Oral Exposure to Polystyrene Microplastics of Mice on a Normal or High-Fat Diet and Intestinal and Metabolic Outcomes

Abstract

Background: Microplastics (MPs) are small particles of plastic ($\le 5\mathrm{mm}$ in diameter). In recent years, oral exposure to MPs in living organisms has been a cause of concern. Leaky gut syndrome (LGS), associated with a high-fat diet (HFD) in mice, can increase the entry of foreign substances into the body through the intestinal mucosa.

Objectives: We aimed to evaluate the pathophysiology of intestinal outcomes associated with consuming a high-fat diet and simultaneous intake of MPs, focusing on endocrine and metabolic systems.

Methods: C57BL6/J mice were fed a normal diet (ND) or HFD with or without polystyrene MP for 4 wk to investigate differences in glucose tolerance, intestinal permeability, gut microbiota, as well as metabolites in serum, feces, and liver.

Results: In comparison with HFD mice, mice fed the HFD with MPs had higher blood glucose, serum lipid concentrations, and nonalcoholic fatty liver disease (NAFLD) activity scores. Permeability and goblet cell count of the small intestine (SI) in HFD-fed mice were higher and lower, respectively, than in ND-fed mice. There was no obvious difference in the number of inflammatory cells in the SI lamina propria between mice fed the ND and mice fed the ND with MP, but there were more inflammatory cells and fewer anti-inflammatory cells in mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. The expression of genes related to inflammation, long-chain fatty acid transporter, and ${\mathrm{Na}}^{+}/\text{glucose}$ cotransporter was significantly higher in mice fed the HFD with MPs than in mice fed the HFD without MPs. Furthermore, the genus Desulfovibrio was significantly more abundant in the intestines of mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. Muc2 gene expression was decreased when palmitic acid and microplastics were added to the murine intestinal epithelial cell line MODE-K cells, and Muc2 gene expression was increased when IL-22 was added.

Discussion: Our findings suggest that in this study, MP induced metabolic disturbances, such as diabetes and NAFLD, only in mice fed a high-fat diet. These findings suggest that LGS might have been triggered by HFD, causing MPs to be deposited in the intestinal mucosa, resulting in inflammation of the intestinal mucosal intrinsic layer and thereby altering nutrient absorption. These results highlight the need for reducing oral exposure to MPs through remedial environmental measures to improve metabolic disturbance under high-fat diet conditions. https://doi.org/10.1289/EHP11072.

Figure 1.

Body weight, food, and water intake; and iPGTT and ITT in mice exposed to ND or $\mathrm{H}\mathrm{F}\mathrm{D}\pm \mathrm{M}\mathrm{P}\mathrm{s}$ from 8 wk to 12 wk of age; intestinal permeability in mice exposed to ND or $\mathrm{H}\mathrm{F}\mathrm{D}\pm \mathrm{M}\mathrm{P}\mathrm{s}$ at 12 wk of age. (A) Exposure to ND or $\text{HFD}\pm \text{MPs}$ started at 8-weeks of age. (B) Body weight ($n=10$) and (C and D) intake of food and water over the course of the experiment ($n=10$). (E and F) Results of iPGTT ($2\mathrm{g}/\mathrm{kg}$ body weight) for 12-wk-old mice and the AUC analysis ($n=5$). Blood glucose was monitored 0, 15, 30, 60, and 120 min after injection. (G and H) Results of ITT ($0.75\mathrm{U}/\mathrm{kg}$ body weight) for 12-wk-old mice and the AUC analysis ($n=5$). Blood glucose was monitored 0, 15, 30, 60, and 120 min after injection. (I) GFP signals from GFP-MP before and after oral gavage of FITC-Dextran for 12-wk-old mice ($n=10$). Normalized to the levels of ND-fed mice. Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. *$p<0.05$, **$p<0.01$, ***$p<0.001$, and ****$p<0.0001$. Summary data can be found in Table S2. Note: ANOVA, analysis of variance; a.u., arbitrary unit; AUC, area under the curve; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HFD, high-fat diet; iPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; min, minutes; MPs, microplastics; ND, normal diet; SD, standard deviation.

