Upregulated Palmitoleate and Oleate Production in Escherichia coli Promotes Gentamicin Resistance.

Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli. Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli. Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli. Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli. Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli, and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli. This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria.

Impact of Airborne Pathogen-Derived Extracellular Vesicles on Macrophages Revealed by Raman Spectroscopy and Multiomics.

Long-term exposure to the indoor environment may pose threats to human health due to the presence of pathogenic bacteria and their byproducts. Nanoscale extracellular vesicles (EVs) extensively secreted from pathogenic bacteria can traverse biological barriers and affect physio-pathological processes. However, the potential health impact of EVs from indoor dust and the underlying mechanisms remain largely unexplored. Here, Raman spectroscopy combined with multiomics (genomics and proteomics) was used to address these issues. Genomic analysis revealed that Pseudomonas was an efficient producer of EVs that harbored 68 types of virulence factor-encoding genes. Upon exposing macrophages to environmentally relevant doses of Pseudomonas aeruginosa PAO1-derived EVs, macrophage internalization was observed, and release of inflammatory factors was determined by RT-PCR. Subsequent Raman spectroscopy and unsupervised surprisal analysis of EV-affected macrophages distinguished metabolic alterations, particularly in proteins and lipids. Proteomic analysis further revealed differential expression of proteins in inflammatory and metabolism-related pathways, indicating that EV exposure induced macrophage metabolic reprogramming and inflammation. Collectively, our findings revealed that pathogen-derived EVs in the indoor environments can act as a new mediator for pathogens to exert adverse health effects. Our method of Raman integrated with multiomics offers a complementary approach for rapid and in-depth understanding of EVs' impact.

Deep Learning-Enabled Raman Spectroscopic Identification of Pathogen-Derived Extracellular Vesicles and the Biogenesis Process.

Pathogenic bacterial infections, exacerbated by increasing antimicrobial resistance, pose a major threat to human health worldwide. Extracellular vesicles (EVs), secreted by bacteria and acting as their "long-distance weapons", play an important role in the occurrence and development of infectious diseases. However, no efficient methods to rapidly detect and identify EVs of different bacterial origins are available. Here, label-free Raman spectroscopy in combination with a new deep learning model of the attentional neural network (aNN) was developed to identify pathogen-derived EVs at Gram±, species, strain, and even down to physiological levels. By training the aNN model with a large Raman data set from six typical pathogen-derived EVs, we achieved the identification of EVs with high accuracies at all levels: exceeding 96% at the Gram and species levels, 93% at the antibiotic-resistant and sensitive strain levels, and still above 87% at the physiological level. aNN enabled Raman spectroscopy to interrogate the bacterial origin of EVs to a much higher level than previous methods. Moreover, spectral markers underpinning EV discrimination were uncovered from subtly different EV spectra via an interpretation algorithm of the integrated gradient. A further comparative analysis of the rich Raman biochemical signatures of EVs and parental pathogens clearly revealed the biogenesis process of EVs, including the selective encapsulation of biocomponents and distinct membrane compositions from the original bacteria. This developed platform provides an accurate and versatile means to identify pathogen-derived EVs, spectral markers, and the biogenesis process. It will promote rapid diagnosis and allow the timely treatment of bacterial infections.

Widespread of Potential Pathogen-Derived Extracellular Vesicles Carrying Antibiotic Resistance Genes in Indoor Dust.

Extracellular vesicles (EVs) are newly recognized as important vectors for carrying and spreading antibiotic resistance genes (ARGs). However, the ARGs harbored by EVs in ambient environments and the transfer potential are still unclear. In this study, the prevalence of ARGs and mobile genetic elements (MGEs) in EVs and their microbial origins were studied in indoor dust from restaurants, kindergarten, dormitories, and vehicles. The amount of EVs ranged from 3.40 × 107 to 1.09 × 1011 particles/g dust. The length of EV-associated DNA fragments was between 21 bp and 9.7 kb. Metagenomic sequencing showed that a total of 241 antibiotic ARG subtypes encoding resistance to 16 common classes were detected in the EVs from all four fields. Multidrug, quinolone, and macrolide resistance genes were the dominant types. 15 ARG subtypes were exclusively carried and even enriched in EVs compared to the indoor microbiome. Moreover, several ARGs showed co-occurrence with MGEs. The EVs showed distinct taxonomic composition with their original dust microbiota. 30.23% of EV-associated DNA was predicted to originate from potential pathogens. Our results indicated the widespread of EVs carrying ARGs and virulence genes in daily life indoor dust, provided new insights into the status of extracellular DNA, and raised risk concerns on their gene transfer potential.

