Citation:

LAZORCHAK, J. M., M. E. SMITH, D. L. LATTIER, T. V. REDDY, D. C. BENCIC, AND A. D. BIALES. INTRODUCTION OF THE VITELLOGENIN GENE IN EARLY LIFE STAGE FATHEAD MINNOWS AS AN EFFECTIVE EXPOSURE INDICATOR FOR ESTROGENIC COMPOUNDS. Presented at SETAC - North America, Milwaukee, WI, November 11 - 17, 2007.

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:

Vitellogenin (Vg) gene expression in adult male fathead minnows (FHM) has previously been used successfully to detect exposures to estrogenic compounds in aquatic systems; however, sample volume(s)required for >24h exposure durations and the logistics of sampling pose some limitations on the use of adult male fish. We have previously established that Vg is indeed expressed in larval FHM and responses were proportional to external concentration. In addition to the minimal experimental scale required to use larval fish, they are readily available, less labor intensive, economical and afford a greater number of experiments that can be performed. Early development is an exquisitely complex biologic sequence in which signaling interactions and changes in gene expression must be orchestrated in a precise spatial and temporal program to ensure that embryogenesis and pattern formation progress faithfully; therefore, a more advantageous rationale to using early developmental stages of teleosts is the would-be predictive biology arising from genomic analysis of ecological exposure. In an initial attempt to use developmental stages as a means to predict adverse outcome and develop consistent exposure biomarkers, we have developed a method for detecting exposure to estrogenic compounds in water samples using early life stage FHM. The objective set for the FHM early life stage estrogen exposure method was to establish an ideal age of larvae/juveniles that exhibited minimal variation in Vg gene expression, within groups exposed to estrogenic endocrine disrupting chemicals(EDCs)(EE2, E2, E1, and NP) and respond to the lowest levels at which adult male fathead minnows are induced to express Vg. To establish an ideal age, different stages of larvae(4,7,14,21,28,and 35 dph,in triplicate) were taken from the same larval pool. Exposures were carried out in 500 ml beakers and renewed daily. For controls, 500 ml beakers containing MHRW with the appropriate amount of DMSO (5 10 µl /L MHRW) were used. Fish were fed for 2 hours before initiation of exposure, with age adjusted amounts of newly hatched brine shrimp and exposed to test chemical(s) for 48 h. At 48 h, fish were fed again for 2 h and exposed to the respective test chemical for an additional 48h. Results will be presented on the various ages using nominal concentrations of EE2 2.5 ng/L, E2 40 ng/L, E1 60 ng/L and NP 50 ¿g/L.

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