Stammzellbasierte Therapieverfahren versprechen neue Lösungen für bisher nur unzureichend behandelbare Erkrankungen. In der Hals-, Nasen- und Ohrenheilkunde ist die Herstellung von Knorpel im Rahmen des Tissue Engineering von besonderem Interesse. Die mesenchymalen Stammzellen des Fettgewebes (ASC) stellen eine vielversprechende Zellpopulation als Ausgangspunkt für die Erzeugung von Gewebe dar. Auf Grund der hohen Zahl an Zellteilungen, oxidativem und mechanischem Stress sowie enzymatischer Verdauung steigt im Rahmen der in vitro Expansion das Risiko für DNA-Schäden. Diese können wiederum der Ausgangspunkt für die maligne Transformation einer Zelle sein. Ziel unserer Studie war es, zu zeigen, ob die Expansion und mehrfache Passagierung zu einer zunehmenden genetischen Instabilität der ASC führt. Es wurden frische ASC aus Liposuktionsaspirat von 8 verschiedenen Patienten isoliert. Mit ASC der Passagen 1, 2, 3, 5 und 10 wurde zur Detektion von Schäden auf DNA-Ebene jeweils eine alkalische Einzelzellgelelektrophorese(Comet Assay) und ein Mikrokerntest durchgeführt. Zur Erfassung von Schäden auf Chromatidebene erfolgte darüber hinaus mit Zellen der selben Passage ein Chromosomenaberrationstest. Mit dem Comet Assay und dem Mikrokerntest konnte keine signifikante Progression der genetischen Instabilität mit zunehmender Passage nachgewiesen werden. Beim Chromosomenaberrationstest zeigte sich im Friedman-Test eine signifikante Zunahme an strukturellen Chromosomenaberrationen mit steigender Passage. Der Wilcoxon-Test hingegen erbrachte kein signifikantes Ergebnis. Die im Rahmen dieser Arbeit gewonnen Daten zeigen, dass eine zunehmende genetische Instabilität der ASC mit zunehmender Dauer der Expansion und steigender Passage nicht vollständig ausgeschlossen werden kann. Aus diesem Grund sollten vor einer Transplantation regelhaft Untersuchungen wie beispielsweise ein Chromosomenaberrationstest oder ein Screening auf typische malignitätsfördernde Mutationen erfolgen. / Stem-cell based therapies promise new solutions for diseases which are insufficiently treatable up to now. In Otorhinolaryngology, the in vitro production of cartilage for tissue engineering approaches is of particular interest. Mesenchymal adipose-derived stem cells (ASCs) are a promising cell population for the production of tissue. Due to a high number of cell divisions, oxidative and mechanical stress as well as enzymatic digestion there is an increasing risk of DNA-damage during in vitro expansion. This DNA-damage can lead to a malignant transformation of the ASCs. The aim of our study was to show whether prolonged in vitro expansion leads to an increased genetic instability of ASCs. Human ASCs were isolated from subcutaneous adipose tissue of different donors (n = 8) undergoing liposuction surgery for aesthetic reasons. To detect DNA-damage, an alkaline single-cell microgel electrophoresis (comet) assay and a micronucleus assay were performed with cells of passage 1,2,3,5 and 10. Moreover, to assess chromosomal damage, a chromosomal aberration test was carried out with cells of the same passage. With the comet assay and the micronucleus assay, no significant progress of DNA-damage could be demonstrated. However, the chromosomal aberration test showed a significant increase of structural chromosomal damage. The results of our study underline the fact that an increasing genetic instability of ASCs during prolonged in vitro expansion cannot be completely excluded. Consequently, tests monitoring malignant transformations or genetic instability should be implemented before transplantation of ASCs.

