Long-term use of etomidate disrupts the intestinal homeostasis and nervous system in mice.

Etomidate (ETO) is used as an anesthetic in surgery, but it is being abused in some populations. The damage caused by long-term intake of ETO to intestinal and brain functions is not yet clear, and it remains to be determined whether the drug affects the central nervous system through the gut-brain axis. This study aimed to investigate the neurotoxic and gastrointestinal effects of ETO at doses of 1 mg/kg and 3 mg/kg in mice over 14 consecutive days. The results showed that long-term injection of ETO led to drug resistance in mice, affecting their innate preference for darkness and possibly inducing dependence on ETO. The levels of 5-hydroxytryptamine in the brain, serum, and colon decreased by 37%, 51%, and 42% respectively, while the levels of γ-aminobutyric acid reduced by 38%, 52%, and 41% respectively. H&E staining revealed that ETO reduced goblet cells in the colon and damaged the intestinal barrier. The expression of tight junction-related genes Claudin4 and ZO-1 was downregulated. The intestinal flora changed, the abundance of Akkermansia and Lactobacillus decreased by 33% and 14%, respectively, while Klebsiella increased by 18%. TUNEL results showed that high-dose ETO increased apoptotic cells in the brain. The expression of Claudin1 in the brain was downregulated. Untargeted metabolomics analysis of the colon and brain indicated that ETO caused abnormalities in glycerophospholipid metabolism. Abnormal lipid metabolism might lead to the production or accumulation of lipotoxic metabolites, causing central nervous system diseases. ETO induced changes in the intestinal flora and metabolism, further affecting the central nervous system through the gut-brain axis. The study unveiled the detrimental effects on the brain and gastrointestinal system resulting from long-term intake of ETO, which holds significant implications for comprehending the adverse impact of ETO abuse on human health.

Neurotoxic effects induced by flunitrazepam and its metabolites in zebrafish: Oxidative stress, apoptosis, and histone hypoacetylation.

Benzodiazepines (BZDs) have been widely detected in aquatic environments, but their neurotoxic effects and potential mechanisms are still unclear. This study focuses on flunitrazepam (FLZ) and its metabolite, 7-aminoflunitrazepam (7-FLZ), as representative psychotropic BZD. We investigated their neurotoxic effects on adult zebrafish following a 30-day exposure to environmentally relevant concentrations. The findings reveal that exposure to these drugs induces anxiety-like and aggressive behaviors in zebrafish. Additionally, notable morphological damage to brain tissue and mitochondrial structures was observed. Through TUNEL staining, an increase in apoptotic cells was detected in the brain tissue of the exposed group, accompanied by marked elevations in ROS and caspase-3/9 levels. The upregulation of apoptosis-related genes Bax, p53, and Bcl-2 confirmed the occurrence of apoptosis. Furthermore, exposure to the drugs resulted in decreased acetylation levels of brain histones H3 and H4. The upregulation of histone deacetylation enzyme genes (HDAC1, HDAC3, HDAC4, and HDAC6) supported this result. Molecular docking results suggest that compared to 7-FLZ, FLZ has a higher binding affinity with HDAC3 and HDAC4, explaining why it causes lower histone acetylation levels. This study in zebrafish elucidates the neurotoxicity and molecular mechanisms induced by FLZ and 7-FLZ, which is significant for further understanding the impact of BZDs on human health and assessing their ecological risks.

Flunitrazepam and its metabolites compromise zebrafish nervous system functionality: An integrated microbiome, metabolome, and genomic analysis.

The psychotropic drug flunitrazepam (FLZ) is frequently detected in aquatic environments, yet its neurotoxicity to aquatic organisms has not received sufficient attention. In this study, microbiome, metabolome, and genome analyses were conducted to study the effects of FLZ and its metabolite 7-aminoflunitrazepam (7-FLZ) on the zebrafish nervous system and understand their toxic mechanisms. The results demonstrated that drug exposure induced gut dysbiosis, decreased short-chain fatty acids and promoted the production of lipopolysaccharides (LPS). LPS entered the brain and interacted with Toll-like receptors to cause neuroinflammation by upregulating the expression of proinflammatory cytokines TNFα and NF-κB. The increased ratio of S-adenosylmethionine to S-adenosylhomocysteine in brain tissues indicated abnormal expression of Dnmt1 gene. Whole-genome bisulfite sequencing displayed an increase in differentially methylated regions (DMRs) associated-genes and pertinent biological pathways encompassed the MAPK signaling pathway, calcium signaling pathway, and Wnt signaling pathway. Correlation analysis confirmed connections between gut microbiota, their metabolites, inflammatory factors, and DNA methylation-related markers in brain tissue. These findings indicate that while the toxicity is somewhat reduced in metabolized products, both FLZ and 7-FLZ can induce DNA methylation in brain tissue and ultimately affect the biological function of the nervous system by disrupting gut microbiota and their metabolites.

