Suchergebnisse

Abstract
Many in vitro studies that focus upon lung toxicology use models that assume, or are specifically based upon adult lung structure, function, and physiology. Yet, the most vulnerable groups of the human population (i.e., young, elderly, and diseased) have an altered lung structure and function. Notably, the developing lung (in utero) and infant lungs exhibit notable differences in term of cell type, number, ratio, organ size and surface area. Recent work has identified the detrimental implications of being exposed to AP while the lung is developing. To elucidate the mechanisms associated with AP exposure to the developing lung, the aim of this study will create and characterise an in vitro model of the foetal human bronchial region composed of cells physiologically appropriate to various stages of development, as well as an older child's airway, notably the developing alveoli. This will allow us to determine the developmental effects of air pollution particles at an air-liquid interface (ALI) utilising a physiologically relevant aerosol exposure and standardised particulate aiming to mimic indoor and outdoor air pollution. For this work, a global approach will be utilised to determine up and down regulation of various pathways associated with both development and exposure to AP. A further aim will include comparison with an "adult" lung to analysis the potential developmental implications exposure to AP may illicit.
This project is funded by the recently initiated RESPIRE project (NE/W002264/1), as part of the SPF Clean Air Programme.

Abstract
In vitro lung models are a valuable tool to assess the inhalation hazard of air pollutants, bioaerosols, chemicals and nanomaterials. Though it is essential that these models are characterised and anatomically relevant to the area of the human lung they seek to represent. Previously within inhalation toxicology, focus has been placed on the use of monocultures of alveolar epithelial cells, however use of alveolar co-culture models have been presented as more realistic and sensitive models. Here, we present the development of a novel triple cell co-culture system, cultured at the air-liquid interface that is sensitive to LPS and particulate matter. This co-culture contains alveolar type 1 cells (hAELVi), alveolar type 2 cells (NCI-H441) and alveolar macrophages (differentiated THP-1 (dTHP-1) using PMA (20 nM)). Using CellTrackers™ with confocal microscopy, the ratio of hAELVi cells to NCI-H441 cells was confirmed to be a histologically relevant 16:1 ratio forming a tight monolayer shown through blue dextran membrane permeability assay. Macrophages were seeded on top of the epithelial co-culture at an anatomically relevant density of 1 macrophage per 18x103 μm2 (confirmed via confocal microscopy) to form the triple cell co-culture. The model, when challenged, released more interleukin-8 and TNF-α than the epithelial mono- and bi-culture systems when exposed to LPS (1 μg/mL for 24 hrs) (p<0.05), suggesting that the macrophages do mount the theoretical (pro-)inflammatory response. This anatomically relevant in vitro model forms the basis to investigate inhaled exogenous substances, such as the differential toxicology impacts of particulate matter and nitrogen dioxide.

Abstract
Production and application of multi-component nanomaterials (MCNMs) and high aspect ratio nanomaterials (HARN) has raised questions as to their inhalation risk. A PRISMA-orientated systematic literature review indicated a key knowledge gap related to the (geno)toxicity of metal oxide MCNMs and HARN. The aim of this study was to conduct a mechanistic-based toxicology assessment of a number of industrially-relevant MCNMs and HARN materials, in order to fill this knowledge gap and provide vital hazard assessment towards these materials' safe and sustainable design. A549 monocultures (2.5x105), and A549/dTHP-1 co-cultures (1:10) were seeded in 12-well transwell-membranes at the air-liquid interface. Cell cultures were exposed, via aerosol, to Printex90, LaCo0.5Ni0.05O3, LaCo0.475Ni0.475Pt0.05O3, LaCo0.475Ni0.475Pd0.05O3, LaCo0.25Ni0.75O3, LaNiO3, NiZnFe4O8, ZnFe2O4, and NiFe2O4 to human exposure-relevant concentrations of 390, 780, 3100 ng/cm2 (24hrs). Aerosolised MCNM and HARN were characterised following acellular deposition via electron microscopy. Cellular cytotoxicity, barrier integrity, (pro)inflammatory/fibrotic response, oxidative stress and mutagenicity/DNA damage was assessed. Results indicate barrier integrity and IL-8 production were unaffected by Printex90, LaCo0.5Ni0.05O3, and ZnFe2O4 exposure. A dose-dependent increase in micronucleus frequency was observed for these four materials. Investigations are continuing to determine the mechanisms associated with this noted genotoxic effect, as well as upon further MCNMs/HARN (i.e. Fe2CoO4(Fe1/Co3), Silica-Ref-Std). Emphasis is being placed upon elucidating specific structure-activity relationships for each MCNM/HARN by testing specific key events of inhalation-based adverse outcome pathways relevant to the in vitro models used. This project was funded by EU H2020 research and innovation programme, grant agreement No. 953183 (HARMLESS).

