Since the discovery of mycotoxins in food, the thrust of biochemical and toxicological research has been carried out on animals which has proven to be uncoordinated and not easily extrapolated to humans. Over the last decade, there have been increasing pressures to review and reduce the use of animals in experimental toxicological studies. Consequently in this study aflatoxin B1 (AFB1), zearalenone (Zea) and their epoxide derivatives have been evaluated using in vitro assays. The HepG2, A549 and Hela cell lines were used for assessing the cytotoxicity, effects on cellular metabolism and sites of action of AFB1, Zea and their derivatives. The cytotoxicity of these mycotoxins was evaluated using the methylthiazol tetrazolium (MTT) reduction assay. Cells, treated with mycotoxins were prepared for transmission electron mlcroscopy (TEM), immunocytochemistry (ICC), scanning electron microscopy (SEM), confocal and light microscopy. From the cytotoxicity assay it was found that the epoxide derivatives were more toxic than the parent toxin when exposed to HepG2 cells with no significant differences in toxicity levels in A549 and Hela treated cells. Both epoxide derivatives displayed a regression of hepatoma cell proliferation at high doses (25ug/ml) while lower concentrations (<12.5ug/ml) enhanced cell growth. Microscopy analyses showed distinct cellular alterations. When exposed to AFB1 (12.5ug/ml) hepatoma cells showed prominent ultrastructural alterations such as areas of cytoplasmic lysis and increased numbers of secondary lysosomes while cells exposed to Zea (l2.5ug/ml) displayed numerous ovoid mitochondria and proliferation of rough endoplasmic reticulum which is indicative of enhanced protein synthesis. The presence of label in toxin treated cells is suggestive of the effects of these mycotoxins. Such cellular changes may lead to altered metabolism and cell function. / Thesis (M.Med.)-University of Natal, Durban, 1996.

Exposure to methyl mercury (MeHg) is a global concern. Increased chronic exposure to MeHg among fish and marine mammal consuming populations will increase the risk of prenatal exposure and as a result, the risk of infant brain damage and neurotoxcity. It is therefore important to understand the role of environmental factors, such as nutrition, in determining susceptibility to MeHg toxicity. Three nutrients (selenium (Se), vitamin C and vitamin E) were selected for examination of their interactions with the mechanisms of McHg cytotoxicity in vitro. Two hybrid neural cell lines (M03.13 and NSC-34) were evaluated for their usefulness in the study of MeHg cytotoxicity. Sixteen toxic endpoints were selected for investigation of growth, viability, structure and biochemistry. Both cell lines responded to MeHg exposure in a dose dependent manner for the majority of endpoints suggesting that both MO3.13 and NSC-34 cells undergo structural and biochemical changes during exposure to McHg, but that MO3.13 cells are more sensitive to DNA, mitochondria) membrane damage and glutathione (GSH) depletion and that NSC-34 cells are more sensitive to protein damage and apoptosis. Se exposure lessened the MeHg-induced decrease in DNA and GSH concentrations in both cell lines. In NSC-34 cells, Se also increased F-actin concentrations and prevented an increase in caspase-3 activity. Se may alter the mechanism of cell death by preventing McHg disruption of DNA replication thus maintaining the production and function of peptides (GSH) and protein (polymerized actin) that aid in MeHg detoxification and neural function. In NSC-34 cells, vitamin C prevented the induction of caspase-3 activity and lessened DNA damage and GSH depletion. Vitamin E lessened GSH depletion and lessened G-actin depletion. Both vitamin C and E improved GSH status, but vitamin C also delayed McHg damage of DNA and prevented early signs of apoptosis suggesting these two vitamins interfere with MeHg metabolism by diffe

