• 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.
    • Multiple anticancer effects of damsin and coronopilin isolated from Ambrosia arborescens on cell cultures.

      Villagomez, Rodrigo; Rodrigo, Gloria C.; Collado, Isidro G.; Calzado, Marco A.; Muñoz, Eduardo; Åkesson, Björn; Sterner, Olov; Almanza, Giovanna R.; Duan, Rui-Dong (2013-09)
      Terpenoids in plants are important sources for drug discovery. In this study, we extracted damsin and coronopilin, two sesquiterpene lactones, from Ambrosia arborescens and examined their anticancer effects on cell cultures. Damsin and coronopilin inhibited cell proliferation, DNA biosynthesis and formation of cytoplasmic DNA histone complexes in Caco-2 cells, with damsin being more potent than coronopilin. Further studies using the luciferase reporter system showed that damsin and coronopilin also inhibited expressions of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription-3 (STAT3), indicating that these sesquiterpenes can interfere with NF-κB and STAT3 pathways. Finally, we examined the effects of two synthetic dibrominated derivatives of damsin, 11α,13-dibromodamsin and 11β,13-dibromodamsin. While bromination appeared to weaken the antiproliferative effects of damsin, the β epimer had strong inhibitory effects on STAT3 activation. In conclusion, the sesquiterpene lactones damsin and coronopilin have inhibitory effects on cell proliferation, DNA biosynthesis and NF-κB and STAT3 pathways, thus being potentially important for discovery of drugs against cancer.
    • 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.
    • Rotating night shift work and polymorphism of genes important for the regulation of circadian rhythm.

      Reszka, Edyta; Peplonska, Beata; Wieczorek, Edyta; Sobala, Wojciech; Bukowska, Agnieszka; Gromadzinska, Jolanta; Lie, Jenny-Anne; Kjuus, Helge; Wasowicz, Wojciech (2013-03-01)
      These results suggest that CRY1 (rs8192440) polymorphism may influence the adaptation to the rotating night shift work among nurses and midwives.
    • 8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients.

      Dziaman, Tomasz; Banaszkiewicz, Zbigniew; Roszkowski, Krzysztof; Gackowski, Daniel; Wisniewska, Ewa; Rozalski, Rafał; Foksinski, Marek; Siomek, Agnieszka; Speina, Elzbieta; Winczura, Alicja; Marszalek, Andrzej; Tudek, Barbara; Olinski, Ryszard (2013-07-05)
      The aim of this work was to answer the question whether the broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair are appropriate prognosis factors of colon cancer (CRC) patients survival? The following parameters were analyzed for 89 CRC patients: concentration of uric acid and vitamins A, E, C in plasma; levels of 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) in DNA of leukocyte and colon tissues; urinary excretion rates of 8-oxodGuo and 8-oxoGua (8-oxo-7,8-dihydroguanine); the activity and mRNA or protein level of repair enzymes OGG1, APE1, ANPG, TDG and PARP1. All DNA modifications and plasma antioxidants were analyzed using high performance liquid chromatography (HPLC) or HPLC/gas chromatography-mass spectrometry techniques. Expression of repair proteins was analyzed by QPCR, Western or immunohistochemistry methods. Longer survival coincided with low levels of 8-oxodGuo/8oxoGua in urine and 8-oxodGuo in DNA as well as with high concentration of uric acid plasma level. In contrast to expectations, longer survival coincided with lower mRNA level in normal colon tissue of the main 8-oxoGua DNA glycosylase, OGG1, but no association was found for PARP-1 expression. When analyzing simultaneously two parameters the discriminating power increased significantly. Combination of low level of urinary 8-oxoGua together with low level of 8-oxodGuo in leukocyte (both below median value) or high concentration of plasma uric acid (above median value) have the best prediction power. Since prediction value of these parameters seems to be comparable to conventional staging procedure, they could possibly be used as markers to predict clinical success in CRC treatment.
    • Ethics and data protection in human biomarker studies in environmental health.

