Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The use of toxicity tests to evaluate the quality of streams affected by mixtures such as acid mine drainage (AMD), adds value to assessments whereby site-specific toxicological data may identify toxicants that pose a threat to humans. To successfully evaluate the risk of combined mixtures, an improved understanding of the individual components, their uptake, metabolism, excretion and mode of action is required. This study aimed to identify the extent of AMD toxicity in a dose dependant manner on the MCF-7 cell line. The first study site associated with gold mining was chosen as the Tweelopies Stream situated in the Gauteng province of South Africa. The AMD effluent (un-neutralised) contaminating the Tweelopies Stream had undergone pH-neutralisation using a reactor-bed limestone technology incorporating the use of both calcium carbonate (CaCO3) powder and limestone beds. The second study site, the Kromdraai River, is situated in the eMalahleni region of South Africa where a predominance of coal mining exists. The pH -neutralisation of the AMD (un-neutralised) contaminated Kromdraai River was performed using a caustic soda (NaOH) precipitation technique. This study demonstrated the rapid and effective application of the comet assay as a screening tool for AMD-associated DNA breakages in the human cell line, MCF-7. Moreover, the study analysed parameters of cellular survival, DNA fragmentation and variations in morphologies indicative of cellular death. Collectively, the cyto-genetic aberrations observed in the MCF-7 cells as a result of exposure to gold and coal mining associated AMD, confirms the urgency of incorporating high-throughput screening in ecological toxicity assessment to evaluate cellular damage at genetic levels in low dose exposures where detection might be missed. / AFRIKAANSE OPSOMMING: Die gebruik van toksisiteitstoetse om die gehalte van strome te evalueer wat geraak word deur mengsels soos suur mynwater (SM), gee waarde aan spesifieke toksikologiese data van gifstowwe wat 'n bedreiging vir die mens kan identifiseer. Om die risiko van gekombineerde mengsels en hul individuele komponente beter te begrip en suksesvol evalueer, is hul opname, metabolisme, uitskeiding en modus van aksie nodig. Hierdie studie het gepoog om die omvang van SM-toksisiteit in 'n dosis afhanklike wyse op die MCF-7-sellyn te identifiseer. Die eerste studie-area wat gekies is, hou verband met goudmyn-ontginning, en is die Tweelopiesspruit, geleë in die Gauteng-provinsie van Suid-Afrika. Die SM-uitvloeisel (on-geneutraliseerde) wat die Tweelopiesspruit besoedel, het pH-neutralisasie ondergaan met behulp van die integrasie van 'n reaktor-bed kalksorpsietegnologie wat gebruik maak van beide kalsiumkarbonaat (CaCO3) poeier en kalksteenbeddens. Die tweede studie-area, is die Kromdraairivier geleë in die eMalahleni-streek van Suid-Afrika, waar steenkoolontginning die oorheersende aktiwiteit is. Die pH-neutralisasie van die SM (on-geneutraliseerde) in die geval van die Kromdraairivier word met behulp van 'n bytsoda (NaOH) neerslag tegniek, uitgevoer. Hierdie studie het die komeet-toets getoon as 'n vinnige en doeltreffende toepassing vir SM-geassosieerde DNA-breekskade in die menslike sel lyn, MCF-7. Verder het die studie parameters van sellulêre oorlewing, DNA-fragmentasie en variasies in sel morfologieë wat ‘n aanduiding van sellulêre dood is, ontleed. Gesamentlik dui die resultate daarop dat die sitogenetiese afwykings wat in die MCF-7-selle waargeneem is, as 'n gevolg van blootstelling aan goud- en steenkool-geassosieerde SM is. Die studie het verder die dringendheid van die integrasie van hoë-deurset tegnologieë in ekologiese toksisiteitstoetse in selle wat genetiese skade mag ondergaan, na 'n lae dosis blootstelling waar opsporing dalk gemis word, ondersteun.

