Research ArticleMARINE POLLUTANTS

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

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Science Advances  15 Apr 2016:
Vol. 2, no. 4, e1600001
DOI: 10.1126/sciadv.1600001
  • Fig. 1 Identification of P-gp–inhibiting POPs.

    Thirty-seven pollutants were tested for interactions with mouse P-gp using two independent assays (see Table 1). Sixteen compounds were identified as inhibitors in both assays. We focused on 10 congeners reported in humans on the basis of the literature (86, 87) and the Fourth National Report on Human Exposure to Environmental Chemicals of the U.S. Centers for Disease Control and Prevention (88). DDD, dichlorodiphenyldichloroethane; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; PCB, polychlorinated biphenyl; PBDE, polybrominated diphenyl ether.

  • Fig. 2 P-gp–inhibiting effects of POPs.

    (A) Representative images showing chemosensitization of P-gp–expressing yeast by POPs. Inhibition of mouse P-gp heterologously expressed in drug (+DOX)–sensitive yeast. Inhibition is indicated by the reduction in yeast growth with increasing POP concentration. None of the POPs were toxic to yeast in the absence of DOX (−DOX). Yeast assays were replicated three times and representative micrographs are shown. (B) Upper panel: Inhibition of verapamil-stimulated P-gp adenosine triphosphatase (ATPase) activity by POPs. Graphs show P-gp ATPase inhibition kinetics with the 10 transporter-inhibiting POPs. ATPase assays were performed with purified, recombinant mouse P-gp protein. Lower panel: Lack of P-gp ATPase activation by POPs. ATPase activation of P-gp–inhibiting organochlorine pesticides (OCPs), PBDEs, and PCBs was determined with increasing concentrations of each compound and without verapamil prestimulation. The black curves show verapamil stimulation. Points were normalized to 100 μM verapamil stimulation (gray dashed line) and represent the average ATPase activity ± SD from three to six experiments. Where not visible, error bars are smaller than symbols; R2 values were all >0.99.

  • Fig. 3 POP interactions at the substrate-binding site of mouse P-gp.

    (A) Structure of mouse P-gp cocrystallized with PBDE-100. (B) Location of PBDE-100 at a distinct binding site in the internal cavity of P-gp, viewed from the intracellular side. TM, transmembrane. (C) 2mFoDFc electron density (where m is the figure of merit and D is the Sigma-A weighting factor) for PBDE-100 (blue; contour level of 1.2σ) and anomalous difference density peaks (purple; contour level of 3.5σ). (D) Stereo view of the binding pocket, with key residues important for the interaction with the diphenyl backbone of PBDE-100 shown as sticks. (E) Conserved binding site for PBDE-100. Top: Side chains found to interact with PBDE-100 are shown in blue (conserved in human and mouse) or green (not conserved). These residues are Y303, Y306, A307, F310, F331, Q721, F724, S725, I727, F728, V731, S752, F755, S975, and F979. Bottom: Amino acid sequence alignment of mouse and human P-gp highlighting the 15 interacting residues with PBDE-100 in TM5, TM6, TM7, TM8, and TM12.

  • Fig. 4 Levels of P-gp inhibitors in yellowfin tuna (T. albacares).

    (A) Sampling site for the eight yellowfin tunas (T. albacares) caught in the GOM. The inset shows a yellowfin tuna with the sampled dorsal muscle tissue marked in red. (B) Lipid-normalized concentrations of the total POPs and the 10 P-gp inhibitors. The red-filled circles represent the minimum and maximum values. The white diamonds represent the mean value. The horizontal lines represent the 50th percentile, and the boxes represent the 25th and 75th percentiles. (C) Range of concentrations of nine inhibitory POPs measured in yellowfin tuna muscle from the GOM.

  • Fig. 5 An environmentally relevant POP mixture inhibits the transport function of human and mouse P-gp.

    (A) Relative ratio of the mean concentrations of P-gp inhibitors found in yellowfin tuna. (B) Inhibition of human P-gp by the POP mixture. Points represent the average percentage of NMQ uptake ± SD relative to the control from nine different experiments and with increasing concentration of the POP mixture. (C) Inhibition of verapamil-stimulated ATPase activity of mouse P-gp by the POP mixture. Shown is the respective dose-response curve as ATPase activity relative to 100 μM verapamil stimulation. The ATPase activity of the purified protein was measured in the presence of increasing concentrations of the POP mixture on the basis of the relative concentration of nine inhibitory POPs identified in this study. All data were fitted using a Hill function [y = v1 + (v2v1) * xn/(kn + xn)]. The R2 value was >0.99.

