Despite being the “gold standard” for toxicity testing, rat in vivo studies are estimated to be only 37-50% accurate at predicting xenobiotic toxicity in humans. If validated, in vitro systems could be incorporated into toxicity testing, allowing direct comparisons to be made between humans and rats and facilitating a reduction of in vivo testing. In this project, a normal rat urothelial (NRU) cell culture system was developed and optimised for comparison against an established normal human urothelial (NHU) cell culture system. NRU cells had a reduced lifespan in culture compared to NHU cells, leading to an investigation of the pathways regulating cellular proliferation. The PI3K/Akt pathway was found to be active in proliferating NRU cells, whereas the EGFR/MAPK and β-catenin pathways were confirmed to regulate NHU cell proliferation. Inhibition of the PI3K/Akt pathway enabled NRU cells to be subcultured to passage 2, most probably by enabling EGFR/MAPK and β-catenin pathway activation, although this requires experimental confirmation. To further increase cell lifespan, the proto-oncogene BMI1 was overexpressed in NRU and NHU cells, with the effects on lifespan and differentiation compared against both human telomerase reverse transcriptase (hTERT) overexpressing and control (empty vector) cells. NRU cell lifespan was not increased by BMI1 or hTERT overexpression. BMI1 overexpression increased NHU cell lifespan in culture, comparable with hTERT overexpression, but further work is required to determine the extent of the effects on lifespan and phenotype beyond passage 12. Finally, the metabolic competence of urothelial cells was explored, with a focus on the cytochrome P450 2B (CYP2B) family of metabolic enzymes. Xenobiotic induction of CYP2B expression by rat urothelium was demonstrated in vivo but not in NRU and NHU cells. Xenobiotic-induced CYP2B protein expression was achieved in a rat ex vivo organ culture model. Together these results identify a previously unknown metabolic capability of urothelial cells that can be modelled ex vivo, and demonstrate fundamental differences in rat and human urothelial cell physiology that support the validation of human tissue-specific in vitro models for assessment of xenobiotic toxicity.

The development and use of engineered nanoparticles (NPs) has continuously expanded over the last two decades. Despite clear beneficial aspects of NPs, their extensive use and hardly regulated dissemination has raised concern regarding their potential adverse effects. Reports on the environmental release of NPs in wastes, waters, and wastewaters have emerged as well as ecotoxicity related information to a diverse range of model microorganisms (e.g. crustaceans, worms, algae, bacteria). However, in spite of growing knowledge in nanoecotoxicology there is limited evidence to draw conclusive statements about the toxicity and fate of NPs, especially in real matrices, in part due to a lack of appropriate methodologies. In this context, this work aimed to investigate the ecotoxicity of widely used and potentially antimicrobial inorganic NPs (Ag, ZnO, CuO, TiO2) to environmentally relevant bacteria (Pseudomonas putida) in various matrices (microbiological growth medium, artificial wastewater, real crude and final wastewaters). Complementary planktonic (i.e. using a luminescent switch-off bioreporter in a microtitre plate format) and biofilm (i.e. using mono and multi-species structures in flow-cell reactors) based assays were used. In addition, the implementation of microcantilever (μCT) and surface plasmon resonance imaging (SPRi) biosensor technologies were piloted. Toxicity of NPs was discussed across approaches (when applicable) in light of their physico-chemical characterisation (using dynamic light scattering, atomic absorption spectroscopy, and ultraviolet-visible spectrophotometry) in used matrices of exposure. This work provides both practical and fundamental insights about water/wastewater related ecotoxicology of NPs using bacteria. Overall outputs highlighted the suitability of original methods for testing of NPs in or with complex (i.e. real) materials and emphasised further the possible limited impact of NPs below the mg L-1 level to bacteria (as planktonic or biofilms) in the environment, especially with ageing of NPs (as reported with the Ag NPs), or considering the potential of recovery of bacterial structures (as shown with the biofilms). In addition, the workability of SPRi for testing of NPs was reported for the first time with bacteria, offering new opportunities of further real-time and high throughput biosensor based applications in nanoecotoxicology.

