Polycyclic aromatic hydrocarbons (PAHs) are produced via the incomplete combustion of organic matter. They are ubiquitously present in the environment, and human exposures typically involve complex PAH mixtures in complex matrices (e.g., soil, urban air). Many PAHs are genotoxic carcinogens; exposures can augment cancer risk and reliable risk assessment of PAH mixtures is a regulatory concern. There is a paucity of in vivo genotoxicity information for most PAHs and PAH mixtures. Risk assessment of PAH mixtures assumes dose addition (i.e., additive, incremental contributions from each PAH); however, there is a lack of evidence to support this assumption. This thesis assessed the in vivo genotoxicity of 9 PAHs and 6 PAH mixtures following sub-chronic oral exposure of transgenic Muta™Mouse (i.e., adduct and lacZ mutant frequency across 5 tissues). The results revealed that PAHs and PAH mixtures induce significant levels of genetic damage; the mixtures induced very high levels of damage and mutations. Differences in the nature and magnitude of the effects in individual tissues appear to be related to the processes that govern PAH metabolism and the processing of genetic damage (e.g., repair and translesion synthesis). Scrutiny of the dose addition assumption revealed more-than-additive effects in tissues proximal to the exposure route (i.e., intestine, liver), but less-than-additive effects in distal tissues (i.e., bone marrow); however, discrepancies between the experimentally-observed and predicted responses were typically small (i.e., within 5-fold). Comparisons of cross-tissue patterns in adduct and mutant frequencies revealed that the frequency of the former is generally inversely related to that of the latter. This appears to be related to the experimental design, and the influence of repair and replication on adduct and mutant frequency. The BMD approach was employed to estimate genotoxic (i.e., adduct) potency and mutagenic (i.e., lacZ mutant) potency for all agent-tissue combinations. The results demonstrate that the mutagenic potency of PAHs and PAH mixtures is empirically related to genotoxic potency; moreover, that there is cross-tissue and cross-compound congruence in the processing of PAH-induced damage. The results obtained significantly advance existing knowledge regarding the genotoxic hazards of PAHs and PAH mixtures; moreover, the empirical relationships between genetic toxicity endpoints.

A Síndrome da Imunodeficiência Adquirida (AIDS) é doença causada pelo vírus da imunodeficiência humana. Apesar dos benefícios proporcionados pela terapia de supressão viral, algumas doenças como lipodistrofia, doenças cardiovasculares e infertilidade aumentaram a sua prevalência. No paciente com AIDS, considera-se a carga viral e o uso de terapia antirretroviral como possíveis agentes de genotoxicidade. Apesar do espermograma não poder detectar a causa precisa da infertilidade, o mesmo ainda é o exame mais realizado para averiguar a qualidade seminal. Por outro lado, a integridade do DNA do espermatozóide tem sido proposta como um parâmetro adicional de qualidade do sêmen. O ensaio cometa alcalino detecta a genotoxicidade de células germinativas humanas e pode ser usado para demonstrar a capacidade de uma substância interagir com o material genético das células gonadais. O presente estudo incluiu 50 pacientes com AIDS, atendidos no Ambulatório de Infectologia do Município de Caxias do Sul e no Ambulatório da Universidade de Caxias do Sul, sendo analisados dois grupos: usuário de terapia antirretroviral e naive. Realizou-se espermograma e o ensaio cometa alcalino, comparando com o número de linfócitos T CD4+, a carga viral, a idade, etc. Os dados demonstraram que o uso de antirretrovirais reduziu a genotoxicidade espermática, mas não se encontrou correlação com o espermograma. O presente trabalho demonstra que o ensaio cometa alcalino é um método eficiente para mensurar a genotoxicidade espermática dos pacientes portadores do vírus da imunodeficiência adquirida. / The Acquired Immune Deficiency Syndrome (AIDS) is a disease caused by the human immunodeficiency virus. Despite all the benefits provided by the viral suppression therapy, some diseases such as lipodystrophy, heart diseases and infertility increased their prevalence. In patients with AIDS, viral load and anti-retroviral therapy are possible agents for genotoxicity. The World Health Organization guidelines are reference for semen analysis and sperm DNA integrity has been proposed as an additionalparameter for semen quality and a potential predictor of fertility. The alkaline comet assay has the potential to detect genotoxic human germ cells and can be used to demonstrate the ability of a substance or metabolite to interact directly with the genetic material of gonadal cells. The study has included 50 patients with AIDS treated at the Outpatient Center for Infectious Diseases and at the University of Caxias do Sul Clinical Center, evaluated as two groups: one treated with anti-retroviral therapy and the other was a naive group. Semen and sperm genotoxicity were analysed by alkaline comet assay, comparing CD4 cell count, viral load, age and other factors of potential genotoxicity between the two groups. Data showed that the use of any anti-retroviral class has reduced sperm genotoxicity. This study shows that the alkaline comet assay is a eficcientmethod to measure sperm genotoxicity in patients infected with the human immunodeficiency virus.