Exposure to air contaminants is significantly associated with both short-term and long-term health effects. However, the precise mechanisms that derive such effects are not always understood. While an extensive background database from in vivo toxicological studies have been developed, most toxicity data is from oral and dermal chemical exposures rather than inhalation exposure. There is a need to explore new alternative approaches to provide toxicity information particularly on this technically demanding area. This research explores the potential of in vitro methods for toxicity assessment of workplace air contaminants. A tiered approach for in vitro toxicity testing of workplace contaminants was designed in which appropriate air sampling and exposure techniques were developed. A diversified battery of in vitro assays including the MTS (tetrazolium salt, Promega), NRU (neutral red uptake, Sigma) and ATP (adenosine triphosphate, Promega) and a multiple human cell system including: A549- lung derived cells; HepG2-liver derived cells, and skin fibroblasts were used. Primarily the application and merits of in vitro methods for prediction of toxicity of selected workplace contaminants including Ammonium hydroxide, Cadmium chloride, Cobalt chloride, Formaldehyde, Glutaraldehyde, Manganese chloride, Mercuric chloride, Sodium dichromate, Sulphureous acid and Zinc chloride was confirmed. To study the toxicity of airborne contaminants an indirect exposure method was established using air sampling techniques followed by static and dynamic direct exposure methods by culturing cells on porous membranes to reveal representative data relating to human airborne exposures. The static method enabled the measurement of an airborne IC50 (50% inhibitory concentration) value for selected volatile organic compounds (VOCs) including: Xylene (IC50 = 5,350-8,200 ppm) and Toluene (IC50 = 10,500- 16,600 ppm) after 1 hr exposure. By implementing the dynamic method, airborne IC50 values were calculated for gaseous contaminants including: NO2 (IC50 = 11 ?? 3.54 ppm; NRU), SO2 (IC50 = 48 ?? 2.83 ppm; ATP) and NH3 (IC50 = 199 ?? 1.41 ppm; MTS). A higher sensitivity of in vitro methods was observed compared to in vivo published data. A range of in vitro bioassays in conjunction with exposure techniques developed in this thesis may provide an advanced technology for a comprehensive risk assessment of workplace air contaminants.

Bibliography: leaves 116-138 / [14], 138 leaves, 5 leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical & Experimental Pharmacology, 1988

Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.

No description available.

This study examined the effects of low to moderate doses of manganese (0, 250, or 750 _g per day from PD 1-21) on a comprehensive battery of behaviors in rats during the neonatal period, preweanling period, and in adulthood.

In vitro models are often used both to characterize and test therapeutics for neurodevelopmental disorders (‘NDDs’). While in vitro models have extraordinary potential to develop therapies for patients, they have historically been confounded by absence of robust phenotypes and/or in vitro phenotypes that fail to translate from laboratory bench to bedside. Within this thesis work, we attempt to address three areas in which in vitro models may be improved – gene selection, model validation and identification of disease-relevant functional assays suited for therapeutic testing. Publicly available databases aggregating identified and annotated disease-causing variants for Mendelian diseases have rapidly expanded over the past two decades. Elucidating mechanisms of disease and developing therapies using in vivo model systems often is both time and cost intensive. Thus, determining which subsets of genes are more likely to generate addressable signals in a dish may lead to more effective drug development. In chapter 1, we identify a set of genes ideally suited for therapeutic inhibition. Specifically, we leverage the aforementioned large genetic databases to identify a set of genes likely to act through a gain-of-function mechanism that are both tolerant to loss-of-function mutations and in the druggable genome. In chapter 2, we aim to characterize the degree of conservation of transcriptomic dysregulation between a human in vitro cortical organoid (‘hCOs’) model, and two mouse models of a severe neurodevelopmental disorder resulting from HNRNPU deficiency. Human model systems may improve upon animal models when human pathogenesis and patient phenotypes are divergent from animal models due to species-specific etiology. However, human model systems often lack the heterogeneity and cell-type specificity and maturity seen in primary fetal samples. Importantly, some mouse models of HNRNPU deficiency have muted phenotypes compared with human patients. We hypothesized that while there are distinctions between humans and mice with HNRNPU deficiency, there will be overlap in transcriptomic dysregulation between human and mouse models. In fact, we find 45-day-old HNRNPU+/- hCOs have consistent transcriptomic dysregulation to embryonic mouse models, but not to perinatal mice. Our findings suggest hCOs are a viable model for characterizing HNRNPU deficiency; however, such models may only be appropriate for elucidating a transcriptomic disease state at a specific developmental time period. Functional assays for neurodevelopmental disorders can aid in understanding whether transcriptomic dysregulation is relevant to patient symptoms, as genomic findings may not always correlate to disease-relevant phenotypes. Further, relevant functional phenotypes can then be utilized for testing potential therapeutics. Importantly, seizures are commonly present in a significant subset of neurodevelopmental disorders and seizure phenotypes have been described as driven by aberrant synchrony in neuronal networks. Using a multielectrode array platform, investigators can use a variety of computational methods to quantify aspects of synchrony in vitro. In chapter 3a, we introduce topological approaches capable of identifying novel synchrony phenotypes in primary neuronal networks from mouse models of neurodevelopmental disorders. Certain mouse models will be confounded by species-specific pathogenesis and/or vastly different developmental timelines and fail to generalize to human patients, motivating the need for functionally active and physiologically relevant human in vitro models. In chapter 3b, we attempt to generate human networks with balanced levels of excitation and inhibition and find confounding lack of functional maturation of inhibitory neuronal subtypes in 90-day-old stem cell-derived neuronal networks. Future work generating in vitro human neuronal networks with functionally mature inhibitory neurons would complement the findings in chapters 1 and 2 and allow for more efficient therapeutic development strategies that may lead to improved patient outcomes.

