Inner-Wall Coated Nanopipette Microextraction for Quantitative Analysis of Per- and Polyfluoroalkyl Substances in Single Cells Using Mass Spectrometry.

Per- and polyfluoroalkyl substances (PFASs) are a series of organic pollutants with potential cytotoxicity and biotoxicity. Accurate and sensitive detection of trace PFASs in single cells can provide insights into investigating their cytotoxicity, carcinogenicity, and mutagenicity. Here we report the development of an inner-wall coated nanopipette microextraction coupled with induced nanoelectrospray ionization mass spectrometry (InESI-MS) method and its application for rapid, sensitive, and accurate analysis of trace PFASs in single cells. A specially designed inner-wall coated nanopipette was prepared for sampling of the cytoplasm from a single cell, and the trace PFASs in the cytoplasm were selectively enriched into the coating via reversed-phase adsorption, ion bonding adsorption, and π-π interaction mechanisms. After the extraction, the cytoplasm was removed, and the enriched PFASs were then desorbed into some organic solvent, applying an alternating current (AC) voltage to the inner-wall coated nanopipette for InESI-MS analysis. The inner-wall coated nanopipette showed an exhaustive extraction to the trace PFASs in one single cell, and thus, the mass of each target analyte in the cytoplasm can be calculated via an internal standard calibration curve method, avoiding the measurement of ultrasmall volume cytoplasm for one single cell. By using the inner-wall coated nanopipette microextraction coupled with InESI-MS method, trace PFASs accumulated in the LO2 cells with pollutant exposure were successfully detected, and the accumulative behaviors and heterogeneities of PFASs in single cells were explored.

Portable Mass Spectrometry for On-site Detection of Hazardous Volatile Organic Compounds via Robotic Extractive Sampling.

Various hazardous volatile organic compounds (VOCs) are frequently released into environments during accidental events that cause many hazards to ecosystems and humans. Therefore, rapid, sensitive, and on-site detection of hazardous VOCs is crucial to understand their compositions, characteristics, and distributions in complex environments. However, manual handling of hazardous VOCs remains a challenging task, because of the inaccessible environments and health risk. In this work, we designed a quadruped robotic sampler to reach different complex environments for capturing trace hazardous VOCs using a needle trap device (NTD) by remote manipulation. The captured samples were rapidly identified by portable mass spectrometry (MS) within minutes. Rapid detection of various hazardous VOCs including toxicants, chemical warfare agents, and burning materials from different environments was successfully achieved using this robot-MS system. On-site detection of 83 typical hazardous VOCs was examined. Acceptable analytical performances including low detection limits (at subng/mL level), good reproducibility (relative standard deviation (RSD) < 20%, n = 6), excellent quantitative ability (R2 > 0.99), and detection speed (within minutes) were also obtained. Our results show that the robot-MS system has excellent performance including safety, controllability, applicability, and robustness under dangerous chemical conditions.

Chlorinated Anthracenes Induced Pulmonary Immunotoxicity in 3D Coculture Spheroids Simulating the Lung Microenvironment.

Chlorinated anthracenes (Cl-Ants), persistent organic pollutants, are widely detected in the environment, posing potential lung toxicity risks due to frequent respiratory exposure. However, direct evidence and a comprehensive understanding of their toxicity mechanisms are lacking. Building on our prior findings of Cl-Ants' immunotoxic risks, this study developed a three-dimensional coculture spheroid model mimicking the lung's immune microenvironment. The objective is to explore the pulmonary immunotoxicity and comprehend its mechanisms, taking into account the heightened immune reactivity and frequent lung exposure of Cl-Ants. The results demonstrated that Cl-Ants exposure led to reduced spheroid size, increased macrophage migration outward, lowered cell viability, elevated 8-OHdG levels, disturbed anti-infection balance, and altered cytokine production. Specifically, the chlorine substituent number correlates with the extent of disruption of spheroid indicators caused by Cl-Ants, with stronger immunotoxic effects observed in dichlorinated Ant compared to those in monochlorinated Ant. Furthermore, we identified critical regulatory genes associated with cell viability (ALDOC and ALDOA), bacterial response (TLR5 and MAP2K6), and GM-CSF production (CEBPB). Overall, this study offers initial in vitro evidence of low-dose Cl-PAHs' pulmonary immunotoxicity, advancing the understanding of Cl-Ants' structure-related toxicity and improving external toxicity assessment methods for environmental pollutants, which holds significance for future monitoring and evaluation.

