[pt] Para a avaliação da toxicidade aguda através do método respirométrico, foram empregados quatro compostos intoxicantes: Cu2+, fenol, surfactante e antibiótico, em distintas concentrações, os quais foram mantidos em contato durante 15 minutos com quatro tipos de lodo ativado: um oriundo de uma indústria farmacêutica, dois provenientes de indústria alimentícia e o outro aclimatado com efluente produzido sinteticamente. O reator biológico experimental foi montado e operado de modo contínuo para simular as condições encontradas nas indústrias. Os testes consistiram da medição da taxa de consumo de oxigênio pelos micro-organismos durante 5 minutos. Os valores obtidos pelos ensaios eram comparados de modo a entender o comportamento dos microorganismos entre os contaminantes e os diferentes lodos ativados utilizados. O lodo da indústria farmacêutica, por conter diversos compostos tóxicos, possui uma maior adaptabilidade dos micro-organismos, apresentando maior resistência aos compostos tóxicos introduzidos no teste; já o lodo ativado de uma indústria alimentícia, por conter concentrações significativas de óleos e graxas, sugere que haja bloqueio das células, apresentando comportamento similar ao da indústria farmacêutica, indicando que compostos tóxicos e óleos e graxas atuam da mesma forma na respiração do lodo ativado. O método respirométrico é eficaz para a determinação de toxicidade em processos de lodos ativados; foi verificado, também, que quando há descarga de compostos tóxicos e/ou óleos e graxas nas ETEs, os micro-organismos tornam-se mais resistentes, dificultando a detecção da presença do elemento intoxicante. / [en] The main aim of this work was to investigate the behaviour of industrial wastewater activated sludges exposed to sudden contamination by copper ions, phenol, surfactant and amoxicillin, in various concentrations, in order to attempt to obtain a better understanding of the respiration changes of the sludge. Four sets of assays were carried out with samples of sludge from: pharmaceutical industry; two types of food industries; an, a synthetic effluent fed with microorganisms. The contact period between the wastewater and microorganisms was about 15 minutes and the respiration time was 5 minutes. The experiments were conducted and operated continuously in order to simulate real conditions found in industry. All the samples were taken for respiration rate measuring. The values obtained from the assays were used to study the microorganisms behavior by comparing intoxicating compounds and all types of activated sludge tested. The sludge from the pharmaceutical industry has higher adaptability to sudden contact with toxic compounds, as the microorganisms in this type of sludge have normally acquired resistance to many toxic compounds that are commonly present in this type of effluent. However, the activated sludge from food industry, that has high concentrations of oil and grease, presented a similar respiration behavior in comparison to pharmaceutical industry sludge, leading to the conclusion that the oil and grease promote cell blockage, preventing contact between food and microorganisms, and causing respiration interferences. The respirometric method was significantly tested and the result is that this technique promotes efficient determination of activated sludge even under adapted activated sludge to toxic compounds, by which measure become slightly rugged.

Little is known of tissue carbon monoxide (CO) changes after acute exposure because tissue levels are in the order of picomoles per milligram and the technology to measure such low concentrations has only become available relatively recently. This study tested three hypotheses: That tissue CO levels (1) vary among tissues after acute poisoning; (2) change over time; but (3) cannot be predicted by measuring carboxyhaemoglobin (COHb) levels. Twenty four healthy male Sprague-Dawley rats were exposed to 2500 ppm CO in air for 45 min. This non-lethal exposure achieved reproducible COHb values of 66 to 72%. Animals were allowed to recover breathing air and were sacrificed at 30 minute intervals for 150 minutes. An additional nine male animals served as unexposed controls. Accurate measurements of tissue CO levels were made in blood, brain, heart, and skeletal muscle samples. All samples were prepared using the validated technique described by Vreman et al., and Reduction Gas Analysis was used to determine the pmol CO per wet weight tissue. Co-oximetry and gas chromatography were performed on all blood samples. Predictably, blood CO content dropped following exposure, but tissue CO content did not follow the same trend in all tissues. This study supports the hypothesis of (1) tissue and (2) time-related variability of CO concentration in three body tissues after exposure and (3) documents lack of utility of COHb for predicting tissue CO tissue values. / Dissertation (MSc)--University of Pretoria, 2007. / Nuclear Medicine / Unrestricted

