Tetrabromobisphenol A (TBBPA) Alters ABC Transport at the Blood-Brain Barrier

Abstract

Tetrabromobisphenol A (TBBPA, CAS No. 79-94-7) is a brominated flame retardant used in 90% of epoxy coated circuit boards. Exposures to TBBPA can induce neurotoxicity and disrupt MAPK, estrogen, thyroid, and PPAR-associated signaling pathways. Because these pathways also regulate transporters of the central nervous system barriers, we sought to determine the effect of TBBPA on the expression and activity of 3 ATP binding cassette (ABC) transporters of the blood-brain barrier (BBB). Using a confocal based assay, we measured the ex vivo and in vivo effects of TBBPA on P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) transport activity in rat brain capillaries. Our rationale for using a rat model was based on tissue availability, ease of handling, and availability of historical TBBPA toxicokinetic data. We found that TBBPA (1-1000 nM) exposure had no significant effect on multidrug resistance-associated protein 2 transport activity in either sex, suggesting TBBPA does not compromise the physical integrity of the BBB. However, low concentrations of TBBPA (1-100 nM) significantly decreased breast cancer resistant protein transport activity in both sexes. Additionally, TBBPA exposures (1-100 nM), elicited a sex-dependent response in P-gp transport: increasing transport activity in males and decreasing transport activity in females. All TBBPA dependent changes in transport activity were dose- and time-dependent. Inhibitors of either transcription or translation abolished the TBBPA dependent increases in male P-gp transport activity. Western blot and immunofluorescent assays confirmed the TBBPA dependent P-gp increases expression in males and decreases in females. Antagonizing PPAR-γ abolished the TBBPA dependent increases in males but not the decreases in females. However, the decreases in female P-gp transport were blocked by an ER-α antagonist. This work indicates that environmentally relevant concentrations of TBBPA (1-100 nM) alter ABC transporter function at the BBB. Moreover, permeability changes in the BBB can alter brain homeostasis, hinder central nervous system drug delivery, and increase the brain's exposure to harmful xenobiotic toxicants.

Keywords: ABC transporters; P-glycoprotein; PPAR gamma; blood-brain barrier; brominated flame retardants; estrogen receptor alpha; tetrabromobisphenol A.

Figure 1.

Changes in ABC transport activity in male and female rat brain capillaries after 6-h exposure to 100 nM TBBPA; VC—vehicle control, TBBPA—100 nM TBBPA. A, (left) Male P-gp transport activity with (right) confocal images. B, (left) Female P-gp transport activity with (right) confocal images. C, (left) Male BCRP transport activity with (right) confocal images. D, (left) Female BCRP transport activity with (right) confocal images. E, (left) Male MRP2 transport activity with (right) confocal images. F, (left) Female MRP2 transport activity with (right) confocal images. Each bar represents the mean value for 15–20 capillaries from a single preparation (pooled tissue from 3 to 7 rats, 15–20 weeks old); variability is shown as SE bars. Units are arbitrary fluorescence intensity. Statistical comparisons: ***significantly lower than control, p < .001. Scale bar in each panel equals 10 µm in length. Abbreviations: BCRP, breast cancer resistant protein; MRP2, multidrug resistance-associated protein 2; P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.

Figure 2.

In vivo effects of TBBPA on P-gp, BCRP, and MRP2 transport in male and female rat brain capillaries; VC—vehicle control, TBBPA—250 mg/kg by gavage. A, Male P-gp transport activity. B, Female P-gp transport activity. C, Male BCRP transport activity. D, Female BCRP transport activity. E, Male MRP2 transport activity. F, Female MRP2 transport activity. Each bar represents the mean value for 15–20 capillaries from a single preparation (pooled tissue from 5 to 7 rats, 15–20 weeks old); variability is shown as SE bars. Units are arbitrary fluorescence. Statistical comparisons: ***significantly different than control, p < .001. Abbreviations: BCRP, breast cancer resistant protein; MRP2, multidrug resistance-associated protein 2; P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.