Development of a Repair Enzyme Fluorescent Probe to Reveal the Intracellular DNA Damage Induced by Benzo[a]pyrene in Living Cells.

Pollutant exposure causes a series of DNA damage in cells, resulting in the initiation and progression of diseases and even cancers. An investigation of the DNA damage induced by pollutants in living cells is significant to evaluate the cytotoxicity, genotoxicity, and carcinogenicity of environmental exposure, providing critical insight in the exploration of the etiologies of diseases. In this study, we develop a repair enzyme fluorescent probe to reveal the DNA damage caused by an environmental pollutant in living cells by single-cell fluorescent imaging of the most common base damage repair enzyme named human apurinic/apyrimidinic endonuclease 1 (APE1). The repair enzyme fluorescent probe is fabricated by conjugation of an APE1 high affinity DNA substrate on a ZnO2 nanoparticle surface to form a ZnO2@DNA nanoprobe. The ZnO2 nanoparticle serves as both a probe carrier and a cofactor supplier, releasing Zn2+ to activate APE1 generated by pollutant exposure. The AP-site in the DNA substrate of the fluorescent probe is cleaved by the activated APE1, releasing fluorophore and generating fluorescent signals to indicate the position and degree of APE1-related DNA base damage in living cells. Subsequently, the developed ZnO2@DNA fluorescent probe is applied to investigate the APE1-related DNA base damage induced by benzo[a]pyrene (BaP) in living human hepatocytes. Significant DNA base damage by BaP exposure is revealed, with a positive correlation of the damage degree with exposure time in 2-24 h and the concentration in 5-150 µM, respectively. The experimental results demonstrate that BaP has a significant effect on the AP-site damage, and the degree of DNA base damage is time-dependent and concentration-dependent.

Exploring the Accumulation Behavior and Heterogeneity of Perfluorooctanesulfonic Acid in Zebrafish Primary Organ Cells by Single-Cell Mass Cytometry.

Perfluorooctanesulfonic acid (PFOS) is a commonly found environmental pollutant with potential toxicity and health risks to biosystems and ecosystems. Study of the accumulation behavior and heterogeneity of PFOS in biological primary organ cells provides us significant insights to explore its cytotoxicity, carcinogenicity, and mutagenicity. Here a single-cell mass cytometry system was established for the high-throughput analysis of trace PFOS and the exploration of its accumulation behavior and heterogeneity in zebrafish primary organ cells. The single-cell mass cytometry system applied a ∼25 µm constant-inner-diameter capillary as the single-cell generation and transportation channel with an etched tip-end of 40 µm as the nanoelectrospray emitter for mass spectrometric analysis. The single-cell mass cytometry system showed satisfactory semiquantitative performance and sensitivity for analysis of PFOS in single cells, with a high detection throughput of ∼35 cells/min. Subsequently, the liver, intestine, heart, and brain from PFOS-exposed zebrafish (100 pg/µL, 28 days) were dissociated and prepared as cell suspensions, and the cell suspensions were introduced into the single-cell mass cytometry system for high-throughput analysis of PFOS in individual primary organ cells. Significant cellular accumulation heterogeneities were observed, with the highest content in liver cells, followed by intestine cells, then heart cells, and the lowest in brain cells. In addition, the dynamics of PFOS in the zebrafish liver, intestine, heart, and brain cells showed typical violin plot distributions and were well-described using a gamma (γ) function.

Nanospray Laser-Induced Plasma Ionization Mass Spectrometry for Rapid and Sensitive Analysis of Polycyclic Aromatic Hydrocarbons and Halogenated Derivatives.

