Incorporation of nanoparticles (NPs) into consumer products is on the rise and human exposure to NPs is unavoidable. Currently, there is insufficient data to assess the safety of nanoparticles. I conducted a series of five studies using the zebrafish model to determine which NP components (i.e., core material or surface functionalization) contribute to biological responses and how ionic strength influences these results. The first study employed a systematic, rapid embryonic zebrafish assay to identify specific responses to precisely engineered lead sulfide (PbS-NPs) and gold nanoparticles (AuNPs) functionalized with different surface ligands. Lead sulfide nanoparticles functionalized with either 3-mercaptopropanesulfane (MT) or sodium 2,3-dimercaptopropanesulfonate (DT) ligands with nearly identical core sizes caused differential responses at the same concentration. I determined that the different responses were because MT-functionalized NPs released more soluble lead ions than DT-functionalized NPs due to different decomposition and oxidation rates. The second study investigated the different biological responses of three NPs identified during toxicity screening of a gold nanoparticle library. AuNPs functionalized with 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (MEEE), induced differential biological responses in embryonic zebrafish at the same concentration. Exposure to MES-AuNPs induced sublethal effects, while TMAT-AuNPs were embryo-lethal and MEEE-AuNPs were benign. Gold tissue concentration was confirmed to be similar in exposed embryos using inductively coupled-mass spectrometry. Microarrays were used to gain insight to the causes of the different responses. This approach identified that MES- and TMAT-AuNPs perturbed inflammatory and immune responses. These differential biological responses may be due to misregulated transport mechanisms causing numerous downstream defects unique to each surface functional group‟s property. In the next study, I tested the long-term consequences of developmental exposure to TMAT-, MES, and MEEE-AuNPs, and showed that MES- and TMAT-AuNPs affected larval behavior that persisted into adulthood. During the course of these investigations, I found that high ion concentration in exposure solutions results in NP agglomeration, presenting a problem for NP testing in the zebrafish model. For the fourth study, I focused on solving this by determining that zebrafish can be raised in nearly ion-free media without adverse consequences. When 3-MPA-AuNPs were dispersed in this new low ionic media, I observed adverse responses in the embryonic zebrafish toxicity assay, but not when the NPs were suspended in high ionic media. Thus, I demonstrated that the media greatly influences both agglomeration rates and biological responses, but most importantly, that the zebrafish is insensitive to external ions. The fifth study focused on the adverse response observed when embryonic zebrafish were exposed to 3-MPA-AuNPs. Exposed larvae failed to respond to a touch in the caudal fin at 120 hours post fertilization (hpf). Addition of a neuromuscular stimulus, nicotine, revealed the exposed embryos were not paralyzed, but experienced a reduction in axonal projections. A global genomic analysis (RNA-seq) using embryos exposed to 3-MPA-AuNP and MEEE-AuNPs (non-toxic control) from 6 to 120 hpf suggested that neurophysiological and signal transduction processes were perturbed. Functional analysis of the data led to the hypothesis that the most elevated gene, early growth response 1 (EGR-1), impacts axonogenesis in the caudal fin, interfering with glutaminergic synapses and preventing the connection of sensory neurons and touch perception. Although MEEE-AuNPs did not cause morphological defects, the RNA-seq analysis identified that these NPs perturbed immune and inflammatory system processes. Collectively, these results suggest that surface functional groups drive the differential responses to nanomaterials. The five studies summarized here confirm that a systems toxicological approach using the zebrafish model enables the rapid identification of structure-activity relationships, which will facilitate the design of safer nano-containing products. / Graduation date: 2013

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

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.

Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1), potentially cytotoxic and carcinogenic mycotoxins are common contaminants of agricultural commodities in South Africa and thus could be detrimental to the human immune system. Many of the cytotoxic effects of AFB1 require its bioactivation to an epoxide, which will bind covalently to macromolecules to form protein and DNA adducts. Fumonisin B1 is a competitive inhibitor of sphingosine and sphinganine N aceyltransferase, which are key components in the pathways for sphingolipid biosynthesis. Accumulation of free sphingoid bases, which are both cytotoxic and mitogenic, could provide a plausible explanation for the toxicity and carcinogenicity of FB1. The cytotoxic effects of AFB1 and FB1 on normal human lymphocytes, individually and in combination were assessed using the methylthiazol tetrazolium (MTT) bioassay. Two different methods of treatment were used, the treatment of isolated normal human lymphocytes for 12, 24, 48, 72 and 96 hours and whole blood treated for 12 hours. Flow cytometry and fluorescent microscopy were used to determine whether AFB1 and FB1 (5uM and 50uM), individually or in combination, were capable of inducing apoptosis, necrosis or nuclear fragmentation in isolated lymphocytes and whole blood treated for 12 hours. DNA damage was evaluated using the comet assay. The results showed that AFB1routinely induced higher levels of cytotoxicity in isolated lymphocytes than FB1. In the combination treatment, the mitogenic properties of FB1 appeared to partially counteract the cytotoxic effect exerted by AFB1. When whole blood was treated with the same concentration and ratio of toxin, FB1 was shown to be more cytotoxic than AFB1. The combination treatment of whole blood was shown to be cytotoxic in a dose dependent manner. The toxins appeared to exert a greater cytotoxic effect, when treated in combination than individually at higher concentrations. Aflatoxin B1 induced increased levels of apoptosis and necrosis in isolated lymphocytes while treatment with the FB1 resulted in increased levels of apoptosis at both concentrations. Treatment with the combination also resulted in increased levels of apoptosis. The levels of apoptosis were reduced in whole blood lymphocytes when compared to isolated lymphocytes. However, treatment with AFB1 and FB1 resulted in increased levels of apoptosis. Both AFB1 and FB1 are capable of inducing nuclear fragmentation. Treatment with FB1 (5uM and 50uM) resulted in greater degree of fragmentation than AFB1. The most nuclear fragmentation was induced by the 5uM combination treatment. The 50uM combination treatment of isolated lymphocytes induced the most DNA damage. As both toxins are common contaminants and have been known to coexist, this could be a potential area of concern for public health. / Thesis (M.Med.)-University of KwaZulu-Natal, 2004.