      Casteleyn, Ludwine; Dumez, Birgit; Van Damme, Karel; Anwar, Wagida A. (2013-08)
      Human biomarker studies in environmental health are essential tools to study the relationship between health and environment. They should ultimately contribute to a better understanding of environmentally induced adverse health effects and to appropriate preventive actions. To ensure the protection of the rights and dignity of study participants a complex legal and ethical framework is applied, consisting of several international directives, conventions, and guidelines, whether or not translated in domestic laws. Main characteristics of ethics and data protection in studies using biomarkers in the field of environmental health are summarized and current discussions on related questions and bottlenecks highlighted. In the current regulatory context, dominated by the protection of the individual study participant, difficulties are reported due to the different interpretation and implementation of the regulations of concern within and across borders. Advancement of consistency and compatibility is recommended and efforts are ongoing. An increasing demand for secondary use of data and samples poses additional challenges in finding a right balance between the individual rights of the study participants on the one hand and the common interest of, and potential benefit for the public or community at large on the other. Ethics committees could play a key role in assessing problems originating from the sometimes competing needs at individual and societal level. Building trust in science amongst (potential) study participants and within the community allows the inclusion of arguments from the societal perspective. This requires increased attention for respectful communication efforts. Striving for public participation in decision making processes may promote policy relevant research and the related translation of study results into action.
    • Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

      Caboux, Elodie; Lallemand, Christophe; Ferro, Gilles; Hémon, Bertrand; Mendy, Maimuna; Biessy, Carine; Sims, Matt; Wareham, Nick; Britten, Abigail; Boland, Anne; Hutchinson, Amy; Siddiq, Afshan; Vineis, Paolo; Riboli, Elio; Romieu, Isabelle; Rinaldi, Sabina; Gunter, Marc J.; Peeters, Petra H. M.; van der Schouw, Yvonne T.; Travis, Ruth; Bueno-de-Mesquita, H Bas; Canzian, Federico; Sánchez, Maria-José; Skeie, Guri; Olsen, Karina Standahl; Lund, Eiliv; Bilbao, Roberto; Sala, Núria; Barricarte, Aurelio; Palli, Domenico; Navarro, Carmen; Panico, Salvatore; Redondo, Maria Luisa; Polidoro, Silvia; Dossus, Laure; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Françoise; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Lagiou, Pagona; Boeing, Heiner; Fisher, Eva; Tumino, Rosario; Agnoli, Claudia; Hainaut, Pierre (2012)
      The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.
    • Effect of blood storage conditions on DNA repair capacity measurements in peripheral blood mononuclear cells.

      Allione, Alessandra; Porcedda, Paola; Russo, Alessia; Ricceri, Fulvio; Simonelli, Valeria; Minoprio, Anna; Guarrera, Simonetta; Pardini, Barbara; Mazzei, Filomena; Dogliotti, Eugenia; Giachino, Claudia; Matullo, Giuseppe (2013-05-30)
      Due to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p<0.0001) and as benzo[a]pyrene diol epoxide (BPDE)-induced damage repair by the comet assay (p=0.05). No apparent effect of the blood storage conditions on the outcome of γ-ray induced H2AX phosphorylation assay was reported. These results prompted us to further analyze the effects of blood storage conditions by performing a validation study. Three blood samples were simultaneously taken from ten healthy donors, PBMCs were isolated and cryopreserved as follows: immediately after blood collection (day 1); on the following day, after blood storage at RT (day 2-RT); or after blood storage at 4°C (day 2-4°C). DRC was then evaluated using phenotypic assays. The γ-ray induced H2AX phosphorylation assay has been confirmed as the only assay that showed good reproducibility independently of the blood storage conditions. The measurement of OGG assay was most affected by the blood storage conditions.
    • DNA-repair measurements by use of the modified comet assay: An inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG).

