Transparency in Non-Technical Project Summaries to Promote the Three Rs in Respiratory Disease Research.

Non-Technical Project Summaries (NTS) are legal documents that were first introduced by the Directive 2010/63/EU to enhance transparency within scientific animal experimentation. Researchers intending to conduct biological research on animal models must fulfil the NTS requirements by outlining their proposed use of animals and how they plan to implement the Three Rs (replacement, reduction and refinement of animal use) in their experiments. This study outlines a novel systematic analysis approach that enables the assessment of NTS transparency based on the accuracy of reporting of certain Three Rs-specific information. This potentially customisable strategy could help toward the development of practical guidelines for use by Animal Welfare and Ethical Review Bodies (AWERBs) in establishments conducting animal research, in the process of scrutinising NTS during their pre-submission review of proposed licence applications. This could help to identify gaps in reporting of Three Rs-specific information relating to the planned animal experiments, which represents a remarkable step toward achieving greater openness in scientific communication. This study supports the concept that NTS transparency can promote the implementation of non-animal alternatives in fields where this is currently lacking, such as respiratory disease research. Although NTS were originally conceived as informative documents for a lay audience, we can conclude that data in NTS can be successfully used as a basis for systematic analysis. By reviewing the NTS, the experimental limitations of the currently available replacement strategies can also be highlighted, potentially pinpointing where there is a need for future method development.

Multiple relationships between aerosol and COVID-19: A framework for global studies.

COVID-19 (Corona Virus Disease 2019) is a severe respiratory syndrome currently causing a human global pandemic. The original virus, along with newer variants, is highly transmissible. Aerosols are a multiphase system consisting of the atmosphere with suspended solid and liquid particles, which can carry toxic and harmful substances; especially the liquid components. The degree to which aerosols can carry the virus and cause COVID-19 disease is of significant research importance. In this study, we have discussed aerosol transmission as the pathway of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), and the aerosol pollution reduction as a consequence of the COVID-19 lockdown. The aerosol transmission routes of the SARS-CoV-2 can be further subdivided into proximal human-exhaled aerosol transmission and potentially more distal ambient aerosol transmission. The human-exhaled aerosol transmission is a direct dispersion of the SARS-CoV-2. The ambient aerosol transmission is an indirect dispersion of the SARS-CoV-2 in which the aerosol acts as a carrier to spread the virus. This indirect dispersion can also stimulate the up-regulation of the expression of SARS-CoV-2 receptor ACE-2 (Angiotensin Converting Enzyme 2) and protease TMPRSS2 (Transmembrane Serine Protease 2), thereby increasing the incidence and mortality of COVID-19. From the aerosol quality data around the World, it can be seen that often atmospheric pollution has significantly decreased due to factors such as the reduction of traffic, industry, cooking and coal-burning emissions during the COVID-19 lockdown. The airborne transmission potential of SARS-CoV-2, the infectivity of the virus in ambient aerosols, and the reduction of aerosol pollution levels due to the lockdowns are crucial research subjects.

Human airway construct model is suitable for studying transcriptome changes associated with indoor air particulate matter toxicity.

In vitro models mimicking the human respiratory system are essential when investigating the toxicological effects of inhaled indoor air particulate matter (PM). We present a pulmonary cell culture model for studying indoor air PM toxicity. We exposed normal human bronchial epithelial cells, grown on semi-permeable cell culture membranes, to four doses of indoor air PM in the air-liquid interface. We analyzed the chemokine interleukin-8 concentration from the cell culture medium, protein concentration from the apical wash, measured tissue electrical resistance, and imaged airway constructs using light and transmission electron microscopy. We sequenced RNA using a targeted RNA toxicology panel for 386 genes associated with toxicological responses. PM was collected from a non-complaint residential environment over 1 week. Sample collection was concomitant with monitoring size-segregated PM counts and determination of microbial levels and diversity. PM exposure was not acutely toxic for the cells, and we observed up-regulation of 34 genes and down-regulation of 17 genes when compared to blank sampler control exposure. The five most up-regulated genes were related to immunotoxicity. Despite indications of incomplete cell differentiation, this model enabled the comparison of a toxicological transcriptome associated with indoor air PM exposure.

Oxidative capacity and hemolytic activity of settled dust from moisture-damaged schools.

