Thesis (Ph.D.)--University of California, Davis, 2010
Studies of multidrug resistance (MDR), the removal of target compounds from cells by ATP binding cassette proteins, in a diverse range of marine invertebrates have revealed a surprising capacity for MDR-mediated transport and a complexity of MDR protein utilization in the cells of early life history stages. The comparative physiology of MDR in the early life history stages of the white sea urchin Lytechinus pictus and the fat innkeeper worm Urechis caupo was examined using confocal microscopy in combination with the calcein-AM dye accumulation assay of MDR activity and the vinblastine cell cleavage assay of MDR function. The oocytes and embryos of U. caupo exhibited sequestration of MDR substrates within cytoplasmic vesicles, a secondary MDR phenotype, in addition to the removal of substrates at the plasma membrane. The eggs and embryos of L. pictus did not appear to employ the secondary MDR phenotype. The vesicles in U. caupo were identified as acidic and lysosome-like using the fluorescent probe Lysotracker RedRTM. It was concluded that inward-pumping multidrug resistance-associated proteins (MRPs) in the membranes of the lysosome-like vesicles are likely responsible for the sequestration mechanism observed in U. caupo based on the absence of evidence for pH gradient-induced sequestration and on the exclusion of calcein from vesicles only after treatment with MK571, a MRP inhibitor. Quantitative comparisons of Lytechinus and Urechis indicated that early life history stages of the sea urchin have greater MDR activity than those of Urechis and that the majority of the MDR activity in both species can be attributed to MRPs
The contamination of the aquatic environment by pharmaceuticals and personal care products (PPCPs) is an issue of emerging concern. The effects of pharmaceuticals like fluoxetine (Prozac(TM)), chemicals that are designed to be biologically active at very low concentrations, on non-target organisms are almost completely unknown. The potential sublethal effects of PPCPs on marine invertebrates are difficult to conceptualize, let alone quantify. However, there are definable endpoints with a focus on the interactions between pharmaceuticals and specific biological mechanisms like MDR. Using the dye accumulation assay, it was found that fluoxetine (Prozac(TM)) can inhibit MDR activity in L. pictus embryos at very high concentrations, but can have a stimulatory effect on MDR at lower, more environmentally relevant, concentrations. This stimulation of MDR activity also appeared to be functionally relevant as fluoxetine-treated embryos were rescued from the effects of vinblastine, a cell cleavage inhibitor and MDR substrate. Fluoxetine was also capable of attenuating the effect of the MRP inhibitor MK571
