Para- and ortho-substitutions are key determinants of polybrominated diphenyl ether activity toward ryanodine receptors and neurotoxicity

Abstract

Background: Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants that bioaccumulate in human tissues. Their neurotoxicity involves dysregulation of calcium ion (Ca(2+))signaling; however, specific mechanisms have yet to be defined.

Objective: We aimed to define the structure-activity relationship (SAR) for PBDEs and their metabolites toward ryanodine receptors type 1 (RyR1) and type 2 (RyR2) and to determine whether it predicts neurotoxicity.

Methods: We analyzed [3H]ryanodine binding, microsomal Ca(2+) fluxes, cellular measurements of Ca(2+) homeostasis, and neurotoxicity to define mechanisms and specificity of PBDE-mediated Ca(2+) dysregulation.

Results: PBDEs possessing two ortho-bromine substituents and lacking at least one para-bromine substituent (e.g., BDE-49) activate RyR1 and RyR2 with greater efficacy than corresponding congeners with two para-bromine substitutions (e.g., BDE-47). Addition of a methoxy group in the free para position reduces the activity of parent PBDEs. The hydroxylated BDEs 6-OH-BDE-47 and 4´-OH-BDE-49 are biphasic RyR modulators. Pretreatment of HEK293 cells (derived from human embryonic kidney cells) expressing either RyR1 or RyR2 with BDE-49 (250 nM) sensitized Ca2+ flux triggered by RyR agonists, whereas BDE-47 (250 nM) had negligible activity. The divergent activity of BDE-49, BDE-47, and 6-OH-BDE-47 toward RyRs predicted neurotoxicity in cultures of cortical neurons.

Conclusions: We found that PBDEs are potent modulators of RyR1 and RyR2. A stringent SAR at the ortho and para position determined whether a congener enhanced, inhibited, or exerted nonmonotonic actions toward RyRs. These results identify a convergent molecular target of PBDEs previously identified for noncoplanar polychlorinated biphenyls (PCBs) that predicts their cellular neurotoxicity and therefore could be a useful tool in risk assessment of PBDEs and related compounds.

Figure 1

Ortho-bromines are essential for PBDE activity toward FKBP12-RyR1 complex. Abbreviations: Rap, rapamycin; RR, ruthenium red. (A) Microsomes enriched in RyR1 protein were incubated in the presence of either BDE-4 or BDE-15 in [³H]Ry binding buffer. (B) Addition of 20 μM BDE-4 (arrow) to microsomal vesicles subsequent to SERCA-dependent Ca²⁺ accumulation in the presence of ATP (loading phase) resulted in net Ca²⁺ release. Inhibition of RyR1 by 1μM RR blocked the Ca²⁺ release triggered by BDE-4 (10 μM). (C) Concentration–effect curve for BDE-4–triggered Ca²⁺ release (EC₅₀ = 9.6 ± 0.2 μM; n = 4). Addition of vehicle to the Ca²⁺-loaded vesicles did not cause Ca²⁺ release (Vehicle). (D) Concentration-dependent inhibition of BDE-4–enhanced [³H]Ry binding to RyR1 by Rap (5 μM BDE-4 with 0–200 μM Rap); Rap alone had negligible effects on [³H]Ry binding to RyR1. n = 3 independent experiments in triplicate. (E) Pretreatment of microsomal vesicles with Rap 2 min before loading vesicle with Ca²⁺ (arrow) inhibited Ca²⁺ release triggered by subsequent addition (dotted arrow) of 5 μM BDE-4 but did not inhibit caffeine-induced Ca²⁺ release. (F) Concentration–effect relationship of Rap inhibition of BDE-4 triggered Ca²⁺ release; n = 4.

Figure 2

Substitution of both para positions with bromine reduces PBDE activity toward RyR1 and RyR2. (A) PBDEs possessing one para-bromine substituent (BDE-17, BDE-42, and BDE-49) enhanced [³H]Ry binding to RyR1 microsomes in a concentration-dependent manner (maximum of 446% for BDE-17, 206% for BDE-42, and 527% for BDE-49 compared with vehicle); in contrast, BDE-47, with two para-bromine substituents, showed significantly lower efficacy (maximum of 145% compared with vehicle). (B) SAR for PBDEs (each at 10 μM) as assessed using [³H]Ry binding analysis of microsomes enriched in RyR2; n = 3 or 4 independent experiments, each in triplicate. **p < 0.01.

Figure 3

Complex activity of 6-OH-BDE-47 and 4′-OH-BDE-49 toward RyR1. (A) [³H]Ry binding analysis under equilibrium conditions (3 hr at 37°C) indicates that 6-OH-BDE-47 inhibits RyR1, whereas 4′-OH-BDE-49 alters [³H]Ry binding in a nonmonotonic manner (n = 3 independent experiments in triplicate). (B) Microsomal vesicles actively loaded with Ca²⁺ release their accumulated Ca²⁺ when acutely challenged with 6-OH-BDE-47 (10 μM), whereas RR (1 μM) blocked 6-OH-BDE-47–induced Ca²⁺ release (n = 3). (C) Preincubation (> 30 min) with 6-OH-BDE-47 significantly attenuates 4-CmC (1 mM)-induced Ca²⁺ release from microsomes (n = 3). **p < 0.01.

Figure 4

Cortical neurons show excitoxicity to BDE-49 and 6-OH-BDE-47, but not BDE-47. (A) Primary cultured rat cortical neurons on MEA (21 days in vitro); bar = 100 μm. (B) Representative raster plot of spike trains over a 6-sec period in neurons exposed acutely to vehicle, BDE-47, or BDE-49. (C) BDE-49, but not BDE-47, increases spontaneous spike activity in a concentration-dependent manner; data are presented as mean ± SE (n = three arrays per treatment group). (D) Micrographs of neurons after 48 hr exposure to vehicle, BDE-47 (10 μM), or BDE-49 (5 μM or 10 μM) between 6 and 8 days in vitro. Compared with neurons exposed to vehicle and BDE-47, BDE-49–exposed neurons showed pronounced morphological changes, including fasciculation and decreased soma diameter; bar = 150 μm. (E) BDE-49 (5 and 10 μM) and 6-OH-BDE-47 (10 μM), but not BDE-47, significantly decreased cell viability as assessed using the MTS assay. *p < 0.05 compared with vehicle control by ANOVA with post hoc Tukey’s test. **p < 0.01.