Silica Nanoparticle Size Determines the Mechanisms Underlying the Inhibition of Iron Oxide Nanoparticle Uptake by Daphnia magna.

Aquatic environments are complicated systems that contain different types of nanoparticles (NPs). Nevertheless, recent studies of NP toxicity, and especially those that have focused on bioaccumulation have mostly investigated only a single type of NPs. Assessments of the environmental risks of NPs that do not consider co-exposure regimes may lead to inaccurate conclusions and ineffective environmental regulation. Thus, the present study examined the effects of differently sized silica NPs (SiO2 NPs) on the uptake of iron oxide NPs (Fe2O3 NPs) by the zooplankton Daphnia magna. Both SiO2 NPs and Fe2O3 NPs were well dispersed in the experimental medium without significant heteroaggregation. Although all three sizes of SiO2 NPs inhibited the uptake of Fe2O3 NPs, the underlying mechanisms differed. SiO2 NPs smaller than the average mesh size (∼200 nm) of the filtering apparatus of D. magna reduced the accumulation of Fe2O3 NPs through uptake competition, whereas larger SiO2 NPs inhibited the uptake of Fe2O3 NPs mainly by reducing the water filtration rate of the daphnids. Overall, in evaluations of the risks of NPs in the natural environment, the different mechanisms underlying the effects of NPs of different sizes on the uptake of dissimilar NPs should be considered.

Stimulated Raman Scattering Microscopy Reveals Bioaccumulation of Small Microplastics in Protozoa from Natural Waters.

Microplastics (MPs) are pollutants of global concern, and bioaccumulation determines their biological effects. Although microorganisms form a large fraction of our ecosystem's biomass and are important in biogeochemical cycling, their accumulation of MPs has never been confirmed in natural waters because current tools for field biological samples can detect only MPs > 10 µm. Here, we show that stimulated Raman scattering microscopy (SRS) can image and quantify the bioaccumulation of small MPs (<10 µm) in protozoa. Our label-free method, which differentiates MPs by their SRS spectra, detects individual and mixtures of different MPs (e.g., polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate, polystyrene, and poly(methyl methacrylate)) in protozoa. The ability of SRS to quantify cellular MP accumulation is similar to that of flow cytometry, a fluorescence-based method commonly used to determine cellular MP accumulation. Moreover, we discovered that protozoa in water samples from Yangtze River, Xianlin Wastewater Treatment Plant, Lake Taihu and the Pearl River Estuary accumulated MPs < 10 µm, but the proportion of MP-containing cells was low (∼2-5%). Our findings suggest that small MPs could potentially enter the food chain and transfer to organisms at higher trophic levels, posing environmental and health risks that deserve closer scrutiny.

Label-Free Imaging of Humic Substance Bioaccumulation by Pump-Probe Microscopy.

Humic substances (HS) are the most abundant forms of natural organic matter on the earth surface. Comprised of decomposed plant and animal materials rich in carbon, oxygen, hydrogen, nitrogen, and sulfur complexes, HS facilitate global carbon and nitrogen cycling and the transport of anthropogenic contaminants. While it is known that HS also interact with organisms at different trophic levels to produce beneficial and harmful effects whether HS exert these biological effects through accumulation remains unknown. Current radiolabeling techniques, which only detect the amount of accumulated radiolabels, cannot visualize the transport and accumulation behavior of HS. Here, using a label-free method based on pump-probe microscopy, we show HS entered the protozoan Tetrahymena thermophila, zebrafish embryos, and human cells and exerted direct effects on these organisms. HS accumulated in the nucleus of T. thermophila, chorion pore canals of zebrafish embryos, and nucleus of intestinal and lung cells in a concentration- and time-dependent way. Epigenetic and transcriptomics assays show HS altered chromatin accessibility and gene transcription in T. thermophila. In zebrafish larvae, HS induced neurotoxicity, altering spontaneous muscle contraction and locomotor activity. Detailed images showing HS accumulation in our study reveal new insights on the ecological and environmental behavior of HS.

Pre-exposure to Fe2O3 or TiO2 Nanoparticles Inhibits Subsequent Biological Uptake of 55Fe-Labeled Fe2O3 Nanoparticles.

