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Dietary flavonoids are ubiquitous and are marketed as supplements. Characterized as antioxidants, they offer protection against a number of degenerative diseases. Flavonoid mechanics involve free radical scavenging, metal chelation, and substrate association. The skeletal structure of flavonoids is a fused ring system modified by hydroxyl, sugar, and carbohydrate additions. Flavone is a structurally simple flavonoid. Quercetin and its glycosidic analog rutin are complex structures. Using a DNA oxidation/cleavage assay, flavone reduces DNA nicking by 91%. Depending on the solvent system used, quercetin can either increase or decrease DNA oxidation. Rutin exhibits neither pro- nor antioxidant activity. The molecular interactions responsible for these results are defined for flavone. 1) Flavone intercalates into DNA and saturates DNA at a 1/3.5 flavone:DNA molar ratio. 3) Flavone reduces iron-dependent DNA oxidation. 4) Flavone interacts with quercetin to enhance DNA protection. These results characterize the primary activities of a simple flavonoid.

Adriamycin is a first line cancer treatment drug for breast cancer and many soft tissue carcinomas. Adriamycin is a highly effective anti-cancer drug. It has a fused ring system that is planar, hydrophobic and electron rich. These features allow intercalation into DNA which, along with its topoisomerase inhibition, results in its anti-cancer effectiveness. Despite its success, its use is limited by a dose-dependent cardiotoxicity. Adriamycin forms tight complexes with iron in varying iron:drug ratios. The chelated complex [Fe+3-Adriamycin (1:2)] oxidatively cleaves DNA via the generation of reactive oxygen species (ROS). Enzymes associated specifically with heart mitochondria increase ROS formation explaining why adriamycin is selectively cardiotoxic. Pretreatment of mice with the neural hormone melatonin eliminates the cardiotoxic effectiveness of adriamycin therapy without reducing the drugÆs antitumor effect (Wahab et al. 2000, Tumori 86: 157-162). This has recently been verified in human cancer patients (Lissoni et al. 1999, European Journal of Cancer 35: (12) 1688-1692). Melatonin is soluble in both lipid and aqueous environments and has no known side-effects. This study analyzes the mechanistic features of DNA damage by Fe+3-Adriamycin and how melatonin ameliorates this damage. An Fe+3-Adriamycin (1:2) complex + glutathione cleaves DNA; this oxidative DNA cleavage does not occur with adriamycin +/- glutathione or with FeADR in the absence of glutathione. Melatonin reduces this oxidative DNA cleavage by 67%. Using a supercoiled-to-nicked-circular-conversion assay in conjunction with spectroscopic analyses give results revealing : 1) Fe+3-Adriamycin (1:2) complex + glutathione forms a reactive intermediate. 2) Melatonin protects the DNA from cleavage by this reactive intermediate. 3) Enzymatic assays show that H2O2 is the primary ROS formed with downstream production of the hydroxy radical via the Fenton reaction. 4) Fe+3-Adriamycin (1:2) intercalates into DNA to the same degree as uncomplexed adriamycin. 5) Melatonin also binds to DNA but not by intercalation. These experiments indicate that the cardioprotective effect of melatonin in adriamycin therapy may stem from melatoninÆs interaction with a reactive intermediate from Fe+3-Adriamycin(1:2) complex + glutathione and/or a direct interaction of melatonin with DNA.

Studies described in this thesis were aimed at characterizing the mechanism(s) of aflatoxin B1 (AFB1) pulmonary carcinogenesis by addressing the formation, prevention, and repair of AFB1-induced oxidative DNA damage. The ability of AFB1 to cause oxidative DNA damage in different lung cell types of the A/J mouse was examined. The formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells, was assessed by high performance liquid chromatography with electrochemical detection. An increase in 8-OHdG formation occurred in macrophage and Clara cell preparations isolated from A/J mice two hours following in vivo treatment with a single tumourigenic dose of AFB1. Prior treatment with polyethylene glycol-conjugated catalase (PEG-CAT) prevented the AFB1-induced increase in 8-OHdG levels in all mouse lung cell preparations. These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB1 carcinogenicity. Mouse lung tumourigenesis was assessed following treatment of A/J mice with PEG-CAT and/or AFB1. Unexpectedly, the mean number of tumours per mouse and tumour size in the PEG-CAT + AFB1 group were greater than those of the group treated with AFB1 alone. There was no difference in K-ras exon 1 mutation spectrum or in the histological diagnosis of tumours between treatment groups. In vitro incubation with mouse liver catalase (CAT) resulted in conversion of [3H]AFB1 into a DNA-binding species, a possible explanation for the results observed in vivo. These results demonstrate that PEG-CAT is not protective against AFB1 carcinogenicity in mouse lung despite preventing DNA oxidation. The effect of in vivo treatment of mice with AFB1 on pulmonary and hepatic base excision repair (BER) activities and levels of 8-oxoguanine DNA glycosylase (OGG1) was investigated. AFB1 treatment increased 8-OHdG levels and BER activity in mouse lung, but did not significantly affect either in liver. Levels of OGG1 immunoreactive protein were increased in both mouse lung and liver. These results indicate that oxidative DNA damage may be an important mechanism in the carcinogenicity of AFB1. However, BER activity is increased by AFB1 treatment, possibly representing a compensatory response to the production of oxidative DNA damage. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2008-12-12 10:00:44.81

