Dissertations and Theses @ UNI
Availability
Open Access Thesis
Keywords
Liver cells--Metabolism; Biochemical markers; Ecological risk assessment; Fishes--Effect of pollution on;
Abstract
Aquatic organisms such as fish, sponges and mussels are exposed to mixtures of chemicals in polluted waters including polycyclic aromatic hydrocarbons, pesticides and heavy metals. The tissues of some of these organisms are found to have a lower concentration of pollutants compared to the surrounding waters and/ or sediments. This suggests that some aquatic organisms may possess a means of detoxifying environmental pollutants. For this study, primary hepatocytes of the marine winter flounder (Pleuronectes americanus) and freshwater rainbow trout (Salmo gairdnen) hepatomas were exposed to environmental pollutants to determine whether a multidrug-resistance (MDR) p-glycoprotein (pgp) extrusion pump is involved in detoxification. Such a mechanism exists in mammalian tumor cells in response to chemotherapy. If an MDR mechanism exists in the fish, its differential expression could be used as an ecosystem indicator (diagnostic biochemical marker) for aquatic pollution. Using the reverse transcriptase polymerase chain reaction {RT-PCR) technique, DNA coding for isotypes A and B of the pgp gene were found to be expressed in winter flounder primary hepatocytes. Messenger RNA levels (measured with RT-PCR) of the putative pgp gene(s) expressed in the cells were then compared under the influence of several environmental toxins. Results indicated an increased level of the mRNA for the two isotypes in cells cultured with all toxins compared to the control cells containing only culture medium. Protein levels in subcellular fractions of winter flounder and freshwater trout hepatocytes were assayed using immunocytochemistry. The results showed that anti-human polyclonal antibody PC03 recognized a ~ 65 kDa polypeptide band in both trout and winter flounder hepatocytes and that the ~ 65 kDa polypeptide is induced in a dose dependent fashion when toxins are added to the medium. Results also showed that trout hepatocytes cultured in medium supplemented with 5% CO2 had no detectable ~ 65 kDa band when incubated in the presence and absence of toxins. This study describes for the first time the establishment of a monolayer cell line of winter flounder primary hepatocytes. This study also shows that these cells and trout hepatomas express mRNA and synthesize pgp polypeptide in response to exposure to environmental pollutants. The fact that the polypeptide synthesis is inducible and dose dependent suggests that this MDR mechanism could be used as an ecosystem indicator for aquatic pollution.
Year of Submission
1998
Degree Name
Master of Science
Department
Department of Biology
First Advisor
Roney Laine
Second Advisor
Edward Brown
Third Advisor
James Jurgenson
Date Original
1998
Object Description
1 PDF file (121 leaves)
Copyright
©1998 Marty C. Braud
Language
en
File Format
application/pdf
Recommended Citation
Braud, Marty C., "Differential Expression of the Putative Multidrug Resistance (MDR) P-Glycoprotein in Marine Hepatocytes" (1998). Dissertations and Theses @ UNI. 2174.
https://scholarworks.uni.edu/etd/2174
Comments
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