Polycyclic aromatic hydrocarbons (PAHs) and halogenated derivatives are a series of environmental pollutants with potential toxicity and health risks on biosystems and the ecosystem. Rapid and sensitive analysis of trace PAHs and halogenated PAHs in complex environmental samples is a challenging topic for analytical science. Here we report the development of a nanospray laser-induced plasma ionization MS method for rapid and sensitive analysis of trace PAHs and halogenated PAHs under ambient and open-air conditions. A nanospray tip was applied for loading samples and placed pointing to the MS inlet, being a nanospray emitter with the application of a high voltage. A beam of laser was focused to induce energetic plasma between the nanospray emitter and the MS inlet for ionization of PAHs and halogenated PAHs for mass spectrometric analysis. Meanwhile, an inner-wall naphthyl-coated nanospray emitter was developed and applied as a solid-phase microextraction (SPME) probe for highly selective enrichment of trace PAHs and halogenated PAHs in complex environmental samples, and some organic solvent was applied to desorb the analytes for nanospray laser-induced plasma ionization MS analysis. Satisfactory linearity for each target PAH and halogenated PAH was obtained, with correlation coefficient values (r) no less than 0.9917. The method showed extremely high sensitivity for analysis of trace PAHs and halogenated PAHs in water, with limits of detection (LODs) and quantification (LOQs) of 0.0001-0.02 and 0.0003-0.08 µg/L, respectively. By using the inner-wall naphthyl-coated nanospray laser-induced plasma ionization MS method, sensitive detection of trace PAHs and halogenated PAHs in real sewage and wastewater samples was successfully achieved.

Slug-Flow Microextraction Mass Spectrometry for Enhanced Detection of Analytes in Human Tear Fluids using Noninvasive Microsampling and Nanoelectrospray Ionization via a Capillary.

In vivo noninvasive sampling and sensitive analysis of human tear fluids at the microliter level is an important but challenging task in investigating eye health. In this work, capillary microsampling coupled with slug-flow microextraction mass spectrometry (SFME-MS) was developed for enhanced detection of analytes in human tear fluids. As low as 1.0 µL of human tear fluid could be directly sampled using a capillary, and extraction/spray solvent was then loaded into the capillary to perform slug-flow microextraction and direct nanoelectrospray ionization (nESI) of analytes. All analytical procedures, including tear microsampling, microextraction, and ionization of analytes, were performed using a capillary. Enhanced detection of therapeutic drugs and disease biomarkers in human tear fluids was successfully demonstrated. Acceptable analytical performances including sensitivity, reproducibility, and quantitation were obtained. It is found that the use of SFME could improve the nESI-MS detection of trace analytes over 100-fold that depends on the chemical properties of analytes. Overall, this study showed that SFME-nESI-MS is a highly effective method for enhanced detection of trace analytes in tear fluids and is expected to be a potentially powerful tool in significant biological and clinical applications.

Discovery of Potential Lipid Biomarkers for Human Colorectal Cancer by In-Capillary Extraction Nanoelectrospray Ionization Mass Spectrometry.

Discovering cancer biomarkers is of significance for clinical medicine and disease diagnosis. In this article, we develop an in-capillary extraction nanoelectrospray ionization mass spectrometry (ICE-nanoESI-MS) method to rapidly and in situ investigate human colorectal cancer for discovering lipid biomarkers. The ICE-nanoESI-MS method is performed using a tungsten microdissecting probe for in situ microsampling of surgical human colorectal cancer tumors and their paired distal noncancerous tissues during/after surgery. After sampling, the tungsten probe and the adhered tissues are inserted into a nanospray tip prefilled with some solvent for simultaneous in-capillary extraction and nanoESI-MS detection under ambient and open-air conditions. Online coupling of the Paternò-Büchi reaction and radical-direct fragmentation with ICE-nanoESI-MS is easily realized, which provides the opportunity to precisely determine carbon-carbon double bond (C═C) locations and stereospecific numbering (sn) positions of lipid biomarkers. Subsequently, a total of 12 pairs of colorectal cancer tumors and distal noncancerous tissues from different patients are investigated by our proposed ICE-nanoESI-MS method. A significant increase in lysophospholipids and fatty acids as well as a significant decrease in ceramides are discovered, and lysophospholipids are found as the potential biomarkers related to the formation and pathogenesis of human colorectal cancer.

