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Organic xenobiotic compounds in the environment are causing widescale concern. Knowledge of the toxicity of organic xenobiotic compounds to the soil microbial biomass is essential as soil health and sustainability are essential for ecosystem function. Traditionally, chemical analysis has been used to assess contamination impacts in soils, however, compound residue cannot provide information on toxicity in environmental matrices, the toxicity of complex chemical mixtures and bioavailability. Bioluminescence bioassays, utilising the naturally bioluminescent bacterium Vibrio fischeri have been widely used to investigate the toxicity of contaminants in soil, sediment and freshwater environments. To utilise the V. fischeri bioassay all samples must be adjusted to a near neutral pH and a salinity of 2 % NaCl. Application of this assay to assess toxicity in terrestrial environments means that samples are not assessed under natural pH and saline conditions. Bioluminescent terrestrial bacteria have been constructed by inserting the lux genes, encoding of bioluminescence in marine bacterium, into the plasmid or chromosomal genome of terrestrial bacteria. The plasmid lux marked strain studied in this thesis, Pseudomonas fluorescens 10586s pUCD607, was used to develop a bioassay which could assess the toxicity of substituted benzenes in aqueous solution. The EC50 values determined for benzene and 1,2-dichlorobenzene were comparable between lux marked P. fluorescens and V. fischeri. Bioluminescence responses to substituted benzenes were investigated with a view to understanding modes of toxic action. Observed stimulation of bioluminescence in response to a number of compounds was thought to be caused by the uncoupling of proton gradients by low organic xenobiotic concentrations. Viable cell counts confirmed that stimulation of bioluminescence was not as a result of an increase in the number of viable cells. At high substituted benzene concentrations an inhibition of bioluminescence was observed. Application of quantitative structure-activity relationships (QSARs) for chlorobenzenes, utilising P. fluorescens toxicity data and physiochemical characteristics, showed that the toxic responses of these non-polar compounds were a function of compound solubility and lipophicity. QSARs applied to assess chlorobenzene toxicity could not be used to predict the toxicity of polar compounds due to the differing modes of toxic action. Correlating P. fluorescens with QSARs developed for C. meneghiniana and Pimephales promelas showed good correlation between the freshwater organisms but a poor correlation between marine bacterium V. fischeri.

Cholera toxin, obtained as crude culture filtrates of the bacterium Vibrio cholerae, was purified by a combination of ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose, and gel filtration cn Ultrogel AcA-44. The purified toxin migrated as a homogeneous protein on SD5 and native polyacrylamide gels, and activated / adenylate cyclase in rat liver homogenates. Activation of rat liver adenylate cyclase by cholera toxin was strictly dependent upon the presence of NAD +. In the absence of exogenous NAD+, homogenates or membrane preparations were refractory to the toxin as a result of high levels of endogenous NAD+ - utilising activities. Under conditions in which competition for NAD+ by endogenous enzymes was minimised, the activity of cholera toxin as a stimulator of adenylate cyclase was markedly enhanced and activation of the enzyme occurred with a detectable toxin-specific % incorporation of [~^c] into TCA-precipitable material from ^adenine U-^cj- NAD+. The results were consistent with the hypothesis that cholera toxin acts by catalysing the ADP-ribosylation of an intracellular membranebound acceptor protein, and that this modification is responsible for the toxin-induced stimulation of adenylate cyclase activity. Results are also presented which argue against the validity of extrapolating from studies of the NADase activity of cholera toxin to the mechanism of toxin action on adenylate cyclase. Under a variety of conditions, NADase activity appeared to proceed independently of the physiologically important ADP-ribosyltransferase activity. furthermore, culture filtrates of V. cholerae were shown to contain a highly active NADase distinct from cholera toxin, and the possibilities of NADase contamination, even in apparently pure toxin preparations, are emphasised.

Renal proximal tubular cells (&'62 90&'37) were isolated from the rat in high yield by collagenase digestion of renal cortical tissue, followed by isopycnic centrifugation in a Percoll density gradient. The cells were of high viability and their identity verified by morphology and PT specific enzyme activities. Cytochrome P450 and dependent monooxygenase activities were maintained and the rapid decline in reduced glutathione prevented by addition of glycine, glutamate and cystine to the buffers used. These parameters were also maintained in culture (24h). Cytochrome P450-dependent monooxygenase activity was induced in proximal tubular cells isolated after pretreatment of rats with 3-methylcholanthrene, particularly with ethoxyresorufin as substrate, indicating the presence of inducible P450c isoenzyme(s) in PT cells. In contrast, treatment with phenobarbitone showed no induction. Treatment in vivo with the renal toxins, gentamicin and cyclosporin A (CsA), resulted in altered glomerular function and renal parenchymal damage. Treatment with gentamicin in vivo and in vitro resulted in increased PAH uptake and decreased gamma glutamyl transferase activity, suggesting a primary effect of gentamicin on cell membranes. CsA treatment in vivo produced an effect on GSH, lipid peroxidation and succinate dehydrogenase activity in the isolated PT cells. On treatment in suspension, no significant effects were observed. Treatment with gentamicin in culture (24h) did not result in a significant effect on the cell parameters studied. In conclusion, PT cells can be isolated in high yield with good viability from the renal cortex. Cell function can be maintained both in suspension and in culture. Therefore, this system may be used as a model for the investigation of PT-specific drug-induced nephrotoxicity as well as renal cytochrome P450 isoenzyme composition and induction characteristics.

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