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Background: Occupational exposure to xenobiotic aerosols may occur within different settings, with a common example being the unintentional inhalation of pesticide aerosols sprayed to protect various crops. Current regulatory guidelines do not exploit emerging in vitro toxicity models and make the unvalidated assumption that 100% of the inhaled pesticide is absorbed into systemic circulation, in stark contrast to oral or dermal routes of exposure, which use established evidence-based in vitro and in silico methods, in addition to in vivo models. The assumption of 100% respiratory bioavailability overlooks several established clearance mechanisms, such as mucociliary clearance and lung metabolism. Data-driven approaches for predicting toxicity and estimating respiratory bioavailability based on experimental evidence are therefore urgently needed and have important implications for future risk assessments. This body of work is focused on (i) the suitability of in vitro respiratory models, (ii) the use of in vitro and in silico data for toxicokinetic predictions and (iii) NMR metabolomics as a sensitive measure of changes to lung cell phenotype following pesticide exposure at subtoxic concentrations. Methods: To address these knowledge gaps regarding regional respiratory toxicity throughout the airway and bioavailability of xenobiotics in an occupational exposure setting, several experimental methods were employed alongside in silico modelling approaches. Epithelial cell models (RPMI-2650, Calu-3, 16HBE14o-, BEAS-2B, TT1 and A549), reflecting different regions of the airway were utilised to investigate the toxicity, permeability and metabolism of a range of pesticides. The results from these respiratory models were compared against the gastrointestinal epithelial cell line Caco-2, which has been more widely employed in the field. Techniques used included various cytotoxicity assays, Phase I and II metabolism studies to highlight enzyme activity in the different models, NMR metabolomics to assess the changes to lung cell phenotype at subtoxic xenobiotic concentrations, and permeability studies using various Transwell models (Calu-3, Caco-2, PAMPA and lung lipid extract), alongside in vitro protein binding assays. Additionally, in vitro data was paired with in silico predictions for lung deposition and pharmacokinetics to predict the bioavailability of inhaled xenobiotics in an occupational exposure scenario. Results: Significant differences were found in the suitability of the different biological models for assessing toxicity, metabolism and permeability within the lungs. The effect of the physicochemical properties of different xenobiotics on the predicted bioavailability following inhalation was also shown to be substantial. Finally, in addition to cytotoxicity, several changes to cell phenotype were observed following realistic (and non-cytotoxic) pesticide exposure concentrations of 1-10 µM. Cytotoxicity, mitochondrial inhibition, CYP1A1 induction and increased intracellular glutathione were observed at within this concentration range for various pesticides, whilst chlorothalonil 20 µM was found to be capable of causing epithelial damage to Calu-3 cells cultured at air-liquid interface. Conclusion: This study confirmed that the assumption of 100% bioavailability for inhaled pesticides is often an overestimate and regional airway models may better predict the actual dose fraction reaching the systemic circulation. These in vitro data combined with in silico approaches highlight the potential to improve predictions of respiratory toxicity and bioavailability and improve future occupational risk assessments for respiratory exposure to xenobiotics.

