• Accelerated (32)P-HPLC for bulky DNA adducts.

      Nagy, Eszter; Cornelius, Michael G.; Moller, Lennart (2009-03)
      Many compounds can react with DNA forming covalent modifications, so-called DNA adducts, which can influence crucial biological processes. DNA adducts from various DNA-damaging agents can act both as biomarkers and as a measurement of the actual damage of the genome. Therefore, they are of value in determining exposed or sensitive individuals or populations and in identifying agents that can induce DNA damage. A common method to measure DNA adducts is through DNA hydrolysis, adduct enrichment, (32)P-post-labelling and chromatographic separation. High-performance liquid chromatography (HPLC) with online radioactivity detection, the (32)P-HPLC (direct injection of the (32)P-labelled mixture into the HPLC with online (32)P-detection) method, gives both high sensitivity and good resolution of complex mixtures of DNA adducts. One limitation with this method is the capacity when dealing with large numbers of samples. The aim of this study was therefore to increase the analytical capacity by reducing analysis time of the (32)P-HPLC method. A change of HPLC columns to low backpressure columns and adaptation of elution conditions enabled a reduction in time per analysis from 100 to 20 min. This did not affect sensitivity but lowered chromatographic resolution, although bulky DNA adducts were still well resolved from other DNA components. This is useful when the total amount of DNA adducts is the primary interest. When high resolution is required, this can be achieved by gradient modifications, which increase the time required per analysis to 30 min. The accelerated (32)P-HPLC increases the capacity in number of samples 3- to 5-fold, depending on resolution requirements, without any negative effect on sensitivity in both in vitro and in vivo samples.
    • Activated neutrophils inhibit nucleotide excision repair in human pulmonary epithelial cells: role of myeloperoxidase.

      Güngör, Nejla; Godschalk, Roger W.L.; Pachen, Daniëlle M.; Van Schooten, Frederik J.; Knaapen, Ad M. (The Federation of American Societies for Experimental Biology, 2007-08)
      Neutrophils are thought to affect pulmonary carcinogenesis by promoting the metabolism of inhaled chemical carcinogens, causing enhanced formation of promutagenic DNA adducts. We hypothesized that neutrophils interfere with the removal of such DNA adducts by inhibiting nucleotide excision repair (NER) in target cells. Human alveolar epithelial cells (A549) were cocultured with activated neutrophils, and we observed a significant reduction of NER in the A549 cells, which was abrogated by addition of the myeloperoxidase (MPO) inhibitor 4-aminobenzoic acid hydrazide. The inhibitory effect of neutrophils could be mimicked by the MPO product hypochlorous acid (HOCl), which caused an acute, dose-dependent inhibition of NER in A549 cells. This was independent of cytotoxicity or ATP loss and persisted up to 24 h. These data were supported by showing that HOCl caused a delayed removal of DNA adducts in benzo[a]pyrene-diolepoxide-exposed A549 cells. The acute HOCl-induced inhibition of NER can only partly be explained by oxidative modification of repair proteins. To explain the more persistent effects of HOCl, we analyzed the expression of NER genes and found that HOCl significantly reduced XPC expression. In conclusion, these data indicate that neutrophils are potent inhibitors of nucleotide excision repair. This may provide a further biological explanation for the association between inflammation and lung cancer development.
    • Acute hypoxia and reoxygenation-induced DNA oxidation in human mononuclear blood cells.