Hintergrund: Die Studienlage zu Kautabak und Zigarettenrauch ist eindeutig und zeigt karzinogenes Potential. Über Schnupftabak ist hingegen wenig bekannt, vor allem auf zellulärer Ebene gibt es keine ausreichenden wissenschaftlichen Publikationen. Somit lässt sich die eventuell mutagene Wirkung von Schnupftabak nur schwer einschätzen. In Konsequenz stützt sich die WHO in ihrer Einstufung des Schnupftabaks als nicht karzinogen auf eine sehr eingeschränkte Datenlage. Ziel: Ziel der vorliegenden Arbeit war es, Schnupftabak auf mögliche zyto- und genotoxische Effekte auf humane Lymphozyten und Nasenschleimhautzellen zu untersuchen um ggf. tumorinitiierende Effekte darzustellen. Material und Methoden: Es kam eine Schnupftabaksorte ohne Menthol und eine Sorte mit Mentholzusatz zum EInsatz. Die benötigten Nasenschleimhautzellen und Lymphozyten wurden von 10 Probanden gewonnen und eine Stunde lang mit einem Schnupftabak-DMSO-Gemisch (2000µg/ml bis 0,01µg/ml) inkubiert. Zur Analyse wurde der Trypanblautest, dee Comet Assay und der Mikrokerntest verwendet. Ergebnis: Der Trypanblautest zeigte keinen Abfall der Vitalität. Beim Comet Assay ergab sich bei Lymphozyten ein signifikanter Anstieg der DNA-Fragmentierung ab 100µg/ml, bei Nasenschleimhautzellen ab 1000µg/ml. Der Mikrokerntest wies keine signifikante Zunahme der Mikrokerne auf. Es konnte kein Unterschied zwischen den beiden Tabaksorten aufgezeigt werden. Diskussion: Es zeigte sich eine Schädigung der Erbsubstanz im Comet Assay, die möglicherweise reparabel ist. Irreparable DNA-Schäden im Sinne von Mikrokernen wurden nicht gefunden. Nach diesen Ergebnissen muss die Einstufung der WHO in Zweifel gezogen werden. Untersuchungen mit weiteren Endpunkten der Genotoxizität sind somit gerechtfertigt, um zu einer fundierten Beurteilung des Risikopotentials von Schnupftabak zu gelangen. / Background: While an abundant number of studies concerning tobacco smoke and chewing tobacco show carcinogenic potential, there is little data on the consequences of snuff, especially on the cellular level. Therefore, the mutagenic effect of snuff is hard to estimate and the WHO assessment of snuff being not carcinogenic bases on very limited data. Objectives: This paper investigates potential cytotoxic and genotoxic effects of snuff on human lymphocytes and nasal mucosa cells. Materials and methods: Two kinds of snuff were used, one with a high degree of essential oil. The necessary nasal mucosa cells and lymphocytes were taken from 10 subjects undergoing nasal obstruction surgery and incubated with a snuff mixture (from 0,01µg/ml to 2000µg/ml). Methods included the trypan blue test, the comet assay and the micronucleus test. Results: The trypan blue test showed no decrease in cell viability for both cell types. The comet assay revealed a significant increase in the Olive Tail Moment for lymphocytes starting at 100µg/ml and 1000µg/ml for nasal mucosa cells. There was no significant increase in micronuclei according to the micronucleus test. Conclusion: The present study demonstrated genotoxic damage, such as DNA strand breaks, which may be repaired, but no non-repairable elevated micronuclei. The present findings cast doubts the WHO assessment that snuff is not carcinogenic, however, further research on various genotoxic endpoints in human cells are warranted.

Lifestyle diseases represent a large burden on developed societies and account for much morbidity worldwide. Research has shown that eating a diet rich in fruit and vegetables helps to ameliorate and prevent some of these diseases. Antioxidants found in fruits and vegetables may provide a substantial benefit in reducing disease incidence. This thesis examines the antioxidant properties of resveratrol, piceatannol, and pterostilbene, and the ability of Burkitt's Lymphoma (Raji) cells to uptake these three antioxidants. It also studies the effect of the antioxidants in protecting against DNA damage and their role in DNA repair following oxygen radical exposure in Raji cells. The Oxygen Radical Absorbance Capacity (ORAC) assay was used to measure overall antioxidant contribution as well as the ability of Raji cells to uptake antioxidant following exposure to 2,2’-Azobis(2-methyl-propionamide) dihydrochloride (AAPH). The single cell gel electrophoresis (Comet) assay was used to assess DNA damage and DNA repair rates of cells. Results showed that Raji cells, following oxygen radical exposure, significantly uptake pterostilbene (p < 0.0001), but not piceatannol or resveratrol. Piceatannol provided protection against hydrogen peroxide induced DNA damage, but pterostilbene and resveratrol increased DNA damage following hydrogen peroxide treatment. None of the compounds showed any effect on DNA repair. Overall, this study indicates there is merit for further research into the bioactive roles, including antioxidant capacity, of all three compounds. Such research may provide evidence for the more widespread use of these and other food based compounds for preventing lifestyle diseases.

