Dissertations / Theses: 'Mutagenicity' – Grafiati (2024)

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Mutageniciteten för olika ämnen bestäms vanligtvis med hjälp av konventionella in vivo och in vitro metoder. Att övergå till in silico metoder vore både etiskt och miljö- mässigt gynnsamt. Flera olika parametrar kan användas för att förutse mutagenicitet. Bland dessa ingår energin för lägsta oockuperade molekylorbitalen (LUMO), aktiveringsenergin, lokala min/max i elektrostatiska potentialen (Vs,min/Vs,max), lokala min i ”electron attachment energy” (Es,min ), med flera. Aktiveringsenergin är en parameter som kan förutspå mutagenicitet med hög noggrannhet, förutsatt att reaktionsmekanismen antingen är känd eller kan antas. Däremot är beräkningskostnaden för aktiveringsenergin hög. Mutagen-X (MX) och dess analoger (föreningar med samma grundstruktur men olika funktionella grupper, totalt 29 olika föreningar), är föreningar vars mutagenicitet har bestämts med hjälp av traditionella metoder men har nyligen också kunnat bestämmas genom kvantkemiskmodellering. Kvantkemiskmodellering har med framgång implementerats i studien av Kari Tuppurainen [1]; där LUMO har bestämts för MX och dess analogier. Utöver detta visades även en statistiskt signifikant korrelation mellan LUMO för MX och dess analoger och den biologiska aktiviteten som bestämdes med hjälp av Ames test. Målet med detta arbete har varit att undersöka vare sig mutageniciteten för MX och dess analogier kan även bestämmas genom att beräkna Es,min, Vs,max och aktiveringsenergin. Reaktionen som undersöktes var en Michael additionsreaktion mellan amingruppen på kvävebasen guanin och betapositionen på MX och dess analogier. Av den anledningen utvärderades parametrarna (Es,min, Vs,max och aktiveringsenergin) vid betapositionen. De beräknade värdena för Es,min var korrelerade med biologiska aktiviteten. Även aktiveringsenergierna för MX och dess analogier beräknades vid betapositionen och korrelerades sedan med biologiska aktiviteten och Es,min värdena. Om en statistiskt signifikant korrelation observerades mellan aktiveringsenergin och Es,min värdena, hade detta varit en indikation att Es,min värden kan användas för att ersätta aktiveringsenergin. Es,min värden har en jämförelsevis låg beräkningskostnad. Dock observerades ingen statistiskt signifikant korrelation mellan Es,min värdena och biologiska aktiviteten. Vidare observerades ingen statistiskt signifikant korrelation mellan aktiveringsenergin och biologiska aktiviteten och/eller Es,min värdena. Därmed fanns flera indikationer att den tänkta reaktionsmekanismen var felaktig. Under efterforskningen hittades en studie som visade att en-elektronreduktionsmekanismen var den mest troliga reaktionsmekanismen för den undersökta reaktionen. Detta kan vara en förklaring till varför en statistiskt signifikant korrelation kunde observeras mellan LUMO och biologiska aktiviteten, medan ingen korrelation observerades för Es,min, Vs,max och aktiveringsenergin mot biologiska aktiviteten. Avsaknaden av en korrelation för dessa parametrar anses bero på att den föreslagna reaktionsmekanismen var felaktig. Vidare studier hade behövts för att undersöka dessa parametrars förmåga att kunna förutse mutagenicitet.
Mutagenicity of various compounds is traditionally predicted by conventional in vivo and in vitro methods. However, transitioning to in silico methods would be beneficial both ethically and environmentally. The descriptors that can be used to predict mutagenicity are the lowest unoccupied molecular orbital (LUMO) energy, activation energy, the local minimum / maximum electrostatic potential energy (Vs,min/Vs,max), the minimum local electron attachment energy (Es,min), etc. The activation energy is a descriptor that can predict mutagenicity accurately provided that the reaction mechanism is known or can be assumed. However, determining the activation energy is computationally costly. Mutagen-X (MX) and its analogues (compounds with the same backbone but different functional groups, 29 compounds in total), are compounds of which the mutagenicity had been characterized by traditional means but recently also using an in silico method – molecular modeling. Molecular modeling had been successively employed in the study by Kari Tuppurainen [Source]; the LUMO of MX and its analogues had been computed and, importantly, the obtained values demonstrated a statistically significant correlation with the biological activity determined using Ames test. The aim of this thesis was to investigate whether the mutagenicity of MX and its analogues could also be determined by computing Es,min, Vs,max and the activation energy. The studied reaction was a Michael addition reaction between an amine group on the guanine nucleobase and the beta position of MX and its analogues. Therefore, the studied parameters (Es,min, Vs,max and the activation energy) were evaluated at the beta position. The computed Es,min values were correlated with the biological activity. Activation energies for MX and its analogues were also computed at the beta position and then correlated with the biological activity and Es,min values. If a highly statistically significant correlation between the activation energy and Es,min values at the beta position would have been observed, that would indicate that Es,min values could be used as a substitute for the activation energy. Es,min values have comparatively low computational cost. However, no statistically significant correlation between Es,min values and the biological activity was observed. Furthermore, no statistically significant correlation was observed between the activation energy and biological activity and/or Es,min values, respectively. Thus, there were several indications that the proposed reaction mechanism was incorrect. After consulting literature, we learned that the one electron reduction mechanism would be a more probable reaction mechanism. This could be an explanation as to why a highly statistically significant correlation could be observed for LUMO vs. the biological activity, whereas no correlation was observed for Es,min, Vs,max, the activation energy versus the biological activity. The absence of a correlation for these parameters is thought to be due to the proposed reaction mechanism being inaccurate. Additional studies would have to be performed to further investigate the predictive abilities for mutagenicity of the studied parameters.

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This thesis describes a computational investigation of the mutagenicity of 5-bromouracil (BrU). In Chapter 1, three models of spontaneous and BrU-induced base mispairing (rare tautomer, wobble pair, and ion) are reviewed. Chapter 2 presents the computational techniques used: electronic structure methods (Hartree–Fock-based and density functional theory) and molecular dynamics. Chapter 3 presents optimisations of the keto and enol tautomers of BrU and uracil (U) in water clusters. The enol tautomer of BrU is found to be more stable than that of U. Chapter 4 is a molecular dynamics study of the keto-enol tautomerism of BrU and U in a periodic water box. The pKₐ of BrU at N3 is found to be lower than that of U. Chapter 5 is a study of stacked base dimers containing BrU, U, or thymine (T) stacking with natural bases. Some structures were taken from the Protein Data Bank, while others were generated using an in-house methodology. BrU is found to stack more strongly than T in vacuo, but solvation and thermal effects nullify this difference. Chapter 6 discusses the significance of the results in Chapters 3–5 in terms of BrU-induced mutagenesis. Appendices A and B–D provide supplementary material to Chapters 2 and 5, respectively. Appendix E is an investigation of the “base flipping” pathway of 2-aminopurine (2AP). Both 2AP/N and A/N dinucleosides (N = thymine or guanine) are found to adopt a wide range of energy-minimum conformations – not only stacked and “flipped”, but also intermediate – and the stacked are not the most favourable by free energy. Appendix F is a list of publications and papers in preparation. One publication concerns BrU stacking. The other is a conformational study of the dipeptide tyrosine-glycine: the theoretical results are shown to be consistent with experiment (R2PI spectra) if thermal effects are taken into account.

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Diesel emissions have been shown to elicit a variety of toxicological effects, and alternative fuels (i.e., biodiesel) are currently being assessed to determine their ability to reduce the risks of adverse health effects. Exhaust emissions were generated using ULSD and biodiesel blended fuels and extracts of diesel PM (i.e., filters and PUFs) were separated into polar aromatic and non-polar neutral compounds. Mutagenic activity was assessed using the Salmonella mutagenicity assay, and Ah-receptor agonism was assessed using the DRCALUX assay. Results indicate that organic extracts of diesel/biodiesel particles contain direct- and indirect-acting polar aromatic mutagens as well as polar and non-polar Ah-receptor agonists. The mutagenicity of direct-acting compounds decreases with increasing concentrations of biodiesel in the fuel; however, there is no change in the indirect-acting mutagenicity. Furthermore, the ability of polar and non-polar compounds to induce the Ah-receptor increases with increasing concentrations of biodiesel in the fuel. These results provide an initial framework for evaluating the toxicological hazards of biodiesel emissions.

