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This study aimed to investigate the potential of dichlorvos (DCV), chlorpyrifos (CPF) and sarin (GB) to induce DNA damage in vitro. DNA damage was measured by the alkaline comet assay in the neuroblastoma SHSY-5Y and lymphoblastoid TK6 cells and corroborated using phosphorylation of H2AX. Cytotoxicity was measured using the MTT assay in TK6 cells. The activation of DNA damage signalling pathways was investigated using western blotting. TK6 cells were used to investigate p53 levels and cleaved PARP-1 after exposure to OPs. Cell cycle effects were investigated in A549 cells by measuring the phosphorylation of the retinoblastoma and cdc2. Whether a slowing of the cell cycle allowed for initiation of repair mechanisms was investigated in cells lacking the DNA repair pathways base excision repair (BER) and non homologous end joining (NHEJ) after DCV. DCV induced DNA damage, the genotoxic potential was also corroborated by increased expression of γH2AX. DCV was cytotoxic at high concentrations. DCV results in increased p53 levels but no apparent phosphorylation on Ser15, which is known to be phosphorylated when exposed to other genotoxins. A G2/M block was found after exposure to dichlorvos as well as the possible initiation of DNA repair mechanisms NHEJ and BER. CPF and CPF oxon induced DNA damage in TK6 cells with a decrease in cell viability. Western blot analysis did not show a p53 response to the DNA damage with CPF. A small p53 effect at 24hours was seen with chlorpyrifos oxon but p53 may have been induced at an earlier time point. It is possible that the DNA damage observed was due to both direct DNA damage and other cellular effects, as the cells undergo apoptosis or necrosis and subsequent DNA degradation. Sarin caused low levels of DNA damage after 1 hour, which was partially repaired at 24 hours. This resulted in low level cytotoxicity and a rapid but transient increase in p53 levels and a G2/M cell cycle arrest.

These studies were aimed at identifying potential surrogate compounds for dermal absorption of bioactive agents in vitro using flow through and static cell diffusion systems, and also to generate novel data on the effects of application vehicle on chemical absorption following low level exposure. For protection and risk assessment purposes, the influence of ‘everyday’ clothing, and skin surface decontamination on dermal chemical absorption was also assessed. The model compounds (caffeine and benzoic acid), and the surrogate compounds (chlorpyrifos, dichlorvos and phorate) were generally found to be poor marker chemicals for comparison with HD absorption through human and pig skin in vitro. However, benzoic acid absorption from a finite dose in IPA more closely matched the absorption profile of HD applied as a finite dose in IPA. Dichlorvos absorption was greatest from all vehicles compared with chlorpyrifos and HD absorption in vitro. Dermal absorption of chlorpyrifos was enhanced when applied in PG compared with absorption from IPA or IPM. No differences were observed between absorption of neat HD and HD in IPA in terms of percentage of applied dose absorbed at 24 hours. Absorption of HD through full thickness pig skin more closely matched absorption through full thickness human skin, split thickness pig skin overestimated absorption of HD in vitro in comparison. ‘Everyday’ clothing (cotton shirt) significantly reduced absorption of dichlorvos, chlorpyrifos and HD through human skin. Chemicals were applied to clothed skin in IPA to mimic finite exposure, and left in contact for 30 minutes (dichlorvos), 4 hours (chlorpyrifos), and 1 hour (HD). For all chemicals, removal of clothing followed by immediate skin surface decontamination with 0.5% (v/v) soap solution further reduced absorption compared with removal of clothing alone. Despite these differences not being significant, in terms or civilian exposure, it would be recommended to remove clothing and decontaminate as early as possible postexposure to minimise the potential for dermal absorption and localised toxicity within the skin. In conclusion, the organophosphate compounds used in these studies (chlorpyrifos and dichlorvos) could potentially be useful surrogates for organophosphate agents such as VX or sarin, however, further work is needed to make these comparisons. The vehicle in which a chemical is applied to the skin can have a profound effect on dermal absorption, and this knowledge is important for risk assessment for exposure to a range of chemicals. Cotton shirt material significantly reduced dermal absorption of all chemicals used compared with 24 hour exposure. Despite this clothing not being designed for protective purposes, this may have a significant impact for reducing dermal absorption and toxicity in vivo as a result of chemical exposure. Further investigation is needed to assess absorption of a wider range of chemicals and application vehicles for risk assessment purposes, and to identify chemicals that more closely mimic dermal absorption of bioactive agents in vitro for extrapolation to in vivo exposure scenarios

Parkinson’s disease (PD) is an age-related neurodegenerative disorder, characterised by progressive degeneration of dopaminergic neurons in the substantia nigra, with the formation of α-synuclein rich, intracytoplasmic Lewy bodies. Several genetic and environmental factors, including pesticides are linked with sporadic PD. The aim of this study was to investigate the effect of selected pesticides on dopaminergic neuroblastoma SH-SY5Y cells and differentiated human neural precursor cells. Several parameters of toxicity were successfully measured including cell-viability in SH-SY5Y cells and estimation of sub-cytotoxic doses which were used to study the effects of signalling inhibitors, measurement of mitochondrial transmembrane potential, reactive oxygen species formation, inhibitory activity towards mitochondrial complex I/II, protein expression after acute and chronic toxin treatment and changes in gene expression. Twenty nine commonly used pesticides were screened for potential PD involvement, using cell viability and Alamar Blue reduction assay in SH-SY5Y cells. Most chemicals showed low toxicity using this system. Chemicals known (MPTP or MPP+) or thought to be involved with PD (e.g. paraquat) showed significant toxicity at the highest chosen dose i.e. 1mM (MPTP/MPP+ caused 20-30% reduction in cell-viability at 1mM whereas paraquat caused 60-70% reduction at 1mM). Significant toxicity was observed at concentrations as low as 0.01mM (60-70% reduction in cell-viability after maneb and mancozeb exposure) and 0.1mM (60%, 50%, 80% and 40% reduction in cell-viability after diquat, epoxiconazole, fluroxypyr-ester and mecoprop-methyl ester treatment respectively). Toxin exposure of human midbrain neurones (hNPCs) derived from embryonic neural stem cells showed that hNPCs were more vulnerable at 0.01mM and 0.1mM than SH-SY5Y cells (except maneb, mancozeb and fluroxypyr ester). Pharmacological inhibition of apoptosis showed a marginal but insignificant reduction in toxicity for most chemicals whereas macroautophagy inhibition had no effect. The absence of any effect of caspase inhibitors, with the exception of diquat, may indicate caspase independent induction of cell death markers like PARP-1 suggesting that toxin treatment seems to cause caspase independent cell death involving RIP. This was shown by using Necrostatin-1, a RIP1 and necroptosis inhibitor, which significantly increased viability (greater than 90% recovery vs. untreated cells) in diquat (0.1mM), mancozeb (0.05mM) and maneb (0.05mM) treated cells. Other results suggested possible involvement of chaperone-mediated autophagy (CMA) with diquat, maneb and mancozeb toxicity which showed increased lysosomal accumulation. Mitochondrial energetics were not affected after acute and chronic toxin exposure which did not affect the mitochondrial complex I or II activities. Coincidentally, cells exposed chronically to diquat appeared to down-regulate expression of autophagic and apoptotic response genes. It can be concluded that these agrochemicals exert their toxicity through distinct mechanisms including indirect energy depletion and direct damage to cell components and show significant toxicity possibly due to ROS generation causing necroptosis and CMA induction.

A correlation was found between individual metal content and in vitro bioreactivity of particulate samples (in both ambient PM samples from Port Talbot and ROFA samples). Global factors such as particulate origin and seasonal variation were also identified as playing a role. A bioreactivity hierarchy of metals was then established: Fe2+Cu2+Fe3+VO2+Zn 2+ As3+=Pb2+=VO3- indicating the importance of oxidation state and suggesting a role for redox mechanisms. Strong synergistic effects were observed between Zn 2+ and various bioreactive metal ions (Cu2+, Fe 3+, VO2+) in vitro. However, this effect was limited to ions which could be readily reduced to a more active form (no synergy with Fe2+). In vivo, the surrogate water-soluble metal mixtures gave rise to a transient (7-14 day) metal ion concentration and valence dependent increase in lung:body weight ratio, protein levels (pulmonary oedema) and differential cell counts (inflammation) in a dose and time dependent manner. Light microscopy of the lung tissue revealed an acute phase response to the installation of the metal mixture with peripheral damage and inflammation followed by bronchiolisation, hyperplasia and increased collagen deposition in repair. There was evidence of organ-specific changes in RNA production in both the heart and lung. In both organs, the primary responses revolved around up regulating redox-specific, stress mechanisms and metabolism pathways, suggesting activation of xenobiotic biotransformation systems.

Recent legislation has accentuated the importance of developing novel in vitro toxicology testing strategies which avoid the use of animals. The focus of this study was to use in vitro epithelial models to characterize the point of change between protective and deleterious responses in the lung following exposure to well-characterized commercial polymers. The first step was to develop and characterize the Normal Human Bronchial Epithelial (NHBE) primary cell model, which resulted in an organotypic, multi-differentiated bronchial model which was then used to elucidate the phenotypic and genotypic responses to polymers. The NHBE model exhibited evidence of ciliogenesis and mucus production comparable to native human bronchial epithelium. Conventional toxicology techniques were utilized to establish the doses required to induce sub-toxic responses in the model a lower (TD05) dose to induce a protective response and a higher (TD20) dose to induce a more injurious response. Transmission (TEM) and Scanning Electron Microscopy (SEM) were employed to examine changes in the inter-cellular morphology and revealed the TD05 dose led to regional hypertrophy but that the TD20 dose resulted in cellular degradation and general degeneration of tissue integrity. Transcriptomic tools (GeneSpring, Metacore) were applied to microarray data to compare gene expression profiles across NHBE cultures (multiple donors) and with a commercial tracheal model (EpiAirway, single donor), as well as to characterize the molecular pathways implicated in response to each dose. This demonstrated that the TD05 dose induced alterations of genes and pathways representative of protective and inflammatory responses but the TD20 dose induced airway injury, wounding and remodeling. It was also found that although the nature of responses were similar, the EpiAirway tissues were over-sensitive and presented a shorter viable experimental window, therefore the NHBE was considered the optimal in vitro model to assess toxicological responses of the pulmonary epithelium.

The respiratory tract is the primary site of exposure to inhaled substances. A growing need exists for high throughput in vitro models of the respiratory epithelium, which can provide rapid, reliable safety and effective screening in preclinical drug development applications. Normal human bronchial epithelial (NHBE) cells were cultured at an air-liquid interface in order to produce an in vitro model of the respiratory epithelium for toxicological testing. Extensive biochemical and morphological characterisation during construct development revealed that the NHBE model formed a pseudo-stratified, fully-differentiated culture of muco-ciliary phenotype. Histochemical and immunohistochemical techniques allowed the identification of basal, Clara, goblet and ciliated cells. Developmental characterisation revealed a toxicological dosing window of 7 days, where the model was deemed to be fully-established. Fully-developed NHBE cultures were then exposed to classical pulmonary toxicants (CPT) Lipopolysaccharide, cadmium chloride, paraquat, Amiodarone and cigarette smoke. Conventional toxicology techniques (culture viability, trans-epithelial electrical resistance TEER and morphology) were utilised to monitor the NHBE response to each CPT. The NHBE model responded with both general and toxicant-specific defence/irritancy mechanisms, observed to take place in the human bronchial epithelium and as such, reflective of in vivo toxicity. The in vitro model was finally challenged with candidate respiratory drugs (AstraZeneca AZ ) to test the utility of the cell system as a drug pre-screening tool. Blind-exposure of AZ compounds were characterised (physicochemical/ biochemically/morphologically) in the in vitro model and compared to AZ in vivo (rat) parallel exposure, focusing on irritancy end-points. A comparison of in vitro to in vivo exposures resulted in a 76.9 - 85% correlation of irritancy responses.