Enhancing knowledge of chemical exposures and fate in honey bee hives: Insights from colony structure and interactions.

Honey bees are unintentionally exposed to a wide range of chemicals through various routes in their natural environment, yet research on the cumulative effects of multi-chemical and sublethal exposures on important caste members, including the queen bee and brood, is still in its infancy. The hive's social structure and food-sharing (trophallaxis) practices are important aspects to consider when identifying primary and secondary exposure pathways for residential hive members and possible chemical reservoirs within the colony. Secondary exposures may also occur through chemical transfer (maternal offloading) to the brood and by contact through possible chemical diffusion from wax cells to all hive members. The lack of research on peer-to-peer exposures to contaminants and their metabolites may be in part due to the limitations in sensitive analytical techniques for monitoring chemical fate and dispersion. Combined application of automated honey bee monitoring and modern chemical trace analysis techniques could offer rapid progress in quantifying chemical transfer and accumulation within the hive environment and developing effective mitigation strategies for toxic chemical co-exposures. To enhance the understanding of chemical fate and toxicity within the entire colony, it is crucial to consider both the intricate interactions among hive members and the potential synergistic effects arising from combinations of chemical and their metabolites.

TICBase: Integrated Resource for Data on Drug and Environmental Chemical Interactions with Mammalian Drug Transporters.

Environmental health science seeks to predict how environmental toxins, chemical toxicants, and prescription drugs accumulate and interact within the body. Xenobiotic transporters of the ATP-binding cassette (ABC) and solute carrier (SLC) superfamilies are major determinants of the uptake and disposition of xenobiotics across the kingdoms of life. The goal of this study was to integrate drug and environmental chemical interactions of mammalian ABC and SLC proteins in a centralized, integrative database. We built upon an existing publicly accessible platform-the "TransPortal"-which was updated with novel data and searchable features on transporter-interfering chemicals from manually curated literature data. The integrated resource TransPortal-TICBase (https://transportal.compbio.ucsf.edu) now contains information on 46 different mammalian xenobiotic transporters of the ABC- and SLC-type superfamilies, including 13 newly added rodent and 2 additional human drug transporters, 126 clinical drug-drug interactions, and a more than quadrupled expansion of the initial in vitro chemical interaction data from 1,402 to 6,296 total interactions. Based on our updated database, environmental interference with major human and rodent drug transporters occurs across the ABC- and SLC-type superfamilies, with kinetics indicating that some chemicals, such as the ionic liquid 1-hexylpyridinium chloride and the antiseptic chlorhexidine, can act as strong inhibitors with potencies similar or even higher than pharmacological model inhibitors. The new integrated web portal serves as a central repository of current and emerging data for interactions of prescription drugs and environmental chemicals with human drug transporters. This archive has important implications for predicting adverse drug-drug and drug-environmental chemical interactions and can serve as a reference website for the broader scientific community of clinicians and researchers.

Transporter-interfering chemicals inhibit P-glycoprotein of yellowfin tuna (Thunnus albacares).

Marine pollutants bioaccumulate at high trophic levels of marine food webs and are transferred to humans through consumption of apex species. Yellowfin tuna (Thunnus albacares) are marine predators, and one of largest commercial fisheries in the world. Previous studies have shown that yellowfin tuna can accumulate high levels of persistent organic pollutants, including Transporter Interfering Chemicals (TICs), which are chemicals shown to bind to mammalian xenobiotic transporters and interfere with their function. Here, we examined the extent to which these same compounds might interfere with the activity of the yellowfin tuna (Thunnus albacares) ortholog of this transporter. To accomplish this goal we identified, expressed, and functionally assayed tuna ABCB1. The results demonstrated a common mode of vertebrate ABCB1 interaction with TICs that predicts effects across these species, based on high conservation of specific interacting residues. Importantly several TICs showed potent inhibition of Ta-ABCB1, such as the organochlorine pesticides Endrin (EC50 = 1.2 ± 0.2 µM) and Mirex (EC50 = 2.3 ± 0.9 µM). However, unlike the effects observed on mouse ABCB1, low concentrations of the organochlorine pesticide TICs p,p'-DDT and its metabolite p,p'-DDD co-stimulated verapamil-induced Ta-ABCB1 ATPase activity possibly suggesting a low transport activity for these ligands in tuna. These results provide a mechanistic basis for understanding the potential vulnerability of tuna to these ubquitous pollutants.

Interactions of Environmental Chemicals and Natural Products With ABC and SLC Transporters in the Digestive System of Aquatic Organisms.

An organism's diet is a major route of exposure to both beneficial nutrients and toxic environmental chemicals and natural products. The uptake of dietary xenobiotics in the intestine is prevented by transporters of the Solute Carrier (SLC) and ATP Binding Cassette (ABC) family. Several environmental chemicals and natural toxins have been identified to induce expression of these defense transporters in fish and aquatic invertebrates, indicating that they are substrates and can be eliminated. However, certain environmental chemicals, termed Transporter-Interfering Chemicals or TICs, have recently been shown to bind to and inhibit fish and mammalian P-glycoprotein (ABCB1), thereby sensitizing cells to toxic chemical accumulation. If and to what extent other xenobiotic defense or nutrient uptake transporters can also be inhibited by dietary TICs is still unknown. To date, most chemical-transporter interaction studies in aquatic organisms have focused on ABC-type transporters, while molecular interactions of xenobiotics with SLC-type transporters are poorly understood. In this perspective, we summarize current advances in the identification, localization, and functional analysis of protective MXR transporters and nutrient uptake systems in the digestive system of fish and aquatic invertebrates. We collate the existing literature data on chemically induced transporter gene expression and summarize the molecular interactions of xenobiotics with these transport systems. Our review emphasizes the need for standardized assays in a broader panel of commercially important fish and seafood species to better evaluate the effects of TIC and other xenobiotic interactions with physiological substrates and MXR transporters across the aquatic ecosystem and predict possible transfer to humans through consumption.

