Serum Metabolomic Profiles in Neonatal Mice following Oral Brominated Flame Retardant Exposures to Hexabromocyclododecane (HBCD) Alpha, Gamma, and Commercial Mixture
Hexabromocyclododecane (HBCD) is a high production volume brominated flame retardant added to building insulation foams, electronics, and textiles. HBCD is a commercial mixture (CM-HBCD) composed of three main stereoisomers: α-HBCD (10%), β-HBCD (10%), and γ-HBCD (80%). A shift from the dominant stereoisomer γ-HBCD to α-HBCD is detected in humans and wildlife.
Considering CM-HBCD has been implicated in neurodevelopment and endocrine disruption, with expected metabolism perturbations, we performed metabolomics on mice serum obtained during a window-of-developmental neurotoxicity to draw correlations between early-life exposures and developmental outcomes and to predict health risks. METHODS: Six female C57BL/6 mice at postnatal day (PND) 10 were administered a single gavage dose of α-, γ-, or CM-HBCD at 3, 10, and 30 mg/kg. Nuclear magnetic resonance metabolomics was used to analyze 60 μL serum aliquots of blood collected 4 days post-oral exposure.
Infantile mice exposed to α-, γ-, or CM-HBCD demonstrated differences in endogenous metabolites by treatment and dose groups, including metabolites involved in glycolysis, gluconeogenesis, lipid metabolism, citric acid cycle, and neurodevelopment. Ketone bodies, 3-hydroxybutyrate, and acetoacetate, were nonstatistically elevated, when compared with mean control levels, in all treatment and dose groups, while glucose, pyruvate, and alanine varied. Acetoacetate was significantly increased in the 10 mg/kg α-HBCD and was nonsignificantly decreased with CM-HBCD. A third ketone body, acetone, was significantly lower in the 30 mg/kg α-HBCD group with significant increases in pyruvate at the same treatment and dose group. Metabolites significant in differentiating treatment and dose groups were also identified, including decreases in amino acids glutamate (excitatory neurotransmitter in learning and memory) and phenylalanine (neurotransmitter precursor) after α-HBCD and γ-HBCD exposure, respectively.
We demonstrated that 4 days following a single neonatal oral exposure to α-, γ-, and CM-HBCD resulted in different serum metabolomic profiles, indicating stereoisomer- and mixture-specific effects and possible mechanisms of action.
Figure 1. 950 MHz 1H NMR spectrum of metabolites.
950 MHz 1H NMR spectrum of metabolites in a representative 60-μL mouse serum preparation. Signals for metabolites at higher concentration are labeled.
- Figure 1 (126 KB)
Figure 2. Multivariate analysis (PLS-DA score plot) of bin data.
Multivariate analysis (PLS-DA score plot) of bin data obtained for serum from individual (n = 6/treatment group) mice exposed to vehicle controls (green), or 30 mg/kg α-HBCD (blue), γ-HBCD (orange), or CM-HBCD (red) [R2X = 0.713; R2Y = 0.379; Q2 = 0.0307].
- Figure 2 (284 KB)
Figure 3. Score plot of PLS-DA analysis of metabolite concentration data for serum samples.
Score plot of PLS-DA analysis of metabolite concentration data for serum samples from mice administered (A) 0, 3, 10, and 30 mg/kg doses of α-HBCD, indicating separation between control (green) and dose groups [R2X = 0.664; R2Y = 0.693; Q2 = –0.11]; (B) vehicle control or 30 mg/kg α-HBCD showing clear separation of study groups (green, left, control; blue, right, α-HBCD) [R2X = 0.76; R2Y = 0.814; Q2 = 0.13]; (C) vehicle controls or 30 mg/kg α-HBCD showing improvement of separation of dose group from control group (green, left, control; blue, right, α-HBCD) [R2X = 0.504; R2Y = 0.915; Q2 = 0.538] was achieved using subset of metabolites that best defined groups (VIP ≥ 1.0 with 95% confidence interval that did not include 0). See Table 1 for numbers of mice per group.
- Figure 3 (367 KB)
Figure 4. PLS-DA plot of metabolite concentrations derived from serum samples from mice.
PLS-DA plot of metabolite concentrations derived from serum samples from mice administered vehicle control (green), or high dose (30 mg/kg) CM-HBCD (red), α-HBCD (blue), or γ-HBCD (orange) [R2X = 0.642; R2Y = 0.345; Q2 = –0.186] (n = 6 for all groups).
- Figure 4 (196 KB)
Figure 5. Endogenous serum metabolite increase and decrease.
Endogenous serum metabolite increase and decrease after administration of CM-HBCD, α-HBCD, or γ-HBCD at 30 mg/kg, 3 mg/kg and 30 mg/kg, respectively, measured 4 days after an acute dose in infant mice. Contributions of each stereoisomer to the mixture at CM-HBCD relevant ratio ~ 10%:90%::3 mg/kg:30 mg/kg::α-HBCD:γ-HBCD. Arrows indicate metabolites that were consistently increased (↑) or decreased (↓), or that had mixed responses (↑↓).
- Figure 5 (168 KB)
Table 1. Metabolites that were significantly higher or lower than controls.
Metabolites that were significantly higher or lower than controls in 60 μL serum aliquots from at least one group of HBCD-exposed mice.
- Table 1 (154 KB)
Table 2. Metabolites important to the differentiation of treatment and control groups.
Metabolites important to the differentiation of treatment and control groups, using PLD-DA analysis (VIP ≥ 1.0 with a jack-knife confidence interval that did not include 0).
- Table 2 (93 KB)
- Supplemental Material (436 KB)