Flunitrazepam and its metabolites induced brain toxicity: Insights from molecular dynamics simulation and transcriptomic analysis.

Psychoactive drugs frequently contaminate aquatic environments after human consumption, raising concerns about their residues and ecological harm. This study investigates the effects of flunitrazepam (FLZ) and its metabolite 7-aminoflunitrazepam (7-FLZ), benzodiazepine-class psychoactive drugs, on brain accumulation, blood-brain barrier (BBB), and neuroinflammation of the model organism zebrafish. Molecular dynamics simulation and transcriptome sequencing were used to uncover their toxic mechanisms. Results demonstrate that both FLZ and 7-FLZ can accumulate in the brain, increasing Evans blue levels by 3.4 and 0.8 times, respectively. This increase results from abnormal expression of tight junction proteins, particularly ZO-1 and Occludin, leading to elevated BBB permeability. Furthermore, FLZ and 7-FLZ can also induce neuroinflammation, upregulating TNFα by 91% and 39%, respectively, leading to pathological changes and disrupted intracellular ion balance. Molecular dynamics simulation reveals conformational changes in ZO-1 and Occludin proteins, with FLZ exhibiting stronger binding forces and greater toxicity. Weighted gene co-expression network analysis identifies four modules correlated with BBB permeability and neuroinflammation. KEGG enrichment analysis of genes within these modules reveals pathways like protein processing in the endoplasmic reticulum, NOD-like receptor signaling pathway, and arginine and proline metabolism. This study enhances understanding of FLZ and 7-FLZ neurotoxicity and assesses environmental risks of psychoactive substances. ENVIRONMENTAL IMPLICATION: With the increasing prevalence of mental disorders and the discharge of psychoactive drugs into water, even low drug concentrations (ng/L-µg/L) can pose neurological risks. This study, utilizing molecular dynamic (MD) simulations and transcriptome sequencing, investigate the neurotoxicity and mechanisms of flunitrazepam and 7-aminoflunitrazepam. It reveals that they disrupt the blood-brain barrier in zebrafish and induce neuroinflammation primarily by inducing conformational changes in tight junction proteins. MD simulations are valuable for understanding pollutant-protein interactions. This research offers invaluable insights for the environmental risk assessment of psychoactive drugs and informs the development of strategies aimed at prevention and mitigation.

Flunitrazepam and its metabolites exposure disturb the zebrafish gut-liver axis: Combined microbiome and metabolomic analysis.

Due to clinical treatment and illegal use, psychoactive substances have been widely detected in the aquatic environment. In this study, we investigated the effects of the benzodiazepine drug flunitrazepam (FLZ) and its metabolite 7-aminoflunitrazepam (7-FLZ) on the gut-liver axis of zebrafish. Zebrafish were exposed to two concentrations of FLZ and 7-FLZ (0.05 and 1 µg/L) for 30 days. Results showed that both FLZ and 7-FLZ exposure altered the relative abundance of Proteobacteria at the phylum level, with significant differences observed at the genus level for pathogenic bacteria such as Paracoccus, Shewanella, and Aeromonas. Metabolomics results showed both exposures significantly interfered with nucleotide and amino acid metabolism. The imbalance of gut microbiota and metabolic disorder increased the level of malondialdehyde, which in turn heightened the permeability of the gut mucosal barrier. FLZ and 7-FLZ induced oxidative stress in the liver via the gut-liver axis, leading to decreased levels of glucose, total cholesterol, and triglyceride, as well as the down-regulation of glycolipid metabolism-related genes (PPARα, PPARγ, FABP2, Fabp11, PFKFB3, and LDHA). Metabolomics results revealed that FLZ and 7-FLZ significantly affected the biosynthesis of amino acids and arginine, and other metabolic pathways such as nucleotide, nicotinate and nicotinamide, and purine in the liver. Our results unveiled the mechanisms behind the toxicological effects of psychoactive substances on the gut-liver axis, providing valuable data for ecological and environmental risk assessments.

Flunitrazepam induces neurotoxicity in zebrafish through microbiota-gut-brain axis.