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Thesis (S.M. in Molecular Systems Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.
Includes bibliographical references (leaves 38-43).
Recent epidemiological data has indicated that chronic exposure to metals such as arsenic and cadmium increases the risk of cancer and other diseases. These metals may have negative biological effects on cells by disrupting homeostatic cellular processes and altering normal signal transduction. One possible mechanism for many of these negative effects may involve overproduction of reactive oxygen species that damage proteins, lipids, carbohydrates, and nucleic acids. To compound this oxidative damage, there is evidence consistent with the inhibition of repair of damaged DNA by these metals. As a result, there is an increase in mutagenicity and toxicity in the organisms. This thesis reviews the current literature relevant to the biochemistry and biology of arsenic and cadmium.
by Leslie Lai.
S.M.in Molecular Systems Toxicology

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Mestrado em Estudos Ambientais
Polycyclic aromatic hydrocarbon (PAH) extracts of PM2.5 collected fromcombustion of seven wood species and briquettes were tested for mutagenicactivities using Ames test with Salmonella typhimurium TA98 and TA100. Thewoods werePinuspinaster (maritime pine), Eucalyptus globulus (eucalypt),Quercussuber (cork oak), Acacia longifolia (golden wattle), Quercusfa*ginea(Portuguese oak), Oleaeuropea (olive), and Quercus ilex rotundifolia (Holmoak). Burning experiments were done using woodstove and fireplace, hot startand cold start. A mutagenic/weak mutagenic response was recorded for allspecies except golden wattle. The extracts with indirect acting mutagenicitywere mainly obtained from fireplace and cold start conditions. The strongmutagenic extracts were not correlated with high emission factors ofcarcinogenic PAHs. Several samples were weak mutagens at lowconcentration of PAHs. The negative result recorded for the golden wattleextracts is positive since after confirmation, this species can be recommendedfor domestic use.
(FCT) - PTDC/AMB/65706/2006 (BIOEMI)

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Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds remains problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. I have constructed several mutagenicity tester strains that co-express combinations of full-length human cytochrome P450 2E1, rat cytochrome P450 reductase, and human cytochrome b5 in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt-; and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. Coexpressing human cytochrome b5 with cytochrome P450 2E1 and cytochrome P450 reductase potentiates the mutagenicity observed with dialkylnitrosamines. These strains were sensitive to nitrosamines with varying alkyl side chains, including dimethylnitrosamine, diethylnitrosamine, dipropylnitrosamine, and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples, and may serve as useful tools in investigating the molecular properties of proteins in the cytochrome P450 monooxygenase system.

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Mechanisms by which carcinogens induce mutations in human cells can be investigated using carcinogen exposed shuttle vector plasmids. In particular, the pSP189 plasmid can be treated with carcinogens in vitro and transfected into human fibroblasts. Following replication, where DNA repair or mutagenesis occurs, recovered plasmids can be screened in indicator bacteria for induced mutations in the supF gene. The aim of this study was to establish and utilise assays to investigate the mutagenicity of genotoxic agents. Specifically, two model reactive intermediates of the cancer drug tamoxifen, α-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM), along with binary treatments of BPDE with UVB or UVC radiation were assessed for their mutagenic potential in human cells. The quantitatively minor DNA adduct of tamoxifen formed by 4-OHtamQM is more mutagenic than the major tamoxifen adduct, formed by a-acetoxytamoxifen in Ad293 cells. The majority of mutations in α-acetoxytamoxifen treated plasmid were GC→TA transversions, while GC→AT transitions were predominant in 4-OHtamQM treated plasmid. Mutational hotspots were observed for both compounds. In GM04429 cells mutations induced by α-acetoxytamoxifen were mainly GC→TA, whereas in GM00637 fibroblasts GC→AT transitions were more common. Treatment of plasmid with BPDE preferentially induced GC→TA transversions whilst UVB and UVC induced GC→AT transitions. Binary treatments were more mutagenic than single treatments, with BPDE then UV being more mutagenic than UV then BPDE. This suggests a synergistic mechanism of activation of BPDE DNA adducts by UV radiation. In the final part of this work, a system for investigating the site-selectivity and consequences of mutagen reaction with DNA was developed. In the future this methodology can be used to detect mutations arising from specific DNA adducts inserted at known locations in important genes. This assay builds on the results provided by the standard supF assay and enables a more detailed investigation of the mechanisms involved in mutagenesis.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Division of Bioengineering and Environmental Health, 2002.
Vita.
Includes bibliographical references.
Aflatoxin B1 (AFB1) is a fungal metabolite that contaminates the food supply in certain areas of the world. It is produced by Aspergillusflavus and related fungi that grow on improperly stored foods such as corn, rice, and peanuts. Epidemiological studies have shown a correlation between exposure to AFBI and incidence of hepatocellular carcinoma (HCC). Mutations in p53 are observed in over 50% of the HCC samples studied, and a unique mutational hotspot occurs at the third position of codon 249 in this gene, yielding almost exclusively GC to TA transversions. It is of interest to evaluate the mutagenic properties of specific chemical structures of AFBI adducts in order to determine which of these may be responsible for the mutations that may play a role in the formation of HCC. The primary DNA adduct formed by the epoxide of AFB is the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFBI-N7-Gua) adduct, which can lead to two secondary lesions, an apurinic site or a ring opened formamidopyrimidine (FAPY) adduct, which itself has two rotameric forms. This study focuses on of the determination of how well cells tolerate each of the AFB1-FAPY rotamers and of the type and frequency of mutations caused by the persistent AFB I-FAPY adduct in a site specifically modified M13 viral vector transfected into E. coli. Four major results were concluded from this work. First, one of the rotamers of AFBI-FAPY is a strong block to DNA replication, even when bypass polymerases are employed by the cell.
(cont.) Second, the G to T mutation frequency of the AFB I-FAPY adduct is at least six fold greater than that observed for the AFBi-N7-Gua adduct. Third, a spectrum of mutations that is unique to the AFBI-FAPY adduct was observed. Fourth, cell strains expressing different bypass polymerases responded differently when challenged with the AFB1-FAPY and AFBI-N7-Gua DNA adducts. These results show that AFB I-FAPY is the most toxic and mutagenic species of aflatoxin adduct studied to date.
by Maryann E. Smela.
Ph.D.

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Gastric aspirate specimens were collected from patients w~th clinically diagnosed gastric carcinoma and from non-carcinoma patients. The nitrite concentration and pH values of the aspirates were measured, the microorganisms present in selected specimens were isolated and identified, and the mutagenicity ratios of the aspirates were determined. The median nitrite concentration of the gastric aspirates from the carcinoma patients was significantly higher than that obtained for the non-carcinoma patients. A positive correlation was found between the nitrite concentration and the pH values of all the specimens tested, and a marked increase in nitrite levels at pH values above 6,0 was evident in specimens from the coloured ethnic "normal" subgroup. Gastric aspirate nitrite concentrations did not correlate with salivary values. The presence of microorganisms in gastric aspirates was shown to be pH dependent. Gastric aspirates with a pH < 2,0 were sterile, below pH 4,0 only acidophilic bacteria survived, whereas above pH 4,0, numerous species, predominantly members of the oral microflora, were isolated. The mean mutagenicity ratio of the gastric aspirates from the carcinoma patients was found to be significantly higher than that found for the control group. There was a positive correlation between the mutagenicity ratios of all the gastric specimens and pH with a maximum at a pH value of approximately 6,0.

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In-vitro mutagenicity assays have traditionally been used for first line identification of potential genotoxic hazard, purporting to chemical carcinogenesis and heritable genetic damage. The recent advances m combinatorial chemistry and high throughput screening technologies have led to a massive explosion in numbers of possible therapeutic candidates being produced at the early stages of drug discovery. This rapid increase in the number of chemicals to be classified results in a greater need for to acquire alternative methods for the prediction of toxicity. Quantitative StructureActivity Relationships (QSAR) can till this need for early hazard identifications by elucidating the physicochemical basis of biological activity. The assumption with predictive QSARs for toxicity is that "biological activity may be described as a function of chemical constitution". This thesis focuses on the Ames mutagenicity assay data for two compound sets; one of 90 compounds, with limited structural flexibility, comprising a range of chemical classes (non-congeneric series), the second, a set of 30 flavonoid compounds. Three physicochemical descriptor sets were generated: EV A, a theoretical molecular descriptor based on the normal co-ordinate modes of vibration; WHIM, derived from weighting functions applied to the 3D-structural molecular co-ordinates; and TSAR, a series of hydrophobic, electronic and steric parameters traditionally associated with the production of biological QSARs. Various "unsupervised" data pre-treatment methods were adopted, to reduce the level of degeneracy within the individual descriptor sets, prior to the calculation of stepwise linear discriminant classification functions. Good predictive models for Ames mutagenicity, as determined by leave-one-out (jackknife) cross-validation, were obtained with each of the three physicochemical descriptor sets. An increase in the predictive ability was observed following the combination of variables from the individual descriptor sets, inferring that some unique information associated with mutagenic activity is contained within each descriptor set. The predictive stability of the models produced was assessed via independent compound predictions, with a poor overall success rate determined. This failure in external prediction was investigated and fundamental differences in physicochemical data space occupancy revealed. Conclusions on training set composition and general model applicability are made with consideration to individual model physicochemical data space coverage.

