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(PH065) P-glycoprotein expression levels as a function of pesticide exposure in the amphipod, Leptocheirus plumulosus.
Block, D1, Griffitt, R1, Chandler, G1, 1 University of South Carolina, Norman J Arnold School of Public Health, Dept. of Environmental Sciences, Columbia, SC, USA
ABSTRACT- P-glycoproteins (P-gps) are members of the evolutionarily well-conserved ATP Binding Cassette (ABC) family of membrane bound proteins. These proteins have the ability to actively transport a wide variety of unrelated chemicals across a cell membrane against a concentration gradient, and are known to be inducible by many xenobiotics. P-gp induction confers increased resistance to these xenobiotics by reducing intracellular concentrations before they reach harmful levels. The resistance conferred by increased P-gp activity can also be inhibited chemically, thus artificially reducing P-gp mediated xenobiotic efflux. The present study demonstrates that both upregulation and inhibition of P-gp in the amphipod, Leptocheirus plumulosus can be measured in vivo by quantitative assessment of the relative cellular efflux of the known P-gp substrate Calcein AM (C-AM). Specifically, P-gp upregulation was observed in amphipods following a 24-hour exposure to 0.5, 1, and 2 ppm concentrations of the insect growth regulating hormone mimic, tebufenozide. Retention of C-AM was significantly lower in individuals exposed to 2 ppm tebufenozide than in unexposed individuals, indicating increased P-gp activity. The decreased retention in exposed individuals was mitigated by the addition of the known P-gp inhibitor, verapamil, indicating that P-glycoproteins are the mode by which the C-AM is removed. A series of pesticides were then tested for their ability to inhibit P-gp activity in tebufenozide exposed amphipods. The fungicide, fenarimol was found to reduce intracellular accumulation of C-AM. P-gp proteins have also been purified from bulk proteins using affinity chromatography, and used to generate a standard curve as a step in developing an ELISA for more precise quantification of P-gp production in amphipods and other crustaceans.
Key words: Calcein AM, P-glycoprotein, amphipod, up-regulation
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