Correction: Effects of nano-cerium dioxide on intestinal microflora in rats by oral subchronic exposure.

[This corrects the article DOI: 10.1371/journal.pone.0298917.].

Effects of nano-cerium dioxide on intestinal microflora in rats by oral subchronic exposure.

OBJECTIVE: To investigate intestinal toxicity in rats and the effects of Nano-cerium dioxide on intestinal flora in rats after oral sub-chronic exposure. METHOD: Forty healthy male SD rats were randomly divided into four groups: a control group (deionized water) and three groups treated with different doses of Nano-ceria (e.g., 20 mg/kg, 100 mg/kg, and 500 mg/kg), with 10 rats in each group. The rats were given intragastric administrations (every other day) for 90 days. After the last intragastric administration, fresh fecal samples were collected by pressing the abdomen, and the animals were sacrificed. Jejunum, ileum and cecum tissues were retained for pathological analysis by Hematoxylin-eosin staining. The stool samples of rats were sequenced by the Illumina NovaSeq sequencing platform, and the sequencing results were further analyzed by QIIME2 software. RESULTS: The histopathology results show that compared with the control group, in the middle- and high-dose groups, epithelial tissue was shed, lamina propria glandular structures were damaged or disappeared, and large numbers of inflammatory cells were distributed in the mucosa. The intestinal flora results show that there were no significant differences in the α-/ß-diversities in each Nano-ceria-treated group compared with the control group (P>0.05). Compared with the control group, the intestinal pathogenic bacteria, Mucispirillum and Streptococcus increased significantly after Nano-cerium dioxide ingestion, while Weissella decreased. The abundances of Akkermansia in all Nano-ceria-treated groups were higher than those in the control group, but the abundances decreased with increasing dose. MetagenomesSeq analysis show that, compared with the control group, the abundances of S24-7, Lactobacillus and Clostridiales in all experimental groups significantly decreased. CONCLUSIONS: The sub-chronic toxicity of Nano-cerium dioxide to rats can affect the structure and abundance of intestinal microflora, long-term exposure to high doses (>100 mg/kg) causes enteritis, but there was no significant difference in the diversity of gut microbiota. Therefore, we infer that the enteritis in rats may be associated with the relative ratios of the pathogenic bacteria and intestinal probiotics, and increased of the intestinal pathogenic bacteria can disrupted intestinal homeostasis.

Single and combined genotoxicity of metals and fluoroquinolones to zebrafish embryos at environmentally relevant concentrations.

Fluoroquinolones (FQs) are known to have genotoxicity to aquatic organisms. However, their genotoxicity mechanisms, individually and in combination with heavy metals, are poorly understood. Here, we investigated the single and joint genotoxicity of FQs, ciprofloxacin (CIP) and enrofloxacin (ENR), and metals (Cd and Cu) at environmentally relevant concentrations (0.2 µM) to zebrafish embryos. We found that FQs or/and metals induced genotoxicity (i.e., DNA damage and cell apoptosis) to zebrafish embryos. Compared with their single exposure, the combined exposure of FQs and metals elicited less ROS overproduction but higher genotoxicity, suggesting other toxicity mechanisms may also act in addition to oxidation stress. The upregulation of nucleic acid metabolites and the dysregulation of proteins confirmed the occurrence of DNA damage and apoptosis, and further revealed the inhibition of DNA repair by Cd and binding of DNA or DNA topoisomerase by FQs. This study deepens the knowledge on the responses of zebrafish embryos to exposure of multiple pollutants, and highlights the genotoxicity of FQs and heavy metals to aquatic organisms.

Exposure to trace levels of metals and fluoroquinolones increases inflammation and tumorigenesis risk of zebrafish embryos.

Exposure to trace-level heavy metals and antibiotics may elicit metabolic disorder, alter protein expression, and then induce pathological changes in zebrafish embryos, despite negligible physiological and developmental toxicity. This study investigated the single and combined developmental toxicity of fluoroquinolones (enrofloxacin [ENR] and ciprofloxacin [CIP]) (≤0.5 µM) and heavy metals (Cu and Cd) (≤0.5 µM) to zebrafish embryos, and molecular responses of zebrafish larvae upon exposure to the single pollutant (0.2 µM) or a binary metal-fluoroquinolone mixture (0.2 µM). In all single and mixture exposure groups, no developmental toxicity was observed, but oxidative stress, inflammation, and lipid depletion were found in zebrafish embryos, which was more severe in the mixture exposure groups than in the single exposure groups, probably due to increased metal bioaccumulation in the presence of ENR or CIP. Metabolomics analysis revealed the up-regulation of amino acids and down-regulation of fatty acids, corresponding to an active response to oxidative stress and the occurrence of inflammation. The up-regulation of antioxidase and immune proteins revealed by proteomics analysis further confirmed the occurrence of oxidative stress and inflammation. Furthermore, the KEGG pathway enrichment analysis showed a significant disturbance of pathways related to immunity and tumor, indicating the potential risk of tumorigenesis in zebrafish larvae. The findings provide molecular-level insights into the adverse effects of heavy metals and antibiotics (especially in chemical mixtures) on zebrafish embryos, and highlight the potential ecotoxicological risks of trace-level heavy metals and antibiotics in the environment.

