Multi-walled Carbon Nanotubes Exposure Research Articles

Multiwalled Carbon Nanotubes-Reprogrammed Macrophages Facilitate Breast Cancer Metastasis via NBR2/TBX1 Axis.

In recent years, carbon nanotubes have emerged as a widely used nanomaterial, but their human exposure has become a significant concern. In our former study, we reported that pulmonary exposure of multiwalled carbon nanotubes (MWCNTs) promoted tumor metastasis of breast cancer; macrophages were key effectors of MWCNTs and contributed to the metastasis-promoting procedure in breast cancer, but the underlying molecular mechanisms remain to be explored. As a follow-up study, we herein demonstrated that MWCNT exposure in breast cancer cells and macrophage coculture systems promoted metastasis of breast cancer cells both in vitro and in vivo; macrophages were skewed into M2 polarization by MWCNT exposure. LncRNA NBR2 was screened out to be significantly decreased in MWCNTs-stimulated macrophages through RNA-seq; depletion of NBR2 led to the acquisition of M2 phenotypes in macrophages by activating multiple M2-related pathways. Specifically, NBR2 was found to positively regulate the downstream gene TBX1 through H3k27ac activation. TBX1 silence rescued NBR2-induced impairment of M2 polarization in IL-4 & IL-13-stimulated macrophages. Moreover, NBR2 overexpression mitigated the enhancing effects of MWCNT-exposed macrophages on breast cancer metastasis. This study uncovered the molecular mechanisms underlying breast cancer metastasis induced by MWCNT exposure.

Immunomodulation of susceptibility to pneumococcal pneumonia infection in mouse lungs exposed to carbon nanoparticles via dysregulation of innate and adaptive immune responses

Carbon nanotubes (CNTs) are emerging pollutants of occupational and environmental health concern. While toxicological mechanisms of CNTs are emerging, there is paucity of information on their modulatory effects on susceptibility to infections. Here, we investigated cellular and molecular events underlying the effect of multi-walled CNT (MWCNT) exposure on susceptibility to Streptococcus pneumoniae infection in our 28-day sub-chronic exposure mouse model. Data indicated reduced phagocytic function in alveolar macrophages (AMs) from MWCNT-exposed lungs evidenced by lower pathogen uptake in 1-h infection assay. At 24-h post-infection, intracellular pathogen count in exposed AMs showed 2.5 times higher net increase (2-fold in vehicle- versus 5-fold in MWCNT-treated), indicating a greater rate of intracellular multiplication and/or survival due to MWCNT exposure. AMs from MWCNT-exposed lungs exhibited downregulation of pathogen-uptake receptors CD163, Phosphatidyl-serine receptor (Ptdsr), and Macrophage scavenger receptors class A type 1 (Msr1) and type 2 (MSr2). In whole lung, MWCNT exposure shifted the macrophage polarization state towards the immunosuppressive phenotype M2b and increased the CD11c+ dendritic cell population required to activate the adaptive immune response. Notably, the MWCNT pre-exposure dysregulated T-cell immunity, evidenced by diminished CD4 and Th17 response, and exacerbated Th1 and Treg responses (skewed Th17/Treg ratio), thereby favoring the pneumococcal infection. Overall, these findings indicated that MWCNT exposure compromises both innate and adaptive immunity leading to diminished host lung defense against pneumonia infection. To our knowledge, this is the first report on an immunomodulatory role of CNT pre-exposure on pneumococcal infection susceptibility due to dysregulation of both innate and adaptive immunity targets.

