Activation of multidrug efflux transporter activity at fertilization in sea urchin embryos (Strongylocentrotus purpuratus)

AbstractThis study presents functional and molecular evidence for acquisition of multidrug transporter-mediated efflux activity as a consequence of fertilization in the sea urchin. Sea urchin eggs and embryos express low levels of efflux transporter genes with homology to the multidrug resistance associated protein (mrp) and permeability glycoprotein (p-gp) families of ABC transporters. The corresponding efflux activity is low in unfertilized eggs but is dramatically upregulated within 25 min of fertilization; the expression of this activity does not involve de novo gene expression and is insensitive to inhibitors of transcription and translation indicating activation of pre-existing transporter protein. Our study, using specific inhibitors of efflux transporters, indicates that the major activity is from one or more mrp-like transporters. The expression of activity at fertilization requires microfilaments, suggesting that the transporters are in vesicles and moved to the surface after fertilization. Pharmacological inhibition of mrp-mediated efflux activity with MK571 sensitizes embryos to the toxic compound vinblastine, confirming that one role for the efflux transport activity is embryo protection from xenobiotics. In addition, inhibition of mrp activity with MK571 alone retards mitosis indicating that mrp-like activity may also be required for early cell divisions

Molecuilar Physiology of Stress Tolerance in Marine Invertebrates: The Heat Shock Response and Multidrug Resistance

A number of molecular mechanisms have been implicated in biological tolerance of marine environmental stress. Among the best characterized of these is the heat shock response, which has been shown to play an important role in determining the distribution of marine invertebrates along natural gradients of stress (eg. thermal stress in the intertidal). The heat shock response also appears to play a critical role in determining the susceptibility of invertebrates to anthropogenic stress. In a parallel context, it has been proposed that multidrug resistance may play an important role in determining the survival of invertebrates along natural and anthropogenic gradients of toxic organic compounds.This study characterized the role of both of these systems in the tolerance of natural stressors in model marine invertebrate systems. ln the first section of the dissertation I demonstrated that, although Pacific oysters are capable of significant adaptive regulation of heat shock protein (HSP) expression, adaptive plasticity occurs at a physiologic cost to inducible stress tolerance. Moreover. although adaptive regulation of HSPs is via a transcriptional mechanism during conditions of acute thermal stress. the response to chronic stress may not require continual transcriptional activation of HSP genes. ln the second section of the dissertation I focused on the role ofmultidrug resistance (MDR) transporters in oocytes. embryos and larvae in model (sea urchin, starfish and urechis) systems. In the first chapter of this section l described species-specific differences in transporter expression and consequent differences in susceptibility to naturally degraded hydrocarbons. In the second chapter I hypothesized that the reason for organic susceptibility in echinoderms is not due to the absence of a transporter. but rather due to the specificity of the predominant transporter family expressed in each species. Consistent with this hypothesis I showed that the predominant type of transporter expressed in echinoderm oocytes and embryos bear homology to the multidrug resistance associated protein (MRP) family rather than the multidrug resistance (P-gp/MDR) family. I examined the potential consequences of this difference for transporter substrate specificity and we measured the level of MRP expression during early development. I show that echinoderms possess a characteristic MRP transport phenotype that is activated at fertilization

Disruption of small molecule transporter systems by Transporter-Interfering Chemicals (TICs).

Small molecule transporters (SMTs) in the ABC and SLC families are important players in disposition of diverse endo- and xenobiotics. Interactions of environmental chemicals with these transporters were first postulated in the 1990s, and since validated in numerous in vitro and in vivo scenarios. Recent results on the co-crystal structure of ABCB1 with the flame-retardant BDE-100 demonstrate that a diverse range of man-made and natural toxic molecules, hereafter termed transporter-interfering chemicals (TICs), can directly bind to SMTs and interfere with their function. TIC-binding modes mimic those of substrates, inhibitors, modulators, inducers, and possibly stimulants through direct and allosteric mechanisms. Similarly, the effects could directly or indirectly agonize, antagonize or perhaps even prime the SMT system to alter transport function. Importantly, TICs are distinguished from drugs and pharmaceuticals that interact with transporters in that exposure is unintended and inherently variant. Here, we review the molecular mechanisms of environmental chemical interaction with SMTs, the methodological considerations for their evaluation, and the future directions for TIC discovery

Evaluation of the global impacts of mitigation on persistent, bioaccumulative and toxic pollutants in marine fish

Although persistent, bioaccumulative and toxic pollutants (PBTs) are well-studied individually, their distribution and variability on a global scale are largely unknown, particularly in marine fish. Using 2,662 measurements collected from peer-reviewed literature spanning 1969–2012, we examined variability of five classes of PBTs, considering effects of geography, habitat, and trophic level on observed concentrations. While we see large-scale spatial patterning in some PBTs (chlordanes, polychlorinated biphenyls), habitat type and trophic level did not contribute to significant patterning, with the exception of mercury. We further examined patterns of change in PBT concentration as a function of sampling year. All PBTs showed significant declines in concentration levels through time, ranging from 15–30% reduction per decade across PBT groups. Despite consistent evidence of reductions, variation in pollutant concentration remains high, indicating ongoing consumer risk of exposure to fish with pollutant levels exceeding EPA screening values. The temporal trends indicate that mitigation programs are effective, but that global levels decline slowly. In order for monitoring efforts to provide more targeted assessments of risk to PBT exposure, these data highlight an urgent need for improved replication and standardization of pollutant monitoring protocols for marine finfish

Phenotypic Plasticity of HSP70 and HSP70 Gene Expression in the Pacific Oyster (Crassostrea gigas): Implications for Thermal Limits and Induction of Thermal Tolerance

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Mercury levels of yellowfin tuna (Thunnus albacares) are associated with capture location.