Figure 3.

TBBPA dependent changes in P-gp transport activity in rat brain capillaries are sex-, dose-, and time dependent. P-gp transport activity in capillaries exposed to 100 nM TBBPA for 4 h; (A) male and (B) female capillaries. Time-dependent response of 100 nM TBBPA on (C) male and (D) female P-gp transport activity. Each bar represents the mean value for 15–20 capillaries from a single preparation (pooled tissue from 5 to 7 rats, 15–20 weeks old); variability is shown as SE bars. Units are arbitrary fluorescence. Statistical comparisons: ***significantly different than control, p < .001. Abbreviations: P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.

Figure 4.

TBBPA dependent decreases in BCRP transport activity are dose- and time dependent. Dose dependent decreases at 4 h of TBBPA on (A) male and (B) female BCRP activity. (C) Male and (D) female BCRP transport activity exhibits time-dependent decreases from 100 nM TBBPA exposure. Shown are mean ± SEM for 15–20 capillaries from single preparation (pooled brains from 4 to 7 rats, 15–20 weeks old). ***p < .001, significantly different than control. Abbreviations: BCRP, breast cancer resistant protein; TBBPA, tetrabromobisphenol A.

Figure 5.

TBBPA induced P-gp transport requires transcription, translation and PPAR-γ. A, In males, pretreatment with an inhibitor of transcription, 1 μM actinomycin D (AMD), or translation, 1.8 mM cycloheximide (CHD), blocks TBBPA increases in P-gp activity. B, In males, co-treatment with 10 nM GW9662 and 100 nM TBBPA blocks P-gp induction. C, In females, co-treatment with 10 nM GW9662 and 100 nM TBBPA does not reverse TBBPA dependent decreases in P-gp induction. Shown are mean ± SEM for 10–20 capillaries from single preparation (pooled brains from 3 to 5 rats, 15–20 weeks old). ***p < .001, significantly different than control. Abbreviations: P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.

Figure 6.

Inhibition of PPAR-γ blocks TBBPA mediated increases in P-gp expression in males. A, Western blot containing proteins from male (left) and female (right) rat brain capillaries. Non-treated vehicle controls (NT), TBBPA 100 nM exposed capillaries (TBBPA), and co-treated with TBBPA + 10 nM GW 9668 (+ GW). B, (top) Immunofluorescent images showing TBBPA increases are blocked by inhibition of PPAR-γ in males. Band density was determined by densitometry using ImageJ software. White numbers under P-gp bands in blot were normalized to the density of actin bands to control for minor loading variances. Graphs depict normalized density values of P-gp band in Western blot. B, (bottom) Immunofluorescent images showing TBBPA decreases in females are insensitive to PPAR-γ inhibition. Graphical analysis of P-gp immunofluorescence in males (C, top) and females (C, bottom). Shown are mean fluorescence ± SEM for 10–20 capillaries from single preparation (pooled brains from 3 to 5 rats, 15–20 weeks old). ***p < .001, significantly different than control. Scale bar equals 10 µm in length. Abbreviations: P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.

Figure 7.

ER-α signaling suppresses PPAR-γ mediated TBBPA induction of P-gp transport. A, 1 nM E2 blocks TBBPA induction of P-gp transport in male rat brain capillaries. B, Antagonizing ER-α signaling with 10 nM ICI blocks TBBPA mediated decreases in female rat brain capillaries. Graphs are mean fluorescence ± SEM for 10–20 capillaries from single preparation (pooled brains from 3 to 5 rats, 15–20 weeks old). ***p < .001, significantly different than control. Abbreviations: P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.

Figure 8.

Proposed model of sex-dependent effects of TBBPA on P-gp transport. Left-Males, TBBPA activation of PPAR-γ leads to increased expression and transport. Right-Females, ER-α signaling in females represses PPAR-γ causing a decrease in P-gp expression and transport. Abbreviations: P-gp, P-glycoprotein; TBBPA, tetrabromobisphenol A.