14 research outputs found
Chemical and Biological Characterization of Southern Ontario Urban Air Particulate
No full textAmbient concentrations of polycyclic aromatic compounds [PAC], other air pollutants and the mutagenic potency of respirable air particulate extracts were used to study air quality in Hamilton from May of 1990 to June of 1991. Concentrations of polycyclic aromatic hydrocarbons [PAH], thiaPAH, oxygenated PAC and in some cases nitroPAH as well as the mutagenic activity caused by these compounds were determined in 68 samples of air particulate, collected over a number of days with widely varying air pollutant concentrations and atmospheric conditions. Chemical analyses of the non-polar aromatic fractions of the air particulate extracts showed a 550-fold range in PAC concentrations, from 0.31 to 170 ng/cubic metre air, while biological assays showed a 140-fold range of mutagenic potencies. The non-polar aromatic fraction represented about three-quarters of the sum of the mutagenic responses in the polar and non-polar aromatic fractions. The mutagenicity and PAC concentrations of air particulate collected in Hamilton are low to average compared to air particulate samples collected in other cities around the world. Relationships between mutagenic potencies, PAC concentrations and atmospheric condistions were examined using principal component analysis, which revealed good positive correlation between the mutagenic potencies of the non-polar aromatic fractions, nitrogen diozide and sulfur dioxide concentrations. Average windspeed and ozone concentrations were inversely correlated with mutagenicity. Principal component analysis also show that sprigtime thermal inversion episodes correlated with conditions favoring the atmospheric transformation of PAH to mutagenic nitroPAH. The mutagenic potency of the non-polar aromatic fraction of air particulate extract rose in direct proportion to nitroPAH concentrations arising from atmospheric transformation. Enhanced-sensitivity strains of Salmonella typhimurium were used in all bioassays of air particulate extract mutagenicity, thereby making more extract available for extensive chemical analyses. For example, bioassay analyses of a pooled sample of air particulate extract were carried out using six different strains of S. typhimurium. Strains containing increased levels of enzymes responsible for metabolizing compounds such as nitroPAH showed 7- to 10-fold enhance mutagenic responses compared to standard tester strains. Normal-phse HPLC seperation of the non-polar aromatic fraction of the pooled air particulate extract was combined with bioassays utilizing three different enhanced-sensitivity S. typhimurium strains, allowing quantitation of individual mutagens through analytical chemical methods as well as accurate determination of the mutagenic activity arising from each compound. Normal-phase HPLC separation was also used in the bioassay-directed fractionation of an extremely mutagenic air particulate sample collected at the side of Highway 404 in Toronto, Ontario. NitroPAH arising from combustion sources were determined to be responsible for virtually all of the mutagenic activity detected in this particular sample, from which was isolated a potent mutagen not previously described in air particulate extracts -3.8-dinitrofluoranthene.Doctor of Philosophy (PhD
Determination of carbon black in urban air
No full textThe City of Hamilton is one of the most heavily industrialized cities in Canada. In recent years, residents of communities in the east-end of Hamilton have complained about "black fallout" on their properties; this air particulate deposition takes the form of a fine, greasy black film that coats their houses, cars, etc. A carbon black production company has been suspected as the primary source of this black particulate, although there are other potential sources of black particulate, including emissions from two large steel industries, vehicular traffic and other sources. One of the major problems that has faced the Ministry of the Environment (MOE) in dealing with public complaints regarding "black fallout" has been the lack of an analytical procedure that would allow carbon black and other types of "black fallout" to be distinguished. My focus has been to develop the first analytical methodology to identify and quantify carbon black in ambient air. This approach to the determination of ambient levels of carbon black is based upon a sequential extraction methodology and the gas chromatography-mass spectrometric (GC-MS) quantification of an unusual polycyclic aromatic compound (PAC) I have identified on carbon blacks (thiacoronene) as a source tracer for carbon black. In an air monitoring study carried out from 1995 to 1998, I have been able to identify and quantify carbon black in ambient air samples collected downwind of a carbon black production plant, which varied from 0.01 to 1.43 μg carbon black/m3 of air. All concentrations of carbon black were well above the method detection limit of 0.004 μg/m3 with an uncertainty estimate of less than 5%. In the evaluation of sources of "black fallout" other than carbon black, I investigated a new source apportionment strategy based on a manganese-tin (Mn-Sn) metal index to differentiate levels of steel industry impacts in ambient air.Doctor of Philosophy (PhD