Aquatic organisms are frequently exposed to various nanoparticles (NPs) in the natural environment. Thus, studies of NP bioaccumulation should include organisms that have been previously exposed to NPs. Our study investigated the effects of pre-exposure of Tetrahymena thermophila (T. thermophila) to Fe2O3 or TiO2 NPs on the protozoan's subsequent uptake of 55Fe-labeled Fe2O3 (55Fe2O3) NPs. Molecular mechanisms underlying the pre-exposure effects were explored in transcriptomic and metabolomic experiments. Pre-exposure to either NPs inhibited the subsequent uptake of 55Fe2O3 NPs. The results of the transcriptomic experiment indicated that NP pre-exposure influenced the expression of genes related to phagosomes and lysosomes and physiological processes such as glutathione and lipid metabolism, which are closely associated with the endocytosis of 55Fe2O3 NPs. The differentially expressed metabolites obtained from the metabolomic experiments showed an enrichment of energy metabolism and antioxidation pathways in T. thermophila pre-exposed to NPs. Together, these results demonstrate that the pre-exposure of T. thermophila to Fe2O3 or TiO2 NPs inhibited the protozoan's subsequent uptake of 55Fe2O3 NPs, possibly by mechanisms involving the alteration of endocytosis-related organelles, the induction of oxidative stress, and a lowering of the intracellular energy supply. Thus, NP pre-exposure represents a scenario which can inform increasingly realistic estimates of NP bioaccumulation.

Unbound Natural Organic Matter Competes with Nanoparticles for Internalization Receptors During Cell Uptake.

Natural organic matter (NOM) that forms coronas on the surface of engineered nanoparticles (NPs) affects their stability, bio-uptake, and toxicity. After corona formation, a large amount of unbound NOM remains in the environment and their effects on organismal uptake of NPs remain unknown. Here, the effects of unbound NOM on the uptake of polyacrylate-coated hematite NPs (HemNPs) by the protozoan Tetrahymena thermophila were examined. HemNPs were well-dispersed without any detectable NOM adsorption. Kinetics experiments showed that unbound NOM decreased the uptake of HemNPs with greater inhibition at lower concentrations of the particles in the presence of NOM of higher molecular weight. The unbound NOM suppressed clathrin-mediated endocytosis but not the phagocytosis of HemNPs. Confirmation of these events was obtained using label-free hyperspectral stimulated Raman spectroscopy imaging and dissipative particle dynamics simulation. Overall, the present study demonstrates that unbound NOM can compete with HemNPs for internalization receptors on the surface of T. thermophila and inhibit particle uptake, highlighting the need to consider the direct effects of unbound NOM in bioapplication studies and in safety evaluations of NPs.

Combined Toxicity of Silver Nanoparticles with Hematite or Plastic Nanoparticles toward Two Freshwater Algae.

In the natural environment, the interactions of different types of nanoparticles (NPs) may alter their toxicity, thus masking their true environmental effects. This study investigated the toxicity of silver NPs (AgNPs) combined with hematite (HemNPs) or polystyrene (PsNPs) NPs toward the freshwater algae Chlamydomonas reinhardtii and Ochromonas danica. The former has a cell wall and cannot internalize these NPs, while the latter without a cell wall can. Therefore, the toxicity of AgNPs toward C. reinhardtii was attributed to the released Ag ions, while AgNPs had direct toxic effects on O. danica. Moreover, nontoxic HemNPs ameliorated AgNP toxicity toward C. reinhardtii, by decreasing the bioavailability of Ag ions through adsorption. Despite their role as Ag-ion carriers, HemNPs still reduced the toxicity of AgNPs toward O. danica by competitively inhibiting AgNP uptake. In both algae, Ag accumulation fully accounted for the combined toxicity of AgNPs and HemNPs. However, the combined toxicity of AgNPs and PsNPs was complicated by their significant individual toxicities and the synergistic interactions of these particles with the algae, regardless of differences in Ag accumulation. Overall, in environmental assessments, considerations of the combined toxicity of dissimilar NPs will allow more accurate assessments of their environmental risks.

Algal Foods Reduce the Uptake of Hematite Nanoparticles by Downregulating Water Filtration in Daphnia magna.