Metabolomics Insights into Oleate-Induced Disorders of Phospholipid Metabolism in Macrophages.

BACKGROUND: Diet-induced disordered phospholipid metabolism and disturbed macrophage metabolism contribute to the pathogenesis of metabolic diseases. However, the effects of oleate, a main dietary fatty acid, on macrophage phospholipid metabolism are unclear. OBJECTIVES: We aimed to discover oleate-induced disorders of macrophage phospholipid metabolism and potential therapeutic targets for treating diet-related metabolic diseases. METHODS: RAW 264.7 cells were exposed to 65 µg oleate/mL, within the blood concentration range of humans and mice, to trigger disorders of phospholipid metabolism. Meanwhile, WY-14643 and pioglitazone, 2 drugs widely used for treating metabolic diseases, were employed to prevent oleate-induced disorders of macrophage phospholipid metabolism. Subsequently, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry was used to discover relevant metabolic disorders and potential therapeutic targets. RESULTS: We showed that 196 metabolites involved in phospholipid metabolism were altered upon oleate treatment and interventions of WY-14643 and pioglitazone (P < 0.05, 2-tailed Mann-Whitney U test). Notably, most lysophospholipids were decreased, whereas most phospholipids were increased in oleate-treated macrophages. Phosphatidylethanolamines accumulated most among phospholipids, and their acyl chain polyunsaturation increased in oleate-treated macrophages. Additionally, saturated fatty acids were decreased, whereas polyunsaturated fatty acids were increased in oleate-treated macrophages. Furthermore, changes in phosphatidylglycerols, phosphatidylinositols, cardiolipins, phosphatidates, lysophosphatidylglycerols, and acylcarnitines in oleate-treated macrophages could be attenuated or even abolished by WY-14643 and/or pioglitazone treatment. CONCLUSIONS: Oleate induced accumulation of various phospholipids, increased acyl chain polyunsaturation of phosphatidylethanolamines, and decreased lysophospholipids in RAW 264.7 macrophages. This study suggests macrophage phospholipid and fatty acid metabolism as potential therapeutic targets for intervening diet-related metabolic diseases.

Perfluorinated compounds disrupted osmoregulation in Oryzias melastigma during acclimation to hypoosmotic environment.

Perfluorinated compounds (PFCs) are a type of ubiquitous contaminants spreading in the estuarine and coastal areas. Anadromous fish should deal with hypoosmotic challenge with PFCs stress during their migration from seawater to estuaries. However, few studies have been carried out to investigate the adverse impact of PFCs on fish osmoregulation and the underlying mechanism. In this study, Oryzias melastigma, an euryhaline fish model, were exposed to four representative PFC congeners including perfluorobutane sulfonate (PFBS), perfluorooctane sulfonates (PFOS), perfluorooctanoic acid (PFOA), and perfluorododecanoic acid (PFDoA) separately under both seawater and freshwater conditions. Histopathological changes in gills, ion homeostasis, Na+/K+-ATPase (NKA) activity, as well as the expression of related genes was detected upon exposure. Results showed that PFCs induced morphological changes in gills, disturbed the levels of major ions (Na+, Ca2+, Mg2+), and inhibited the NKA activity. Transcriptome analysis in fish gills during the acclimation to freshwater revealed that PFCs influenced the osmoregulation mainly by interfering with the endocrine system, signal transduction, as well as cellular community and motility. Validation with qRT-PCR confirmed that the mRNA expressions of osmoregulatory genes encoding hormones and receptors, as well as ion transmembrane transporters were disturbed by PFCs. Longer chain homolog (PFOS) showed a greater impact on osmoregulation than the shorter chain homolog (PFBS). Within the same carbon chain, sulfonic congener (PFOS) induced more serious injury to gills than carboxylic congener (PFOA). The interaction between PFCs and salinity varied in different adverse outcome. These results help to further understand the mechanism of how PFCs influence osmoregulation and elicit the need to assess the ecological risk of PFCs and other pollutants on anadromous migration.