Personal exposure studies have found that individuals are exposed to the highest levels of traffic-related pollution while commuting. This is a concern as there is strong evidence that exposure to traffic-related air pollution, particularly diesel exhaust, has greater adverse health effects than pollutants from other sources. However, while people often spend one to two hours in the commuting environment each day, few studies have considered the exposures experienced by professional drivers who are required to drive in this environment for the majority of their working day. Due to the scarcity of studies and the likelihood that professional drivers are disproportionately exposed to traffic-related air pollution, a better understanding of how much pollution these drivers are exposed to and what can be done to reduce their exposure is needed. This thesis aimed to address this research gap by undertaking a comprehensive monitoring campaign to characterise professional drivers' exposure to traffic-related pollution with a specific focus on diesel exhaust across a range of sectors. This exposure data were then analysed to identify the most important determinants of professional drivers' exposure to develop realistic and cost-effective exposure reduction strategies based on real-world evidence. The baseline monitoring campaign collected personal black carbon (BC) exposures as a proxy for diesel exhaust for 141 drivers across seven sectors in London. Each participant's personal BC exposures were continuously measured using real-time monitors for 96 hours, incorporating four shifts per participant. Results found 'at work' BC exposures (median: 2.3 µg/m³) were 2.3 times higher compared to when participants were 'not at work' (1.0 µg/m³). Workers spent 19% of their time 'at work driving', however this activity contributed 36% of total BC exposure, highlighting the disproportionate effect that driving had on professional drivers' daily exposure. Taxi drivers experienced the highest BC exposures at work due to the time they spent working in congested central London, while emergency services had the lowest, due to the reduced time spent driving during their shift. Spikes in exposure were observed while driving and were at times greater than 100 µg/m³. Further quantitative analysis and mixed effects models highlighted the most significant determinants of drivers' BC exposures. These were driving in tunnels, the sector the driver worked in, congestion, driving on roads with higher numbers of buses and HGVs, window position, driving location, background BC concentrations, day of the week and time of day. Two further real-world intervention monitoring campaigns on a subset of 44 drivers were conducted investigating the effectiveness of portable in-cabin filters and reduced vehicle numbers on roads in reducing professional drivers' exposure to traffic-related pollution. A further controlled test on 92 unique vehicles assessed the effect that vehicle type and ventilation settings had on in-cabin pollutant concentrations. Results of these campaigns found that setting ventilation settings to recirculate had the most immediate effect in reducing drivers' exposures, although care needs to be taken when utilising this setting due to the accumulation of potentially adverse carbon dioxide levels from driver respiration. The in-cabin filter intervention only resulted in a slight reduction in exposure for drivers and therefore was not thought to be as effective as utilising the recirculate ventilation setting. The controlled test suggested that the filter quality built into vehicles' ventilation systems likely resulted in differences in the infiltration of pollutants into the cabin, highlighting that different vehicle types could be important in reducing drivers' exposure. The campaign which investigated the effect of reduced vehicle numbers during the COVID-19 lockdown suggested that potentially the most effective intervention to reduce exposures for professional drivers would be local or national policy to reduce the number of vehicles on roads. The results of this thesis provide practical recommendations on interventions and strategies which drivers, employers, vehicle manufacturers and local policy makers can implement to reduce professional drivers' exposure to traffic-related air pollution.

Airborne particulate matter has been defined as a heterogenous collection of solid or liquid droplets of varying size suspended in the air. Epidemiological studies have shown an association between an increase in the concentration of particulate matter (PM) and exacerbation of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). A central mechanism for adverse health outcomes has been attributed to primary combustion particles from vehicle exhausts (residing exclusively within the fine but not coarse mode) causing a pro-inflammatory cytokine milieu production by PM exposed dendritic cells (DC). Hence, I hypothesised that fine mode PM₂.₅ from high traffic sites in London would have a greater capacity to drive inflammatory DC activation than coarse mode PM₁₀. PM₂.₅ and PM₁₀ filters collected over a whole year (2013) were sourced from the Marylebone Road (representing high traffic roadside) and North Kensington (representing urban background). Each individual filter was extracted, and the contents pooled to create a unique annual PM sample of PM₂.₅ and PM₁₀ for each site. Standard reference material-1648a (SRM; urban particulate matter), SRM-2975 (diesel) and brake abrasion dust (BAD) were included since the SRMs are prevalent in air pollution studies. To investigate the compositional metal and metalloid differences between the PMs, an inductively coupled plasma mass spectrometry was used. This showed noticeable differences in metal contents (Fe and Ca) between the different sites. PM from RS had higher metal and metalloid content for both PM₂.