      Risom, Lotte; Lundby, Carsten; Thomsen, Jonas Juhl; Mikkelsen, Lone; Loft, Steffen; Friis, Gitte; Moller, Peter (2007-12-01)
      Research indicates that exposure to hypoxia is associated with oxidative stress. In this investigation, healthy subjects were exposed to hypoxia by inhalation of 10% oxygen for 2h (corresponding to 5500m above sea level). The levels of strand breaks and oxidatively damaged purine bases, measured by the comet assay, and the expression of genes involved in DNA repair of oxidatively damaged DNA were investigated in mononuclear blood cells (MNBC) at baseline, after 2h of hypoxia, 2h of reoxygenation, and 1 day and 8 days after the exposure. The level of strand breaks and oxidized purine bases in MNBC increased following both the 2h of hypoxia and the 2h reoxygenation period, whereas this effect was not observed in unexposed subjects. The expressions of oxoguanine DNA glycosylase 1 (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1), nei endonuclease VIII-like 1 (NEIL1), and mutY homolog (MUTYH) were unaltered throughout the experiment in both groups of subjects, indicating that DNA repair genes are not up-regulated by the hypoxia and reoxygenation treatment. Taken together, this report shows that inhalation of 10% oxygen for 2h is associated with increased number of oxidized DNA lesions in MNBC, but acute hypoxia may not inflict upon the regulation of genes involved in repair of oxidized DNA.
    • Adherence to a Mediterranean diet and risk of gastric adenocarcinoma within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study.

      Buckland, Genevieve; Agudo, Antonio; Luján, Leila; Jakszyn, Paula; Bueno-de-Mesquita, H. Bas; Palli, Domenico; Boeing, Heiner; Carneiro, Fátima; Krogh, Vittorio; Sacerdote, Carlotta; et al. (2010-02)
      BACKGROUND: The Mediterranean dietary pattern is believed to protect against cancer, although evidence from cohort studies that have examined particular cancer sites is limited. OBJECTIVE: We aimed to explore the association between adherence to a relative Mediterranean diet (rMED) and incident gastric adenocarcinoma (GC) within the European Prospective Investigation into Cancer and Nutrition study. DESIGN: The study included 485,044 subjects (144,577 men) aged 35-70 y from 10 European countries. At recruitment, dietary and lifestyle information was collected. An 18-unit rMED score, incorporating 9 key components of the Mediterranean diet, was used to estimate rMED adherence. The association between rMED and GC with respect to anatomic location (cardia and noncardia) and histologic types (diffuse and intestinal) was investigated. A calibration study in a subsample was used to control for dietary measurement error. RESULTS: After a mean follow-up of 8.9 y, 449 validated incident GC cases were identified and used in the analysis. After stratification by center and age and adjustment for recognized cancer risk factors, high compared with low rMED adherence was associated with a significant reduction in GC risk (hazard ratio: 0.67; 95% CI: 0.47, 0.94). A 1-unit increase in the rMED score was associated with a decreased risk of GC of 5% (95% CI: 0.91, 0.99). There was no evidence of heterogeneity between different anatomic locations or histologic types. The calibrated results showed similar trends (overall hazard ratio for GC: 0.93; 95% CI: 0.89, 0.99). CONCLUSION: Greater adherence to an rMED is associated with a significant reduction in the risk of incident GC.
    • Adulthood lifetime physical activity and breast cancer.

      Peplonska, Beata; Lissowska, Jolanta; Hartman, Terryl J.; Szeszenia-Dabrowska, Neonila; Blair, Aaron; Zatonski, Witold; Sherman, Mark E.; Garcia-Closas, Montserrat; Brinton, Louise A. (2008-03)
      BACKGROUND: Epidemiologic studies have shown that breast cancer risk is reduced 30% to 40% in highly physically active compared with inactive women. However, the effects of moderate activities, timing of activities, and intervening effects of other risk factors remain less clear. METHODS: We analyzed data on physical activity patterns in 2176 incident breast cancer cases and 2326 controls in a population-based breast cancer case-control study in Poland conducted in 2000-2003. Using unconditional logistic regression analyses, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) associated with physical activity levels (measured by average metabolic equivalents of energy expenditure hours per week), controlling for potential confounders. RESULTS: Total adult lifetime activity reduced risk of breast cancer, with individuals in the highest quartile having an OR of 0.80 (CI = 0.67-0.96) compared with the lowest quartile. Reduced risks were most consistent for the highest quartiles of moderate-to-vigorous activities: moderate/vigorous recreational activities (OR = 0.74; CI = 0.62-0.89), outdoor activities (0.81; 0.68-0.97), heavy physical work (0.60; 0.42-0.87), and combined high intensity (metabolic equivalent >6.0) activities (0.75; 0.63-0.90). These relations were not modified by body mass index, menopausal status, or family history of breast cancer. Reductions in risk with moderate/vigorous recreational activities were stronger for larger tumors and those with nodal involvement. Women who increased their recreational activity in their 50s had significantly reduced risk, with those in the highest tertile of change being at a 27% lower risk. CONCLUSIONS: Leisure-time moderate-to-vigorous activities reduce breast cancer risk irrespective of underlying host characteristics.
    • 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.
    • Aging and defense against generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA.