Potent lung tumor promotion by inhaled MWCNT

In the lung, carcinogenesis is a multi-stage process that includes initiation by a genotoxic agent, promotion that expands the population of cells with damaged DNA to form a tumor, and progression from benign to malignant neoplasms. We have previously shown that Mitsui-7, a long and rigid multi-walled carbon nanotube (MWCNT), promotes pulmonary carcinogenesis in a mouse model. To investigate the potential exposure threshold and dose-response for tumor promotion by this MWCNT, 3-methylcholanthrene (MC) initiated (10 μg/g, i.p., once) or vehicle (corn oil) treated B6C3F1 mice were exposed by inhalation to filtered air or MWCNT (5 mg/m3) for 5 h/day for 0, 2, 5, or 10 days and were followed for 17 months post-exposure for evidence of lung tumors. Pulmonary neoplasia incidence in MC-initiated mice significantly increased with each MWCNT exposure duration. Exposure to either MC or MWCNT alone did not affect pulmonary neoplasia incidence compared with vehicle controls. Lung tumor multiplicity in MC-initiated mice also significantly increased with each MWCNT exposure duration. Thus, a significantly higher lung tumor multiplicity was observed after a 10-day MWCNT exposure than following a 2-day exposure. Both bronchioloalveolar adenoma and bronchioloalveolar adenocarcinoma multiplicity in MC-initiated mice were significantly increased following 5- and 10-day MWCNT exposure, while a 2-day MWCNT exposure in MC-initiated mice significantly increased the multiplicity of adenomas but not adenocarcinomas. In this study, even the lowest MWCNT exposure promoted lung tumors in MC-initiated mice. Our findings indicate that exposure to this MWCNT strongly promotes pulmonary carcinogenesis.

Estrogen contributes to sex differences in M2a macrophages during multi-walled carbon nanotube-induced respiratory inflammation.

Lung diseases characterized by type 2 inflammation are reported to occur with a female bias in prevalence/severity in both humans and mice. This includes previous work examining multi-walled carbon nanotube (MWCNT)-induced eosinophilic inflammation, in which a more exaggerated M2a phenotype was observed in female alveolar macrophages (AMs) compared to males. The mechanisms responsible for this sex difference in AM phenotype are still unclear, but estrogen receptor (ER) signaling is a likely contributor. Accordingly, male AMs downregulated ERα expression after MWCNT exposure while female AMs did not. Thus, ER antagonist Fulvestrant was administered prior to MWCNT instillation. In females, Fulvestrant significantly attenuated MWCNT-induced M2a gene expression and eosinophilia without affecting IL-33. In males, Fulvestrant did not affect eosinophil recruitment but reduced IL-33 and M2a genes compared to controls. Regulation of cholesterol efflux and oxysterol synthesis is a potential mechanism through which estrogen promotes the M2a phenotype. Levels of oxysterols 25-OHC and 7α,25-OHC were higher in the airways of MWCNT-exposed males compared to MWCNT-females, which corresponds with the lower IL-1β production and greater macrophage recruitment previously observed in males. Sex-based changes in cholesterol efflux transporters Abca1 and Abcg1 were also observed after MWCNT exposure with or without Fulvestrant. Invitro culture with estrogen decreased cellular cholesterol and increased the M2a response in female AMs, but did not affect cholesterol content in male AMs and reduced M2a polarization. These results reveal the modulation of (oxy)sterols as a potential mechanism through which estrogen signaling may regulate AM phenotype resulting in sex differences in downstream respiratory inflammation.

Effects of advanced nanomaterials on the respiratory system of a murine COPD model

This study assessed the effects of cellulose nanofibrils (CNFs) and multi-walled carbon nanotubes (MWCNTs) on lung inflammation in a cigarette smoke-induced chronic obstructive pulmonary disease (COPD) mouse model. Prior to instillation, COPD model mice displayed distinctive cellular compositions and elevated cytokine levels in bronchoalveolar lavage fluid (BALF). After intratracheal instillation of 80 μg CNFs, no significant histopathological changes, BALF composition alterations, or cytokine level shifts were observed on day 28. This suggests minimal lung impact and no interference with reducing smoke-induced inflammation. In contrast, the instillation of 80 μg MWCNTs resulted in significant histopathological changes, increased cellular composition, and elevated cytokine levels in BALF on day 28. These findings indicate that CNF exposure had little effect on the lungs and did not impede the reduction of smoke-induced inflammation, while MWCNT exposure hindered the attenuation of pulmonary inflammatory response. The study emphasizes the importance of considering diverse cases, including individuals with pre-existing respiratory conditions, when assessing occupational safety and health risks associated with advanced nanomaterial exposure.