The abuse of psychoactive substances has led to their frequent detection in the environment, with unknown effects on the nervous system. In this study, zebrafish were exposed to benzodiazepine drug flunitrazepam (FLZ, 0.2 and 5 µg/L) for 30 days to assess its neurotoxicity. Results revealed that FLZ disrupted the balance of gut microbiota and caused an increase in pathogenic bacteria, such as Paracoccus and Aeromonas, leading to pathological damage to the intestine. The upregulation of intestinal pro-inflammatory factors, IL-1ß and TNF-α, by 2.4 and 6.3 times, respectively, along with the downregulation of tight junction proteins, Occludin and zonula occludens 1 (ZO-1), by 80 % and 50 %, increased in intestinal permeability. Moreover, untargeted metabolomics demonstrated that FLZ interfered with intestinal nucleotide metabolism and amino acid biosynthesis. FLZ could also increase the levels of lipopolysaccharide (LPS) and malondialdehyde (MDA) in the brain by 0.9 and 3.4 times, respectively, leading to pathological changes in brain tissue. Furthermore, FLZ significantly disturbed nucleotide metabolism and amino acid biosynthesis and metabolism pathways in the brain. Correlation analysis between gut microbiota and neurochemicals confirmed that FLZ can induce neurotoxicity through the microbiota-gut-brain axis. These findings elucidate the molecular mechanisms of psychoactive drugs on microbiota-gut-brain axis and provide a theoretical basis for the ecological environmental risk assessment of various psychoactive substances.

Fish isotopic niches associated with environmental indicators and human disturbance along a disturbed large subtropical river in China.

Stable isotopes are increasingly used to detect and understand the impacts of environmental changes on riverine ecological properties. The δ13C and δ15N signatures of fish with different feeding habits were measured in a large subtropical river to evaluate how fish isotopic niches respond to environmental gradients and human disturbance. From basal resources to fish consumers, the high values of epilithic periphyton (biofilm) δ13C and suspended particulate organic matter δ15N concurrently determined the niche ranges and space (e.g., convex hull area) of fish communities. Along a longitudinal gradient (except in the industrial zone), the number of fish trophic guilds identified by Bayesian ellipses continuously increased; meanwhile, higher trophic diversity and less redundancy were observed near the lower reaches and estuary. Variance inflation factors were estimated to detect the multicollinearity of 40 environmental variables, 14 of which were selected as indicators. Relative importance (RI) analysis was used to evaluate the explanatory power of these indicators for the spatial variation in isotopic niche metrics; the results showed that riffle habitat area, water nitrate concentration, gravel-cobble substrate, and riparian buffer width were the 4 key environmental indicators (average RI > 12%) that determined the longitudinal pattern of fish isotopic niches. These findings suggested that community-level δ13C signatures are more responsive to changes in habitats (e.g., riffle) and substrates (e.g., gravel-cobble) supporting the productivity of autochthonous diatoms while δ15N signatures respond to water quality altered by nitrogen pollution from manure-fertilized farming and poultry livestock effluent. Furthermore, δ15N may be more robust and interpretable than δ13C as an isotopic indicator of ecosystem change in rivers exposed to multiple or complex anthropogenic stressors.

Stable isotopes reveal effects of natural drivers and anthropogenic pressures on isotopic niches of invertebrate communities in a large subtropical river of China.

Isotopic niches reflect the basic structure and functioning of river food webs; however, their response to riverine environments remains unclear. We used stable isotope analysis and community-wide metrics to quantify how invertebrate niches vary with environmental changes along a large subtropical river in China. Eight niche metrics, which had higher values in the wet than in the dry season, increased from headwaters to the middle river and decreased sharply near the estuarine industrial zones. The δ13C value of > - 23.8‰, which indicated consumption of epilithic diatoms, separated the invertebrates between the upper and mid-lower reaches. The δ15N values > 9.4‰ identified site-specific nitrogen sources from manure (e.g., animal effluent) and domestic sewage in agricultural area. The output of mixing models showed a downstream shift in carbon utilization by invertebrates from autochthonous periphyton and submerged hydrophytes to allochthonous C3 plants. Principle component (PC) and cluster analysis decomposed and grouped 40 environmental variables into 4 PCs that explained 84.5% of the total variance. Hierarchical partitioning revealed that the second and first PCs, which were driven mainly by biological indicators and habitat characteristics, had the highest explanatory power for niche ranges and areas (e.g., Bayesian ellipse), respectively. Our results suggest that reducing anthropogenic pressures (e.g., habitat loss and water pollution) on river ecosystems through measures, such as protecting diatom-dominated biofilms in riffles and controlling nitrogen loading in rural regions, may produce the greatest impact for river management. Graphical abstract.