• ECNIS-sponsored workshop on biomarkers of exposure and cancer risk: DNA damage and DNA adduct detection and 6th GUM-32P-postlabelling workshop, German Cancer Research Center, Heidelberg, Germany, 29-30 September 2006.

      Arlt, Volker M.; Frei, Eva; Schmeiser, Heinz H. (Oxford University Press, 2007-01)
      Of all the chemicals classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC), 90% exert their biological effects through binding of their metabolically activated intermediates to DNA forming covalent DNA adducts. As a consequence DNA adducts are generally considered to be causative and directly related to tumour formation. DNA adduct analyses reflect tissue-specific rates of adduct formation and removal, which depend on carcinogen uptake, metabolic activation, DNA repair, adduct instability and tissue turnover and are thus useful markers of carcinogen exposure. The measurement of carcinogen-DNA adduct levels is central to the understanding of chemical carcinogenesis both in animals and humans to determine molecular mechanisms and exposure. Sensitive methods for DNA adduct analysis used to date are based on (32)P-post-labelling, immunoassay, mass spectrometry and laser-induced fluorescence. The aim of this workshop held over 2 days (29-30 September 2006) at the German Cancer Research Center (DKFZ) in Heidelberg, Germany, was to discuss methodological improvements of DNA adduct detection with emphasis on the (32)P-post-labelling procedure as well as new findings achieved by applying the methods to studies on understanding human cancer mechanisms and to elucidate the relationship between adduct formation and human cancer risk.
    • 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.; et al. (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.
    • An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay.

      Johansson, Clara; Moller, Peter; Forchhammer, Lykke; Loft, Steffen; Godschalk, Roger W. L.; Langie, Sabine A. S.; Lumeij, Stijn; Jones, George D. D.; Kwok, Rachel W. L.; Azqueta, Amaya; et al. (2010-03)
      The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.
    • 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; et al. (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.
    • Effect of hepatic cytochrome P450 oxidoreductase deficiency on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adduct formation in P450 reductase conditional null mice.

      Arlt, Volker M.; Singh, Rajinder; Stiborova, Marie; Gamboa da Costa, Goncalo; Frei, Eva; Evans, James D.; Farmer, Peter B.; Wolf, C. Roland; Henderson, Colin J.; Phillips, David H. (2011-09-22)
      2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic CYPs in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to CYPs, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic CYP function. RCN mice were treated orally with 50 mg/kg body weight PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e. N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts per 10(8) deoxynucleosides), while adduct levels in liver were ~3.5-fold lower. While no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average ~2-fold lower in extra-hepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was ~8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extra-hepatic tissues and further activation resulting in the formation of dG-C8-PhIP. Besides hepatic CYPs, PhIP may be metabolically activated mainly by a non-CYP pathway in liver.
    • Effect of sonication and serum proteins on copper release from copper nanoparticles and the toxicity towards lung epithelial cells.

      Cronholm, Pontus; Midander, Klara; Karlsson, Hanna L.; Elihn, Karine; Wallinder, Inger Odnevall; Moller, Lennart (2011-06)
      Abstract Different methodological settings can influence particle characteristics and toxicity in nanotoxicology. The aim of this study was to investigate how serum proteins and sonication of Cu nanoparticle suspensions influence the properties of the nanoparticles and toxicological responses on human lung epithelial cells. This was investigated by using methods for particle characterization (photon correlation spectroscopy and TEM) and Cu release (atomic absorption spectroscopy) in combination with assays for analyzing cell toxicity (MTT-, trypan blue- and Comet assay). The results showed that sonication of Cu nanoparticles caused decreased cell viability and increased Cu release compared to non-sonicated particles. Furthermore, serum in the cell medium resulted in less particle agglomeration and increased Cu release compared with medium without serum, but no clear difference in toxicity was detected. Few cells showed intracellular Cu nanoparticles due to fast release/dissolution processes of Cu. In conclusion; sonication can affect the toxicity of nanoparticles.
    • Effects of ambient air particulate exposure on blood-gas barrier permeability and lung function.