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Thesis (Civ. E.)--Massachusetts Institute of Technology, Dept. of Civil Engineering, 1991.
Includes 3-1/2 inch computer disc in pocket.
Includes bibliographical references.
by John Laighton Durant.
Civ.E.

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The potential mutagenicity and clastogenicity (ability to cause chromosomal damage) of five South African traditional medicinal plants: Aco*kanthera oppositifolia (Lam.); Pelargonium sp. cf. inquinans ( L.) L Herit; Pteridium aquilinum subsp aquilinum; Rumex lanceolatus Thunb. and Zantedeschia aethiopica (L.) Sg, were investigated using two in vitro tests in both a bacterial and a mammalian cell system. The Salmonella reverse mutation assay and chromosomal aberration test, two frequently used and accepted pharmacological bioassays, were selected for the investigation. The rat liver extract (S9), containing CYP P450 and other liver enzymes, was added to the in vitro cell system to detect pro-mutagens that require metabolic activation in order to exert mutagenicity or clastogenicity - directly acting mutagens do not require metabolic activation. A significant mutagenic potential (p 0.01) was evident with the Salmonella reverse mutation assay for three of the aqueous plant extracts: (i) R. lanceolatus (in strains TA97a, TA98, TA100 and TA102) with and without metabolic activation (S9), (ii) P. aquilinum in TA100 with S9 and in TA102 without S9 and (iii) Pelargonium (in TA102) without S9. Furthermore, R. lanceolatus and P. aquilinum were clastogenic in the chromosomal aberration test but this effect was reduced with S9. Z. aethiopica demonstrated clastogenicity, which was reduced with S9, but the extract was not mutagenic. Since the chromosomal aberration test is dependent on cells entering the cell cycle (Go-G1, S and G2-M) and chromosome visibility with light microscopy only occurs at metaphase, the clastogenicity of A. oppositifolia and Pelargonium could not be detected because these extracts inhibited mitosis (M). A DNA analysis of cultures treated with A. oppositifolia and Pelargonium by Flow Activated Cell Sorting (FACS) indicated a blockage in the Go/G1 phase of the cell cycle. The in vitro mutagenicity and clastogenicity tests served as a preliminary investigation into the safety of five traditional plants. In addition to mutagenicity testing, it is suggested that further scientific evaluation, validation, standardisation and regulation of South African traditional medicine is essential in order to prevent the adverse acute and chronic effects of plant ingestion. iii

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Thesis (M.S.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains xi, 61 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 34-37).

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Young children are particularly sensitive to environmental pollutants. They can directly ingest soil by putting dirty hands and objects in their mouths. The reliance on animal derived models for human health risk and exposure assessment has several limitations. In this investigation, a tool-kit was developed and optimised to facilitate more accurate, reliable and representative predictions of soil contaminants that might pose a significant hazard to young children. The tool-kit was developed and optimised using an in vitro human digestion bioassay. This procedure was followed by the optimisation of several mutagenicity bioassays to link to the bioaccessible fraction which quantified by the in vitro bioassay. The application of novel and sensitive environmental-based biosensors requires them to work in parallel with effective and proven extraction techniques. In this study, chemical analysis was used to quantify the bioaccessible (human assimilated portion) of pollutants in soils. Acute toxicity was measured using constitutively marked bioluminescent bacterial biosensors and these were indicative of the total contaminant burden. A range of mutagenic assays were applied and optimised. In the Ames assay, any compound exhibiting a greater than two-fold increase in the number of revertants colonies over the number of spontaneous revertants was considered as a mutagen. Mutagenic-responsive SOS-lux based microbial biosensors were compared to the Ames assay. Mutagenicity assessment of a broad range of environmental pollutants (i.e. B[a]P, DiB(a,h)A, B[a]A, Ni and Cu), was performed using four SOS-lux microbial biosensors; E. coli DPD1718, E. coli K12C600, S. aureus pAmiUmuC and S. aureus pAmiRecA. The results substantiated that the four biosensors were unable to be induced by these pollutants. Nevertheless, E. coli DPD1718 and E. coli K12C600 were successfully induced by Mitomycin C (MMC) in a dose response manner. The Ames assay was performed for the above pollutants in the absence and the presence of the metabolic activation S9 mix. The standard plate incorporation assay and a modification protocol for the Ames assay were applied. Results reported from the Ames assay confirmed mutagenicity responses of the tested pollutants except Cu and Ni. MMC was selected and introduced into soil samples as a case study to assess the performance of the developed tool-kit. Soils amended with MMC were extracted by the in vitro human digestion bioassay, and the mutagenicity of the bioaccessible fraction was measured using the Ames assay and the biosensors. A comparison was made between the permissible concentrations of MMC obtained from the developed tool-kit and RISC4 derived concentrations. The four microbial biosensors applied in this study were incapable to detect the mutagenicity of the tested pollutants. On the other hand, the Ames assay was more robust and sensitive to a broad range of environmental pollutants. The in vitro human digestion bioassay enabled the quantification of the human bioaccessible fraction of the tested pollutants. This fraction posed a concern due to its estimation of the doses that would reach the blood circulation and cause harm to human. While the permissible concentration of MMC measured by the developed tool-kit was less than 10 μg MMC/g, the RISC4 model calculated that it should be 40 μg MMC/g. This revealed that, in this situation, risk assessment model was less conservative than empirical study for human health risk assessment. This study enabled the assessment of the permissible concentrations of environmental pollutants that could remain in a soil and pose permissible harm to humans. This approach also enabled a comparison of modelled and empirical data to allow a measure of sensitivity to be judged. There is a need to develop bioassay techniques more able to assess the potency of hydrophobic compounds both in isolation and combination.

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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Chronic myelogenous leukemia (CML) is a myeloproliferative disease of hematopoietic stem cells, characterized by the presence of the Philadelphia (Ph) chromosome, originating from a reciprocal translocation between the long arms of chromosomes 9 and 22, forming the gene BCR-ABL, which encodes a BCR-ABL oncoprotein with constitutive tyrosine kinase activity. The clinical course of CML is often divided into three phases: chronic, accelerated, and blast. The treatment of choice for the chronic phase is the first-generation tyrosine kinase inhibitor (TKI), imatinib mesylate, and for refractory patients, second-generation TKIs (dasatinib or nilotinib) are used. Studies have shown that residual leukemia may persist even in the best responders to TKI, since therapy is not curative. In this context, the present study aimed to evaluate the genotoxicity and mutagenicity of TKI in patients with CML followed at the hematology clinic of the Walter CantÃdio University Hospital (HUWC). It is a cross-sectional study with 44 patients with clinical and molecular diagnosis of CML. Patients were stratified into three groups: diagnosis (CML D) (n = 5), use of first generation TKI (CML) (n = 31) and use of second generation TKI (CML) (n = 8). The control group (CG) consisted of apparently healthy individuals. Genotoxicity and mutagenicity were analyzed by the comet assay and micronucleus test. Statistical analysis of the data was performed using the GraphPad Prism 6.0 program using the Kruskal-Wallis or ANOVA, Mann Whitney or T-student tests, depending on the normality of the data and the level of significance was 5% (p < 0.05). Patients with CML had a statistically higher ADN damage index (DI) compared to CG (p < 0.0001). When the patients were stratified, a progressive increase of the DNA ID was verified in the groups: CML D, CML G1 and CML G2, respectively, relative to GC (p < 0.05). Patients with CML had a statistically higher micronucleus index (NMI), nucleoplasmic bridge index (NPI) and nuclear bud index (NBI) compared to the CG (p < 0.05). By stratifying patients with CML, it was found that patients in the G1 and G2 CML groups had statistically higher NMI and NPI compared to CG (p <0.001). NMI was also elevated in the CML G2 group in relation to the patients in the CML D group (p <0.01). The nuclear bud index (NBI) did not present statistical difference in the analyzes performed after the stratification of the groups. The TKI revolutionized CML therapy, improving patient survival. However, these results point to the relevance of studies that evaluate the possible genotoxic and mutagenic effects of this therapy in the long term. The mechanisms involved should be elucidated for the purpose of improving treatment as well as assessing the clinical impact this harm may cause.
A leucemia mielÃide crÃnica (LMC) à uma doenÃa mieloproliferativa das cÃlulas-tronco hematopoÃticas, caracterizada pela presenÃa do cromossomo Philadelphia (Ph), originado a partir de uma translocaÃÃo recÃproca entre os braÃos longos dos cromossomos 9 e 22, formando o gene BCR-ABL, que codifica uma oncoproteÃna BCR-ABL com atividade tirosino-quinase constitutiva. O curso clÃnico da LMC à frequentemente dividido em trÃs fases: crÃnica, acelerada e blÃstica. O tratamento de escolha para a fase crÃnica à o inibidor de tirosino-quinase (ITK) de primeira geraÃÃo, mesilato de imatinibe, e para os pacientes refratÃrios, utiliza-se os ITK de segunda geraÃÃo (dasatinibe ou nilotinibe). Estudos tÃm demonstrado que a leucemia residual pode persistir mesmo nos melhores respondedores aos ITK, uma vez que a terapia nÃo à curativa. Nesse contexto, o presente estudo objetivou avaliar a genotoxicidade e mutagenicidade dos ITK em pacientes com LMC acompanhados no ambulatÃrio de hematologia do Hospital UniversitÃrio Walter CantÃdio (HUWC). Trata-se de um estudo transversal com 44 pacientes com diagnÃstico clÃnico e molecular de LMC. Os pacientes foram estratificados em trÃs grupos: ao diagnÃstico (LMC D) (n=5), em uso de ITK de primeira geraÃÃo (LMC G1) (n=31) e em uso de ITK de segunda geraÃÃo (LMC G2) (n=8). O grupo controle (GC) foi composto por indivÃduos aparentemente saudÃveis. A genotoxicidade e mutagenicidade foram analisadas atravÃs do ensaio cometa e teste de micronÃcleos. A anÃlise estatÃstica dos dados foi realizada atravÃs do programa GraphPad Prism 6.0 utilizando-se os testes de KruskalâWallis ou ANOVA, Mann Whitney ou T-student, dependendo da normalidade dos dados e o nÃvel de significÃncia foi de 5% (p < 0,05). Pacientes com LMC apresentaram Ãndice de dano (ID) no DNA estatisticamente mais elevado em comparaÃÃo ao GC (p < 0,0001). Quando os pacientes foram estratificados, foi verificado um aumento progressivo do ID no DNA nos grupos: LMC D, LMC G1 e LMC G2, respectivamente, em relaÃÃo ao GC (p < 0,05). Pacientes com LMC apresentaram Ãndice de micronÃcleos (IMN), Ãndice de pontes nucleoplasmÃticas (IPN) e Ãndice de buds nucleares (IBN) estatisticamente mais elevados em comparaÃÃo com o GC (p < 0,05). Ao se estratificar os pacientes com LMC, foi verificado que pacientes dos grupos LMC G1 e G2 apresentaram IMN e IPN estatisticamente mais elevados em comparaÃÃo ao GC (p < 0,001). O IMN tambÃm foi elevado no grupo LMC G2 em relaÃÃo aos pacientes do grupo LMC D (p < 0,01). O Ãndice de bud nuclear (IBN) nÃo apresentou diferenÃa estatÃstica nas anÃlises realizadas apÃs a estratificaÃÃo dos grupos. Os ITK revolucionaram a terapia da LMC, melhorando a sobrevida dos pacientes. No entanto esses resultados alertam para a relevÃncia de estudos que avaliem os possÃveis efeitos genotÃxicos e mutagÃnicos dessa terapia a longo prazo. Os mecanismos envolvidos devem ser elucidados com a finalidade de aprimorar o tratamento, bem como avaliar o impacto clÃnico que esse dano pode causar.