Aluminium is the third most abundant element in the lithosphere and yet no biological function has been elucidated. The ubiquity and pH-dependent chemical speciation of aluminium provides multiple routes of exposure to organisms, inducing neurotoxicity, tissue necrosis and organelle dysfunction. However, many studies of aluminium toxicity lack consideration of the speciation and relevant concentration of aluminium and the route of exposure. The aim of this thesis was to examine the accumulation, distribution, excretion and toxicity of aluminium following a common route of exposure (ingestion) at a concentration likely to be encountered by the model organism (freshwater crayfish, Pacifastacus leniusculus) in the wild. Crayfish are sediment dwelling omnivorous crustaceans distributed worldwide and as such are vulnerable to multiple routes of aluminium exposure. They play a central role in aquatic food webs and are becoming increasingly popular for human consumption, raising concern about food chain transfer. Crayfish were fed aluminium chloride-spiked artificial food pellets for either 20 days, 28 days + 10 day aluminium-free clearance period, or 22 weeks + 4 week aluminium-free clearance period. In addition, systemic administration of aluminium citrate was undertaken to draw comparisons with previous mammalian work and compare the two routes of exposure. Tissue distribution and accumulation was measured in the gills, hepatopancreas, flexor muscle and antennal gland. Stress and tissue damage were analysed using biochemical and histopathological techniques. Behavioural toxicity tests and measurements of the neurophysiological parameters of the crayfish medial giant neuron were used to assess aluminium-induced neurotoxicity. In vitro neurotoxicity tests with aluminium chloride were also carried out on isolated nerve tissue to assess the suitability of in vitro studies. The key site of aluminium accumulation following ingestion was the hepatopancreas. Excretion was observed via the gills, antennal glands (in the urine) and hepatopancreas (for incorporation into the faeces). However, physiological consequences such as tissue damage, inflammation and altered neuronal activity were observed and persisted even after cessation of aluminium ingestion. Consequently there are implication for crayfish fitness and survival, the aquatic food web and human toxicity following ingestion of aluminium.

This work detailed the physical and chemical characterization of oil water mixtures prepared using fresh and weathered Macondo related oils under different conditions of mixing energy/time and in the presence/absence of chemical dispersants. The results indicated that WAFs produced consistent, droplet free solutions for both source and weathered oils with concentration ranges that represented the soluble components of the oil used. Chemically enhanced WAFs prepared with the source oil generated a large amount of micron-size droplets; however the viscosity of the weathered oils were a limiting factor for the preparation of CEWAFs with weathered oils. Droplet size distributions were influenced by the amount of energy in the system and the oil weathering stage, when high energy WAFs were made the increase in weathering of the oil resulted in the formation of smaller droplets that were more stable over time.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.

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.

To calculate the sensitivity and specificity of multifocal electroretinography (mfERG) in detection of chloroquine and hydroxychloroquine retinal toxicity, 120 eyes of 63 patients were evaluated using the currently recommended diagnostic tests. The results were compared to those of 54 eyes of 28 control subjects. The sensitivity and specificity of mfERG relative to the combination of automated visual fields and optical coherence tomography (the reference test) were calculated to be 87% and 86.5% respectively. However, analysis of the “false positive” cases proved that mfERG was more sensitive than the reference test and the actual sensitivity and specificity values were higher than the results of this study. Reduction of mfERG amplitude was a strong and reliable sign of early retinal toxicity and was correlated with the cumulative dose of hydroxychloroquine. This correlation was not observed with the reference test quantitative values.

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.

