The large use of perfluorinated compounds (PFCs) to produce fluoropolymers in consumer and industrial applications, including insecticides, plastics, non-stick surfaces and fire fighting foams has led to a well known widespread occurrence and high concentrations are found in wild life including avian species. For instance, concentrations of perfluorooctane sulfonate (PFOS) in eggs from the common guillemot in the Baltic Sea are among the highest in the Nordic environment. In our laboratory studies, PFOS has caused early mortality in chicken at doses close to concentrations found in eggs of the Baltic guillemot. The mechanisms behind the avian toxicity are unclear but many studies suggest mechanisms including lipid homeostasis. We have designed a method in which hepatic embryonic tissue from chicken (Gallus domesticus) is used to investigate the effects of PFCs on the β-oxidation of fatty acids.  The purpose of this project was to assess the effects of PFOS, perfluorooctanoic acid (PFOA) or perfluorobutane sulfonate (PFBS) on the hepatic fatty acid oxidation using an egg injection technique followed by the use of a tritium release assay with palmitate (16:0) as substrate. The embryos were exposed in ovo and on day 10 of incubation embryo livers are incubated in vitro with tritiated fatty acids. The β-oxidation was significantly induced after exposed to 1 mg/kg PFOS (p = 0.003) and 10 mg/kg PFOS (p = 0.04), and difference in oxidation values was 39% and 34% respectively compared to control. The oxidation effect was not significant (p > 0.05) in samples exposed to PFOA (4 mg/kg) or PFBS (20 mg/kg), however noted, the difference in oxidation values was 18% and 30.5% respectively, compared to control calculated on current average. The results show that in ovo exposure in combination with an in vitro method, using a tritium release assay to detect effects on the β-oxidation of fatty acids in avian embryo hepatic tissue could be a useful method to elucidate possible mechanisms behind avian developmental toxicity.

Non-steroidal anti- inflammatory drugs (NSAIDs) have been in use for over 100 years to treat pain, exerting their analgesic effect by inhibiting prostaglandin (PG) synthesis via the COX pathway. Some of the NSAIDs have adverse side effects including ulceration of the stomach and cardiovascular events which are associated with bleeding. Search is still going on to find a safe NSAID. Two new coxib NSAIDs, namely celecoxib and etoricoxib have been developed and they exert marked beneficial effects in reducing pain in humans and other small animals with little or no side effects. No such study has been done on horses to see if they can tolerate the drug as an analgesic pain killer. This study was designed to investigate the effects of the two coxib NSAIDs, celecoxib and etoricoxib in six retired race horses to determine any adverse side effects of the drugs, the time course changes in their metabolism and elimination once administered orally in known physiological doses and the metabolites produced by each drug over time. The study employed well established clinical and biochemical techniques to measure blood-borne parameters and the metabolism of each drug. The results show that either etoricoxib or celecoxib had no adverse side effects on blood borne parameters and the stomach of the horses. Pharmacokinetic study following oral administration of 2 mg/kg b wt of either celecoxib or etoricoxib to the six race horses showed a Cmax of 1.15 ± 0.3 µg/ml, tmax, to be 4.09 ± 1.60 hr and a terminal half- life of 15.52 ± 1.99hr for celecoxib and a Cmax of 1.0± 0.09 µg/ml, tmax of 0.79 ± 0.1 hr and, terminal half- life of 11.51 ± 1.56 hr, respectively for etoricoxib. The results also show that each coxib is metabolized in the horse and both the parent drug and its metabolites are found in the urine, plasma and faeces. The results have also shown that even small traces of either drug or its metabolites can be measured in urine samples even 120 hours following oral administration. The main metabolites found in plasma, urine and faeces are hydroxyl celecoxib and carboxycelecoxib when celecoxib was administered orally to the 6 retired race horses. Similarly, hydroxymethyletoricoxib, carboxylic etoricoxib, hydroxymethyl-1-N-oxide metabolite of etoricoxib and hydroxymethyletoricoxib glucuronide were also found in plasma, urine and faeces following oral administration etoricoxib .to the animals. The results for either horse haeptocytes or camel liver show to some extend similar metabolites. In conclusion, the results show that both drugs have no adverse side effects in the horse and their metabolites are completely eliminated within 120 hours following oral administration.