      Brauner, Elvira Vaclavik; Mortensen, Jann; Moller, Peter; Bernard, Alfred; Vinzents, Peter; Wahlin, Peter; Glasius, Marianne; Loft, Steffen (2009-01)
      Particulate air pollution is associated with increased risk of pulmonary diseases and detrimental outcomes related to the cardiovascular system, including altered vessel functions. This study's objective was too evaluate the effects of ambient particle exposure on the blood-gas permeability, lung function and Clara cell 16 (CC16) protein release in healthy young subjects. Twenty-nine nonsmokers participated in a randomized, two-factor crossover study with or without biking exercise for 180 min and with 24-h exposure to particle-rich (6169-15,362 particles/cm(3); 7.0-11.6 microg/m(3) PM(2.5); 7.5-15.8 microg/m(3) PM(10-2.5)) or filtered (91-542 particles/cm(3)) air collected above a busy street. The clearance rate of aerosolized (99m)Tc-labeled diethylenetriamine pentaacetic acid ((99m)Tc-DTPA) was measured as an index for the alveolar epithelial membrane integrity and permeability of the lung blood-gas barrier after rush-hour exposure. Lung function was assessed using body plethysmography, flow-volume curves, and measurements of the diffusion capacity of carbon monoxide. CC16 was measured in plasma and urine as another marker of alveolar integrity. Particulate matter exposure had no significant effect on the epithelial membrane integrity using the methods available in this study. Exercise increased the clearance rate of (99m)Tc-DTPA indicated by a 6.8% (95% CI: 0.4-12.8%) shorter half-life and this was more pronounced in men than women. Neither particulate matter exposure nor exercise had an effect on the concentration of CC16 in plasma and urine or on the static and dynamic volumes or ventilation distribution of the lungs. The study thus demonstrates increased permeability of the alveolar blood-gas barrier following moderate exercise, whereas exposure to ambient levels of urban air particles has no detectable effects on the alveolar blood-gas barrier or lung function.
    • Effects of an acute exposure to toluene on the DNA repair activity of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) in healthy subjects.

      Finkenwirth, P.; Spelmeyer, U.; Hommel, G.; Rose, D.-M.; Jung, D.; Rossbach, B.; Mayer-Popken, O.; Platt, K.-L.; Oesch, F.; Muttray, A. (2009-08)
      The structure and previous studies on the biotransformation of toluene lead to the suspicion that metabolites may be formed which preferentially react with strongly nucleophilic partners such as sulfhydryl groups of cysteines in proteins. Human 8-oxoguanine DNA glycosylase 1 removes the major oxidative DNA damage and possesses eight cysteines. Its potential inactivation may lead to accumulation of DNA damage by reactive oxygen species formed by exogenous agents or by ubiquitous endogenous processes. The goal of the present investigation was to study the in vivo effect in humans of an acute toluene exposure on hOGG1 activity. Twenty healthy, non-smoking males were exposed to 50 ppm toluene and to filtered air in an exposure chamber for 270 min, using a cross-over design. Before and 30 min after the end of exposure, blood samples were taken and toluene concentrations and the hOGG1 activity were measured. hOGG1 activity was determined in peripheral mononuclear blood cells. Thirty minutes after exposure to toluene, we found a median blood concentration of 0.25 mg toluene/l. Compared with the activity before exposure, upon exposure to toluene a statistically insignificant median increase of hOGG1 activity by +0.4% and upon exposure to air by +2.3% was determined. Thus, no reduction of the hOGG1 repair activity after acute exposure to 50 ppm toluene was observed.
    • Effects of basal level of antioxidants on oxidative DNA damage in humans.

