Complex chemical mixtures are ubiquitous in the environment. Humans are frequently exposed to these mixtures; therefore, it is important to understand potential interactions of chemical mixtures. Mixture interactions may influence the absorption, distribution, metabolism or excretion of the components of a complex mixture. The research conducted for this dissertation has coupled chemical fractionation with in vitro and in vivo bioassays to assess the potential carcinogenic risk of complex mixtures. A non-aqueous phase liquid from a wood treatment plant was separated into acid (AF), base (BF) and neutral fractions (NF). The NF was further enriched using column chromatography to produce a polychlorinated dinbenzo-p-dioxin (PCDD) and a polycyclic aromatic hydrocarbon (PAH) fraction. The genotoxicity of these mixtures were assessed via analytical quantification, in vitro (Salmonella microsome and E. coli prophage induction) and in vivo (32P-postlabeling) bioassays. The NF was further tested to measure bulky DNA adducts and induction of tumor formation. The AF contained the highest level of pentachlorophenol and the highest concentration of total PAHs. Although the carcinogenic PAHs were highest in the PCDD fraction, the highest concentrations of benzo(a)pyrene (BAP), indeno(1,2,3-cd)pyrene and dibenz(a,h)anthracene were detected in the PAH fraction. A positive genotoxic response in Salmonella was induced by the crude extract, the PAH and BF, whereas the AF and BF induced a positive response in the E. coli assay. In vivo, the PAH fraction induced the highest DNA adduct frequencies in the lung. The NF, reconstituted mixture (RM) (which includes equivalent concentrations of seven carcinogenic PAHs in the NF), BAP and the NF amended with BAP (NF+BAP) were all tested in an infant mouse model. At the highest dose, after a 24 hr exposure, NF+BAP had the highest total DNA adducts measured in liver which was three to seven times higher than with other treatments. Adduct levels were comparable to the control after 280 days. The highest incidence of tumors was observed in the liver. At the high dose, NF+BAP elicited the highest incidence of tumors. The results of this research confirm previous studies and indicate that the carcinogenic potential of PAH mixtures may be greater than predicted by chemical analysis.

No description available.

Recent studies have highlighted the poor specificity of a battery of in vitro genotoxicity tests when predicting rodent carcinogenicity, in some cases leading to misleading predictions of genotoxic carcinogenicity. GreenScreen HC (GSHC) is a highly sensitive human cell-based genotoxicity assay that, in contrast to the battery in vitro mammalian cell tests, also exhibits high specificity. GSHC employs the response of the human DNA damage-inducible gene GADD45a as a marker of genotoxic stress, using a GFP reporter construct hosted by the TK6 cell line. In order to better understand the biological basis for the high specificity of GSHC, three approaches were taken. Firstly, the relevance of the choice of a p53-competent host cell line, TK6, was investigated. A database was compiled consisting of published genotoxicity data from the in vitro battery tests performed in TK6 cells, and comparative GSHC data generated. This work revealed that discordance existed between the tests analysed, therefore the specificity of GSHC was not dictated by the choice of cell line alone. To confirm that misleading positive results could still be generated in the GSHC TK6 cell line, mutation assays were performed for compounds with pre-existing misleading positive battery test data, and a dose-dependent increase in mutation frequency was observed for 1 of 3 compounds. Secondly, the importance of wild-type p53 to the specificity of GSHC was investigated by generating p53-deficient GADD45a-GFP reporter cell lines. This work identified wild-type p53 as a key regulator of GADD45a in the context of GSHC, with reduced expression levels observed in response to genotoxic stimuli. Finally NF-kB, a putative direct regulator of GADD45a in response to DNA damage, was shown to contribute to the expression of GADD45a at high doses of 4-nitroquinoline-N-oxide via mutational analysis of two NF-kB binding sites in the promoter and intron 3 of the GADD45a-GFP reporter. This implicates NF-kB in the direct regulation of GADD45a in response to genotoxic stress in the context of GSHC. The work presented here demonstrates that the high specificity of GSHC is not dictated solely by the use of the p53-competent TK6 cell line, but is due to the use of a unique biosensor of genotoxic stress; GADD45a expression. Interference with the function of two key stress response signalling nodes, p53 and NF-kB, revealed that these regulators dictate the magnitude of expression of GADD45a, directly impacting upon the specificity of GSHC.

