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

Abstract

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.

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) 2mF_o − DF_c 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 + (v₂ − v₁) * xⁿ/(kⁿ + xⁿ)]. The R² value was >0.99.