Covalent Organic Frameworks-Based Solid-Phase Microextraction Probe for Rapid and Ultrasensitive Analysis of Trace Per- and Polyfluoroalkyl Substances Using Mass Spectrometry.

Rapid and ultrasensitive analysis of trace pollutants in complex matrices is of significance for understanding their environmental behaviors and toxic effects. Here a novel method based on the integration of solid-phase microextraction (SPME) and nanoelectrospray ionization mass spectrometry (nanoESI-MS) was developed for rapid and ultrasensitive analysis of trace per- and polyfluoroalkyl substances (PFASs) in environmental and biological samples. A novel SPME probe with F-functionalized covalent organic frameworks (COFs) coating was designed for highly selective enrichment of trace PFASs from complex samples. After extraction, the loaded COFs-SPME probe was directly appplied to nanoESI-MS analysis under ambient and open-air conditions. The method showed satisfactory linearities between 1 and 5000 ng/L for 14 investigated PFASs in water, with correlation coefficient values no less than 0.9952. The limits of detection and quantification varied from 0.02 to 0.8 ng/L and 0.06 to 3 ng/L, respectively. By using the proposed method, ultrasensitive detection of PFASs in environmental water and whole blood was successfully achieved.

Coupling Paternò-Büchi Reaction with Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for In Vivo and Microscale Profiling of Lipid C═C Location Isomers in Complex Biological Tissues.

Lipids are important structural components of biological systems, and lipid C═C locations play important roles in their biophysical and biochemical properties. Rapid, in vivo, in situ, and microscale lipidomics investigation (including precise identification of lipid C═C locations and isomers) of biological specimen has great potential for clinical diagnosis, biological studies, and biomarker discovery. Here we report a novel lipidomics methodology by coupling Paternò-Büchi (PB) reaction with surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) for in vivo, in situ, and microscale analysis of lipid species and C═C location isomers in complex biological tissues. The proposed SCP-PB-nanoESI-MS method was performed by application of a biocompatible solid-phase microextraction (SPME) probe for in vivo, in situ, and microscale sampling and extraction of lipids from biological tissues, and then some spray solvent containing PB reagent was applied to desorb lipid species enriched on SPME probe within a nanospray tip. Subsequently, ultraviolet irradiation was employed to initiate PB reaction for unsaturated lipids within the nanospray tip. After that, a high voltage was applied on the SPME probe to induce nanoESI for MS analysis under ambient and open-air conditions, and collision-induced dissociation was performed to the PB reaction product ions for determination of lipid C═C locations and isomers. By using our proposed SCP-BP-nanoESI-MS method, microscale investigation of lipid compositions and C═C location isomers for lipid droplet of Perilla seed and human intestinal tissue were successfully achieved, and in vivo analysis of lipid species and C═C locations for zebrafish was accomplished.

A microscale solid-phase microextraction probe for the in situ analysis of perfluoroalkyl substances and lipids in biological tissues using mass spectrometry.

The simultaneous analysis of perfluoroalkyl substances (PFASs) and lipids in biological tissues is of importance, especially for in situ and microscale analysis, because it provides significant information to understand the relevance of content, composition, and distribution of lipids to the bioaccumulation of PFASs as well as lipid metabolism affected by the biotoxicity of PFASs. In this study, we report the development of a novel ambient mass spectrometry method for the rapid, in situ, and microscale analysis of PFASs and lipids simultaneously in biological tissues for the investigation of their biological correlation. A microscale solid-phase microextraction (SPME) probe with a probe-end diameter of several-µm was employed for in situ and microscale sampling of biological tissues after PFAS exposure. The SPME probe showed a desirable capacity for the enrichment of PFASs and lipid species simultaneously. After sampling and extraction, the loaded SPME probe was directly applied for nanoESI-MS analysis under ambient and open-air conditions. A high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated in the field-induced mode was introduced to record mass spectra using fast polarity switching between positive and negative ion detection. Most of the lipid species were recorded in the positive ion mass spectrum, and PFASs were recorded in the negative ion mass spectrum. By using the developed method, the in situ analysis of PFASs and lipids in the muscle, brain, heart, kidney, liver, and intestine of zebrafish was realized. In addition, simultaneously imaging PFASs and lipids in individual Daphnia magna was successfully achieved for the investigation of their biological correlation.