Extracellular vesicles in toxicological studies: key roles in communication between environmental stress and adverse outcomes.

External stressors, especially environmental toxicants can disturb biological homeostasis and thus lead to adverse health effects. However, there is limited understanding of how cells directly exposed to stressors transmit the signals to cells indirectly in contact with stressors. Extracellular vesicles (EVs) are receiving increasing attention as signal transductors between various types of cells in organisms. Cargo in EVs, including RNAs, proteins, lipids, and other signal molecules can be transferred between cells and become critical determining factors of intercellular communication. EVs can be a powerful mediator of environmental stimuli. It has been shown that external stressors reshape the secretion of EVs, modify the composition of EVs, and thus influence the mediating function of EVs. These abnormal EVs can lead to dysfunction of recipient cells, and even the pathogenesis of diseases. In this review, we first summarized current knowledge about the responses of EVs to external stimuli, including chemicals and chemical mixtures. Then we explained how these altered EVs regulate signal pathways in recipient cells, thus mediating physio-pathological responses in detail. The most up-to-date evidence from molecular, cellular, animal and human levels was synthesized to systematically address the mediating roles of EVs. EVs can be regarded as a bridge to link external stressors and internal response. Further toxicological and molecular epidemiological studies are expected to provide further insight into the roles of EVs in toxicology. The gaps in the engulfment of toxicants into EVs are listed as the priority to be solved in future studies.

Resveratrol inhibits lipid accumulation in the intestine of atherosclerotic mice and macrophages.

Disordered intestinal metabolism is highly correlated with atherosclerotic diseases. Resveratrol protects against atherosclerotic diseases. Accordingly, this study aims to discover novel intestinal proatherosclerotic metabolites and potential therapeutic targets related to the anti-atherosclerotic effects of resveratrol. An untargeted metabolomics approach was employed to discover novel intestinal metabolic disturbances during atherosclerosis and resveratrol intervention. We found that multiple intestinal metabolic pathways were significantly disturbed during atherosclerosis and responsive to resveratrol intervention. Notably, resveratrol abolished intestinal fatty acid and monoglyceride accumulation in atherosclerotic mice. Meanwhile, oleate accumulation was one of the most prominent alterations in intestinal metabolism. Moreover, resveratrol attenuated oleate-triggered accumulation of total cholesterol, esterified cholesterol and neutral lipids in mouse RAW 264.7 macrophages by activating ABC transporter A1/G1-mediated cholesterol efflux through PPAR (peroxisome proliferator-activated receptor) α/γ activation. Furthermore, we confirmed that PPARα and PPARγ activation by WY14643 and pioglitazone, respectively, alleviated oleate-induced accumulation of total cholesterol, esterified cholesterol and neutral lipids by accelerating ABC transporter A1/G1-mediated cholesterol efflux. This study provides the first evidence that resveratrol abolishes intestinal fatty acid and monoglyceride accumulation in atherosclerotic mice, and that resveratrol suppresses oleate-induced accumulation of total cholesterol, esterified cholesterol and neutral lipids in macrophages by activating PPARα/γ signalling.

MiR-26a functions as a tumor suppressor in ambient particulate matter-bound metal-triggered lung cancer cell metastasis by targeting LIN28B-IL6-STAT3 axis.

Exposure to ambient particulate matter (PM) has been linked to the increasing incidence and mortality of lung cancer, but the principal toxic components and molecular mechanism remain to be further elucidated. In this study, human lung adenocarcinoma A549 cells were treated with serial concentrations of water-extracted PM10 (WE-PM10) collected from Beijing, China. Our results showed that exposure to 25 and 50 µg/ml of WE-PM10 for 48 h significantly suppressed miR-26a to upregulate lin-28 homolog B (LIN28B), and in turn activated interleukin 6 (IL6) and signal transducer and activator of transcription 3 (STAT3) in A549 cells, subsequently contributing to enhanced epithelial-mesenchymal transition and accelerated migration and invasion. In vivo pulmonary colonization assay further indicated that WE-PM10 enhanced the metastatic ability of A549 cells. In addition, luciferase reporter assay demonstrated that 3' untranslated region of LIN28B was a direct target of miR-26a. Last but not the least, the key toxic contribution of metals in WE-PM10 was confirmed by the finding that removal of metals through chelation significantly rescued WE-PM10-mediated inflammatory, carcinogenic and metastatic responses. Taken together, miR-26a could act as the tumor suppressor in PM10-related lung cancer, and PM10-bound metals promoted lung cancer cell metastasis through downregulation of miR-26a that directly mediated LIN28B expression.