Exposure to moisture-damaged indoor environments is associated with adverse respiratory health effects, but responsible factors remain unidentified. In order to explore possible mechanisms behind these effects, the oxidative capacity and hemolytic activity of settled dust samples (n = 25) collected from moisture-damaged and non-damaged schools in Spain, the Netherlands, and Finland were evaluated and matched against the microbial content of the sample. Oxidative capacity was determined with plasmid scission assay and hemolytic activity by assessing the damage to isolated human red blood cells. The microbial content of the samples was measured with quantitative PCR assays for selected microbial groups and by analyzing the cell wall markers ergosterol, muramic acid, endotoxins, and glucans. The moisture observations in the schools were associated with some of the microbial components in the dust, and microbial determinants grouped together increased the oxidative capacity. Oxidative capacity was also affected by particle concentration and country of origin. Two out of 14 studied dust samples from moisture-damaged schools demonstrated some hemolytic activity. The results indicate that the microbial component connected with moisture damage is associated with increased oxidative stress and that hemolysis should be studied further as one possible mechanism contributing to the adverse health effects of moisture-damaged buildings.

The 2017 Lush Prize Awards.

This year saw eighteen prizewinning projects from eleven countries, and a new award in recognition of the work of Andrew Tyler.

A normal and biotransforming model of the human bronchial epithelium for the toxicity testing of aerosols and solubilised substances.

In this article, we provide an overview of the experimental workflow by the Lung and Particle Research Group at Cardiff University, that led to the development of the two in vitro lung models - the normal human bronchial epithelium (NHBE) model and the lung-liver model, Metabo-Lung™. This work was jointly awarded the 2013 Lush Science Prize. The NHBE model is a three-dimensional, in vitro, human tissue-based model of the normal human bronchial epithelium, and Metabo-Lung involves the co-culture of the NHBE model with primary human hepatocytes, thus permitting the biotransformation of inhaled toxicants in an in vivo-like manner. Both models can be used as alternative test systems that could replace the use of animals in research and development for safety and toxicity testing in a variety of industries (e.g. the pharmaceutical, environmental, cosmetics, and food industries). Metabo-Lung itself is a unique tool for the in vitro detection of toxins produced by reactive metabolites. This 21st century animal replacement model could yield representative in vitro predictions for in vivo toxicity. This advancement in in vitro toxicology relies on filter-well technology that will enable a wide-spectrum of researchers to create viable and economic alternatives for respiratory safety assessment and disease-focused research.

Hemolytic Properties of Fine Particulate Matter (PM2.5) in In Vitro Systems.

Epidemiological studies have suggested that inhalation exposure to particulate matter (PM) air pollution, especially fine particles (i.e., PM2.5 (PM with an aerodynamic diameter of 2.5 microns or less)), is causally associated with cardiovascular health risks. To explore the toxicological mechanisms behind the observed adverse health effects, the hemolytic activity of PM2.5 samples collected during different pollution levels in Beijing was evaluated. The results demonstrated that the hemolysis of PM2.5 ranged from 1.98% to 7.75% and demonstrated a clear dose-response relationship. The exposure toxicity index (TI) is proposed to represent the toxicity potential of PM2.5, which is calculated by the hemolysis percentage of erythrocytes (red blood cells, RBC) multiplied by the mass concentration of PM2.5. In a pollution episode, as the mass concentration increases, TI first increases and then decreases, that is, TI (low pollution levels) < TI (heavy pollution levels) < TI (medium pollution levels). In order to verify the feasibility of the hemolysis method for PM toxicity detection, the hemolytic properties of PM2.5 were compared with the plasmid scission assay (PSA). The hemolysis results had a significant positive correlation with the DNA damage percentages, indicating that the hemolysis assay is feasible for the detection of PM2.5 toxicity, thus providing more corroborating information regarding the risk to human cardiovascular health.

Medical waste tissues - breathing life back into respiratory research.

With the advent of biobanks to store human lung cells and tissues from patient donations and from the procurement of medical waste tissues, it is now possible to integrate (both spatially and temporally) cells into anatomically-correct and physiologically-functional tissues. Modern inhalation toxicology relies on human data on exposure and adverse effects, to determine the most appropriate risk assessments and mitigations for beneficial respiratory health. A point in case is the recapitulation of airway tissue, such as the bronchial epithelium, to investigate the impact of air pollution on human respiratory health. The bronchi are the first point of contact for inhaled substances that bypass defences in the upper respiratory tract. Animal models have been used to resolve such inhalation toxicology hazards. However, the access to medical waste tissues has enabled the Lung Particle Research Group to tissue-engineer the Micro-Lung (TM) and Metabo-Lung(TM) cell culture models, as alternatives to animals in basic research and in the safety testing of aerosolised consumer goods. The former model favours investigations focused on lung injury and repair mechanisms, and the latter model provides the element of metabolism, through the co-culturing of lung and liver (hepatocyte) cells. These innovations represent examples of the animal-free alternatives advocated by the 21st century toxicology paradigm, whereby human-derived cell/tissue data will lead to more-accurate and more-reliable public health risk assessments and therapeutic mitigations (e.g. exposure to ambient air pollutants and adverse drug reactions) for lung disease.