Biocompatible Surface-Coated Probe for in Vivo, in Situ, and Microscale Lipidomics of Small Biological Organisms and Cells Using Mass Spectrometry.

Lipidomics is a significant way to understand the structural and functional roles that lipids play in biological systems. Although many mass spectrometry (MS)-based lipidomics strategies have recently achieve remarkable results, in vivo, in situ, and microscale lipidomics for small biological organisms and cells have not yet been obtained. In this article, we report a novel lipidomics methodology for in vivo, in situ, and microscale investigation of small biological organisms and cells using biocompatible surface-coated probe nanoelectrospray ionization mass spectrometry (BSCP-nanoESI-MS). A novel biocompatible surface-coated solid-phase microextration (SPME) probe is prepared, which possesses a probe-end diameter of less than 5 µm and shows excellent enrichment capacity toward lipid species. In vivo extraction of living biological organisms (e.g., zebrafishes), in situ sampling a precise position of small organisms (e.g., Daphnia magna), and even microscale analysis of single eukaryotic cells (e.g., HepG2) are easily achieved by the SPME probe. After extraction, the loaded SPME probe is directly applied for nanoESI-MS analysis, and a high-resolution mass spectrometer is employed for recording spectra and identifying lipid species. Compared with the conventional direct infusion shotgun MS lipidomics, our proposed methodology shows a similar result of lipid profiles but with simpler sample pretreatment, less sample consumption, and shorter analytical times. Lipidomics of zebrafish, Daphnia magna, and HepG2 cell populations were investigated by our proposed BSCP-nanoESI-MS methodology, and abundant lipid compositions were detected and identified and biomarkers were obtained via multivariate statistical analysis.

Surface-Modified Wooden-Tip Electrospray Ionization Mass Spectrometry for Enhanced Detection of Analytes in Complex Samples.

Replacement of capillary with solid substrates for sample loading and ionization has created many new possibilities for electrospray ionization mass spectrometry (ESI-MS). Surface modification is an attractive strategy to enhance the analytical capability of solid-substrate ESI-MS and allow understanding the relationship between surface activity of solid substrates and analytical properties. In this study, we performed surface modification of wooden tips with hydrophobic (-C18), basic (-NH2), and acidic (-SO3H) functional groups and applied various sampling methods, i.e., extractive sampling and direct loading, to comprehensively investigate the analytical properties of solid-substrate ESI-MS. Our results showed that, for the direct loading method, analytes with weak interactions with solid-substrate surface could be readily sprayed out for detection. While for the extractive sampling method, analytes strongly retained on solid-substrate surface could be selectively enriched and detected, and a washing step after sample loading could effectively remove unbound components for reducing interference. Overall, the insights on the effects of surface-analyte interactions on the analytical features obtained in this study could aid the development of surface-modified strategies for enhancing the analytical capability of solid-substrate ESI-MS.

Surface-coated probe nanoelectrospray ionization mass spectrometry for analysis of target compounds in individual small organisms.

Analysis of target compounds in individual small organisms is of significant importance for biological, environmental, medicinal, and toxicological investigation. In this study, we reported the development of a novel solid-phase microextraction (SPME) based ambient mass spectrometry (MS) method named surface-coated probe nanoelectrospray ionization (SCP-nanoESI)-MS for analysis of target compounds in individual small organisms with sizes at micrometer-to-millimeter level. SCP-nanoESI-MS analysis involves three procedures: (1) modification of adsorbent at the surface of a fine metal probe to form a specially designed surface-coated SPME probe with probe-end diameter at several-micrometer level, (2) application of the surface-coated SPME probe for enrichment of target analytes from individual small organisms, and (3) employment of a nanospray tip and some solvent to desorb the analytes and induce nanoESI for mass spectrometric analysis under ambient condition. A SCP-nanoESI-MS method for determination of the perfluorinated compounds (PFCs) in individual Daphnia magna was developed. The method showed satisfactory linearities for analysis of real Daphnia magna samples, with correlation coefficient values (R(2)) of 0.9984 and 0.9956 for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), respectively. The limits of detection were 0.02 and 0.03 ng/mL for PFOS and PFOA, respectively. By using the proposed method, the amount, bioaccumulation kinetics, and distribution of PFOS and PFOA in individual Daphnia magna were successfully investigated.