Sediments alleviate the inhibition effects of antibiotics on denitrification: Functional gene, microbial community, and antibiotic resistance gene analysis.

Both antibiotics and sediments can affect the denitrification in aquatic systems. However, little is known how antibiotics influence the denitrification in the presence of sediments. Here, the effects of antibiotics (sulfamethoxazole, tetracycline and ofloxacin) on denitrification in the absence and presence of sediments were investigated. The influencing mechanisms were revealed by quantifying the denitrification functional genes (DNGs), 16S-seq of bacteria, and antibiotic resistance genes (ARGs). The results showed that the presence of antibiotics inhibited NO3-N reduction by decreasing the abundances of narG, nirK, nosZ, total DNGs, and denitrifying bacteria. However, the inhibition effect was alleviated by sediments, which promoted the growth of bacteria and decreased the selective pressure of antibiotics as the vector of bacteria and antibiotics, thus increasing the abundances of denitrifying bacteria and all the DNGs. Partial least-squares path model disclosed that antibiotics had negative effects on bacteria, ARGs and DNGs, while sediments had negative effects on ARGs but positive effects on bacteria and DNGs. The network analysis further revealed the close relation of the genera Bacillus, Acinetobacter, and Enterobacter with the ARGs and DNGs. The findings are helpful to understand the denitrification in antibiotic-polluted natural waters.

Toxicity of gabapentin-lactam on the early developmental stage of zebrafish (Danio rerio).

Gabapentin-lactam (GBP-L) is a transformation product (TP) of gabapentin (GBP), a widely used anti-epileptic pharmaceutical. Due to its high persistence, GBP-L has been frequently detected in the surface water. However, the effects of GBP-L on aquatic organisms have not been thoroughly investigated. In the present study, zebrafish (Danio rerio) embryos as a model organism were used to study the impacts of GBP-L in terms of embryos LC50, spontaneous movement at 24 hpf (hours post fertilization), heartbeat rates at 48 hpf, and body length at 72 hpf, with the concentrations of GBP-L down to 0.01 µg/L, covering its environmental concentrations. Various biomarkers from nervous, antioxidant and immune systems of zebrafish larvae were analyzed, including acetylcholinesterase, acetylcholine, dopamine, gamma-aminobutyric acid, superoxide dismutase, catalase, glutathione S-transferase, C reactive protein, and lysozyme, to assess its toxicity on these systems. RT-qPCR was then used to further verify the results and explain the toxicological mechanism at the gene level. The results demonstrated that GBP-L is much more toxic than its parent compound, and could lead to adverse impacts on the aquatic organisms even at every low concentrations.

Impaired learning and memory in mice induced by nano neodymium oxide and possible mechanisms.

A growing number of individuals are now exposed to neodymium (Nd) owing to its extensive applications. However, the biological effects of Nd on humans, especially on learning and memory, remain elusive. To investigate whether Nd exposure affects learning and memory, in this study female ICR mice were exposed to nano Nd2 O3 via intranasal instillation at doses of 50, 100, and 150 mg/kg body weight, daily for 45 days. According to Morris water maze data, learning and memory parameters were significantly reduced in the 150 mg/kg nano-Nd2 O3 group than the sham control. Furthermore, inductively coupled plasma-mass spectroscopy analysis revealed that Nd levels were significantly higher in the hippo campus of the 100 and 150 mg/kg exposed group than the sham control; however, no significant differences were observed in the hippocampal histopathology between these groups. Furthermore, reactive oxygen species were elevated in hippocampal tissues of experimental groups than the sham control, 447.3 in high dose group and 360.0 in control group; however, malondialdehyde levels were significantly increased and superoxide dismutase activities were decreased only in mice exposed to 100 and 150 mg/kg Nd2 O3 . High-performance liquid chromatography data demonstrated that levels of glutamic acid, glycine, and gamma-aminobutyric acid were higher in the hippocampus of mice exposed to 150 mg/kg Nd2 O3 than the sham control. Our findings indicated that the neuronal injury was induced by disruption of the oxidation-antioxidation homeostasis and altered amino acid neurotransmitter levels in the hippocampus, which could result in the poor cognitive performance demonstrated by exposed mice.