Drug induced liver injury is a leading cause of human illness and a major cause of drug withdrawals from the market. A systems biology approach has the potential to aid toxicology research since toxicological responses are a consequence of multiple non-linear and interdependent biological responses. Here such an approach is developed.The glutathione pathway is a key hepatic defence mechanism and deactivates reactive metabolites before they have the chance to damage cellular proteins. However, glutathione availability is limited and can vary between individuals. As hepatic glutathione levels cannot be measured directly, two serum-based biomarkers, i.e. 5-oxoproline and ophthalmic acid, have been proposed in literature as a means of tracking glutathione depletion. This thesis aims to test the reliability of the correlation between biomarker concentration and decreasing glutathione concentration.In this study a spiral between experiments, model predictions and falsifications, model improvement, and experimental design is described. Using this approach a kinetic model of the hepatic glutathione pathway and biomarker metabolism was constructed and subsequently expanded by adding physiologically based pharmacokinetic (PBPK) models of paracetamol and the proposed biomarkers. These models have increased the understanding of the glutathione pathway. For example, the model predicted that Glutamyl-Cysteine Synthetase induction should be a highly effective way to increase the robustness of the liver to a paracetamol challenge.In addition, it was possible to qualify with increasing precision, the correlation between biomarkers and hepatic glutathione depletion. 5-Oxoproline and ophthalmic acid provide different information about the status of the glutathione pathway. 5-Oxoproline is correlated with paracetamol-glutathione conjugate formation, but not with extreme toxicity. Ophthalmic acid is a biomarker of a more advanced stage of toxicity, where the cell is unable to protect against glutathione depletion. However, care must be taken when inferring hepatic glutathione concentration. Both models demonstrate that the sensitivity of biomarkers to exposure of paracetamol, depends on the dynamics of exposure as well as on the concentrations of intracellular metabolites, such as methionine.I discuss how the methodology of biomarker assessment could be personalised with regards to individual patients and how systems toxicology could be further developed towards reliable tools for the pharmaceutical industry.

A five-year study was carried out to test a sensitive, nondestructive, new method for determining sublethal and long-term consequences of fenitrothion applications on forest songbirds. I used censuses and territory mapping of singing males to provide indices of relative abundance on sprayed and control plots. Mist-netting was used to colour-band individuals of 3 indicator species (the chestnut-sided warbler, magnolia warbler and white-throated sparrow), to determine "their breeding condition, and to follow their fates after a fenitrothion application. Time-budget observations were made of the behaviour of some marked individuals. Fenitrothion was applied aerially in 1985, and by ground in 1986. I first studied the effect of fenitrothion on the songbird community; comparing treatment and control plots, before and after the application. Data on the bird populations showed that fewer young were caught in 1985, and birds returned at a lower rate the year following treatment; a lower proportion of the 1986 catch in mist nets were males. Nonetheless, other factors (budworm cycles, for example) had a greater influence on population trends than the treatment. Behavioural observations indicated that individuals of the 3 indicator species did not abandon the treated area after either application, although white-throated sparrows and magnolia warblers moved away from foliage regions with higher deposits of spray. There was no significant change in the allotment of time to social, maintenance, or feeding behaviours, for any of the indicator species. This observation is not consistent with the known symptoms of organophosphate poisoning (i.e. increased time spent sitting, bill-wiping, and preening). Chestnut-sided warblers and white-throated sparrows continued to forage actively on sprayed plots or nearby. Magnolia warblers decreased their foraging effort after both applications. In part 2 of the thesis, I studied the effect of fenitrothion on the invertebrate food of forest songbirds. Branch samples were taken as a measure of the food available to foliage-gleaning birds. Drop trays were placed under sample trees to measure the amount of food fallen from the tree after the application. Following treatment, there was a lower density of invertebrates on white birch and balsam fir branches than on control trees. This reduction was not observed until 5 days after the application, although drop tray samples indicated an immediate kill of invertebrates. These techniques sampled different types of invertebrates such that a large proportion of the drop tray samples were not associated with the tree (eg. flying Diptera). In addition to a decreased abundance, a large portion of the remaining invertebrates on white birch treated trees were dead. This suggests a further decrease in food availability to birds like warblers that specialize on moving prey. The depression of food available to birds was probably short-lived, assuming movement of invertebrates into depopulated areas from unsprayed foliage nearby. Behavioural responses of birds to spraying were also short-lived, apparent only within 5 days of the application. At the maximum allowable single dose, fenitrothion appears to have little effect on forest songbirds, even when tested with a highly-sensitive method. / Science, Faculty of / Zoology, Department of / Graduate