This thesis presents a study on possible genetic consequences of the exposure to the space environment during space missions The present study was undertaken in co-operation with the Canadian Space Agency, and involved the analysis of the lymphocyte samples taken from experienced cosmonauts and trainees. For the analysis of genotoxicity of the space environment, a T-lymphocyte hprt clonal assay has been employed. In order to distinguish between artefacts associated with this method and the spaceflight-related effects, we have conducted a series of in vitro reconstruction experiments. In these experiments we have analysed interactions between plating efficiency (PE) of T-lymphocytes and efficiency of mutant recovery. Using 12 pairs of independent wild type (WT) and mutant clones, we have demonstrated an inverse correlation between initial viability of the WT cells and survival of mutant cells (r = 0.3496, p < 0.05). Our data suggest that the presence of WT cells in the selection plates does suppress the recovery of mutants in HPRT assay. This effect is stronger in samples with high PE, and may be a source of large error in estimation of mutant frequencies (approx. 3-fold in the range of PEs from 10% to 60%), which is especially relevant when samples with different PEs are compared. Analysis of samples from cosmonauts was conducted in two experiments. The first experiment involved 5 samples taken in 1992 from cosmonauts who have completed spaceflights ranging in duration from 7 to 365 days. Hprt mutant frequencies (MF) in these samples were 2.5–5 times higher than the age-corrected values for healthy, unexposed subjects in Western countries (Tates et al., 1991; Branda et al., 1993), and 2-3-fold higher than those determined for unexposed individuals residing in Russia (Jones et al., 1995). The cosmonaut mutational spectrum differed from that of unexposed healthy subjects (p = 0.042), and showed a higher incidence of splicing errors, frameshifts, and complex mutations. Distribution of base substitutions was remarkably similar to that observed in Russian twins sampled at the same period (Curry et al., 1998), thus suggestive of possible environmental, diet, or life-style related exposures. The second study was conducted on samples taken 5 years later and involved trainees and a group of cosmonauts with more uniform (at least 6 months) and recent flight experience. Hprt MFs in both cosmonaut and trainee groups were virtually identical (17.2 ± 0.6 and 17.6 ± 4.7 × 10⁻⁶ respectively), and approximately 2-fold higher than in matching Western controls, although considerably lower than in our previous observations. Mutational spectra in both datasets were very similar to that observed in our earlier study, and were significantly different from spontaneous data (p = 0.031–0.038). Distribution of base substitutions, however, did not show any differences. Our data indicate that the space environment is not genotoxic at the hprt locus. At the same time, uniformly high MFs observed in all studied groups suggest that the level of the mutagenic burden in at least megalopolis areas of Russia may be considerably larger than in the West. Also, there are some indications of a possible restructuring of mutagenic burden in post-transitional Russia. / Graduate

M.Sc. (Biochemistry) / Manganese (Mn) is an essential trace element. Although it is vital for the normal development of mammals, too much Mn can be harmful. Most reported cases of toxicity have been found in occupational settings, such as welding, mining and ferro-manganese (FeMn) production plants. Long-term overexposure to Mn can result in lung epithelial necrosis and the development of a neurological disease, manganism. Even though evidence of Mn-associated diseases exists, some epidemiological studies have found no association between occupational exposure levels and possible indicators of neurotoxic effects. It is, therefore, important to establish Mn toxicity and the mechanisms involved in this toxicity, for a possible identification of biomarkers of exposure and effect. The hypothesis formulated states that, FeMn particulate matter consists of nano and micro sized particles that, upon inhalation, may cause injury to the lungs and translocate to the brain. Since Mn-induced injury to the brain and lungs is a possibility, this study aimed to investigate the effects of FeMn dust, which was collected from a FeMn smelter works, on primary rat astrocytes and human bronchial epithelial (BEAS-2B) cells. This was achieved by first characterizing the physicochemical properties of the particles by using a scanning mobility particle sizer (SMPS) and aerodynamic particle sizer (APS) for size distribution, Brunauer-Emmett-Teller (BET) for surface area determination and inductively coupled plasma atomic emission spectroscopy (ICP-AES) for elemental composition analysis. Cells were treated with 5, 10, 25 μg/cm2 FeMn, and particle uptake, by astrocytes and BEAS-2B cells, was confirmed using dark field microscopy e.g. Cytoviva® hyperspectral imaging system. The viability and toxicity of FeMn was studied using the conventional toxicity assay systems, including 3-bis [2-Methoxy-4- nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide salt (XTT), adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) assays. It was, however, established that FeMn particles interfere with the final read-out produced by some of these assay systems. Therefore, a rare application of the xCELLigence real time cell analysis (RTCA) system was implemented, as a better option, in the assessment of the toxicity and viability of cells in the presence of FeMn particles. The ability of FeMn particles to cause deoxyribonucleic acid (DNA) damage in both cell types was also determined using the alkaline comet assay. Finally, the nuclear translocation of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and inflammatory transcription factor nuclear factor kappa B (Nf-κB), was studied using Western blotting. The results showed that FeMn, in a dose dependent manner, could enter the cell, decrease the viability, induce DNA damage, and initiate nuclear transport of the studied transcription factors. The same methodologies were implemented to determine the physicochemical properties of Min- U-Sil 5 crystalline silica, used as a positive control, to assess its toxicity and effect on cellular viability. As well as its ability to induce DNA damage and initiate nuclear translocation of the two transcription factors, in astrocytes and BEAS-2B cells. Similar to FeMn particles, crystalline silica also enters the cells with subsequent reduction in cellular viability. It results in increased DNA damage and increased nuclear translocation of the studied transcription factors. The effects of crystalline silica on these cellular effects were, however, always higher than those produced by FeMn particles. To conclude, these results indicate that depending on the size distribution of particles in the work environment, they may enter different regions of the lungs. However, for those particles in the nano size region, direct access to the brain is a possibility. These results also indicate that after deposition in the target organ, these particles will produce cellular changes through oxidative stress. This would lead to inflammation, decreased cellular viability and increased toxicity.

