Polybrominated Diphenyl Ethers and Gut Microbiome Modulate Metabolic Syndrome-Related Aqueous Metabolites in Mice

Abstract

Polybrominated diphenyl ethers (PBDEs) are persistent environmental toxicants associated with increased risk for metabolic syndrome. Intermediary metabolism is influenced by the intestinal microbiome. To test the hypothesis that PBDEs reduce host-beneficial intermediary metabolites in an intestinal microbiome-dependent manner, 9-week old male conventional (CV) and germ-free (GF) C57BL/6 mice were orally gavaged once daily with vehicle, BDE-47, or BDE-99 (100 μmol/kg) for 4 days. Intestinal microbiome (16S rDNA sequencing), liver transcriptome (RNA-Seq), and intermediary metabolites in serum, liver, as well as small and large intestinal contents (SIC and LIC; LC-MS) were examined. Changes in intermediary metabolite abundances in serum, liver, and SIC, were observed under basal conditions (CV vs. GF mice) and by PBDE exposure. PBDEs altered the largest number of metabolites in the LIC; most were regulated by PBDEs in GF conditions. Importantly, intestinal microbiome was necessary for PBDE-mediated decreases in branched-chain and aromatic amino acid metabolites, including 3-indolepropionic acid, a tryptophan metabolite recently shown to be protective against inflammation and diabetes. Gene-metabolite networks revealed a positive association between the hepatic glycan synthesis gene α-1,6-mannosyltransferase (Alg12) mRNA and mannose, which are important for protein glycosylation. Glycome changes have been observed in patients with metabolic syndrome. In LIC of CV mice, 23 bacterial taxa were regulated by PBDEs. Correlations of certain taxa with distinct serum metabolites further highlight a modulatory role of the microbiome in mediating PBDE effects. In summary, PBDEs impact intermediary metabolism in an intestinal microbiome-dependent manner, suggesting that dysbiosis may contribute to PBDE-mediated toxicities that include metabolic syndrome.

Fig. 1.

Two-way hierarchical clustering dendrogram of aqueous metabolites measured using LC-MS that were differentially regulated across treatment groups in serum of GF and CV mice following exposures to BDE-47 and BDE-99. Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized mean metabolite relative abundances (z-scores) and blue lower. Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE treated groups within enterotype (adjusted P value <0.05, Tukey’s HSD post-hoc test). Caret signs (^) represent statistically significant baseline differences between CV and GF mice. Percent change from CVCO are shown for treatment groups with significant changes.

Fig. 2.

Two-way hierarchical clustering dendrogram of aqueous metabolites measured using LC-MS that were differentially regulated across treatment groups in liver of GF and CV mice following exposures to BDE-47 and BDE-99. Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized treatment group mean metabolite relative abundances (z-scores) and blue lower. Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE treated groups within enterotype (adjusted P value <0.05, Tukey’s HSD post-hoc test). Caret signs (^) represent statistically significant baseline differences between CV and GF mice. Percent change from CVCO are shown for treatment groups with significant changes.

Fig. 3.

Two-way hierarchical clustering dendrogram of aqueous metabolites measured using LC-MS that were differentially regulated across treatment groups in SIC of GF and CV mice following exposures to BDE-47 and BDE-99. Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized treatment group mean metabolite relative abundances (z-scores) and blue lower. Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE-treated groups within enterotype (adjusted P value <0.05, Tukey’s HSD post-hoc test). Caret signs (^) represent statistically significant baseline differences between CV and GF mice. Percent change from CVCO are shown for treatment groups with significant changes.

Fig. 4.

Two-way hierarchical clustering dendrogram of aqueous metabolites measured using LC-MS that were differentially regulated across treatment groups in LIC of GF and CV mice following exposures to BDE-47 and BDE-99. Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized treatment group mean metabolite relative abundances (z-scores) and blue lower. Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE treated groups within enterotype (adjusted P value <0.05, Tukey’s HSD post-hoc test). Caret signs (^) represent statistically significant baseline differences between CV and GF mice. Percent change from CVCO are shown for treatment groups with significant changes.

Fig. 5.

Two-way hierarchical clustering dendrogram of carbohydrate and glycan metabolism genes that were differentially regulated across treatment groups in liver of GF and CV mice following exposures to BDE-47 and BDE-99 (A). Gene expression was quantified using RNA-Seq as previously reported (Li et al., 2017) and can be accessed through NCBI Gene Expression Omnibus (GSE101650). Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized mean metabolite relative abundances and blue lower. Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE-treated groups (FDR adjusted P value <0.05). Caret signs (^) represent statistically significant baseline differences between CV and GF mice. Network of differentially regulated carbohydrate (GCK) and glycan (ALG12) metabolism genes (circles) and the significantly altered carbohydrate metabolite mannose (square), which was quantified using LC-MS, in liver (B). Mouse orthologs for human genes are listed in mixed case. Pearson correlation of log2 abundances of mannose and Alg2 across treatment group means (C). Means and S.E. of Alg12 and mannose for each treatment group (D). Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE-treated groups (FDR adjusted P value <0.05, Cuffdiff). Caret signs (^) represent statistically significant baseline differences between CV and GF mice.

Fig. 6.

Two-way hierarchical clustering dendrogram of amino acid–metabolism genes that were differentially regulated across treatment groups in liver of GF and CV mice following exposures to BDE-47 and BDE-99 (A). Gene expression was quantified using RNA-Seq as previously reported (Li et al., 2017) and can be accessed through NCBI Gene Expression Omnibus (GEO; GSE101650). Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized mean metabolite relative abundances and blue lower. Network of differentially regulated amino acid metabolism genes [dopa decarboxylase (DDC), NQO1, GNMT, CYP1A2, and CYP3A5] (circles) and significantly altered aqueous metabolites (glycine and glutathione) (squares), which were measured using LC-MS, in liver (B). Mouse orthologs for human genes are listed in mixed case. Two-way hierarchical clustering dendrogram of vitamin and cofactor metabolism genes that were significantly altered across treatment groups in liver of GF and CV mice following exposures to BDE-47 and BDE-99 (C). Gene expression was measured using RNA-Seq as previously reported (Li et al., 2017) and can be accessed through NCBI GEO (GSE101650). Euclidian distance and complete linkage were used to generate the dendrogram. Red indicates higher standardized mean metabolite relative abundances and blue lower. Network of differentially regulated vitamin metabolism genes (CYP2D6 and CYP3A4) (circles) and the significantly altered aqueous metabolite nicotinamide (square), which was measured using LC-MS, in liver (square) (D). Asterisks (*) represent statistically significant differences between corn oil-treated and PBDE-treated groups (FDR adjusted P value <0.05). Caret signs (^) represent statistically significant baseline differences between CV and GF mice.

Fig. 7.

Pearson correlation matrix of differentially regulated gut-microbiome operational taxonomical units (identified and quantified using 16S rRNA sequencing) and serum metabolites (quantified using LC-MS). Red indicates stronger positive correlations and blue stronger negative.