Nano Today
Nanosafety evaluation through feces: A comparison between selenium nanoparticles and selenite in rats
Graphical abstract
Nanosafety evaluation is necessary not only for nanomaterials R&D but also as a cornerstone for legal regulation. In this study, we proposed a non-invasive method for nanosafety evaluation using Se0NPs and Na2SeO3 as examples. 16S rRNA and LC–MS were used to compare their impacts on intestinal microbiota and metabolites in fecal samples, which agreed well with their toxicity. ICP-MS, XRF and XAS were used to confirm these results. This method may also be used for the screening of the nanosafety of other nanomaterials.
Introduction
Nanomaterials show unique physical and chemical properties, which have been widely used in electronics, engineering, optics, environmental remediation and medical systems, etc [1,2]. On the other hand, safety concerns are also raised about their potential adverse effects and a new discipline regarding the safety of nanomaterials, i.e. nanotoxicology is formed [3]. Nanosafety evaluation is necessary not only for nanoscience research and development but also as a cornerstone for legal regulation [1]. Cellular, tissue models as well as chemoinformatics like adverse outcome pathways can be used for nanosafety evaluation but animal models are generally required to prove these in vitro results [4]. Nanosafety evaluation using animal models usually requires the sacrifice of animals. Non-invasive methods for nanosafety evaluation in animal models without sacrificing animals are always desired.
Selenium (Se) is an essential trace element for animals and human beings [5,6]. However, high dose of Se are also toxic [7]. The toxicity of Se depends its chemical form: organic Se such as selenomethionine (SeMet) is less toxic than inorganic Se such as selenide (Se2−), selenite (SeO32−) and selenate (SeO42−) [50]. The nano-sized elemental Se (Se0NPs) are even less toxic than other inorganic Se [8]. For example, the median lethal dose (LD50) of Se0NPs (113.0 mg/kg bw) in mice is seven times lower than that of selenite (15.7 mg/kg bw), and four times lower than that of SeMet (25.6 mg/kg bw) [9]. This has also been found in other animals, such as rats [10], chickens [11,12] and goats [10,13]. More importantly, comparable bioavailability of Se0NPs in rats with selenite was found as indicated through Se-dependent enzymes [8]. Due to its low toxicity and comparable bioavailability, Se0NPs have been used in the treatment of many diseases, such as cancers [14] and Huntington's disease [15].
The gastrointestinal system is the first physiological barrier to the circulation of blood, which includes mechanical barriers (made up of tight junctions between epithelial cells), chemical barriers (stomach acid, bile, digestive enzymes, lysozyme, etc.), biological barriers (microecosystems), and immune barriers (gut-associated lymphoid tissue and associated immunoglobulins) [16]. In recent years, more and more attention has been paid to intestinal microbiota, which play an important role in immune and cognitive development, metabolic processes and so on. It has been found that microbiota, such as sulfur-removing Monospora and Vibrio desulfidum, are directly involved in the oxidation, reduction and methylation of Se. Therefore, the interaction of Se with the intestinal flora may contribute to its toxicity. In addition, the gut flora produces half of the body's dopamine (DA) [17], as well as many neurotransmitters, such as GABA [18] and short-chain fatty acid [19], so it is also interesting to study the metabolites of Se after oral administration.
The distinct difference in toxicity between Se0NPs and other forms of Se made it an ideal candidate for nanosfety evaluation. Understanding the low toxicity with comparable bioavailability of Se0NPs may shed light on finding ways for nanosafety evaluation. In this study, a non-invasive protocol for nanosafety evaluation through feces using metagenomics and metabolomics together with metallomics was proposed using Se0NPs and Na2SeO3 as model compounds. Firstly, the changes of intestinal microbial community structure and metabolic products were studied using 16S rRNA and LC–MS; Secondly, the pathological morphology of intestinal tract (including large intestine and small intestine) and ADME were analyzed with ICP-MS, SR-XRF and XAS. Through these efforts, a non-invasive protocol using fecal samples as the testing matrix for nanosafety evaluation was developed.
Section snippets
Animals and Se exposure
To rule out the sex difference, only male SD rats were used in this study, which were purchased from Beijing Keyu Animal Breeding Center (weight 200 ± 20 g, 4 weeks old, certificate No.: SCXK-2012-0004). A total of 15 male rats were raised in 15 metabolic cages (1 rat/cage) with 12:12 h light-dark cycle. The room temperature was 20−23℃ with humidity of 40–70 %. The rats were given a standard diet and tap water ad libitum. The rats were randomly divided into 3 groups, ie, Na2SeO3, Se0NPs and the
Perturbance to intestinal microbiota by Se0NPs or Na2SeO3
It was found that 24 h after exposure with equal moles of Se0NPs and Na2SeO3, rats in the Na2SeO3 group had erected hairs with about 10 g of weight loss compared to the control group while rats in the Se0NPs group performed normally.
The changes of intestinal microbiota are shown in Fig. 1. Using the Wayne diagram to count the number of common and unique OTUs in the sample, it was showed that the composition of the number of OTUs in samples was similar, i.e. Se0NPs had 1068 OTUs and Na2SeO3 had
Different changes in intestinal microbes and metabolites in rats feces caused by Se0NPs and Na2SeO3
Compared with the control group, Na2SeO3 caused the much decreased abundance of beneficial bacteria than Se0NPs, such as Proteobacteria (Control 42 %, Se0NPs 31 %, Na2SeO3 27 %, Fig. 1C) [31], and the increased abundance of harmful bacteria, such as Eluaimicrobia (Control 8%, Se0NPs 40 %, Na2SeO3 52 %) [32], and the uncalssified (Control 9 %, Se0NPs 18 %, Na2SeO3 73 %). Se0NPs induced the increased abundance of beneficial bacteria, such as Firmicutes (Control 22 %, Se0NPs 40 %, Na2SeO3 38 %) [33
Conclusion
In all, through the proposed method combined with metagenomics, metabolomics and metallomics in this study as illustrated in Fig. 6, the two forms of Se could be distinctively differentiated: 1) Through 16S rRNA sequencing in feces, it was found that Na2SeO3 was more toxic than Se0NPs on gut microbial barrier with a large number of bacteria involved in the reduction of Na2SeO3 increased; 2) Through metabolomics study on fecal samples, it was found that Na2SeO3 brought more disrupted metabolites
CRediT authorship contribution statement
Xiaoying Lin: Data curation, Writing - original draft, Visualization, Investigation. Liming Wang: Data curation, Writing - original draft, Visualization, Investigation, Software, Validation. Jiating Zhao: Data curation, Writing - original draft, Visualization, Investigation. Lina He: Visualization, Investigation, Software, Validation. Liwei Cui: Visualization, Investigation, Software, Validation. Yuxi Gao: Supervision. Chunying Chen: Conceptualization, Methodology, Software, Supervision,
Declaration of Competing Interest
All authors declare there is no copyright disputes.
Acknowledgements
The authors are grateful for the financial support from the Ministry of Science and Technology of China (2016YFA0201600), National Natural Science Foundation of China (11975247, 11475496), Guizhou Department of Science and Technology (No. QKH-2016-2804), and Jilin Medical College Doctoral Research Startup Fund Project (JYBS2019011). We gratefully acknowledged the staff from BL15U1 beamline at Shanghai Synchrotron Radiation Facility and 4W1B at Beijing Synchrotron Radiation Facility for beam
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