L'objectif de cette thèse est d'améliorer la compréhension de la toxicité de diverses nanoparticules de synthèse (ENPs) pour l'homme et l'écosystème. Les travaux réalisés s’appuient sur la combinaison de données toxicologiques et d’un modèle environnemental - le modèle USEtox. En tant qu'élément important de l'évaluation de l'impact du cycle de vie, le facteur de caractérisation (CF) a été utilisé, dans ce travail, comme indicateur de toxicité pour l'homme et l'écosystème. Pour avoir accès aux courbes dose-réponse et à différentes données toxicologiques, des expériences in vitro ont été réalisées en exposant des neutrophiles porcins fraîchement isolés à trois types de nanoparticules de synthèse. Les modifications morphologiques, les taux de mortalité et la chimioluminescence des neutrophiles ont été évaluées. De plus, pour estimer le temps de persistance des nanoparticules de synthèse dans l'écosystème eau douce, un modèle basé sur la science des colloïdes a été développé. Il prend en compte les comportements spécifiques des nanoparticules de synthèse et inclut des recommandations sur le choix des paramètres hydrologiques régionaux. Enfin, une enquête documentaire exhaustive a été réalisée pour recueillir les données écotoxicologiques de diverses nanoparticules de synthèse. Dans le cadre du modèle USEtox, le CF toxicologique non cancérogène pour cuivre NPs et les CF écotoxicologiques pour 14 ENPs sont recommandés. Ces valeurs des CF pourraient être utiles à l'avenir pour évaluer les impacts environnementaux des produits contenant des ENPs. / The objective of this thesis is to improve understandings of toxicity of various engineered nanoparticles (ENPs) to human and ecosystem. It is realized via coordinating toxicological data and a scientific consensus environmental model -- the USEtox model. As an important element in life cycle impact assessment, the characterization factor (CF) is employed as a toxicity indicator for human and ecosystem in this work. To obtain the firsthand dose-response phenomena and human toxicological data, in vitro experiments have been conducted by exposing freshly isolated porcine neutrophils to three kinds of ENPs (i.e. copper, nickel and aluminum oxide nanoparticles). The morphologies, mortality rates, and chemiluminescence, of neutrophils are observed or monitored. Additionally, to estimate the persistence time of ENPs in freshwater ecosystem, a fate model on the basis of colloid science is developed. It takes nano-specific behaviors of ENPs into account and includes recommendations of regionalized hydrological parameters. Finally, a comprehensive literature survey is accomplished to collect the ecotoxicological data of various ENPs. Under the framework of USEtox model, the non-carcinogenic human toxicological CFs for Copper NPs and the ecotoxicological CFs for 14 ENPs are recommended. These CF values could be useful in the future when evaluating the environmental impacts of products containing ENPs.