Lipidomics as a Tool for Functional Genomics in Sinorhizobium Meliloti
No full text This thesis focused on the development of comprehensive, rapid and
simple methodologies for the analysis of fatty acids by gas chromatography mass
spectrometry (GC/MS) and intact lipids by electrospray ionization tandem mass
spectrometry (ESIIMS/MS). The methodologies were applied as a tool for
functional genomics in the soil bacterium Sinorhizobium meliloti. The effects of
inorganic phosphate (Pi)-starvation and acidity on lipid composition were studied. A micro-scale, one-vial method for the analysis of fatty acids as their fatty
acid methyl esters by GC/MS was developed. The method required small sample
sizes, involved minimum handling and avoided tedious extraction steps, which
increased sample throughput. A series of quality controls were included to
measure losses due to handling, derivatization efficiencies and the extent of side
reactions. The method was suitable for the analysis of sensitive bacterial fatty
acids such as cyclopropane fatty acids. A shotgun lipidomics approach was developed for the analysis of intact lipids by ESIIMS/MS. Fatty acid distributions were obtained for eight lipid
classes and up to 58 individual lipids were identified in crude lipid extracts
without sample cleanup or chromatography. For the first time, fatty acid
distributions were provided for non-phosphorus containing lipids using shotgun
lipidomics. Fatty acid distributions within lipid classes suggested that
phospholipids and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine lipids
(TMHSs) were both synthesized from phosphatidic acid while
sulfoquinovosyldiacylglycerol (SLs) had a different biosynthetic origin. The methodologies were applied to study knockout mutants of five genes
thought to participate in lipid metabolism in S. meliloti. It was demonstrated that:
(1) cfa2 gene coded for the main cyclopropane fatty acyl synthase; (2) the plsC
gene coded for a fatty acy 1 transferase specific for C 16 fatty acids in the sn-2
position of phospholipids; (3) a metabolic phenotype was revealed for knockout
mutants of dme and tme genes (DME and TME, malic enzymes) when succinate
was the carbon source. ThesisDoctor of Philosophy (PhD
A multi-media bioassay-directed investigation of the hamilton harbour area of western lake ontario
No full textA methodology for the extraction, clean-up and compound class fractionation of complex environmental mixtures was developed and tested using standard reference materials and sediment from Hamilton Harbour. Samples were extracted using a Soxhlet apparatus or an ultrasonication apparatus and the resulting organic solvent extracts were fractionated into compound classes using an alumina/Sepahadex LH20 clean-up procedure and high performance liquid chromatographic techniques. In the next phase of the study, sediment samples, sediment trap samples, and air particulate material from the Hamilton Harbour area of western Lake Ontario were fractionated using the developed methodology. These samples were chosen with the aim of evaluating the contributions of a variety of sources to chemical and genotoxic contamination in the harbour. The resulting fractions were analyzed by chromatographic techniques and tested for genotoxicity using Ames Salmonella/microsome assay with TA98-like and TA100-like bacterial strains modified by the inclusion of genes for the activating enzymes nitroreductase and O-acetyltransferase. These data were used to construct chemical and biological profiles of the samples and to identify specific compounds and compound classes responsible for mutagenic activity observed in the sample extracts. The majority of the mutagenic activity displayed by a Randle Reef sediment sample extract was found to be present in the fraction containing the polycyclic aromatic hydrocarbons (PAH). Extracts of the PAH-containing fraction displayed dramatically higher responses with the TA100-type strains with metabolic activation. The PAH fraction was further fractionated