At birth, the mammalian ovary has a finite number of dormant primordial follicles. Repeated daily dosing of rats with the occupational chemical, 4- vinylcyclohexene diepoxide (VCD), depletes the ovary of small pre-antral follicles (primordial and primary follicles) through an increase in the natural process of atresia (apoptosis). In addition, in vitro exposure of postnatal day 4 (PND4) rat ovaries to VCD causes a similar depletion of ovarian follicles. Since many growth factors play crucial roles in the promotion of early folliculogenesis and follicle survival, it is possible that any number of factors and subsequent signaling pathways could be disrupted in response to VCD exposure. Therefore, the studies in this work address the hypothesis that VCD disrupts oocyte-granulosa cell survival pathways in the rat ovary, thereby compromising cell-cell communication and causing follicle cell death. The results from the first aim reveal that through the use of genomic analyses a subset of genes were determined to be affected via in vivo and in vitro exposure routes to VCD. The results of the second aim show that two transforming growth factor β (TGFβ) growth factors, growth and differentiation factor-9 (GDF-9), and bone morphogenetic factor-4 (BMP-4), are not likely involved in VCD-induced ovotoxicity as they were unable to prevent ovarian follicle loss in the presence of VCD. The results of the third aim reveal that expression of the c-Kit receptor, present on the oocytes, is decreased and its ligand, Kit Ligand (KL), produced from the granulosa cells, is increased in response to in vitro VCD exposure. In addition, attenuation of VCD-induced follicle loss occurs in the presence of exogenous KL. Finally, the results of the fourth aim examines the involvement of the AKT signaling molecule in response to VCD exposure, in which the active phosphorylated AKT is determined to be down-regulated by VCD. Taken together, these studies show that VCD is able to disrupt at least one of the cellular survival pathways that are crucial to maintain the ovarian follicle. As a result, a breakdown in cell-cell communication may occur at that level and contribute to an increase in follicular atresia and eventual cell death.

No description available.

Nanoparticles (particles less than 100 nm in at least one dimension) exhibit chemical properties that differ from their bulk counterparts. Furthermore, they exhibit increased potential for systemic toxicities due to their deposition deep within pulmonary tissue upon inhalation. Thus, standard regulatory assays alone may not always be appropriate for evaluation of their full spectrum of toxicity. Systems biology (e.g., the study of molecular processes to describe a system as a whole) has emerged as a powerful platform proposed to provide insight in potential hazard, mode of action and human disease relevance. This work makes use of systems biology to characterize carbon black nanoparticle-induced toxicities in pulmonary and extra-pulmonary tissues (i.e., liver and heart) in mice over dose and time. This includes investigations of gene expression profiles, microRNA expression profiles, tissue-specific phenotypes and plasma proteins. The data are discussed in the context of potential use in human health risk assessment. In general, the work provides an example of how toxicogenomics can be used to support human health risk assessment.

The aim of this study was to develop a suitable monitoring protocol for mediated amperometric whole-cell biosensors for in situ assessment of heavy metals in soil. E. coli 8277, Pseudomonas 9773, Pseudomonas 9046 and Pseudomonas 8917 were screened as biosensor catalysts to select the sensitive biosensor configurations to heavy metals. A new protocol was developed for monitoring heavy metals in defined solution, soil pore water, and in situ in soil. This study also demonstrated the applications of mediated amperometric bacterial biosensors for in situ assessing the bioavailability and toxicity of heavy metals in freshly spiked soils or historically contaminated soils, and mixture toxicities of heavy metals. It was found that the biosensors incorporating selected bacterial strains were appropriately sensitive to copper, but less sensitive to Zn, Pb, and Hg, compared to Microtox assay. The advantage of the mediated amperometric bacterial biosensor system is its in situ application in soils. The present study demonstrated that soil pore water does not accurately reflect conditions of soil ecosystem, and that in situ bioassays are more reliable for determining the bioavailability and toxicity of heavy metals. This is the first reported use of disposable whole cell biosensors for in situ heavy metal bioavailability and toxicity assessment. The biosensor protocol developed here can be adapted to allow the incorporation of dfferent bacterial biocatalysts for applications in soil quality assessment, screening of sites for contamination ‘hot spots’, and the evaluation of soil degradation or rehabilitation from metal pollution. Mediated amperometric bacterial biosensors are not analyte specific, their response reflecting the metabolic impact of the combined chemical and physical properties of the environment to which they are exposed. In assessing the toxicity of soil samples from fields using these biosensors, it is vital to get appropriate control soil samples. The conditions of soil samples also need to be well defined. The sensitivity of the mediated amperometric whole-cell biosensors to heavy metals need to be further improved. Investigations are also required to determine how the natural conditions affect the application of the biosensor system in the field.

