• STrengthening the Reporting of OBservational studies in Epidemiology: Molecular Epidemiology STROBE-ME. An extension of the STROBE statement.

      Gallo, Valentina; Egger, Matthias; McCormack, Valerie; Farmer, Peter B.; Ioannidis, John P. A.; Kirsch-Volders, Micheline; Matullo, Giuseppe; Phillips, David H.; Schoket, Bernadette; Stromberg, Ulf; et al. (2012-09)
      Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility, and clinical outcomes are used as proxies for investigating the interactions between external and/or endogenous agents and the body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as STrengthening Reporting of Observational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE Statement implementing 9 existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.
    • Validation of the nucleotide excision repair comet assay on cryopreserved PBMCs to measure inter-individual variation in DNA repair capacity.

      Allione, Alessandra; Russo, Alessia; Ricceri, Fulvio; Vande Loock, Kim; Guarrera, Simonetta; Voglino, Floriana; Kirsch-Volders, Micheline; Matullo, Giuseppe (2013-01)
      Inter-individual susceptibility to mutagens/carcinogens can be assessed by either genotyping DNA repair genes in different pathways or phenotyping DNA repair capacity (DRC) at the molecular or cellular level. Due to the large number of known DNA repair genes, and the interactions between repair pathways, phenotyping is becoming the preferred approach to measure DRC, and reliable assays are therefore increasingly needed. The use of a cellular phenotype comet assay for the nucleotide excision repair (NER) pathway using benzo[a]pyrene diol epoxide (BPDE) has been described in previous papers, but no thorough evaluation of its applicability in large genotype-phenotype studies has been presented. Our aim was to evaluate the possibility of using cryopreserved instead of fresh peripheral blood mononuclear cells (PBMCs) to evaluate intra- and inter-assay variation, and inter-individual variation, for the aphidicolin (APC)-block NER comet assay. Moreover, we measured the variation for the designated internal standard (K562 erythroleukaemia cell line) and we evaluated the feasibility to use lymphoblastoid cell lines (LCLs) as surrogate of PBMCs. Our results showed a low intra-assay [coefficient of variation (CV) 19.9%] and inter-assay (CV 32.3%) variation, with a good inter-individual variation (122 subjects, mean ± standard deviation 7.38 ± 4.99; range 0.66-26.14; CV 67.63%). A significant correlation between results derived from cryopreserved and fresh PBMCs from the same individuals was found (10 subjects, r = 0.62, P = 0.05). Results from LCLs and cryopreserved PBMCs from the same subjects showed an inverse significant correlation (10 subjects, r = -0.712, P = 0.02). K562 cells as internal standard showed low intra-assay variation. In the present study the APC-block NER comet assay on cryopreserved PBMCs seemed to be a reliable method to measure DRC variation in epidemiological studies; LCLs were not a good surrogate in this assay.
    • The effects of hemodialysis treatment on the level of DNA strand breaks and oxidative DNA lesions measured by the comet assay.

      Ersson, Clara; Odar-Cederlof, Ingegerd; Fehrman-Ekholm, Ingela; Möller, Lennart (2012-12-20)
      Hemodialysis patients have a higher risk for oxidative stress-related complications, such as cardiovascular disease and cancer. The increased level of oxidative stress is due to several factors, e.g., the hemodialysis treatment itself and the uremic state. In the present study, the effects of dialysis treatment on the level of DNA breaks and oxidative DNA lesions in mononuclear cells were measured with the comet assay. Factors possibly affecting DNA damage (reported as % DNA in tail) such as the duration of dialysis, time since last dialysis session, years of dialysis treatment, nutritional status (measured as protein catabolic rate), age, and diabetes were also investigated. The levels of DNA breaks (13.6 ± 4.7 before dialysis) and oxidative DNA lesions (7.9 ± 4.8 before dialysis) were significantly higher in dialysis patients (n = 31) compared to the levels of DNA breaks (5.8 ± 1.1) and oxidative DNA lesions (3.4 ± 1.7) in 10 healthy controls (P < 0.001). A decrease of DNA breaks was observed after dialysis (P = 0.038), and the level of oxidative DNA lesions was higher when the time between two treatment sessions were 68 hours compared to 44 hours (P < 0.001). Older subjects had a higher level of DNA breaks (P = 0.003), a good nutritional status predicted a lower level of DNA breaks (P < 0.001), and the duration of the dialysis session was inversely correlated with oxidative DNA lesions (P = 0.014). Diabetes or years of dialysis treatment did not affect DNA damage. The observations in the present study suggest that accumulation of uremic toxins induce DNA damage. The hemodialysis treatment seems to change the DNA damage.
    • Quantification of ultraviolet radiation-induced DNA damage in the urine of Swedish adults and children following exposure to sunlight.