Rapid developments in nanotechnology have led to the release of substantial amounts of nanoparticles (NPs) into aquatic environments, where many types of biotic particles are present and could potentially interact with the NPs. Nevertheless, how biotic particles may affect the bioaccumulation and toxicity of NPs remains largely unknown. In the present study, we investigated the effects of the green alga Chlamydomonas reinhardtii on the accumulation kinetics (uptake, assimilation, efflux) and toxicity of polyacrylate-coated hematite NPs (HemNPs), using Daphnia magna as the test organism. As a biotic particle and daphnid food, C. reinhardtii reduced the accumulation and toxicity of HemNPs in D. magna. The HemNPs were well-dispersed with little adsorption to the alga. Their decreased accumulation could thus be partly explained by their low trophic transfer from the alga to the daphnid and by the inductive effects of the alga on HemNP efflux. However, the main cause was the direct inhibition of HemNP uptake from the water phase as a result of the reduced water-filtration activity of D. magna in the presence of C. reinhardtii. Overall, in bioaccumulation studies, the inhibitory effects of biotic particles on NP uptake from the water phase should be paid attention.

Label-Free Imaging of Nanoparticle Uptake Competition in Single Cells by Hyperspectral Stimulated Raman Scattering.

Imaging and quantification of nanoparticles in single cells in their most natural condition are expected to facilitate the biotechnological applications of nanoparticles and allow for better assessment of their biosafety risks. However, current imaging modalities either require tedious sample preparation or only apply to nanoparticles with specific physicochemical characteristics. Here, the emerging hyperspectral stimulated Raman scattering (SRS) microscopy, as a label-free and nondestructive imaging method, is used for the first time to investigate the subcellular distribution of nanoparticles in the protozoan Tetrahymena thermophila. The two frequently studied nanoparticles, polyacrylate-coated α-Fe2 O3 and TiO2 , are found to have different subcellular distribution pattern as a result of their dissimilar uptake routes. Significant uptake competition between these two types of nanoparticles is further discovered, which should be paid attention to in future bioapplications of nanoparticles. Overall, this study illustrates the great promise of hyperspectral SRS as an analytical imaging tool in nanobiotechnology and nanotoxicology.

Waterborne and Dietborne Toxicity of Inorganic Arsenic to the Freshwater Zooplankton Daphnia magna.

Waterborne and dietborne exposure are both important sources for the accumulation of inorganic arsenic (iAs) in aquatic organisms. Although the waterborne toxicity of iAs has been extensively investigated, its dietborne toxicity has received little attention. The present study examined the acute and chronic toxicity of arsenate (iAsV) and arsenite (iAsIII) to the freshwater zooplankton species Daphnia magna under both waterborne and dietborne exposure scenarios. The bioaccumulation, speciation, and tissue and subcellular distributions of arsenic were analyzed to understand the mechanisms accounting for differences in toxicity related to different arsenic species, exposure scenarios, and exposure duration. The toxicity of iAs increased with exposure time, and iAsIII was more toxic than iAsV. Moreover, although dietborne iAs had no acute effect on D. magna, it incurred significant toxicity in the chronic-exposure experiment. Nevertheless, the toxicity of dietborne iAs was still lower than that of waterborne iAs regardless of the exposure duration. This difference was found to be caused by the lower bioaccumulation of dietborne iAs, its higher distribution in the gut and in the biologically detoxified subcellular fraction, and greater transformation to the less toxic dimethylarsinic acid. Overall, the dietborne toxicity of iAs should be considered when evaluating the environmental risks posed by arsenic.

Aggregation Reverses the Carrier Effects of TiO2 Nanoparticles on Cadmium Accumulation in the Waterflea Daphnia magna.

Our previous study reported that the Ca-dependent aggregation of polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) determines their routes of uptake by the waterflea Daphnia magna. Besides the effects of aggregation on NP bioaccumulation, how this process may influence the bioavailability of NP-adsorbed pollutants remains obscure. In the present study, the aggregation of PAA-TiO2-NPs was also adjusted through Ca. Then the accumulation and toxicity of Cd in D. magna were investigated in the presence and absence of the NPs. Although PAA-TiO2-NPs ameliorated Cd toxicity at both low and high Ca concentrations, the underlying mechanisms differed completely. At low Ca, the metal-NP complexes were accumulated by endocytosis and passive drinking, with both pollutants distributed throughout the daphnid. Nevertheless, Cd accumulation was reduced due to its rapid dissociation from the NPs during the endocytosis of the metal-NP complexes. At high Ca, the metal-NP complexes were actively ingested, Cd accumulation was induced, and both pollutants were concentrated in the daphnid gut. The aggregation-dependent effects of PAA-TiO2-NPs on Cd bioaccumulation were further evidenced by the distinct patterns of metal efflux from D. magna at different Ca concentrations. Overall, Cd adsorption by PAA-TiO2-NPs may either increase or reduce its bioaccumulation, as determined by the aggregation of the NPs.

TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via Different Routes is Calcium-Dependent.

Calcium plays versatile roles in aquatic ecosystems. In this study, we investigated its effects on the uptake of polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) by the water flea (cladoceran) Daphnia magna. Particle distribution in these daphnids was also visualized using synchrotron radiation-based micro X-ray fluorescence spectroscopy, transmission electron microscopy, and scanning electron microscopy. At low ambient Ca concentrations in the experimental medium ([Ca]dis), PAA-TiO2-NPs were well dispersed and distributed throughout the daphnid; the particle concentration was highest in the abdominal zone and the gut, as a result of endocytosis and passive drinking of the nanoparticles, respectively. Further, Ca induced PAA-TiO2-NP uptake as a result of the increased Ca influx. At a high [Ca]dis, the PAA-TiO2-NPs formed micrometer-sized aggregates that were ingested by D. magna and concentrated only in its gut, independent of the Ca influx. Our results demonstrated the multiple effects of Ca on nanoparticle bioaccumulation. Specifically, well-dispersed nanoparticles were taken up by D. magna through endocytosis and passive drinking whereas the uptake of micrometer-sized aggregates relied on active ingestion.

TiO2 nanoparticles act as a carrier of Cd bioaccumulation in the ciliate Tetrahymena thermophila.

When nanoparticles can enter a unicellular organism directly, how may they affect the bioaccumulation and toxicity of other pollutants already present in the environment? To answer this question, we conducted experiments with a protozoan Tetrahymena thermophila. The well-dispersed polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) were used as a representative nanomaterial, and Cd as a conventional pollutant. We found that PAA-TiO2-NPs could get into Tetrahymena cells directly. Such internalization was first induced by low concentrations of Cd, but later suppressed when Cd concentrations were higher than 1 µg/L. Considering its significant adsorption on PAA-TiO2-NPs, Cd could be taken up by T. thermophila in the form of free ion or metal-nanoparticle complexes. The latter route accounted for 46.3% of Cd internalization. During the 5 h depuration period, 4.34-22.1% of Cd was excreted out, which was independent of the concentrations of intracellular Cd and PAA-TiO2-NPs. On the other hand, both free and intracellular Cd concentrations only partly predicted its toxicity at different levels of PAA-TiO2-NPs. This may have resulted from PAA-TiO2-NPs' synergistic effects and the distinct subcellular distribution of Cd taken up via the two routes above. Overall, we should pay attention to the carrier effects of nanoparticles when assessing their environmental risks.

Cell-excreted proteins mediate the interactions of differently sized silica nanoparticles during cellular uptake.

Exposure to different types of nanoparticles (NPs) results in their deposition in human bodies. While most studies have examined the cellular uptake of only one type of NP at a time, how the dynamics of NP uptake may change in the presence of other types of NPs remains unclear. We therefore investigated the interplay of two differently sized SiO2 NPs during their uptake by A549 human lung carcinoma cells. Both NPs contained a CdSeTe core, which was labeled with different Cd isotopes to differentiate between them. Our study showed that the uptake of one size of SiO2 NPs either increased or decreased with the concentration of the other size of SiO2 NPs. This variation in uptake was attributable to the concentration-dependent aggregation of SiO2 NPs, as determined by the amount of cell-excreted proteins adsorbed on the NP surface. Further, the effects of the protein corona on the attachment of SiO2 NPs to the cell surface and uptake competition between differently sized SiO2 NPs also played important roles. Cell-excreted proteins were then analyzed by proteomics. Overall, the complex interactions between coexisting NPs of different physicochemical properties and cell-excreted proteins should be considered during bio-applications and bio-safety evaluations of NPs.

Silica nanoparticles inhibit cadmium uptake by the protozoan Tetrahymena thermophila without the need for adsorption.