and analysed to identify the compound(s) responsible for the mutagenic activity. The biological activity of this PAH-containing fraction was found to co-elute with compounds of molecular mass 252, 276, 278, and 302 amu. In contrast to the results obtained from the investigation of the Randle Reef sediment sample, extracts of sediment trap samples displayed significantly higher responses with the TA98-type strains. The compound(s) responsible for this biological activity were contained in the more polar polycyclic aromatic compound (PAC) fractions and did not require oxidative metabolism to manifest their activity. Further separation of the most biologically active fraction from the sediment trap samples revealed that the biological activity was contained in a very narrow elution time range in the reversed-phase high performance liquid chromatography (RP-HPLC) chromatogram. This narrow band of activity was collected and analysed usign gas chromatography-mass spectrometry (GC-MS). While alkyl-benzocarbazole derivatives were identified in these fractions, they were not considered to be the agents responsible for the observed biological activity. The use of ultrasonic extraction and the alumina/Sephadex LH2- clean-up and normal phase HPLC compound class fractionation procedures were found to be effective for the preparation of a number of complex environmental samples. The chemical and biological profiles of the harbour sediment and suspended sediments indicate that PAH from resuspended coal tar contaminated sediment is a principal contributor to the chemical profile of suspended sediments in the harbour and a significant contributor to the genotoxicity of extracts of these samples. However, the biological profiles of the suspended sediments indicate the presence of additional mutagens other than PAH that are not present in the Randle Reef sediment sample. A comparison of the biological profiles of extracts of suspended sediments and air particulate material indicate that there may be a common mutagenic contaminant in both of these sample matrices.Doctor of Philosophy (PhD
Synthesis and Testing of Neurological Produgs for the Gamma-Aminobutyric Acid System
No full textThe purpose of this work was to synthesize derivatives of a GABA agonist to be used as prodrugs. These compounds would be chemically liable and thus would not depend on enzymatic activation to release the parent drug. Two of the most potent GABA agonists, muscimoi and isoguvacine, were synthesized in 19% and 29% overall yield from propargyl chloride and isonicotinic acid, respectively. A series of prodrugs of muscimol, phenyl carbamate derivatives, were synthesized in 42-66% yields from the corresponding chloroformates. Depending on the substituents on the aromatic ring, a wide range of hydrolysis rates (2.8 to 380 mins) and partition coefficients (0.05 to over 15) were observed for these carbamates. These prodrugs were evaluated as sedatives and anticonvulsants. All carbamates with partition coefficients greater than 0.1 induced dose-dependent sedation which was found to increase with increasing partition coefficient and with increasing hydrolysis rate. Sedation times as long as 12 hours were observed in mice. While one of these carbamates, the p-carboethoxyphenyl carbamate, showed no inhibition of seizures induced by GABA antagonists, a significant delay (about 80%) in the onset of these seizures was observed. Muscimol itseIf had no effect on these antagonist-induced seizures. Some carbamates radiolabelled in the muscimol portion were administered to mice i.p. and the amount of free [³H]-muscimol in the brain was determined after 1 hour. The amounts of muscimol found ranged from 2.3 to 10.5 nmols/brain. These quantities paralleled the amount of sedation produced by these carbamates. A phenyl acetamide of muscimol with a partition coefficient identical to a sedative carbamate was prepared, as a non-labile counter-part to the labile carbamate. No sedation was observed for this acetamide even at high doses. Thus it appears that intact carbamates are not responsible for the sedative effects. • However, seven per cent of a radiolabelled form of this acetamide was found in the brain, indicating that agents with similar partition coefficients have ready access to the brain by passive diffusion.Doctor of Philosophy (PhD
The Use of Fluorinated Maleimides as Protein Sulfhydryl Reagents for ¹⁹F-nmr Studies