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke. To exert its carcinogenic effect, NNK is metabolically activated to reactive intermediates that can damage DNA by alkylation or pyridyloxobutylation. NNK also has the ability to induce DNA oxidation and alter DNA repair activities that can result in deficient repair and potentially exacerbate carcinogenesis. Base excision repair (BER) is a ubiquitous DNA repair system that mainly repairs oxidative DNA damage. The goal of this study was to determine the effect of NNK on DNA oxidation status and BER activity in A/J mouse lung and liver. Female mice were treated with 10 µmol of NNK i.p. and lung and liver were isolated 1, 2 and 24 hours post administration. DNA was isolated from lung and liver, and the formation of 8-hydroxydeoxyguanosine (8-OHdG, a biomarker of DNA oxidation) was assessed by high-performance liquid chromatography with electrochemical detection. At 1, 2 and 24 hours in both murine lung and liver, there was no statistically significant difference in 8-OHdG levels (n = 4, P > 0.05) between control and NNK-treated mice. To assess BER, cell-free whole tissue nuclear protein extracts from liver and lung were prepared and incubated with a plasmid substrate containing oxidative DNA damage. In vivo treatment with NNK did not alter BER activity in lung or liver compared to control mice (n=3 or 4, P > 0.05). These experiments indicate that acute treatment with a tumourigenic dose of NNK does not significantly stimulate oxidative DNA damage or significantly alter BER activity in murine lung and liver. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2011-04-28 17:42:08.172

Background: The human organism is exposed daily to many endogenous and exogenous substances that are the source of oxidative damage. Cell structures, including DNA (deoxyribonucleic acid) in the nucleus are damaged due to high concentrations of these substances and accumulation of oxidative stress in cells. The predominance of these damaging processes may later be responsible for human diseases such as cancer, neurodegenerative diseases or heart failure. In our study, we observed oxidative damage at the DNA level due to spinal anesthesia. Methods: Sample processing was performed by comet analysis. The principle consists in fixation of cells (lymphocytes) in the agarose gel, lysis of cell structures for nucleotide release, incubation with specific enzymes and exposure to electrophoresis. Damaged, negatively charged parts of the DNA in the electric field are directed to the positive charged anode, creating a typical comet shape. For visualization, the gels were stained with ethidium bromide (DNA intercalating dye). Results: We have quantified single-strand breaks, oxidized purines and pyrimidines (use of enzymes to detect specific damages). The results are reported in percentage of DNA in the comet's tail. The principle is to compare the intensity of the comet's tail with the total comet intensity....

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The studies described in this thesis investigated the relationship between DNA repair mechanisms, aflatoxin B1 (AFB1)-induced DNA damage and carcinogenesis. Mice deficient in 8-oxoguanine glycosylase (OGG1, the rate-limiting enzyme in repair of oxidized guanine), mice heterozygous for OGG1, and wild type mice, were exposed to a single tumourigenic dose (50 mg/kg) of AFB1. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidized guanine in lung or liver 2 h post-treatment. ogg1 (-/-) mice had increased susceptibility to AFB1 toxicity, as reflected by increased mortality within one week of AFB1 exposure. AFB1 treatment did not significantly increase lung or liver tumourigenesis compared to DMSO controls. No difference was observed between ogg1 genotypes, although a non-significant trend towards AFB1-treated ogg1 (-/-) mice being more susceptible to tumourigenicity was apparent. Overall, deletion of ogg1 did not significantly affect AFB1-induced DNA damage or tumourigenicity, suggesting that oxidized guanine may not be a major contributor to AFB1-induced tumourigenesis. The effects of AFB1 on DNA repair were assessed in p53 (a protein implicated in regulation of DNA repair) wild type and heterozygous mice. p53 (+/+) mice treated with 0, 0.2 or 1.0 ppm AFB1 for 26 weeks had increased nucleotide excision repair (NER) activities in lung and liver compared to control, which may represent an adaptive response to AFB1-derived DNA adducts. In p53 (+/-) mice, the AFB1-induced increase in NER was significantly attenuated, suggesting that loss of one allele of p53 limits the ability of NER to up-regulate in response to AFB1-induced DNA damage. Twenty-six week exposure to AFB1 did not affect base excision repair (BER) in p53 (+/+) mouse lung or liver compared to control. BER was significantly decreased in livers from mice exposed to 1.0 ppm AFB1 compared to those exposed to 0.2 ppm AFB1, a result that was not due to liver cell death or to altered levels of OGG1 protein. In lungs and livers of p53 (+/-) mice, BER activity was unchanged by AFB1. As such, the difference in BER response between 0.2 ppm and 1.0 ppm AFB1 treatment seen in the p53 (+/+) mice appears to be p53 dependent. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-10-17 22:24:31.577