• Biological activities of natural and semi-synthetic pseudo-guaianolides: Inhibition of transcription factors.

      Villagomez, Rodrigo (Media-Tryck, Lund University, Sweden, 2014, 2014-06)
      Damsin (1) is a natural sesquiterpene lactone (SL) isolated from Ambrosia arborescens Mill., a plant used in the Andes as antiinflammatory medicine. This natural product is an inhibitor of NF-κB, a protein complex that controls the transcription of many genes in mammalian cells, and has a potential for standing model for the development of new anti-cancer lead structures. In order to improve the anti-cancer activity, the chemistry of 1 was explored and in the process, dozens of derivatives were prepared. Damsin (1) inhibited cell proliferation, DNA biosynthesis and formation of cytoplasmic DNA histone complex in Caco-2 cells and further studies using the luciferase reporter system showed that it also inhibited expressions of NF-κB and STAT3. Therefore, the NF-κB inhibitory capacity of some derivatives was evaluated and two analogues, 31 and 32, were found to be more potent. In order to have a preliminary evaluation method of the derivatives, we developed fast and cheap biochemical assay to study the effect of SLs in the binding capacity of NF-κB (heterodimer RelA/p50) to the DNA recognition target. In this assay the compounds 21, 22, 24, 25 and 26 had a high dissociation capacity of the complex NF-κB/DNA. Finally, four compounds were selected for MS characterization studies with recombinant NF-κB, the most selective compound was 26 (compared with 1) by selective alkylation of Cys-38 and Cys-120 in RelA. The Cystein-38 is crucial for the transcriptional activity of NF-κB.
    • Effects of dietary fibre on the human metabolism and metabolome.

      Johansson-Persson, Anna (Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, Sweden, 2014, 2014-09)
      It is well-known that dietary fibre can have a positive effect on the development of lifestyle-dependent diseases such as cardiovascular disease and type 2 diabetes. However, the effect may be different for different kinds of fibre and different sub-populations at risk. Therefore, the effects of three different kinds of fibre on postprandial and long-term response in healthy subjects were investigated. Consumption of rye bran, oat powder, sugar beet fibre or a mixture of all three in a meal study, led to lower postprandial glucose levels for all meals except oat powder, although the difference was only significant for rye bran. The outcome seemed to be determined not only by the amount of soluble fibre, but also by the total dietary fibre content. The combined effect of an intake of oat bran, rye bran and sugar beet fibre was also investigated in a 5-week randomised cross-over intervention study in healthy, mildly hypercholesterolaemic subjects. Subjects were given a high-fibre (HF) diet (48 g) and a low-fibre (LF) diet (30 g). Despite the high fibre intake, no significant effects were observed on glucose, insulin, or lipid metabolism. However, low-grade inflammatory response was reduced by the HF diet, as reflected by decreased C-reactive protein and fibrinogen levels. Moreover, markers from the high intake of oat, rye and sugar beet fibre were observed in plasma and 24-h urine samples using an untargeted metabolomic profiling approach. After the HF diet, different benzoxazinoids and their metabolites 2-aminophenol sulphate, HPAA (N-(2-hydroxyphenyl)acetamide) and HHPAA (2-hydroxy-N-(2-hydroxyphenyl)acetamide), together with the alkylresorcinol metabolite DHPPA (3-(3,5-dihydroxyphenyl)-1-propanoic acid), were found to be specific for the rye intake, whereas enterolactone was related to rye and oat fibre intake. Some specific markers for oat intake were found, however, their identity needs further validation. One identified marker, 2,6-DHBA (dihydroxybenzoic acid), has not previously been reported as a marker related to dietary fibre intake. Whether there is a specific marker for sugar beet fibre intake remains unclear. These markers of the intake of specific dietary fibre sources could, if validated, serve as markers in intervention studies and larger observational studies, to provide more accurate data on general dietary fibre intake, apart from the subjects’ self-reported values, which are normally used. The effect of a healthy Nordic diet based on the Nordic Nutrition Recommendations, including a high dietary fibre intake, was investigated in obese subjects with metabolic syndrome. This was a randomised, parallel multi-centre study in which the Nordic diet was compared to a control diet. No effects were observed on the glucose and insulin metabolism, however, reductions in lipoproteins were found together with an indication of reduction in the inflammatory response after the intake of the Nordic diet. In conclusion, these studies confirm that a high dietary fibre intake has a beneficial effect on glucose and lipid metabolism. The intervention studies also indicated a reduction in low-grade inflammation markers that are associated with overweight and type 2 diabetes.
    • Bioactivity of Medicinal Bolivian Andean plants. Effects on cell proliferation and related processes.