No description available.

Chemical-induced nephrotoxicity is a major cause of acute kidney injury. My dissertation reveals that all-trans-retinoic acid (ATRA) affords cytoprotection against renal cell injury. Pretreatment with ATRA (25 μM, 24 hr) affords selective cytoprotection against p-aminophenol (PAP), iodoacetamide (IDAM), and 2-(glutathion-S-yl)-hydroquinone-induced necrosis. In contrast, pretreatment of cells with ATRA provides no protection against cisplatin-induced apoptosis. Inhibition of protein synthesis blunts ATRA-mediated cytoprotection, suggesting that critical cell survival signaling pathways are activated prior to toxicant exposure. Oxidative stress is a major contributor to cellular damage. To investigate the mechanism(s) by which ATRA affords cytoprotection, we determined its effects on ROS generation using a DCFDA assay. ATRA did not alter PAP or MGHQ-induced ROS levels. Moreover, ATRA had no effect on GSH levels nor Nrf2 expression, suggesting that other cytoprotective mechanisms are engaged by ATRA. Elevated ROS disrupt endoplasmic reticulum protein folding guided by the molecular chaperone Grp78. During ATRA-mediated pretreatment, the ER stress proteins Grp78 and p-eIF2α were induced (2-fold) in a time-dependent manner (24 and 4 hr respectively). In addition to influencing organelle stress proteins in the ER, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT with maximum levels achieved at 30 min. Moreover, induction of these stress kinases was observed at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Inhibition of p-ERK with PD98059 reduced the ability of ATRA to provide protection against PAP toxicity, implying a role for p-ERK and downstream target genes in the protective effects of ATRA. Gene ontology analysis of a microarray experiment of cells treated with ATRA revealed that ATRA rapidly (0.5, 1 hr) induced growth factors and genes involved in cell proliferation, with subsequent (4, 8, 12 hr) induction of genes involved in ribosome biogenesis, DNA replication and repair, and cell cycle regulation. Complementary data from a cell stress protein array and western blot analyses indicated that ATRA induced HIF1α 3-fold at 8 hr. Furthermore, the microarray data indicated the HIF1α target gene BHLHE40 (which encodes a basic-helix-loop-helix protein involved in cell differentiation) was increased 3-fold. As ATRA induced genes that were associated with cell proliferation, related assays were employed. ATRA had a small effect on cell cycle distribution demonstrated by an increase in the population of cells in the S and G2 phases between 8 and 24 hr. In addition, ATRA markedly increased total DNA content and cell number at 24 hr suggesting that mitogenic/proliferative effects contribute to ATRA cytoprotection. The present studies indicate that a signaling cascade of proteins downstream of p-ERK associated with mitogenesis work cooperatively to afford ATRA protection against renal cell injury. Understanding the mechanism of ATRA-mediated cytoprotection will provide insights into the development of novel therapeutic strategies for renal pathological conditions.

