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Journal of Peking University (Health Sciences) ›› 2022, Vol. 54 ›› Issue (3): 468-476. doi: 10.19723/j.issn.1671-167X.2022.03.011

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Effects of nano titanium dioxide on gut microbiota based on human digestive tract microecology simulation system in vitro

Jia-he ZHANG,Jia-qi SHI,Zhang-jian CHEN,Guang JIA*()   

  1. Department of Occupational and Environmental Health Sciences, Peking University School of Public Health; Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, China
  • Received:2022-01-15 Online:2022-06-18 Published:2022-06-14
  • Contact: Guang JIA E-mail:jiaguangjia@bjmu.edu.cn
  • Supported by:
    National Key Research and Development Program of the Ministry of Science and Technology of China(2017YFC1600200);National Natural Science Foundation of China(81703257)

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Abstract:

Objective: To explore the effects of oral exposure to titanium dioxide nanoparticles (TiO2 NPs) on the composition and structure of human gut microbiota. Methods: The particle size, shape, crystal shape and degree of agglomeration in ultrapure water of TiO2 NPs were characterized. The in vitro human digestive tract microecological simulation system was established by simulating the fluid environment and physical conditions of stomach, small intestine and colon, and the stability of the simulation system was evaluated. The bacterial communities were extracted from human feces and cultured stably in the simulated system. They were exposed to 0, 20, 100 and 500 mg/L TiO2 NPs, respectively, and the bacterial fluids were collected after 24 h of exposure. The effect of TiO2 NPs on the composition and structure of human gut microbiota was analyzed by 16S rRNA sequencing technology. Linear discriminant analysis effect size (LEfSe) was used to screen differential bacteria, and the Kyoto encyclopedia of genes and genomes (KEGG) database for functional prediction. Results: The spherical and anatase TiO2 NPs were (25.12±5.64) nm in particle size, while in ultra-pure water hydrated particle size was (609.43±60.35) nm and Zeta potential was (-8.33±0.22) mV. The in vitro digestive tract microecology simulation system reached a relatively stable state after 24 hours, and the counts of Enterococci, Enterobacte-rium, and Lactobacillus reached (1.6±0.85)×107, (5.6±0.82)×107 and (2.7±1.32)×107, respectively. 16S rRNA sequencing results showed that compared with the control group, the number and evenness of gut microbiota were not significantly affected at phylum, class, order, family and genus levels in TiO2 NPs groups (20, 100 and 500 mg/L). The relative abundance of some species was significantly changed, and a total of 42 different bacteria were screened between the TiO2 NPs groups (20, 100 and 500 mg/L) and the control group [linear discriminant analysis(LDA) score>3], represented by Enterobacter, Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae and Clostridium. Further predictive analysis of gut microbiota function showed that TiO2 NPs might affect oxidative phosphorylation, energy meta-bolism, phosphonate and phosphonate metabolism, and methane metabolism (P < 0.05). Conclusion: In human digestive tract microecological simulation system, TiO2 NPs could significantly change the composition and structure of human gut microbiota, represented by Enterobacter and probiotics, and may further affect a variety of metabolism and function of the body.

Key words: Nanoparticles, Titanium dioxide, Gastrointestinal microbiome, Models, biological, 16S rRNA sequencing technology

CLC Number: 

  • R155.5

Figure 1

The flow diagram of the in vitro digestive tract microecological simulation system TiO2 NPs, titanium dioxide nanoparticles. CNC, computer numerical control."

Figure 2

The growth curves of three main bacterial in vitro digestive tract simulation system The mean and standard deviation of the bacterial count at each time point are marked."

Figure 3

Venn diagram (A) and petal diagram (B) of OTUs distribution of each sample The sample names and operational taxonomic units (OTUs) quantity of each part are marked in the figure. Low, 20 mg/L TiO2 NPs; Middle, 100 mg/L TiO2 NPs; High, 500 mg/L TiO2 NPs."

Figure 4

The relative abundance distribution and clustering of all samples in the TiO2 NPs exposed groups and control group were observed at phylum (A), class (B), order (C), family (D) and genus (E) levels In the proportional bar charts, each color represents a species of bacteria at the corresponding level (the same color represents different bacteria at diffe-rent levels), and the length of each color block represents the relative abundance of the corresponding bacteria. On the left side of the bar charts it is showed the results of clustering each sample according to the similarity degree of strain composition."

Figure 5

Alpha diversity index of TiO2 NPs exposed group and control group Low, 20 mg/L TiO2 NPs; Middle, 100 mg/L TiO2 NPs; High, 500 mg/L TiO2 NPs. *P < 0.01, #P < 0.001, vs. control."

Figure 6

PCA score (A) and PLS-DA score (B) of metabolites in the control group and the dose groups Low, 20 mg/L TiO2 NPs; Middle, 100 mg/L TiO2 NPs; High, 500 mg/L TiO2 NPs; PC, principal component; PCA, principal component analysis; PLS-DA, partial least squares discrimination analysis."

Figure 7

LEfSe analysis histogram (A) and branching evolution diagram (B) A, bacteria with significant difference in LEfSe analysis between TiO2 NPs exposed groups and control group (LDA score>3). The abscissa is LDA score, and the ordinate is bacterial name (sorted by group, in descending order of score). B, branching evolution of bacteria detected in the TiO2 NPs exposed groups and control group. The concentric circles from inside to outside represent different classification levels from the phylum to the species, in which each small circles represent the level and relative abundance of the classification bacteria. The pale-yellow dots said there was no significant diffe-rence between groups, while the rest of the color dots said significant differences. The colored areas represent all bacteria with significant differences on the branches of that region. Low, 20 mg/L TiO2 NPs; Middle, 100 mg/L TiO2 NPs; High, 500 mg/L TiO2 NPs; Phylum (p), class (c), order (o), family (f), genus (g), species (s) are marked in the brackets."

Figure 8

Differential function and pathways predicted by PICRUSt between TiO2 NPs exposed groups and control group The ordinate represents the 14 functions and pathways with statistical differences, and the ordinate shows the corresponding-lgP values."

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