The simultaneous presence of nanoparticles (NPs) and heavy metals in the environment may affect their mutual biological uptake. Although previous studies showed that NPs could alter the cellular uptake of heavy metals by their adsorption of heavy metals, whether they could affect metal uptake without the need for adsorption is unknown. This study examined the effects of silica (SiO2) NPs on the uptake of Cd ion by the protozoan Tetrahymena thermophila. We found that, even with negligible levels of adsorption, SiO2 NPs at concentrations of 3 to 100 mg/L inhibited Cd uptake. This inhibitory effect decreased as the ambient Cd concentration increased from 1 to 100 µg/L, suggesting the involvement of at least two transporters with different affinities for Cd. The transporters were subsequently identified by the specific protein inhibitors amiloride and tariquidar as NCX and ABCB1, which are responsible for the uptake of Cd at low and high Cd levels, respectively. RT-qPCR and molecular dynamics simulation further showed that the inhibitory effects of SiO2 NPs were attributable to the down-regulated expression of the genes Ncx and Abcb1, steric hindrance of Cd uptake by NCX and ABCB1, and the shrinkage of the central channel pore of the transporters in the presence of SiO2 NPs. SiO2 NPs more strongly inhibited Cd transport by NCX than by ABCB1, due to the higher binding affinity of SiO2 NPs with NCX. Overall, our study sheds new light on a previously overlooked influence of NPs on metal uptake and the responsible mechanism.

Bioaccumulation of CdTe quantum dots in a freshwater alga Ochromonas danica: a kinetics study.

The bioaccumulation kinetics of thioglycolic acid stabilized CdTe quantum dots (TGA-CdTe-QDs) in a freshwater alga Ochromonas danica was comprehensively investigated. Their photoluminescence (PL) was determined by flow cytometry. Its cellular intensity increased hyperbolically with exposure time suggesting real internalization of TGA-CdTe-QDs. This hypothesis was evidenced by the nanoparticle uptake experiment with heat-killed or cold-treated cells and by their localization in the vacuoles. TGA-CdTe-QD accumulation could further be well simulated by a biokinetic model used previously for conventional pollutants. Moreover, macropinocytosis was the main route for their internalization. As limited by their diffusion from the bulk medium to the cell surface, TGA-CdTe-QD uptake rate increased proportionally with their ambient concentration. Quick elimination in the PL of cellular TGA-CdTe-QDs was also observed. Such diminishment resulted mainly from their surface modification by vacuolar biomolecules, considering that these nanoparticles remained mostly undissolved and their expulsion out of the cells was slow. Despite the significant uptake of TGA-CdTe-QDs, they had no direct acute effects on O. danica. Overall, the above research shed new light on nanoparticle bioaccumulation study and would further improve our understanding about their environmental behavior, effects and fate.

Seasonal variation of phytoplankton nutrient limitation in Lake Taihu, China: a monthly study from year 2011 to 2012.

Lake Taihu is the third largest freshwater lake in China with severe eutrophication issues. However, it remains ambiguous how its phytoplankton growth is limited by various nutrients in different seasons. A series of bottle-enrichment assays in Meiliang Bay was thus performed once a month from July, 2011 to June, 2012 in the present study. The initial chlorophyll a concentration and phytoplankton cell density ranged from 4.70 to 34.6 µg/l and from 1.25×10(6) to 6.72×10(8) cells/l with three peaks in July, November, and March. Although Cyanophyta was dominant (30.9-99.2 percent) in most cases, other phyla like Chlorophyta, Bacillariophyta, and Cryptophyta could account for as much as 69.1 percent of total phytoplankton in cold seasons. The microcystin-LR content in the particulate phase followed a similar seasonal pattern as Cyanophyta. It further went up exponentially with the proportion of cyanobacteria in phytoplankton suggesting more toxigenic species and (or) upregulated microcystin synthesis when the contribution of Cyanophyta was enhanced. On the other hand, the dissolved concentrations of various nitrogen and phosphorus species reached their maxima in late spring and autumn, respectively. According to its growth response to nutrient addition, phytoplankton in Meiliang Bay was restricted by nitrogen in August, October, and November. No nutrient limitation occurred in July, September, and April, whereas phosphorus deficiency prevailed in the other months. Overall, nutrient limitation in Lake Taihu and possibly other aquatic ecosystems worldwide may be more dynamic than what we thought before, which should be considered to eliminate eutrophication.

Pre-exposure to titanium or iron oxide nanoparticles suppresses the subsequent cellular uptake of gold nanoparticles.