      Godschalk, Roger W. L.; Ersson, Clara; Riso, Patrizia; Porrini, Marisa; Langie, Sabine A. S.; van Schooten, Frederik-Jan; Azqueta, Amaya; Collins, Andrew R.; Jones, George D. D.; Kwok, Rachel W. L.; Phillips, David H.; Sozeri, Osman; Allione, Alessandra; Matullo, Giuseppe; Möller, Lennart; Forchhammer, Lykke; Loft, Steffen; Møller, Peter (2013-09)
      The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.
    • Association between 8-oxo-7,8-dihydro-2'-deoxyguanosine Excretion and Risk of Postmenopausal Breast Cancer: Nested Case-Control Study.

      Loft, Steffen; Olsen, Anja; Møller, Peter; Poulsen, Henrik E.; Tjønneland, Anne (2013-07)
      Our results suggest that oxidative stress with damage to DNA is important for the development of breast cancer. Cancer Epidemiol Biomarkers Prev; 22(7); 1289-96. ©2013 AACR.
    • Urinary excretion of 8-oxo-7,8-dihydroguanine as biomarker of oxidative damage to DNA.

      Loft, Steffen; Danielsen, Pernille; Løhr, Mille; Jantzen, Kim; Hemmingsen, Jette G.; Roursgaard, Martin; Karotki, Dorina Gabriela; Møller, Peter (2012-02-15)
      Oxidatively damaged DNA may be important in carcinogenesis. 8-Oxo-7,8-dihydroguanine (8-oxoGua) is an abundant and mutagenic lesion excised by oxoguanine DNA glycosylase 1 (OGG1) and measurable in urine or plasma by chromatographic methods with electrochemical or mass spectrometric detectors, reflecting the rate of damage in steady state. A common genetic OGG1 variant may affect the activity and was associated with increased levels of oxidized purines in leukocytes without apparent effect on 8-oxoGua excretion or major change in cancer risk. 8-OxoGua excretion has been associated with exposure to air pollution, toxic metals, tobacco smoke and low plasma antioxidant levels, whereas fruit and vegetable intake or dietary interventions showed no association. In rodent studies some types of feed may be source of 8-oxoGua in collected urine. Of cancer therapies, cisplatin increased 8-oxoGua excretion, whereas radiotherapy only showed such effects in experimental animals. Case-control studies found high excretion of 8-oxoGua in relation to cancer, dementia and celiac disease but not hemochromatosis, although associations could be a consequence rather than reflecting causality of disease. One prospective study found increased risk of developing lung cancer among non-smokers associated with high excretion of 8-oxoGua. Urinary excretion of 8-oxoGua is a promising biomarker of oxidatively damaged DNA.
    • 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.
    • A high intake of dietary fiber influences C-reactive protein and fibrinogen, but not glucose and lipid metabolism, in mildly hypercholesterolemic subjects.

      Johansson-Persson, Anna; Ulmius, Matilda; Cloetens, Lieselotte; Karhu, Toni; Herzig, Karl-Heinz; Onning, Gunilla (2013-02-07)
      PURPOSE: The aim of the study was to investigate how a diet high in dietary fiber, with several fiber sources included, modulates glucose and lipid metabolism and the inflammatory response in humans. METHODS: Subjects (n = 25) aged 58.6 (1.1) years (mean and SD) with a BMI of 26.6 (0.5) kg/m(2) and a total cholesterol (TC) of 5.8 (0.1) mmol/L (mean and SEM) were given a high fiber (HF) and low fiber (LF) diet, in a randomized controlled 5-week crossover intervention, separated by a 3-week washout. The HF diet consisted of oat bran, rye bran, and sugar beet fiber incorporated into test food products; one bread roll, one ready meal, and two beverages consumed daily. Equivalent food products, without added fibers, were provided in the LF diet. RESULTS: Total dietary fiber intake was 48.0 g and 30.2 g per day for the HF and LF diet, respectively. Significant reduction in C-reactive protein (CRP) was observed between the diets (P = 0.017) and a significant reduction in fibrinogen within the HF diet (P = 0.044). There were no significant effects in other measured circulating cytokines or in glucose, insulin, and lipid levels. CONCLUSIONS: Our study suggests that a 5-week high dietary fiber intake of oat bran, rye bran, and sugar beet fiber might reduce the low-grade inflammatory response measured as CRP which could, together with reduced fibrinogen, help to prevent the risk of cardiovascular disease.
    • LC-QTOF/MS metabolomic profiles in human plasma after a 5-week high dietary fiber intake.