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Thesis (Ph. D.)--Illinois State University, 1993.
Title from title page screen, viewed March 7, 2006. Dissertation Committee: Herman E. Brockman (chair), Alan J. Katz, Brian J. Wilkinson, David F. Weber, Radheshyam K. Jayaswal. Includes bibliographical references (leaves 162-177) and abstract. Also available in print.

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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.

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Orientador: Maria Aparecida Marin-Morales
Banca: Jonas Contiero
Banca: Edson Luis Maistro
Banca: Mário Sérgio Mantovani
Banca: Silvia Regina Batistuzzo de Medeiros
Resumo: 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 sem*ntes, 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... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: 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)
Doutor

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Quantum Chemical Topology (QCT) descriptors, calculated from ab initio wave functions, have been utilised to model pKa and mutagenicity for data sets of pharmaceutically relevant compounds. The pKa of a compound is a pivotal property in both life science and chemistry since the propensity of a compound to donate or accept a proton is fundamental to understanding chemical and biological processes. The prediction of mutagenicity, specifically as determined by the Ames test, is important to aid medicinal chemists select compounds avoiding this potential pitfall in drug design. Carbocyclic and heterocyclic aromatic amines were chosen because this compounds class is synthetically very useful but also prone to positive outcomes in the battery of genotoxicity assays.The importance of pKa and genotoxic characteristics cannot be overestimated in drug design, where the multivariate optimisations of properties that influence the Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) profiles now features very early on in the drug discovery process.Models were constructed using carboxylic acids in conjunction with the Quantum Topological Molecular Similarity (QTMS) method. The models produced Root Mean Square Error of Prediction (RMSEP) values of less than 0.5 pKa units and compared favourably to other pKa prediction methods. The ortho-substituted benzoic acids had the largest RMSEP which was significantly improved by splitting the compounds into high-correlation subsets. For these subsets, single-term equations containing one ab initio bond length were able to accurately predict pKa. The pKa prediction equations were extended to phenols and anilines.Quantitative Structure Activity Relationship (QSAR) models of acceptable quality were built based on literature data to predict the mutagenic potency (LogMP) of carbo- and heterocyclic aromatic amines using QTMS. However, these models failed to predict Ames test values for compounds screened at GSK. Contradictory internal and external data for several compounds motivated us to determine the fidelity of the Ames test for this compound class. The systematic investigation involved recrystallisation to purify compounds, analytical methods to measure the purity and finally comparative Ames testing. Unexpectedly, the Ames test results were very reproducible when 14 representative repurified molecules were tested as the freebase and the hydrochloride salt in two different solvents (water and DMSO). This work formed the basis for the analysis of Ames data at GSK and a systematic Ames testing programme for aromatic amines. So far, an unprecedentedly large list of 400 compounds has been made available to guide medicinal chemists. We constructed a model for the subset of 100 meta-/para-substituted anilines that could predict 70% of the Ames classifications. The experimental values of several of the model outliers appeared questionable after closer inspection and three of these have been retested so far. The retests lead to the reclassification of two of them and thereby to improved model accuracy of 78%. This demonstrates the power of the iterative process of model building, critical analysis of experimental data, retesting outliers and rebuilding the model.

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Tetramethylthiuram disulfide (42-S Thiram), a carbamate fungicide was studied for its mutagenic potential on the germ cell stages of wild-type male Drosophila melanogaster. The mutagenicity was tested using the sex-linked recessive lethal assay (SLRL). Any lethals induced in the F2 generation were evidenced by the absence of wild-type males. Although there was an increase in mutation rates in the 42-S Thiram treated wild-type males over the control wild-type males, it was not significantly higher. According to the laboratory conditions in this preliminary study, tetramethylthiuram disulfide failed to produce mutagenic effect.

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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 sem*ntes, 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)