Orientador: Luciane Dias de Oliveira / Banca: Jônatas Rafael de Oliveira / Banca: Luana Marotta Reis de Vasconcellos / Resumo: Stryphnodendron barbatiman (Vell.) Mart (barbatimão) tem sido utilizado popularmente como anti-inflamatório e antimicrobiano. Este estudo se propôs a realizar a análise fitoquímica do extrato de S. barbatiman (Vell.) Mart. (barbatimão) e verificar in vitro algumas de suas atividades biológicas, como: antimicrobiana, sobre culturas planctônicas e biofilmes de Fusobacterium nucleatum, Porphyromonas endodontalis, Porphyromonas gingivalis, e Parvimonas micra; citotoxicidade e genotoxicidade em macrófagos murinos (RAW 264.7), fibroblastos murinos (L929) e queratinócitos humanos (HACAT); e ação anti-inflamatória sobre RAW 264.7 estimuladas por lipopolissacarídeo (LPS) de Escherichia coli. A atividade antimicrobiana sobre culturas planctônicas foi avaliada pelo método de microdiluição em caldo. Após, as concentrações efetivas foram avaliadas sobre biofilmes monomicrobianos. A análise de citotoxicidade foi verificada pelo teste de redução do brometo de 3(4,5-dimetiltiazol-2-yl)2,5-difeniltetrazólio (MTT) e a de genotoxicidade pelo teste de micronúcleos. A atividade anti-inflamatória foi analisada quantificando os níveis de citocinas pró-inflamatórias (TNF-α, IL-1β, IL-6 e IL-17) e anti-inflamatória (IL-10), por enzyme-linked immunosorbent assay (ELISA). Os dados foram analisados por ANOVA e Teste de Tukey, ou Kruskal-Wallis e teste de Dunns, com nível de significância de 5% (P≤0,05). O extrato foi eficaz sobre os biofilmes, redução entre 54 e 100%. O extrato não foi citotóxico para R... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Anti-inflammatory and antimicrobial are popular uses of Stryphnodendron adstringens (Mart.) Coville (barbatimão). This study proposed to perform the phytochemical analysis of S. barbatiman (Vell.) Mart. (barbatimão) extract and to verify in vitro some of its biological activities as antimicrobial on planktonic cultures and biofilms of Fusobacterium nucleatum, Porphyromonas endodontalis, Porphyromonas gingivalis, and Parvimonas micra; cytotoxicity and genotoxicity on human keratinocytes (HACAT), murine macrophages (RAW 264.7) and fibroblasts (L929), and anti-inflammatory action on RAW 264.7. The antimicrobial activity on planktonic cultures was evaluated by broth microdilution. Afterwards, the effective concentrations were evaluated on mono microbial biofilms. The cytotoxicity analysis was verified by the MTT test and the genotoxicity by the micronucleus test. Anti-inflammatory activity was evaluated in RAW 264.7 stimulated by lipopolysaccharide (LPS) of Escherichia coli and treated with the extract, quantifying the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-17) and anti-inflammatory (IL-10), by enzyme-linked immunosorbent assay (ELISA). The data that presented normal distribution were analyzed by ANOVA and Tukey's test, and those that were not, were analyzed by Kruskal-Wallis and Dunns test, with a significance level of 5% (p≤0.05). Barbatimão extract was effective on biofilms, reduction between 54 and 100%. The extract was not cytotoxic to RAW 264.7 and ... (Complete abstract click electronic access below) / Mestre

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Assessment of chemical mutagenicity is essential to protecting human health from genetic disease. Current assays are limited in their ability to provide mechanistic insight into the endogenous and exogenous processes involved in mutagenesis. Duplex Sequencing (DS), a novel error-corrected sequencing technology, overcomes many of the limitations faced by conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. Furthermore, customizable panels enable assessment of the endogenous genomic features that drive mutagenesis. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. The objectives of this study were to demonstrate the potential of DS as a robust in vivo mutagenicity test and to explore its rich data to gain a better understanding of spontaneous and chemically-induced mutagenicity in somatic and germ cells. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) and germ cells of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and tissues were sampled at least 28 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in MF and distinct spectra consistent with the known mutagenic mechanisms of PRC in both tissues. Mouse PRC doses at which significant effects were observed are in range with those used for chemotherapy, suggesting that similar effects may be observed in human patients. This supports the contribution of PRC towards secondary cancers following treatment. DS results were comparable to those obtained using the gold-standard lacZ TGR assay, with DS showing greater sensitivity to detect smaller changes in MF. Analysis of mutation spectra and the genomic features that drive the mutational response revealed intrinsic differences between BM and germ cells that may underlie differences in endogenous mutagenic mechanisms and/or DNA repair pathways. The results suggest that germ cells may have intrinsic mechanisms to reduce mutation burden relative to somatic cells. While historically analysis of germ cell mutagenicity has been neglected in favour of somatic cells, our work supports the independent assessment of germ cell mutagenicity during regulatory testing. Finally, we conducted power analyses to inform the optimal DS study designs for the two tissues. We found that low intra-group variability within BM samples allows a reduction in sample size to three animals per group whilst still maintaining 80% power to detect an effect. In contrast, the relatively high intra-group variability and low background MF in germ cells suggests a minimum of eight animals per group to detect an effect. Overall, our results support the use of DS as a mutagenicity test and highlight many of the advantages it holds over conventional assays. Moreover, our study reveals the potential for mutagenic effects in PRC-treated cancer patients. Further work to test DS with more chemicals and across a wider range of tissues is recommended for future implementation as a mutagenicity test.

Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation presents a body of research that attempts to tackle the ‘overfitting’ problem for gene signature and biomarker development in two different aspects (mechanistically and computationally). In achievement of a deeper understanding of cancer molecular mechanisms, this study presents new approaches to derive gene signatures for various biological phenotypes, including breast cancer, in the context of well-defined and mechanistically associated biological pathways. We identified the pattern of gene expression in the cell cycle pathway can indeed serve as a powerful biomarker for breast cancer prognosis. We further built a predictive model for prognosis based on the cell cycle gene signature, and found our model to be more accurate than the Amsterdam 70-gene signature when tested with multiple gene expression datasets generated from several patient populations. Aside from demonstrating the effectiveness of dimensionality reduction, phenotypic dissection, and prognostic or diagnostic prediction, this approach also provides an alternative to the current methodology of identifying gene expression markers that links to biological mechanism. This dissertation also presents the development of a novel feature selection algorithm called Predictive Power Estimate Analysis (PPEA) to computationally tackle on overfitting. The algorithm iteratively apply a two-way bootstrapping procedure to estimate predictive power of each individual gene, and make it possible to construct a predictive model from a much smaller set of genes with the highest predictive power. Using DrugMatrix™ rat liver data, we identified genomic biomarkers of hepatic specific injury for inflammation, cell death, and bile duct hyperplasia. We demonstrated that the signature genes were mechanistically related to the phenotype the signature intended to predict (e.g. 17 out of top 20 genes for inflammation selected by PPEA were members of NF-kB pathway, which is a key pre-inflammatory pathway for a xenobiotic response). The top 4 gene signature for BDH has been further validated by QPCR in a toxicology lab. This is important because our results suggest that the PPEA model not largely deters the over-fitting problem, but also has the capability to elucidate mechanism(s) of drug action and / or of toxicity.

