Assessing DNA Damage in Hemocytes of the Freshwater Mussel Elliptio complanata with the Comet Assay

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Title: Assessing DNA Damage in Hemocytes of the Freshwater Mussel Elliptio complanata with the Comet Assay
Author: Prochazka, Sharon Tencza
Advisors: W. Gregory Cope, Committee Chair
Heather M. Cheshire, Committee Member
Leslie Recio, Committee Member
David B. Buchwalter, Committee Member
Abstract: The single cell gel electrophoresis or comet assay is widely used to detect DNA damage following exposure to genotoxic compounds with cells isolated from tissue or circulatory fluids from various organisms. The cell sampling method can often lead to the fatality of the test organism, depending on the type of tissue sampled. The objective of this study was to assess genotoxicity with the comet assay from in vitro and in vivo studies using a non-lethal hemolymph sampling method on Elliptio complanata, a native freshwater mussel found abundantly in North Carolina. The hemolymph was withdrawn from the anterior adductor muscle sinus and exposed in vitro to various concentrations (previously tested for cell viability) of selected pesticide formulations, Aatrex 4L herbicide (atrazine), Roundup herbicide (glyphosate), Lorsban 4E insecticide (chlorpyrifos), and Thionex 3EC insecticide (endosulfan), technical grade copper sulfate and a mixture of 46 different PAHs for 4-hours at 4°C. After exposure, the hemocytes were isolated and the comet assay was performed on the treated cells, control cells, and positive control cells exposed to 160 uM hydrogen peroxide (H2O2). For quality assurance purposes, CometAssay Control Cells™ were analyzed throughout each comet assay procedure in the laboratory to ensure the validity of results. The levels of DNA damage measured were expressed as % tail DNA and olive tail moment (OTM). Of all the compounds and concentrations tested during the in vitro exposures, excluding the positive H2O2 controls, only the mixture of PAHs yielded statistically significant (P < 0.05) levels of DNA damage compared to the controls for both DNA damage parameters. The DNA damage was observed at 50 and 100 mg⁄L PAHs, with % tail DNA of 40.70 % and 38.55 %, and OTM of 12.41 and 11.03, respectively. The remaining test compounds yielded no detectable genotoxic effects, regardless of the DNA damage parameter, under the specified concentrations and test conditions. Because genotoxicity was detected during the in vitro exposure with PAHs, an in vivo exposure with PAHs was performed to assess the predictive capabilities of the in vitro test. The in vitro PAH exposure produced a much greater genotoxic response with both parameters then was detected in vivo, in which only the positive control yielded statistically significant levels of DNA damage. Thus, under the conditions tested in this study, in vitro exposures with freshwater mussel hemolymph were unable to predict a similar in vivo response, based on the presented data. Nonetheless, a method with a high degree of accuracy was demonstrated during this study with consistent and repeatable levels of DNA damage measured in the CometAssay Control cells™ and positive control treatments. Therefore, we are confident that if the chemicals tested had been genotoxic under the conditions tested, the effects would have been detected. This research investigated the use of a non-lethal genotoxicity screening tool using freshwater mussel hemolymph. Further testing and evaluation is needed before this tool could be widely implemented. Moreover, there is need for a better understanding of freshwater mussel hemolymph and the functions and capabilities of hemocytes in the defense of genotoxic compounds.
Date: 2007-12-21
Degree: MS
Discipline: Toxicology
URI: http://www.lib.ncsu.edu/resolver/1840.16/1920


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