      Liljendahl, Tove Sandberg; Kotova, Natalia; Segerback, Dan (2012-11)
      DNA damage following exposure to ultraviolet radiation (UVR) is important in skin cancer development. The predominant photoproduct, cyclobutane thymine dimer (T=T), is repaired and excreted in the urine, where it provides a biomarker of exposure.
    • Human and Methodological Sources of Variability in the Measurement of Urinary 8-Oxo-7,8-dihydro-2'-deoxyguanosine.

      Barregard, Lars; Moller, Peter; Henriksen, Trine; Mistry, Vilas; Koppen, Gudrun; Rossner, Pavel; Sram, Radim J.; Weimann, Allan; Poulsen, Henrik E.; Nataf, Robert; et al. (2013-01-31)
      Abstract Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r(p) 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.
    • Rotating night shift work and mammographic density.

      Peplonska, Beata; Bukowska, Agnieszka; Sobala, Wojciech; Reszka, Edyta; Gromadzinska, Jolanta; Wasowicz, Wojciech; Lie, Jenny Anne; Kjuus, Helge; Ursin, Giske (2012-07)
      An increased risk of breast cancer has been observed in night shift workers. Exposure to artificial light at night and disruption of the endogenous circadian rhythm with suppression of the melatonin synthesis have been suggested mechanisms. We investigated the hypothesis that rotating night shift work is associated with mammographic density.
    • Detection of acetaldehyde derived N(2)-ethyl-2'-deoxyguanosine in human leukocyte DNA following alcohol consumption.

      Singh, Rajinder; Gromadzinska, Jolanta; Mistry, Yogita; Cordell, Rebecca; Juren, Tina; Segerback, Dan; Farmer, Peter B. (2012-09-01)
      Epidemiological studies have shown an association between alcohol (ethanol) consumption and increased cancer risk. The effect of alcohol consumption on the levels and persistence of N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) formed by acetaldehyde, the oxidative metabolite of ethanol, in human leukocyte DNA was investigated. DNA was isolated from venous blood samples obtained from 30 male non-smoking individuals before consumption of alcohol (0h) and subsequently at 3-5h following the consumption of 150mL of vodka (containing 42% pure ethanol). Additional samples were collected 24h and 48h post-alcohol consumption. The levels of N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) in the DNA were determined following reduction of N(2)-ethylidene-dG with sodium cyanoborohydride using a liquid chromatography-tandem mass spectrometry selected reaction monitoring method. A slight time-dependent trend showing an increase and decrease in the levels of N(2)-ethyl-dG was observed following consumption of alcohol compared to time 0h, however, the differences were not statistically significant. The average levels of N(2)-ethyl-dG observed at 0h, 3-5h, 24h and 48h time points following ingestion of alcohol were 34.6±21.9, 35.1±21.0, 36.8±20.7 and 35.6±21.1 per 10(8) 2'-deoxynucleosides, respectively. In conclusion, alcohol consumption that could be encountered under social drinking conditions, does not significantly alter the levels of the acetaldehyde derived DNA adduct, N(2)-ethyl-dG in human leukocyte DNA from healthy individuals.
    • Inter-laboratory variation in DNA damage using a standard comet assay protocol.

      Forchhammer, Lykke; Ersson, Clara; Loft, Steffen; Moller, Lennart; Godschalk, Roger W. L.; van Schooten, Frederik J.; Jones, George D. D.; Higgins, Jennifer A.; Cooke, Marcus; Mistry, Vilas; et al. (2012-07-27)
      There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.
    • Oxidative stress and DNA damage caused by the urban air pollutant 3-NBA and its isomer 2-NBA in human lung cells analyzed with three independent methods.

      Nagy, Eszter; Johansson, Clara; Zeisig, Magnus; Moller, Lennart (2005-11-15)
      The air pollutant 3-nitrobenzanthrone (3-NBA), emitted in diesel exhaust, is a potent mutagen and genotoxin. 3-NBA can isomerise to 2-nitrobenzanthrone (2-NBA), which can become more than 70-fold higher in concentration in ambient air. In this study, three independent methods have been employed to evaluate the oxidative stress and genotoxicity of 2-NBA compared to 3-NBA in the human A549 lung cell line. HPLC-EC/UV was applied for measurements of oxidative damage in the form of 8-oxo-2'-deoxyguanosine (8-oxodG), (32)P-HPLC for measurements of lipophilic DNA-adducts, and the Comet assay to measure a variety of DNA lesions, including oxidative stress. No significant oxidative damage from either isomer was found regarding formation of 8-oxodG analysed using HPLC-EC/UV. However, the Comet assay (with FPG-treatment), which is more sensitive and detects more types of damages compared to HPLC-EC/UV, showed a significant effect from both 3-NBA and 2-NBA. (32)P-HPLC revealed a strong DNA-adduct formation from both 3-NBA and 2-NBA, and also a significant difference between both isomers compared to negative control. These results clearly show that 2-NBA has a genotoxic potential. Even if the DNA-adduct forming capacity and the amount of DNA lesions measured with the (32)P-HPLC and Comet assay is about one third of 3-NBA, the high abundance of 2-NBA in ambient air calls for further investigation and evaluation of its health hazard.
    • Immunologic profile of excessive body weight.