      Rodrigo, Gloria C. (Lund University, 2012, 2012-11-22)
      Colon cancer is common in both developed and developing countries, and is responsible for at least 600,000 deaths globally every year. It is therefore the second most common cause of cancer-related mortality. Extensive studies are being conducted worldwide to find more effective drugs that can be used in cancer treatment. In these studies, phytochemicals have proven to be good sources for drug discovery. In Bolivia, there is a long tradition of using plants for medicinal purposes. The objective of the present thesis was to study the effects of extracts and compounds from medicinal plants in Bolivia on the growth of colon cancer (Caco-2) cells. Firstly, a survey of many plant extracts and some isolated compounds for their antiproliferative activity was performed. Sixty-six extracts from thirty-two medicinal plants and 15 extracts from 8 food plants were evaluated for antiproliferative activity in Caco-2 cells. Extracts from 7 plant species showed antiproliferative activity but in most of the preparations tested no cytotoxic activity was observed at the concentrations used. Secondly, some assays including DNA replication, DNA degradation, oligonucleosomal formation, and caspase-3 activity were performed to understand the mechanism by which the compounds isolated affect cell proliferation and cell death. Curcuphenol, isolated from Baccharis genistelloides and Myrmekioderma styx, and damsin and coronopilin, isolated from Ambrosia arborescens, were found to inhibit cell proliferation and to induce cell death in colon cancer cells. Further studies are needed to find new anti-cancer compounds in medicinal plants in Bolivia.
    • Development and application of biomarker methods in molecular environmental epidemiology for the detection of exposure to polycyclic aromatic hydrocarbons.

      Kovács, Katalin (University of Pécs, 2012, 2012)
      My research aimed at the further development of the 32P-postlabelling method by which, whilst maintaining the adduct-labelling efficiency, the amount of the radioisotope per sample can be reduced, and thereby the sample processing capacity of the laboratory is increased. The regular and the modified 32P-postlabelling methods were used for comparative determinations of the levels of DNA adducts from various types of DNA samples, i.e. BPDE-DNA adduct standard, DNA samples from MCF-7 cell line treated with B[a]P, DNA samples from human non-tumorous peripheral lung tissue, human peripheral blood lymphocytes and human buffy coat samples. In the modified 32P-postlabelling method, the final reaction volume of the radio-labelling was reduced with an evaporation-to-dryness step to one-third of the volume that was used in the regular method. This facilitated the reduction of the amount of [γ-32P]ATP substrate from 50 μCi per sample – depending on the DNS isolation method – by 50% for the Qiagen-isolated samples (i.e., 25 μCi per sample) and by 80% for the DNA samples isolated with the classic phenol extraction procedure (i.e.,10 μCi) for both experimental and human samples. The newly-developed BPDE-DNA direct sandwich chemiluminescence immunoassay (BPDE-DNA SCIA) for the determination of PAH-DNA adducts has been published in 2012. Whereas in the earlier competitive immunoassays the signal to be measured is coupled to the DNA adducts, in the SCIA the chemiluminescent signal is coupled to the non-adducted nucleotides. In SCIA, the strong end-point signal derives from the many orders of magnitude difference between the number of the non-adducted and the adducted mononucleotides. The limit of detection of the method is ~ 1.5 adducts/109 nucleotides from 5 μg DNA sample. For the validation of BPDE-DNA SCIA, I performed comparative measurements between the immunoassay and the 32P-postlabelling method. The DNA samples were obtained from MCF 7 cells treated with B[a]P, from the liver of mice, which were treated in vivo with several doses of B[a]P, B[b]F, and DB[a,h]A, respectively, and from human maternal peripheral blood and newborn cord blood samples. For the B[a]P-DNA adduct levels measured by SCIA and 32P-postlabelling from the MCF 7 cells, the ratio between the adduct values was about 0.5. For the animal samples, the adduct levels were several times lower by the immunoassay than by the 32P-postlabelling method (the ratios were ≈1:5 for B[a]P, ≈ 1:30 for B[b]F and ≈ 1:5 for DB[a,h]A). All the same, there was a very strong, highly significant positive correlation between the DNA adduct measurements of the dose-response curves by SCIA and 32P-postlabelling for each PAH compound (r = 0.87-0.99). For the human samples, the ratio between the SCIA and 32P-postlabelling values was approximately 1:10, but there was not correlation between the data-pairs measured by the two methods. For the human samples, the lack of correlation between the two methods may be explained by the different efficiency of detection of different structural types of DNA adducts that are derived from complex human environmental exposure.
    • Investigation of the associations of smoking-related DNA damages with biomarkers in a human lung cancer population.