<p> Sigma-2 receptors are a new target in cancer research. These receptors tend to be overexpressed in cancers compared to normal tissue, this expression is also high during proliferation. Fluorescent and radio-labeled sigma-2 receptors show increased uptake in tumors, and this uptake seems to be largely endocytotic. Many sigma-2 receptor ligands demonstrated anticancer properties either as sole treatments or adjunct therapies where they act as chemosensitizers that increase the activity of DNA-targeting drugs. Some sigma-2 receptors ligands have been utilized as cancer-selective, cargo-delivering moieties, showing potent cytotoxicity both in vitro and in vivo with minimal off target toxicity.</p><p> In this research, we synthesize sigma-2 receptor ligands based on previously discovered bicycloheptyl- and cyclohexylamines with high selectivity and affinity towards the sigma-2 receptors. The new novel ligands fall into two categories, fluorescent sigma-2 ligands, and doxorubicin-conjugated ligands. The compounds were synthesized, purified and analyzed in our laboratory. after which they were sent to the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) where they underwent receptor binding studies to determine binding affinities on a panel of a 45 central nervous system receptors. The three synthesized fluorescent compounds displayed varying affinities to sigma-2 receptors ranging from high (32 nM) to medium (557 nM). Absorption and emission spectra for the fluorescent compounds were determined and showed consistency with other compounds in literature containing the same fluorescent moiety (NBD-chloride, i.e. 4-chloro-7-nitrobenzofurazan). Uptake into cancer cells (PANC-1, HT-29, and HCT-116 cells) known to express the sigma-2 receptors was assessed. Results show that higher sigma-2 affinity translates into increased uptake into these cancer cells. The doxorubicin-conjugated ligands showed superior anticancer activity compared to either doxorubicin alone or in combination with another sigma-2 ligand. This effect was significantly reduced by using a pan-caspase inhibitor (Z-VAD-FMK). Furthermore, the higher affinity conjugate (22) (MA-28C) was more active than the low affinity one (25) (MA-30C). These two conjugated ligands where submitted to the NCI-60 Human Tumor Cell Line Screen program. The results showed that in eight out of the nine cell lines known to overexpress sigma-2 receptors, the high affinity conjugate was much more potent than doxorubicin at reaching LC50 (lethal dose to 50% of cells), while both drugs were mostly comparable in their IG50, and TGI concentrations (concentration that reduce growth by 50% and total growth inhibition concentration, respectively).</p><p>

Cyanobacteria are known to produce a variety of toxic compounds. β-N-methylamino-L-alanine (BMAA) is one of the neurotoxins produced by most cyanobacteria. BMAA has been implicated in amyotrophic lateral sclerosis / Parkinsonism dementia complex (ALS / PDC) and was suggested to contribute to this pathology after biomagnification and slow release of BMAA from a protein associated form. The uptake and accumulation of BMAA by the aquatic macrophyte Ceratophyllum demersum has recently been shown, but the consumption of aquatic macrophytes by humans is not typical. The uptake by, and accumulation in, crop plants (Nasturtium officinale and Daucus carota) was therefore investigated so as to establish the existence of any risk to humans from the consumption of plants irrigated with water from dams with high cyanobacterial biomass and therefore high BMAA levels. After the exposure to the BMAA through the growth medium, BMAA had no effect on growth and development of N. officinale and D. carota. The uptake and bioaccumulation of BMAA was observed in N. officinale and D. carota, and was found to be concentration-dependent. Both free and bound cellular BMAA was detected following BMAA exposure through the growth medium. The photosynthetic apparatus of N. officinale was not significantly damaged. The uptake and accumulation of BMAA in edible terrestrial plants may constitute another route of human exposure to BMAA; it may now be prudent to avoid spray irrigation of edible plants with waters from dams with high cyanobacterial biomass and therefore high BMAA levels. After uptake by plants, the cyanotoxins may induce oxidative stress. A recent study showed that BMAA has a significant inhibitory effect on the oxidative stress enzymes in C. demersum. Therefore, the toxicological effects on selected plants were investigated by a range of biochemical enzyme assays in order to establish the plant stress response to exogenous BMAA. The inhibition of antioxidant enzymes upon exposure of N. officinale to BMAA through the growth medium was observed. The inhibition of antioxidant defence enzymes by BMAA correlated with the BMAA bioaccumulation in N. officinale. Further investigations are needed to analyze the uptake, accumulation, and ecotoxicology of BMAA in other crop plants, and to examine the fate of BMAA in these plants particularly its distribution and metabolism.

<p>The intensive drilling and extraction of fossil fuels in the Gulf of Mexico (GoM) result in a considerable risk for oil spills impacting coastal ecosystems in the GoM. Two ecologically-important macroinvertebrate species on the northern GoM coast are the gulf marsh fiddler crab Uca longisignalis and the gulf sand fiddler crab Uca panacea. These decapod crustaceans are ecosystem engineers as their burrowing and feeding activities modify biogeochemical processes. They are also important as prey for a wide variety of other crustaceans, fish, birds and mammals. The present study used mesocosm and microcosm designs to investigate effects of crude oil on fiddler crab burrowing and to assess cellular and tissue damage by the oil. Fiddler crabs were exposed for periods of 5 or 10 days to oil concentrations up to 55 mg/cm2 on the surface of the sediment. Burrowing was delayed, burrows were smaller, and movement of sediment transport was less efficient in oil-contaminated sediment. The hepatopancreas had elevated levels of lipid peroxidation and showed an increase in the relative abundance of specific cells (blister cells) that play a role in secretory processes. Interspecific differences were also observed for most endpoints, suggesting higher oil-sensitivity in U. panacea than in U. longisignalis. These new insights into the effects of crude oil on fiddler crabs demonstrate that oil spills are likely to impact both the fiddler crabs themselves, as well as the many species that depend on them for their diet or for the ecological changes that result from their burrowing.

No description available.