Abstract
Cell passage number is of high importance in experimental design and can cause biochemical, genetic, and/or morphological changes to a cell. This project aimed to understand the impact of increasing cell passage number on the structure and function of epithelial cells from different organs. Two epithelial cell types were studied, NCI-H441 lung cells (type II alveolar cells), and HepG2 liver cells. Predefined ranges of 11-20, 21-30, 31-40, and 41-50 were studied. Cells were exposed to 1µg/mL tumour necrosis factor α (TNFα) and 15µM hydrogen peroxide (H2O2) via submerged or quasi-air liquid interface, according to GIVIMP guidelines. Endpoints tests included cytotoxicity (lactate dehydrogenase (LDH) release), cell death (BCL2, BAX, FAS), (pro-) inflammatory mediators (IL6 & IL8), oxidative stress (SOD1) and genetic toxicology (DNA damage (p21 & p53)). In addition, confocal microscopy was used to observe any changes to cell morphology. Preliminary data indicates the NCI-H441 epithelial cell passage range has an effect on the secretion of (pro-)inflammatory mediators, with an increase in IL8 and IL6 in higher passage ranges (41-50) compared to lower passage ranges (11-20) when stimulated with TNFα as a positive control. In HepG2 cells there is also an increase in IL8 production across passage range, however, it is considerably less than NCI-H441 cells. In addition, IL6 production in HepG2 cells was negligible across all passage ranges. LDH assay showed minimal variation between cell type and passage number. Data obtained suggests passage range, depending on cell type, has an influence on the effect level of the end point measured.

Abstract
Air pollution (AP) is a significant environmental risk to human health. Historically, the impact of AP exposure has focused upon the physical health effects, yet the implications of AP on mental health have received limited attention. Despite this, recent research has highlighted emerging evidence supporting a possible aetiological link. The purpose of this study, therefore, was to investigate the potential consequences of AP upon mental health and well-being. In a PRISMA based systematic review multiple databases were searched from January 2012 to 2022 for peer-reviewed, English-language, human based primary research. Of the 2,224 studies identified in the literature search, 94 met the inclusion criteria. The mental health and wellbeing issues explored were psychosis, anxiety, suicide, mania, overall diagnosed conditions, and self-reported wellbeing symptoms. Depression was omitted due to the volume of attention the condition has received in the literature. Key air pollutants, particulate matter (PM10 and PM2.5), sulphur oxides (SOx), carbon monoxide (CO) and nitrogen oxides (NOx) were investigated. Data revealed a positive association between psychotic disorders and NOx exposure in ten studies with only one contradictory finding. Whereas the other conditions received multiple contradictory findings. To compare these results to real-life situations, currently a pilot study is exploring associations between psychotic disorder diagnoses and AP, specifically NOx, in Wales using the Secure Anonymised Information Linkage (SAIL) database. There is a large body of evidence showing associations between mental health and AP. However, due to research complexities and some contradictory findings more high-quality research is required to elucidate these relationships.

Abstract
When considering air pollution particles (AP), they are often assessed in isolation, when this is not how the population is exposed to them. Within the indoor environment, there are bioaerosols present, which contain various allergens including house dust mite (HDM) and lipopolysaccharides (LPS). This RESPIRE study aims to apply a well-characterised in vitro model of the alveolar barrier (type I and II epithelial cells (NCI-H441 and hAELVi at relevant in vivo ratios) and macrophage-like cells) to determine the effects of these bioaerosols on the toxicological and immunological responses to AP. To achieve this, we will investigate cytotoxicity, inflammatory responses, morphology, oxidative stress, membrane integrity and genetic toxicology of these models with specific focus on responses from human allergic responses and airway diseases (i.e., bronchiolitis-interstitial lung disease and sarcoidosis) to determine the additional health considerations the addition of bioaerosols to AP has on our models. We will utilise a physiologically relevant aerosolization exposure method) and a standardised indoor (NIST2583) particulate at an environmentally relevant concentration to mimic air pollution along with HDM and LPS to determine if the addition of these environmentally appropriate bioaerosols influence the responses to IAP.
This project is funded by the recently initiated RESPIRE project (NE/W002264/1), as part of the SPF Clean Air Programme.