Hypermethylation and low expression of FANCC involved in multi-walled carbon nanotube-induced toxicity on ARPE-19 cells

Multi-walled carbon nanotube (MWCNT) exposure was observed to cause damages on the viability of ocular cells, however, the underlying mechanisms remain not well understood. Epigenetic alterations that regulate gene expression have been identified as a major responsiveness to environmental challenge. Thus, the aim of this study was to screen methylation-regulated genes involved in MWCNT exposure. The Illumina Human Methylation 850 K array was employed to determine the genome-wide DNA methylation profile of human retinal pigment epithelial cell line (ARPE-19) exposed to 50% inhibition concentration of MWCNTs (100 μg/ml) for 24 h or without (n = 3 for each group). Then, the transcriptome data obtained by high-throughput RNA sequencing previously were integrated with DNA methylome to identify the overlapped genes. As a result, the integrative bioinformatics analysis identified that compared with controls, FA complementation group C (FANCC) was hypermethylated and downregulated in MWCNT-exposed ARPE-19 cells. Quantitative real-time polymerase chain reaction analysis confirmed the mRNA expression level of FANCC was significantly decreased following MWCNT treatment and the addition of DNA methylation inhibitor 5-Aza-deoxycytidine (10 μM) reversed this decrease. Pyrosequencing analysis further validated the hypermethylation status at the 5′-untranslated promoter region of FANCC (cg14583550) in MWCNT-exposed ARPE-19 cells. Protein-protein interaction network and function analyses predicted that FANCC may contribute to MWCNT-induced cytotoxicity by interacting with heat shock protein 90 beta family member 1 and then upregulating cytokine interleukin-6 and apoptosis biomarker caspase 3. In conclusion, the present study links the epigenetic modification of FANCC with the pathogenesis of MWCNT-induced retinal toxicity.

Nitro-oxidative response to internalized multi-walled carbon nanotubes in Brassica napus and Solanum lycopersicum

In addition to their beneficial effects on plant physiology, multi-walled carbon nanotubes (MWCNTs) are harmful to plants in elevated concentrations. This study compared the effects of two doses of MWCNT (10 and 80 mg/L) in Brassica napus and Solanum lycopersicum seedlings focusing on nitro-oxidative processes. The presence of MWCNTs was detectable in the root and hypocotyl of both species. Additionally, transmission electron microscopy analysis revealed that MWCNTs are heavily transformed within the root cells forming large aggregates. The uptake of MWCNTs negatively affected root viability and root cell proliferation of both species, but more intense toxicity was observed in S. lycopersicum compared to B. napus. The presence of MWCNT triggered more intense protein carbonylation in the relative sensitive S. lycopersicum, where increased hydrogen peroxide levels were observed. Moreover, MWCNT exposure increased the level of physiological protein tyrosine nitration which was more intense in S. lycopersicum where notable peroxynitrite accumulation occurred. These suggest for the first time that MWCNT triggers secondary nitro-oxidative stress which contributes to its toxicity. Moreover, the results indicate that the extent of the nitro-oxidative processes is associated with the extent of MWCNT toxicity.

Preparation and characterization of polyhydroxyamide composite fibers reinforced with carbon nanotubes—Experiment and computation collaborative approach

AbstractAn experiment‐computation collaborative approach is conducted to investigate and understand the mechanical reinforcement effect of carbon nanotubes (CNTs) on polyhydroxyamide (PHA) composite fibers. The experimental studies provide results of PHA composite fibers with enhanced mechanical properties based on well‐dispersed multi‐walled CNTs (MWCNTs) inside the PHA matrix, suggesting optimal conditions for the experiment‐computation collaborative approach in the polymer/CNT composite systems. For instance, adding MWCNTs up to 2.0 wt% results in an improvement of 48.0% in tensile strength, 36.7% in initial modulus, and 69.9% in breaking strain of the composite fibers. FE‐SEM images show that many MWCNTs are partially exposed or pulled out to the outside of the PHA matrix through the tensile fracture in a very homogeneous dispersion state. In addition, the molecular dynamics (MD) simulation study with a single‐walled CNT (SWCNT) exhibits that the overall displacements of PHA molecules near the CNT surface decrease gradually, which indicates that the PHA molecules form an effective interaction with the CNT. Therefore, it is clear that the excellent interaction between PHA and CNTs, obtained from the results of well‐dispersed CNTs (experiment) and the reduction of the overall displacement of PHA molecules near the CNT surface (computation), is a very important clue to support the improved mechanical characteristics of the PHA fibers reinforced with the CNTs. Moreover, we are confident that the combined experimental‐computational approach used this study will yield valuable insights into the design of composite systems.Highlights Experiment‐computation collaboration reveals enhanced mechanical properties of PHA composite fibers with well‐dispersed CNTs. Addition of MWCNTs up to 2.0 wt% improves tensile strength by 48.0%, initial modulus by 36.7%, and breaking strain by 69.9%. FE‐SEM images demonstrate homogeneous dispersion and partial exposure of MWCNTs in the fractured PHA matrix. MD simulation shows reduced displacements of PHA molecules near SWCNT surface, indicating effective interaction. Combined experimental‐computational approach offers valuable insights for designing composite systems.