Coupling solid-phase microextraction with ambient mass spectrometry using surface coated wooden-tip probe for rapid analysis of ultra trace perfluorinated compounds in complex samples.

Coupling solid-phase microextraction (SPME) with ambient mass spectrometry using surface coated wooden-tip probe was achieved for the first time and applied in the analysis of ultra trace perfluorinated compounds (PFCs) in complex environmental and biological samples. We modified n-octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride on the surface of sharp wooden tip via silanization to form a novel SPME probe, which was then used for highly selective enrichment of PFCs from complex matrices and applied as a solid substrate to induce electrospray ionization for mass spectrometric analysis. The porous structural surface together with the dual extraction mechanisms (reversed phase adsorption and ion exchange adsorption) demonstrated that the SPME probe has an outstanding enrichment capacity, enhancing sensitivity by approximately 4000-8000 folds for the detection in aqueous samples, and 100-500-fold in whole blood and milk samples. The method showed good linearity, with correlation coefficient values (r(2)) of no less than 0.9931 for eight target PFCs. The limits of detection and qualification of the eight PFCs were 0.06-0.59 and 0.21-1.98 ng/L, respectively. Quantification of real samples was achieved by isotope internal standard calibration curve method or isotope dilution method, and ultratrace levels of PFCs present in lake water, river water, whole blood, and milk samples had been successfully detected and qualified.

Rapid and sensitive determination of legacy and emerging per- and poly-fluoroalkyl substances with solid-phase microextraction probe coupled with mass spectrometry.

This study was designed to develop a rapid and sensitive method for quantifying legacy and emerging per- and polyfluoroalkyl substances (PFASs) in environmental samples with solid-phase microextraction (SPME) coupled with mass spectrometry (MS). An innovative SPME probe was fabricated via in situ polymerization, and the probe coating was optimized with response surface methodology to maximize the fluorine-fluorine interactions and electrostatic properties and ensure high selectivity for the target PFASs with enrichment factors of 48-491. The coupled SPME and MS provided a rapid and sensitive method for analyses of PFASs, with excellent linearity (r ≥ 0.9962) over the concentration range 0.001-1 µg/L and remarkably low detection limits of 0.1-13.0 ng/L. This method was used to analyze trace PFASs in tap water, river water, and wastewater samples and proved to be a simple and efficient analytical method for selective enrichment and detection of contaminants in the environment.

Environmentally relevant concentrations of 2,3,7,8-TCDD induced inhibition of multicellular alternative splicing and transcriptional dysregulation.

Alternative splicing (AS), found in approximately 95 % of human genes, significantly amplifies protein diversity and is implicated in disease pathogenesis when dysregulated. However, the precise involvement of AS in the toxic mechanisms induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) remains incompletely elucidated. This study conducted a thorough global AS analysis in six human cell lines following TCDD exposure. Our findings revealed that environmentally relevant concentration (0.1 nM) of TCDD significantly suppressed AS events in all cell types, notably inhibiting diverse splicing events and reducing transcript diversity, potentially attributed to modifications in the splicing patterns of the inhibitory factor family, particularly hnRNP. And we identified 151 genes with substantial AS alterations shared among these cell types, particularly enriched in immune and metabolic pathways. Moreover, TCDD induced cell-specific changes in splicing patterns and transcript levels, with increased sensitivity notably in THP-1 monocyte, potentially linked to aberrant expression of pivotal genes within the spliceosome pathway (DDX5, EFTUD2, PUF60, RBM25, SRSF1, and CRNKL1). This study extends our understanding of disrupted alternative splicing and its relation to the multisystem toxicity of TCDD. It sheds light on how environmental toxins affect post-transcriptional regulatory processes, offering a fresh perspective for toxicology and disease etiology investigations.

