The development and characterization of fish gene expression bioassays for detecting aquatic endocrine disruptors and other emerging contaminants

• Main Author: Moffatt, Lauren T
• Language: ENG
• Published: ScholarWorks@UMass Amherst 2008
• Subjects: Molecular biology|Environmental science
• Online Access: https://scholarworks.umass.edu/dissertations/AAI3325132

As availability of clean water resources decreases globally, evaluating water quality sensitively and reliably is becoming critical. Emerging contaminants, especially those categorized as endocrine disruptors, are of concern because of limited knowledge regarding non-target effects. Quantifying changes in the expression of vitellogenin, a gene normally expressed only in female fish but known to be induced in male fish exposed to estrogens, is a sensitive biomarker of estrogenic contamination in water. However, the utility and limitations of vitellogenin-gene-induction as a reliable bioassay are poorly defined. The work described here characterizes the assay by examining its sensitivity to experimental variables. After exposure to low levels of estradiol, two species demonstrated robust time- and concentration-dependent induction of vitellogenin mRNA. Exposure to a concentration gradient of environmental estrogens demonstrated that the Japanese medaka bioassay detects low, environmentally relevant levels of ethynylestradiol in a short laboratory assay, but does not detect low concentrations of a weakly estrogenic contaminant, bisphenol A. Although the expression of other genes was altered, the changes were not as robust as the vitellogenin response. The robustness of the vitellogenin bioassay was further investigated by manipulating the volume of test-water, the length of time between exposure and assessment, housing conditions, and access to food. The volume of test-water had no effect on the bioassay as all groups exposed to 100pM of estradiol expressed similar levels of vitellogenin. Furthermore, estradiol-induced vitellogenin levels remained high even after 72 hours of depuration in clean water and despite 72 hours of food deprivation while confined in field cages. These results demonstrate that the vitellogenin response is robust, reliable, and resistant to stresses associated with field applications. The effects of diclofenac, a pharmaceutical frequently detected in the environment, were assessed with the vitellogenin bioassay and cDNA microarray. Analyses revealed no robust differential regulation of gene expression and suggested that previous reports as to mechanisms of the drugs pathology are likely misleading. The information gained from this work forms a foundation of knowledge on gene expression bioassays in fish that will help guide implementation of the assays and interpretation of their data in a useful and appropriate way.