Made available in DSpace on 2014-06-11T19:31:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-31Bitstream added on 2014-06-13T18:41:52Z : No. of bitstreams: 1 duarte_ra_dr_arafcf.pdf: 4649782 bytes, checksum: 99bc00374a88b89ba36ff755fc732fa3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O câncer de mama é a maior causa de morbidade e mortalidade entre as mulheres no mundo. Pesquisas revelam vários fatores prognósticos e preditivos para a identificação de pacientes com alto risco de agressividade, metastases e doença recorrente na condição de combater estas estatísticas. Por esta razão, é evidente a necessidade do desenvolvimento de estratégias de tratamento mais eficazes. Estudos prévios com Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), uma planta nativa do Brasil resultou o isolamento de dois alcalóides guanidínicos. Exibiram atividade seletiva direcionada a DNA deficiente de reparo, sugerindo potencial atividade anticâncer. Objetivo: O objetivo do presente estudo foi avaliar a citotoxicidade e apoptose induzidas pelos alcalóides pteroginina (PGN) and pteroginidina (PGD) em linhagem de adenocarcinoma (MCF-7) e carcinoma ductal invasivo (ZR-7531). Materiais e Métodos: As duas linhagens celulares foram tratadas pelos alcalóides em várias concentrações (0.25 – 10 mM) em dois tempos, t0 (24h) e t24 (24h seguido por 24h pós-tratamento). O ensaio de citotoxicidade foi determinado pelo teste de MTT; a morte celular (apoptose e necrose) foi analisada usando os métodos Hoechst 33342/iodeto propídio, Kit de Anexina V-FITC e atividade de Caspases 3/7. Resultados: Os tratamentos com os alcalóides demonstraram citotoxicidade concentração-resposta nas linhagens de câncer de mama. Para avaliação da apoptose foi observado um intense efeito concentraçãoresposta em apoptose tardia/necrose e discreto sinal para apoptose precoce em todas concentrações (p<0,01). No ensaio Hoechst/iodeto, observou diferença significante entre os estágios de apoptose precoce e tardia de ambas linhagens. A pteroginina no período t0 e t24, e pteroginidina no período t0 demonstraram possuir intenso efeito concentração... / Breast cancer is a major cause of morbidity and mortality among women worldwide. Research has elucidated several specific prognostic and predictive factors to identify patients at high risk of the aggressive disease, metastasis and recurrence of the disease in order to combat these statistics. For this reason, there is an obvious need to develop more efficacious treatment strategies. Previous studies on Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), a native plant of Brazil, resulted in the isolation of two guanidine alkaloids, exhibited selective activity towards a DNA repair-deficient, suggesting potential anti-cancer activity. Objective: The aim of the present study was to evaluate the citotoxicity and apoptosis induced by alkaloids pterogynine (PGN) and pterogynidine (PGD) in human adenocarcinoma cell line (MCF-7) and human invasive ductal carcinoma cell line(ZR-7531). Material and Methods: The two cell lines were treated by both alkaloids at several concentrations (0.25 – 10 mM) and two time points, 24h (t0) and 24h followed by 24h pos-treatment (t24). The cytotoxicity assay was determined by MTT assay; the cell death (apoptosis and necrosis) were analyzed using the dye Hoechst 33342/propidium iodide, Annexin V-FITC and Caspase 3/7 activity. Results: The treatments with the alkaloids demonstrated citotoxicity effect concentrationresponse in breast cell lines. Apoptosis evaluation, pterogynine and pterogynidine has an intense effect concentration-response of late apoptosis/necrosis and a discrete signal of early apoptosis in all of the concentrations (p<0.01). Hoechst/iodide assay, it was observe significant difference among the stages of early and late apoptosis in the both cells lines. Pterogynine for the period of t0 and t24, and pterogynidine for the period of t0 demonstrated to possess an intense concentrationresponse... (Complete abstract click electronic access below)