₅ and PM₁₀ than BG. Noticeably, PM₁₀ contained a higher degree of metal components, had a significant oxidative profile. As bacterial antigens can trigger an immune response, lipopolysaccharides (LPS) levels for all PMs in the study was measured. There was a higher concentration of LPS for SRM-1648a (30.54pg/ml), BG PM₁₀ (9.91pg/ml) and RS PM₁₀ (6.15pg/ml). However, all these values were noted as below the capacity to stimulate human DC maturation. Human monocyte-derived DC (MDDC) was used to review the immunogenicity of various PM samples by measuring their effects on cell surface maturation markers and cytokine secretion. However, contrary to the hypothesis, coarse mode PM₁₀ but not fine mode PM₂.₅ caused a greater increase in the expression of maturation markers CD83, CD86 and MHC Class II. Among the SRMs, SRM-1648a was found to have the capacity to mature MDDC. The MDDCs exposed to PM₁₀ (not PM₂.₅) had a significant increase in the secretion of cytokines TNFα, IL-6, IL-8, IL-10, and IL-23p40. To elucidate the mechanistic relationship between PM and MDDC response, putative pathways including Fe, oxidative stress, and polycyclic aromatic hydrocarbons (PAH) were tested. There was a significant increase in transcripts related to Fe, oxidative stress and PAHs when measured by quantitative polymerase chain reaction (qPCR). Nevertheless, when measuring oxidative stress via (glutathione: glutathione disulphide ratio) on PM exposed MDDC, there was no significant difference, suggesting the PM exposed MDDC was able to tolerate the increased in oxidative stress. In the presence of the inhibitor for the aryl hydrocarbon receptor (PAH receptor), there was no significant change in the maturation response by PM exposed MDDCs. While the inhibitor was able to reduce the secretion of IL-23 by SRM-1648a, a similar response was not observable in ambient PM. Results generated from this PhD project indicate the initial hypothesis was rejected- MDDC maturation and cytokine production were largely restricted to the coarse mode in PM₁₀, with little evidence for an effect from PM₂.₅. The increased expression of antigen-presenting molecules and cytokine production is likely to have a potential impact on the activation of CD4 T cells. The cytokine profile (IL-6 and IL-23) secreted by PM₁₀ stimulated MDDCs, suggests that CD4 T cells may be driven to polarise into pathogenic TH17 cells which are associated with steroid-refractory asthma as previously demonstrated by experiments with SRM-1648a. These findings corroborate with findings from the London low emission zone, which suggest PM₁₀ may have a higher association with negative respiratory outcomes than originally perceived. Analysis of the transcripts results from PCR indicates Fe, oxidative and PAH pathways were affected by the presence of PM regardless of the size fraction. Although the components within the coarse mode that are responsible for the maturation of MDDCs remain to be determined, the results presented in this thesis have narrowed down the potential pathways for future studies.

The respiratory tract lining fluid (RTLF) is the first physical interface with which inhaled materials and aerosolised drugs come into contact in the airways. Surrounding the underlying epithelial cells, the RTLFs are composed of a thin amphipathic layer, providing crucial physiological and protective roles across the airways. The aim of this thesis was to investigate the composition of this extracellular compartment using human lavage samples from healthy volunteers, asthmatics and patients with Chronic Obstructive Pulmonary Disease (COPD) to guide the development of a series of lung lining fluid simulants for the testing of inhaled drugs. Analysis across nasal, bronchial and alveolar compartments revealed marked compositional differences throughout the respiratory tract in healthy individuals, with significant differences also observed with age, smoking status and the presence of established respiratory disease. Within the asthmatic group I observed evidence of an impaired microbicidal defense network at the air-lung interface, with significant depression of innate immunity proteins in the bronchial RTLFs associated with elevated concentrations of bacterial cell wall components. In the lavage samples obtained from healthy aged populations a general dysregulation of immunity and inflammatory processes was observed, alongside a pro-oxidant shift in the redox balance. In COPD, characteristic pathological changes were identified, from evidence of chronic inflammation, to protease-antiprotease imbalance and oxidative stress. In addition, I obtained preliminary evidence of dysregulated metal homeostasis, superimposed on compositional protein signatures characteristic of normal physiologic ageing. Clear differences in the composition of the RTLF could be attributed to smoking; with the greatest number of proteins identified (234) in the RTLFs of COPD smokers, in stark contrast to only 58 proteins accounted for within the COPD ex-smoking proteome. This extensive characterization of the RTLFs was used to successfully develop a physiologically representative ‘base model’; the suitability of which was assessed in a series of preliminary studies, focusing on particle and cellular interactions (nanoparticle characterization and biocompatibility with in vitro cell lines). These fundamental applications will be significant in evolving both understanding the behaviour of inhaled particles at the surface of the lung and improving the biopharmaceutical screening of aerosolized medicines.