Emerging roles of long non-coding RNAs in the toxicology of environmental chemicals.

Environmental chemicals (ECs) are drawing great attention to their effects on health and their toxicological mechanisms are being investigated. Long non-coding RNA (lncRNA) is a class of RNA with more than 200 nucleotides and does not have protein coding potential. Recently, it is emerging as a star molecule that participates in a wide range of physiological and pathological processes. It has been reported to be abnormally expressed in diseases. As an epigenetic factor, lncRNAs play an important role in the response of organisms to environmental stress. Their roles in the toxicity of ECs are being identified. Altered expression profiles of lncRNAs have been explored after exposure to ECs. Various kinds of ECs are reported to disturb the expression of lncRNAs in vitro and in vivo. Then, dysregulated lncRNAs can affect the expression of target genes directly or indirectly via regulating the level of microRNAs. The network among lncRNAs, microRNAs and mRNAs can initiate or impede specific signaling pathway and lead to adverse outcome upon exposure to ECs. Recovery of the lncRNAs level by overexpression or knockdown technology diminished the effect induced by ECs. In the review, biological roles of lncRNAs are depicted. The lncRNAs involved in the toxicology are summarized. Types of ECs that have been reported to affect the expression of lncRNAs are categorized. The interaction between various types of ECs and lncRNAs is discussed.

Environmental risk assessment of selected organic chemicals based on TOC test and QSAR estimation models.

Environmental risks of organic chemicals have been greatly determined by their persistence, bioaccumulation, and toxicity (PBT) and physicochemical properties. Major regulations in different countries and regions identify chemicals according to their bioconcentration factor (BCF) and octanol-water partition coefficient (Kow), which frequently displays a substantial correlation with the sediment sorption coefficient (Koc). Half-life or degradability is crucial for the persistence evaluation of chemicals. Quantitative structure activity relationship (QSAR) estimation models are indispensable for predicting environmental fate and health effects in the absence of field- or laboratory-based data. In this study, 39 chemicals of high concern were chosen for half-life testing based on total organic carbon (TOC) degradation, and two widely accepted and highly used QSAR estimation models (i.e., EPI Suite and PBT Profiler) were adopted for environmental risk evaluation. The experimental results and estimated data, as well as the two model-based results were compared, based on the water solubility, Kow, Koc, BCF and half-life. Environmental risk assessment of the selected compounds was achieved by combining experimental data and estimation models. It was concluded that both EPI Suite and PBT Profiler were fairly accurate in measuring the physicochemical properties and degradation half-lives for water, soil, and sediment. However, the half-lives between the experimental and the estimated results were still not absolutely consistent. This suggests deficiencies of the prediction models in some ways, and the necessity to combine the experimental data and predicted results for the evaluation of environmental fate and risks of pollutants.

Epithelial-mesenchymal transition effect of fine particulate matter from the Yangtze River Delta region in China on human bronchial epithelial cells.

Epidemiological studies have demonstrated that fine particulate matter (PM2.5) exposure causes airway inflammation, which may lead to lung cancer. The activation of epithelial-mesenchymal transition (EMT) is assumed to be a crucial step in lung tumor metastasis and development. We assessed the EMT effect of low concentrations (0, 0.1, 1.0, and 5.0µg/mL) of PM2.5 organic extract on a human bronchial epithelial cell line (BEAS-2B). PM2.5 samples were collected from three cities (Shanghai, Ningbo, and Nanjing) in the Yangtze River Delta (YRD) region in autumn 2014. BEAS-2B cells were exposed to the PM2.5 extract to assess cell viability, invasion ability as well as the relative mRNA and protein expressions of EMT markers. Our findings revealed that BEAS-2B cells changed from the epithelial to mesenchymal phenotype after exposure. In all groups, PM2.5 exposure dose-dependently decreased the expression of E-cadherin and increased the expression of Vimentin. The key transcription factors, including ZEB1 and Slug, were significantly up-regulated upon exposure. These results indicated that the PM2.5 organic extract induced different degrees of EMT progression in BEAS-2B cells. The cell invasion ability increased in a concentration-dependent manner after 48hr of treatment with the extract. This study offers a novel insight into the effects of PM2.5 on EMT and the potential health risks associated with PM2.5 in the YRD region.