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Rosa roxburghii, also known as "Burr Rose" or "Chestnut Rose", originated insouthwest China and was introduced to the botanic garden in Calcutta around1824. It was named after William Roxburgh who was the superintendent. Theextract of fruit of the Rosa roxburghii plant is the base ingredient of a range ofproducts that is commercially sold under the Cili Bao label. The extract iscomposed of a wide range of substances of nutritional value, in particular arelatively high amount of antioxidants such as ascorbate and plant phenols. Ithas been reported before that supplementation with the fruit extractresulted in increased red blood cell superoxide dismutase, catalase and thereduced form of glutathione. An enhancement of the antioxidant status couldcontribute to the protection against several diseases where oxidative stress is amajor factor in the pathology, such as atherosclerosis, cancer and immunitystress. Several anecdotal reports with little (published) scientific support claimthat human supplementation of the Rosa roxburghii extract to the diet has aprotective effect against several diseases, including the above mentioned.Medicinal and herbal plants are used in large sections in developing countries forprimary care and there is now also an increase in the use of natural therapies indeveloped countries. However, plant extracts can also consist of anti-nutritionaland possible toxic components, such as oxalic acid and nitrates, which couldexpress cytotoxic and genotoxic activities. Therefore, understanding the healthbenefits but also the potential toxicity of these plants is important. The objectiveof this study was to investigate the beneficial properties of Rosa roxburghiiextract from an antioxidant potential perspective and in particular to investigatethe safety of the product for human consumption. For this purpose in vitroevaluation of the cellular toxicity, mutagenicity and genotoxicity was performed.In addition, specific biochemical parameters relating to the antioxidant status ofthe product, i.e. antioxidant capacity, oxidative stress prevention and glutathioneredox state profiles were investigated in vitro as well as in vivo.The results indicated that Rosa Roxburghii fruit extract was not mutagenic whentested with Salmonella typhimurium strains TA 98, TA 100 and TA 102 in theAmes test. The results, however, pointed towards an antimutagenic effect of theextract in these strains against metabolic activated mutagens 2-acetylaminoflurorene (2-AAF) and aflatoxin B1, and the direct-acting mutagen,methanesulfonate (MMS). In primary rat hepatocyte, Rosa roxbughii extract didnot elicit double or single strand DNA damage and cell viability loss using thesingle cell gel electrophoresis (Comet assay), lactate dehydrogenase leakagetest or the mitochondria1 conversion test of MTT to formazan (MTT test). Againthe opposite effect was observed: pre-treatment of hepatocytes with Rosaroxbughii extract significantly reduced the effect of oxidative stress-inducedcellular- and genotoxicity. These results point to a protective effect againstoxidative stress which is reflected in an increase of the antioxidant capacity andglutathione redox state (GSH/GSSG) in vitro (lymphoblasts) and in vivo (humans)reported in this study. This study underlines the previously suggested potential ofthis plant extract as a natural and safe antioxidant supplement.
Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O teste do cometa é utilizado para estimativa de dano ao DNA nos protocolos de avaliação do risco toxicológico. É rápido, de baixo custo, de fácil realização, seguro e pode ser aplicado em diversos tecidos. Corantes fluorescentes são os mais empregados para o ensaio. É também descrita a utilização de sais de prata com vantagens operacionais, carecendo de sistemas automatizados validados para esta análise. Neste trabalho, desenvolveram-se dois algoritmos automatizados para a análise do cometa corado pela prata. Foram realizados ensaios do cometa para sete grupos com dois camundongos Balb/c machos cada, submetidos ao tratamento intraperitoneal com soro fisiológico (G1), e três doses crescentes de genotóxicos conhecidos: MNU (N-nitroso-N-metilurea) (G2 – 5 μg/g, G3 – 25 μg/g, G4 – 50 μg/g) e DEN (N-nitrosodietilamina) (G5 – 20 μg/g, G6 – 80 μg/g, G7 – 180 μg/g). As lâminas dos testes do cometa foram confeccionadas a partir do sangue total e coradas pela prata e SYBR Gold. As coradas pela prata foram analisados por três avaliadores e pelos algoritmos automatizados. Aqueles corados pela fluorescência foram analisados pelo software Comet IV®. Os avaliadores apresentaram alta correlação de suas estimativas. Os algoritmos se correlacionaram fortemente com a análise dos avaliadores, principalmente com os escores obtidos pela classificação visual. Houve alta concordância na avaliação da sua repetitividade. Os algoritmos apresentaram resultados semelhantes, não indicando superioridade entre si para a análise do teste. Todos os métodos avaliados se mostraram capazes de diferenciar as concentrações dos genotóxicos utilizados. Porém, os algoritmos desenvolvidos não apresentaram correlação com os resultados obtidos pelo sistema do teste do cometa corado pela fluorescência, sugerindo que os métodos de coloração discriminem diferentes estruturas...
The comet assay is used to estimate DNA damage in the protocols for toxicological risk assessment. It's fast, inexpensive, easily performed, safe and can be applied in most tissues. Fluorescent dyes are frequently used for testing. Silver salts have been reported with some operational advantages; however automated systems for its analysis were not sufficiently validated for this staining. In this study, we have developed two algorithms for automated analysis of the comet assay silver stained. The assay was performed for seven groups of two (Balb/c) mice each, treated with intraperitoneal saline (G1) and three different genotoxic doses: MNU (N-nitroso-N-methylurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) and DEN (N-nitrosodiethylamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). The slides of the comet assay were made with total blood and were stained with silver and SYBR Gold. The silver stained slides were analyzed by three evaluators and the automated algorithms. Those stained by fluorescence were analyzed through software Comet IV®. Evaluators showed high correlation of their estimates. Both algorithms were correlated strongly with the evaluators' analysis mainly on the scores obtained by visual classification. Moreover, their agreement was high when assessed by repetitivity. The algorithms showed similar results, demonstrating no superiority to each other for the analysis of the test. All methods evaluated proved able of differentiating genotoxic concentrations used. However, the algorithms were not correlated with the results obtained by the automated system of comet assay fluorescence stained, suggesting that the staining methods linkage different genomic structures or with different affinities. The automated algorithms developed proved valid for the direct analysis of comet assay silver stained, allowing their use in genotoxicity research

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Orientador: Gisela de Aragão Umbuzeiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia
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Resumo: O fracionamento de uma mistura complexa, como o extrato orgânico do material particulado de ar, com base nas diferentes características físicas e químicas dos seus componentes acoplada a testes de mutagenicidade e análises cromatográficas, vem sendo um procedimento útil na elucidação de quais substâncias está presentes na mistura. Este trabalho foi realizado em colaboração com outro grupo de pesquisa em um projeto temático financiado pela FAPESP, e seu objetivo foi estudar a mutagenicidade de frações de extratos orgânicos de material particulado de ar, utilizando ensaio Salmonella/microssoma em microssuspensão com os pares de linhagens TA98/YG1041, TA98/TA1538 e TA1538/YG5161 de Salmonella typhimurium que apresentam diferentes sensibilidades para grupos específicos de compostos. Também foi objetivo do trabalho comparar as potências mutagênicas obtidas para cada uma das frações com os níveis de hidrocarbonetos policíclicos aromáticos analisados pelos pesquisadores do projeto temático. Foram avaliadas três frações, Nitro-HPA, Oxi-HPA e HPA utilizando o método Salmonella/microssoma em microssuspensão na presença e ausência de ativação metabólica (mistura S9) em experimentos de dose-resposta, com quantidade máxima de 10 m³ por placa; também foi avaliada a contribuição na mutagenicidade dos hidrocarbonetos policíclicos aromáticos analisados para cada fração. De acordo com a contribuição na mutagenicidade dos compostos identificados, foi possível inferir que esses não explicam totalmente a mutagenicidade observada para algumas linhagens, e que outros compostos podem estar causando esse efeito. A comparação dos perfis observados entre as frações analisadas e os compostos químicos encontrados na literatura utilizando os pares de linhagens foi capaz de fornecer alguns grupos que podem justificar a mutagenicidade observada, porém, faltam estudos com compostos químicos puros. Um estudo utilizando os pares de linhagens com compostos químicos pode ajudar a identificar e entender o perfil das respostas observadas em amostras ambientais de material particulado de ar, bem como em outras amostras ambientais, especialmente em análises químicas direcionadas pelo efeito biológico (ADEB)
Abstract: The fractionation of a complex mixture based on different physical and chemical characteristics of its components coupled to mutagenicity testing and chromatographic analyzes have been useful in elucidating what substances are present in the mixture. This work was performed in collaboration with another research group in a broad thematic project funded by FAPESP. Samples were collected extracted and prepared by the participants and in this work the objective was to study the mutagenicity of fractions of organic extracts of air particulate matter using Salmonella/microsome microsuspension in combination with selective pair of strains TA98/YG1041, TA98/TA1538 and TA1538/YG5161 of Salmonella typhimurium which have different sensitivities to specific types of compounds. Another objective was to compare the mutagenic potencies obtained for each of the fractions with the levels of polycyclic aromatic hydrocarbons analyzed by the researchers of the thematic project. We evaluated three fractions, named as Nitro-PAH, Oxy-PAH and PAH, using the Salmonella/microsome microsuspension protocol in the presence and absence of metabolic activation (S9) in dose-response experiments, with maximum of 10 m³ per plate, was also evaluated the contribution of mutagenicity in polycyclic aromatic hydrocarbons for each fraction analyzed. According to the contribution in the mutagenicity of the compounds identified, it was possible to infer that these do not fully explain the mutagenicity observed for some strains, and that other compounds may be causing this effect. The comparison of the profiles observed between fractions and analyzed the chemical compounds found in the literature using the pairs of strains was able to provide some groups which may justify the mutagenicity observed, however, there are few studies with pure chemicals. A study using pairs of strains with single chemicals could help in the identification and understanding of the profile of the responses seen atmospheric air particulate samples as well as in other environmental samples, and so assist chemical analysis studies in effect directed analysis (EDA)
Mestrado
Tecnologia e Inovação
Mestre em Tecnologia

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Thesis (Ph. D.)--West Virginia University, 2002.
Title from document title page. Document formatted into pages; contains xvi, 148 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 162-183).

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Universidade Estadual Paulista (UNESP)
Os flavonoides exibem uma multiplicidade de atividades biológicas tanto in vivo como in vitro. No entanto, não existem dados suficientes para fornecer provas conclusivas sobre os efeitos benéficos da maioria das subclasses de flavonoides. Dessa maneira, neste estudo tornou-se relevante avaliar a mutagenicidade, antimutagenicidade, estrogenicidade e atividade antibacteriana dos flavonoides, com o objetivo de traçar o perfil relação estrutura-atividade, uma vez que a atividade biológica dos flavonoides depende da sua estrutura química. A mutagenicidade e antimutagenicidade foram avaliadas pelo teste de Ames, em cepas de Salmonella typhimurium TA98, TA100 e TA102, com e sem ativação metabólica. Para comparação do efeito protetor dos flavonoides foram utilizados 4-nitro-o-fenilenodiamina (NPD), azida sódica (AZS) e mitomicina C (MMC) como mutágenos de ação direta e benzo[a]pireno (B[a]P), aflatoxina B1 (AFB1) e 2-aminoantraceno (2-AA) como mutágenos de ação indireta. A atividade estrogênica foi avaliada por meio do ensaio com leveduras recombinantes (RYA - Recombinant Yeast Assay) e pelo ensaio de proliferação de células de câncer de mama humano (MCF-7⁄BUS) responsivas à estrógeno (E-screen). A determinação da atividade antibacteriana in vitro foi realizada neste estudo utilizando a técnica de diluição em microplacas, com as bactérias Staphylococcus aureus ATTC 25923 e Escherichia coli ATCC 25922. Os compostos avaliados foram: quercetina, kaempferol, luteolina, fisetina, crisina, galanina, flavona, 3-hidroxiflavona, 5- hidroxiflavona e 7-hidroxiflavona. No teste de Ames, a quercetina mostrou-se diretamente mutagênica na linhagem TA98 e antimutagênica...
The flavonoids exhibit a wide range of biological activities both in vivo and in vitro. However, there are insufficient data to provide conclusive evidence on the health effects of most flavonoid subclasses. Thus, is relevant to assess the mutagenicity, antimutagenicity, estrogenicity and antibacterial activity of flavonoids with the aim of tracing the structure-mutagenicity relationship profile, since the biological activity of flavonoids is governed by their chemical structure. The mutagenicity and antimutagenicity was assayed by the Ames test, with Salmonella typhimurium strains TA98, TA100 and TA102, carried out with and without metabolic activation. To compare the protective effect of flavonoids were used 4-nitro-o-phenylenediamine (NPD), sodium azide (AZS) and mitomycin C (MMC) as direct acting mutagens and benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1) e 2-aminoanthracene (2-AA) as indirect acting mutagens. The estrogenic activity was assayed by recombinant yeast assay (RYA) and by proliferation assay of cells of human breast cancer (MCF-7⁄ BUS) responsive to estrogen (E-screen). The determination of antibacterial activity in vitro was performed in this study by technique of dilution in microplates, with the bacteria Staphylococcus aureus ATTC 25923 and Escherichia coli ATCC 25922. The evaluated compounds were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavones, 3-hydroxyflavone, 5- hydroxyflavone and 7- hydroxyflavone. In the Ames test, quercetin was directly mutagenic... (Complete abstract click electronic access below)

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
A radiação ultravioleta (UV) induz diversos efeitos nocivos nos organismos e a quantidade desta radiação que atinge a biosfera é afetada pela concentração de ozônio, latitude, altitude, clima e reflexão especular. As respostas de briófitas em relação aos efeitos da radiação UV e a presença de compostos que absorvem esta radiação têm sido estudadas. Sanionia uncinata, Holomitriopsis laevifolia e Leucobryum laevifolium são espécies de musgos encontrados em locais expostos a alta incidência de radiação UV e com habitats distintos. Considerando que as respostas de musgos contra os efeitos da radiação UV e seus mecanismos de proteção ainda são pouco caracterizados, o objetivo deste estudo foi investigar o potencial fotoprotetor e possíveis riscos toxicológicos associados aos extratos dos musgos S. uncinata, proveniente da Antártica e H. laevifolia e L. laevifolium, proveniente do Amazonas. Seus extratos metanólico (EM), aquoso (EA), hidroalcoólico (EH) e etanólico (EE) foram estudados com a caracterização química por absorção ao UV e visível e pela cromatografia líquida de alta eficiência; quantificação do índice total de compostos fenólicos; determinação da capacidade captadora do radical 2,2-difenil-1-picril-hidrazila a fim de avaliar as atividades antioxidantes; avaliação do potencial de fotoproteção cutânea pela determinação do fator de proteção solar; avaliações do potencial mutagênico e citototóxico, através do ensaio de Salmonella/microssoma, utilizando as cepas TA97, TA98, TA100, TA102 e TA104; do potencial fotomutagênico através do ensaio de fotomutagenicidade, usando as cepas TA102 e TA104; e investigação dos efeitos genotóxicos e fotogenotóxicos, pelo ensaio de micronúcleo e fotomicronúcleo, respectivamente, usando diferentes linhagens celulares estabelecidas. Foram encontradas atividades fotoprotetoras e antioxidantes e observou-se que os extratos se apresentaram singulares devido a sua composição química. Os resultados fotoprotetores, além dos mutagênicos/fotomutagênicos, genotóxicos/fotogenotóxicos e suas respectivas avaliações citotóxicas também permitiram selecionar extratos e suas concentrações, como promissores candidatos em fotoproteção Assim, os EA e EH de H. laevifolia e L. laevifolium apresentam, no geral, os resultados mais significativos, tornando-se potenciais para avaliações refinadas em fotoproteção e na separação de componentes que possam levar a futuras aplicações como antioxidantes e protetores solares ou como adjuvantes.
The ultraviolet radiation (UV) induces many harmful effects in all living organisms and the amount of this radiation that reaching the ground is affected by many factors including ozone concentration, latitude, altitude, climate and specular reflection. The responses of bryophytes against the effects of UV radiation and the presence of compounds that absorb the UV region have been studied. Mosses Sanionia uncinata, Holomitriopsis laevifolia and Leucobryum laevifolium are found in locations exposed to UV at high levels of radiation and in different habitats. Whereas that the responses of mosses against the effects of UV radiation and their protection systems are poorly characterized yet, the aim of this study was to investigate photoprotective potential and possible toxicological risks associated with extracts of mosses S. uncinata (from Antarctica) and H. laevifolia and L. laevifolium (from Amazônia). Methanol (ME), aqueous (AE), hydroalcoholic (HE) and ethanolic (EE) were studied by: chemical characterization by UV/visible spectrophotometry and by High performance liquid chromatography; phenolic content estimation; 2,2-diphenyl-1-picrylhydrazyl scavenging activity; potential of skin photoprotection by in vitro determination of sun protection factor; the mutagenic potential, and cytotoxic by Salmonella/microsome assay, using the TA97, TA98, TA100, TA102 and TA104 strains; photomutagenic potential by photomutagenicity test, using TA102 and TA104 strains and; investigation of genotoxic and photogenotoxic effects by micronucleus test and photo-micronucleous assay, respectively, using different established cell lines. Photoprotective and antioxidant activities were found and it was observed that the extracts showed strong uniqueness due to its chemical composition. From the photoprotective, mutagenic/photomutagenic and genotoxic/photogenotoxic results and their cytotoxic evaluations it was possible to select extracts and their concentrations as promising candidates for photoprotection. Thus, the EA and EH of H. laevifolia and L. laevifolium demonstrated the most significant results, becoming potential for refined evaluations in photoprotection and separating components that can lead to future applications such as sunscreens and antioxidants or as adjuvants.

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Orientador: Maria Aparecida Marin Morales
Banca: Silvia Tamie Matsumoto
Banca: Maria Tercília Vilela de Azeredo Oliveira
Resumo: O controle de plantas daninhas é fundamental para a produção agrícola, devido à sua competição pelos fatores limitantes de crescimento (nutrientes, água, luz) e aos seus efeitos alelopáticos. Pesticidas orgânicos sintéticos têm se tornado um importante elemento nas práticas de produção agrícola tecnificada, dando um grande impulso na indústria agroquímica em todo mundo. Os benefícios de tais produtos são claramente visíveis nos substanciais aumentos na produtividade. No entanto, o destino final desses compostos orgânicos, principalmente dos herbicidas, têm causado sérias preocupações, pelo grande volume utilizado. Preocupados principalmente com a possível interação desses produtos com o material genético dos organismos, nosso estudo visou compreender a ação da trifluralina, por ser este herbicida amplamente utilizado na agricultura. O presente estudo aborda avaliações toxicológicas, de mutagenicidade, genotoxicidade e carcinogenicidade do herbicida trifluralina. Também foram abordadas as características químicas, físicas, persistência, mobilidade, processo de degradação e mecanismos de ação do herbicida. Alguns dados foram obtidos junto às pesquisas realizadas na literatura científica disponível sobre o produto, mas a maioria das apresentações, aqui registradas, são decorrentes da própria investigação realizada com a aplicação do produto em organismos testes. Os dados obtidos foram comparados e discutidos, principalmente, com citações de autores especialistas no estudo da ação do produto e nas publicações da Agência de Proteção Ambiental Norte-Americana (USEPA). Aspectos da legislação brasileira dos agrotóxicos também foram sucintamente abordados
Abstract: The controlling of weed plants is fundamental for agricultural production, due to their competition by the limiting factors of growing (nutrients, water, light) and due to their alelopatics effects. Synthetic organic pesticides have becoming an important element in the practice of technical agricultural production, resulting in a great impulse in agrochemical industry around the world. The benefits from these products are clearly visible in the substantial increasing in the productivity. Otherwise, the final destination of these organic composts, mainly of the herbicides, have causing serious worrying because their high volume. Preoccupation mainly with the possible interaction of these products with the genetic organisms material, our study have the aim to examine the trifluralin action because this herbicide is widely utilized in the agriculture. The present work also looks at the toxicological interactions of mutagenicity, genotoxicity and carcinogenicity of the trifluralin herbicide as well the chemical and physical features, persistence, mobility, degradation process, and herbicide action mechanisms. Some data are from researches done in the available scientific literature about the product, but the majority of the presentation here reported is from our own investigation realized with the application of products in test organisms. The data collected were compared and discussed mainly with report of expert authors in the study of the action of the product and using publications in the United States Environmental Protection Agency (USEPA). Aspects of Brazilian agrotoxic legislation were also briefly discussed
Mestre

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Aflatoxins (AFs) are a group of mycotoxins produced by moulds of Aspergillus genus which contaminate food commodities of tropical and sub-tropical countries. The aims of this study are to assess the extent of AF contamination of foods in the UK that originated in Asia and to identify components of Thai foods that may protect against the toxicity of AFs. Examination of 12 commercial, dried Asian foods showed that long grain rice, fragrant rice, peanuts, black beans and black pepper contained Aspergillus spp. which were identified as A. parasiticus (afiatoxigenic), A. versicolor, A. ustus, A.niger and A. ochraceus. These commodities contained undetectable AFs. Jasmine brown rice and crushed chilli contained 14.7 and 11.4 IJglkg of AFs, respectively, in the absence of Aspergillus. AFBl, the most toxigenic AFs was detected in crushed chilli (lO.7IJglkg) so Aspergillus was present at some stage of food production, particularly pre-harvest stage. Cross contamination during food processing is one of possible cause of AFs contamination in these commodities. These results indicate direct and indirect risks of exposure to AFs from these products since AFs fOlmation is possible in Aspergillus-contaminated crops and AFs can be carried throughout the food chain. Hence an alternative strategy to mitigate toxicity of ingested AFs is required. One possibility is by using food components to modulate the harm from ingested AFs by altering AF metabolism and mutagenesis. In this study, the effectiveness of the compounds in Thai culinary herb, fingerroot (Boesenbergia rotunda) were investigated. A crude methanol extract (ME) of fingerroot was analysed and found to contain ~ 15 putative flavonoids as major components of which pinocembrin, pinocembrin chalcone, cardamonin, pinostrobin, 4-hydroxypanduratin A and panduratin A were identified by HPLC and LC-MS. The ME significantly inhibited fonnation of mutagenic/carcinogenic metabolite of AFBl (AFB1-epoxide, AFBO) in a cell-free metabolic system (Model 1) and also suppressed mutagenicity of AFBl in Salmonella/microsomal assay (Model 2). These inhibitory properties of the ME might be related to its indigenous flavonoids which could modulate activities of AFB1 -metabolising enzymes (CYPIA2, 3A4). Purified flavonoids (cardamonin, apigenin, pinostrobin) were found to affect AFB 1 toxicity to some extent in both models, but their potencies were much lower than the ME particularly in the Salmonella model and evidence is provided that suggests that Reactive Oxygen Species rather than AFBO are the mutagenic entities in this assay. This also suggests that fingerroot contains effective metabolic modulators that were not identified. Consumption of fmgerroot could provide a combination of potential phytochemicals that protect against aflatoxin-mediated toxicity by altering AF metabolism at the initial stages of enzymatic activation.

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A hipertensão arterial é uma condição clínica de ocorrência multifatorial, sendo a mais frequente entre as doenças crônicas não transmissíveis no Brasil. É caracterizada pelo aumento e manutenção dos níveis pressóricos acima de 140mmHg (pressão sistólica) e 90mmHg (pressão diastólica). É também o maior agravante nas complicações cardiovasculares e está associada ao surgimento de outras co-morbidades. A OMS estima que 40% da população mundial acima dos 25 anos seja hipertensa, estando localizada majoritariamente em países onde a renda e o nível de escolaridade são menores. Depois de diagnosticada, a hipertensão arterial deve ser tratada com mudanças no estilo de vida e medicamentos que mantenham os níveis pressóricos controlados, sendo assim, várias classes de anti-hipertensivos são usados nesse tratamento. Visando ampliar o acesso a medicamentos básicos e permitir uma melhor adesão ao tratamento, o Governo Federal criou em 2004 o Programa Farmácia Popular do Brasil, uma parceria entre governo e instituições públicas e privadas para fornecer a população medicamentos para o controle da hipertensão, diabetes, asma, dislipidemias, anticoncepcionais, entre outros; de forma gratuita ou subsidiada em até 90% do valor. Para tratar a hipertensão, o Programa Farmácia Popular do Brasil distribui de forma gratuita os anti-hipertensivos – atenolol, captopril, cloridrato de propranolol, hidroclorotiazida, losartana e maleato de enalapril, todos estes sendo lançados no mercado anterior ao ano de 2004, quando testes de segurança genética ainda não eram obrigatórios, de acordo com a Resolução nº 90/2004 da ANVISA. Desta forma, o objetivo deste trabalho foi avaliar o potencial genotóxico de anti-hipertensivos distribuídos pela Farmácia Popular em células leucocitárias humanas, haja vista que são fármacos amplamente utilizados e se faz necessários maiores estudos que garantam a segurança genotoxicológica dos mesmos. Os testes foram desenvolvidos a partir de culturas celulares tratadas com cinco diferentes concentrações dos anti-hipertensivos, sendo avaliados parâmetros genotoxicológicos – viabilidade celular e índice de dano ao DNA (teste Cometa), e parâmetros mutagênicos – teste de micronúcleo e instabilidade cromossômica numérica. Os resultados mostram que captopril e maleato de enalapril foram capazes de diminuir a viabilidade celular e causar danos ao DNA conforme o aumento da dose, e que, o atenolol foi capaz de também diminuir a viabilidade celular de acordo com o aumento da dose. A hidroclorotiazida também mostrou causar dano ao DNA, porém esse dano ocorreu de forma igual para as cinco doses. Quanto aos parâmetros mutagênicos, nenhum dos seis anti-hipertensivos testados e, em nenhuma das cinco concentrações foi capaz de causar alterações mutagências.
Hypertension is an clinical condition of the multifactorial occurrence, the most frequent among chronic diseases not transferable in Brazil. It is characterized by increase and maintenance blood pressure levels above 140mmHg (sistolic pressure) and 90mmHg (diastolic pressure). It i also the most aggravating in the cardiovascular complications and it is associated with the appearance of others comorbidities. The WHO estimes that 40% of the world population over 25 years old is hypertensive, being located mostly in countries where income and educations levels are lower. After the diagnosed, hypertension must be treated with changes in the life style and drugs that maintain pressure levels controled, therefore, several classes of antihypertensive are used in this treatment. Aiming to expand access to basic drugs and allow a better adhesion to treatment, the federal government created in 2004 the “Farmácia Popular do Brasil” Program, an association among government and public and private institutions to provide the people medications for control of the hypertension, diabetes, asthma, dyslipidemia, contraceptives, etc, free or subsidized form by up to 90% of the value. To treat hypertension, the “Farmácia Popular do Brasil” Program distributes of free form antihypertensive – atenolol, captopril, propranolol hydrochloride, hydrochlorotiazhide, losartan and enalapril maleate, all these being released before the year 2004, when genetic safety tests still not required, according to Resolution nº 90/2004 of ANVISA. Thus, the objective this work was to evaluate the genotoxic potencial of the antihypertensives distributed by “Farmácia Popular” in human leukocytes cells, considering that they are widely used drugs and it is necessary larger studies that ensure the genotoxicology their safety. The tests are desenvolved from cell cultures treated with five different concentrations of the antihypertensives, being evaluated genotoxicological parameters – cell viability and DNA damage index, and mutagenic parameters – micronucleus test and numerical chromossomal aberrations. The results showed that captopril and enalapril maleate were able to reduce cell viability and cause damage to DNA wiht increasing dose. The hydrochlorothiazide also showed to cause DNA damage, but, this damage occorred equallity for five doses. As for mutagenic parameters , none of the six antihypertensives tested and, none of the five concentrations were capable to cause mutagenic changes.

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Orientador: Hélio Amante Miot
Banca: Paula Helena Ortiz Lima
Banca: Marco Antonio Rodrigues Fernandes
Resumo: O teste do cometa é utilizado para estimativa de dano ao DNA nos protocolos de avaliação do risco toxicológico. É rápido, de baixo custo, de fácil realização, seguro e pode ser aplicado em diversos tecidos. Corantes fluorescentes são os mais empregados para o ensaio. É também descrita a utilização de sais de prata com vantagens operacionais, carecendo de sistemas automatizados validados para esta análise. Neste trabalho, desenvolveram-se dois algoritmos automatizados para a análise do cometa corado pela prata. Foram realizados ensaios do cometa para sete grupos com dois camundongos Balb/c machos cada, submetidos ao tratamento intraperitoneal com soro fisiológico (G1), e três doses crescentes de genotóxicos conhecidos: MNU (N-nitroso-N-metilurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) e DEN (N-nitrosodietilamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). As lâminas dos testes do cometa foram confeccionadas a partir do sangue total e coradas pela prata e SYBR Gold. As coradas pela prata foram analisados por três avaliadores e pelos algoritmos automatizados. Aqueles corados pela fluorescência foram analisados pelo software Comet IV®. Os avaliadores apresentaram alta correlação de suas estimativas. Os algoritmos se correlacionaram fortemente com a análise dos avaliadores, principalmente com os escores obtidos pela classificação visual. Houve alta concordância na avaliação da sua repetitividade. Os algoritmos apresentaram resultados semelhantes, não indicando superioridade entre si para a análise do teste. Todos os métodos avaliados se mostraram capazes de diferenciar as concentrações dos genotóxicos utilizados. Porém, os algoritmos desenvolvidos não apresentaram correlação com os resultados obtidos pelo sistema do teste do cometa corado pela fluorescência, sugerindo que os métodos de coloração discriminem diferentes estruturas... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The comet assay is used to estimate DNA damage in the protocols for toxicological risk assessment. It's fast, inexpensive, easily performed, safe and can be applied in most tissues. Fluorescent dyes are frequently used for testing. Silver salts have been reported with some operational advantages; however automated systems for its analysis were not sufficiently validated for this staining. In this study, we have developed two algorithms for automated analysis of the comet assay silver stained. The assay was performed for seven groups of two (Balb/c) mice each, treated with intraperitoneal saline (G1) and three different genotoxic doses: MNU (N-nitroso-N-methylurea) (G2 - 5 μg/g, G3 - 25 μg/g, G4 - 50 μg/g) and DEN (N-nitrosodiethylamina) (G5 - 20 μg/g, G6 - 80 μg/g, G7 - 180 μg/g). The slides of the comet assay were made with total blood and were stained with silver and SYBR Gold. The silver stained slides were analyzed by three evaluators and the automated algorithms. Those stained by fluorescence were analyzed through software Comet IV®. Evaluators showed high correlation of their estimates. Both algorithms were correlated strongly with the evaluators' analysis mainly on the scores obtained by visual classification. Moreover, their agreement was high when assessed by repetitivity. The algorithms showed similar results, demonstrating no superiority to each other for the analysis of the test. All methods evaluated proved able of differentiating genotoxic concentrations used. However, the algorithms were not correlated with the results obtained by the automated system of comet assay fluorescence stained, suggesting that the staining methods linkage different genomic structures or with different affinities. The automated algorithms developed proved valid for the direct analysis of comet assay silver stained, allowing their use in genotoxicity research
Mestre

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Abstract:

Thesis (Ph. D.)--Illinois State University, 1992.
Title from title page screen, viewed January 30, 2006. Dissertation Committee: Herman E. Brockman (chair), Alan J. Katz, Brian J. Wilkinson, Lynne A. Lucher, Radheshaym Jayaswal. Includes bibliographical references (leaves 115-123) and abstract. Also available in print.

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Abstract:

Polluted soil can be a problem in urban areas, and can have a direct impact on human health upon exposure. The toxic potential of mixtures can be mediated by constituents and information about toxicity of mixtures is regarded as important, as it represents the real exposure situation. The main purpose of this project was to measure the mutagenic and CYP1A inducing potential in vitro from organic extracts of soil, sampled in nursery schools in Oslo. Selection of soil was mainly based on the content of ∑PAH16 and ∑PCB7, chemical groups known to include CYP1A inducing and/or genotoxic and mutagenic compounds. Generally were Soil 1 considered as a “clean” sample, Soil 2 to Soil 4 contained increasing level of PAHs and Soil 5 contained mainly elevated levels of PCBs. Assessing varying samples in relation to chemical content was valuable due to potential differences in biological responses. The soil samples were of top soil (0- 2 cm depth) and had been collected by NGU in connection to a geological survey of soil at playgrounds in nursery schools and schools in Norway. The soil was sampled in 2005- 2007, dried at 40 °C for one to two weeks, sieved in a 2 mm nylon sieve and stored in the dark at room temperature. Chemical analyses of both inorganic and organic compounds were performed before storage. In relation to this master project the organic pollutants in the selected soil samples were extracted by ultrasonic agitation in dichloromethane (DCM). Before experimental use the solvent was changed into dichloromethane (DMSO) by evaporating off the DCM using a water bath at 25 °C and a flow of nitrogen above. The dried extracts were redissolved in DMSO.The Ames Salmonella typhimurium assay was used for measuring mutagenicity. The presence of primary and secondary mutagens was assessed by conducting the assay both with and without addition of a metabolic S9- mix. Induction of different point mutations was revealed by utilising two bacterial strains, TA98 and TA100, detecting frameshift and base- pair substitutions, respectively. Induction of CYP1A enzymes was assessed in the rat H4IIE hepatoma cell line, and measured immunologically by Western blotting. The exposure concentrations used in the CYP1A assay were based on results of cell viability, assessed by utilising MTT- assay for finding the highest non- cytotoxic exposure concentrations. Concentration ranges of the extracts were tested in both assays. The mutagenic potential of extracts showed presence of secondary mutagenic compounds, and indicated absence or very low levels of primary mutagens. It was a general incidence of higher mutagenic activity with TA98 than TA100, reflecting highest induction of frameshift mutations. The inducing potential of extracts was in accordance with chemical analysis, showing a general increase in the potential of extract from Soil 1 to Soil 4, suggested to partly reflect differences in level of PAHs. The relative low potency of extract from Soil 5 was considered to be a reflection of a low content of PAHs and an expected dominance of PCBs, which have shown not to induce mutagens in the Ames assay.Induction of CYP1A in H4IIE was measured after exposure to extracts of Soil 3, Soil 4 and Soil 5. Results clearly indicated presence of CYP1A inducers in the extracts. A positive concentration- effect relationship was detected from exposure to extract of Soil 5. Extract of Soil 3 and Soil 4 did clearly induce CYP1A, but in a negative concentration dependent manner. These negative responses were suggested to indicate inhibition of CYP1A induction at the higher concentration, which may be linked to antagonism at the Ah- receptor. The biological endpoints measured in the current project reflected the integrated effect from extract exposure, potentially affected by additivity, synergism and/or antagonism. Differences in toxicity between in vitro and in vivo conditions, along with several biological and environmental parameters can affect the biological responses. The results obtained in the current project indicated presence of potential hazards in the soil, but no further conclusion could be drawn about the actual hazard from humans exposure to polluted soil.

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Abstract:

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
Vita.
Includes bibliographical references.
The Escherichia coli AlkB protein is an exceptionally versatile DNA repair enzyme. Its expression is induced upon exposure to alkylating agents as part of the Ada-mediated adaptive response. This member of the ac-ketoglutarate/iron(II)-dependent dioxygenase family was originally discovered to reverse directly methylated lesions formed preferentially in single-stranded regions of DNA, such as 1-methyladenine and 3- methylcytosine. Repair proceeds via an oxidative demethylation pathway, in which the aberrant methyl group is hydroxylated and spontaneously lost as formaldehyde. Since these early studies, the list of lesions repaired by AlkB through this pathway has been extended to include 1-methylguanine, 3-methylthymine, 3-ethylcytosine, and 1-ethyladenine. Furthermore, the protein possesses a second, distinct chemical mechanism through which it can repair another class of lesions, the etheno-adducts formed by the reaction of DNA with metabolites of the carcinogen vinyl chloride or with breakdown products generated by lipid oxidation. In this case, direct repair proceeds through epoxidation of the etheno bond, creating an intermediate that hydrolyzes to a glycol form and finally releases the two-carbon bridge as glyoxal, restoring the unadducted adenine or cytosine. Thus, the AlkB protein bridges the repair of alkylative lesions with those induced by oxidative stress and embodies the multi-faceted protection required to preserve genomic stability and coding information despite the constant threats to which organisms are exposed.
(cont.) Herein, we exploit and characterize a pair of E. coli strains differing only in AlkB status to demonstrate the ability of AlkB to repair the etheno-lesions, the structural analog 1,N6-ethanoadenine (EA), and 3-methyluracil in vivo. Additionally, we establish the ability of the EA "repair product" to form interstrand cross-links in certain sequence contexts of duplex DNA. We also show that although the adaptive response proteins repair lesions generated by oxidative stress, oxidative agents do not induce expression of the response. Finally, we establish that certain hypothesized substrates for AlkB are not in fact repaired by the enzyme, nor are they repaired by another adaptive response protein, AidB. This work extends the current knowledge regarding the amazing ability of AlkB to protect cellular nucleic acids from damage arising from a diverse array of both endogenous and exogenous sources.
by Lauren Elizabeth Frick.
Ph.D.