No description available.

nÃo hà / A exposiÃÃo ocupacional em ambientes de curtumes tem sido objeto de estudo e de investigaÃÃes cientÃficas. As informaÃÃes controversas sobre a toxicidade do cromo III e sua relaÃÃo com a epidemiologia do curtume serviram de subsÃdio para esta pesquisa. O objetivo deste estudo foi avaliar os possÃveis efeitos genotÃxicos e mutagÃnicos da exposiÃÃo de trabalhadores de curtume de Teresina (PI) à substÃncias quÃmicas contendo cromo III atravÃs do uso de biomarcadores de danos ao DNA e aos cromossomos.Trata-se de uma investigaÃÃo transversal, observacional e quantitativa. Foram coletadas amostras de mucosa, sangue e urina de 43 trabalhadores de curtume para anÃlise de micronÃcleo em mucosa (20 trabalhadores), micronÃcleo em linfÃcitos (30 trabalhadores), cometa em mucosa (20 trabalhadores), cometa em linfÃcitos (43 trabalhadores), aberraÃÃes cromossÃmicas (30 trabalhadores) e mensuraÃÃo de cromo na urina (27 trabalhadores) . As mesmas anÃlises foram realizadas em trabalhadores nÃo expostos ao ambiente do curtume, para fins de comparaÃÃo estatÃstica. AlÃm disso, foi aplicado um questionÃrio de saÃde pessoal recomendado pela International Comission for Protection Against Environmental Mutagens and Carcinogens-ICPEMC com intuito de correlacionar suas informaÃÃes aos dados obtidos nos testes. Os resultados indicaram aumentos significativos (p<0,05) do nÃmero (8,100  3,905 vs1,200 1,609) e frequÃncia de micronÃcleo em mucosa (0,403 0,1513vs 0,060 0,04887), do nÃmero (17,73  9,44vs10,90 7,092) e frequÃncia de micronÃcleos em linfÃcitos(0,886 0,472vs 0,306 0,202), para o total (16,3 3,38 vs 0,60  0,16) e frequÃncia (8,01  1,82 vs 0,403 0,09) de aberraÃÃes cromossÃmicas(teste T Student). AlÃm disso, foi observado aumento sigficativo (ANOVA p<0,05 ) para o todas as classes de danos e frequÃncia de danos no teste de cometa em mucosa e em linfÃcitos. O cromo na urina indicou que os trabalhadores estavam com maior concentraÃÃo deste metal na urina que os nÃo expostos (p<0,05 ) ( 1,108  1,277vs 0,089  0,027). As correlaÃÃes (Pearson) entre o cromo na urina e as anÃlises genotÃxicas indicaram forte relaÃÃo positiva com a frequÃncia de aberraÃÃes cromossÃmicas (r=0,841 p= 0,018 ) e frequÃncia de micronÃcleo em linfÃcitos (r=0,730 p < 0,0001).Conclui-se que os trabalhadores de curtume apresentam riscos de toxicidade e de instabilidade genÃtica associados à exposiÃÃo ocupacional a misturas quÃmicas contendo crmo III. / Occupational exposure in tannery environments has been the subject of study and scientific research. Controversial information about the toxicity of chromium III and its relationship with the epidemiology of the tannery employees served as a subsidy for this research. The objective of this study was to evaluate the possible genotoxic and mutagenic effects of the exposure of tannery workers in Teresina (PI) to chemicals containing chromium III through the use of biomarkers of DNA and chromosomal damage. This was a cross-sectional, observational and quantitative study. Samples were taken from the mucosa, blood and urine of 43 tannery workers to analyze mucosal micronuclei (20 workers), lymphocytic micronuclei (30 workers), mucosal comet assays (20 workers), lymphocytic comet assay (43 workers) and chromosomal aberrations (30 workers); the level of urinary chromium was also measured (27 employees).The same analyses were carried out in non-exposed workers of the tannery environment for statistical comparison. In addition, personal health questionnaires were administered as recommended by the International Commission for Protection Against Environmental Mutagens and Carcinogens-ICPEMC with the aim of correlating their information with data obtained in the tests. The results indicated significant increases (p <0.05) in: the number (8.100  3.905 vs 1.200  1.609) and frequency (0.403  0.060 vs. 0.1513  0.04887) of mucosal micronuclei; the number (17.73  9.44 vs 10.90  7.092) and frequency (0.886  0.472 vs 0.306  0.202) of lymphocytic micronuclei; and the total (16.3  3.38 vs 0.60  0.16) and frequency (8.01  1.82 vs 0.403  0.09) of chromosomal aberrations (Studentâs t test). There was a significant increase (ANOVA, p <0.05) for all types of damage and frequency of damage to the mucosal and lymphocytic COMET assay. Urine chromium levels was higher in exposed workers than in non-exposed workers (p <0.05) (1.108  1.277 vs 0.089  0.027).Pearson correlations between the level of chromium in the urine and genotoxic analyses indicated a strong positive correlation with the frequency of chromosomal aberrations (r = 0.841, p = 0.018) and frequency of lymphocytic micronuclei (r = 0.730, p <0.0001). It was concluded that the tannery workers are at risk of toxicity and genetic instability associated with occupational exposure to chemical mixtures containing chromium III.