Lau Wing-Ngar Vivian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 135-144). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Roadside air pollution worldwide and in Hong Kong --- p.2 / Chapter 1.2.1 --- Air quality in Hong Kong --- p.3 / Chapter 1.3 --- Characteristics of particulate matter --- p.9 / Chapter 1.4 --- Composition and sources of particulate matter --- p.11 / Chapter 1.5 --- Toxic effects of particulate matter --- p.12 / Chapter 1.5.1 --- Lung injury --- p.12 / Chapter 1.5.2 --- Cardiovascular injury --- p.15 / Chapter 1.5.3 --- Mutagenesis and carcinogenesis --- p.16 / Chapter 1.6 --- Aims of my study --- p.16 / Chapter 2 --- Toxic Effects of Heavy Metals Ions on Selected Cultured Cell-lines --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1 --- Metals --- p.18 / Chapter 2.1.1.1 --- Cadmium --- p.22 / Chapter 2.1.1.2 --- Chromium --- p.23 / Chapter 2.1.1.3 --- Lead --- p.25 / Chapter 2.1.1.4 --- Zinc --- p.26 / Chapter 2.1.2 --- Metallothioneins --- p.28 / Chapter 2.1.3 --- p53 --- p.31 / Chapter 2.1.4 --- Tumor Necrosis Factor-alpha (TNF-α) --- p.32 / Chapter 2.1.5 --- Aims of this chapter --- p.32 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- Reagents --- p.35 / Chapter 2.2.2 --- Cultured Cell lines --- p.35 / Chapter 2.2.2.1 --- PU5-18 --- p.36 / Chapter 2.2.2.2 --- LL24 --- p.36 / Chapter 2.2.2.3 --- HBE4-E6/E7 --- p.37 / Chapter 2.2.3 --- Cytotoxicity assays --- p.37 / Chapter 2.2.4 --- ELISA assays --- p.40 / Chapter 2.2.4.1 --- ELISA assay ofp53 levels --- p.41 / Chapter 2.2.4.2 --- ELISA assay of TNF-α levels --- p.43 / Chapter 2.2.5 --- MT gene expression studies by Luciferase assay --- p.44 / Chapter 2.2.5.1 --- PCR amplification --- p.44 / Chapter 2.2.5.2 --- 5´ة End modification of PCR amplified DNA --- p.44 / Chapter 2.2.5.3 --- Ligation of DNA fragment to linearized vector --- p.46 / Chapter 2.2.5.4 --- E. coli. transformation by heat shock --- p.46 / Chapter 2.2.5.5 --- PCR sequencing --- p.47 / Chapter 2.2.5.6 --- Transfection of plasmid into HBE4-E6/E7 cells --- p.49 / Chapter 2.2.5.7 --- Data analysis --- p.50 / Chapter 2.3 --- Results and discussion --- p.51 / Chapter 2.3.1 --- Cytotoxicity assays --- p.51 / Chapter 2.3.2 --- Combination effects of metals on cytotoxicity --- p.61 / Chapter 2.3.3 --- p53 --- p.65 / Chapter 2.3.4 --- TNF-α --- p.68 / Chapter 2.3.5 --- MT gene expression studies by Luciferase assay --- p.69 / Chapter 2.4 --- Conclusion --- p.74 / Chapter 3 --- Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Cultured Cell-lines --- p.75 / Chapter 3.1 --- Introduction --- p.75 / Chapter 3.2 --- Materials and methods --- p.79 / Chapter 3.2.1 --- Reagents --- p.79 / Chapter 3.2.2 --- Cell culture --- p.79 / Chapter 3.2.3 --- AlamarBlue assay --- p.80 / Chapter 3.2.4 --- EROD assay --- p.80 / Chapter 3.3 --- Results and discussion --- p.84 / Chapter 3.4 --- Conclusion --- p.88 / Chapter 4 --- Chemical and Biological Assays on Roadside Dust --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.1.1 --- Composition of particulate matter in Hong Kong --- p.89 / Chapter 4.1.2 --- Metal contents of particulate matter in Hong Kong --- p.91 / Chapter 4.1.3 --- Possible adverse health impacts of particulate matter --- p.94 / Chapter 4.1.3.1 --- In vitro studies using different cell models --- p.94 / Chapter 4.1.3.2 --- In vivo studies using rodents --- p.97 / Chapter 4.1.3.3 --- Epidemiological studies --- p.98 / Chapter 4.1.4 --- Aims of this chapter --- p.100 / Chapter 4.2 --- Materials and methods --- p.101 / Chapter 4.2.1 --- Sampling of roadside dust --- p.101 / Chapter 4.2.2 --- Chemical analysis of roadside dust --- p.104 / Chapter 4.2.2.1 --- Reagents --- p.104 / Chapter 4.2.2.2 --- Total metal contents --- p.105 / Chapter 4.2.2.3 --- Extractable metal contents --- p.105 / Chapter 4.2.3 --- Biological assays --- p.105 / Chapter 4.2.3.1 --- Cell models --- p.106 / Chapter 4.2.3.2 --- Pretreatment of roadside dust --- p.106 / Chapter 4.2.3.3 --- AlamarBlue assay --- p.106 / Chapter 4.2.3.4 --- ELISA assays --- p.108 / Chapter 4.2.3.5 --- Luciferase assay --- p.108 / Chapter 4.3 --- Results and discussion --- p.110 / Chapter 4.3.1 --- Total metal contents --- p.110 / Chapter 4.3.2 --- Extractable metal contents --- p.113 / Chapter 4.3.3 --- AlamarBlue assay --- p.116 / Chapter 4.3.4 --- p53 --- p.122 / Chapter 4.3.5 --- TNF-α --- p.122 / Chapter 4.3.6 --- Luciferase assay --- p.126 / Chapter 4.4 --- Conclusion --- p.129 / Chapter 5 --- General discussion and conclusion --- p.130 / Chapter 6 --- References --- p.135