      Anna, Livia (2012)
      The aim of my PhD research was to further explore the associations among smoking status, two different DNA adduct types, the O4-etT and bulky DNA adduct, the TP53 tumour suppressor gene mutations and lung cancer in a molecular epidemiological study in a Hungarian lung cancer study population. The levels of O4-etT and bulky DNA adducts were significantly higher in the combined group of subjects who smoked until surgery or gave up smoking at most one year before surgery than in the combined group of those subjects who gave up smoking more than one year before the surgery or never smoked. O4-etT appeared to be a highly persistent DNA damage. There was no statistically significant correlation between the individual levels of O4-etT and of bulky DNA adducts. The TP53 mutation frequency and the variety of mutation types were higher in the present study population as compared to the IARC database. 45% of the samples carried TP53 mutation. The mutation frequency was significantly higher in squamous cell carcinoma than in adenocarcinoma, and in the cases with more than 20 years of smoking history. The most common mutations were G→A (19%), G→T (19%) and G→C (16%) base changes. The mutation pattern was influenced by the smoking status. G→T transversion was detected exclusively in smokers, and most carriers of the G→T transversions had also high level of bulky DNA adducts. My results confirm that O4-etT level is increased by smoking in the lung. O4-etT is persistent in human lung, and the activation and elimination pathways of O4-etT and bulky DNA adducts are not closely linked. I consider O4-etT a suitable biomarker of smoking exposure for comparison of exposure groups in molecular epidemiological studies. For the first time at international level, I demonstrated strong association between G→T mutation of TP53 and high level of bulky DNA adducts in a human study, which is a significant scientific progress from the in vitro studies in the exploration of the causal relationship between a carcinogen-DNA adduct and a gene mutation.
    • Lymphoma; Pre-diagnostic Blood Markers and Occupational and Environmental Exposures.

      Saberi Hosnijeh, Fatemeh (Utrecht University, 2012, 2012)
      The overall aim of this thesis is to study the possible perturbations of the immune system by occupational and environmental risk factors of Non-Hodgkin lymphoma (NHL) and to study these changes in relation to NHL risk in prospective cohorts. In the first section, we validated the application of single blood cytokine measurements as a biomarker of immune status in prospective epidemiological studies. Potential utility of stored blood samples of a prospective cohort was evaluated by the effect of different blood sample types and freeze-thaw cycles on cytokine levels. This study showed strong correlations between different sample types. Moreover, freeze-thaw cycles did not markedly change cytokine levels. The intra-individual variance in cytokine levels was much smaller than the inter-individual variance which supports the notion that a single cytokine measurement can be used to characterize an individual's immune profile prospectively. Subsequently, we assessed the levels of these cytokines in pre-diagnostic blood samples of participants of a case–control study nested into the Italian European Prospective Investigation into Cancer and Nutrition cohort (EPIC) and determined their association with the risk of developing NHL. The results suggest a possible association between increased/decreased plasma levels of interleukin (IL)2, inter-cellular adhesion molecule, interferon gamma (IFN-γ), and tumor necrosis factor alpha with NHL risk. In the second section, we studied the possible immunological effects of two possibly lymphomagens: an occupational exposure (i.e. 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD)) and an environmental risk factor (i.e. obesity). We assessed a broad range of immunologic parameters directed towards detecting abnormalities in the humoral and cellular arms of the immune system among workers exposed to chlorophenoxy herbicides, chlorophenols and dioxins in particular TCDD. These studies showed that plasma TCDD levels were not associated with markers of humoral immunity with the possible exception of a decrease in complement factor 4 levels. Most lymphocyte subsets, in particular the B cell compartment, showed a decrease in cell counts with increasing levels of TCDD. Moreover, blood levels of most cytokines, chemokines and growth factors had a negative association with TCDD levels with a formal statistical significance for fractalkine, transforming growth factor alpha and fibroblast growth factor 2. These changes were independent from the changes in blood cell counts. Our findings support the notion that dioxin exposure can have an adverse impact on the immune system and likely suppresses the immune system. To identify immunologic hallmarks of obesity, we measured plasma levels of cytokines in pre-diagnostic blood samples of participants of a case-control study nested in the Italian EPIC cohort. IL8, IL10, IFN-γ, and interferon-induced protein 10 (IP10) were related to obesity. However, the set of obesity predictors (IL8, IL10, IFN-γ, IP10) were not associated with future NHL risk. Our studies support that subtle perturbations in the immune system may precede lymphoma development and suggest that B cell activation, chronic inflammation and/or an unbalance in Th1/Th2-responses are likely important phenomena in lymphomagenesis. Although, possible occupational and environmental risk factors of NHL influence the immune system they did not pertubate immune markers shown to be associated with future NHL risk.
    • International Validation of the Comet Assay and a Human Intervention Study.