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.

2,4,6-Tribromophenol Exposure Decreases P-Glycoprotein Transport at the Blood-Brain Barrier.

2,4,6-Tribromophenol (TBP, CAS No. 118-79-6) is a brominated chemical used in the production of flame-retardant epoxy resins and as a wood preservative. In marine environments, TBP is incorporated into shellfish and consumed by predatory fish. Food processing and water treatment facilities produce TBP as a byproduct. 2,4,6-Tribromophenol has been detected in human blood and breast milk. Biologically, TBP interferes with estrogen and thyroid hormone signaling, which regulate important transporters of the blood-brain barrier (BBB). The BBB is a selectively permeable barrier characterized by brain microvessels which are composed of endothelial cells mortared by tight-junction proteins. ATP-binding cassette (ABC) efflux transporters on the luminal membrane facilitate the removal of unwanted endobiotics and xenobiotics from the brain. In this study, we examined the in vivo and ex vivo effects of TBP on two important transporters of the BBB: P-glycoprotein (P-gp, ABCB1) and Multidrug Resistance-associated Protein 2 (MRP2, ABCC2), using male and female rats and mice. 2,4,6-Tribromophenol exposure ex vivo resulted in a time- (1-3 h) and dose- (1-100 nM) dependent decrease in P-gp transport activity. MRP2 transport activity was unchanged under identical conditions. Immunofluorescence and western blotting measured decreases in P-gp expression after TBP treatment. ATPase assays indicate that TBP is not a substrate and does not directly interact with P-gp. In vivo dosing with TBP (0.4 µmol/kg) produced decreases in P-gp transport. Co-treatment with selective protein kinase C (PKC) inhibitors prevented the TBP-mediated decreases in P-gp transport activity.

Geographic Differences in Persistent Organic Pollutant Levels of Yellowfin Tuna.

BACKGROUND: Fish 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. OBJECTIVES: Yellowfin 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. METHODS: We 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. RESULTS: POP 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. CONCLUSIONS: Capture 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.

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.

Conformational Changes and Competitive Adsorption between Serum Albumin and Hemoglobin on Bioceramic Substrates.

Traditional methods to analyze interactions and conformational changes of proteins adsorbed onto biomaterials are limited by the protein's associations with the substrate material and the complexity of the surrounding media. We have used EPR spectroscopy in combination with site-directed spin labeling (SDSL) to investigate single protein and competitive adsorption kinetics of horse hemoglobin (Hgb) and bovine serum albumin (BSA) on a silica-calcium-phosphate bioceramic substrate. Combined continuous wave and pulsed (DEER) EPR techniques were employed to monitor local mobility/flexibility changes within the proteins and tertiary structure dynamics upon adsorption. An alternate labeling technique was introduced to allow for specific quantification of each protein adsorbed to the bioceramic surface. We show that at buffer pH 7.4 and 4.7 the amount of adsorbed hemoglobin was increased by a factor of 4-5 compared with BSA. The tertiary structure of hemoglobin was strongly affected upon adsorption, leading to a dissociation of the tetrameric molecule into monomers or αß dimers. When the bioceramic substrate was previously functionalized with a layer of BSA, dissociation was reduced by 71 % compared with the untreated surface, indicating a "primer" effect of BSA for better adhesion of the globular hemoglobin.

Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure.

The world's oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)-100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals.

Redox Capacity of an Extracellular Matrix Protein Associated with Adhesion in Mytilus californianus.

Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Foot-extracted Mfp-6 has a reducing capacity of ~17 e(-) per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ~500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced ß-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6.

Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins.

The 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay is well established for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility: Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.Maximal concentration of DPPH was limited to 100 µM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs.

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.

Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein-6.

Mytilus foot protein type 6 (mfp-6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp-6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp-6 showed high purification yields of 5-6 mg L(-1) cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp-6.1 protein showed high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano-Newton range, we show that rmfp-6.1 is also able to rescue the oxidation-dependent adhesion loss of mussel foot protein 3 (mfp-3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4-dihydroxyphenyl-l-alanine in mfp-3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp-6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587-1593, 2013.

Mini-review: the role of redox in Dopa-mediated marine adhesion.

3, 4-Dihydroxyphenylanine (Dopa)-containing proteins are key to wet adhesion in mussels and possibly other sessile organisms also. However, Dopa-mediated adhesive bonding is a hard act to follow in that, at least in mussels, bonding depends on Dopa in both reduced and oxidized forms, for adhesion and cohesion, respectively. Given the vulnerability of Dopa to spontaneous oxidation, the most significant challenge to using it in practical adhesion is controlling Dopa redox in a temporally- and spatially defined manner. Mussels appear to achieve such control in their byssal attachment plaques, and factors involved in redox control can be measured with precision using redox probes such as the diphenylpicryl hydrazyl (DPPH) free radical. Understanding the specifics of natural redox control may provide fundamentally important insights for adhesive polymer engineering and antifouling strategies.