Gene and protein responses of human lung tissue explants exposed to ambient particulate matter of different sizes.

CONTEXT: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size. OBJECTIVES: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model. MATERIALS AND METHODS: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24 h to washed ambient PM of different sizes (size 1: 7-615 nm; size 2: 616 nm-2.39 µm; size 3: 2.4-10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics. RESULTS: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways. DISCUSSION AND CONCLUSIONS: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.

A multidisciplinary approach to characterise exposure risk and toxicological effects of PM10 and PM2.5 samples in urban environments.

Urban aerosol samples collected in Barcelona between 2008 and 2009 were toxicologically characterised by means of two complementary methodologies allowing evaluation of their Reactive Oxidative Stress (ROS)-generating capacity: the plasmid scission assay (PSA) and the dichlorodihydrofluorescin assay (DCFH). The PSA determined the PM dose able to damage 50% of a plasmid DNA molecule (TD(50) values), an indication of the ability of the sample to exert potential oxidative stress, most likely by formation of ·OH. This toxicity indicator did not show dependency on different air mass origins (African dust, Atlantic advection), indicating that local pollutant sources within or near the city are most likely to be mainly responsible for PM health effect variations. The average TD(50) values show PM(2.5-0.1) samples to be more toxic than the PM(10-2.5) fraction, with doses similar to those reported in previous studies in polluted urban areas. In addition, the samples were also evaluated using the oxidant-sensitive probe DCFH confirming the positive association between the amount of DNA damage and the generation of reactive oxidant species capable of inducing DNA strand break. Results provided by the PSA were compared with those from two other different methodologies to evaluate human health risk: (1) the toxicity of particulate PAHs expressed as the calculated toxicity equivalent of benzo[a]pyrene (BaPteq) after application of the EPA toxicity factors, and (2) the cancer risk assessment of the different PM sources detected in Barcelona with the receptor model Positive Matrix Factorisation (PMF) and the computer programme Multilinear Engine 2 (ME-2) using the organic and inorganic chemical compositions of particles. No positive associations were found between PSA and the toxicity of PAHs, probably due to the inefficiency of water in extracting organic compounds. On the other hand, the sum of cancer risk estimates calculated for each of the selected days for the PSA was found to correlate with TD(50) values in the fine fraction, with fuel oil combustion and industrial emissions therefore being most implicated in negative health effects. Further studies are necessary to determine whether toxicity is related to PM chemical composition and sources, or rather to its size distribution.

Microplastic atmospheric dustfall pollution in urban environment: Evidence from the types, distribution, and probable sources in Beijing, China.

Airborne microplastics (MPs) pollution is an environmental problem of increasing concern, due to the ubiquity, persistence and potential toxicity of plastics in the atmosphere. In recent years, most studies on MPs have focused on aquatic and sedimentary environments, but little research has been done on MPs in the urban atmosphere. In this study, a total of ten dustfall samples were collected in a transect from north to south across urban Beijing. The compositions, morphologies, and sizes of the MPs in these dustfall samples were determined by means of Laser Direct Infrared (LDIR) imaging and Field Emission Scanning Electron Microscopy (FESEM). The number concentrations of MPs in the Beijing dustfall samples show an average of 123.6 items/g. The MPs concentrations show different patterns in the central, southern, and northern zones of Beijing. The number concentration of MPs was the highest in the central zone (224.76 items/g), as compared with the southern zone (170.55 items/g), and the northern zone (24.42 items/g). The LDIR analysis revealed nine compositional types of MPs, including Polypropylene (PP), Polyamide (PA), Polystyrene (PS), Polyethylene (PE), Polyethylene Terephthalate (PET), Silicone, Polycarbonate (PC), Polyurethane (PU) and Polyvinylchloride (PVC), among which PP was overall dominant. The PP dominates the MPs in the central zone (76.3%), and the PA dominates the MPs in the southern zone (55.86%), while the northern zone had a diverse combination of MPs types. The morphological types of the individual MPs particle include fragments, pellets, and fibers, among which fragments are dominant (70.9%). FESEM images show the presence of aged MPs in the Beijing atmosphere, which could pose a yet unquantified health risk to Beijing's residents. The average size of the MPs in the Beijing samples is 66.62 µm. Our study revealed that the numbers of fibrous MPs increase with the decrease in size. This pollution therefore needs to be carefully monitored, and methods of decreasing the sources and mitigations developed.

