Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references. Also available via the Internet.

Thesis (PhD. (Paraclinical Studies))--University of Pretoria, 2004. / Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 2005. / Printout. Includes bibliographical references (leaves 53-56). Also available on the World Wide Web.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 125-126). Also available via the Internet.

Freshly isolated hepatocytes from fed and starved rats were used as a model in the investigation of the mechanisms by which cadmium chloride exerts its toxic effects at the cellular level. Exposure to cadmium chloride resulted in a slight decrease in viability, more pronounced in hepatocytes from starved rats. Morphological changes preceded the increase in membrane permeability. Hepatocytes exhibited a rapid initial uptake of cadmium chloride, followed by a second slower phase. The accumulation of more metal in hepatocytes from starved rats may contribute to their enhanced susceptibility to cadmium chloride. Adverse metabolic effects of cadmium chloride included an increase in the lactate:pyruvate ratio in hepatocytes from fed rats, with a concomitant decrease in the 3-hydroxybutyrate:acetoacetate ratio in hepatocytes from fed and starved rats. Incubation with cadmium chloride resulted in increased glycogenolysis and glycolysis. Decreased rates of gluconeogenesis from lactate and pyruvate reflected the decreased uptake of gluconeogenic precursors. Studies of intracellular lactate concentrations could not resolve whether the decrease in gluconeogenesis was due to an inhibition of lactate transport into the hepatocyte or due to a decrease in its metabolism. Cadmium chloride caused a slight decrease in the basal and pyruvate-stimulated rates of cellular respiration, a marked dose-related decrease with lactate, and no significant effects with succinate. Carbonyl- cyanide-m-chlorophenylhydrazone was less effective in stimulating respiration in hepatocytes incubated with cadmium chloride, this effect being more pronounced with lactate and pyruvate than with succinate. Cadmium chloride had little effect on the uncoupled rates of FADH2 oxidation with succinate suggesting that electron transport from succinate dehydrogenase to cytochrome a/a3 was not impaired. The results from these studies suggest a primary effect of cadmium chloride on mitochondrial function and cellular energy production, resulting in secondary metabolic changes in an attempt to overcome the declining levels of ATP within the cell.

1. The molecular mechanism and the reactive species involved in the inactivation of microsomal cytochrome P-450 during the reductive metabolism of carbon tetrachloride (CCl[4]) were studied. Results obtained with spectrophotometric, fluorimetric and chromatographic methods and using strictly anaerobic conditions indicate that the prosthetic group of the cytochrome, haem, is both the site and the target of CCl4 activation. 2. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when CCl[4] was incubated anaerobically with NADPH- or sodium dithionite-reduced rat liver microsomes. A rapid loss of haem was also observed during reductive incubation of CCl[4] with soluble haem preparations (methaemalbumin) in the absence of the apoprotein. In both cases the loss of haem was accompanied by an equimolar loss of its tetrapyrrolic structure and was prevented by carbon monoxide. Similar results were obtained using halothane as a suicidal substrate. 3. A dichlorocarbene-cytochrome P-450 ligand complex was found to be responsible for the difference spectrum obtained on addition of CCl[4] to anaerobically reduced rat liver microsomes. A molar extinction coefficient of 56.2 for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene did not prevent, however, the CCl[4]-dependent loss of microsomal haem indicating that the carene is not involved. 4. Inactivation of haem by CCl[4], both in microsomes and the non-enzymic system is a typical suicide reaction which follows saturable, pseudo first-order kinetics. A partition ratio of 26 was calculated between molecules of CCl[4] metabolised and molecules of cytochrome P-450 haem lost. The loss of haem in the chemical system is due to covalent binding of CCl[4] reactive metabolites in a 1:1 stoichiometry.

The main aims of this thesis were to establish whether an acute inflammatory response is provoked in rats by the subcutaneous administration of cadmium, and to evaluate the possible role that such a response might play in the alterations in metal homeostasis and the development of anaemia which accompanies the use of this model of cadmium intoxication. An intense local reaction to the subcutaneous administration of cadmium was found. Many of the systemic changes, most notably in haematological parameters and in levels of iron, copper and plasma proteins, mimicked those seen in the acute inflammatory response. Possible causes of the resultant anaemia are discussed and inflammation is implicated as a predominant factor in its development. The results suggest that many of the effects which in previously published work have been attributed to a direct interaction of cadmium with the system under investigation, may in fact be secondary consequences of cadmium-induced inflammation. Comparison of the effects of subcutaneous administration of cadmium and other selected metal salts with changes occurring in two recognised models of acute inflammation revealed marked differences in the local tissue reaction to different substances as well as in the magnitude of various components of the systemic response. The oedematous, necrotic and extensively destructive nature of the cadmium-induced lesion has been highlighted and shown to be partially alleviated by pre- and simultaneous treatment with zinc. Explanations for this protective phenomenon are offered, based on possible target sites of cadmium, particularly in terms of interaction with zinc-dependent processes. With a view to understanding the mechanisms involved in acute cadmium toxicity, luminol-amplified chemiluminescence, which is indicative of free radical formation and the production of reactive oxygen species, was measured directly from intact tissue samples. Inflamed tissue sampled from subcutaneous sites of cadmium administration emitted substantially more chemiluminescence than noninflamed tissue or tissue from sites to which other metals or turpentine was administered. It was demonstrated that intact tissue samples can also be used to assess free radical generation, as detected by chemiluminescence, during in vitro treatment. A pronounced dose-related response was seen with cadmium which could be inhibited by various pretreatment procedures, such as incubation with zinc or certain metal chelators. The significance of these results in relation to the mechanism of toxic action of cadmium as well as to the potential use of this chemiluminescence technique is discussed.

