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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 CellsÅsgård, Rikard January 2014 (has links)
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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