Downregulation of miR-192 causes hepatic steatosis and lipid accumulation by inducing SREBF1: Novel mechanism for bisphenol A-triggered non-alcoholic fatty liver disease.

Exposure to Bisphenol A (BPA) has been associated with the development of nonalcoholic fatty liver disease (NAFLD) but the underlying mechanism remains unclear. Given that microRNA (miRNA) is recognized as a key regulator of lipid metabolism and a potential mediator of environmental cues, this study was designed to explore whether exposure to BPA-triggered abnormal steatosis and lipid accumulation in the liver could be modulated by miR-192. We showed that male post-weaning C57BL/6 mice exposed to 50µg/kg/day of BPA by oral gavage for 90days displayed a NAFLD-like phenotype. In addition, we found in mouse liver and human HepG2 cells that BPA-induced hepatic steatosis and lipid accumulation were associated with decreased expression of miR-192, upregulation of SREBF1 and a series of genes involved in de novo lipogenesis. Downregulation of miR-192 in BPA-exposed hepatocytes could be due to defective pre-miR-192 processing by DROSHA. Using HepG2 cells, we further confirmed that miR-192 directly acted on the 3'UTR of SREBF1, contributing to dysregulation of lipid homeostasis in hepatocytes. MiR-192 mimic and lentivirus-mediated overexpression of miR-192 improved BPA-induced hepatic steatosis by suppressing SREBF1. Lastly, we noted that lipid accumulation was not a strict requirement for developing insulin resistance in mice after BPA treatment. In conclusion, this study demonstrated a novel mechanism in which NAFLD associated with BPA exposure arose from alterations in the miR-192-SREBF1 axis.

Dioxin-like rather than non-dioxin-like PCBs promote the development of endometriosis through stimulation of endocrine-inflammation interactions.

Polychlorinated biphenyls (PCBs) contain 209 congeners with various structure-activities. Exposure to PCBs was related to disorders of female reproduction. Endometriosis (EM) is an estrogen- and inflammation-dependent disease with high prevalence and severe health outcomes. Epidemiological studies have shown the effects of PCBs exposure on EM in regard to various structures of PCBs. However, little evidence is available from the toxicology considering the structure of PCBs. In the study, environmentally relevant concentrations of PCBs were used to treat primary cultured endometrial cells and an EM mouse model. Dioxin-like CB126, but not non-dioxin-like CB153, significantly enhanced 17ß-estradiol (E2) biosynthesis in a dose-dependent manner. Among the genes related to estrogen metabolism, the level of 17ß-hydroxysteroid dehydrogenase 7 (HSD17B7) showed significant increase following CB126 exposure. We further found that CB126 exposure decreased the methylation of the HSD17B7 promoter. Elevated expression of HSD17B7 was observed in the eutopic endometrium of EM patients. CB126 rather than CB153 triggered the inflammatory response by directly stimulating the secretion of inflammatory factors and indirectly reducing the level of lipoxin A4 (LXA4). Furthermore, the inflammation enhanced the expression of HSD17B7. Antagonism of the aryl hydrocarbon receptor (AhR) diminished the effects induced by CB126. In vivo, the PCB-treated EM mouse model confirmed that CB126 rather than CB153 increased the levels of both E2 and inflammatory factors in peritoneal fluid and promoted the development of endometriotic lesions. In all, CB126, but not CB153, triggered EM development by stimulating estrogen biosynthesis, inflammation and their interactions and that these effects were mediated by the AhR receptor.

Accumulation of di(2-ethylhexyl) phthalate causes endocrine-disruptive effects in marine medaka (Oryzias melastigma) embryos.