In vitro genetic toxicity tests using cultured bacterial or mammalian cells provide a cost- and time-effective alternative to animal tests. Unfortunately, existing in vitro assays are not always reliable. This is in part due to the limited metabolic capacity of the cells used, which is often critical to accurately assess chemical genotoxicity. This limited metabolic capacity necessitates the use of exogenous sources of mammalian metabolic enzymes that can simulate in vivo mammalian metabolic activation reactions. In response to this, and other limitations, alongside the worldwide trend to reduce animal testing, there is an acute need to consider various strategies to improve in vitro mutagenicity assessment. This thesis first examined the utility of exogenous metabolic activation systems based on human hepatic S9, relative to conventional induced rat liver S9, for routine genetic toxicity assessment. This was accomplished by critically evaluating existing literature, as well as new experimental data. The results revealed the limitations of human liver S9 for assessment of chemical mutagenicity. More specifically, the analyses concluded that, due to the increased risk of false negative results, human liver S9 should not be used as a replacement for induced rat liver S9. To address the limitations of conventional mammalian cell genetic toxicity assays that require exogenous hepatic S9, the thesis next evaluated the utility of an in vitro mutagenicity assay based on metabolically-competent primary hepatocytes (PHs) derived from the transgenic MutaMouse. Cultured MutaMouse PHs were thoroughly characterized, and found to temporarily retain the phenotypic attributes of hepatocytes in vivo; they express hepatocyte-specific proteins, exhibit the karyotype of typical hepatocytes, and maintain metabolic activity for at least the first 24 hours after isolation. Preliminary validation of the in vitro MutaMouse PH gene mutation assay, using a panel of thirteen mutagenic and non-mutagenic chemicals, demonstrated excellent sensitivity and specificity. Moreover, inclusion of substances requiring a diverse array of metabolic activation pathways revealed comprehensive metabolic competence. Finally, the thesis further investigated the applicability domain of the in vitro MutaMouse PH assay by challenging the assay with selected azo compounds. Comparison of these results with those obtained using the in vivo MutaMouse TGR (transgenic rodent) assay revealed that MutaMouse PHs can carry out some forms of reductive metabolism. Overall, this thesis demonstrated that a gene mutation assay based on MutaMouse PHs holds great promise for routine assessments of chemical mutagenicity.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-21T18:53:49Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) GABRIELA TAGLIANI LOPEZ.pdf: 1929634 bytes, checksum: 65b59bcc7ee2dd96fa3d6a96b37b9882 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-21T18:54:04Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) GABRIELA TAGLIANI LOPEZ.pdf: 1929634 bytes, checksum: 65b59bcc7ee2dd96fa3d6a96b37b9882 (MD5) / Made available in DSpace on 2016-09-21T18:54:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) GABRIELA TAGLIANI LOPEZ.pdf: 1929634 bytes, checksum: 65b59bcc7ee2dd96fa3d6a96b37b9882 (MD5) Previous issue date: 2015-08-14 / A leishmaniose é uma doença infecciosa que pode acometer a pele, as mucosas e os órgãos internos. Apesar desta doença atingir, anualmente, cerca de 1,5 milhão de pessoas em todo o mundo, ainda possui poucas alternativas terapêuticas. O fármaco recomendado pela Organização Mundial de Saúde (OMS) para o tratamento das leishmanioses é o antimoniato de N-metilglucamina (Sb-AMG). O tratamento com esse fármaco pode induzir o indivíduo a um quadro de toxicidade, já que é derivado do antimônio (Sb), um metal encontrado livremente na crosta terrestre, principalmente sob a forma trivalente e pentavalente de oxidação. A forma trivalente mostrou-se clastogênica e com potencial cancerígeno tanto in vitro quanto in vivo. Dentro dessa problemática, estudos envolvendo o Sb-AMG são escassos, levando-se em consideração parâmetros genotóxicos. Este trabalho, portanto, teve como objetivo, avaliar comparativamente os efeitos de cinco diferentes concentrações (5-50 μg/mL) de Sb-AMG e de SbV sobre parâmetros citotóxicos, genotóxicos e mutagênicos em leucócitos humanos. Para os testes de citotoxicidade e genotoxicidade aplicamos a análise estatística