The in vitro immunomodulatory effect of multi-walled carbon nanotubes by multilayer analysis

The study of multi-walled carbon nanotube (MWCNT) induced immunotoxicity is crucial for determining hazards posed to human health. MWCNT exposure most commonly occurs via the airways, where macrophages are first line responders. Here we exploit an in vitro assay, measuring dose-dependent secretion of a wide panel of cytokines, as a measure of immunotoxicity following the non-lethal, multi-dose exposure (IC5, IC10 and IC20) to 7 MWCNTs with different intrinsic properties. We find that a tangled structure, and small aspect ratio are key properties predicting MWCNT induced immunotoxicity, mediated predominantly by IL1B cytokine secretion. To assess the mechanism of action giving rise to MWCNT immunotoxicity, transcriptomics analysis was linked to cytokine secretion in a multilayer model established through correlation analysis across exposure concentrations. This reinforced the finding that tangled MWCNTs have greater immunomodulatory potency, displaying enrichment of immune system, signal transduction and pattern recognition associated pathways. Together our results further elucidate how structure, length and aspect ratio, critical intrinsic properties of MWCNTs, are tied to immunotoxicity.

Inflammatory Genes Associated with Pristine Multi-Walled Carbon Nanotubes-Induced Toxicity in Ocular Cells.

The wide application of multi-walled carbon nanotubes (MWCNTs) in various fields has raised enormous concerns regarding their safety for humans. However, studies on the toxicity of MWCNTs to the eye are rare and potential molecular mechanisms are completely lacking. This study was to evaluate the adverse effects and toxic mechanisms of MWCNTs on human ocular cells. Human retinal pigment epithelial cells (ARPE-19) were treated with pristine MWCNTs (7-11 nm) (0, 25, 50, 100 or 200 μg/mL) for 24 hours. MWCNTs uptake into ARPE-19 cells was examined using transmission electron microscopy (TEM). The cytotoxicity was evaluated by CCK-8 assay. The death cells were detected by Annexin V-FITC/PI assay. RNA profiles in MWCNT-exposed and non-exposed cells (n = 3) were analyzed using RNA-sequencing. The differentially expressed genes (DEGs) were identified through the DESeq2 method and hub of which were filtered by weighted gene co-expression, protein-protein interaction (PPI) and lncRNA-mRNA co-expression network analyses. The mRNA and protein expression levels of crucial genes were verified using quantitative polymerase chain reaction (qPCR), colorimetric analysis, ELISA and Western blotting. The toxicity and mechanisms of MWCNTs were also validated in human corneal epithelial cells (HCE-T). TEM analysis indicated the internalization of MWCNTs into ARPE-19 cells to cause cell damage. Compared with untreated ARPE-19 cells, those exposed to MWCNTs exhibited significantly decreased cell viabilities in a dose-dependent manner. The percentages of apoptotic (early, Annexin V positive; late, Annexin V and PI positive) and necrotic (PI positive) cells were significantly increased after exposure to IC50 concentration (100 μg/mL). A total of 703 genes were identified as DEGs; 254 and 56 of them were, respectively, included in darkorange2 and brown1 modules that were significantly associated with MWCNT exposure. Inflammation-related genes (including CXCL8, MMP1, CASP3, FOS, CXCL2 and IL11) were screened as hub genes by calculating the topological characteristics of genes in the PPI network. Two dysregulated long non-coding RNAs (LUCAT1 and SCAT8) were shown to regulate these inflammation-related genes in the co-expression network. The mRNA levels of all eight genes were confirmed to be upregulated, while caspase-3 activity and the release of CXCL8, MMP1, CXCL2, IL11 and FOS proteins were demonstrated to be increased in MWCNT-treated ARPE-19 cells. MWCNTs exposure also can induce cytotoxicity and increase the caspase-3 activity and the expression of LUCAT1, MMP1, CXCL2, and IL11 mRNA and protein in HCE-T cells. Our study provides promising biomarkers for monitoring MWCNT-induced eye disorders and targets for developing preventive and therapeutic strategies.