Abstract
The impact of micro-, nanoplastics (MNPs) and particulate matter (PM) upon human health are escalating concerns worldwide. MNPs released from surgical face masks, and PM from ambient air pollution, can both be inhaled and consequently have potentially significant negative impacts upon human health, particularly on our lung epithelial cell structure and function. Currently, the impact of the realistic co-exposure of these samples is unknown. Therefore, the purpose of this study was to determine the potential inhaled hazardous health effects of the co-exposure of MNPs and ambient (indoor and outdoor) air PM. Using an advanced in vitro testing model combined with a specific mechanistic toxicology approach, it is hypothesised that the co-exposure approach will elucidate important information regarding the differential toxicology of ambient (indoor and outdoor) air PM. The in vitro system will consist of alveolar epithelial type-II cells (-NCI-H441) combined with differentiated monocytes (dTHP-1), cultured at the relevant in vivo ratio and at the air-liquid interface. Cell cultures will be exposed for both 4 and 24hrs to MNP (commercially available polypropylene (PP) and blue face mask extracted PP), PM (NIST 2584 and NIST 1649a respectively) and then subsequent MNP+PM (both NIST 2584 and NIST 1649a respectively) via aerosol (VITROCELL Cloud12), using alternative dosimetry patterns. The biochemical endpoints of cytotoxicity, oxidative stress, (pro-)inflammatory response and genotoxicity, will then be assessed to provide the basis for future global analytical techniques to examine the currently unknown human hazard of inhaled MNPs (+/- PM).

Abstract
Exposure to indoor air pollutants (IAP) has increased recently, with people spending more time indoors. Increased exposures of IAP on a healthy population are poorly understood, and those with allergic respiratory conditions less so. The objective of this study, therefore, was to implement a well-characterised in vitro model of the human alveolar epithelial barrier (A549+PMA differentiated THP-1 incubated with/without IL-13, IL-5 and IL-4) to determine the effects of a standardised indoor particulate (NIST2583) on both a healthy lung model and modelling a type-II inflammatory response. Using concentrations from the literature, and an environmentally appropriate exposure we investigated 232, 464 and 608ng/cm2 of NIST2583 respectively. Membrane integrity (blue dextran), viability (trypan blue), genotoxicity (micronucleus (Mn) assay) and (pro-) and (anti-)inflammatory effects (IL-6, IL-8, IL-33, and IL-10) were then assessed 24hours post exposure to both models. No changes in Mn frequency or membrane integrity in either model was noted when exposed to any concentration of NIST2583 tested. Though, a significant decrease (p<0.05) in viability at the highest concentration was observed in the healthy model. Whilst cell viability in the "inflamed" model significantly decreased (p<0.05) at the lower concentrations. A significant reduction (p<0.05) in IL-10 and increase in IL-33 was seen after exposure to NIST2583 in the "inflamed" model. Collectively, the results indicate the potential for IAP to cause the onset of an allergic response as well as exacerbating pre-existing allergic conditions. Furthermore, the data imposes the importance of considering unhealthy individuals when investigating the potential health effects of IAP.

