Fathead minnow. Photo credit: USGS Photo Library Archive

The herbicide atrazine is widely applied in farming areas and detected in surface waters of the Midwestern United States, and has been implicated in reproductive dysfunction of exposed organisms. We observed decreased egg production in Japanese medaka fish and fathead minnows during 35-42 day exposures to 0.5, 5, and 50 μg/L atrazine. This study evaluated the molecular mechanisms underlying the loss in egg production in these fish. We measured gene expression of a series of biomarkers in steroidogenesis pathways and the hypothalamus-pituitary-gonad axis of male and female fish. We observed a shift in the population of females in an active reproductive state with atrazine exposure. Females with low vitellogenin (Vtg) expression also had low expression of other genes involved in reproduction, including steroidogenesis genes in the gonad, estrogen receptor and vitellogenin in the liver, and gonadotropins in the brain. In the medaka, the number of females in a tank that fell into the high Vtg category was significantly correlated with higher egg production. The number of females in the low vitellogenin category increased with atrazine exposure. Thus, the decline in egg production observed in response to atrazine exposure may be the result of a general downregulation of genes required for reproduction in a subpopulation of exposed females.

For more information contact Donald Tillitt, Columbia Environmental Research Center.

Atlantic salmon parr. Photo credit: E. Peter Steenstra, courtesy of USFWS National Digital Library

Two-color microarray enlarged to show detail. Photo credit: L. Robertson, Genomics Lab, Aquatic Ecology Branch, Leetown Science Center. Larger view

The transformation of Atlantic salmon from parr (adapted to freshwater) to smolt (adapted to seawater) is a complicated process that involves numerous physiological changes.  Exposure to environmental contaminants like the estrogen mimic nonylphenol can disrupt smolt development and may be a contributing factor in salmon population declines.  We are using microarray analysis to investigate the parr-smolt transformation and to assess the possible disruption of this transformation by nonylphenol.

For more information contact Laura S. Robertson, Leetown Science Center.

Largemouth bass. Photo credit: Eric Engbretson, courtesy of USFWS National Digital Library

The coincidence of intersex and fish lesions in smallmouth bass in the Potomac and Shenandoah Rivers suggests a putative relationship between the presence of estrogenic endocrine-disrupting compounds (EEDCs), endocrine disruption, and altered immune function. Estrogen or EEDCs, which may cause intersex in smallmouth bass, may also interfere with the expression of genes involved in innate immunity, thus increasing susceptibility of fish to bacterial infection. Using real-time PCR, we are investigating the effects of estrogen and EEDCs on the expression of genes involved in immunity.

For more information contact Laura S. Robertson, Leetown Science Center.

One-color microarray. Photo credit: Barbara Carter, EcoArray Inc., Gainesville FL

Microarray analysis allows measurement of the expression of thousands of genes at once, leading to the development of gene expression profiles associated with different physiological states.  To help realize the potential benefits of microarray analysis to environmental toxicology, we are conducting microarray analysis of the gene expression profiles induced in fathead minnow and largemouth bass tissues by acute exposure to a variety of contaminants with different modes of action.  Contaminants to be studied include metals, pesticides, endocrine disruptors, organochlorines, and surfactants.  By comparing the common and unique properties of each gene expression profile, we will gain understanding of distinct aspects of the modes of action of these classes of contaminants.  The ultimate goal of this line of research is to use gene expression profiling in the field to identify specific contaminant exposures causing adverse effects in natural populations.

For more information contact Catherine A. Richter, Columbia Environmental Research Center.

Largemouth bass. Photo credit: USGS Picturing Science

Chemical disruption of endocrine systems in fish has been a topic of intense public interest over the past two decades.  As a consequence of more intense research and monitoring, biologists have documented endocrine and reproductive dysfunction in wild populations of fish and wildlife across the country and across the world.  In parallel, environmental chemists have documented the presence of endocrine disrupting chemicals (EDCs) in both ground and surface waters of the US.  Through these efforts, EDCs have been observed in numerous locations, associated with municipal wastewaters, industry, agriculture, animal production operations, and urbanization.  Measures of endocrine function in fish and EDCs in aquatic environments have both been important to document the potential for endocrine disruption in fish (and wildlife) from chemicals.  Yet, the presence of these chemicals and the presence of endocrine-related disruption in fish, even together, do not provide the required evidence to establish causal linkages among these factors. The studies in this work unit will begin to assess the biological mechanisms for the development of EDC-induced intersex in fish.  The specific objectives for the studies are: 1) Induce intersex in largemouth bass with an endocrine disrupting chemical; 2) Monitor the development of the intersex in the gonads of the LMB over a chronic exposure period through histological examinations; 3) Measure global gene expression profiles in target tissues of the HPG-axis with a LMB-specific DNA microarray; 4)  Select gene expression biomarkers indicative of the genotypic and phenotypic changes observed in intersex in largemouth bass; and 5)  Develop testable hypotheses for potential biochemical pathways and cellular mechanisms leading to the condition of intersex in largemouth bass.