      Johansson-Persson, Anna; Barri, Thaer; Ulmius, Matilda; Onning, Gunilla; Dragsted, Lars Ove (2013-05)
      The objective was to investigate the alterations of plasma metabolome profiles to identify exposure and effect markers of dietary fiber intake. Subjects (n = 25) aged 58.6 (1.1) years (mean and SD) with a body mass index of 26.6 (0.5) kg/m(2) were given a high fiber (HF) and a low fiber (LF) diet, in a 5-week randomized controlled crossover intervention. The HF diet consisted of oat bran, rye bran, and sugar beet fiber incorporated into test food products, whereas the LF diet was made of equivalent food products to the HF diet, but without adding fibers. Blood plasma samples were collected at the start and end of each intervention period and analyzed by LC-QTOF/MS. In total, 6 features in positive mode and 14 features in negative mode were significantly different between the HF and the LF diet (p < 0.01, q < 0.05). Two markers, 2,6-dihydroxybenzoic acid and 2-aminophenol sulfate, were increased after HF diet, along with a tentatively identified saponin derived from oat avenacosides. The untargeted metabolomics approach enabled the identification of two new markers of dietary fiber intake in human plasma. Further studies will be needed to verify if these markers could serve as compliance markers of fiber intake.
    • 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.
    • An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells.

      Ersson, Clara; Møller, Peter; Forchhammer, Lykke; Loft, Steffen; Azqueta, Amaya; Godschalk, Roger W. L.; van Schooten, Frederik-Jan; Jones, George D. D.; Higgins, Jennifer A.; Cooke, Marcus S.; Mistry, Vilas; Karbaschi, Mahsa; Phillips, David H.; Sozeri, Osman; Routledge, Michael N.; Nelson-Smith, Kirsty; Riso, Patrizia; Porrini, Marisa; Matullo, Giuseppe; Allione, Alessandra; Stepnik, Maciej; Ferlinska, Magdalena; Teixeira, João Paulo; Costa, Solange; Corcuera, Laura-Ana; López de Cerain, Adela; Laffon, Blanca; Valdiglesias, Vanessa; Collins, Andrew R.; Möller, Lennart (2013-02-27)
      The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.
    • Evidence of exposure to aristolochic acid in patients with urothelial cancer from a Balkan endemic nephropathy region of Romania.

      Schmeiser, Heinz H.; Kucab, Jill E.; Arlt, Volker M.; Phillips, David H.; Hollstein, Monica; Gluhovschi, Gheorghe; Gluhovschi, Cristina; Modilca, Mirela; Daminescu, Liviu; Petrica, Ligia; Velciov, Silvia (2012-10)
      Recently, chronic Aristolochia poisoning was found responsible for the aetiology of Balkan endemic nephropathy (BEN) in Croatia, Serbia, and Bosnia, and diet was the likely route of exposure to aristolochic acid (AA). BEN, often associated with an increased incidence of upper urinary tract carcinoma (UUC), also affects residents of certain rural villages in Romania. AA is a nephrotoxin and human carcinogen that forms DNA adducts after metabolic activation, which induce characteristic TP53 mutations in urothelial tumours. Here we present the first evidence linking AA exposure to UUC in residents of an endemic region in the Romanian Mehedinti County. DNA was extracted from kidney and tumour tissue of seven patients who underwent nephroureterectomy for UUC and resided in BEN villages (endemic group). Five patients with UUC from nonendemic villages served as controls. AA-DNA adducts (7-(deoxyadenosin-N(6) -yl)-aristolactam I), established biomarkers of AA exposure, were identified by (32)P-postlabelling in renal DNA of six patients from the endemic group and in one of the nonendemic group (adduct levels ranged from 0.3 to 6.5 adducts per 10(8) nucleotides). Additionally, an A to T transversion in TP53, a base substitution characteristic of AA mutagenic activity was found in urothelial tumour DNA of one patient from the endemic group. Our results provide a molecular link to the cause of urothelial tumours in BEN regions of Romania indicating that AA is the common aetiological agent for BEN across its numerous geographical foci.
    • Variation in PAH-related DNA adduct levels among non-smokers: The role of multiple genetic polymorphisms and nucleotide excision repair phenotype.