Oxidative potential and water-soluble heavy metals of size-segregated airborne particles in haze and non-haze episodes: Impact of the "Comprehensive Action Plan" in China.

Air pollution is a major environmental health challenge in megacities, and as such a Comprehensive Action Plan (CAP) was issued in 2017 for Beijing, the capital city of China. Here we investigated the size-segregated airborne particles collected after the implementation of the CAP, intending to understand the change of oxidative potential and water-soluble heavy metal (WSHM) levels in 'haze' and 'non-haze' days. The DNA damage and the levels of WSHM were analyzed by Plasmid Scission Assay (PSA) and High-Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS) techniques. The PM mass concentration was higher in the fine particle size (0.43-2.1 µm) during haze days, except for the samples affected by mineral dust. The particle-induced DNA damage caused by fine sized particles (0.43-2.1 µm) exceeded that caused by the coarse sized particles (4.7-10 µm). The DNA damage from haze day particles significantly exceeded those collected on non-haze days. Prior to the instigation of the CAP, the highest value of DNA damage decreased, and DNA damage was seen in the finer size (0.43-1.1 µm). The Pearson correlation coefficient between the concentrations of water-soluble Pb, Cr, Cd and Zn were positively correlated with DNA damage, suggesting that these WSHM had significant oxidative potential. The mass concentrations of water-soluble trace elements (WSTE) and individual heavy metals were enriched in the finer particles between 0.43 µm to 1.1 µm, implying that smaller sized particles posed higher health risks. In contrast, the significant reduction in the mass concentration of water-soluble Cd and Zn, and the decrease of the maximum and average values of DNA damage after the CAP, demonstrated its effectiveness in restricting coal-burning emissions. These results have demonstrated that the Beijing CAP policy has been successful in reducing the toxicity of 'respirable' ambient particles.

COVID-19 mortality and exposure to airborne PM2.5: A lag time correlation.

COVID-19 has escalated into one of the most serious crises in the 21st Century. Given the rapid spread of SARS-CoV-2 and its high mortality rate, here we investigate the impact and relationship of airborne PM2.5 to COVID-19 mortality. Previous studies have indicated that PM2.5 has a positive relationship with the spread of COVID-19. To gain insights into the delayed effect of PM2.5 concentration (µgm-3) on mortality, we focused on the role of PM2.5 in Wuhan City in China and COVID-19 during the period December 27, 2019 to April 7, 2020. We also considered the possible impact of various meteorological factors such as temperature, precipitation, wind speed, atmospheric pressure and precipitation on pollutant levels. The results from the Pearson's correlation coefficient analyses reveal that the population exposed to higher levels of PM2.5 pollution are susceptible to COVID-19 mortality with a lag time of >18 days. By establishing a generalized additive model, the delayed effect of PM2.5 on the death toll of COVID-19 was verified. A negative correction was identified between temperature and number of COVID-19 deaths, whereas atmospheric pressure exhibits a positive correlation with deaths, both with a significant lag effect. The results from our study suggest that these epidemiological relationships may contribute to the understanding of the COVID-19 pandemic and provide insights for public health strategies.

Proteomic profiling of human respiratory epithelia by iTRAQ reveals biomarkers of exposure and harm by tobacco smoke components.

Historically, it has been challenging to go beyond epidemiology to investigate the pathogenic changes caused by tobacco smoking. The EpiAirway-100 (MatTek Corp., Ashland, MA) was employed to investigate the effects of cigarette smoke components. Exposure at the air-liquid-interface represented particle and vapour phase components of cigarette smoke. A proteomic study utilising iTRAQ labelling compared expression profiles. The correlative histopathology revealed focal regions of hyperplasia, hypertrophy, cytolysis and necrosis. We identified 466 proteins, 250 with a parameter of two or more peptides. Four of these proteins are potential markers of lung injury and three are related to mechanistic pathways of disease.

Characterisation of airborne particles and associated organic components produced from incense burning.

Airborne particles generated from the burning of incense have been characterized in order to gain an insight into the possible implications for human respiratory health. Physical characterization performed using field-emission scanning electron microscopy showed incense particulate smoke mainly consisted of soot particles with fine and ultrafine fractions in various aggregated forms. A range of organic compounds present in incense smoke have been identified using derivatisation reactions coupled with gas chromatography-mass spectrometry analysis. A total of 19 polar organic compounds were positively identified in the samples, including the biomass burning markers levoglucosan, mannosan and galactosan, as well as a number of aromatic acids and phenols. Formaldehyde was among 12 carbonyl compounds detected and predominantly associated with the gas phase, whereas six different quinones were also identified in the incense particulate smoke. The nano-structured incense soot particles intermixed with organics (e.g. formaldehyde and quinones) could increase the oxidative capacity. When considering the worldwide prevalence of incense burning and resulting high respiratory exposures, the oxygenated organics identified in this study have significant human health implications, especially for susceptible populations.

