exposure indicator research stands to change markedly with the ability to connect molecular biological technologies with cellular or tissue effects and outcomes. Three focal areas of ecological research aim to develop a sequence of approaches where "the earliest recognizable signatures of exposure" (i.e., unique patterns of up- and down-regulated genes and proteins) are identified for numerous stressors, demonstrable in case studies and incorporated into Agency, State and Regional studies supported by EMAP and other programs.
Area 1, Computational Toxicology Research: Exposure assessment has historically been based on use of chemical analysis data to generate exposure models. While biological activity of chemicals has been recognized to be important for exposure risk assessments, measurement of such activity has been limited to whole organism toxicity tests. Use of molecular approaches will:
improve extrapolation between components of source-to-outcome continuum (source , exposure , dose , effect , outcome)
Using a systems modeling approach, gene and protein expression data, in small fish models (fathead minnow and zebrafish), will be integrated with metabolomic and histopathological data. This will assist in prediction of environmental transformation and chemical effects based on structural characteristics, and enhance quantitative risk assessments, including areas of uncertainty such as a basis for extrapolation of effects of endocrine disrupting chemicals, interspecies extrapolation, complex chemical mixtures and dose-response assessment.
Area 2, Ecological Research-Environmental Diagnostics: Development of molecular diagnostic indicators contributes to several of the GPRA Diagnostic Research Goals. Methods will employ DNA microarray technology and expression proteomics, focusing on species of relevance to aquatic ecosystem risk assessment. Significantly, these diagnostic indicators will open the door to understanding subcellular interactions resulting from exposure to complex chemical mixtures.
define relationship between genetic disposition of populations and degree/specificity of stressor-specific gene transcriptional response in aquatic organisms (fish and invertebrates)
identify of chemical mixture induced transcriptional "patterns" using microarrays and hyperspectral scanning - via collaboration with DOE Sandia National Labs
apply molecular indicators to watershed level stressor study, including pilot studies with targeted pesticides and toxins indicators
develop molecular indicators of exposure for invertebrates (Daphnia, Lumbriculus, Chironomus)
Area 3, Exposure Research in Endocrine Disruptors:
Subobjective 1: Develop exposure methods, measurement protocols, and models for assessment of risk management practices of endocrine disrupting compounds. As risk management approaches are identified and developed, there will be a need to identify, adapt and develop bioassay screening tools and other analytical methods to assess their efficacy. Measurements research will be performed to define management needs. This effort will entail cross-lab participation from NRMRL, NERL and NHEERL.
Subobjective 2: Determine extent of environmental and human exposures to EDCs, characterize sources and factors influencing these exposures, develop and evaluate risk management strategies to reduce exposures. In order to develop effective risk management strategies, it is important to understand the extent of exposures to endocrine disrupting compounds and factors influencing source-to-exposure-to-dose relationships.
apply molecular indicators of exposure to estrogenic compounds in selected wastewater treatment plants located in ten USEPA Regions
identify differential gene expression following exposure of fathead minnows to environmental androgens and androgen-like compounds
apply molecular indicators of exposu
Description:
The endocrine disruptor risk assessment process is being delayed without more robust data on the estrogenic and androgenic activity of chemicals found in the environment such as trenbolone and di(2-ethylhexyl) phthalate (DEHP). Trenbolone is an androgenic compound known to reduce vitellogenin (Vtg) blood protein levels in female fathead minnows. Our preliminary results showed that the plasticizer di (2-ethylhexyl) phthalate (DEHP) induced vitellogenin gene transcription in the livers of adult male fathead minnows (Pimephales Promelas). In this study, we used fluoro differential display to investigate the global gene expression pattern in the liver of male and female fathead minnows exposed to trenbolone or DEHP. Adult (8-10 months old) male and female fathead minnows were continuously exposed to moderately hard reconstituted water (MHRW) as control, and MHRW with 50 ppt trenbolone or 10 ppb of DEHP, for one week. Total liver RNA was isolated, and reverse transcribed using an anchored oligo-dT primer. The cDNAs were subsequently amplified in a PCR reaction using fluorescent TMR-labeled anchored oligo-dT primers, in conjunction with four separate arbitrary decamers. The PCR products were separated on a 5.6% polyacrylamide gel, and visualized with a fluorescent scanner. Several bands were identified that exhibited differential expression among fathead minnows exposed to trenbolone , DEHP and controls. These bands were excised from the gel, and the cDNAs were eluted into buffer, re-amplified, and sequenced using an MJ Research BaseStation sequencer. Acquired nucleotide sequences were compared to sequences in GenBank using Blastn and Blastx programs to identify fathead minnow gene homologs.