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Methotrexate is a folic acid analogue widely used as a chemotherapeutic agent. It is known to be a potent inhibitor of the enzyme dihydrofolate reductase, therefore, perturbing intracellular pools of purine and pyrimidine bases for DNA synthesis, as well as pools of reduced folates used in a variety of metabolic reactions. It has been postulated, and subsequently widely accepted, that methotrexate kills cells by perturbing the intracellular ratio of dUTP:dTTP thereby leading to dUMP misincorporation into DNA. This would initiate an excision repair pathway designed to rid cellular DNA of this aberrant base. However, because of the imbalance of nucleotide pools, dUMP may well be re-incorporated during repair thus initiating a futile cycle of dUMP misincorporation and repair eventually leading to single-strand breaks in the DNA. From the results presented in this thesis, no evidence for dUMP misincorporation could be found in the two human cell lines studied (HeLa and CCRF-HSB2), despite the drug exhibiting dose-dependent cytotoxicity to both cell lines. This was true after a variety of methotrexate treatment times and at two different drug concentrations. Subsequent analysis of the drug treated cells, using the nucleoid sedimentation technique, for evidence of single-strand breaks in DNA yielded some anomalous results. Single-strand breaks, in the form of slower sedimenting nucleoids, were easily detectable after exposure of cells to low doses of methotrexate. However, treatment with higher doses resulted in the creation of faster sedimenting nucleoids. Subsequent analysis using other techniques showed that this faster sedimentation was occurring in the presence of DNA single-strand breaks. Collaborative work involving electron microscopy revealed methotrexate induced gross morphological changes in chromatin structure. Analogies with other unrelated anti-tumour agents interacting with topoisomerase enzymes are discussed.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisors: Serrine S. Lau and Shawn B. Bratton. Vita. Includes bibliographical references.

Orientador: Carlos Alberto de Souza Costa / Banca: Elisa Maria Aparecida Giro / Banca: Denise Madalena Palomari Spolidorio / Banca: Maria Cristina Borsatto / Banca: Mario Fernando de Góes / Resumo: O objetivo da presente pesquisa foi avaliar o efeito citotóxico direto e transdentinário de diferentes concentrações de clorexidina (CHX), bem como sua atividade antibacteriana. Para isto, concentrações de 0,06%; 0,12%; 0,2%; 1% e 2% de CHX foram aplicadas sobre células odontoblastóides MDPC-23 em cultura (teste direto). Além disto, as concentrações de CHX também foram aplicadas sobre a superfície oclusal de discos de dentina de 0,5mm ou 0,2mm de espessura, os quais apresentavam células MDPC-23 cultivadas sobre sua superfície pulpar (teste indireto). O metabolismo e morfologia celular foram avaliados pelo teste de MTT e pela análise em MEV, respectivamente. O potencial antibacteriano da CHX foi avaliado sobre S.mutans com e sem interposição de discos de dentina. Os dados numéricos obtidos dos experimentos realizados foram submetidos à análise estatística. A redução do metabolismo celular provocado pela CHX mostrou-se dose e tempo dependentes. Foi demonstrado ainda que quanto menor a espessura do disco de dentina e maior a concentração da CHX, mais intensos são os efeitos tóxicos deste agente químico sobre as células pulpares em cultura, e mais efetiva é a atividade antibacteriana da CHX sobre S.mutans. Foi possível concluir que o intenso efeito tóxico direto observado para a CHX é notavelmente reduzido pela interposição de barreiras de dentina, cuja espessura interfere no metabolismo das células pulpares MDPC-23. Além disso, a CHX difunde através dos túbulos dentinários exercendo efeito antibacteriano contra S.mutans. / Abstract: The aim of this in vitro study was to evaluate the direct and transdentinal cytotoxicity of different concentrations of chlorhexidine (CHX) and its antibacterial activity. The following concentrations of CHX: 0.06%; 0.12%; 0.2%; 1%; and 2% were applied directly on odontoblast-cell line MDPC-23 or on the occlusal surface of dentin discs (0.5mm or 0.2mm thick) which presented pulp cells seeded on their pulpal surface. Cell metabolic activity and morphology were evaluated by MTT assay and SEM, respectively. The influence of dentin thickness on the antibacterial activity of CHX against S. mutans was also assessed. The data were submitted to the statistical analysis of Mann-Whitney. Reduction in the cell metabolism from 42% to 78% was observed according to the concentration of CHX and its period of application on the cells. Therefore, the direct cytotoxicity of CHX is dose and time dependent. It was also observed that the most intense toxic effects occurred as thicker was the dentin disc and as higher was the concentration of CHX. Antibacterial activity against S. mutans was observed to the highest concentration of CHX. It was concluded that the intense cytotoxicity of all concentrations of CHX applied directly on the MDPC-23 cells is notably reduced by the presence of dentin discs interposed between this chemical agent and the cultured cells. In addition, the transdentinal diffusion of CHX causes antibacterial activity against S. mutans. / Doutor