Humans are exposed to a wide variety of natural and engineered nanoparticles (NPs) during their lifetime. However, the effects of pre-exposure to NPs on subsequent uptake of other NPs have not been investigated. In the present study, we investigated the effects of pre-exposure to three NPs (TiO2, Fe2O3, and SiO2 NPs) on the subsequent uptake of gold NPs (AuNPs) by hepatocellular carcinoma cells (HepG2). When HepG2 cells were pre-exposed to TiO2 or Fe2O3 NPs, but not SiO2 NPs for 2 days, their subsequent uptake of AuNPs was inhibited. Such inhibition was also observed in human cervical cancer (HeLa) cells, suggesting that this phenomenon is present in different cell types. The mechanisms underlying the inhibitory effect of NP pre-exposure include altered plasma membrane fluidity due to changes in lipid metabolism and reduced intracellular ATP production due to decreased intracellular oxygen. Despite the inhibitory effects of NP pre-exposure, full recovery was observed after transferring the cells to medium without NPs, even when the pre-exposure time was extended from 2 days to 2 weeks. Overall, the pre-exposure effects observed in the present study should be considered in the biological application and risk evaluation of NPs.

Bioaccumulation determines the toxicity of carbon dots to two marine dinoflagellates.

With the ever-increasing application of carbon dots (CDs), a substantial amount will be released and assemble in the aquatic environment. Nevertheless, potential photodegradation of CDs in the aquatic environment, their accumulation and impacts in aquatic organisms remain unclear. Our study examined the toxicity of CDs to two marine dinoflagellates Prorocentrum micans and Prorocentrum donghaiense. Their bioaccumulation including the uptake and elimination kinetics was also determined. Significant photodegradation of CDs in seawater was observed. Moreover, both the degraded CDs and their photodegradation products were toxic to the dinoflagellates. Although P. donghaiense was more sensitive to CDs than P. micans with the median effect concentration 17.0 and 99.0 mg L-1, respectively, such sensitivity difference disappeared when the toxicity data were plotted against cellularly accumulated CDs instead of their concentration in the experimental medium. Therefore, the higher sensitivity of P. donghaiense was attributable to its higher accumulation of CDs. Overall, the photodegradation and bioaccumulation of CDs should be considered when evaluating their environmental risks.

Accumulation of nanoplastics in human cells as visualized and quantified by hyperspectral imaging with enhanced dark-field microscopy.

Nanoplastic (NP) pollution is receiving increasing attention regarding its potential effects on human health. The identification and quantification of intracellular NPs are prerequisites for an accurate risk assessment, but appropriate methods are lacking. Here we present a label-free technique to simultaneously visualize and quantify the bioaccumulation of NPs based on hyperspectral imaging with enhanced dark-field microscopy (HSI-DFM). Using polystyrene NPs (PS NPs) as representative particles, the construction of a hyperspectral library was optimized first with more accurate NP identification achieved when the library was based on intracellular instead of extracellular PS NPs. The PS NPs used herein were labeled with a green fluorescent dye so that the accuracy of HSI-DFM in identifying and quantifying intracellular NPs can be evaluated, by comparing the results with those obtained by fluorescence microscopy and flow cytometry. The validation of HSI-DFM for use in determinations of the NP concentration at the single-cell level allows analyses of the accumulation kinetics of NPs in single living cells. The utility of HSI-DFM in different cell lines and with NPs differing in their chemical composition was also demonstrated. HSI-DFM therefore provides a new approach to studies of the accumulation and distribution of NPs in human cells.

Potential effects of Ag ion on the host by changing the structure of its gut microbiota.

Silver (Ag) can change the structure of the gut microbiota (GM), but how such change may affect host health is unknown. In this study, mice were exposed to silver acetate daily for 120 days. During this period, Ag accumulation in the liver was measured, its effects on GM structure were analyzed, and potential metabolic changes in liver and serum were examined. Although Ag accumulation remained unchanged in most treatments, the ratio of Firmicutes to Bacteroidetes at the phylum level increased and changes in the relative abundance of 33 genera were detected, suggesting that Ag altered the energy metabolism of mice via changes in the gut GM. In serum and liver, 34 and 72 differentially expressed metabolites were identified, respectively. The KEGG pathways thus enriched mainly included those involving the metabolism of amino acids, organic acids, lipids, and purine. Strong correlations were found between 33 % of the microorganisms with altered relative abundances and 46 % of the differentially expressed metabolites. The resulting clusters yielded two communities responsible for host inflammation and energy metabolism. Overall, these results demonstrate potential effects of Ag on the host, by changing its GM structure, and the need to consider them when evaluating the health risk of Ag.