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Master's Dissertation
DOI
Document
Author
Full name
Amanda Lucila Medeiros da Silva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2010
Supervisor
Committee
Loureiro, Ana Paula de Melo (President)
Cruz, Aurea Silveira
Nascimento, Elizabeth de Souza
Title in Portuguese
Mecanismos de toxicidade do conteúdo de sinalizadores luminosos (light-sticks) -- formação de adutos com DNA e dano oxidativo em células em cultura
Keywords in Portuguese
8-oxodGuo
Aduto de DNA
Células HepG2
Citotoxicidade
Estresse oxidativo
Light sticks
Abstract in Portuguese
Bastões plásticos quimioluminescentes, chamados light sticks, são usados por companhias pesqueiras e descartados nas praias. Moradores locais utilizam seus conteúdos como repelentes, óleo bronzeador e medicamento para dores nas articulações. Nós investigamos a reatividade das soluções de bastões de light sticks coletados em praias brasileiras e a toxicidade celular de seus conteúdos e também de soluções de light stick de bastões novos. Produtos da reação dos conteúdos de light stick descartado com 2'-desoxiguanosina (dGuo) foram analisados por HPLC/UV/ESI-MS/MS. Um aduto foi purificado e caracterizado em espectrômetro de massas por Dissociação Induzida por Colisão (CID) e através de experimentos de 1H RMN. A estrutura do aduto revelou que o produto de degradação de bis(triclorofenil)oxalato é reativo para nucleófilos in vitro. O mesmo aduto foi detectado em DNA de timo de bezerro incubado in vitro com a solução do light stick descartado pelo uso HPLC/ESI-MS/MS. Além do DNA, albumina também foi modificada pelos conteúdos de light stick descartado. Células HepG2 foram incubadas por 16 h com 0.0125% - 0.12% (v/v) das soluções de light sticks: (i) coletadas na praia, (ii) obtidas imediatamente após a reação quimioluminescente no laboratório, e (iii) previamente a reação, contendo ou n-butil-ftalato, difenilantraceno, e bis(triclorofenil)oxalato (solução 1), ou dimetil-ftalato, H2O2, e salicilato de sódio (solução 2). A sobrevivência celular foi avaliada pelo teste do XTT, corante cristal violeta e lactato desidrogenase realizados em placas de 96 poços. Com as concentrações testadas foram obtidas significativamente a morte celular. O dano oxidativo ao DNA celular foi avaliado pela análise da 8-oxo-2'-desoxiguanosina através do equipamento HPLC/ESI-MS/MS, que revelou aumento de alterações nas células tratadas com 0.006% das soluções de light stick de bastões descartados e novos. Nossos dados apontam genotoxicidade e citotoxicidade das soluções de light sticks e podem contribuir como alerta a autoridades públicas para proibição do uso incontrolado.
Title in English
Mechanisms of toxicity of the contents of beacons (light-sticks) DNA addcts formation and oxidative damage in cultured cells
Keywords in English
8-oxodGuo
Cytotoxicity
DNA adduct
HepG2 cells
Light sticks
Oxidative stress
Abstract in English
Chemiluminescent plastic rods, called light sticks, are used by fishery companies and littered on the shores. Local inhabitants use their contents as repellents, tanning oil, and medicine for joint pain. We have investigated the reactivity of spent light stick solutions collected on brazilian beaches and the cellular toxicity of their contents as well as that of brand-new light stick solutions. Products of the reaction of the spent light stick contents with 2'-deoxyguanosine (dGuo) were analyzed by HPLC/UV/ESI-MS/MS. A dGuo adduct was purified and characterized in a Collision Induced Dissociation (CID) mass spectrometer and through 1H NMR experiments. The adduct structure revealed that a degradation product of bis(trichlorophenyl)oxalate is reactive towards nucleophiles in vitro. The same adduct was detected in calf thymus DNA incubated in vitro with spent light stick solution, by using HPLC/ESI-MS/MS. Besides DNA, albumin was also modified by spent light stick contents. HepG2 cells were incubated for 16 h with 0.0125% 0.12% (v/v) of light stick solutions: (i) collected on the beaches, (ii) obtained immediately after the chemiluminescent reaction in the laboratory, and (iii) previously the reaction, containing either n-butyl-phthalate, diphenylanthracene, and bis(trichlorophenyl)oxalate (solution 1), or dimethyl-phthalate, H2O2, and sodium salicylate (solution 2). Cell survival was evaluated by the XTT, crystal violet dye, and lactate dehydrogenase assays performed in 96 well plates. The concentrations tested were found to significantly kill cells. Oxidative damage to cellular DNA was assessed by 8-oxo-2'-deoxyguanosine analysis via an HPLC/ESI-MS/MS equipment, which revealed increased changes in cells treated with 0.006% of spent and brand-new light stick solutions. Our data point to important genotoxicity and cytotoxicity of the light stick solutions and may contribute to alert public policies to ban their uncontrolled use.
 
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Publishing Date
2011-06-03
 
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