No full textThe overall objectives of this work were the development of a) fluorinated maleimides for the specific modification of protein sulfhydryl groups and subsequent ¹⁹F- nmr studies and b) fluorinated phospholipids for the study of protein-lipid interactions by ¹⁹F-nmr. Two fluorinated analogues of N-ethylmaleimide, N-2,2,2-trifluoroethylmaleimide (FEM) and a deuterated analogue N-2,2,2-trifluoro-1,1-dideuteroethyl-maleimide (FEM-d₂) were synthesized from trifluoroacetamide in three steps. A detailed kinetic study showed that FEM reacted at least 10 times faster with thiols than with either other amino acid side chains namely imidazoles amines, alcohols or water. The rates of reaction between FEM and thiols increased ten-fold for each unit increase in pH, in addition, apparent activation parameters were determined for these reactions at pH 6.65. The FEM-thiol adducts were also isolated and fully characterized. Bovine serum albumin (BSA) on exposure to FEM-d₂ reacted rapidly at the single sulfhydryl residue exclusively. On the other hand, 3-bromo-1,1,1-trifluoropropanone reacted with BSA at the sulfhydryl group and at other sites on the protein to the extent of 20%. The neutral to fast (N-F) transition of BSA was examined by ¹⁹F-NMR following modification of the protein with FEM-d₂ (BSA-FEM-d₂) or Br-TFP (BSA-TFP). Over the pH range 3.0-6.0 at least three distinct ¹⁹F-NMR resonances were observed for both modified proteins; the linewidths, chemical shifts and peak areas of these resonances changed as a function of pH. Circular dichroism and fluorescence spectra of modified and unmodified BSA proteins in the pH 3.0-6.0 range were indistinguishable indicating that the sulfhydryl labels caused little structural perturbation to the protein. At pHs above 8.0, BSA-FEM-d₂ underwent an irreversible chemical reaction; likely opening of the succinimide ring by an amino group of the protein. Chemical shift anisotropy contributions to the ¹⁹F-nmr linewidths for both BSA-FEM-d₂ and BSA-TFP increased linearly on going from 84.66 to 235.36 MHz. Two phospholipids fluorinated in the acyl chains were synthesized for the purpose of incorporating them into vesicles with lipophilin, an integral membrane protein; bis-8-fluoro, 8-deutero- and bis-12-fluoro, 12-deuterodimyristoylphosphatidyl-choline were synthesized and characterized by ²H-nmr and calorimetric studies. However, ¹⁹F-nmr studies of lipophilin/fluorolipid mixtures showed no evidence for the presence of any immobilized or "boundary lipid" around the protein. In addition, lipophilin was modified with FEM-d₂ but ¹⁹F-nmr experiments provided no information about the environment of the FEM-d₂ labelled cysteine residues of the protein.Doctor of Philosophy (PhD
Thia-arenes as pollution source tracers in urban air particulate
No full textA method to distinguish coke oven emissions from diesel exhaust emissions in ambient samples has been developed. Sulfur-containing polycyclic aromatic compounds (thia-arenes) and polycyclic aromatic hydrocarbons (PAH) were determined in source samples, standard reference samples and ambient samples using gas chromatography/mass spectrometry. A total of 92 ambient respirable air particulate samples that were collected at four locations upwind and downwind of the steel industries in Hamilton, Ontario in 1995, were analyzed for their total particulate mass, 15 PAH and a number of thia-arene compounds. The steel industries were found to contribute the majority of PAH pollution in Hamilton air particulate above the urban background of approximately 1.5 ng/m3 . Particulate concentrations in Hamilton air indicate several major sources of particulate, including vehicle emissions. Profiles of alkylated dibenzothiophene isomers, 234 amu this-arene isomers and 258 amu thia-arene isomers were examined in Hamilton air particulate, Toronto air particulate, coke oven condensate, Diesel Exhaust Particulate Standard Reference Material (SRM 1650), Urban Dust Standard Reference Material (SRM 1649) and Coal Tar Standard Reference Material (SRM1597). Selected thia-arenes were also examined in a variety of other environmental samples. Principal component analysis (PCA) was used to select suitable thia-arene tracer compounds and examine the relationship between a variety of environmental samples. A thia-arene index was developed using a combination of ratios of selected 234 amu and 258 amu isomers. The suitability of thia-arene tracers for distinguishing coke oven emissions from diesel emissions is demonstrated with Hamilton air particulate and source samples. Ambient samples with particulate bound PAH levels below 1.6 ng/m3 were classified as petrogenic in origin in all cases, while samples with PAH levels above about 3.6 ng/m3 were shown to have significant coke oven impacts. The quality of the thia-arene criteria is discussed in comparison to PAH criteria found in the literature. Four gas chromatography stationary phases were evaluated for the separation of thia-arenes and PAH. Retention index values are reported for 49 PAH and 66 thia-arene compounds. The 50% phenyl-substituted methylpolysiloxane stationary phase provided the best overall separation of thia-arenes and PAH.Doctor of Philosophy (PhD