Alternatives for lung research: stuck between a rat and a hard place.

The respiratory system acts as a portal into the human body for airborne materials, which may gain access via the administration of medicines or inadvertently during inhalation of ambient air (e.g. air pollution). The burden of lung disease has been continuously increasing, to the point where it now represents a major cause of human morbidity and mortality worldwide. In the UK, more people die from respiratory disease than from coronary heart disease or non-respiratory cancer. For this reason alone, gaining an understanding of mechanisms of human lung biology, especially in injury and repair events, is now a principal focus within the field of respiratory medicine. Animal models are routinely used to investigate such events in the lung, but they do not truly reproduce the responses that occur in humans. Scientists committed to the more robust Three Rs principles of animal experimentation (Reduction, Refinement and Replacement) have been developing viable alternatives, derived from human medical waste tissues from patient donors, to generate in vitro models that resemble the in vivo human lung environment. In the specific case of inhalation toxicology, human-oriented models are especially warranted, given the new REACH regulations for the handling of chemicals, the rising air pollution problems and the availability of pharmaceutically valuable drugs. Advances in tissue-engineering have made it feasible and cost-effective to construct human tissue equivalents of the respiratory epithelia. The conducting airways of the lower respiratory system are a critical zone to recapitulate for use in inhalation toxicology. Three-dimensional (3-D) tissue designs which make use of primary cells, provide more in vivo-like responses, based on the targeted interactions of multiple cell types supported on artificial scaffolds. These scaffolds emulate the native extracellular matrix, in which cells differentiate into a functional pulmonary tissue. When 3-D cell cultures are employed for testing aerosolised chemicals, drugs and xenobiotics, responses are captured that mirror the events in the in situ human lung and provide human endpoint data.

Human lung tissue engineering: a critical tool for safer medicines.

In the field of human tissue-engineering, there has been a strong focus on the clinical aspects of the technology, i.e. repair, replace and enhance a given tissue/organ. However, much wider applications for tissue engineering (TE) exist outside of the clinic that are often not recognised, and include engineering more relevant models than animals in basic research and safety testing. Traditionally, research is initially conducted on animals or cell lines, both of which have their limitations. With regard to cell lines, they are usually transformed to enable indefinite proliferation. These immortalised cell lines provide the researcher with an almost limitless source of material. However, the pertinence of the data produced is now under scrutiny, with the suggestion that some historical cell lines may not be the cell type originally reported. By engineering normal, biomimetic (i.e. life-mimicking), human tissues with defined physiology (i.e. human tissue equivalents), the complex 3-dimensional (3-D) tissue/organ physiology is captured in vitro, providing the opportunity to directly replace the use of animals in research/testing with more relevant systems. Therefore, it is imperative that testing strategies using organotypic models are developed that can address the limitations of current animal and cellular models and thus improve drug development, enabling faster delivery of drugs which are safer, more effective and have fewer side effects in humans.

The oxidative capacity of indoor source combustion derived particulate matter and resulting respiratory toxicity.

Indoor air pollution sources with emissions of fine particles (PM2.5), including environmental tobacco smoke (ETS) and incense smoke (IS) deteriorate indoor air quality and may cause respiratory diseases in humans. This study characterized the emission factors (EFs) of five types of tobacco and incense in Hong Kong using an environmental chamber. Human alveolar epithelial cells (A549) were exposed to PM2.5 collected from different indoor sources to determine their cytotoxicity. The PM2.5 EF of ETS (109.7±36.5 mg/g) was higher than IS (97.1±87.3 mg/g). The EFs of total polycyclic aromatic hydrocarbons (PAHs) and carbonyls for IS were higher than ETS, and these two combustion sources showed similar distributions of individual PAHs and carbonyls. Oxidative damage and inflammatory responses (i.e. DNA damage, 8-hydroxy-desoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6)) of A549 cells was triggered by exposure to PM2.5 generated from ETS and IS. Different indoor sources showed different responses to oxidative stress and inflammations due to the accumulation effects of mixed organic compounds. High molecular weight PAHs from incense combustion showed higher correlations with DNA damage markers, and most of the PAHs from indoor sources demonstrated significant correlations with inflammation. Exposure to anthropogenic produced combustion emissions such as ETS and IS results in significant risks (e.g. lung cancer) to the alveolar epithelium within the distal human respiratory tract, of which incense emissions posed a higher cytotoxicity.