Di (2-ethylhexyl) phthalate (DEHP) is extensively distributed in marine environments. However, limited research on the toxicological and molecular effects of DEHP on marine organisms has been conducted. Our study investigated the accumulation, elimination, and endocrine-disruptive effects of DEHP on embryonic marine medaka (Oryzias melastigma). The medaka embryos were continuously exposed to DEHP (0.01, 0.1, and 1 mg/L) or 17ß-estradiol (E2, 0.01 mg/L) until hatching, and the newly hatched larvae were then transferred to clean sea water for 12 days of depuration. DEHP and E2 appeared to have no significant effects on the mortality and hatching rates of medaka embryos, but E2 exposure significantly delayed the hatching. Significantly higher DEHP embryonic burdens were detected in the group treated with higher DEHP (0.1 and 1 mg/L) at 10 dpf (days post fertilization). The recovered larvae showed an elimination tendency of DEHP during the recovery period. DEHP had no significant effects on the transcriptional responses of endocrine-disrupting biomarker genes in the 3-dpf embryos. Treatment with 0.1 and 1 mg/L DEHP elicited a significant induction of transcriptional responses of ER, PPAR, and the CYP19 genes in a concentration-dependent manner at 10 dpf, indicating endocrine disruption may be due to bioaccumulation of DEHP. With the elimination of DEHP during the depuration period, all of the effects on these genes showed no significant effects. However, 0.1 mg/L E2 significantly affected the expression of ER, PPAR, and the CYP19 genes in the exposed embryos at both 3 and 10 dpf and recovered larvae. Therefore, these results demonstrate that accumulation of DEHP caused endocrine disruption in medaka embryos and that recovery in clean sea water may weaken the endocrine-disrupting effects.

In situ combined chemical and biological assessment of estrogenic pollution in a water recycling system.

Estrogenic pollution and its control in aquatic systems have drawn substantial attention around the world. The chemical and biological assessment approaches currently utilized in the laboratory or field cannot give an integrated assessment of the pollution when used separately. In this study, in situ chemical and biological methods were combined to detect pollution in a water recycling system. Data for the water quality index (WQI) demonstrated that the water treatment resulted in the decline of pollution from upstream to downstream. Wild male Nile tilapia, Oreochromis niloticus, was sampled in June and September. The concentrations of four common endocrine disrupting chemicals (EDCs) were determined in the tilapia liver by chromatographic analysis methods. The level of 17ß-estradiol (E2) declined from upstream to downstream in both months. In contrast, the levels of bisphenol A (BPA), di-(2-ethylhcxyl) phthalate (DEHP), and perfluorooctane sulfonate (PFOS) did not display this declining tendency. The highest relative expression of vitellogenin 1 (VTG1) was observed in tilapia from upstream, then the level significantly decreased along the water system. The relative expression levels of CYP1A1 in the water system were also significantly higher than that of the control. However, no declining trend could be observed along the water system. The change of VTG1 expression corresponded well with that of E2 levels in the tilapia liver. Overall, our study assessed the pollution by endocrine disruptors using chemical and biological data with good correspondence. This study also demonstrated the effectiveness of the water recycling system in eliminating estrogen pollution in municipal sewage.

Di(2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection.

Di(2-ethylhexyl) phthalate (DEHP) is used as plasticizer and is ubiquitously found in the environment. Exposure to DEHP has been linked to an increased incidence of type 2 diabetes. Pancreatic ß-cell dysfunction is a hallmark of type 2 diabetes; however, it is unknown whether DEHP exposure contributes to this risk. Here, we aimed to investigate the cytotoxic effects of DEHP on INS-1 cells and to further explore the related underlying mechanisms. INS-1 cells were exposed to 0, 5, 25, 125 or 625 µM DEHP for 24 hrs. Cell viability, glucose-stimulated insulin secretion, reactive oxygen species (ROS) generation, cellular antioxidant response, Ca(2+) homoeostasis and the levels of genes and proteins involved in endoplasmic reticulum (ER) stress were measured. The results showed that DEHP decreased insulin secretion and content and induced apoptosis in INS-1 cells in a dose-dependent manner. Furthermore, ROS generation was increased and Nrf2-dependent antioxidant defence protection was dysregulated in INS-1 cells after DEHP exposure. Most importantly, DEHP effectively depleted ER Ca(2+) and triggered the ER stress response as demonstrated by the elevated transcription and translation of the ER chaperone GRP78 and GRP94, the increased phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and its downstream substrate eukaryotic translation initiation factor 2α (eIF2α), as well as the increased levels of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Taken together, DEHP exerted toxic effects on INS-1 cells by inducing apoptosis, which is dependent on the activation of the PERK-ATF4-CHOP ER stress signalling pathway and the suppression of Nrf2-dependent antioxidant protection.