      Foksinski, Marek; Gackowski, Daniel; Rozalski, Rafal; Siomek, Agnieszka; Guz, Jolanta; Szpila, Anna; Dziaman, Tomasz; Olinski, Ryszard (2007-04)
      BACKGROUND: Vitamins A, E and C, and uric acid, which can scavenge free radicals should also protect DNA from the damage. It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease subsequent cancer development. AIM OF THE STUDY: A relationship between basal level of antioxidants (vitamins A, C and E and uric acid) and oxidative DNA damage was assessed. For the first time, the broad spectrum of oxidative DNA damage biomarkers: urinary excretion of 8-oxodG, 8-oxoGua and 5HMUra as well as the level of oxidative DNA damage in leukocytes was analyzed in healthy subjects (n = 158). METHODS: Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of oxidative DNA damage products excreted into urine and the amount of 8-oxodG in leukocytes' DNA (with HPLC/EC technique). The level of antioxidant vitamins and uric acid was estimated by HPLC technique with fluorimetric and UV detection. RESULTS: Analyses of relationship between the most common antioxidants (vitamins A, C, E and uric acid) and oxidative DNA damage products reveal weak, statistically significant negative correlation between retinol and all the measured parameters except 5HMUra. Vitamin C negatively correlates with urinary excretion of 8-oxodG and 8-oxoGua. Uric acid revealed statistically significant negative correlation with 8-oxodG in cellular DNA and urinary excretion of 5HMUra, while alpha-tocopherol correlates negatively only with 8-oxodG in cellular DNA. Good, significant (P < 0.0001), positive correlation (r = 0.61) was noted between urinary levels of the base, 8-oxoGua and the deoxynucleoside, 8-oxodG. CONCLUSION: Our results suggest that oxidative DNA damage shows limited but significant response to antioxidants analyzed in this study and is more affected by many other cellular functions like antioxidant enzymes or DNA repair enzymes as well as genetics.
    • 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.
    • Effects of nitrated-polycyclic aromatic hydrocarbons and diesel exhaust particle extracts on cell signalling related to apoptosis: possible implications for their mutagenic and carcinogenic effects.

      Landvik, Nina E.; Gorria, Morgane; Arlt, Volker M.; Asare, Nana; Solhaug, Anita; Lagadic-Gossmann, Dominique; Holme, Jorn A. (2007-03-07)
      Nitrated-polycyclic aromatic hydrocarbons (nitro-PAHs) and diesel exhaust particle extracts (DEPE) induced apoptosis in Hepa1c1c7 cells with the following potency: 1,3-dinitropyrene (1,3-DNP)>1-nitropyrene (1-NP) >> DEPE >> 1,8-dinitropyrene (1,8-DNP). The compounds induced cyp1a1, and activated various intracellular signalling pathways related to apoptosis. The CYP inhibitor alpha-naphthoflavone strongly reduced 1,3-DNP-induced cell death, whereas cell death induced by 1-NP was rather increased. Toxic 1,3-DNP and 1-NP were found to induce a concentration-dependent lipid peroxidation. 1,3-DNP caused pro-apoptotic events, including increased phosphorylation and accumulation of p53 in the nucleus, cleavage of bid and of caspases 8 and 3, down-regulation of bcl-x(L) and phosphorylation of p38 and JNK MAPK. Furthermore, 1,3-DNP increased the activation of survival signals including phosphorylation of Akt and inactivation (phosphorylation) of pro-apoptotic bad. Although less potent, rather similar effects were observed following exposure to DEPE, compared to 1-NP. The most important finding was that the most mutagenic and carcinogenic compound tested, 1,8-DNP, induced little (if any) cell death, despite the fact that this compound seemed to give the most DNA damage as judged by DNA adduct formation, increased phosphorylation of p53 and accumulation of cells in S-phase. Immunocytochemical studies revealed that the p53 protein did not accumulate into the nucleus suggesting that 1,8-DNP inactivated the pro-apoptotic function of the p53 protein by a non-mutagenic event. These results suggest that after exposure to 1,8-DNP more cells may survive with DNA damage, thereby increasing its mutagenic and carcinogenic potential.
    • Effects of selenium status and polymorphisms in selenoprotein genes on prostate cancer risk in a prospective study of European men.