Figure 1.

Body weight, food, and water intake; and iPGTT and ITT in mice exposed to ND or $\mathrm{H}\mathrm{F}\mathrm{D}\pm \mathrm{M}\mathrm{P}\mathrm{s}$ from 8 wk to 12 wk of age; intestinal permeability in mice exposed to ND or $\mathrm{H}\mathrm{F}\mathrm{D}\pm \mathrm{M}\mathrm{P}\mathrm{s}$ at 12 wk of age. (A) Exposure to ND or $\text{HFD}\pm \text{MPs}$ started at 8-weeks of age. (B) Body weight ($n=10$) and (C and D) intake of food and water over the course of the experiment ($n=10$). (E and F) Results of iPGTT ($2\mathrm{g}/\mathrm{kg}$ body weight) for 12-wk-old mice and the AUC analysis ($n=5$). Blood glucose was monitored 0, 15, 30, 60, and 120 min after injection. (G and H) Results of ITT ($0.75\mathrm{U}/\mathrm{kg}$ body weight) for 12-wk-old mice and the AUC analysis ($n=5$). Blood glucose was monitored 0, 15, 30, 60, and 120 min after injection. (I) GFP signals from GFP-MP before and after oral gavage of FITC-Dextran for 12-wk-old mice ($n=10$). Normalized to the levels of ND-fed mice. Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. *$p<0.05$, **$p<0.01$, ***$p<0.001$, and ****$p<0.0001$. Summary data can be found in Table S2. Note: ANOVA, analysis of variance; a.u., arbitrary unit; AUC, area under the curve; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HFD, high-fat diet; iPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; min, minutes; MPs, microplastics; ND, normal diet; SD, standard deviation.

Figure 2.

Histological evaluation of jejunum and immune cells involved in innate immunity in LPL of small intestine. (A) Representative images of HE- and PAS-stained, GFP-positive, and Muc2-immunostained jejunum sections. Jejunum tissue was collected at 12 wk of age. In the GFP fluorescence image, MPs are enlarged and indicated by arrows. The scale bars show $100\mathrm{\mu }\mathrm{m}$ ($50\mathrm{\mu }\mathrm{m}$ for Muc2 image). (B) Villus height ($n=10$). (C) Villus width ($n=10$). (D) Crypt depth ($n=10$). (E) Total goblet cells/area (${\mathrm{mm}}^2$ of jejunum) ($n=10$). (F) Mucus layer thickness ($n=10$). (G) GFP-positive area ($n=10$). Ratio of (H) ILC1s to CD45-positive cells, (I) T-bet positive ILC3s to CD45-positive cells, (J) M1 macrophages to M2 macrophages in the small intestine, and (K) ILC3s to CD45-positive cells ($n=10$ in each case). Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. Summary data can be found in Table S2. *$p<0.05$, **$p<0.01$, ***$p<0.001$, and ****$p<0.0001$. Note: ANOVA, analysis of variance; GFP, green fluorescent protein; H&E, hematoxylin and eosin; HFD, high-fat diet; ILCs, innate lymphoid cells; MPs, microplastics; ND, normal diet; PAS, periodic acid Schiff; SD, standard deviation.

Figure 2.

Histological evaluation of jejunum and immune cells involved in innate immunity in LPL of small intestine. (A) Representative images of HE- and PAS-stained, GFP-positive, and Muc2-immunostained jejunum sections. Jejunum tissue was collected at 12 wk of age. In the GFP fluorescence image, MPs are enlarged and indicated by arrows. The scale bars show $100\mathrm{\mu }\mathrm{m}$ ($50\mathrm{\mu }\mathrm{m}$ for Muc2 image). (B) Villus height ($n=10$). (C) Villus width ($n=10$). (D) Crypt depth ($n=10$). (E) Total goblet cells/area (${\mathrm{mm}}^2$ of jejunum) ($n=10$). (F) Mucus layer thickness ($n=10$). (G) GFP-positive area ($n=10$). Ratio of (H) ILC1s to CD45-positive cells, (I) T-bet positive ILC3s to CD45-positive cells, (J) M1 macrophages to M2 macrophages in the small intestine, and (K) ILC3s to CD45-positive cells ($n=10$ in each case). Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. Summary data can be found in Table S2. *$p<0.05$, **$p<0.01$, ***$p<0.001$, and ****$p<0.0001$. Note: ANOVA, analysis of variance; GFP, green fluorescent protein; H&E, hematoxylin and eosin; HFD, high-fat diet; ILCs, innate lymphoid cells; MPs, microplastics; ND, normal diet; PAS, periodic acid Schiff; SD, standard deviation.