For more information contact Donald Tillitt, Columbia Environmental Research Center.

Zebrafish. Photo credit: ZFIN and Oregon Zebrafish Laboratories

Mercury is a widespread contaminant in aquatic systems throughout the United States, due to direct inputs and atmospheric deposition.  In aquatic systems, elemental mercury is transformed to methylmercury, enhancing its toxicity to the brain and reproductive system.  We are conducting three related investigations of functional genomics of methylmercury exposure in fish, using goldfish, fathead minnow, and zebrafish, and defining gene expression profiles in the brain, liver, and gonad.

Agarose gel electrophoresis of zebrafish genes for biomarker development. Photo credit: Marie Pope, USGS Columbia Environmental Research Center. Larger view

Gene expression profiles were analyzed in liver and gonad of male fathead minnow in response to acute and chronic exposure to environmentally relevant doses of methylmercury.  In both tissues, genes involved in apoptosis and the immune system were altered, suggesting that these processes may mediate the toxicity of methylmercury.  A manuscript describing this work has been published in the Journal of Fish Biology: Klaper, R., Carter, B.J., Richter, C.A., Drevnick, P.E., Sandheinrich, M.B., Tillitt, D.E., 2008. Use of a 15 k gene microarray to determine gene expression changes in response to acute and chronic methylmercury exposure in the fathead minnow Pimephales promelas Rafinesque. J. Fish Biol. 72, 2207-2280.

              We have completed microarray analysis of gene expression profiles in brain of female zebrafish in response to an acute exposure to an environmentally relevant dose of methylmercury and are beginning quantitative PCR validation of the results.  Our results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of methylmercury in the female brain.  A manuscript describing this work is in preparation for submission to Environmental Toxicology and Chemistry.

These studies have provided mechanistic insights into the toxicity of methylmercury and will allow comparisons of the gene expression profile induced in response to methylmercury with that induced by other toxins with similar and distinct modes of action, as well as development of responsive genes as biomarkers of methylmercury exposure.

For more information contact Catherine A. Richter, Columbia Environmental Research Center.

Brown bullhead adherent anterior kidney leukocytes. Photo credit: Luke Iwanowicz, Leetown Science Center, Fish Health Branch

The presence of endocrine disrupting compounds and other emerging contaminants in local rivers and streams has become a contentious issue of escalating interest during recent years. It is now clear that myriad chemical compounds manufactured and applied for pharmaceutical, agricultural and other purposes transit to local watersheds and exist detectable concentrations. While significant advances in chemical analyses permit the detection of hundreds of contaminants present at low concentrations, such analyses cannot predict their biological significance. This is particularly the case in the instance of chemical mixtures. Not surprisingly, chemical contaminants tend to exist as mixtures in locations of aquatic environmental contamination. These mixtures may have relevant biological effects on hormone systems, immune function, or other physiological processes that require biological screening approaches to resolve.

Currently research is in progress to assess the biological effects of aquatic environmental contaminants using in vitro screening platforms.  This work is an ongoing collaboration with the USGS Water Resources, Toxic Substances Hydrology Program and will help assign biological relevance to environmentally relevant chemical mixtures. Water samples have been collected from multiple sites in the Chesapeake Bay watershed and out-of basin-reference sites using polar organic chemical integrative samplers (POCIS). These are sites from which centrarchids have been identified with a low to high incidence of intersex (1). Fish kills have been reported at some of these sites as well and immune suppression in these populations has been suggested (2).  Assays in development will assess endocrine disrupting potential, aryl hydrocarbon receptor activation, and immunotoxicity using mRNA expression, protein synthesis and functional responses as end-points

(1) Blazer,V.S., L.R. Iwanowicz, D.D. Iwanowicz, D.R. Smith, J.A. Young, J. Hedrick, S. W. Foster, S. J. Reeser. (2007) Intersex (Testicular Oocytes) in Smallmouth Bass Micropterus dolomieu From the Potomac River and Selected Nearby Drainages.  Journal of Aquatic Animal Health. 19: 242-253.

(2) Ripley, J.L, L.R. Iwanowicz, V.S. Blazer and C.M. Foran. (2008) Utilization of Protein Profiles as Indicators of Environmental Impairment of Smallmouth Bass (Micropterus dolomieu) from the Shenandoah River, VA, USA.  Environmental Toxicology and Chemistry. 27: 1756-1767.

For more information contact Luke R. Iwanowicz, Leetown Science Center, Fish Health Branch.

Standard curve for real-time PCR. Photo credit: L. Robertson, Genomics Lab, Aquatic Ecology Branch, Leetown Science Center. Larger view

TGF-beta is an immune gene whose expression is used as a marker for stress.  We are using real-time PCR assays to measure TGF-beta expression in several different species of fish, which will be used to monitor stress in fish at different areas of concern. 

For more information contact Laura S. Robertson, Leetown Science Center, and Chris Ottinger, Leetown Science Center.