The use of nanoscale materials is growing exponentially, but there are also concerns about the environmental hazard to aquatic biota. Metal-containing engineered nanoparticles (NPs) are an important group of these new materials, and whilst there are undoubtedly a plethora of beneficial uses for these NPs, it is essential that an appropriate risk assessment is carried out in order to protect the environment and human health, with the consumption of contaminated fish a distinct possibility. The current study aimed to assess the bioavailability, uptake and toxicological effects of two metal-NPs (TiO2 NPs and Cu-NPs) to fish from both dietary and waterborne exposure routes and where appropriate compare them to their bulk counterpart. Whole body system effects were assessed along with the influences of the life stage of exposed fish and abiotic factors on toxicity. A technique to improve the quantification of Ti from TiO2 NPs in fish tissue was also developed. Effects from exposure to dietary TiO2 NPs manifested similarly to traditional dietary metal exposure, with no reduction in growth, but some sublethal affects. Exposure to waterborne Cu-NPs showed that rainbow trout were more acutely sensitive to CuSO4 than the NPs, but that despite limited uptake several body systems were affected (most notably ionoregulation). Larvae were more sensitive to CuSO4 than Cu-NPs, but no differences were seen with embryos, whilst larvae were more sensitive than embryos. Abiotic factors did have an effect on acute Cu-NP toxicity, though not always in a predictable manner, with some effects more pronounced than with CuSO4. Overall, it appears that metal-NPs are not as acutely toxic as their bulk counterparts, but sublethal effects, were routinely observed. As TiO2 NPs appear more toxic than its bulk counterpart, current legislation governing safe environmental limits may have to be adjusted, though the situation with Cu-NPs isn’t as clear and further investigation is required. However, the risk of human exposure via the consumption of NP contaminated fish fillets is extremely low.

The population of harbour ragworms, Nereis diversicolor, inhabiting the upper reaches of Restronguet Creek in the Fal Ria, UK, are known to be resistant to the acute, toxic effects of copper, zinc and cadmium. This research aimed to establish whether metabolic and reproductive performance costs were associated with the resistance phenomenon and what the biochemical mechanisms of resistance were, by comparing animals from the Creek with two comparable study sites in the South West of England: Froe Creek and the Teign estuary. There was a significant metabolic cost, measured as a reduction in Scope for Growth, associated with the resistance phenomenon. As no differences in energy intake or uptake were found between resistant and non-resistant animals it was concluded that this cost was covered by demand-side regulation of energetics. A reduction in the amount of biochemical energy reserves in the form of lipids and sugars was also observed in resistant animals suggesting that resource allocation had been shifted away from this endpoint and towards maintenance and activity. The perturbed metabolism and physiology of resistant N. diversicolor is shown to have detrimental consequences for their life-history. Sexually mature, resistant females were significantly smaller than non-resistant ones, indicating that they had either matured at a younger age or grown more slowly. Both total fecundity and mass-specific fecundity were significantly reduced in resistant females, likely as a direct result of the metabolic costs of resistance reducing the resources available to fuel gametogenesis. No differences were found in the energetic reserves stocked within each gamete by resistant and non-resistant animals, suggesting that this trait is heavily selected for. Synthesis of reduced glutathione [GSH] in resistant animals’ tissues appeared to be elevated. Glutathione peroxidase activity was also increased, likely to remediate the effects of Reactive Chemical Species [RCS] that result from the inevitably incomplete binding of intracellular metals. As GSH functions in metal detoxification to covalently binding metals entering resistant animals’ cells, preventing their involvement in toxic interactions and their catalysis of the production of RCS, it is proposed that together there two phenomena comprise the fundamental mechanism of resistance to metal toxicity. Ultimately, this research revealed a hierarchy of health and reproductive performance across the three study populations, with significant associations evident between measured biological endpoints and the degree of metal contamination, illustrating the consequences of anthropogenic pollution for the biology of wild animals.

III-V materials such as indium arsenide (InAs) and gallium arsenide (GaAs) are increasingly used in electronic and photovoltaic devices. The extensive application of these materials may lead to release of III-V ionic species during semiconductor manufacturing or disposal of decommissioned devices into the environment. Although arsenic is recognized as an important contaminant due to its high toxicity, there is a lack of information about the toxic effects of indium and gallium ions. In this study, acute toxicity of As(III), As(V), In(III) and Ga(III) species was evaluated using two microbial assays testing for methanogenic activity and 02 uptake, as well as two bioassays targeting aquatic organisms, including the marine bacterium Aliivibrio fischeri (bioluminescence inhibition) and the crustacean Daphnia magna (mortality). The most noteworthy finding was that the toxicity is mostly impacted by the element tested. Secondarily, the toxicity of these species also depended on the bioassay target. In(III) and Ga(III) were not or only mildly toxic in the experiments. D. magna was the most sensitive organism for In(III) and Ga(III) with 50% lethal concentrations of 0.5 and 3.4 mM, respectively. On the other hand, As(III) and As(V) caused clear inhibitory effects, particularly in the methanogenic toxicity bioassay. The 50% inhibitory concentrations of both arsenic species towards methanogens were about 0.02 mM, which is lower than the regulated maximum allowable daily effluent discharge concentration (2.09 mg/L or 0.03 mM) for facilities manufacturing electronic components in the US. Overall, the results indicate that the ecotoxicity of In (III) and Ga(III) is much lower than that of the As species tested. This finding is important in filling the knowledge gap regarding the ecotoxicology of In and Ga.

