Toxic effects of the food additives titanium dioxide and silica on the murine intestinal tract: Mechanisms related to intestinal barrier dysfunction involved by gut microbiota

https://doi.org/10.1016/j.etap.2020.103485Get rights and content

Highlights

  • Food grade TiO2 micro-/nanoparticles and SiO2 nanoparticles can cause toxic effects to the murine intestine.

  • All three particles caused inflammatory damage to the intestine.

  • The particles altered the gut microbiota, especially mucus-associated bacteria.

  • Disruption of the intestinal mucus barrier and increased LPS production are key mechanisms.

  • Nano-TiO2 activated the intestinal PKC/TLR4/NF-κB signalling pathway.

Abstract

This study aimed to compare the effects of three food-grade particles (micro-TiO2, nano-TiO2, and nano-SiO2) on the murine intestinal tract and to investigate their potential mechanisms of action. A 28-day oral exposure murine model was established. Samples of blood, intestinal tissues and colon contents were collected for detection. The results showed that all three particles could cause inflammatory damage to the intestine, with nano-TiO2 showing the strongest effects. Exposure also led to changes in gut microbiota, especially mucus-associated bacteria. Our results suggest that the toxic effects on the intestine were due to reduced intestinal mucus barrier function and an increase in metabolite lipopolysaccharides which activated the expression of inflammatory factors downstream. In mice exposed to nano-TiO2, the intestinal PKC/TLR4/NF-κB signalling pathway was activated. These findings will raise awareness of toxicities associated with the use of food-grade TiO2 and SiO2.

Introduction

Titanium dioxide (TiO2) is a white pigment commonly used as an additive in human foods to improve the color, quality and taste of food. Food-grade TiO2 in the form of E171 and INS171 exists in a number of food products, including confectionery items, candy coatings, cheeses, sauces, skim milk, ice-cream and chewing gum (Baan et al., 2006; Skocaj et al., 2011; Bachler et al., 2015; Rompelberg et al., 2016; Dudefoi et al., 2017a). Food-grade TiO2 particles range from nanometre-sized (nanoparticles) to micrometre-sized (microparticles), and approximately 36 % of TiO2 particles are less than 100 nm in at least one dimension (Weir et al., 2012; Chen et al., 2013; Yang et al., 2014; Dudefoi et al., 2017b). Exposure to TiO2 in the United Kingdom has been estimated at 2−3 mg/kg body weight (bw)/day for children under the age of 10 years and 1 mg/kg bw/day for other consumer age groups (Weir et al., 2012; Bachler et al., 2015). Based on food portion sizes, an individual’s daily intake of TiO2 could exceed 200 mg from a single product containing detectable titanium (Miranda et al., 2000). Although daily intake would vary greatly depending on an individual’s diet, 5.4 mg per adult in the United Kingdom has also been estimated (Ministry of Agriculture, Fisheries and Food, 1993; Miranda et al., 2000). At present, the TiO2 additive E171 is registered and labelled as an inactive ingredient in foods (Jovanović et al., 2016). However, based on possible toxicity of TiO2 nanoparticles (Gao et al., 2012; M. Wang et al., 2013; Y. Wang et al., 2013), the health risks associated with food-grade TiO2 particles have attracted the attention of scientists and relevant governing bodies worldwide (European Commission, 2013; Martirosyan and Schneider, 2014; Jovanović, 2015; Ismael et al., 2016).

Silicon dioxide (SiO2) is another typical food-grade nanoparticle registered within the European Union as food additive E551. It acts as an anticaking agent to maintain flow properties in powder products and to thicken different types of paste (OECD, 2004; EU Official Journal of the European Union, 2008; Peters et al., 2012). SiO2 nanoparticles are produced in bulk for food applications, and a large portion of SiO2 contained in foods is within the nanometre size range (Dekkers et al., 2011; Peters et al., 2012). The intake of SiO2 nanoparticles from food products has been estimated at 124 mg/day, which equates to 1.8 mg/kg bw/day for an adult of 70 kg (Dekkers et al., 2011).