      Mikkelsen, Lone; Bialkowski, Karol; Risom, Lotte; Lohr, Mille; Loft, Steffen; Moller, Peter (2009-09-01)
      The imbalance between the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA and the efficiency of cellular systems of DNA protection and repair is considered an important factor in the age-dependent development of cancer. This study investigated the relationship between oxidatively damaged DNA and the activity of the DNA repair system and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxodGTPase) activity in liver and lung tissue from mice at 10-100 weeks of age. The level of 8-oxodG increased with age, whereas the level of formamidopyrimidine DNA glycosylase sites was unaltered. The enzyme activity toward single oxygen-induced DNA damage and mRNA expression levels of Ercc1, Neil1, and Ogg1 remained unaltered with age. However, the 8-oxodGTPase activity in the liver was 18% (95% CI: 0.2-37%) lower in mice at 25 and 50 weeks than in 10-week-old mice. The 10- and 100-week-old mice had similar 8-oxodGTPase activity. In contrast, the mRNA expression of Nudt1 was statistically unaltered that likely resulted from higher variation of measurements. The accumulation of 8-oxodG with age is not a direct consequence of decreased enzyme activity toward singlet oxygen-induced substrate DNA. An age-related higher level of 8-oxodG even occurs concomitantly with high 8-oxodGTPase activity.
    • AHR- and DNA-damage-mediated gene expression responses induced by benzo(a)pyrene in human cell lines.

      Hockley, Sarah L.; Arlt, Volker M.; Brewer, Daniel; Te Poele, Robert; Workman, Paul; Giddings, Ian; Phillips, David H. (2007-12)
      Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene- trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP.
    • Air pollution, oxidative damage to DNA, and carcinogenesis.

      Moller, Peter; Folkmann, Janne Kjaersgaard; Forchhammer, Lykke; Brauner, Elvira Vaclavik; Danielsen, Pernille Hogh; Risom, Lotte; Loft, Steffen (2008-07-18)
      There is growing concern that air pollution exposure increases the risk of lung cancer. The mechanism of action is related to particle-induced oxidative stress and oxidation of DNA. Humans exposed to urban air with vehicle emissions have elevated levels of oxidized guanine bases in blood cells and urine. Animal experimental studies show that pulmonary and gastrointestinal exposure is associated with elevated levels of oxidized guanines in the lung and other organs. Collectively, there is evidence indicating that exposure to traffic-related air pollution particles is associated with oxidative damage to DNA and this might be associated with increased risk of cancer.
    • Annex 1. State of validation of biomarkers.

      The Nofer Institute of Occupational Medicine, 2007
    • Antiapoptotic effects of dietary antioxidants towards N-nitrosopiperidine and N-nitrosodibutylamine-induced apoptosis in HL-60 and HepG2 cells.

      Garcia, Almudena; Morales, Paloma; Arranz, Nuria; Delgado, Ma Eugenia; Rafter, Joseph; Haza, Ana I. (2009-07)
      The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced apoptosis in human leukemia (HL-60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. None of the vitamin C (5-50 microm), DADS and DPDS (1-5 microm) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL-60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2',7'-dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N-nitrosamine was reduced to control levels by vitamin C (5-50 microm) in a dose-dependent manner. However, DADS (5 microm) increased ROS levels induced by NPIP and NDBA in HL-60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 microm) in both HL-60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP- and NDBA-induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP- and NDBA-induced apoptosis in HL-60 and HepG2 cells.
    • Anticarcinogenic compounds of olive oil and related biomarkers.