Genetic toxicity tests used for regulatory screening must be rigorously validated to ensure accuracy, reliability and relevance. Hence, prior to establishment of an internationally-accepted test guideline, a new assay must undergo multi-stage validation. An in vitro transgene mutagenicity assay based on an immortalized cell line derived from MutaMouse lung (i.e., FE1 cells) is currently undergoing formal validation. FE1 cells retain a lacZ transgene in a λgt10 shuttle vector that can be retrieved for scoring of chemically-induced mutations. This work contributes to validation of the in vitro transgene (lacZ) mutagenicity assay in MutaMouse FE1 cells. More specifically, the work includes an intra-laboratory variability study, and a follow-up study to assess the endogenous metabolic capacity of FE1 cells. The former is essential to determine assay reliability, the latter to define the range of chemicals that can be reliably screened without an exogenous metabolic activation mixture (i.e., rat liver S9). The intra-laboratory variability assessment revealed minimal variability; thus, assay reproducibility can be deemed acceptable. Assessment of metabolic capacity involved exposure of FE1 cells to 5 known mutagens, and subsequent assessment of changes in the expression of genes involved in xenobiotic metabolism; induced transgene mutant frequency (±S9) was assessed in parallel. The results revealed that the FE1 cell line is capable of mobilising several Phase I and Phase II gene products known to be involved in the bioactivation of mutagens. Collectively, the results presented support the contention that the FE1 cell mutagenicity assay can be deemed reliable and reproducible. Consequently, the assay is an excellent candidate for continued validation, and eventual establishment of an OECD (Organization for Economic Cooperation and Development) Test Guideline.

Corantes são utilizados na coloração de diferentes substratos, incluindo papel, couro e plásticos, mas o uso mais importante é o têxtil e 1 a 5% destes corantes podem ser descartados no ambiente. Em geral, os corantes do tipo azo são tóxicos para os organismos aquáticos e alguns tipos de corantes podem ser mais tóxicos que outros. Mas, embora estes compostos e seus produtos de transformação reduzidos e/ou clorados podem ser encontrados no ecossistema aquáticos, não existem dados sobre genotoxicidade em organismos aquáticos até o momento. Muitos estudos têm demonstrado que avaliar danos ao DNA representa um biomarcador de exposição muito sensível em espécies aquáticas, que pode ser estudado utilizando ensaios in vivo e in vitro, como no caso das linhagens de células de peixe. Os objetivos deste trabalho foram: avaliar a ocorrência de corantes dispersos em amostras ambientais; avaliar a mutagenicidade dessas amostras utilizando o ensaio de Salmonella/microssoma com as linhagens TA98 e YG1041, e a genotoxicidade com o ensaio do cometa em culturas celulares de peixe RTL-W1. HPLC-MS/MS foi utilizada para verificar a ocorrência de corantes em amostras do Rio Piracicaba à montante e à jusante do Ribeirão Quilombo e do descarte de uma Estação de Tratamento de Efluentes (ETE), localizados no Estado de São Paulo, Brasil. Foram detectados seis corantes dispersos nas amostras de águas superficiais e efluentes. O corante Disperse Red 1 foi o composto mais frequente, detectado em 8 das 16 amostras, porém sua contribuição para a mutagenicidade total foi baixa; os corantes Disperse Blue 373 e Disperse Violet 93 foram os que mais contribuíram. A genotoxicidade do Rio Piracicaba, avaliada pelo ensaio de Salmonella/microssoma e ensaio do cometa, aumentou após o lançamento do Ribeirão Quilombo e do efluente ETE, mostrando uma possível contribuição destes na genotoxicidade do Rio Piracicaba. / Dyes are used in the coloration of different substrates, including paper, leather and plastics, but the most important use is on textiles and 1 to 5% of these dyes might be lost into the environment. Azo dyes are the most important class, accounting for over 50% of all commercial dyes, and this class has been the most studied. In general, azo dyes are toxic to aquatic organisms and some types of dyes are more toxic than others. But although these compounds as well as their reduced/chlorinated transformation products can be found in aquatic ecosystems, no mutagenicity data are available until now in aquatic