  • Table 1 Summary of the interaction kinetics of environmental chemicals with mouse P-gp.

    Compounds that were inhibitors in both assays and present in humans are in boldface. Compounds that were inhibitors in both assays but not known to be detected in humans are italicized. Y, yes; N, no; HCH, hexachlorocyclohexane; DDD, dichlorodiphenyldichloroethane; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; DEHP, di(2-ethylhexyl)phthalate; NI, noninteraction; DL, dioxin-like PCB congeners.

    ChemicalIC50 (μM)*IMax (%)IYeast
    Organochlorine pesticides
      Aldrin26.2 (±1.9)0.93Y
      Dieldrin21.8 (±4.2)0.69Y
      Endrin1.1 (±0.7)0.51Y
      α-HCH26.8 (±18.0)0.09N
      β-HCHNI0.00N
      γ-HCH (lindane)82.6 (±9.9)0.58N
      Heptachlor10.4 (±0.9)0.98N
      HexachlorobenzeneNI0.00N
      Methoxychlor21.7 (±2.4)0.57N
      Mirex3 (±0.2)0.24N
      4,4′-DDD72.5 (±5.7)0.45Y
      4,4′-DDE31.3 (±3.7)0.63Y
      4,4′-DDT25.6 (±4.8)0.61Y
    Polybrominated diphenyl ethers
      PBDE-3NI0.00N
      PBDE-4722.6 (±6.2)0.52Y
      PBDE-4935.6 (±5.4)0.52Y
      PBDE-10023.2 (±2.9)0.68Y
      PBDE-2096.5 (±0.4)0.94N
    Perfluorochemicals
      PFOA156.5 (±6.1)1.20N
      PFOSNI0.00N
    Plastic-related compounds
      Bisphenol ANI0.00N
      DEHPNI0.00N
    Polychlorinated biphenyls
      PCB-118DL15.9 (±1.0)0.89N
      PCB-134§12.5 (±0.8)0.82Y
      PCB-1426.1 (±0.7)0.90Y
      PCB-1454.4 (±0.4)0.92Y
      PCB-14612.8 (±1.9)0.50Y
      PCB-147§23.6 (±3.1)0.82Y
      PCB-15222 (±4.2)0.84N
      PCB-153§21.8 (±3.1)0.46N
      PCB-154§14.3 (±1.1)0.54N
      PCB-16143.2 (±8.3)0.40N
      PCB-168§25.8 (±3.7)0.66N
      PCB-169DL9.7 (±0.5)0.93N
      PCB-1709.2 (±0.8)0.73Y
      PCB-1866.9 (±0.5)0.91N
      PCB-18711.6 (±0.6)0.56Y

    *Inhibition coefficients (IC50) (±SD) of verapamil-stimulated ATPase activity.

    †Percent maximum inhibition (IMax).

    ‡Inhibition in the yeast cytotoxicity assay (IYeast).

    §Co-elusion with other PCB congeners.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/4/e1600001/DC1

      fig. S1. ATPase activity of purified, recombinant mouse P-gp.

      fig. S2. Functional expression of mouse P-gp in yeast cells.

      fig. S3. Comparison of PBDE-100 interacting residues.

      fig. S4. Amino acid sequence alignment of human, mouse, zebrafish, and sea urchin P-gp.

      fig. S5. Levels of POPs in wild-caught yellowfin tuna.

      fig. S6. Individual POP congeners from the mixture inhibiting human P-gp.

      table S1. Data collection and refinement statistics of the mouse P-gp/PBDE-100 cocrystal structure.

      table S2. Metadata on yellowfin tuna specimens used in this study.

      table S3. Physical and chemical properties of the 10 POP inhibitors.

      References (8994)

    • Supplementary Materials

      This PDF file includes:

      • fig. S1. ATPase activity of purified, recombinant mouse P-gp.
      • fig. S2. Functional expression of mouse P-gp in yeast cells.
      • fig. S3. Comparison of PBDE-100 interacting residues.
      • fig. S4. Amino acid sequence alignment of human, mouse, zebrafish, and sea urchin P-gp.
      • fig. S5. Levels of POPs in wild-caught yellowfin tuna.
      • fig. S6. Individual POP congeners from the mixture inhibiting human P-gp.
      • table S1. Data collection and refinement statistics of the mouse P-gp/PBDE-100 cocrystal structure.
      • table S2. Metadata on yellowfin tuna specimens used in this study.
      • table S3. Physical and chemical properties of the 10 POP inhibitors.
      • References (89–94)

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