The teratogenicity of methoxyacetic acid (MAA) was studied using rat whole-embryo and limb bud micromass cultures. MAA and some related alkoxy acids were direct-acting teratogens in both systems. Structure-activity investigations produced the same rank-order of potency; methylthioacetate > MAA > ethoxyacetate. All other compounds tested were either much less effective or inactive. In whole embryo culture the uptake of MAA was rapid, appeared to be by simple diffusion, and to be determined by pH partitioning. MAA accumulated in the relatively alkaline conceptus; for example, the exocoelomic fluid concentration was 1.7 fold that of the medium. In micromass cultures uptake was also dependent on pH partitioning, hence MAA was more effective in Hams F-12 (pH 7.1) medium than in DMEM (pH 7.6). MAA did not appear to be metabolised by the rat embryo in culture. After a 48 h exposure, radiolabel derived from MAA was not incorporated into macromolecular fractions (lipid, RNA, DNA, or protein) of the developing conceptus. Also, HPLC analysis of the acid-soluble phase of embryonic tissue and media samples did not reveal methoxyacetylglycine or any other known MAA metabolite. Using both culture systems it was shown that MAA did not evoke its teratogenic response via an initial inhibitory effect on either DNA or protein synthesis, glycolysis, nor the incorporation of acetate or sulphate. In addition, MAA did not inhibit yolk-sac pinocytosis. The first day of a 6 day micromass culture was most sensitive to MAA treatment. The protein content and cell number of treated cultures were severely reduced by 24 h, indicating cell death and/or cell loss. Thus, biochemical measurements taken at 24 h or later may reflect secondary, rather than initial, insults. Replating of MAA-treated micromass cultures (after a 24 h exposure), to re-establish normal cell density, did not restore the ability to form chondrogenic foci. The inhibition of differentiation was not, therefore, freely reversible nor the result of a decreased cell density, but may be due to selective cytotoxicity against the sub-population of pre-chondrogenic cells.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / BenzotiazÃis sÃo compostos com anel benzeno ligados ao anel tiazol, que apresentam atividade antitumoral significante. Devido à alteraÃÃo de ligaÃÃes quÃmicas de molÃculas ser um mÃtodo comum para desenho de fÃrmacos em quÃmica medicinal e uma tÃcnica Ãtil para o desenvolvimento de novos medicamentos, a sÃntese de BenzotiazÃis substituÃdos apresentou muita vantagem para o tratamento de tipos especÃficos de cÃncer. No presente estudo foi avaliado o potencial citotÃxico de quatro anÃlogos de benzotiazÃis substituÃdos frente a linhagens tumorais e nÃo tumorais. Os resultados demonstraram que todos os anÃlogos apresentaram atividade citotÃxica, porÃm o composto 1 (CI50 = 1,65ÂM) foi escolhido para dar continuidade aos ensaios por ser protÃtipo dos demais, e o composto 3 (CI50 = 1,01ÂM) foi escolhido por apresentar maior citotoxicidade quando comparado aos demais e ainda por apresentar uma seletividade para linhagem nÃo tumoral avaliada, cÃlulas mononucleadas do sangue perifÃrico humano (CMSPH). Foi entÃo realizada uma sÃrie de ensaios in vitro, onde se buscou esclarecer os efeitos envolvidos nessa atividade citotÃxica. A linhagem HL60 foi a escolhida para dar continuidade ao restante dos experimentos por ser a linhagem mais sensÃvel à exposiÃÃo dos compostos. Nos ensaios de viabilidade foi observado uma reduÃÃo no nÃmero de cÃlulas viÃveis jà na concentraÃÃo de 1ÂM para o composto 1 (56,54% cÃlulas viÃveis, p<0,001) e para o composto 3 (38,45% cÃlulas viÃveis, p<0,001). A morfologia das cÃlulas HL-60 avaliadas atravÃs do uso da coloraÃÃo May-GrÃnwald-Giemsa revelou morte celular com caracterÃsticas de apoptose que ainda foi confirmado com ensaio da coloraÃÃo diferencial de laranja de acridina e brometo de etÃdio (LA/BE), onde foi considerada estatisticamente significante a partir da concentraÃÃo de 2ÂM do composto 1, com 79,92% (p<0,001) de cÃlulas com caracterÃsticas de apoptose e para o composto 3 na concentraÃÃo de 1ÂM com 28,86% (P<0,001). Nos ensaios de citometria de fluxo foi revelado que, quando tratados com os compostos, as cÃlulas HL60 promoviam a externalizaÃÃo da fosfatidil serina no qual na concentraÃÃo de 1ÂM foi considerado estatisticamente significante para o composto 1 (12,38% de cÃlulas em apoptose, p<0,001) e para o composto 3 (42,67%, p<0,001). Foi obervado ainda que os compostos ativam caspase 8 (via extrÃnseca), com 21,78% (P<0,001) de cÃlulas em apoptose para o composto 1 (2ÂM) e 47,5% (p<0,001) para o composto 