Bisphenol F exposure induced vascular toxicity through intestinal microbiota imbalance

Abstract

Introduction: Bisphenol F (BPF), a common substitute for bisphenol A (BPA), has documented toxicity in multiple organs, but its vascular effects remain unclear. This study investigated BPF's role in vascular calcification (VC) and underlying mechanisms.

Methods: Differences in the intestinal microbiota were analyzed by 16S ribosomal RNA gene sequencing. Metabolites were analyzed using liquid chromatography-mass spectrometry. Faecal microbiota transplantation and antibiotic treatment experiments were performed to evaluate the functions of the intestinal microbiota in VC.

Results: We enrolled consecutively 57 patients. Patients were assigned to a calcification group (30 patients) and a non-calcification group (27 patients) based on the presence or absence of calcification in the thoracic aorta wall. The results showed that patients with vascular calcification (VC) had higher levels of bisphenol F (BPF), bisphenol S (BPS) and bisphenol A (BPA) in the fecal samples than patients without VC. The thoracic aortic calcification score was significantly positively correlated with the BPF (Spearman r = 0.4935, p < 0.001), BPA (Spearman r = 0.2860, p < 0.05) and BPS (Spearman r = 0.2650, p < 0.05). We then explored the effects of BPF exposure on normal and vitamin D3 + nicotine (VDN)-treated rats. BPF exposure induced mild VC in normal rats and aggravated VC in VDN-treated rats. BPF exposure disturbed the gut microbiota and promoted inflammatory responses.

Conclusion: The results here elucidate the mechanism underlying BPF-triggered or BPF-aggravated VC through the gut-vascular axis and provide a theoretical basis for cardiovascular disease risk assessment in humans.

Keywords: bisphenol F; faecal microbiota transplantation; gut microbiota; inflammation; short-chain fatty acids; vascular calcification.

Figure 1

Fecal bisphenols in patients with VC. (A) Comparison of the abundances of bisphenols in fecal samples from patients with and without VC. (B–D) Spearman’s correlation analyses of BPF, BPA and BPS levels in fecal samples with TAC scores, respectively. (E) Predictive ability of BPF, BPA and BPS levels in fecal samples for vascular calcification, respectively. Continuous and non-normal data were described as median (interquartile range). The Mann–Whitney U test was used to analyze the differences of two groups. **p < 0.01, ***p < 0.001. BPA, Bisphenol A; BPF, Bisphenol F; BPS, Bisphenol S; TAC, Thoracic aortic calcification; VC, Vascular calcification.

Figure 2

Association between fecal bisphenols and gut microbiota in patients with VC. (A) PCoA plot showing changes in the β-diversity of the gut microbiota in patients with and without VC. (B) α-diversity of the gut microbiota (Shannon index). (C) α-diversity of the gut microbiota (Observed ASVs). (D) LEfSe (linear discriminant analysis (LDA) coupled with exact size measurements) for the analysis of differences between the gut microbiota of VC group and Non-VC group (LDA > 3.5). (E) Spearman’s correlation analysis of the relationship of the intestinal microbiota with BPF, BPA, BPS, acetate, propionate, butyrate and TAC scores. Negative and positive correlations are denoted in blue and red, respectively. The adjusted p-value was calculated with the Benjamini–Hochberg false discovery rate (FDR) method to correct the multiple comparisons and Spearman’s correlations. (F) Comparison of the differences in fecal acetate, propionate, and butyrate levels, respectively. Continuous data were described as mean ± standard deviation or median (interquartile range) as appropriate. The t-test for normally distributed data and with the Mann–Whitney U test for non-normally distributed data. ^#p < 0.25, ^##p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns > 0.05.

Figure 3

BPF exposure promoted VC and disturbed the gut microbiota. (A) Alizarin red staining of ascending aorta calcification (original magnification, 40×) (n = 5). (B) Alizarin red staining of ascending aorta calcification (original magnification, 200×) (n = 5). (C) Relative quantification of calcium content for the vessel sections of the ascending aorta (n = 5). (D) PCoA plot showing changes in the β-diversity of the gut microbiota after BPF treatment (n = 5). (E) α-diversity of the gut microbiota (Observed ASVs) (n = 4 ~ 5). (F) α-diversity of the gut microbiota (Shannon index) (n = 5). (G) LEfSe [linear discriminant analysis (LDA) coupled with exact size measurements] for the analysis of differences between the gut microbiota of the normal group and the normal + BPF group (LDA > 3.5) (n = 5). (H) LEfSe analysis of the differences between the gut microbiota of the VC group and the VC + BPF group (LDA > 3.5) (n = 5). The t-test was used to analyze the differences of two groups. ns > 0.05, **p < 0.01, ***p < 0.001.

Figure 4

BPF exposure promoted systemic inflammatory responses. (A) Comparison of the differences in plasma LPS levels (n = 5). (B–D) Comparison of the differences in plasma levels of IL-6, IL-1β and TNF-α, respectively (n = 5). (E–G) Comparison of the differences in fecal acetate, propionate, and butyrate levels, respectively (n = 5). The t-test for normally distributed data and with the Mann–Whitney U test for non-normally distributed data. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

BPF promotes VC through the gut microbiota. (A) Alizarin red staining of ascending aorta calcification (original magnification, 40×) (n = 4). (B) Alizarin red staining of ascending aorta calcification (original magnification, 200×) (n = 4). (C) Relative quantification of calcium content for the vessel sections of the ascending aorta (n = 4). (D) Comparison of the differences in plasma LPS levels (n = 4). (E–G) Comparison of the differences in plasma levels of IL-6, IL-1β and TNF-α, respectively (n = 4). The t-test was used to analyze the differences of two groups. *p < 0.05, **p < 0.01, ***p < 0.001.