Substituted 2-amino-tetrahydronaphthalenes as affinity and photoaffinity probes for dopamine receptors
No full textIn the current investigation several biochemical techniques, including solubilization, affinity chromatography and photoaffinity labelling, were used to purify the D-1 dopamine receptor. The design and synthesis of novel analogues of the dopamine agonist ADTN (2-amino-5,6-dihydroxy-tetrahydronaphthalene) to be used as affinity and photoaffinity probes was an integral part of this investigation. Solubilization of the D-1 receptor was achieved with several detergents although cholic acid proved to be the most effective for receptor solubilization prior to affiinty chromatography. Using this procedure, yields of solubilized receptors of greater than 30% were consistently obtained. An affinity chromatography protocol utilizing an ADTN analogue covalently coupled to an affinity matrix was established for cholate-solubilized D-1 receptor. The affinity protocol developed during this investigation purified the D-1 receptor approximately 50-fold, while an average of 8% of the receptors were recovered. These results were superior to any previous literature reports of D-1 receptor purification. Several photoactive compounds were synthesized and used to crosslink D-1 receptors. One compound in particular, a photoactive derivative of the dopamine agonist ADTN, proved to be a useful ligang for this purpose. A tritiated derivative of this compound was covalently and specifically incorporated into a protein of M.W. = 79 kDa. This was the first report that the D-1 receptor had a M.W. of greater than 70 kDa was determined by affinity crosslinking. Several other investigators have subsequently confirmed this observation. Other photoactive compounds radiolabelled with ¹²⁵I were synthesized and examined for activity as D-1 and D-2 receptor probes. These compounds were not as useful as photoaffinity labels for dopamine receptors as had been originally proposed. The compounds did label and a 50 kDa protein which was determined to be neither the D-1 nor D-2 receptor. This protein (originally designated Apo-50 and later CatNAP) has very interesting properties asa it possesses binding activities with several catecholamines which are without precedent in the literature.Doctor of Philosophy (PhD
A MULTI-DIMENSIONAL METHOD FOR THE ANALYSIS OF HUMAN BLOOD PLASMA METABOLOME
No full textThe comprehensive analysis of human blood plasma metabolome has been completed using derivatization gas chromatography-mass spectrometry (GC-MS) analysis, liquid chromatography-mass spectrometry (LC-MS) analysis and a comprehensive LC-GC-MS analysis approach wherein LC fractions were collected, derivatized and analyzed using GC-MS. In all cases blood plasma samples were deproteinized using solvent precipitation prior to chromatography and MS analysis. In GC-MS analyses, the progress of all derivatization reactions was monitored by adding 9-anthracenemethanol and 1,3-diphenylacetone to all reaction mixtures; their conversions to 9-anthracenemethanol trimethylsilyl ether and the oxime derivative of 1,3-diphenylacetone were used as measures of the completion of these derivatization reactions. Any reactions with completions less than 99% were repeated. GC-MS analysis of blood plasma samples detected 100 peaks; 44 were positively identified by comparing retention indices and mass spectra with those of authentic standards. LC-MS analyses were conducted on a HILIC column (aminopropyl phase) with MS detection in both negative ion and positive ion modes and resulted in the identification of 97 peaks; 47 were observed in the positive ion mode, 58 in the negative ion mode with 8 peaks observed in both modes. The multi-dimensional LC-GC approach was not designed as a routine analytical method; rather the purpose of this approach was to see how many compounds could be observed in the sample and to obtain better quality mass spectra and retention index values. The LC separation afforded 16 fractions which upon derivatization GC-MS analysis gave an additional 176 peaks from a total of 276 peaks. The MS data from these additional spectra can be used to develop selected ion monitoring GC-MS or tandem mass spectrometry analytical methods. This thesis has demonstrated the power of off-line comprehensive methods to identify compounds that neither the GC nor the LC methods detected.Master of Science (MSc