Single and combined effects of carbamazepine and copper on nervous and antioxidant systems of zebrafish (Danio rerio).

Various pollutants co-exist in the aquatic environment such as carbamazepine (CBZ) and copper (Cu), which can cause complex effects on inhabiting organisms. The toxic impacts of the single substance have been studied extensively. However, the studies about their combined adverse impacts are not enough. In the present study, zebrafish were exposed to environmental relevant concentrations of CBZ (1, 10, and 100 µg/L), Cu (0.5, 5, and 10 µg/L) and the mixtures (1 µg/L CBZ + 0.5 µg/L Cu, 10 µg/L CBZ + 5 µg/L Cu, 100 µg/L CBZ + 10 µg/L Cu) for 45 days, the effects on nervous and antioxidant systems of zebrafish were investigated. The results demonstrated that, in comparison with single exposure group, the combined presence of CBZ and Cu exacerbated the effect of antioxidant system (the ability of inhibition of hydroxyl radicals (IHR), superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST)) but not nervous system (Acetylcholinesterase [AChE]). The qPCR results supported the changes of corresponding enzymes activities. Hepatic histopathological analysis verified the results of biomarkers. Our work illustrated that the toxicity of mixed pollutants is very complicated, which cannot simply be inferred from the toxicity of single pollutant, and calls for more co-exposure experiments to better understanding of the co-effects of pollutants on aquatic organisms.

A transcriptomics-based analysis of the toxicity mechanisms of gabapentin to zebrafish embryos at realistic environmental concentrations.

Gabapentin (GPT) has become an emerging contaminant in aquatic environments due to its wide application in medical treatment all over the world. In this study, embryos of zebrafish were exposed to gabapentin at realistically environmental concentrations, 0.1 µg/L and 10 µg/L, so as to evaluate the ecotoxicity of this emergent contaminant. The transcriptomics profiling of deep sequencing was employed to illustrate the mechanisms. The zebrafish (Danio rerio) embryo were exposed to GPT from 12 hpf to 96 hpf resulting in 136 and 750 genes differentially expressed, respectively. The results of gene ontology (GO) analysis and the Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis illustrated that a large amount of differentially expressed genes (DEGs) were involved in the antioxidant system, the immune system and the nervous system. RT-qPCR was applied to validate the results of RNA-seq, which provided direct evidence that the selected genes involved in those systems mentioned above were all down-regulated. Acetylcholinesterase (AChE), lysozyme (LZM) and the content of C-reactive protein (CRP) were decreased at the end of exposure, which is consistent with the transcriptomics results. The overall results of this study demonstrate that GPT simultaneously affects various vital functionalities of zebrafish at early developmental stage, even at environmentally relevant concentrations.

Neural Activity-Dependent Regulation of Radial Glial Filopodial Motility Is Mediated by Glial cGMP-Dependent Protein Kinase 1 and Contributes to Synapse Maturation in the Developing Visual System.

UNLABELLED: Radial glia in the developing optic tectum extend highly dynamic filopodial protrusions within the tectal neuropil, the motility of which has previously been shown to be sensitive to neural activity and nitric oxide (NO) release. Using in vivo two-photon microscopy, we performed time-lapse imaging of radial glial cells and measured filopodial motility in the intact albino Xenopus laevis tadpole. Application of MK801 to block neuronal NMDA receptor (NMDAR) currents confirmed a significant reduction in radial glial filopodial motility. This reduction did not occur in glial cells expressing a dominant-negative form of cGMP-dependent protein kinase 1 (PKG1), and was prevented by elevation of cGMP levels with the phosphodiesterase type 5 inhibitor sildenafil. These results suggest that neuronal NMDAR activation results in the release of NO, which in turn modulates PKG1 activation in glial cells to control filopodial motility. We further showed that interfering with the function of the small GTPases Rac1 or RhoA, known to be regulated by PKG1 phosphorylation, decreased motility or eliminated filopodial processes respectively. These manipulations led to profound defects in excitatory synaptic development and maturation of neighboring neurons. SIGNIFICANCE STATEMENT: Radial glia in the developing brain extend motile filopodia from their primary stalk. Neuronal NMDA receptor activity controls glial motility through intercellular activation of cGMP-dependent protein kinase 1 (PKG1) signaling in glial cells. Manipulating PKG1, Rac1, or RhoA signaling in radial glia in vivo to eliminate glial filopodia or impair glial motility profoundly impacted synaptogenesis and circuit maturation.