The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes. Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified. Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects. Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. vi The human body has evolved certain defence mechanisms to cope with the high occurrence of free radicals. These radicals are obtained endogenously from the mitochondria, peroxisomes, the cytochrome P450 (CYP 450) system and neutrophils, or exogenously from the environment. Lack of antioxidants and/or increased production of free radicals will result in oxidative stress, which has been implicated in certain human diseases such as hypertension, inflammation, ageing, autoimmunity, atherosclerosis, Parkinson?s disease, cancer and diabetes. Although the initial aim was to standardise a single assay to quantify both 8–OHdG and DHN–MA, this could not be achieved in this study due to the vast difference in the chemical properties of these two metabolites. Following the decision to use two separate assays for the quantification of the mentioned biomarkers, the 8–OHdG assay was standardised and validated. The intrabatch variation of the assay was 4.18% and the interbatch variation was 17.37%. Unfortunately, the DHN–MA assay could not be standardised within the time frame of this study due to experimental difficulties. Therefore, only urinary 8–OHdG and serum ROS levels were quantified. Urinary 8–OHdG levels were measured in 409 participants (209 Caucasians, 101 males and 108 females and 200 Africans, 100 males and 100 females) from the SABPA study. After removal of outliers from the data matrix, the effect of gender and ethnicity was investigated on the measured urinary 8–OHdG levels. No significant difference in the urinary 8–OHdG levels between Caucasian males (n=87) and females (n=96) were observed (p = 0.68). A similar observation was made for the African males (n=86) and females (n=84), where no significant difference in 8–OHdG levels was detected (p = 0.053). Thus, from the results obtained in this study, it seems that urinary 8–OHdG levels are not influenced by gender. However, 8–OHdG levels were dramatically influenced by ethnicity. Caucasian males (n=87) excreted 70% higher amounts of 8–OHdG compared to African males (n=86) (p < 0.001). Caucasian females (n=96) also excreted larger urinary 8–OHdG amounts (42%) compared to African females (n=84) (p < 0.001). Therefore, it seems that urinary 8–OHdG levels are dramatically influenced by ethnicity. Finally, urinary 8–OHdG levels were compared to serum ROS levels, but no significant correlation between the measured metabolites was observed (r = –0.045). Hence, urinary 8–OHdG and serum ROS levels are not related in these subjects. Even though the initial aim of this study was to standardise an analytical method to quantify both urinary 8–OHdG and DHN–MA, this could not be achieved due to time constraints. However, an LC–MS/MS analytical assay was standardised and validated for the quantification of urinary 8–OHdG. The method proved reliable for the quantification of 8–OHdG from urine samples and can thus be used for further studies on oxidative DNA damage. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