Abstract
A primary toxicological focus has been the development of cellular models and exposure systems representative of nanomaterial inhalation due to the inevitable risk of environmental or occupational exposure. This current study compared the genotoxic and immunogenic potential of two nanomaterials: copper oxide (CuO) and zinc oxide (ZnO) and a positive-particle control tungsten-carbide cobalt (WC-Co), in type-II alveolar epithelial cells (A549) utilising cell culture and exposure systems of increasing complexity. Firstly, treatment was undertaken in submerged-culture at a dose range of 1–100 μg/cm2 for all materials. This was repeated with A549s established at the air-liquid interface using a quasi-ALI exposure technique. Finally, complexity of the quasi-ALI exposure was increased with the addition of a dynamic-flow system (Kirkstall Quasi-Vivo,600) allowing media circulation on the basal side of A549 cultures. For all exposures, cytotoxicity, chromosomal damage, and immunogenicity were assessed by relative population doubling, cytokinesis blocked micronucleus assay and interleukin-8 quantification respectively. Compared to the untreated control, significant cytotoxicity (~50%;p<0.05)) was observed in A549s treated with CuO at 100 μg/cm2 following static quasi-ALI exposure. Moreover, both CuO and ZnO demonstrated a significantly increased micronucleus frequency and IL-8 response at 20 and 100 μg/cm2. WC-Co did not promote any (pro-)inflammatory response under any exposure scenario, however genotoxicity was observed in the static and dynamic-flow ALI cultures. Subsequently the variation between the applied exposure scenarios across the chosen test materials has highlighted the importance of considering the impact of the culture environment and exposure system applied when undertaking nanomaterial genotoxicity assessment in vitro.

Abstract
Surgical face masks are routinely used in clinical settings, but also extensively by the public during the SARS-CoV-2 pandemic. These masks contain a high proportion of plastic, specifically polypropylene (PP), which could shed microscopic particles to the environment or nasal cavity. However, to-date, despite focus upon an increased environmental burden, the (human) hazard relevant to inhaled MNPs remains largely unknown. The aim of this project therefore, was to determine the toxicity of inhaled PP (commercially available powders, or extracted from surgical masks; either (i) all layers, or (ii) the innermost layer only), using an advanced in vitro approach. Type-II lung epithelial cells (NCI-H441) were cultured at the air-liquid interface and exposed to respirable PP via aerosol for 24hrs at depositions of 500, 100 and 2000 ng/cm2 (Vitrocell Cloud12). Carbon black (Printex 90) was used as a positive particle control. Respirable MNP samples required cryomilling and cryotome slicing, and were analysed using pyrolysis-GCMS. In addition to acellular deposition, as well as particle and cellular morphology (electron microscopy) assessed endpoints of cytotoxicity (trypan blue exclusion), barrier integrity (dextran blue), (pro-)inflammatory response (IL-1β/IL-6/IL-8), oxidative stress (SOD-1), and genotoxicity (mononucleate micronucleus) were conducted. So far, a concentration-dependent decrease in cell viability was observed following exposure to commercial PP and complete mask PP samples, whilst a concentration-dependent increase in all (pro-)inflammatory mediators assessed was noted. Further, initial genotoxicity assessment suggests a minor increase to micronucleus formation for both exposure samples. Preliminary data therefore suggests that aerosolised MNPs instigate a hazardous response in vitro.

Abstract
Statement from the Committee on the Medical Effects of Air Pollutants (COMEAP) on the differential toxicity of particulate matter according to source or constituents
Particulate matter in outdoor air comes from a wide range of different sources and contains many different chemical components. Understanding which of these sources and components are particularly harmful would help to focus policies and interventions to reduce impacts on health. COMEAP recently (July 2022) published an updated statement on this topic, drawing on recent reviews of the available evidence. It noted that particulate matter, and constituents of particulate matter, associated with combustion and road traffic have received the most research attention. However, there was insufficient coherence in the epidemiological evidence to conclude that these sources of particulate matter play a significantly greater role in the health effects of particulate air pollution than other sources or constituents. A comprehensive review of the evidence reveals that toxicity has been attributed to a wide variety of constituents and sources, possibly acting by a number of different mechanisms. However, the complexity of sources, sizes and chemical composition of particulate matter makes it extremely difficult to identify which characteristics of particles might make them less harmful or more toxic than others. Future research assessing the influence of chemical composition of particulate matter should offer greater insights into toxicity. But, at present, PM2.5 (the mass concentration of fine particulate matter in air) remains the most suitable metric for evaluating health impacts and regulating particulate air pollution.