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

The objective of this thesis was to construct general models to predict pollutant fluxes in limnoplankton by incorporating characteristics of the organism and the structures of the chemical. A two-compartmental pharmacokinetic model was used to quantify the pollutant uptake, depuration and intercompartmental exchanges. The model pollutants were phosphorus and 22 organic chemicals. / The rate constants of phosphorus uptake, excretion and intercompartmental changes by algae and cladocerans decreased with cell volume or body size raised to a power close to $-$0.25, except the intercompartmental exchanges for cladocerans which showed more negative slopes. In contrast, uptake, excretion and internal exchange rates per individual increased with cell size or body weight to a power similar to 0.75 with a similar exception for the cladoceran intercompartmental exchanges, which had slopes $<$0.75. / Bioconcentration factors, rate constants and flux rates of uptake and intercompartmental exchange from metabolic pool to structural pool of 22 $ sp{14}$C-labelled organic toxicants by Chlorella pyrenoidosa and Daphnia magna were positively correlated with the octanol/water partition coefficient, molecular weight, parachor, connectivity index, boiling point and melting point, and negatively with aqueous solubility. However, those of elimination and internal transfer from structural pool to metabolic pool showed opposite changes. Comparisons of pharmacokinetic parameters between Daphnia and Chlorella demonstrated that, although all kinetic parameters displayed similar patterns, the relative magnitudes of each corresponding parameters were significantly different between two species.