Magister Scientiae Dentium - MSc(Dent) / Objectives:Substances leaching from denture- base polymers have been associated with cytotoxicity and allergic reactions. This study examined the effect of polishing,mixing ratios, water immersion temperatures and different thicknesses on the sorption and solubility of a heat-polymerized, denture-base polymer. The effect of different water immersion temperatures on the flexural strength of the denture base, was tested as well. The next component of this study, is the testing of the most significant sorption and solubility findings on in vitro cell viability. Materials and Methods:Disc shaped specimens from a heat-polymerized, denture-base polymer (Vertex®) were prepared, based on ISO 1567 specifications for sorption and solubility testing, following the manufacturers’ instructions. The following tests were performed: 1) Sorption and solubility of two groups (n = 12 each) of polished and unpolished discs were established and compared by means of the Mixed procedure; 2) Sorption and solubility of three groups (n = 12 each) with different mixing ratios were compared by means of the Mixed procedure; 3) Four groups (n = 14 each) were immersed in water at different temperatures, sorption and solubility were compared by means of pairwise comparison and the Median test; 4) Specimens with different thicknesses (n = 36) were compared, again, by means of pairwise comparison and the Median test; 5) To test the influence of different water-soaking temperatures on the flexural strength of the disc, strips were prepared from the disc used in test no. 3. The flexural strength was compared, by means of the Median test; 6) To test the influence of no postpolymerization treatment, polishing and water immersion on the cytotoxicity of mouse fibroblast cells, (n = 9) for each test group, were prepared. A preliminary test was performed beforehand, over a period of 24 hours, up to a maximum period of four weeks. The Balb/c 3T3 mouse fibroblast cells were cultured and incubated for 24 hours in Eagles medium. Eluates prepared from the disc and medium without any disc (control) replaced the medium. Cytotoxicity was assessed by MTT-assay. Optical density values were obtained at 24 and 48 hour intervals. The data was analyzed by means of the Means procedure.Results:In the entire thesis, the data was analyzed using SAS on a 0.01 probability level.Between polished and unpolished groups, no significant difference in water sorption (p> 0.01) was found, but there was a difference in solubility (p<0.01).Different mixing ratios did not alter sorption (p = 0.34) or solubility (p = 0.68).However, a difference (p<0.01) in sorption and solubility was found among the different temperature and thickness groups. Soaking the denture base in water at different temperatures did not alter its flexural strength (p = 0.48). Cell viability levels were noted in all the experimental groups in the MTT assay test. The analysis was a two-factor study, with one factor being the group, and the other, being time. The interaction between these factors was found to be significant, indicating that the effect of the groups varied by time (and vice versa).Conclusion:The processes of the soaking in warm water and the polishing of a denture-base polymer, reduce its solubility. Therefore, it is recommended that dentures are soaked in warm water before polishing. Within the limits of this study, the mixing ratios may be changed without affecting sorption or solubility. As solubility increases within the increasing denture-base thickness, it is recommended that unnecessarily thick dentures be avoided.Short- and long-term exposure to eluates of a PMMA, has a negative effect on cell viability. For water-stored and polished discs, this effect is time-dependent, with a higher viability for 48 hours’, than for 24 hours eluates. Polishing is associated with lower solubility. At 24 hours, the polished discs, indeed, had a lower cytotoxic effect than the untreated discs: it may be recommended that dentures be polished on the fitting surface as well.The cytotoxic potential of PMMA-eluates appears to fluctuate over time.