      Ersson, Clara (Karolinska Institutet, 2011, 2011)
      The comet assay is an established, sensitive method extensively used in biomonitoring studies. The methods' advantages include; a) only a small cell sample is required, b) possibility to measure damage in practically any cell type, c) ability to measure heterogeneity in response within a cell population, d) relatively fast and economical procedure, and e) various applications of the method, which allow measurement of a range of different DNA lesions as well as DNA repair. Several guidelines for the comet assay have been published, but no standardised protocol exists as yet. There are considerable differences between the protocols used by different research groups, which negatively affect inter-laboratory comparisons of results. Several experts in the field have highlighted the need for multi-laboratory, international validation studies, to assess intra- and inter-laboratory reproducibility and to investigate sources of variability in the results. The papers in this thesis can be divided into two parts; one part that deals with international inter-laboratory validation studies and methodological aspects of the comet assay (paper I-III), and the other part covers a human intervention study with antioxidant capsules consisting of many different antioxidants in low doses for which the comet assay has been applied (paper IV-V). The inter-laboratory validation trials in papers I-II indicate that the participants can detect dose-responses of both DNA breaks and oxidatively damaged DNA in coded cells, but that there is a large inter-laboratory variation in the reported values. This variation can in part be explained by differences in comet assay protocols and in image analysis. The inter-laboratory variation was decreased, but not completely removed, by calibration with ionising radiation. In paper III we verified that several protocol steps significantly affected the outcome of the comet assay, including a) density of the agarose gel, b) extent of enzymatic incubation, c) duration of alkaline treatment, and d) time of electrophoresis, as well as the strength of the electric field applied. In a parallel placebo-controlled, double-blind intervention study, overweight middle-aged men were supplemented for six weeks with a multivitamin supplement consisting of a range of antioxidants in doses resembling those achieved by a healthy diet (paper IV-V). In spite of elevated levels of seven out of eight measured antioxidants in the blood, the intervention did not affect the level of oxidation of lipids or DNA. Many intervention studies with good design report similar null findings. It is preferred to consume antioxidants through a healthy diet, and dietary supplements are not recommended for cancer prevention.
    • Advancing the Contribution of Occupational Epidemiology to Risk Assessment.

      Vlaanderen, J.J. (Utrecht University, 2011, 2011)
      The identification and quantification of risk factors that are characterized by low exposure levels, moderately increased risks, and unspecific exposure-disease relations is a major challenge facing risk assessment today. Occupational epidemiological studies can play a role in addressing this challenge. The main advantage of occupational epidemiological studies over other potential sources of information for risk assessment (primarily animal bioassays) is that in these studies humans are being investigated, rendering the extrapolation of study results from animals to humans unnecessary. However this advantage is also a disadvantage because occupational epidemiological studies are mostly observational by nature which makes them prone to bias. Although some limitations of the use of occupational epidemiological studies in risk assessment are inherent to the discipline, improvements in the design, conduct and interpretation of studies will likely enhance their use in risk assessment. Furthermore, recent developments in the field of molecular biology and the related increase in the understanding of carcinogenesis and other adverse health effects have opened opportunities to further advance the contribution of occupational epidemiological studies to risk assessment. This thesis consists of a set of approaches that can be used as a framework to advance the use of occupational epidemiological studies in risk assessment. The approaches focus on the evaluation of the quality of occupational epidemiological studies for risk assessment, the incorporation of differences in study quality in methods for evidence synthesis, and the incorporation of biomarkers in occupational epidemiological studies. Some of the approaches are ready to be applied in risk assessment. Other approaches need further development before their actual value for risk assessment can be assessed. Further progress in the use of occupational epidemiological studies in risk assessment should come from tailoring study designs to the needs of risk assessment. A strong focus on high quality quantitative exposure assessment in the design of new occupational epidemiological studies and transparency of the steps undertaken to develop quantitative exposure estimates would significantly contribute to an increased weight of evidence for risk assessment and would likely improve the overall quality of risk assessment for many exposures.