Mercury is a toxic compound to which humans are exposed by consumption of fish. Current fish consumption advisories focus on minimizing the risk posed by the species that are most likely to have high levels of mercury. Less accounted for is the variation within species, and the potential role of the geographic origin of a fish in determining its mercury level. Here we surveyed the mercury levels in 117 yellowfin tuna caught from 12 different locations worldwide. Our results indicated significant variation in yellowfin tuna methylmercury load, with levels that ranged from 0.03 to 0.82 μg/g wet weight across individual fish. Mean mercury levels were only weakly associated with fish size (R2 < 0.1461) or lipid content (R2 < 0.00007) but varied significantly, by a factor of 8, between sites. The results indicate that the geographic origin of fish can govern mercury load, and argue for better traceability of fish to improve the accuracy of exposure risk predictions

Xenobiotic transporter activity in zebrafish embryo ionocytes.

Ionocytes are specialized cells in the epidermis of embryonic zebrafish (Danio rerio) that play important roles in ion homeostasis and have functional similarities to mammalian renal cells. Here, we examined whether these cells might also share another functional similarity with renal cells, which is the presence of efflux transporter activities useful for elimination of toxic small molecules. Xenobiotic transporters (XTs), including the ATP-Binding Cassette (ABC) family, are a major defense mechanism against diffusible toxic molecules in aquatic embryos, including zebrafish, but their activity in the ionocytes has not previously been studied. Using fluorescent small molecule substrates of XT, we observed that specific populations of ionocytes uptake and efflux fluorescent small molecules in a manner consistent with active transport. We specifically identified a P-gp/ABCB1 inhibitor-sensitive efflux activity in the H+-ATPase-rich (HR) ionocytes, and show that these cells exhibit enriched expression of the ABCB gene, abcb5. The results extend our understanding of the functional significance of zebrafish ionocytes and indicate that these cells could play an important role in protection of the fish embryo from harmful small molecules

Geographic Differences in Persistent Organic Pollutant Levels of Yellowfin Tuna.

BackgroundFish are a source of persistent organic pollutants (POPs) in the human diet. Although species, trophic level, and means of production are typically considered in predicting fish pollutant load, and thus recommendations of consumption, capture location is usually not accounted for.ObjectivesYellowfin tuna (Thunnus albacares) are harvested from across the world's oceans and are widely consumed. Here, we determined geographic variation in the overall mass, concentration, and composition of POPs in yellowfin and examined the differences in levels of several POP congeners of potential relevance to human health.MethodsWe sampled dorsal muscle of 117 yellowfin tuna from 12 locations worldwide, and measured POP levels using combined liquid or gas chromatography and mass spectrometry according to U.S. Environmental Protection Agency standard procedures.ResultsPOP levels varied significantly among sites, more than 36-fold on a mass basis. Individual fish levels ranged from 0.16 to 138.29 ng/g wet weight and lipid-normalized concentrations from 0.1 to 12.7 μM. Levels of 10 congeners that interfere with the cellular defense protein P-glycoprotein, termed transporter interfering compounds (TICs), ranged from 0.05 to 35.03 ng/g wet weight and from 0.03 to 3.32 μM in tuna lipid. Levels of TICs, and their individual congeners, were strongly associated with the overall POP load. Risk-based analysis of several carcinogenic POPs indicated that the fish with the highest levels of these potentially harmful compounds were clustered at specific geographic locations.ConclusionsCapture location is an important consideration when assessing the level and risk of human exposure to POPs through ingestion of wild fish. https://doi.org/10.1289/EHP518

Transport in technicolor: mapping ATP-binding cassette transporters in sea urchin embryos.

One quarter of eukaryotic genes encode membrane proteins. These include nearly 1,000 transporters that translocate nutrients, signaling molecules, and xenobiotics across membranes. While it is well appreciated that membrane transport is critical for development, the specific roles of many transporters have remained cryptic, in part because of their abundance and the diversity of their substrates. Multidrug resistance ATP-binding cassette (ABC) efflux transporters are one example of cryptic membrane proteins. Although most organisms utilize these ABC transporters during embryonic development, many of these transporters have broad substrate specificity, and their developmental functions remain incompletely understood. Here, we review advances in our understanding of ABC transporters in sea urchin embryos, and methods developed to spatially and temporally map these proteins. These studies reveal that multifunctional transporters are required for signaling, homeostasis, and protection of the embryo, and shed light on how they are integrated into ancestral developmental pathways recapitulated in disease