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Genotoxicology involves the assessment of a substance’s ability to induce DNA damage after exposure to humans. DNA damage is an underlying cause of mutations that are likely to initiate carcinogenesis. Furthermore, the investigation of low dose responses in genotoxicology testing helps to improve health risk assessment by establishing whether DNA reactive compounds follow linear or non-linear (thresholded) dose response relationships. The current assumption for direct acting genotoxins is that the relationship between exposure to genotoxic chemicals, DNA damage formation and the induction of mutagenic changes is linear. However, it is known that mutations are not produced directly by DNA adducts as DNA repair activity limits the proportion of adducts processed into mutations. It is therefore possible, that no observed effect levels (NOEL) may exist for some genotoxins. The main aim of this thesis was to improve in vitro genotoxicity testing by assessing the low dose response relationships for the genotoxic agents mitomycin-C (MMC), 4-nitroquinoline 1-oxide (4NQO) and cytosine arabinoside (araC). Furthermore, the automated micronucleus slide scoring system Metafer was validated and used for these studies. In addition, the mechanism of action of each test component was further investigated by follow up experiments to gain a better understanding of the processes involved in this type of damage. The in vitro micronucleus assay for the detection of chromosomal damage revealed non­linear dose response relationships following low dose exposure of MMC and araC, while 4NQO revealed a weak clastogenic potential. The semi-automated scoring protocol for the Metafer-System proved to be a rapid and accurate system for scoring micronuclei. DNA repair plays most likely a major role in these non-linear responses by removing genetic damage induced at low levels. Furthermore, p53 was shown to be involved in the DNA damage response in human lymphoblastoid cells, through cell cycle delay and the induction of apoptosis. In addition, this work confirmed that a proper dosing regime, accurate toxicity measurements and the appropriate choice of cell type are cmcial criteria for defining the dose response relationships and the induction of genotoxicity and cytotoxicity.

No / In the past two decades comparative genomic hybridization (CGH) and array CGH have become crucial and indispensable tools in clinical diagnostics. Initially developed for the genome-wide screening of chromosomal imbalances in tumor cells, CGH as well as array CGH have also been employed in genotoxicology and most recently in toxicogenomics. The latter methodology allows a multi-endpoint analysis of how genes and proteins react to toxic agents revealing molecular mechanisms of toxicology. This chapter provides a background on the use of CGH and array CGH in the context of genotoxicology as well as a protocol for conventional CGH to understand the basic principles of CGH. Array CGH is still cost intensive and requires suitable analytical algorithms but might become the dominating assay in the future when more companies provide a large variety of different commercial DNA arrays/chips leading to lower costs for array CGH equipment as well as consumables such as DNA chips. As the amount of data generated with microarrays exponentially grows, the demand for powerful adaptive algorithms for analysis, competent databases, as well as a sound regulatory framework will also increase. Nevertheless, chromosomal and array CGH are being demonstrated to be effective tools for investigating copy number changes/variations in the whole genome, DNA expression patterns, as well as loss of heterozygosity after a genotoxic impact. This will lead to new insights into affected genes and the underlying structures of regulatory and signaling pathways in genotoxicology and could conclusively identify yet unknown harmful toxicants.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-08T02:37:18Z No. of bitstreams: 1 127110041.pdf: 1450512 bytes, checksum: bbda08f02d9cddbd1397c133ddee4bbe (MD5) / Made available in DSpace on 2015-05-08T02:37:18Z (GMT). No. of bitstreams: 1 127110041.pdf: 1450512 bytes, checksum: bbda08f02d9cddbd1397c133ddee4bbe (MD5) Previous issue date: 2014 / A utilização de produtos naturais na medicina popular como recurso terapêutico é uma tendência generalizada pela população brasileira. Esta tendência tem contribuído significativamente para o consumo não só de produtos naturais, como também de medicamentos fitoterápicos. Neste contexto, o Manjericão (Ocimum basilicum L.) surge como um destes recursos terapêuticos, sendo amplamente utilizado como planta ornamental e como condimento. Originário da Ásia tropical, hoje é cultivado no mundo todo. Entre os usos populares do O. basilicum L., está o emprego como antioxidante, anti-inflamatório, anti-cancerígeno, antimicrobiano, agente cardiovascular, fatores associados ao envelhecimento, dentre outros. Diversos metabólitos secundários com atividade antioxidante e anti-inflamatória já foram identificados nesta espécie e trabalhos relatam que o Ácido Rosmarínico é o composto biologicamente mais ativo. Sabendo da importância econômica e da enorme difusão mundial de seus usos, tanto na culinária, quanto na medicina popular, torna-se importante a comprovação dos efeitos do Manjericão, visando garantir sua eficácia e sua segurança, já que estudos de toxicidade para esta espécie são raros e