At present, only a few reports have described the potential risks and adverse effects associated with food-grade micro- and nanoparticles in vivo. Oral toxicity studies of food-grade TiO2 and SiO2 are particularly limited, and there remains a lack of sufficient toxicological data to address the safety concerns of these nanoparticles for human consumption (Bergin and Witzmann, 2013; Dekkers et al., 2013; European Commission, 2013; Bouwmeester et al., 2014, 2018). Recent studies analysing changes in gut microbiota caused by disease have received significant attention due to direct implications for fields such as microbiology, medicine, genetics and physiology (Arumugam et al., 2011; Lepage et al., 2011; Yan et al., 2011; Karlsson et al., 2012; Rubin et al., 2012; Qin et al., 2012).

Our previous findings suggested that gut microbiota data could be used to evaluate the toxicology of nanoparticles since it serves as a highly-sensitive target for assessing adverse effects. In this study, we aimed to evaluate the toxicities of oral food-grade micro- and nanoparticles (TiO2 micro-/nanoparticles and SiO2 nanoparticles) in mice, and their impact on the intestinal systems were measured and compared between the three types of micro-/nanoparticles. We also investigated potential mechanisms of action. This information will supplement toxicological data for risk assessment of micro- and nanoparticles used in human foods.

Section snippets

Characterisation of particles and suspension preparation

Food-grade TiO2 micro-/nanoparticles in the form of anatase (purity > 99.1 %, 0.25 μm and 20 nm, respectively) were purchased from Jianghu Co. Ltd (Shang Hai, China) and SiO2 nanoparticles as amorphous silica (purity > 99.8 %, 12 nm) were purchased from Evonik Industries AG (Essen, Germany). All were in the form of white powder. Their physicochemical properties were assessed (Fig. 1 and Fig. S1) and their morphological characteristics were observed using a scanning electron microscope (SEM,

Changes in serological indicators after particle exposure

IL-1α levels in the serum in all particle exposure groups increased compared to those in the control group, and were significantly elevated in the 160 mg/kg micro- and nano-TiO2 groups (p < 0.05; Table 1). IL-1α levels were also significantly higher in the 160 mg/kg nano-TiO2 group than in groups exposed to micro-TiO2 and nano-SiO2 at the same dose. CRP levels in the serum tended to increase after oral exposure to the three particle types and were significantly higher in the 160 mg/kg nano-TiO2

Discussion

In this study, key serological markers were selected to evaluate the possible toxicity of the particles, including IL-1α, CRP, Glob, TP, TG and GLU. Except for GLU, the other indicators in the 160 mg/kg nano-TiO2 group were significantly higher than those in the control group, and IL-1α, TG and GLU increased significantly in the 160 mg/kg micro-TiO2 group, while nano-SiO2 group at 160 mg/kg did not show any significant changes in the indicators tested. When the exposure dose of nano-SiO2

Conclusions

This study explains the effects of three varieties of food-grade micro-/nanoparticles in terms of changes to gut microbiota and effects on the intestinal mucosal barrier in mice. Our results suggest that TiO2 and SiO2 food-grade micro-/nanoparticles have specific toxic effects on the intestines of mice by oral exposure. They can activate intestinal infection and inflammatory responses by diminishing the function of intestinal mucus barrier. This would likely occur via disordered gut microbiota,

CRediT authorship contribution statement

Jun Yan: Writing - original draft. Degang Wang: Investigation. Kang Li: Visualization. Qi Chen: Data curation. Wenqing Lai: Investigation. Lei Tian: Investigation. Bencheng Lin: Data curation. Yizhe Tan: Data curation. Xiaohua Liu: Supervision. Zhuge Xi: Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21607172), the grant (No. AWS16J004) and the National Key Research and Development Program (2016YFC0206900). We want to thank Genergy Biotechnology Limited Corporation (Shanghai, China) for the 16S rDNA Illumina Miseq analysis.s

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