Abstract
The HARMLESS project focusses upon assessment approaches to support Safe-and-Sustainable-by-Design (SSbD) development of advanced materials (AdMa). Case studies comprise several industry sectors. Specifically for the construction sector we study façade insulation by fibre-aerogel-mats. Beyond the relatively simpler mono-component organic aerogels presented earlier (Keller et al. 2021), here we deal with multi-component materials: Glass fibers provide structural integrity for the silica-based aerogel that provides the thermal insulation performance, though is coated by organosilane as a hydrophobizing agent to prevent moisture uptake. Optionally, an additional inorganic coating can be applied to enhance mechanical stability. Here we explored this wide SSbD design space by four industrially relevant SSbD-versions of fibre-aerogel-mats; these were ranked and compared against each other as well as conventional insulation material (i.e. stone wool). We assessed the release by performing several typical occupational operations under clean-air conditions within a laboratory setting. Up to 106/ccm particles are released, with respirable aerodynamic diameters. We collected the dust and assessed the structure and behavior with respect to its biological reactivity and biodissolution, focusing on the screening and ranking of inhalation hazards. The data on exposure and hazard was complemented by data on functionality in the intended use, where all SSbD versions performed significantly better than the conventional alternative. The new data refines the risk screening and categorisation schemes for advanced materials, including Early4AdMa (RIVM 2022), AdmGRID (Kennedy et al. 2019), InnoMat.Life (Wohlleben et al. 2022) and an environmental risk screening (Arvidsson et al. 2022).

Abstract
The prevalent application of engineered nanomaterials (ENMs) has led to an extensive effort to develop tools facilitating their risk assessment and management. This interlaboratory trial (encompassing two laboratories) sought to investigate the genotoxic impact of ENMs using the hypoxanthine phosphoribosyltransferase (HPRT) forward mutation assay and in vitro cytokinesis-blocked micronucleus (CBMN) assay, utilising harmonised protocols. In the CBMN assay, human lymphoblast (TK6) cells were exposed to zinc oxide (ZnO), titanium dioxide (TiO2) and tungsten carbide-cobalt (WC/Co) for 24-hours after which 1000 binucleated cells were scored for micronuclei (n=2). For the HPRT assay, TK6 and mouse fibroblast (V79) cells were exposed to ZnO, TiO2, WC/Co, CuO and Nanocyl-CNTs for 24-hours after which 600 wells were scored for point mutations (n=2). Significant cytotoxicity and micronuclei frequency was reported following ZnO exposure (20µg/ml). Significant micronuclei induction was reported following WC/Co exposures (2-fold over control, 100µg/ml). No significant mutagenicity was detected with ZnO or TiO2; both laboratories observed significant cytotoxicity following exposure to ZnO (20µg/ml; 37% reduction in cell viability). In the second interlaboratory trial CuO induced significant cytotoxicity (36% reduction in viability: 0.5µg/ml), and a 32-fold increase in mutagenicity. The Nanocyl induced a 6-fold increase in mutagenicity. The data generated here has shown good statistical concordance between laboratories and has highlighted a promising route forward in supporting risk decision making for ENMs. The authors would like to acknowledge this research has received funding from the European Union's Horizon 2020 research and innovation program for the RiskGONE project, grant agreement #814425.

Abstract
Ambient nitrogen dioxide (NO2) and particulate matter (PM) concentrations are often found to be proportional, such that inhalation of one pollutant is expected to be accompanied by the inhalation of the other. Epidemiological evidence consistently demonstrates the health burdens associated with the inhalation of NO2 and PM, though has difficulty deciphering the inhalation hazard of either pollutant individually. Airway models can be exposed to NO2 and PM separately or as a co-exposure in vitro, allowing mechanisms of toxicity to be elucidated. Herein, NCI-H441 cells were exposed to either 780 ng/cm2 Printex 90 carbon black (CB) (a surrogate model of PM) using a VitroCell Cloud12, 5 ppm NO2 (within a modified Coy hypoxic cabinet) or a combination of NO2+CB over 24 hours. Cytotoxicity was not altered by NO2 or CB (trypan blue exclusion assay), however CB, and to a lesser extent CB+NO2 exposure resulted in a non-significant increase in membrane permeability (p>0.05). qPCR revealed no alterations in expression of superoxide dismutase 1 (SOD1) for any exposure condition. Both CB and NO2 exposures resulted in significantly increased IL6 and IL8 release (ELISA) (p<0.05), although co-exposure of the two pollutants did not augment the detected IL6 or IL8 levels. These data indicate NO2 and CB produce an inflammatory response in this model, though little evidence suggests that NO2 and CB act in an additive or synergistic manner via this pathway. Additional work remains ongoing to establish the mechanisms of toxicity utilising an anatomically relevant triple cell co-culture of the alveolar epithelial barrier.