      Azar Sharabiani, Mansour Taghavi; Vermeulen, Roel; Scoccianti, Chiara; Hosnijeh, Fatemeh Saberi; Minelli, Liliana; Sacerdote, Carlotta; Palli, Domenico; Krogh, Vittorio; Tumino, Rosario; Chiodini, Paolo; et al. (2011-05)
      The purpose of this paper is to identify immunologic hallmarks of excessive bodyweight. The analysis is based on 176 adults (106 women, 70 men) who participated in a nested case-control study in Italy. All participants were healthy at the time of blood collection and aged between 36 and 75 years. We employed multivariate analysis of variance and a nonparametric Bayesian additive regression tree approach along with a receiver operating characteristic (ROC) curve analysis to determine the immunologic signature of excessive body weight (i.e., obesity and overweight). Interleukin 8 (IL-8), IL-10, interferon γ, and inducible protein 10 were shown to be predictive of excessive body weight with an area under the ROC curve of 71% (p < 0.0002). We propose that by using this profile-based approach to define immunologic signatures, it might be possible to identify unique immunologic hallmarks of specific types of obesity.
    • Plasma cytokines and future risk of non-Hodgkin lymphoma (NHL): a case-control study nested in the Italian European Prospective Investigation into Cancer and Nutrition.

      Saberi Hosnijeh, Fatemeh; Krop, Esmeralda J.M.; Scoccianti, Chiara; Krogh, Vittorio; Palli, Domenico; Panico, Salvatore; Tumino, Rosario; Sacredote, Carlotta; Nawroly, Niga; Portengen, Lutzen; et al. (2010-06)
      Recently, biological markers related to the immune system such as cytokines have been studied to further understand the etiology of non-Hodgkin Lymphoma (NHL). However, to date, there are no studies that have studied cytokine levels prospectively in relation to NHL risk in the general population.
    • Stability and reproducibility of simultaneously detected plasma and serum cytokine levels in asymptomatic subjects.

      Saberi Hosnijeh, Fatemeh; Krop, Esmeralda J. M.; Portengen, Lutzen; Rabkin, Charles S.; Linseisen, Jakob; Vineis, Paolo; Vermeulen, Roel (2010-03)
      Blood levels of cyto- and chemokines might reflect immune deregulations which might be related to lymphomagenesis. 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 analyte levels. Bead-based immunoassays were performed on two fresh samples (serum, citrate and heparin plasma) of 10 asymptomatic adults collected 14 days apart and on aliquots of the first samples which were put through one to three freeze-thaw cycles to measure 11 cytokines, four chemokines and two adhesion molecules. Median coefficients of variation (CVs) of the measured analytes were 20%, 24% and 32% in serum, citrate and heparin plasma, respectively. Strong correlations (rank correlation coefficient 0.74-0.98) were observed between sample types, although small differences in analyte levels were observed for most analytes. Freeze-thaw cycles did not markedly change analyte levels. Our study supports the use of this assay among asymptomatic subjects in epidemiological studies.
    • TP53 mutaciok es aromas DNS addukt szintek osszefuggesei tudorakban. (in Hungarian)

      Anna, Livia; Reetta, Holmila; Kovacs, Katalin; Gyorffy, Erika; Gyori, Zoltan; Segesdi, Judit; Minarovits, Janos; Soltesz, Ibolya; Kostic, Szilard; Csekeo, Attila; et al. (2011-11)
    • Modszer osszehasonlito vizsgalatok policiklusos aromas szenhidrogen (PAH) modellvegyuletek DNS adduktjainak meghatarozasara. (in Hungarian)

      Kovacs, Katalin; Panagiotis, Georgiadis; Stella, Kaila; Anna, Livia; Schoket, Bernadette; Soterios, Kyrtopoulos (2011-11)
    • Gastrointestinal release of β-glucan and pectin using an in vitro method.