      Sotiroudis, Theodore G.; Kyrtopoulos, Soterios A. (2008-05)
      Olive oil, one of the oldest vegetable oils consumed without any refining, is associated with a reduced risk of a number of common cancers. Minor constituents of virgin olive oil have been suggested to be among the major chemopreventive components. A brief overview is presented of recent findings concerning the bioavailability of certain important olive oil minor components including efficient antioxidant polyphenols, the triterpene hydrocarbon squalene and beta-sitosterol, considered as putative nutritional biomarkers, in relation to the incidence of cancer.
    • Antioxidant vitamins and cancer risk: is oxidative damage to DNA a relevant biomarker?

      Loft, Steffen; Moller, Peter; Cooke, Marcus S.; Rozalski, Rafal; Olinski, Ryszard (2008-05)
      Oxidative damage to DNA is regarded as an important step in carcinogenesis. These lesions may arise as a consequence of exposure to xenobiotics, but are also generated as a consequence of endogenous generation of oxidizing compounds. Measurements of oxidative damage to guanines, such as 8-oxo-7, 8-dihydroguanine (8-oxodG) are increasingly being regarded as reliable biomarkers of oxidative stress and they may have a predictive value of cancer risk, although this needs to be established independently in several cohort studies. A survey of intervention studies of the ingestion of antioxidant-containing foods or tablets of antioxidants indicate that about one-third of the studies reported a protective effect in terms of lower levels of oxidative damage to DNA in white blood cells or decreased urinary excretion of 8-oxodG. Although firm conclusions cannot be reached, there appears to be links between ingestion of antioxidants, oxidative damage to DNA, and risk of cancer.
    • 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.
    • Antiproliferative effects of curcuphenol, a sesquiterpene phenol.

      Rodrigo, Gloria; Almanza, Giovanna R.; Cheng, Yajun; Peng, Jiangnan; Hamann, Mark; Duan, Rui-Dong; Akesson, Bjorn (2010-10)
      Curcuphenol is a sesquiterpene isolated from sponges and plants having several significant biological activities. The present study explored its effect on cell proliferation and apoptosis in Caco-2 human colon cancer cells. It was demonstrated that curcuphenol in concentrations in the range of 29-116 µg/ml inhibited cell proliferation and DNA replication and induced cell death in a dose-dependent manner. The induction of apoptosis was associated with a stimulation of the activity of caspase-3. The findings presented here suggest that curcuphenol has antiproliferative and pro-apoptotic properties.
    • Appendix 1. DNA adducts.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 2. Heterocyclic aromatic amines.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 3. 1-Hydroxypyrene.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 4. Oxidative damage to DNA.

      The Nofer Institute of Occupational Medicine, 2007
    • Application of OMICS technologies in occupational and environmental health research; current status and projections.

      Vlaanderen, J.; Moore, L. E.; Smith, M. T.; Lan, Q.; Zhang, L.; Skibola, C. F.; Rothman, N.; Vermeulen, R. (2010-02)
      OMICS technologies are relatively new biomarker discovery tools that can be applied to study large sets of biological molecules. Their application in human observational studies (HOS) has become feasible in recent years due to a spectacular increase in the sensitivity, resolution and throughput of OMICS-based assays. Although, the number of OMICS techniques is ever expanding, the five most developed OMICS technologies are genotyping, transcriptomics, epigenomics, proteomics and metabolomics. These techniques have been applied in HOS to various extents. However, their application in occupational environmental health (OEH) research has been limited. Here, we will discuss the opportunities these new techniques provide for OEH research. In addition we will address difficulties and limitations to the interpretation of the data that is generated by OMICS technologies. To illustrate the current status of the application of OMICS in OEH research, we will provide examples of studies that used OMICS technologies to investigate human health effects of two well-known toxicants, benzene and arsenic.