Aryl hydrocarbon receptor-mediated up-regulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier

Abstract

Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

Figure 1.

Increased P-glycoprotein transport activity in isolated rat brain capillaries following exposure to TCDD. A) Representative confocal images showing luminal accumulation of NBD-CSA, a fluorescent substrate for P-glycoprotein. Luminal fluorescence was reduced by 5 μM PSC833, a specific inhibitor of P-glycoprotein, and was increased following 3 h of exposure to 0.5 nM TCDD. Scale bar = 5 μm. B) Exposing rat brain capillaries to TCDD increased luminal accumulation of NBD-CSA in a concentration-dependent manner. Values are means ± se for 8–12 capillaries from a single preparation (each containing pooled brain tissue from 5–10 rats). ***P < 0.001 vs. control.

Figure 2.

A) Inhibiting transcription by 1 μM actinomycin or translation by 100 μg/ml cycloheximide blocked the effects of 0.5 nM TCDD on P-glycoprotein transport activity. B, C) TCDD-induced increase in transport activity was abolished when capillaries were pretreated with the specific AhR antagonists, resveratrol (RES; B) or α-naphthoflavone (α-NF; C). Values are means ± se for 8–12 capillaries from a single preparation (pooled brains from 5–10 rats). ***P < 0.001 vs. control.

Figure 3.

Increased protein expression of P-glycoprotein, Cyp1a1, and Cyp1b1 in isolated rat brain capillaries following 3 h of exposure to 0.5–1 nM TCDD in vitro. A) Western blots with β-actin as a loading control. B–D) Measured band intensities in membranes from 3 preparations: P-glycoprotein (B), Cyp1a1 (C), and Cyp1b1 (D). *P < 0.05, **P < 0.01 vs. control.

Figure 4.

Increased transport activity of P-glycoprotein, Mrp2, and BCRP in brain capillaries following in vivo dosing of rats with 1 μg/kg or 5 μg/kg TCDD (single i.p. injection, tissues collected 2 d later). A) Representative confocal images showed luminal accumulation of fluorescent substrates NBD-CSA (for P-glycoprotein), TX red (for Mrp2), and BODIPY-prazosin (for Bcrp) in capillaries from control and TCDD-dosed rats. Scale bar = 5 μm. B–D) Dose-dependent increases of transport activity in isolated rat brain capillaries for NBD-CSA (B), Texas Red (C), and BODIPY-prazosin (D). Values are means ± se for 8–12 capillaries from a single preparation (pooled brains from 10 rats). ***P < 0.001 vs. control.

Figure 5.

Up-regulation of transporter and enzyme protein expression in brain capillary (A) and liver (B) membranes following in vivo dosing of rats with 1 μg/kg or 5 μg/kg TCDD (single i.p. injection, tissues collected 2 d later).

Figure 6.

EMSA assay showing binding of AhR to dioxin response element (DRE) following TCDD exposure in vitro and in vivo. A) EMSA gel. Lane 1: negative control (no nuclear protein); lane 2: control (in vitro exposure); lane 3: 0.5 nM TCDD, 3 h in vitro exposure; lane 4: 1 nM TCDD, 3 h in vitro exposure; lane 5: control (in vivo exposure); lane 6: 1 μg/kg TCDD; lane 7: 5 μg/kg TCDD; lane 8: 5 μg/kg TCDD plus 200× excess of unlabeled probe. B) Supershift with antibody against AhR. Lane 1: negative control; lane 2: 5 μg/kg TCDD; lane 3: 5 μg/kg TCDD plus antibody.

Figure 7.

Reduced brain uptake of P-glycoprotein substrate verapamil in TCDD-dosed rats. A) Rats received a single injection of TCDD (5 μg/kg). After 2 d, rat brains were perfused via the common carotid arteries with Ringer solution containing either [¹⁴C]-sucrose or [³H]-verapamil. TCDD dosing significantly reduced brain distribution of the P-glycoprotein substrate verapamil compared to control, indicating increased P-glycoprotein activity. B) Brain distribution of sucrose in TCDD-treated rats did not differ significantly from controls, indicating that neither tight junction-restricted permeability nor vascular volume was altered by TCDD.