      Ulmius, Matilda; Johansson-Persson, Anna; Nordén, Tina Immerstrand; Bergenstahl, Bjorn; Onning, Gunilla (2011-07)
      The release of soluble dietary fiber is a prerequisite for viscous effects and hence beneficial health properties. A simple in vitro method was adapted to follow the release during gastrointestinal digestion, and the percentage of solubilized fiber was measured over time. β-Glucan from oat bran was mainly released during gastric digestion while the release of pectin from sugar beet fiber continued in the small intestine. Unmilled fractions of sugar beet fiber released more soluble fiber than oat bran flakes, probably due to the porous structure of sugar beet fiber as a result of manufacturing processes, but also due to differences in source. Milling to smaller fiber particles significantly improved releasability (from 20 to 55% released β-glucan and from 50 to 70% released pectin, respectively, after digestion). When milled fibers were included in individual food matrices, the release was reduced by protein and starch matrices (5% β-glucan and 35% pectin released, respectively) and slowed by fat (45% β-glucan and 60% pectin released). This may result in a too low or too late release in the upper small intestine to be able to interfere with macronutrient uptake. The method may be suitable for predicting the gastrointestinal release of soluble dietary fibers from food matrices in the development of healthy food products.
    • Gastrointestinal conditions influence the solution behaviour of cereal β-glucans in vitro.

      Ulmius, Matilda; Adapa, Srimannarayana; Onning, Gunilla; Nilsson, Lars (2012-02)
      The solution behaviour of β-glucans in a gastrointestinal model was investigated in order to explore the mechanisms explaining the physiological effects of the soluble fibre. Asymmetrical flow field-flow fractionation was used to determine the molar mass distribution, and in-line calcofluor labelling allowed specific detection of β-glucans in complex samples. When dispersed in water, weight-average molar mass (Mw) was determined to 1 × 106 g/mol for pure oat and barley β-glucans, and 200 × 106 g/mol for β-glucans in oat bran, indicating that the β-glucans were aggregating. Samples from the gastric digestion displayed disrupted aggregates, while samples from the small intestinal digestion contained re-formed aggregates. Additionally, the aggregates from pure β-glucans were considerably denser after intestinal digestion. This may be construed as gel-formation in the small intestine, which should be tested for its relevance to health effects. Our results signal the difficulties in predicting β-glucan activity in the gastrointestinal tract purely from analysis of the fibre-rich product.
    • Antiproliferative activity of extracts of some Bolivian medicinal plants.

      Rodrigo, Gloria C.; Almanza, Giovanna R.; Akesson, Bjorn; Duan, Rui-Dong (2010-11-04)
      Twenty-six plant species from the native flora used in Bolivian folk medicine were selected for studies of bioactivity. They belonged to the following families namely: Asteraceae (11), Brassicaceae (1), Cactaceae (1), Caesalpinaceae (2), Chenopodiaceae (1), Frankeniaceae (1), Geraniaceae (1), Laureaceae (1), Oxalidaceae (1), Piperaceae (1), Plantaginaceae (1), Rosaceae (1), Solanaceae (1) and Verbenaceae (2). Their effects on the proliferation of colon cancer cells (Caco-2) were studied using the WST-1 reagent. The results indicated that four out of 26 ethanolic extracts had a significant antiproliferative activity in this assay from (Schkuhria pinnata, Piper longestylosum, Parastrephia lepidophylla and Erodium cicutarium). The bioactivity of the extracts was correlated with the phytochemical characterization. Further studies of the mechanism of action of the bioactive extracts are needed.
    • STrengthening the reporting of OBservational studies in Epidemiology-Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement.

      Gallo, Valentina; Egger, Matthias; McCormack, Valerie; Farmer, Peter B.; Ioannidis, John P. A.; Kirsch-Volders, Micheline; Matullo, Giuseppe; Phillips, David H.; Schoket, Bernadette; Stromberg, Ulf; et al. (2011-10)
      Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility, and clinical outcomes are used as proxies for investigating the interactions between external and/or endogenous agents and the body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as STrengthening Reporting of Observational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology-Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE Statement implementing 9 existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.
    • STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement.

      Gallo, Valentina; Egger, Matthias; McCormack, Valerie; Farmer, Peter B.; Ioannidis, John P. A.; Kirsch-Volders, Micheline; Matullo, Giuseppe; Phillips, David H.; Schoket, Bernadette; Stromberg, Ulf; et al. (2012-01)
      Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility and clinical outcomes are used as proxies for investigating interactions between external and/or endogenous agents and body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as the STrengthening Reporting of OBservational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology -Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE statement implementing nine existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.