Exposure to multi-walled carbon nanotubes causes suppression in octopamine signal associated with transgenerational toxicity induction in C.elegans

Multi-walled carbon nanotube (MWCNT), a kind of carbon-based nanomaterials, has been extensively utilized in a variety of fields. In Caenorhabditis elegans, MWCNT exposure can result in toxicity not only at parental generation (P0-G) but also in the offspring. However, the underlying mechanisms remain still largely unknown. DAF-12, a transcriptional factor (TF), was previously found to be activated and involved in transgenerational toxicity control after MWCNT exposure. In this study, we observed that exposure to 0.1–10 μg/L MWCNTs caused the significant decrease in expression of tbh-1 encoding a tyramine beta-hydroxylase with the function to govern the octopamine synthesis, suggesting the inhibition in octopamine signal. After exposure to 0.1 μg/L MWCNT, the decrease in tbh-1 expression could be also detected in F1-G and F2-G. Moreover, in germline cells, the TF DAF-12 regulated transgenerational MWCNT toxicity by suppressing expression and function of TBH-1. Meanwhile, exposure to 0.1–10 μg/L MWCNTs induced the increase in octr-1 expression and the decrease in ser-6 expression. After exposure to 0.1 μg/L MWCNT, the increased octr-1 expression and the decreased ser-6 expression were further observed in F1-G and F2-G. Germline TBH-1 controlled transgenerational MWCNT toxicity by regulating the activity of octopamine receptors (SER-6 and OCTR-1) in offspring. Furthermore, in the offspring, SER-6 and OCTR-1 affected the induction of MWCNT toxicity by upregulating or downregulating the level of ELT-2, a GATA TF. Taken together, these findings suggested possible link between alteration in octopamine related signals and MWCNT toxicity induction in offspring in organisms.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels.

Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications. Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure. Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.

Continuous infiltration of small peritoneal macrophages in the mouse peritoneum through CCR2-dependent and -independent routes during fibrosis and mesothelioma development induced by a multiwalled carbon nanotube, MWNT-7.

Although toxicities of multiwalled carbon nanotube (MWCNT) have been found to be related with activities of macrophages phagocytosing the fibers, the exact relationship between macrophage population and pathogenesis of fibrosis and mesotheliomas induced by MWCNTs is largely unknown. CCL2-CCR2 axis, a major monocyte/macrophage infiltration route, is thought to be involved in not only acute inflammation but also the formation of tumor microenvironment. We therefore described a time-course of alteration of macrophage population in an attempt to clarify the contribution of the Ccr2 gene to mesotheliomagenesis. Wild-type (WT) C57BL/6 mice and Ccr2-knockout (KO) mice were intraperitoneally administered with MWNT-7 and were sequentially necropsied at 1, 7, 28, 90, and 245 day(s) after the injection. Peritoneal fibrosis was prominent in all MWCNT-treated mice, with a lower severity in the KO mice. No differences were observed in the incidences of neoplastic lesions of mesothelia between WT and KO mice. A flow cytometric analysis revealed that after gross disappearance of macrophages after MWCNT exposure, small peritoneal macrophages (SPMs) were exclusively refurbished by the CCR2-dependent route at day 1 (as Ly-6C+MHC class II- cells), followed by additional CCR2-independent routes (as Ly-6C-MHC class II- cells); i.e., the only route in KO mice; with a delay of 1-7 days. The SPMs derived from both routes appeared to differentiate into maturated cells as Ly-6C-MHC class II+, whose ratio increased in a time-dependent manner among the total SPM population. Additionally, most macrophages expressed M1-like features, but a small fraction of macrophages exhibited an M1/M2 mixed status in MWCNT-treated animals. Our findings demonstrate a long-persistent activation of the CCL2-CCR2 axis after MWCNT exposure and enable a better understanding of the participation and potential roles of SPMs in fibrous material-induced chronic toxicities.

Multi-walled carbon nanotubes induce transgenerational toxicity associated with activation of germline long non-coding RNA linc-7 in C.elegans.

With the increase in application, multi-walled carbon nanotubes (MWCNTs) are potentially bioavailable to environmental organisms. However, the potential transgenerational effect of MWCNTs and underlying mechanisms remains still unclear. Here, we examined transgenerational MWCNT toxicity and the underlying mechanism mediated by germline long non-coding RNAs (lncRNAs) in Caenorhabditis elegans. Exposure to 0.1-10μg/L MWCNT caused transgenerational toxicity reflected by endpoints of brood size and locomotion behavior. Meanwhile, among germline lncRNAs, expression of 5 lncRNAs were dysregulated by MWCNT exposure. Among these 5 dysregulated lncRNAs, only germline RNAi of linc-7 affected MWCNT toxicity. Increase in germline linc-7 expression was observed transgenerationally, and transgenerational MWCNT toxicity was prevented in linc-7(RNAi) nematodes. Moreover, germline linc-7 controlled transgenerational MWCNT toxicity by activating downstream DAF-12, a transcriptional factor. Therefore, our data indicated the association between induction of transgenerational MWCNT toxicity and increase in germline linc-7 expression in organisms.

Interactions between multi-walled carbon nanotubes and plankton as detected by Raman spectroscopy

Raman spectroscopy has been commonly used in materials science to detect chemicals. Based on inelastic scattering of light after incident photons interact with a molecule, it has high potential for non-destructive detection of specific contaminants in living biological specimens. The increasing use of carbon nanotubes (CNTs) increases its chance to enter the aquatic habitats through direct discharge, surface runoff and atmospheric deposition, but their potential environmental impacts remain poorly known. We tested the use of Raman spectroscopy to investigate the interactions between multi-walled CNTs (MWCNTs) and aquatic plankton in vivo. For phytoplankton cells (Scenedesmus obliquus) that were exposed to MWCNTs, Raman spectroscopy was able to distinguish between background biological material and MWCNTs that adhere to the cells (G-band peak at 1590 cm−1 and D-band peak at 1350 cm−1 in the Raman spectra that were unique to MWCNTs). Harmful effects of MWCNT exposure manifested as lower photosynthetic efficiency and/or lower specific growth rate in the phytoplankton. MWCNT particles also adhered to the body surface of zooplankton, especially the carapace. Both Ceriodaphnia sp. and Daphnia sp. ingested MWCNTs directly, which was verified by the signature G-band and D-band Raman peaks in the zooplankton gut region. MWCNTs remained in the gut overnight after the zooplankton had been returned to clean water, showing that the zooplankton retained MWCNTs inside their body for an extended time, thereby increasing the chance to disperse and transfer the contaminants throughout the aquatic food web. Our results demonstrate that Raman spectroscopy is a promising method for non-destructive investigation of the uptake and dynamic fate of CNTs and other contaminants in aquatic organisms.

Aging influence on pulmonary and systemic inflammation and neural metabolomics arising from pulmonary multi-walled carbon nanotube exposure in apolipoprotein E-deficient and C57BL/6 female mice

Objective Environmental exposures exacerbate age-related pathologies, such as cardiovascular and neurodegenerative diseases. Nanoparticulates, and specifically carbon nanomaterials, are a fast-growing contributor to the category of inhalable pollutants, whose risks to health are only now being unraveled. The current study assessed the exacerbating effect of age on multiwalled-carbon nanotube (MWCNT) exposure in young and old C57BL/6 and ApoE−/− mice. Materials and methods Female C57BL/6 and apolipoprotein E-deficient (ApoE−/−) mice, aged 8 weeks and 15 months, were exposed to 0 or 40 µg MWCNT via oropharyngeal aspiration. Pulmonary inflammation, inflammatory bioactivity of serum, and neurometabolic changes were assessed at 24 h post-exposure. Results Pulmonary neutrophil infiltration was induced by MWCNT in bronchoalveolar lavage fluid in both C57BL/6 and ApoE−/−. Macrophage counts decreased with MWCNT exposure in ApoE−/− mice but were unaffected by exposure in C57BL/6 mice. Older mice appeared to have greater MWCNT-induced total protein in lavage fluid. BALF cytokines and chemokines were elevated with MWCNT exposure, but CCL2, CXCL1, and CXCL10 showed reduced responses to MWCNT in older mice. However, no significant serum inflammatory bioactivity was detected. Cerebellar metabolic changes in response to MWCNT were modest, but age and strain significantly influenced metabolite profiles assessed. ApoE−/− mice and older mice exhibited less robust metabolite changes in response to exposure, suggesting a reduced health reserve. Conclusions Age influences the pulmonary and neurological responses to short-term MWCNT exposure. However, with only the model of moderate aging (15 months) in this study, the responses appeared modest compared to inhaled toxicant impacts in more advanced aging models.

Nanoparticle-Induced Airway Eosinophilia Is Independent of ILC2 Signaling but Associated With Sex Differences in Macrophage Phenotype Development.

The majority of lung diseases occur with a sex bias in terms of prevalence and/or severity. Previous studies demonstrated that, compared with males, female mice develop greater eosinophilic inflammation in the airways after multiwalled carbon nanotube (MWCNT) exposure. However, the mechanism by which this sex bias occurs is unknown. Two immune cells that could account for the sex bias are type II innate lymphoid cells (ILC2s) and alveolar macrophages (AMs). In order to determine which immune cell type was responsible for MWCNT-induced airway eosinophil recruitment and subsequent sex differences in inflammation and disease, male and female C57BL/6 mice were exposed to MWCNTs (2 mg/kg) via oropharyngeal aspiration, and the respiratory immune response was assessed 7 d later. Greater eosinophilia and eotaxin 2 levels were observed in MWCNT-treated females and corresponded with greater changes in airway hyperresponsiveness than those in MWCNT-treated males. In MWCNT-treated females, there was a significant increase in the frequency of ILC2s within the lungs compared with control animals. However, depletion of ILC2s via α-CD90.2 administration did not decrease eosinophil recruitment 24 h and 7 d after MWCNT exposure. AMs isolated from control and MWCNT-treated animals demonstrated that M2a macrophage phenotype gene expression, ex vivo cytokine production, and activation of (p)STAT6 were upregulated to a significantly greater degree in MWCNT-treated females than in males. Our findings suggest that sex differences in AM phenotype development, not ILC2 signaling, are responsible for the observed female bias in eosinophilic inflammation after MWCNT inhalation.

Exposure to a combination of MWCNTs and DBP causes splenic toxicity in mice

The large conjugated π bond in the molecular structure of carbon nanotubes (CNTs) interacts with the benzene ring structure in di (n-butyl) phthalates (DBP) through a π - π bond. Compounds of CNTs and DBP form easily, becoming another environmental pollutant of concern. We explore whether CNTs entering animals slow down the degradation of the DBP adsorbed in the CNT cavity, thereby prolonging the "hormonal activity" of DBP. In our study, male BALb/c mice were used as experimental subjects divided into four groups: the control group; the multi-walled carbon nanotubes (MWCNTs) exposure group (10mg/kg/d); the DBP exposure group (2.15 mg/kg/d); and the compound exposure group (MWCNTs + DBP). After 30 days of exposure, the mice were sacrificed and their spleens used for immunotoxicology study. The results showed that the exposure groups exhibited splenomegaly and suffered severe oxidative damage to the spleen. In the compound exposure group: levels of IgA and IgG in the serum of the mice changed, and were significantly different from levels in both the MWCNTs and DBP exposure groups (p <0.05); the pathological sections of the spleen showed that the boundary between the white pulp area (WP) and the red pulp area (RP) was blurred, that the cell arrangement was loose, and that more red blood cells were retained in the spleen. Proteomics mass spectrometry analysis showed that compared with the control group, 70 proteins were up-regulated and 27 proteins were down-regulated in the MWCNTs group, 36 proteins were up-regulated and 23 proteins were down-regulated in the DBP group, 87 proteins were up-regulated and 21 proteins were down-regulated in the compound exposure group. The results of GO enrichment analysis and KEGG enrichment analysis of the differentially expressed proteins showed that the compound exposure harmed the spleen antigen recognition, processing, and presentation, inhibited the activation and proliferation of B cells and T cells, and hindered the adaptive immune responses. Our results showed that MWCNTs and DBP compounds can damage the spleen, and impair the innate and adaptive immune functions of the body.

