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Site-specific impacts on gene expression and behavior in fathead minnows exposed to streams adjacent to STPs

ABSTRACT: Environmental monitoring for pharmaceuticals and endocrine disruptors in the aquatic environment traditionally employs a variety of methods including analytical chemistry, as well as a variety of histological and biochemical endpoints that correlate with the fish fitness. It is now clear that analytical chemistry alone is insufficient to identify aquatic environments that are compromised because these measurements do not identify the biologically available dose. The biological endpoints that are measured are important because they relate to known impairments, however, they are not specific to the contaminants and often focus on only a few known endpoints. These studies can be enhanced by looking more broadly at changes in gene expression, especially if the analysis focuses on biochemical pathways. The present study was designed to obtain additional information on well-characterized sites adjacent to sewage treatment plants in MN that are thought to be impacted by endocrine disruptors. Here we examine five sites that have been previously characterized and examine changes in gene expression in fathead minnows (Pimephales promelas) that have been caged for 48 h in each of the aquatic environments. We find that the gene expression changes are characteristic and unique at each of the five sites. Also, fish exposed to two of the sites, 7 and 12, display a more aggressive behavior compared to control fish. Our results show that short-term exposure to sewage treatment plant effluents was able to induce a site-specific gene expression pattern in the fathead minnow gonad and liver. The short-term exposure was also sufficient to affect fish sexual behavior. Our results also show that microarray analysis can be very useful for determining potential exposure to chemicals, and could be used routinely as a tool for environmental monitoring.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE16645 | GEO | 2009/06/19

SECONDARY ACCESSION(S): PRJNA116229

REPOSITORIES: GEO

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Project description:Diethylstilbestrol (DES) is a synthetic estrogen that has been banned for use in humans, but still is employed in livestock and aquaculture operations in some parts of the world. Detectable concentrations of DES in effluent and surface waters have been reported to range from slightly below 1 to greater than 10 ng/L. Little is known, however, concerning the toxicological potency of DES in fish. In this study, sexually-mature fathead minnows (Pimephales promelas) of both sexes were exposed to 1, 10 or 100 ng DES/L water in a flow-through system. Tissue concentrations of DES and changes in a number of estrogen-responsive endpoints, including alterations to the hepatic transcriptome in females, were measured in the fish at the end of a 4-d exposure, and after a 4-d depuration/recovery period in clean water. The objectives of the study were to measure accumulation of DES in fish tissues, characterize effects of the chemical on physiological endpoints related to reproductive performance, and to evaluate DES's potency relative to concentrations reported in aquatic environments. The current series includes n=11 microarrays associated with recovery-phase samples (collected from fish exposed continuously for 4 d, and then held in control water for an additional 4 days before tissues were collected). Fish were exposed to 0, 1, 10, or 100 ng DES/L delivered in a continuous flow (45 ml/min) of sand filtered, UV treated, Lake Superior Water, without the use of carrier solvents. Flow of DES into the test system was initiated without fish present and continued for 1 d. Exposures then were started by placing three male and three female sexually-mature fathead minnows into each tank. There were eight replicate tanks for the control and 100 ng/L treatments, and six replicate tanks for the 1 and 10 ng/L groups. The fish were held at 25+1M-BM-:C under a 16:8 L:D photoperiod and were fed frozen adult brine shrimp twice daily (San Francisco Bay Brand, Newark, CA, USA). Fish used for the experiment were from an on-site culture, and all procedures involving animals conformed to guidelines approved by the local Animal Care and Use Committee. After the 4-d exposure, fish from three tanks from each treatment (n=9 per sex) were sampled for determination of various biological endpoints. At this time fish from two additional tanks from the control and 100 ng/L treatments also were sampled for determination of DES tissue residues (n=6 per sex). Delivery of DES to the test system was subsequently stopped, and fish from the remaining three tanks per treatment group were sampled 4 d later (n= 9 per sex). The fathead minnows were euthanized with buffered MS-222 (Argent, Redmond, VA, USA) and weighed. Fish were scored for occurrence and relative expression of nuptial tubercles, which in males can be reduced by ER agonists. Blood was collected from the caudal vein/artery with a microhematocrit tube, and plasma was separated by centrifugation and stored at -80M-BM-:C. Gonads were removed from males and a subsample (8.5M-BM-13.0 mg; meanM-BM-1SD) was immediately immersed in tissue culture media for determination of ex vivo steroid production. Livers were removed from both sexes and flash-frozen for gene expression analyses using microarray (females) or real-time QPCR (males). Hepatic transcripts from three females per treatment group, in each phase of the experiment (except n=2 from recovery phase 10 ng/L) were analyzed using a custom 15,000 feature microarray (GEO Platform Accession GPL9248). Data sets for the exposure phase (n=12 microarrays) and recovery phase (n=11) were normalized independently using Fastlo (Ballman et al., 2004) implemented in R (http://www.r-project.org/), but analyzed using parallel approaches.

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Site-specific impacts on gene expression and behavior in fathead minnows exposed to streams adjacent to STPs

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