de variância (ANOVA) de uma via e para os testes de mutagenicidade a ANOVA de duas vias. Entre as múltiplas comparações utilizamos o teste de Tukey e consideramos um resultado estatisticamente significativo quando p<0,05. Testes de proliferação celular, instabilidade cromossômica, teste do cometa alcalino e ensaio de quebra de fita dupla de DNA, demonstraram que ambas as formas de Sb testadas foram capazes de induzir alterações genotóxicas significativas, quando comparado ao controle negativo em, pelo menos, uma das concentrações ensaiadas. Os testes de viabilidade celular e micronúcleo foram utilizados para avaliar, respectivamente, a citotoxicidade e a mutagenicidade. Adicionalmente, foi avaliado o número de células apoptóticas e necróticas, bem como o índice de divisão nuclear citotóxico. Todos esses parâmetros demonstraram significantes alterações. De uma forma sem precedentes, o nível de Sb intracelular foi quantificado e relacionado com as diferentes concentrações de exposição e, a partir disso, nós sugerimos que o influxo de Sb ocorre de forma concentração dependente, mas com cinética de ordem zero. Tomando conjuntamente os dados obtidos, os resultados evidenciam claramente que o Sb-AMG possui efeitos citotóxicos, genotóxicos e mutagênicos em leucócitos humanos nas concentrações testadas. / Leishmaniasis is an infectious disease that can affect the skin, mucosa and internal organs. Although affecting about 1.5 million people each year, leishmaniasis has few therapeutic alternatives. N-methylglucamine antimoniate (Sb-GAM) is the drug of choice to treat leishmaniasis according to the World Health Organization. The treatment with this drug can induce the individual to a frame of toxicity, since it is derived from the antimony (Sb), a metal free found in the earth's crust, principally in the form trivalent and pentavalent oxidation. The trivalent form has clastogenic and carcinogenic potential both in vitro and in vivo. However, there are few genotoxic studies involving Sb-GAM. The main contribution of this study was to evaluate in vitro the cytotoxic, genotoxic and mutagenic parameters of five different concentrations (5, 10, 20, 40 and 50 μg/mL) of Sb-GAM and SbV in human leukocyte. For the cytotoxicity and genotoxicity tests we apply statistical one-way analysis of variance (ANOVA) and for mutagenicity tests the two-way ANOVA. Among the multiple comparisons we used the Tukey test and consider a statistically significant result when p <0.05. Cell proliferation, chromosomal instability, the alkaline comet assay and double- stranded DNA breaks assays, showed that both forms of Sb were genotoxic when compared to the negative control, at least, in one of the concentrations tested. Cell viability and micronucleus tests were used to evaluate, respectively, cytotoxicity and the mutagenicity. Additionally, there was assessed the number of apoptotic and necrotic cells, as well as nuclear division cytotoxicity index. All these parameters showed significant changes. In an unprecedented strategy, the intracellular Sb level was quantified and correlated with the different exposure concentrations and, from this, we could induce that the Sb inflow occurs in a concentration-dependent, but with a zero-order kinetics. Altogether, our results show clearly that Sb-GAM has cytotoxic, genotoxic and mutagenic effects on human leukocytes at the concentrations tested.

DNA damage is important to understand since it has the potential to lead to disease if unrepaired. In particular, bulky C8 guanine adducts (addition products) are known to induce a variety of mutations due to their conformational flexibility. C-linked C8-phenoxyl-deoxyguanosine adducts (PhOH-dG) have been poorly understood despite their potential for genotoxicity. This thesis systematically develops a computational model to predict the conformational and base-pairing preferences of PhOH-dG by gradually increasing the size of the system. The structure of PhOH-dG in DNA is determined, where the bulky C8 group induces a syn conformation of the base similar to other C8-adducts. A stabilized guanine mismatch is identified for the syn adducts, which implies that the primary mechanism of genotoxicity may be base-substitution mutations resulting in G→C transversions. This thesis has contributed to a growing body of literature dedicated to understanding the role of conformational heterogeneity in the mutagenicity of bulky C8-adducts. / xix, 192 leaves : ill. (some col.) ; 29 cm