A strong correlation exists between acute and chronic exposure to traffic derived pollutants and poor respiratory health. Specifically, diesel exhaust (DE) components such as NO2 and fine particles (PM2.5) have been related to impaired lung growth and increased respiratory and allergic symptoms in children and adults living near busy roads. On this basis, implementation of strategies to reduce diesel emissions and improve air quality should provide a measureable improvement in the respiratory health of populations resident in high traffic areas. The introduction of the London Low Emission Zone (LEZ), the largest of its kind in the world, covering an area of 2,644 km2 and a resident population of more than 8 million, provided a unique opportunity to examine this, as well to quantify the impact of DE emissions on the respiratory health of London's population. London’s Low Emission Zone was introduced as part of the Mayor of London’s Air Quality Strategy, with the aim of improving public health through targeted reductions in tail pipe emissions from the most polluting vehicles entering the city. The objective of decreasing PM10 concentrations was to be achieved by restricting the entry of the oldest and most polluting diesel vehicles (heavy goods vehicles (HGVs), buses and coaches, larger vans and minibuses) into Greater London by providing incentives to operators to upgrade their fleets to lower emission vehicles. The Low Emission Zone was enacted as a phased tightening of emission standards for each vehicle class, with the first phase coming into force at the beginning of February 2008. This applied to HGVs greater than 12 tonnes and restricted entry to the zone for those vehicles not meeting the Euro III emissions standard for PM10. Phase 2 followed in July 2008 widening restrictions to include HGVs between 3.5 and 12 tonnes, buses and coaches. Vehicles failing to meet these emissions standards within the zone were initially charged £200 (£100 for vans an minibuses) per day, with enforcement achieve using cameras to identify the registration numbers of vehicles and the Driver and Vehicle Licensing Agency (DVLA) database to identify a vehicle’s emissions standard. In its initial configuration phase 3, restricting access to heavier LGVs and mini-buses not meeting Euro III PM standard was planned for October 2008. In our initial four year study design we planned to examine the respiratory health of cross sectional panels of 8-9 year old school children living within the zone from November 2008; encompassing the first two years post phases 1 and 2, and two years post phase 3 (see Figure s1). As the subject recruitment and health assessments began in November 2008, this afforded us the opportunity of addressing the impact of the third phase of the LEZ, by comparing lung function and respiratory symptoms in the two years before and after phase 3. We also planned to examine year-on-year changes related to projected reductions in vehicle emissions as newer cleaner vehicles entered the fleet, independent of the LEZ, and the increased period the children had lived within the zone, from 11-15 months (11.5-13.9% of lifetime) in year 1, to 44-60 months (45.8-55.6%) in year 4. During annual school visits, children were asked to perform spirometry and provide a urine sample for the assessment of exposure (metals, reflective of defined traffic sources) and response biomarkers (oxidative damage). In addition, the parents/guardians of the children completed a questionnaire on respiratory / allergic symptoms and the children provided DNA samples to investigate genetic susceptibility to the detrimental effects of air pollution, focusing on a panel of antioxidant and xenobiotic genes, as well as a genetic marker associated with the onset of childhood asthma. In May 2008 Boris Johnson was elected the new Mayor of London, with a manifesto commitment to review ongoing traffic management schemes within the city, including the LEZ, and on the 2nd February 2009 he announced intention to cancel the third phase of the LEZ, subject to the outcome of a public consultation later in the year. This political decision therefore robbed us of the original intervention we were planning to address in our original design. Following a further consultation, the scheme was finally fully implemented