organisms. This remark remains of value, as well, regarding genotoxicity potential of such dyes towards aquatic organisms. Many studies have demonstrated that DNA damage measurement represents a very sensitive biomarker of exposure in aquatic species that can be studied both in vivo and in vitro using for example fish cell lines. The objectives of this work were evaluate the occurrence of disperse dyes in environmental samples; evaluate the mutagenicity of this samples using the Salmonella/microsome assay with strains TA98 and YG1041; evaluate the genotoxicity using the comet assay with fish cell lines RTL-W1. HPLC-MS/MS was used to verify the occurrence of dyes in samples of Piracicaba River upstream and downstream the discharge of Quilombo River and Wastewater Treatment Plant (WWTP) effluent, located in São Paulo State, Brazil. Six dyes were detected in samples of water and effluents. Disperse Red 1 dye was detected in 8 of 16 samples, but its contribution for the mutagenicity was low. Disperse Blue 373 and Disperse Violet 93 were the major contributors for the mutagenicity found in the samples. The genotoxicity of Piracicaba River, evaluated with Salmonella/microsome assay and comet assay, increased after the discharges of Quilombo River and the effluent of WWTP, showing a contribution of this discharges on the river genotoxicity.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-13Bitstream added on 2014-06-13T21:01:39Z : No. of bitstreams: 1 fernandes_tcc_dr_rcla.pdf: 2200644 bytes, checksum: 409c85d73c03ec2d26288cc8ef322eaa (MD5) / O ramnolipídio é um biossurfactante da classe dos glicolipídios, produzido pela bactéria Pseudomonas aeruginosa. Estudos têm mostrado que, mesmo com um grande potencial de uso do ramnolipídio na indústria cosmética e na área da saúde, este composto é, principalmente, promissor para uso na indústria do petróleo. Apesar dos biossurfactantes serem considerados menos tóxicos e mais biodegradáveis que os surfactantes sintéticos, estes compostos ainda não foram investigados quanto a sua ação genotóxica direta ou indireta sob o material genético de organismos expostos. Desta maneira, o objetivo deste trabalho foi investigar os possíveis danos celulares promovidos pela ação do biossurfactante ramnolipídio e suas implicações no material genético quando utilizado como agente biorremediador de águas impactadas por petróleo, por meio dos bioindicadores Allium cepa, Oreochromis niloticus e as células humanas mantidas em cultura - HepG2. Para a realização da investigação, o biossurfactante ramnolipídio foi produzido em condições de laboratório e a partir da concentração indicada para o uso em processos de biorremediação (1g/L), foram estabelecidas as concentrações utilizadas no estudo. Para avaliar o potencial tóxico, genotóxico e mutagênico das concentrações de ramnolipídio, assim como seu feito sinérgico com o petróleo, foram realizados os testes de germinação de sementes, aberrações cromossômicas e teste do micronúcleo em A. cepa; teste do micronúcleo em eritrócitos circulantes, ensaio do cometa com sangue periférico e células de brânquias e análise ultraestruturais de eritrócitos circulantes em O. niloticus; e teste do MTT, teste do micronúcleo e ensaio do cometa em células HepG2. Nossos resultados mostraram que as concentrações 1g/L e 2g/L de ramnolipídio são tóxicas para A. cepa. No entanto, quando as concentrações 1g/L e 10g/L... / Rhamnolipids belong to the glycolipid class of biosurfactants, which are produced by the bacterium Pseudomonas aeruginosa. Studies have shown that, even being potentially used in the cosmetics industry, this compound is especially promising in the petroleum industry. Although the biosurfactants are considered less toxic and more biodegradable than the synthetic surfactants, these compounds have not yet been investigated when it comes to their either direct or indirect genotoxic action upon the genetic material of exposed organisms. Thus, the purpose of this study was to investigate the possible cell damage promoted by the action of rhamnolipid biosurfactants and their implications for the genetic material when they are used as a bioremediation agent of petroleum-impacted water, by using Allium cepa, Orechromis niloticus and human cells kept in culture – HepG2. In order to carry out this investigation, the rhamnolipid biosurfactant was produced under laboratory conditions, and