Citation:

BULMAN, A., D. C. BENCIC, A. D. BIALES, R. W. FLICK, G. ROSS, AND D. L. LATTIER. SELDI PROTEINCHIP-BASED LIVER BIOMARKERS IN FUNGICIDE EXPOSED ZEBRAFISH. Presented at Society of Toxicology, Seattle, WA, March 16 - 20, 2008.

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 research presented here is part of a three-phased small fish computational toxicology project using a combination of 1) whole organism endpoints, 2) genomic, proteomic, and metabolomic approaches, and 3) computational modeling to (a) identify new molecular biomarkers of exposure to endocrine disrupting compounds (EDCs) representing several modes of action (MOA) and (b) link these biomarkers to effects that are relevant for both diagnostic and predictive risk assessments. The effects of prochloraz (PCZ) on reproductively mature male and female zebrafish were examined following 48 h continuous exposure in a flow-through system to control (water only), low (100 µg/l), and high (500 µg/l) doses. PCZ is an imidazole fungicide used to protect food crops by weakening fungal cell membranes through CYP51 inhibition. PCZ is known to have a mixed MOA in vertebrates, functioning as an aromatase (CYP19) inhibitor as well as an androgen receptor antagonist. Zebrafish liver lysates were profiled on three SELDI ProteinChip array types (weak cation exchange, strong anion exchange, and metal affinity), focusing on the 1.5 kDa to 25 kDa mass range. Over 300 individual m/z peak clusters were analyzed across the six sample groups (n=8 each). Gender related protein expression differences (p<0.05) were observed, with a majority of these markers exhibiting higher expression in males. Both up- and down-regulated markers were detected upon PCZ exposure. PCZ response in the females was observed primarily as up-regulation of candidate markers; in contrast, considerably more changes, primarily down-regulation of candidate markers, were observed in males. These differential expression profiles will be used to help identify new molecular biomarkers of exposure and integration of these data with whole organism endpoints will provide more definitive description of the mixed MOA of PCZ.

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