Date of Award

12-2011

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Ecology and Environmental Sciences

Advisor

Adria A. Elskus

Second Committee Member

Julie Gosse

Third Committee Member

Michael Kinnison

Abstract

There is great need for non-lethal, biologically relevant screening tools to assess the effects of surface water contaminants on threatened or endangered fish species, as typical screening procedures, such as liver sampling and skin plugs, are lethal or highly invasive. Evidence suggests that fish scales biochemically respond to a range of contaminants and, therefore, could serve as non-lethal, rapid biosensors of fish exposure to contaminants. Cytochrome P4501A (CYP1A) is a monooxygenase biomarker commonly measured in organisms to determine exposure to organic contaminants, and evidence suggests that CYP1A is expressed in bone cells (and fish scales). Vitellogenin β (VtgB) is a glycoprotein biomarker commonly measured in organisms to determine exposure to estrogenic contaminants, and evidence suggests that VtgB is expressed in fish scales. We were interested in determining the location of CYP1A protein in and around the fish scales. We were also interested in establishing if scale CYP1A and VtgB would respond to contaminant exposures. We aqueously exposed Atlantic salmon (Salmo salar) parr for 48-hours to 40 ppb acetone, left them untreated, or exposed them to 0.327, 3.27, or 32.7 ppb 3,4,3',4',5-pentachlorobiphenyl (PCB126), to 0.01, 0.1, or 34 ppb fluoxetine hydrochloride (FLU), to 27.2 or 330 ppb β-naphthoflavone (BNF) to assess responses to organic contaminants. We also aqueously exposed S. salar parr for 96-hours to 40 ppb ethanol-treated or untreated controls or to 10,000 ppb 17α-ethinylestradiol (EE2) to assess responses to an estrogenic contaminant. Regarding CYP1A protein location, we hypothesized that the CYP1A protein is expressed in scale osteoblasts (bone-forming cells). Regarding scale biomarker response to contaminant exposure, we hypothesized that 1) fish scale CYP1A mRNA would be induced by PCB126, BNF, and FLU, and 2) fish scale VtgB mRNA would be induced by EE2.

Through immunohistochemical analysis of skin-scale sections using CYP1A-specific MAb 1-12-3, we observed CYP1A protein location in skin-scale and liver samples from contaminant-treated parr. We demonstrate that the CYP1A protein is expressed and induced in skin-scales (i.e., the epithelium around the scale and the dermal, fat, and muscle layers) of 3.27 and 32.7 ppb PCB126-treated and 330 ppb BNF-treated S. salar parr. We also demonstrate that CYP1A is expressed and significantly induced in liver cells and vasculature from treated fish. We observed CYP1A protein expression in osteoblasts, but determined that CYP1A expression among the osteoblasts is impossible to accurately quantify due to potential artifact staining. We conclude that the majority of CYP1A expression measured in fish scales comes from the epithelial covering surrounding scales.

Through real-time quantitative PCR (qPCR), we measured mRNA expression levels in scale samples (i.e., the epithelial covering around the scale) from the contaminant-treated parr. We measured CYP1A mRNA to determine exposure to organic contaminants, in scale samples from PCB126-, BNF-, and FLU-exposed fish, and we measured VtgB mRNA in scale samples from EE2-exposed fish. We demonstrate that CYP1A mRNA is significantly induced in scale tissues of 32.7 ppb PCB126- and 27.2 and 330 ppb BNF-treated parr, but was not significantly induced in scale tissues of FLU-treated parr at any dose. We also demonstrate that VtgB mRNA is not significantly induced in scale tissues of Atlantic salmon parr aqueously exposed for 96-hours to EE2. However, we confirmed that induction of VtgB did occur in the livers of EE2-exposed parr. We conclude that it is plausible that fish scales could serve as non-lethal, rapid biosensors of some surface water contaminants, but that more research needs to be conducted to evaluate other possible biomarkers and assess biomarker response in contaminant mixtures, before fish scale biosensors can be utilized in the field.

From our research, fish scales show promise as non-lethal, rapid, and biologically relevant biosensors of fish response to contaminants, at least for organic compounds. However, further research needs to be conducted before a fish-scale contaminant screening assay is established. For example, additional biomarkers and biomarker induction in contaminant mixtures should both be investigated.

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