3 (1ÂM); ativaram tambÃm caspase 9 (intrÃnseca), com 17,10% (p<0,001) de cÃlulas em apoptose para o composto 1 (1ÂM) e 33,55% (p<0,001) para o composto 3 (1(P<0,001)M); e conseqÃentemente ativaram as caspases 3 e 7, envolvidas no processo final da morte celular por apoptose, com 30,05% (p<0,001) de cÃlulas em apoptose para o composto 1 (2ÂM) e 53,19% (p<0,001) para o composto 3 (1ÂM). Foi observada intensa fragmentaÃÃo de DNA em cÃlulas tratadas com ambos os compostos, onde jà na concentraÃÃo de 1ÂM foi observado 23,13% (p<0,001, composto 1) e 61,02% (p<0,001, composto 3) de DNA fragmentado. Os composto nÃo foram capazes de induzir a formaÃÃo de espÃcies reativas de oxigÃnio (EROs) e ou causar danos diretos ou indiretos à fita de DNA. Conclui se que ambos podem ser considerados como molÃculas com potencial citotÃxico, destacando o composto 3 por sua maior citotoxicidade e seletividade. E ainda, esses dados reforÃam a importÃncia de sintetizar e estudar anÃlogos sintÃticos com atividades cada vez mais seletivas para o tratamento do cÃncer. / Benzothiazole are compounds consisted of a benzene ring with a thiazole ring that present significant anticancer activity, especially the fenil substituted. Due to molecular modeling, a common method for drug design in medicinal chemistry and a useful technique for the development of new drugs, the substituted synthesis of Benzothiazole presented great advantage for the treatment of specific types of cancer. In the present study it was evaluated the cytotoxic potential of four of synthetic benzothiazole analogues with malignant and non malignant cell lines. The results demonstrated that all the tested analogues presented cytotoxic activity, however compound 1 (CI50 = 1,65ÂM) was chosen to give continuity to the assays because it is the prototype of the others, and compound 3 (CI50 = 1,01ÂM) was chosen due to its higher cytotoxicity compared to the others and for presenting a selectivity towards malignant cell line when compared to a non malignant cell line, peripheral mononuclear blood cells(PMBC). A series of assays was then carried out in vitro, where it aimed to clarify effects involved in this cytotoxic activity. The cell line HL60 was chosen to conduct the experiments for it was the most sensitive cell line to the exposure of compounds. In the viability assay a reduction in the number of viable cells was observed in the concentration of 1ÂM for compound 1 (56.54% viable cells, p < 0.001) and compound 3 (38.45% viable cells, p< 0.001). The morphology of HL-60 cells evaluated with the use of the May-GrÃnwald-Giemsa dye showed cellular death with characteristics of apoptosis which was further confirmed with the orange acridine and ethidium bromide differential count assay (LA/BE), where it was considered statistically significant from the concentration of 2ÂM for compound 1, with 79,92% (p< 0.001) of cells with characteristics of apoptosis and for compound 3 in the concentration of 1ÂM with 28,86% (P< 0.001). In the flow cytometry assays it was observed that, when treated with the tested compounds, HL60 cells promoted the externalizaÃÃo of phosphatidyl serine in which in the concentration of 1ÂM it was considered statistically significant for compound 1 (12.38% of cells in apoptosis, p< 0.001) and for compound 3 (42.67%, p< 0.001). It was observed that the compounds activated caspase 8 (Extrinsic pathway), with 21,78% (P< 0.001) of cells with characteristics of apoptosis for compound 1 (2ÂM) and 47.5% (p< 0.001) for compound 3 (1ÂM); they also activated caspase 9 (intrinsic pathway), with 17,10% (p< 0.001) of cells with characteristics of apoptosis for compound 1 (1ÂM) and 33.55% (p< 0.001) for compound 3 (1ÂM); consequently, it was activated caspases 3 and 7, involved in the final process of apoptosis, with 30,05% (p< 0.001) of cells with characteristics of apoptosis for compound 1 (2ÂM) and 53.19% (p< 0.001) for compound 3 (1ÂM). Intense DNA fragmentation was observed in cells treated with the tested compounds, where in the concentration of 1ÂM it was observed 23.13% (p 0.001<, composition 1) and 61.02% (p 0.001<, composition 3) of fragmented DNA. The compounds were not able to generate reactive oxygen species (ROS) and cause direct or indirect damage to the DNA strand. In conclusion, both tested compounds can be considered as molecules with cytotoxic potential, highlighting compound 3 for its slightly higher cytotoxicity and selectivity. These data strengthen the importance to synthesize and to study synthetic compounds with more selective activities for the treatment of cancer.

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