Effect of oxidative stress induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin on DNA damage.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic persistent organic pollutant (POP) that can induce DNA damage within cells. Although oxidative stress is one of the primary mechanisms causing DNA damage, its role in the process of TCDD-induced DNA damage remains unclear. In this study, the TCDD-induced production of reactive oxygen species (ROS) and the occurrence of DNA damage at the AP site were monitored simultaneously. Further investigation revealed that TCDD impaired the activities of superoxide dismutase (SOD) and catalase (CAT), compromising the cellular antioxidant defense system. Consequently, this led to an increase in the production of O2.- and NO, thus inducing DNA damage at the AP site under oxidative stress. Our findings were further substantiated by the upregulation of key genes in the base excision repair (BER) pathway and the absence of DNA AP site damage after inhibiting O2.- and NO. In addition, transcriptome sequencing revealed that TCDD induces DNA damage by upregulating genes associated with oxidative stress in the mitogen-activated protein kinase (MAPK), cyclic adenosine monophosphate (cAMP), and breast cancer pathways. This study provides important insights into the toxicity mechanisms of TCDD.

Single-cell transcriptomic analysis reveals heterogeneity of the patterns of responsive genes and cell communications in liver cell populations of zebrafish exposed to polystyrene nanoplastics.

Nanoplastics (NPs) are a group of emerging environmental pollutants with potential toxicity and health risk on biosystem and ecosystem. Great efforts have been devoted to describing the uptake, distribution, accumulation, and toxicity of NPs at various aquatic organisms; however, the heterogeneous response patterns in zebrafish (Danio rerio) liver cell populations caused by NP exposure have not yet been clarified. Investigation of the heterogeneous response patterns in zebrafish liver cell populations after NPs exposure provides us significances to explore the NP cytotoxicity. In this article, the heterogeneous response patterns in zebrafish liver cell populations after polystyrene (PS)-NPs exposure were studied. Significantly increased content of malondialdehyde and decreased levels of catalase and glutathione were observed, indicating the oxidative damage of zebrafish liver induced by PS-NPs exposure. Afterwards, the liver tissues were enzymatically dissociated and used for single-cell transcriptomic (scRNA-seq) analysis. Nine cell types were identified based on unsupervised cell cluster analysis followed by their marker genes. Hepatocytes were the cell type most impacted by PS-NP exposure, and heterogeneous response patterns of male and female hepatocytes were observed. The PPAR signaling pathway was up-regulated in hepatocytes from both male and female zebrafish. Lipid metabolism-related functions were altered more notably in male-derived hepatocytes, while female-derived hepatocytes were more sensitive to estrogen stimulus and mitochondria. Macrophages and lymphocytes were also highly responsive cell types, with specific immune pathways activated to suggest immune disruption after exposure. Oxidation-reduction process and immune response were significantly altered in macrophages, and oxidation-reduction process, ATP synthesis, and DNA binding were most altered in lymphocytes. Our study not only integrates scRNA-seq with toxicology effects to identify highly sensitive and specific populations of responding cells, revealing highly specialized interactions between parenchymal and non-parenchymal cells and expanding our current understanding of PS-NPs toxicity, but also highlights the importance of cellular heterogeneity in environmental toxicology.

Polystyrene microplastics significantly facilitate influenza A virus infection of host cells.

Microplastics (MPs) are emerging pollutants which exist in various environments and pose a potential threat to human health. However, the effect of MP on respiratory pathogens-infected organisms is unknown. In order to explore the effect of MP on respiratory pathogen infection, we studied the effect of polystyrene microplastics (PS) on influenza A virus (IAV)-infected A549 cells. Western blot, qPCR, and viral plaque assay demonstrated that PS could promote IAV infection. Further study by bioluminescence imaging showed that a large number of IAV could be enriched on PS and entered cells through endocytosis. Meanwhile, the expression of IFITM3 in cells was significantly reduced. In addition, our results showed that PS down-regulated IRF3 and its active form P-IRF3 by down-regulating RIG-I and inhibiting TBK1 phosphorylation activation, which then significantly reduced IFN-ß expression and affected the cellular innate antiviral immune system. Taken together, our results indicate the potential threat of MPs to respiratory diseases caused by IAV and provide new insights into human health protection.