and expanded on the 3rd of January 2012 (LEZ phase 3 and 4), with Euro III emission standards for minibuses and vans and a further tightening of emission standards (Euro IV) on Lorries over 12 tonnes, between 3.5-12 tonnes, as well as buses and coaches. In light of this development we obtained additional funding to examine furthers panels of school children in Nov 2012 - March 2013 and Nov 2013 - March 2014, extending our study to six years, allowing a formal assessment of the three years pre and two years post LEZ phase 3 and 4, with year 4 straddling the periods of phase 3 and 4 implementation (Figure s2). Children at the conclusion of the study in March 2014, who have been resident within the LEZ since birth will have lived within the zone for 68.8-83.3% of their lives. The data presented in this thesis is therefore based upon the first three years of the study and therefore constitutes a baseline analysis of the relationship between air pollution in London and our key respiratory endpoints prior to the formal evaluation of Phase 3 and 4 in 2014/15. In the first experimental chapter (Chapter 3) I evaluated the associations between traffic-related air pollutants and respiratory/allergic symptoms within our cross-sectional children's cohort. Information on respiratory/allergic symptoms was obtained using a parent-completed questionnaire and linked to modelled annual air pollutant concentrations based on the residential address of each child, using a multivariable mixed effects logistic regression analysis. Exposure to traffic-related air pollutants was associated with current rhinitis (NOx [OR 1.01, 95% CI 1.00-1.02], NO2 [1.03, 1.00-1.06], PM10 [1.16, 1.04-1.28] and PM2.5 [1.38, 1.08-1.78], all per g/m3), but not with other respiratory/allergic symptoms. Furthermore, over the first three years of the operation of London's LEZ I did not observe evidence of reduced ambient air pollution levels, or year-on-year changes in the prevalence of respiratory/allergic symptoms. I found no evidence that these associations were modified by polymorphisms in gasdermin B, located at the chromosome 17q12, associated with the risk of childhood asthma. These data confirm previously reported associations between traffic-related air pollutant exposures and symptoms of current rhinitis. Importantly, whilst the data is largely confirmatory, this remains one of the few studies that has addressed respiratory symptoms in urban children over the period of rapid dieselization within Europe. In Chapter 4 I report evidence of reduced lung volumes (FVC - Forced Vital Capacity) in children living within the study area. This negative association was small and most strongly associated with modeled annual NOx concentrations, at the residential address level. A straightforward method to discriminate between acute versus chronic pollutant effects was developed for the study. Acute exposures were assessed by scaling annual mean concentrations according to a ‘Nowcast’ factor calculated for each pollutant for the period immediately prior to the health assessment. This factor was defined as the ratio between concentrations measured by a local subset of London Air Quality Network monitoring sites in the prior period, and the annual mean measured by the same sites. Using this approach I was able to dissect out whether basal lung function was related to short or long term exposures. In the absence of relationships between FEV1 and FVC with 24 hour and 7 day average exposures, the association between FVC and annual pollutant exposures was interpreted as reflecting evidence of impaired lung growth. In this initial analysis I found no evidence that polymorphisms in the commonly studied glutathione S transferases (GSTM1 and GSTP1) and NADPH quinone oxidoreducatse (NQO1) genes modified the association between lung function and pollutant exposure. In a secondary analysis I examined whether polymorphisms in Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and the Aryl hydrocarbon receptor (AhR) might modify the association between pollutant exposures and lung function, based on their role in the xenobiotic metabolism of Polycyclic aromatic hydrocarbons (PAHs). This is the first time polymorphisms in these genes have been investigated in the context of air pollution – lung function interactions.