Gold compounds have been used for the treatment of rheumatoid arthritis since the mid 20th century as a disease modifying anti-rheumatic drug. Auranofin, an oral anti-rheumatic drug, has been used for many years in the treatment of rheumatoid arthritis (RA). Although the drug has been successful in treating the symptoms of RA, many patients discontinue its use due to severe toxicity over long periods of continued treatment. Since the introduction of auranofin in 1985 there has been no new clinically approved gold drug. Drug discovery research is directing focus on overcoming these toxicity problems. Much of the problems related to the toxicity related to auranofin are due to its lipophilicity. As a result, three compounds (Asa-fin, Mpta-fin and Pta-fin) with varying substituents were synthesised and hence the lipophilic- hydrophilic balance was modulated. All compounds including auranofin were tested against normal cells to determine its toxicity as well as its anti-inflammatory activity. Three novel auranofin derivatives were compared to auranofin with regards to lipophilicity, toxicity and anti-inflammatory properties The lipophilicity of the three compounds were compared to auranofin using the octanol-water partition coefficient method. All the novel compounds showed variable lipophilicity compared to auranofin, with Pta-fin and Mpta-fin being more hydrophilic than auranofin. The cytotoxicity of these novel gold compounds Asa-fin, Mpta-fin and Pta-fin were compared to auranofin using primary porcine hepatocytes and chicken embryo fibroblasts cultures. A metabolic assay based on the reactivity of 3-[4,5-dimethylyhiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) with viable cells was done to measure the effect of the drugs on the growth of cultures. All three novel compounds proved less toxicity at comparable concentrations in primary porcine hepatocytes and in fibroblast proliferation, Asa-fin and Mpta-fin proved less toxic than Auranofin. The Anti-inflammatory activity of the experimental compounds was determined by testing the effects of the experimental compounds on human lymphocyte proliferation. The MTT assay was used to measure the effect of the drugs on the growth of the cell cultures. All three compounds inhibited the proliferation of human lymphocytes with Pta-fin having the least effect. The effect of these drugs was also evaluated on the reactive oxidant production by chemiluminescence and flow cytometry on resting, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and Phorbol Myristate Acetate (PMA) stimulated human neutrophils. Oxidant production by neutrophils was measured after a 45-minute incubation period with luminol enhanced chemiluminescence. Treatment of neutrophils with auranofin and the three compounds showed that auranofin, Asa-fin and Mpta-fin had a biphasic activity on hydrogen peroxide production with higher concentrations decreasing hydrogen peroxide production, possibly leading to the anti-inflammatory action of these drugs. With Pta-fin no decrease in hydrogen peroxide was observed. Using flow cytometry three dyes specific to different reactive oxygen species were used. 2’, 7’-Dichloroflourescein diacetate (DCFH) is specific for detecting nitric oxide, Dihydrorhodamine 123 (DHR) is specific for detecting hydrogen peroxide and Hydroethidine (HE) is specific for detecting superoxide. Oxidant production was measured after a 30 minute incubation period with the relative dyes on a flow cytometer. Auranofin and Asa-fin decreased hydrogen peroxide and superoxide production. None of the drugs had an effect on nitric oxide production. The expression of the â2-integrin adhesion molecule, CR3, on resting and PMA stimulated neutrophils treated with the experimental compounds was measured by flow cytometry. CR3 expression by neutrophils was measured after 10 minute incubation in the dark with CD11b FITC monoclonal antibody. Treatment of neutrophils with auranofin and the three experimental compounds showed a decrease in CR3 expression on resting and stimulated neutrophils, however the effect was more marked in stimulated neutrophils. The Anti-inflammatory activity of the experimental compounds was determined by testing the effects of the experimental compounds on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX 2) in resting and lipopolysaccharide (LPS) stimulated human monocytes. COX 1 and 2 production was measured by flow cytometry. Treatment