There has been an increased global interest in biofuels which provide a renewable and sustainable alternative to fossil fuels. Isobutanol is an attractive and superior alternative to the currently produced bioethanol possessing several key advantages. Previous work focuses on strategies for metabolic optimisation of carbon utilisation. However, existing solutions reach a stage where the amount of alcohol produced reaches toxic thresholds for bacteria. This inhibits growth and reduces carbohydrate consumption resulting in lower product yields rendering the biofuel production process uneconomical. In this project, a novel strategy has been adopted which uses a whole system integrated genomics approach consisting of expression profiling, selection to create isobutanol-adapted lineages, next generation sequencing, and comparative behavioural genomics to interrogate the system thoroughly and identify critical determinants of resistance to isobutanol. These were used in the highly-defined model species, E. coli K12 to deliver results of the adaptive mechanisms which take place across the entire genome. 41 gene candidates (4 previously identified in literature) were identified to play a role in isobutanol tolerance. These candidates belong to a range of functional groups such as carbohydrate metabolism, oxidative stress response, osmotic stress response; but also identified novel membrane-associated functions such as the Tol-Pal system, BAM complex and colanic acid production. The results also identify critical genes with unknown functions. The results support previous notions that central carbon metabolism shifts from aerobic to anaerobic metabolism in the presence of isobutanol, but also shows there is a transitionary phase where mixed acid fermentation pathways are utilised. This shift was previously thought to be mediated by the ArcA-ArcB two-component system. However, these results suggest the inactive 2Fe-2S core of the anaerobic-regulator Fnr is re-activated by Fe2+ to form the 4Fe-4S core transported by the FeoAB ferrous iron transport system. The strategy also identified the Tol-Pal system and show it is essential to grow in the presence of isobutanol, which is responsible for the maintaining the integrity of the cell envelope structure and increasing the rate of cell division. The BAM complex is responsible for folding and assembly of outer membrane proteins (OMP) and OMP membrane permeability- this system was found to be important for growth in isobutanol, and SurA, which is the primary OMP assembly pathway provided tolerance which was specific to isobutanol. Colanic acid, an extracellular polysaccharide is produced when the cell experiences stress, and provides protection by forming a physical barrier around the cell. The results show that the presence of colanic acid plays a large role in allowing E. coli to grow in presence of isobutanol, and its role becomes essential at critical concentrations. The results also show deletion of the negative regulator of the colanic acid gene cluster improves growth at critical and growth-inhibiting concentrations. When consolidated, these results facilitated knowledge-led based design and subsequently led to the identification of components for a synthetic design schedule, which lists the genetic manipulations proposed to exploit E. coli to enhance isobutanol tolerance.

M.Sc. (Aquatic Health) / Sediments can contain a variety of organic and inorganic contaminants. These contaminants accumulate, resulting in extremely high concentrations even once the overlying water concentrations are at or below acceptable water quality guidelines. Any changes in the physical parameters'of the overlying water can cause these pollutants to be released back into solution. Accumulated contaminants can be released at even higher concentrations than previously detected. In recent years, sediment contamination has highlighted the need to monitor these previously overlooked pollutant sources that have accumulated in aquatic ecosystems. South Africa does not currently have standardised methods to assess sediment toxicity. Although international methods exist, they are largely untested in South Africa and the organisms needed to conduct these tests are not readily available. Over the years numerous culture methods have been develop globally for culturing organism to be used for water and sediment toxicity tests. In South Africa, the focus has mainly been on culturing organisms for water. toxicity testing. Sediment toxicity testing with indigenous organism however, was not developed. Established international culture methods from the United States Environmental Protection Agency, the Organisation for Economic Cooperation and Development, and Environment Canada were taken into consideration when developing the laboratory culture method for two (2)of the selected organisms (Chironomus spp. & Hydra sp.) from this study. A preliminary culture method was also developed for the third selected organism, Melanoides tuberculata (gastropod). The organisms cultured in this study were selected based on their extent of contact with the substrate, ease of handling, availability, culture maintenance as well as their reproductive cycle. The Hydra, Chironomids and M. tuberculata cultures were successfully breeding under laboratory conditions and remained stable. The Chironomus sp. and M. tuberculata maintain contact with the sediment making them suitable as ecologically relevant organisms for use in whole sediment toxicity testing in South Africa.

On August 29, 2005 Hurricane Katrina struck the Gulf Coast and storm surges breached levees flooding much of New Orleans, Louisiana. One month after the storm, sediment was collected and toxicity was tested using Japanese medaka (Oryzias latipes) embryos. Sediments with the highest contaminant levels showed the highest embryonic mortality and most delayed development. However, no sediment caused an increased mutant frequency. When the most contaminated site was resampled in February, 2006 contaminant levels and toxicity decreased. During toxicity testing, approximately 20% of embryos incubated with sediment from one of these sites died and turned red. A red bacterium was isolated that is Gram-negative, cocco-baccilus, non-motile, and most similar to Hahella chejuensis based on genetic and metabolic tests. This bacterium caused 100% infection at 108 bacterial cells per ml and variable infection at lower doses. This study was the first to examine biological effects of exposure to post-Hurricane Katrina sediments.