Impact of Plasticizer on the Intestinal Epithelial Integrity and Tissue-Repairing Ability within Cells in the Proximity of the Human Gut Microbiome

Abstract

Toxicological research into the impact of plasticizer on different organs has been reported in the past few decades, while their effects on shifting the gut microbiota and immune cells homeostasis in zebrafish were only studied recently. However, studies on the impact of plasticizer on human gut microbiota are scarce. In this study, we co-incubated healthy human fecal microbiota with different concentrations of Di(2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DINP), analyzed microbial composition by 16S rDNA sequencing, and compared the influence of their derived microbiomes on the human enterocyte (HT-29) and murine macrophage (RAW264.7) cell lines. Microbial diversity is reduced by DEHP treatment in a dose-dependent manner. DEHP treatment reduced the phyla Firmicutes/Bacteroidetes ratio, while DINP treatment promoted Proteobacteria. Expressions of tight/adherens junction genes in HT-29 and anti-inflammatory genes in RAW264.7 were down-regulated by plasticizer-co-incubated microbiota derived metabolites. Overall, it is observed that selected plasticizers at high dosages can induce compositional changes in human microbiota. Metabolites from such altered microbiota could affect the tight junction integrity of the intestinal epithelium and upset macrophage differentiation homeostasis in proximity. Chronic exposure to these plasticizers may promote risks of dysbiosis, leaky gut or the exacerbation of intestinal inflammation.

Keywords: di(2-ethylhexyl) phthalate (DEHP); di-iso-nonyl phthalate (DINP); dysbiosis; gut microbiota; intestinal epithelial cell; intestinal inflammation; leaky gut; macrophage; phthalate plasticizer.

Figure 1

Experimental workflow. After 24 h fermentation, sterilized luminal extracts from fermenters treated with no plasticizer control, DEHP or DINP were used for cell assays in vitro utilizing human intestinal epithelial cell line HT-29 and murine macrophage cell line RAW264.7.

Figure 2

PCoA plot showing no DEHP control, DEHP-D1, DEHP-D2 and DEHP-D3 microbiota compositions at 0 h (DE_0 to DE_3) and 24 h (DE_4 to DE_7).

Figure 3

Relative abundance at (A) phylum level and (B) genus level in resultant microbiota of no DEHP control (DE_4), DEHP-D1 (DE_5), DEHP-D2 (DE_6) and DEHP-D3 (DE_7) fermenters.

Figure 4

PCoA plot showing no DINP control, DINP-D1, DINP-D2 and DINP-D3 microbiota compositions at 0 h (DI_0 to DI_3) and 24 h (DI_4 to DI_7).

Figure 5

Relative abundance at (A) phylum level and (B) genus level in resultant microbiota of no DINP control (DI_4), DINP-D1 (DI_5), DINP-D2 (DI_6) and DINP-D3 (DI_7) fermenters.

Figure 6

Relative quantification of TJ/AJ- and ROS-related gene mRNA levels in HT-29 after treatment of (A) DEHP experiment fermenters’ extracts and (B) DINP experiment fermenters’ extracts. Expression in cells treated with luminal extracts from no plasticizer fermenter was normalized as 1. t-test, * p < 0.05, and ** p < 0.01.

Figure 7

Relative quantification of cytokine mRNA levels in RAW264.7 after treatment of extracts from (A) DEHP fermenters and (B) DINP fermenters. Expression levels of mRNA in cells treated with luminal extracts from no plasticizer fermenter were normalized as 1. t-test, the data were produced in triplicate and t-test was used for statistical analysis. * p < 0.05, and ** p < 0.01.

Figure 8

Expressions of (A) E-cadherin, (B) JAM-A and (C) Claudin-4 in HT-29 after luminal extract treatments. Expression levels of protein in cells treated with luminal extracts from no plasticizer fermenter were normalized as 1. The data were produced in triplicate and t-test was used for statistical analysis. * p < 0.05, and ** p < 0.01.

Figure 9

Claudin-2 expression in HT-29 after (A) DEHP and (B) DINP experimental luminal extract treatments. Expression of Claudin-2 in cells treated with luminal extracts from no plasticizer fermenter was normalized as 1. Image width = 330 µm. t-test, ns: not significant, * p < 0.05, ** p < 0.01 and *** p < 0.005.