of monocytes with the experimental compounds showed a decrease in COX 2 production in stimulated monocytes but an increase in COX 2 production in resting monocytes. No effect on COX 1 production was observed with the experimental compounds. Prostaglandin E2 (PGE2) was measured with a Prostaglandin E2 Enzymeimmunoassay (ELISA) kit on human macrophages. Auranofin, Asa-fin, Mpta-fin and Pta-fin inhibited the production of PGE2. Auranofin and Asa-fin inhibited the PGE2 directly proportional to the drug concentration. The effect of these drugs was also evaluated on various inflammatory cytokines using an inflammatory cytokine kit and measured on a flow cytometer. The cytometric bead array (CBA) human inflammation kit was used to quantitatively measure interlukin-8(IL-8), interlukin-1â (IL-1â), interlukin-6 (IL-6), interlukin-10 (IL-10), tumour necrosis factor alpha (TNFá) and interlukin-12p70 (IL-12p70). Auranofin and Asa-fin decreased IL 10, TNFá, and IL1â in stimulated cells. No effect was observed on IL 8, IL-12p70 and IL 6. With Mpta-fin and Pta-fin, no significant effect was observed in the cytokines tested. Drug toxicity was evaluated in mice using all four compounds in BALB/c inbred mice. Aspartate transaminase (AST), gamma glutamine transferase (GGT), urea and creatine levels were measured in the test mice. The group receiving the highest dose of Asa-fin showed the greatest elevation of AST . The lowest dose of the auranofin treatment group showed the greatest elevation in GGT, however this increase was not seen in the subsequent higher dosing groups. None of the treatment groups indicated an increase in urea levels. Mpta-fin and Pta-fin showed no increase in the liver enzymes or in urea and creatine. The results of this work are indicative that novel gold compounds could play a promising role in anti arthritic applications. Asa-fin exhibited similar anti-inflammatory activity to auranofin but in vivo toxicity was high. Mpta-fin showed slightly inferior anti-inflammatory activity to auranofin but in vivo toxicity profiles were much more promising. Pta-fin showed the least anti-inflammatory activity of the three novel compounds tested with a similar in vivo toxicity profile as Mpta-fin. / Dissertation (MSc)--University of Pretoria, 2009. / Pharmacology / unrestricted

Various plant species have been reported traditionally as well as in scientific literature for cytotoxicity against animal species. Isolation of several poisonous compounds from plant species has been reported previously. Elaeodendron croceum is a well-known poisonous plant species of which the poisonous compounds have not yet been isolated. A phytochemical investigation of E. croceum leaves guided by cytotoxicity against Vero cells, led to the isolation of five known compounds; 20-hydroxy-20-epi-tingenone (1), tingenone (2), tingenine B (3), 11α-hydroxy-β-amyrin (4), and naringenin (5). Compounds 1 and 2 showed the highest toxicity against Vero cells (IC50: 2.651 nM and 8.233 μM respectively). Cytotoxicity of the isolated compounds against three human cancer cell lines, HeLa, MCF-7, and SNO was also determined. Compounds 1 and 2 again showed the highest cytotoxicity with IC50 values ranging between 2.478 – 0.427 μM. This is the first report on the isolation, identification, and in vitro evaluation of poisonous compounds from E. croceum. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Production and Soil Science / unrestricted

The aim of this diploma thesis is the preparation of liposome nanoparticles enriched with PHA and PHA nanofibers. The nanostructures served to encapsulate extracts of lipophilic and hydrophilic nature. The characterization of the properties of nanostructures such as polydispersity, size, colloidal stability, long-term stability after exposure to various environments such as seawater, water from the Brno dam and tap water, and finally the cytotoxicity of fibers with extracts was addressed. The theoretical part is focused on different types of water, human skin, coffee and subsequently carotenoids. It continues by describing of possibilities of extraction and preparation of lipophilic and hydrophilic extracts and possibilities of their determination is discussed. Finally, it describes the possibilities of preparation and characterization of PHA based nanomaterials. The practical part deals with the preparation of liposome particles and fibers enriched with PHA with encapsulated extracts, their characterization, and their subsequent exposure to various environments. Monitoring of their long-term stability was carried out, but the release of the encapsulated extracts into the environment to which the nanoparticles and nanofibers were exposed was also measured. Finally, the interaction of nanofibers with live HaCaT cells was monitored, and cytotoxicity assays determined the viability of the cells after interaction with the nanofibers.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Recently nanoparticles (NPs) have been introduced and used in combination with therapeutic approaches to develop nanotechnology-enabled medicine. These nanostructures allow for the exploitation of the physiochemical properties which may be beneficial in cancer treatment. The use of NPs in nanomedicine has proven successful in modern chemotherapeutics and has demonstrated promising potential in in vivo and in vitro radiosensitization studies. This is a baseline study aimed to determine the cytotoxic effects of AuNPs for potential radiosensitization analysis. The study analysed the effects of different AuNP sizes (30, 50 and 80nm), concentrations (5, 10 and 15 μg/ml) over various time periods in CHOK1 and A549 cells. AuNPs were characterised by DLS and ZP analysis and showed that particles were moderately polydispersed and moderately to highly stable in charge. The effects on viability and metabolic activity of cells were determined using crystal violet and the WST-1 assay.

Certain organophosphorus compounds have the ability to induce a delayed neuropathic condition in sensitive species termed organophosphorus compound-induced delayed neurotoxicity (OPIDN). Esteratic changes associated with OPIDN have been successfully modeled in vitro. The physical characteristics of lesion development in OPIDN including the mode of nerve cell death, cytotoxic initiator and effector molecules, and cytoskeletal involvement have received little in vitro investigation. This dissertation evaluated the mode of cell death (apoptosis versus oncotic-necrosis), and cell cycle, cytoskeletal, nuclear, and mitochondrial alterations induced by OP compounds in SH-SY5Y cultures, an in vitro human neuroblastoma model. The distribution of in vivo neural degeneration in white Leghorn hen models was also assessed as a prelude to validating the mode of OP compound-induced in vivo neural cell death. These endpoints were evaluated by utilizing flow cytometry, spectrophotometry, gel electrophoresis, immunohistochemistry, light, and electron microscopy. Initial data gathered on culture parameters revealed that the mitotic status, basal rates of cell death, and total culture density were dependent on the condition of the media and the initial seeding density. Subsequent in vitro investigations used standardized culture conditions with OP compounds (diisopropylphosphorofluoridate (DFP), paraoxon, parathion, phenyl saligenin phosphate (PSP), tri-ortho-tolyl phosphate (TOTP), and triphenyl phosphite (TPPi); 1uM - 1mM). These studies revealed that OP compounds altered the cell cycle phase, decreased the amount of intracellular f-actin, altered the mitochondrial membrane potential, and induced caspase-3 activation and nuclear partitioning characteristic of apoptosis. The amount of change in these parameters was strongly dependent on the OP compound, the length of incubation time, and the presence of modulators of cytotoxicity such as phenylmethylsulfonyl fluoride (PMSF), carbachol, Ac-DEVD-CHO, Ac-IETD-CHO, and cyclosporin A. Preliminary in vivo experiments designed to validate in vitro results revealed neural degeneration involving fibers, terminals, and cell soma in spinal cord and brain tissue of PSP- and TPPi- exposed hens. The distribution and magnitude of these changes were contingent on the OP compound and length of time post-exposure. Subsequent experiments designed to evaluate the mode of cell death in these tissues revealed little evidence of either necrosis or apoptosis. These results, therefore, did not support or refute in vitro observations. Many OP compound-induced subcellular alterations have been demonstrated in our in vitro SH-SY5Y neuroblastoma model. Even though the mode of cell death observed in SH-SY5Y cells was not validated in in vivo experiments, in vitro observations nonetheless provide stimulating areas to further research the mechanisms of OPIDN and OP compound-induced cell death. / Ph. D.