usualmente não abordam aspectos genéticos. Este trabalho, portanto, teve como objetivo a avaliação da atividade antioxidante pelo teste da peroxidação lipídica, conteúdo de proteína carbonilada, vitamina C; atividade das enzimas Superóxido dismutase (SOD) e Catalase (CAT); análise dos parâmetros inflamatórios pela da avaliação de citocinas anti- inflamatórias e pró-inflamatórias, e a toxicidade genética do extrato hidroalcoólico frente a leucócitos humanos em cultura celular, através da viabilidade e proliferação celular, ensaio cometa, teste de micronúcleo e instabilidade cromossômica. Observou-se que o extrato etanólico de O. basilicum agiu como agente antioxidante reduzindo/revertendo os efeitos causados pelo peróxido de hidrogênio, ações essas que podem ser explicadas pela composição rica em polifenois e flavonoides do extrato, além de seu conteúdo de ácido rosmarínico, um importante antioxidante com atividade comprovada e descrita na literatura. Em relação aos parâmetros genotoxicológicos, para todos os testes, os resultados foram dose-dependentes. Enquanto que para os parâmetros inflamatórios, o extrato apresentou capacidade anti-inflamatória, onde o possível mecanismo envolvido ocorre pela interação de inibição das citocinas mediadoras pró- inflamatórias e o estímulo de citocinas mediadoras anti-inflamatórias. / The use of natural products in folk medicine as a treatment method is a genereal ternd for the Brazilian population. This trend has contributed significantly to the consumption not only of natural products, as well as herbal medicines. In this context, Basil (Ocimum basilicum L.) arises as one of these therapeutical resources. Basil is widely used as an ornamental plant and as a condiment. Original in tropical Asia, is now grown worldwide. Among the popular uses of Ocimum basilicum L., its employment as an antioxidant, anti-inflammatory, anti-carcinogenic, anti-microbial, cardiovascular agent, factors associated with aging, between others. Several secondary metabolites with antioxidant and anti-inflammatory activity have been identified in this species and several studies have reported that rosmarinic acid is the most biologically active compound. Knowing the economic importance and worldwide dissemination of their uses, both in cooking, as in folk medicine, it is important to prove the effects of Basil, to ensure its efficacy and safety, as toxicicty studies for this species are rare and usually do not approach genetic aspects. This study evaluated the antioxidant activity by testing lipid peroxidation, protein carbonyl content, vitamin C; activity of superoxide dismutase (SOD) and catalase (CAT); analysis of inflammatory parameters for the evaluation of anti-inflammatory and pro-inflammatory cytokines, and genetic toxicity of the hydroalcoholic extract in human white blood cells (WBC) using cell culture, through the analysis of cell viability and cellular proliferation, chromosome instability, comet assay and micronucleus test. With our results it is possible to verify that O. basilicum extract acts as an antioxidant and effectively reverts or subjugates the effects of a high oxidizing agent as hydrogen peroxide and, these actions are explained because its composition, which is rich in polyphenols and flavonoids besides several compound as Rosmarinic Acid, who have a well-known antioxidant activity. In toxicological tests, all results were dose-dependent. In the anti-inflammatory aspect, we show that our extract actually presents these properties and the mechanism involved in these particular actions are a composed interaction between the inhibition of pro-inflammatory mediator and the stimulation of anti-inflammatory cytokines.

<p>Hamilton Harbour is one of the most polluted sites on the Great Lakes, affected by airborne and sedimentary contamination as a result of both heavy vehicle traffic and thousands of kilograms of industrial steel emissions. Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous mutagenic byproducts of incomplete organic combustion; they are present at very high concentrations in the air and sediment of Hamilton Harbour. We quantified DNA mutation rates in three different nesting colonies of Double-crested Cormorants (<em>Phalacrocorax auritus</em>) using five microsatellite markers. These colonies were located at various distances from sources of PAHs and other contamination. We compared pollution-exposed and reference colonies, hypothesizing that cormorants living closest to pollution will have higher rates of germline microsatellite mutations than those living farther away from pollution sources. Using a pedigree approach, we identified mutations when chicks showed microsatellite alleles not found in either parent, and other explanations such as extra-pair parentage had been ruled out. Microsatellite mutation rates were 4.4 times higher at the Hamilton Harbour site closest to the industrial sources of PAH contamination than the other Hamilton Harbour site, and both were higher than the reference colony. Metabolites of the PAH benzo[a]pyrene in cormorant tissues from both Hamilton Harbour sites were identified by LC-MS/MS, demonstrating that cormorants in Hamilton Harbour are exposed to, and metabolizing, PAHs. Diet was not substantially different between the two Hamilton Harbour colonies when measured with regurgitated samples and fatty acid analysis. This suggests airborne pollution in Hamilton Harbour induced germline mutations in cormorants.</p> / Master of Science (MSc)