the recommended concentration for use in bioremediation processes (1g/L) was also used; the concentrations used in the study were established. To assess the toxic, genotoxic and mutagenic potential of rhamnolipid concentrations, as well as their synergy with petroleum, seed germination, chromosome aberration tests and micronucleus test in A. ceppa; micronucleus test in circulating red blood cells, comet assay in the peripheral blood and gill cells, and ultrastructural analysis of circulating blood cells in O. niloticus; and MTT test, micronucleus test and comet assay in HepG2 cells. Our studies showed that the 1 g/L and 2 g/L rhamnolipid concentrations are toxic to A. cepa. However, when 1 g/L and 10 g/L concentrations were used in the bioremediation process, these concentrations did not cause toxic, genotoxic or mutagenic damage besides the ones observed for the cells exposed to soluble petroleum... (Complete abstract click electronic access below)

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Alpinia zerumbet, conhecida popularmente como colÃnia no Nordeste do Brasil, Ã uma planta medicinal usada amplamente na medicina popular na forma de chÃs e infusÃes para o tratamento de doenÃas cardiovasculares, como a hipertensÃo arterial. O intenso consumo popular dessas infusÃes levou-nos a avaliar o perfil toxicolÃgico e genotoxicolÃgico do extrato aquoso e do Ãleo essencial das folhas de A. zerumbet. Esse estudo foi avaliado pelos ensaios de curta duraÃÃo in vivo e in vitro. Inicialmente foi avaliada a citotoxicidade e o efeito hemolÃtico in vitro, porÃm nÃo houve resposta tÃxica. A DL50 encontrada para o extrato aquoso foi >5 g/Kg, demonstrando que os princÃpios ativos do extrato apresentam baixa toxicidade. Os estudo de genotoxicidade foram realizados in vivo. Os animais foram tratados, por via oral, com trÃs doses do extrato aquoso (2 g/Kg, 3,5 g/Kg e 5 g/Kg) e com a dose de 400 mg/Kg do Ãleo essencial. ApÃs 24h e 48h, o sangue perifÃrico e a medula Ãssea foram coletados. No ensaio do cometa nÃo houve detecÃÃo de nenhum cometa de grau elevado e as anÃlises estatÃsticas demonstraram um P<0,01 (grau de significÃncia: P<0,05) para todas as amostras em relaÃÃo ao controle positivo e um P>0,05 (grau de significÃncia: P<0,05) para as amostras em relaÃÃo ao controle negativo. No ensaio do micronÃcleo, todas as doses do extrato aquoso e do Ãleo essencial tiveram diferenÃa estatisticamente significante em relaÃÃo a ciclofosfamida, um antineoplÃsico citotÃxico, com um P<0,001 e um P>0,05 em relaÃÃo ao controle negativo (significÃncia: P<0,05). Todos esses resultados indicam que o extrato aquoso e o Ãleo essencial das folhas de A. zerumbet nÃo apresentam aÃÃes citotÃxicas nem genotÃxicas nos modelos testados. / Alpinia zerumbet, known ordinarily as colony in Northwestern Brazil, is a medicinal plant widely used in popular medicine as tea and infusions for the treatment of intestinal and cardiovascular illnesses, such as hypertension. Due to the high levels of consumption of such infusions, we have sought to evaluate the toxicological and genotoxicological profile of the tea and essential oil made from A. zerumbet leaves. This study has been evaluated by short term in vivo and in vitro trials. We initially evaluated the cytotoxicity and the hemolytic effect in vitro; however, there was no toxic response. The DL50 found for the tea was of >5 mg/Kg, which demonstrates that the active principles of the tea present low toxicity. The genotoxicity trials were carried out in vivo. The animal received treatment orally, with three doses of the tea (2 g/Kg, 3,5 g/Kg and 5 g/Kg) and with a dosage of 400 mg/Kg of the essential oil. Peripheral blood and bone marrow were collected after 24 and 48 hours. In the comet trial no high level comets were detected and the statistical analyses demonstrate a P<0,01 (significance: P<0,05) for all samples when compared to the positive control and a P<0,05 (significance: P<0,05) for samples in relation to the negative control. In the micronucleus test, all doses of the tea and essential oil presented a statistically significant difference in relation to cyclofosfamide, with a P < 0.001 and a P > 0.05 compared with the negative control (significance: P<0,05). All those results indicate that the tea and essential oil made from A. zerumbet leaves do not present cytotoxic or genotoxic action in the tested models.