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Phenformin (N[1]-phenethylbiguanide) is an oral hypoglycaemic agent which has been in use in the U. K. since 1959 for the treatment of some forms of diabetes. In recent years there have been reports of lactic acidosis associated with phenformin therapy, particularly in patients with renal or hepatic diseases. Metabolic and pharmacological studies in the rat and guinea-pig have shown that a number of factors are involved in the differences in response to phenformin in these species. Following a low oral dose of [2'-[14]C] phenformin rats excreted the radioactivity more rapidly than guinea-pigs and metabolised the drug more extensively. The rat eliminated almost 88% of a 7 mg/kg dose in the urine and faeces in 24h and the urine contained almost entirely 4-hydroxyphenformin (free and conjugated with glucuronic acid) which had no effects on blood lactate or glucose concentrations in this species. The guinea-pig excreted only 57% of a 25 mg/kg dose and 50$ of the urinary radioactivity (18.5% of the dose) was unchanged phenformin. The rat actively eliminated a large proportion (26% in 6h) of a 20 mg/kg intraduodenal dose of [2'-[14]C] phenformin in the bile, most of the radioactivity being attributable to the parent compound. In the guinea-pig biliary excretion was initially rapid but declined so that only 6% of the same dose was eliminated in 6h. The possibility that active biliary excretion of phenformin is inhibited by the pharmacological effects of the drug in the guinea-pig is discussed. After oral administration of [2'-[14]C] phenformin guinea-pig urine contained no 4-hydroxyphenformin but small amounts were detected after intraperitoneal administration. After both routes of administration guinea-pig urine contained a novel metabolite and its glucuronide. After oral administration a second novel metabolite was detected in guinea-pig faeces. These metabolites have not been completely characterised but in both cases the biguanide portion of the metabolites had been modified. The urinary metabolite is probably the product of aliphatic hydroxylation rather than aromatic hydroxylation. The identification and pharmacological properties of these metabolites remain to be determined. Preliminary in vitro studies using isolated rat and guinea-pig hepatocytes showed a faster rate of metabolism of [2'-[14]C]phenformin in rat cells. Rat cells produced significant amounts of 4-hydroxy-phenformin and its glucuronide but guinea-pig cells did not. In the rat the time taken to eliminate the dose and the extent to which phenformin was excreted unchanged in the urine increased with larger doses of the drug. After a high oral dose (100 mg/kg) 24% of the urinary radioactivity (12.5% of the dose) was attributable to unchanged phenformin. Pharmacological studies showed that the rise in blood lactate concentrations and fall in blood glucose concentrations associated with phenformin were dose related in the rat. The relationship between this observation and the increased elimination of unchanged drug in rat urine after high doses is discussed in terms of a first-pass effect for phenformin in the rat. The dose-response data indicated a critical dose in rats in the region of 120 mg/kg intraperitoneally, at which point marked increases in the pharmacological effects of the drug were seen. Normal and fasted guinea-pigs were more susceptible than rats to both pharmacological actions of phenformin. 4-Hydroxy-phenformin produced hyperlactataemia in guinea-pigs without affecting blood glucose levels. The possibility that the guinea-pig may produce a biologically active metabolite has not been resolved, but the differences in pharmacological response to phenformin in the rat and guinea-pig have been explained by the results in the thesis, which show a slower rate of metabolism and excretion of the drug in the guinea-pig, together with the established biochemical differences in the mechanisms of carbohydrate regulation in these species. Sodium dichloroacetate was effective in reducing phenformin-associated hyperlactataemia in rat and guinea-pig and prevented the increase in blood lactate levels caused by 4-hydroxyphenformin in guinea-pigs.

Static and rotary cultures of adult mammalian hepatocytes have been routinely established from suspensions of monkey, rabbit and pig liver parenchymal cells isolated by means of a simplified, inexpensive recycling perfusion system. Light and electron microscope examination of the cultures showed that the hepatocytes appeared to retain similar morphological chacteristics of fully differentiated hepatic parenchymal cells for up to 5 days in static culture and up to 10 days in rotary culture, after which time they degenerated and were replaced by other cell types. The hepatocytes in both static and rotary culture, exhibited several major metabolic functions characteristic of liver in vivo, these included albumin synthesis, hippuric acid synthesis, urea production, bilirubin conjugation and responsiveness to insulin. The foreign metabolising capability of the cultured hepatocytes was typical of cytochrome P-450, not cytochrome P-448 as reported by other investigators, as witnessed by the metabolite profiles of biphenyl, aminopyrine, ethylmorphine, benzo/alpha/pyrene and 7-ethoxycoumarin. When exposed to inducing agents such as phenobarbitone a normal pattern of drug metabolising enzyme induction was observed. As the findings indicated that the hepatic parenchymal cells in both rotary and static culture were viable and behaved in many respects like normal adult liver in vivo investigations were undertaken to ascertain if such cultures could be used in in vitro bioassays for the detection of hepatotoxins and carcinogens.