The Port of Durban, with its close proximity to industrial, urban and agricultural activities, receives a number of chemical pollutants that settle out and accumulate in sediments. Chemical analysis of these sediments has indicated elevated levels of chemicals that, according to sediment quality guidelines, might cause adverse biological effects. However, elevated concentrations alone do not necessarily imply that chemicals are present in bioavailable concentrations high enough to be harmful to organisms that come into contact with them. Thus, chemical tests alone cannot provide an accurate indication of the potential adverse biological effects of these chemicals. In this regard, toxicity tests of sediment porewaters have been developed using sea urchin gametes to assist in determining the bioavailability of chemicals present in porewaters. Further, procedures such as Toxicity Identification and Evaluation (TIE), which involves the manipulation and/or treatment of toxic porewater, have also been developed to assist in the isolation and identification of chemicals causing porewater toxicity. In this research, on a number of sampling occasions between July 2007 and July 2009, three replicate sediment samples were extracted from a site in the Port of Durban known to contain sediment with potentially toxic porewater. Results of initial toxicity tests, using the sea urchin fertilisation test indicated the presence of toxic porewater although, in some instances, porewater toxicity was highly variable between replicate samples. However, results from TIE procedures performed to reduce potentially toxic concentrations of metals, ammonia and organic compounds did not resolve the primary cause of porewater toxicity. Further research indicated that chemicals including hydrogen sulphide, which can occur naturally in organically enriched sediments, may have been confounding factors that masked the potential toxicity of other chemicals present in the sediment samples. Consequently, a sampling strategy and modified TIE procedure have been recommended. The sampling strategy has been designed to assist with detecting and understanding any sample variability that may occur. The modified TIE procedure, which suggests initial procedures to determine and reduce/remove the possible confounding effects of potential naturally occurring compounds such as hydrogen sulphide from the porewater, could be used in future to understand and evaluate the quality of contaminated sediments from similar environments. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Microorganisms are increasingly used in toxicological studies to determine potential environmental impacts of xenobiotic compounds. A multi-stage laboratory model was developed to facilitate the examination of environmental impacts of selected pollutants on fundamental cycling processes inherent to aquatic ecosystems, namely, the degradation of organic substances and nitrogen transformations under aerobic conditions. A microbial association representative of riverine ecosystems was enriched for, isolated and cultured within the model. Characterisation of the microbial association were undertaken. Scanning electron microscopy and bright field microscopy revealed that a diverse heterogenous community of microorganisms had established within the model. Successional metabolic events, namely organic carbon catabolism, ammonification of organic nitrogen and the process of nitrification were differentiated in time and space with the microbial association integrity still being retained. The establishment of a microbial association within the model was primarily dependent on: dilution rates, specific growth rates and interactions between microorganisms and the prevailing environmental conditions. Growth-rate independent populations of microorganisms established within the model and were thought to contribute significantly to the metabolic processes within the model. Nitrifying activity was identified as a rate-limiting process within the model. Following separation of metabolic events, the ecotoxicological impacts of phenol and 2,4-dichlorophenol on the association were assessed. The biological oxidation of ammonia through to nitrate (nitrification) was found to be a sensitive indicator of perturbation. The model was found to be suitable for testing both acute and chronic intoxication by pollutant compounds as well as for biodegradation testing and the possible evaluation of ecotoxicological impacts of wastewater treatment plants. The main disadvantages of the model arose from its operational complexity, its empirical nature and its impracticality for screening large numbers of compounds. A bioassay based on the inhibition of ammonium oxidation was developed in order to fulfil the requirements for a simple and rapid test protocol for the initial screening of perturbant compounds. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Ethnopharmacological relevance: Indigenous plants such as Tulbaghia violacea(TV) and Allium sativum (garlic) are traditionally used as natural remedies to treat a variety of ailments, including cancer. This study investigated the effects of TV leaf and bulb extracts and garlic extract on a cancerous oesophageal cell line (SNO). Materials and methods: The methylthiazoltetrazolium (MTT) assay was used to determine the IC50 of TV leaf (TVL) (250μg/ml) and TV bulb extracts (TVB) (25μg/ml) and garlic (500μg/ml). Extracts were treated individually and in combination for a period of 24 hours. Oxidative damage and intracellular glutathione levels were assessed using the Thiobarbituric Acid Reactive Substances (TBARS) Assay and GSH-Glo™ Luminometry Assay, respectively. The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess ATP activity. Induction of apoptosis and mitochondrial membrane potential were determined via the Caspase-Glo® 3/7 Assay, Caspase-Glo® 8 Assay, Caspase-Glo® 9 Assay and JC-1 Mitoscreen Assay, respectively. Morphological apoptotic changes were determined using the Hoechst 33342 stain. Expressions of p53, PARP and NFKB activities were determined by western blotting. Results: Bulb and leaf extracts of TV increased lipid peroxidation compared to the control (p>0.05), whilst garlic and combination of TV leaf and bulb (TVB + TVL) extracts significantly decreased lipid peroxidation relative to the control (p< 0.05). Endogenous glutathione levels significantly decreased in all TV treatments compared to the control (p<0.05).However, garlic was accompanied by insignificantly increased intracellular glutathione levels compared to the control (p> 0.05). The percentages of depolarised mitochondria in all treated cells were significantly decreased compared to untreated cells (p< 0.05). ATP levels increased significantly in garlic and combination (TVB + TVL) treated cells as compared to the control (p< 0.05), yet no significant differences were noted in TVL and TVB treatments (p> 0.05). Caspase8 and caspase 9 activities significantly increased in garlic and combination treated cells relative to the control (p<0.05). A similar trend was noted for caspase 3/7 activity in garlic and combination treatments (p< 0.05). However, initiator and executioner activities in TVL (p> 0.05) and TVB (p> 0.05) treatments did not significantly differ from the control (p> 0.05). All treatments (including garlic) resulted in increased DNA fragmentation and condensation. All treatments decreased p53 expression (p< 0.05), PARP expression (p< 0.05) and NFK B expression (p>0.05) compared to the control. Conclusions: All TV extracts and garlic induces apoptosis in the oesophageal cancerous SNO cell line through changes in oxidative stress, antioxidant systems, and nuclear chromatin condensation, as well as through induction of nuclear genes and signalling pathways. Since inhibition of apoptosis is a principal alteration in cancer, induction of apoptosis would result in a decrease in cancer cell growth. Thus, TV could be exploited as a potential anti-cancer agent. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2012.

The current work determined whether there is a constant tissue residue associated with narcotic compounds. In this investigation, the cladoceran, Daphnia magna was exposed to lethal levels (48h LC50) of ten, $ sp{14}$C-labelled, narcotic organic chemicals in a closed system. Exposure times, ambient concentrations, and body sizes were varied to evaluate their effects. The $ sp{14}$C-method developed in current work can detect chemicals in single D. magna in concentrations ranging from 0.02 to 6310 mmol/kg. Moreover, the technique detected phobic and lipophilic chemicals equally well. The technique's sensitivity (nmol/kg) allowed for detection of differences in the internal concentrations of pollutants among the unaffected, immobilized, and dead D. magna. Immobilized D. magna contained between 0.14 mmol/kg and 200 mmol/kg of narcotics. On the average, however, the internal residues were 3.1 mmol/kg (95%CL = 3.1 $ pm$ 2.0). This agreed with literature values. The effects of time of exposure, ambient concentration, and body size on the tissue residues of narcotics varied with the chemical compound.