Les nanoparticules (NP) comportent au moins une dimension entre 1 et 100 nm. Les nanoparticules de dioxyde de cérium (NPCeO2) sont largement utilisées au niveau industriel, en particulier comme additif au carburant diesel, mais font aussi l’objet d’étude en cancérologie. L’OCDE a placé les NPCeO2 dans la liste des nanomatériaux nécessitant une évaluation urgente de leur toxicité.Plusieurs études ont démontré le passage de NP métalliques dans les gonades après exposition in vivo chez l’animal, mais très peu de données sont disponibles concernant les NPCeO2.L’objectif de ce travail a été dans un premier temps d’étudier la génotoxicité des NPCeO2 sur spermatozoïdes et ovocytes murins par exposition in vitro, ainsi que leur impact sur la fécondation in vitro (FIV) murine. Nous avons dans un second temps étudié leur génotoxicité sur spermatozoïdes humains in vitro.Nous avons montré une diminution significative des taux de fécondation chez la souris, lors des FIV réalisées en présence de NPCeO2 à faible concentration (0,01 mg/l). Nous avons également montré une génotoxicité significative des NPCeO2 à faibles doses sur ovocytes, spermatozoïdes de souris, et spermatozoïdes humains par le test des comètes. En microscopie électronique à transmission, nous n’avons pas retrouvé de NP dans les milieux de culture à 0,01 mg/l, mais observé une endocytose dans les cellules du cumulus entourant les ovocytes à forte concentration (100 mg/l) et une accumulation des NP le long des membranes des spermatozoïdes et des zones pellucides des ovocytes. Ces résultats apportent des informations nouvelles et importantes sur les mécanismes d’actions suspectés. / The fast development of nanotechnology gives rise to questions regarding the potential risks on human health. Cerium oxide nanoparticles (CeO2NP) are widely used, as diesel additive, as well as promising therapeutic in cancerology, yet scarce data are currently available on their toxicity. It has been shown that metal nanoparticles can cross biological barriers and accumulate into various organs including the gonads after in vivo exposure. Our objectives were to investigate CeO2NP’s genotoxicity on mouse spermatozoa and oocytes, human spermatozoa, and to study their possible impact on murine IVF.We showed a significant decrease of fertilization rate, during IVF carried out in culture medium containing CeO2NP at very low concentration (0.01mg/l). We also showed significant DNA damage induced in vitro by CeO2NP on mouse spermatozoa and oocytes at 0.01 mg/l using Comet assay. TEM did not detect any nanoparticles in the IVF samples at 0.01 mg/l, but showed, at high concentration (100 mg/l), their endocytosis by the cumulus cells surrounding oocytes and their accumulation along spermatozoa plasma membranes and oocytes zona pellucida. We showed significant DNA damage induced by CeO2NP on human spermatozoa. The genotoxicity was inversely proportional to the concentration. At 0.01 mg/l, the genotoxicity mechanisms involved oxidative stress and close interactions between spermatozoa and CeO2NP.We demonstrate for the first time the impact of CeO2NP on in vitro fertilization, as well as their genotoxicity on mouse gametes and human sperm, at very low nanoparticle concentration exposure. These results support several publications on metal nanoparticles reprotoxicity.