Immunotoxic effects of perfluorooctane sulfonate and di(2-ethylhexyl) phthalate on the marine fish Oryzias melastigma.

Perfluorooctane sulfonate (PFOS) and di(2-ethylhexyl) phthalate (DEHP) have both been reported to induce adverse effects including immunotoxicity. Despite the widespread presence of these two chemicals in estuaries and seawater, their health effects on marine fish have received little attention. Oryzias melastigma is a potential marine fish model for immunological studies. In the present study, immune-related genes in O. melastigma were enriched at the transcriptome level. Three-month-old fish were exposed to PFOS and DEHP (single or combined) for one week. The liver index-hepatosomatic index (HSI) of the fish was higher in the PFOS-exposed group and combined group than in the control group. This result indicates that PFOS might lead to liver toxicity. The mRNA level of interleukin-1 beta (IL1ß) was upregulated after exposure. For catalase (CAT), glutathione peroxidase (GPx) and cluster of differentiation 3 (CD3), single exposure did not affect mRNA levels, but the combined exposure did significantly alter the expression of these genes. In all, our study provides a useful reference for immunotoxicological studies with O. melastigma; it also highlights the importance of assessing the combined effects of pollutant mixtures when determining the risk to aquatic organisms.

The combined effects of DEHP and PCBs on phospholipase in the livers of mice.

Di(2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) are two widely distributed pollutants that are of great concern due to their adverse health effects. However, few studies have investigated the combined effects of DEHP and PCBs. In this study, adult mice were continuously exposed to mixtures of DEHP (15 mg/kg bodyweight/day) and Aroclor 1254 (7.5 mg/kg bodyweight/day) for 12 days to investigate the combined effects of these compounds. The results showed that the ratio of the liver weight to the body weight was higher in the treated group than that in the control group. The effects of combined exposure on three important receptors, the proliferator-activated receptor (PPAR), estrogen receptor (ER), and aryl hydrocarbon receptor (AHR), were investigated. The mRNA level of PPARγ was significantly up-regulated after exposure. The expression level of ERα was decreased in the male treated group. In contrast, the expression levels of AHR and related genes (cyp1a1 and cyp1b1) were not markedly affected. The expression level of phospholipase A (PLA) was significantly down-regulated at both the mRNA and protein levels in male mice after combined treatment. In all, our study demonstrated the combined effects of DEHP and PCBs on the expression levels of key receptors in mice. The combined exposure led to a decrease in phospholipase in male mice.

Temporal dynamics of microbial composition and antibiotic resistome in fermentation bed culture pig farms across various ages.

The discharge from pig farms presents significant challenges to the environment and human health, specifically regarding the dissemination of antimicrobial resistance (AMR). Fermentation bed culture has emerged as an increasingly popular and environmentally friendly pig farming model in China, as it minimizes the release of harmful substances into the environment. However, there remains a limited understanding of the occurrence and dynamics of microbiome and antibiotic resistome in fermentation bed culture. Herein, we collected fermentation bed materials (FBM) from four fermentation bed culture pig farms with varying service ages and investigated their bacterial communities, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), metal resistance genes (MRGs) and potential antibiotic-resistant bacterial hosts through metagenomics. Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota were identified as the dominant phyla present in the FBM. In total, we detected 258 unique ARGs in the FBM samples, with 79 core ARGs shared by all FBM samples, accounting for 95 % of the total ARG abundance. Our results revealed significant variations in microbial communities and ARG profiles across varying service ages of FBM. Compared to long-term FBW, short-term FBM exhibited higher numbers and abundances of ARGs, MRGs and MGEs, along with higher levels of potential bacterial pathogens and high-risk ARGs. Further analysis of metagenome-assembled genome (MAG) indicated that the putative hosts of ARGs primarily belonged to Pseudomonadota, Actinomycetota and Bacillota. Alarmingly, among the 80 recovered ARG-carrying MAGs, 23 MAGs encoded multi-resistance, including clinically significant species that require urgent attention. Overall, this study provided valuable insights into the temporal patterns of antibiotic resistome and bacterial communities within FBM, enhancing our understanding of FBM in pig farming. The findings could potentially contribute to the development of effective strategies for evaluating and regulating fermentation bed culture practices in pig farming.