Multi-Walled Carbon Nano-Tubes (MWCNTs) Accelerate Leukaemia Development in Mice: Implications for Drug Development in Humans

Biomedical applications of nano-technology including carbon nano-tubes (CNTs) are of considerable recent interest. However, relatively little attention is given to potential biological effects including cancer-induction or acceleration resulting from long-term exposures. We investigated whether and how multi-walled carbon nano-tubes (MWCNTs) affected leukaemia development in a mouse model. We found injections of MWCNTs accelerated murine leukaemia virus (MLV)-induced leukaemia development in normal mice with the magnitude of acceleration correlated with extent of MWCNT exposure. In contrast, injection of MWCNTs to immune-deficient mice caused no acceleration. Co-treatment of normal mice with MWCNTs and rapamycin reduced or eliminated accelerated leukaemia development. We found exposure to MWCNTs induced phagocytosis by macrophages and production of pro-inflammatory cytokines in normal mice and in cultured RAW264.7 cells. Methoxy-polyethylene glycol amine modified MWCNTs (PEG-MWCNTs) did not accelerate leukaemia development in normal mice, an effect correlated with reduced macrophage phagocytosis and reduced production of pro-inflammatory cytokines. We also found increased levels of pro-inflammatory cytokines induced by exposure to MWCNTs in normal mice increased intra-cellular reactive oxide species (ROS) levels resulting in increased cell proliferation and enhanced survival of the leukaemia cells. Our data indicate exposure to MWCNTs accelerates leukaemia development in a virus-induced leukaemia model. This effect is associated with activation of macrophages and increased levels of pro-inflammatory cytokines resulting in increased intra-cellar ROS levels. Our data have implications for the application of nano-technology to drug development in humans. For example, some MWCNTs are of the same size as asbestos fibers proved to cause mesothelioma in mice and humans. Also, increased macrophage phagocytosis and production of pro-inflammatory cytokines are reported in these settings. Our data also suggest interventions which might reduce a theoretical increased cancer-risk or acceleration of cancer development resulting from MWCNT exposures. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of the combined toxicity of multi-walled carbon nanotubes and cadmium on earthworms in soil using multi-level biomarkers

The coexistence of multi-walled carbon nanotubes (MWCNTs) with cadmium (Cd) in soil may cause the combined biological effects, but few study reported about their joint toxic effects on earthworms. Therefore, this study investigated the effects of sub-lethal levels of MWCNTs (10, 50, 100 mg/kg) and Cd (2.0, 10 mg/kg) on earthworms Eisenia fetida for 14 days. The changes in multi-level biomarkers of growth inhibition rate, cytochrome P450 isoenzymes (CYP1A2, 2C9 and 3A4), and small molecular metabolites (metabolomics) were determined. The toxic interaction between MWCNTs and Cd was characterized by the combination of the biomarker integration index (BRI), joint effect index concentration addition index (CAI), and the effect concentration addition index (EAI). The results showed that the single MWCNTs exposure caused insignificant change in most biomarkers, while the combined exposure of MWCNTs (50–100 mg/kg) and 10 mg/kg Cd led to significant changes in ten most important metabolites identified by metabolomics and activities of CYP1A2, 2C9, and 3A4. Compared with the toxicity of Cd alone, the combined toxicity of the mixture was significantly reduced. According to the integration of BRI and CAI/EAI, a clearly antagonistic interaction at relatively low effects was observed between MWCNTs and Cd. The responses of multiple biomarkers suggest the toxic action mode of the mixture on earthworms was related to the oxidative injury, and the disruption of amino acid, purine, and pyrimidine metabolism, and the urea cycle.