      Etemadi, Arash; Islami, Farhad; Phillips, David H.; Godschalk, Roger; Golozar, Asieh; Kamangar, Farin; Malekshah, Akbar Fazel-Tabar; Pourshams, Akram; Elahi, Seerat; Ghojaghi, Farhad; Strickland, Paul T.; Taylor, Philip R.; Boffetta, Paolo; Abnet, Christian C.; Dawsey, Sanford M.; Malekzadeh, Reza; van Schooten, Frederik J. (2012-11-23)
      Polycyclic aromatic hydrocarbons (PAHs) likely play a role in many cancers even in never-smokers. We tried to find a model to explain the relationship between variation in PAH-related DNA adduct levels among people with similar exposures, multiple genetic polymorphisms in genes related to metabolic and repair pathways, and nucleotide excision repair (NER) capacity. In 111 randomly selected female never-smokers from the Golestan Cohort Study in Iran, we evaluated 21 SNPs in 14 genes related to xenobiotic metabolism and 12 SNPs in eight DNA repair genes. NER capacity was evaluated by a modified comet assay, and aromatic DNA adduct levels were measured in blood by32P-postlabeling. Multivariable regression models were compared by Akaike's information criterion (AIC). Aromatic DNA adduct levels ranged between 1.7 and 18.6 per 10(8) nucleotides (mean: 5.8 ± 3.1). DNA adduct level was significantly lower in homozygotes for NAT2 slow alleles and ERCC5 non-risk-allele genotype, and was higher in the MPO homozygote risk-allele genotype. The sum of risk alleles in these genes significantly correlated with the log-adduct level (r = 0.4, p < 0.001). Compared with the environmental model, adding Phase I SNPs and NER capacity provided the best fit, and could explain 17% more of the variation in adduct levels. NER capacity was affected by polymorphisms in the MTHFR and ERCC1 genes. Female non-smokers in this population had PAH-related DNA adduct levels three to four times higher than smokers and occupationally-exposed groups in previous studies, with large inter-individual variation which could best be explained by a combination of Phase I genes and NER capacity.
    • On the origins and development of the (32)P-postlabelling assay for carcinogen-DNA adducts.

      Phillips, David H. (2012-11-23)
      The (32)P-postlabelling method for the analysis of carcinogen-DNA adducts originated 30years ago from Baylor College of Medicine in Houston and was the work of a team comprised of Kurt and Erica Randerath, Ramesh Gupta and Vijay Reddy. With subsequent modifications and developments, it has become a highly sensitive and versatile method for the detection of DNA adducts that has been applied in a wide range of human, animal and in vitro studies. These include monitoring human exposure to environmental and occupational carcinogens, investigating genotoxicity of chemicals, elucidating pathways of metabolic activation of carcinogens, mechanistic studies of DNA repair, analysing the genotoxicity of complex mixtures and in ecotoxicology studies. Its use has been instrumental in providing new clues to the aetiology of some cancers and in identifying a new human carcinogen.