Figure 3.

Metabolites in feces of mice exposed to ND or $\text{HFD}\pm \text{MPs}$ at 12 wk of age. The concentrations of (A) palmitic acid, (B) acetic acid, (C) propanoic acid, and (D) butanoic acid in the feces ($n=10$). Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. Summary data can be found in Table S2. **$p<0.01$ and ****$p<0.0001$. Note: ANOVA, analysis of variance; HFD, high-fat diet; MPs, microplastics; ND, normal diet; SD, standard deviation.

Figure 4.

Histological evaluation of liver and palmitic acid concentration in liver of mice exposed to ND or $\text{HFD}\pm \text{MPs}$ at 12 wk of age. (A and B) Absolute and relative liver weight ($n=10$). (C) Representative images of HE- and Oil Red O–stained liver sections. Liver tissue was collected at 12 wk of age. The scale bars show $100\mathrm{\mu }\mathrm{m}$. (D) Nonalcoholic fatty liver disease (NAFLD) activity scores ($n=10$). (E) Area of Oil Red O–stained region ($n=10$). (F) The concentration of palmitic acid in the liver ($n=10$). Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. Summary data can be found in Table S2. *$p<0.05$, **$p<0.01$, and ****$p<0.0001$. Note: HE, hematoxylin & eosin; HFD, high-fat diet; MPs, microplastics; ND, normal diet; SD, standard deviation.

Figure 5.

16s rRNA sequence of gut microbiota of mice exposed to ND or $\text{HFD}\pm \text{MPs}$ at 12 wk of age. (A) The relative abundance of phyla (%). Summary data for this graph shown in Table 2. (B) The number of OTUs ($n=6$). (C) Chao1 index ($n=6$). (D) Shannon index ($n=6$). (E) Gini-Simpson index ($n=6$). (F and H) LDA score (Log10) and LEfSe cladogram of ND and $\text{ND}+\text{MP}$ mice; (Red) taxa enriched in ND mice; (Green) taxa enriched in mice fed the ND with MPs ($n=6$). (G) LDA score and LEfSe cladogram of HFD and mice fed the HFD with MPs; (Red) taxa enriched in HFD mice; (Green) taxa enriched in mice fed the HFD with MPs ($n=6$). Only taxa with a significant LDA threshold value $>2$ are shown. Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. *$p<0.05$ and **$p<0.01$. Summary data can be found in Table S2. Note: ANOVA, analysis of variance; HFD, high-fat diet; LDA, linear discriminant analysis; LEfSe, LDA coupled with effect size measurements; MPs, microplastics; ND, normal diet; OTUs, operational taxonomic units; SD, standard deviation.

Figure 6.

Differences in Muc2 gene expression and MP accumulation in MODE-K cells exposed to MP and saturated fatty acids. (A) Relative mRNA expression of Muc2 in MODE-K cells without/with MP and PA ($n=10$). (B) Relative mRNA expression of Muc2 in MODE-K cells with MP without/with PA and IL-22 ($n=10$). (C) The fold differences in accumulation of MPs in MODE-K cells without/with MP and PA ($n=10$). (D) The fold differences in accumulation of MPs in MODE-K cells with MP without/with PA and IL-22 ($n=10$). Data are presented as $\text{mean}\pm \text{SD}$ values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. Summary data can be found in Table S2. *$p<0.05$, **$p<0.01$, ***$p<0.001$, and ****$p<0.0001$. Note: MP, microplastics; PA, palmitic acid; SD, standard deviation.