Abstract The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens, 3-amino-1-methyl-5 H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), and/or H2O2 was evaluated using single-cell electrophoresis (comet assay). The toxicity of these tested compounds on lymphocytes was not found. These compounds did not cause DNA damage at lower concentration of 0.1-10 &#x00B5;g/ml. At a concentration of 100 &#x00B5;g/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA damage. TA and its related compounds decreased the DNA damage induced by Trp-P-2 or PhIP at a concentration of 0.1-10 &#x00B5;g/ml. Moreover, the inhibition of H2O2-induced DNA damage increased with increasing concentrations up to 10 &#x00B5;g/ml. DNA repair enzymes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG) were used to examine the levels of oxidised pyrimidines and purines in DNA damage induced by H2O2, respectively. All the compounds at 10 &#x00B5;g/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicate that these compounds can enhance lymphocyte resistance towards DNA damage induced by food mutagens or H2O2. Keywords: Tannic acid; Human lymphocyte; Comet assay; Hydrogen peroxide

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Ulm, Univ., Diss., 2008.

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Plants have been utilized as a source of medicines since ancient times. They contain a vast range of secondary metabolites which play important roles in different diseases. The Scope of this study is to define the function of secondary metabolites from Andrographis paniculata (Kalmegh) and Silybum marianum (Milk Thistle) against DNA damage, which initiates many diseases. Sequential extraction of both plant materials was performed with different polarity solvents. Qualitative analysis was performed with Gas (GC) and High Performance Liquid Chromatography (HPLC). Primary extracts screening studies were performed against cytotoxicity, antioxidant and soluble collagen assays (Sircol dye). Further bioactivity was confirmed using the single-cell gel electrophoresis (Comet assay) to estimate levels of DNA damage. Fourier Transform Infrared analysis of bioactive extracts was performed to identify the functional groups present in them . Subsequently, bioactive extracts were further separated into acid, base, phenol and neutral fractions. These fractions and bioactive extracts were screened with the free radical assays to identify the scavenging activity. Chemical mapping of the bioactive fraction was performed with High Performance Liquid Chromatography (HPLC). Preparative-HPLC was performed to separate the compounds which were present in the bioactive fractions. MTT assay, Hydroxyl radical and nitric oxide radical scavenging activity assays were performed to screen the fractions and DNA protective activity of the bioactive fractions was confirmed with single-cell gel electrophoresis. These bioactive fractions were de-replicated with hyphenated techniques like LCMS and LCMS/MS to identify the molecular weight of the compounds and Quadrupole time-of-flight mass spectrometry was performed to identify the accurate mass of the compounds. Sequential extraction separates the non-polar and polar compounds present in the plant material. Qualitative analysis confirmed the presence of fatty acids in the non-polar extracts of both plants using GC and the presence of standard constituents in the polar extracts of both plants using HPLC. It also helps in chemical mapping of the extracts. Acetone, Methanol1 and Methanol2 extracts from either plant are non-cytotoxic. The high antioxidant activity is observed in methanol extracts from Andrographis paniculata and in acetone/methanol2 extracts from Silybum marianum. Extracts that protect against UVA and UVB damage also increased soluble collagen production in Human Dermal Fibroblast (HDF) in culture. Primary Screening helped to select six extracts out of twelve extracts for further analysis. Comet assay confirmed DNA protective activity in Methanol1 extract of Milk thistle and Acetone, Methanol2 extracts from Kalmegh. These three extracts were further fractionated into 38 fractions out of which three fractions that are F1, F13 and F31 fractions confirm the DNA protection activity. De-replication of the bioactive fractions was performed with LC-ESI-MS/MS which confirm twenty one compounds and accurate mass of fifteen compounds was determined using Q-tof mass spectrometry.

Made available in DSpace on 2014-10-09T12:26:48Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:22Z (GMT). No. of bitstreams: 0 / Tendo em vista um mercado cada vez mais exigente na qualidade dos alimentos, faz-se necessário o desenvolvimento de processos que atendam às expectativas do consumidor. Dentre os processos existentes, destacam-se a cadeia do frio e a irradiação. A cadeia do frio compreende todas as etapas de conservação do alimento, desde a produção, resfriamento, congelamento, armazenamento, e transporte até o consumidor final. A irradiação, como processo de conservação de alimentos, estende a vida de prateleira, inibe o brotamento e reduz a contaminação por patógenos, entre outros benefícios. É importante a identificação da degradação dos alimentos em função de falhas nos processos a que foram submetidos. O teste do cometa (DNA Comet Assay) é um método de varredura largamente estudado, considerado rápido e de baixo custo, pelo fato de identificar quebras no DNA, é possível considerar sua utilização como mais uma ferramenta no controle de falhas na cadeia do frio que podem degradar e prejudicar os alimentos. Algumas etiquetas e selos usados no controle dos processos do frio não consideram a situação anterior do alimento, indicando falhas a partir do momento em que forem colocadas em contato com o mesmo, já o teste do cometa verifica a degradação ocorrida no alimento até o momento de sua realização podendo ainda, acompanhar o aumento da degradação. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP