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- Bivehed, Erik, 1990-
(författare)
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Evolving the Methodology for Detection of Primary DNA Damage : Development, adaptation and assessment of the single cell gel electrophoresis (comet) assay
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Deoxyribonucleic acid (DNA) is one of the most important molecules in nature. It is the fundamental carrier of evolutionary information and constitutes the genetic blueprint of all living organisms. Being the sole source of information, it is vital for the cell to transmit the correct genetic information from generation to generation. DNA damage is a critical precursor to cancer development, highlighting the need for tests to predict genotoxicity and mutagenicity of various agents, including pharmaceuticals and environmental factors. This thesis focuses on enhancing the single cell gel electrophoresis (comet assay) for assessing primary DNA damage.The work was concentrated around several fundamental aspects of the methodology where a novel statistical approach, Uppsala Comet Data Analysis Strategy (UCDAS), was developed for data evaluation. A proportional odds model tailored to continuous outcomes was used, accommodating the experimental design's hierarchy, large zero values, and avoiding data transformation. A revisit of the formulation of the electrophoresis medium led to the introduction of a low conductive lithium hydroxide-based solution, enabling higher field strengths, significantly reducing runtimes and increasing sensitivity compared to the conventional comet assay. A lot of work was done on the investigation of the pH's impact on DNA integrity, revealing elevated background DNA damage at higher pH levels. Extended unwinding at pH >13, typical of the most commonly used versions of alkaline comet assays, jeopardizes the integrity of DNA, resulting in greater background DNA damage than at lower pH values. The study underscores pH's significance for DNA stability, highlighting risks associated with extremely alkaline conditions.A new method was developed, the Polymerase Assisted DNA Damage Assay (PADDA), to label and quantify single- and double-strand DNA breaks selectively in comet heads and tails after exposure to established DNA-damaging agents. This approach also allowed detection of DNA damage inside comet heads, an ability lacking in traditional comet assays.In conclusion, this research enhances DNA damage assessment methodologies, introducing new statistical innovations, novel electrophoresis mediums, and a novel technique for the selective detection and quantification of single- and double-strand breaks. These advancements deepen our understanding of DNA damage's complexities and underscore the crucial role of pH in influencing DNA stability and its implications for genotoxicity assessment.
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| 2. |
- Åsgård, Rikard
(författare)
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Effects of Antioxidants and Pro-oxidants on Oxidative Stress and DNA Damage using the Comet Assay : Studies on Blood Cells from Type 2 Diabetes Subjects and Mouse Lymphoma Cells
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diet and oral supplements comprise two distinct sources of antioxidants known to prevent oxidative stress. Beneficial effects from antioxidants have been seen for patients at risk for type 2 diabetes.The aim of this thesis was to evaluate the positive effects of antioxidants against oxidative stress and DNA damage in type 2 diabetes subjects. We also used antioxidants as tools to determine the mechanisms behind genotoxicity induced by mutagenic pro-oxidative agents in mouse lymphoma cells. Several techniques were used to measure oxidative stress and DNA damage, but the main technique used was alkaline comet assay.The results showed that the fruit and vegetable intake was inversely related to oxidative stress in type 2 diabetes subjects. However, oral supplementary intake of 20 antioxidants did not decrease oxidative stress biomarkers.In studies on mouse lymphoma cells, using the alkaline comet assay, DNA damage was induced by catechol and o-phenylenediamine (OPD), while 4-nitro-o-phenylenediamine (4-NOPD) induced only oxidative damage, showing different mechanisms of action behind the mutagenicity of the compounds. Also, oxidative stress was induced by catechol and 4-NOPD, whereas imbalances in the nucleotide pool were seen after exposure to OPD or 4-NOPD. Addition of antioxidants together with these pro-oxidants showed that β-carotene was able to reduce DNA damage at low concentrations of catechol, but increased DNA damage at high concentration. In comparison, addition of α-tocopherol slightly decreased catechol-induced DNA damage at all concentrations of catechol. However, no effect of α-tocopherol was seen on OPD-or 4-NOPD-induced DNA damage.In conclusion, antioxidants from fruits and vegetables, but not from oral supplements, reduced oxidative stress in type 2 diabetes patients, suggesting fruits and vegetables being a healthier source for antioxidant-intake, as compared to oral supplements. Different mechanisms of action for mutagenic pro-oxidants were shown in mouse lymphoma cells, introducing the nucleotide pool as an interesting target for oxidative stress. Reduction of catechol-induced DNA damage by β-carotene or α-tocopherol was shown, with a pro-oxidative action of β-carotene at high concentration of catechol, Interestingly, α-tocopherol was not able to decrease OPD- or 4-NOPD-induced DNA damage, supporting different mechanisms of action behind the genotoxicity from the three pro-oxidants.
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