Orientador: Christiane Pienna Soares / Banca: Christiane Pienna Soares / Banca: Haroldo Wilson Moreira / Banca: Ana Marisa Fusco Almedia / Banca: Raquel Alves dos Santos / Banca: Maria Regina Torqueti Toloi / Resumo: O câncer de mama é a maior causa de morbidade e mortalidade entre as mulheres no mundo. Pesquisas revelam vários fatores prognósticos e preditivos para a identificação de pacientes com alto risco de agressividade, metastases e doença recorrente na condição de combater estas estatísticas. Por esta razão, é evidente a necessidade do desenvolvimento de estratégias de tratamento mais eficazes. Estudos prévios com Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), uma planta nativa do Brasil resultou o isolamento de dois alcalóides guanidínicos. Exibiram atividade seletiva direcionada a DNA deficiente de reparo, sugerindo potencial atividade anticâncer. Objetivo: O objetivo do presente estudo foi avaliar a citotoxicidade e apoptose induzidas pelos alcalóides pteroginina (PGN) and pteroginidina (PGD) em linhagem de adenocarcinoma (MCF-7) e carcinoma ductal invasivo (ZR-7531). Materiais e Métodos: As duas linhagens celulares foram tratadas pelos alcalóides em várias concentrações (0.25 - 10 mM) em dois tempos, t0 (24h) e t24 (24h seguido por 24h pós-tratamento). O ensaio de citotoxicidade foi determinado pelo teste de MTT; a morte celular (apoptose e necrose) foi analisada usando os métodos Hoechst 33342/iodeto propídio, Kit de Anexina V-FITC e atividade de Caspases 3/7. Resultados: Os tratamentos com os alcalóides demonstraram citotoxicidade concentração-resposta nas linhagens de câncer de mama. Para avaliação da apoptose foi observado um intense efeito concentraçãoresposta em apoptose tardia/necrose e discreto sinal para apoptose precoce em todas concentrações (p<0,01). No ensaio Hoechst/iodeto, observou diferença significante entre os estágios de apoptose precoce e tardia de ambas linhagens. A pteroginina no período t0 e t24, e pteroginidina no período t0 demonstraram possuir intenso efeito concentração... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Breast cancer is a major cause of morbidity and mortality among women worldwide. Research has elucidated several specific prognostic and predictive factors to identify patients at high risk of the aggressive disease, metastasis and recurrence of the disease in order to combat these statistics. For this reason, there is an obvious need to develop more efficacious treatment strategies. Previous studies on Pterogyne nitens Tul. (Fabaceae-Caesalpinioideae), a native plant of Brazil, resulted in the isolation of two guanidine alkaloids, exhibited selective activity towards a DNA repair-deficient, suggesting potential anti-cancer activity. Objective: The aim of the present study was to evaluate the citotoxicity and apoptosis induced by alkaloids pterogynine (PGN) and pterogynidine (PGD) in human adenocarcinoma cell line (MCF-7) and human invasive ductal carcinoma cell line(ZR-7531). Material and Methods: The two cell lines were treated by both alkaloids at several concentrations (0.25 - 10 mM) and two time points, 24h (t0) and 24h followed by 24h pos-treatment (t24). The cytotoxicity assay was determined by MTT assay; the cell death (apoptosis and necrosis) were analyzed using the dye Hoechst 33342/propidium iodide, Annexin V-FITC and Caspase 3/7 activity. Results: The treatments with the alkaloids demonstrated citotoxicity effect concentrationresponse in breast cell lines. Apoptosis evaluation, pterogynine and pterogynidine has an intense effect concentration-response of late apoptosis/necrosis and a discrete signal of early apoptosis in all of the concentrations (p<0.01). Hoechst/iodide assay, it was observe significant difference among the stages of early and late apoptosis in the both cells lines. Pterogynine for the period of t0 and t24, and pterogynidine for the period of t0 demonstrated to possess an intense concentrationresponse... (Complete abstract click electronic access below) / Doutor

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