O câncer é a segunda maior causa de morte no mundo, sendo responsável por aproximadamente 7,6 milhões de óbitos. Entretanto, pesquisadores alertam para uma associação inversa entre o consumo de frutas e hortaliças e o desenvolvimento de neoplasias, desta forma a organização mundial da saúde sugere, dentre outras medidas para controle do câncer, o aumento do consumo de frutas e hortaliças. Nesse contexto o objetivo deste trabalho foi avaliar os eventuais efeitos quimiopreventivos das hortaliças Brassicas, couve (C) e repolho (R). Realizaram-se dois experimentos sendo o primeiro, o modelo de hepatocacinogênese de Ito, onde as hortaliças foram fornecidas durante 8 semanas na água de beber (10% p/v), animais que receberam apenas água foram utilizados como controle. Nesse experimento não houve inibição (P>0,05) de lesões pré-neoplásicas hepáticas positivas para glutationa S-transferase forma placental e não houve indução (P>0,05) da apoptose nos grupos tratados com C ou R. Contudo, observou-se redução (P<0,05) de danos em DNA hepático e aumento (P<0,05) da concentração hepática de luteína de ratos tratados com C e R, quando comparados a ratos controle. No segundo experimento as hortaliças foram fornecidas durante 8 semanas na água de beber (20% p/v), e os animais foram submetidos a aplicação do carcinogênico hepático 24h antes da eutanásia. Não houve redução (P>0,05) de danos em DNA, contudo a concentração do aduto de DNA 8-hidroxi-2-deoxiguanosina (8-OHdG) e foi elevada (P<0,05) em animais tratados com R quando comparados a tratados com C e controles. Com relação à expressão diferencial de genes, 29 genes foram diferencialmente expressos em fígado, dentre eles o gene da 8-oxoguanina-DNA-glicosilase (enzima de reparo do DNA), foi hipoexpressa no grupo tratado com R, o que pode explicar o aumentado valor de adutos no mesmo grupo. O cólon apresentou 31 genes com diferença de expressão, onde 5 genes estão relacionados ao metabolismo de xenobióticos. / Cancer is the major cause of death in the world, being responsible for approximately 7.6 million deaths. However, there is a hypothesis of an inverse association between fruit and vegetable consumption and the development of cancer. Therefore, the World Health Organization suggests, among other actions for controlling cancer, the increase in vegetable and fruit consumption. The aim of this work was to evaluate eventual chemopreventive effects of Brassicas vegetables, kale (K) and cabbage (C). Two experiments were done: the first one was Ito´s hepatocarcinogenesis model, where vegetables were provided during 8 weeks in the rats´ drinking water (10% w/v). Animals that received only water were considered control. In this experiment, there was no inhibition (P<0,05) of glutathione S-transferase placental form positive preneoplastic lesions and, also, there was no induction (P<0,05) of apoptosis in the groups treated with K or C However, it was observed a reduction (P<0,05) in hepatic DNA damages and an increase (P<0,05) in lutein hepatic concentration of rats treated with K or C, when compared to the control. In the second experiment, the vegetables were provided during 8 weeks in the rats´ drinking water (20% w/v), and animals were submitted to carcinogenic application 24h before euthanasia. There was no reduction (P<0,05) in DNA damages, however there was an increase (P<0,05) in the concentration of 8-hydroxy-2-deoxyguanosine (8-OHdG) DNA in animals treated with C when compared to the ones treated with K and control. In relation to the differential gene expression, 29 genes were differently expressed in the liver, such as the 8-oxoguanine-DNA-glycosylase gene, which was downregulated in the group treated with C. This might explain the increased value of adducts in the same group. Colon presented 31 genes with difference in expression, whereas 5 genes are related to xenobiotic metabolism.

Point-of-care testing (POCT) is defined as medical testing at or near the site of patient care and has become a critical component of the diagnostic industry. POCT has many advantages over tests in centralized laboratories including small reagent volumes, small size, rapid turnaround time, cost-effectiveness, low power consumption and functional integration of multiple devices. Paper-based POCT sensors are a new alternative technology for fabricating simple, low-cost, portable and disposable analytical devices for clinical diagnosis. The focus of this dissertation was to develop simple, rapid and low cost paper-based POCT sensors with high sensitivity and portability for disease biomarker detection. Lateral flow strips (LFS) were used as the basic platform as it provides several key advantages such as simplicity, fast response time, on site and cost-effectiveness, and it can be used to detect specific substances including small molecules, large proteins and even whole pathogens, in a sample by immunological reactions. Earlier designs of paper strips lacked the quantitative information of the analyte concentration and could only provide single analyte detection at a time. In this study, a series of modifications were made to upgrade the platform to compensate for these limitations. First, we developed a gold nanoparticle based LFS for qualitative colorimetrical detection of bladder cancer related biomarkers in standard solutions and in urine samples. Second, by incorporating an image processing program “ImageJ”, a semi-quantitative LFS platform was established. The capability of the strip was evaluated by testing a small DNA oxidative damage biomarker in urine and cell culture models. Third, we combined the electrochemical method and colorimetrical method for quantitative biomarker detection. Finally, we integrated a commercialized blood glucose meter to quantitatively detection of two non-glucose biomarkers by converting their signals to that of glucose. The upgraded sensor could provide a noninvasive, rapid, visual, quantitative and convenient detection platform for various disease biomarkers. In addition, this platform does not require expensive equipments or trained personnel, deeming it suitable for use as a simple, economical and portable field kit for on-site biomarker monitoring in a variety of clinical settings.

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2′-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and 13C- and 15N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.

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