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The use of the anthracenedione mitoxantrone as an antitumor agent is steadily increasing. While the toxicities associated with its use are significantly less than those observed following treatment with the widely used doxorubicin, mitoxantrone cardiotoxicity is clearly a substantial clinical problem. Current information on the mechanism by which mitoxantrone causes toxicity in heart tissue is limited. Thus, the goal of these studies was to describe a model system in which mitoxantrone cardiotoxicity can be studied, and begin to describe the mechanism by which mitoxantrone exerts its cardiotoxic effect. These experiments have shown that cultured neonatal rat heart cells are an effective model system for studying mitoxantrone-induced cytotoxicity and biochemical changes in heart tissue. Cultured heart cells develop dose- and time-dependent toxicity following a short exposure to near-pharmacologically achievable drug concentrations. Furthermore, histologic changes characteristic of this drug are also observed at the light and electron microscopic level. Initial experiments aimed at defining mitoxantrone mechanism of action showed that mitoxantrone likely does not stimulate a significant production of active oxygen species, or have a specific effect on mitochondrial function. However, there is evidence to support the possibility that mitoxantrone can form a reactive intermediate in vitro. These studies have shown that covalent binding of mitoxantrone to proteins can occur under certain conditions. Mitoxantrone toxicity is lowered with the addition of ICRF-187, a metal chelating agent. Protection is not due to inactivation of mitoxantrone, decreased mitoxantrone uptake, or a delayed increase in cytosolic calcium. Similar protection is observed against doxorubicin and the oxidized form of mitoxantrone, but not against the non-hydroxylated analog of mitoxantrone, ametantrone. Furthermore, in a cell-free system, mitoxantrone can form complexes with both copper (II) and iron (III). Mitoxantrone metal binding is reversible as ICRF-187 as well as other chelators can remove the metals from these complexes. These data suggests that metal chelation is involved in the enhancement of mitoxantrone toxicity in vitro.

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A suite of biomarkers was developed using the crab Carcinus maenas and the mussel Mytilus edulis as test organisms. The ability of the biomarkers to differentiate amongst the major toxic components and to indicate the concentration of chemical mixtures was evaluated in the laboratory. Biomarkers were also applied in a field trial and their potential to monitor environmental water quality in a chemically contaminated estuary investigated. The results from the biomarker assays were compared with and validated against two commonly used toxicity tests (Tisbe battagliai LC-50, and Microtox®). Novel methods for recognising patterns of biomarker responses were developed and assessed. The most sensitive and reliable biomarker assays investigated were neutral red retention time in crabs and mussels and heart rate and glutathione-S-transferase activity in crabs. Effects were observed at environmentally realistic concentrations; for example lysosomal enlargement was observed in mussels exposed to a complex mixture containing chemicals at environmental quality standard concentrations. Exposure concentrations required to illicit biomarker responses were similar to toxicity test EC-50 values. The ease of interpretation and clarity of the results was enhanced when data from suites of biomarkers were pooled and analysed using multivariate statistical techniques (multidimensional scaling and cluster analysis). Multivariate analysis differentiated amongst mixtures containing solely organic chemicals, metals and metal and organic chemical mixtures. Exposure response relationships to complex mixtures were established for some of the individual biomarkers tested (crab heart rate and gill metallothionein) and also for suites of biomarkers when multivariate analysis was carried out. In the field biomarkers, in both transplanted and indigenous animals, were able to differentiate between clean and contaminated sites and indicate a pollution gradient along the Tees Estuary. This was not achieved using toxicity tests. The results were displayed clearly using multivariate analysis, enhancing the power of biomarkers as monitoring tools.

Studies have demonstrated the immune system to be significantly more plastic than previously believed. Multiple external factors have been shown to influence the immune response including alterations to the host's external environment and psychological status. This thesis details an investigation of this influence; exposing male CD1 mice to a two-week environmental enrichment paradigm then subjecting them to one of a range of inflammatory disease models chosen to assess a specific aspect of their immune function. Enriched animals were found to possess significantly higher numbers of circulating innate leukocytes compared to those animals housed in a standard lab environment. This leukocytosis was found to persist when animals were subject to a model of zymosan-induced peritonitis, where enriched animals presented an enhanced neutrophil and macrophage influx into their peritoneal cavity. Similar results were observed in a model of sepsis induced by caecal ligation and puncture where enriched animals were also found possess an enhanced capacity for systemic bacterial clearance. Across both experiments no changes in inflammatory cytokine expression were observed between enriched and standard environment animals. Genomic and proteomic profiling supported these findings, revealing the increased expression of immune-modulatory genes associated with a heightened immune and moderated inflammatory response. Ex vivo analysis of leukocytes extracted from enriched animals showed they also possessed enhanced phagocytic function and an accompanying reduction in gene expression associated with heightened cytotoxic function. When subject to a model of persistent inflammation induced by sponge implantation, enriched animals again presented heightened leukocyte infiltration to the point of immune insult. This was accompanied with a reduction in the release of pro-inflammatory cytokines and the heightened expression of genes associated with a pro-resolving, wound healing phenotype. This study provides novel insights into the mechanisms by which environmental modulation may influence the immune response and of the potentially immune-protective influence of environmental enrichment.

Omega-3 fatty acids (ω-3 FA, including eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), are essential polyunsaturated fatty acids which are correlated with lower incidence of chronic diseases. DHA and EPA can be enzymatically converted to resolvins, protectins and maresins, which play important roles in resolution of inflammation. Additionally, ω-3 FA can also directly activate surface receptors, namely the long-chain free fatty acid receptors GPR40 and GPR120, two GPCRs with poorly investigated biology. Using real-time PCR analysis, GPR40 transcript in human neutrophils was detected; the protein expression was also confirmed by flow cytometry and image stream analysis. Expression of GPR40 protein was up-regulated after stimulation with platelet-activating factor (PAF, 10nM) or leukotriene B4 (LTB4, 10nM) for 10 minutes. I utilised the selective agonist GW9508 to investigate the biology of GPR40. Tested on human neutrophils, GW9508 elevated intracellular calcium when applied within the 0.1-10μM range. The up-regulation of GPR40 expression by pro-inflammatory stimuli suggested to us potential regulatory roles for this receptor during inflammation. I then showed that 1 and 10μM GW9508 increased neutrophil chemotaxis in response to the cytokine IL-8 (30ng/ml). In addition, GPR40 activation by GW9508 enhanced phagocytosis of E. coli by human neutrophils by approximately 50% when tested at 0.1 and 1μM. Moreover, GW9508-neutrophil stimulation augmented microvesicle release and delayed apoptosis after stimulation. Finally, I demonstrated that GPR40 is expressed in inflammatory cells isolated from murine arthritic joints, such as neutrophils, macrophages and inflammatory monocytes. KBN-serum induced arthritic mice developed a more severe disease when treated prophylactically with GW9508 (10mg/kg, i.p. treated from day 0, daily), characterized by a higher clinical score and increased oedema when compared to vehicle control mice. Therapeutic intervention with GW9508 at the peak of the disease (day 5) delayed the resolution of arthritis. In summary, the data suggest that activation of GPR40 by GW9508 enhances neutrophil activation, up regulating the pro-inflammatory properties of this cell type, and therefore, exacerbating experimental inflammatory arthritis.