      Steinbrecher, Astrid; Méplan, Catherine; Hesketh, John; Schomburg, Lutz; Endermann, Tobias; Jansen, Eugene; Akesson, Bjorn; Rohrmann, Sabine; Linseisen, Jakob (2010-11)
      Evidence for an association between selenium status and prostate cancer risk is still inconclusive. Anticarcinogenic effects of selenium are supposedly mediated through cellular protective and redox properties of selenoenzymes in vivo. We evaluated the association between serum selenium status and prostate cancer risk in a population with relative low selenium concentrations considering effect modification by genetic variants in selenoprotein genes.
    • The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments.

      Ersson, Clara; Moller, Lennart (2011-11)
      The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study suggest that (i) there is a significant linear dose-response relationship between the agarose gel's density and DNA migration and that damaged cells are more sensitive to the agarose gel's density; (ii) incubation with formamidopyrimidine DNA glycosylase for 10 min is inadequate, whereas 30 min is sufficient; (iii) the typically used 20 min of alkaline treatment might be to short when analysing samples that contain particular alkali-labile sites (ALS) and (iv) the duration of electrophoresis as well as the strength of the electric field applied affects the DNA migration. By using protocol-specific calibration curves, it is possible to reduce the variation in DNA migration caused by differences in comet assay protocols. This does, however, not completely remove the impact of the durations of alkaline treatment and electrophoresis when analysing cells containing ALS that are relatively resistant to high alkaline treatment.
    • Elaboration of a quantitative job-exposure matrix for historical exposure to airborne exposures in the Polish rubber industry.

      de Vocht, F.; Sobala, Wojciech; Peplonska, B.; Wilczynska, U.; Gromiec, J.; Szeszenia-Dabrowska, Neonila; Kromhout, H. (2008-11)
      BACKGROUND: A job-exposure matrix (JEM) for inhalable aerosols, aromatic amines, and cyclohexane soluble matter (CSM) was elaborated based on measurements collected routinely between 1981 and 1996. METHODS: The data were grouped based on similarities in exposure levels and time trends in different departments, and were analyzed using smoothing splines and mixed effects models. RESULTS: Although higher than in western European countries, inhalable aerosol exposure decreased after changes in production volume and implementation of exposure reduction measures in mid-1980s. Aromatic amines concentrations first increased following the factory's production volume, but subsequently decreased in more recent years. CSM concentrations were uniformly distributed between departments. CONCLUSIONS: This JEM provides an overview of historical exposure levels in a large Polish rubber factory and will enable estimation of lifetime exposure for individual workers in a Polish rubber workers cohort and further investigation of the associations between specific exposures and cancer risk.
    • The environmental pollutant and carcinogen 3-nitrobenzanthrone induces cytochrome P450 1A1 and NAD(P)H:quinone oxidoreductase in rat lung and kidney, thereby enhancing its own genotoxicity.

      Stiborova, Marie; Dracinska, Helena; Mizerovska, Jana; Frei, Eva; Schmeiser, Heinz H.; Hudecek, Jiri; Hodek, Petr; Phillips, David H.; Arlt, Volker M. (2008-05-02)
      3-Nitrobenzanthrone (3-NBA) is a carcinogen occurring in diesel exhaust and air pollution. Using the (32)P-postlabelling method, we found that 3-NBA and its human metabolite, 3-aminobenzanthrone (3-ABA), are activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and kidney. Each compound generated identical five DNA adducts. We have demonstrated the importance of pulmonary and renal NAD(P)H:quinone oxidoreductase (NQO1) to reduce 3-NBA to species that are further activated by N,O-acetyltransferases and sulfotransferases. Cytochrome P450 (CYP) 1A1 is the essential enzyme for oxidative activation of 3-ABA in microsomes of both organs, while cyclooxygenase plays a minor role. 3-NBA was also investigated for its ability to induce NQO1 and CYP1A1 in lungs and kidneys, and for the influence of such induction on DNA adduct formation by 3-NBA and 3-ABA. When cytosols from rats treated i.p. with 40mg/kg bw of 3-NBA were incubated with 3-NBA, DNA adduct formation was up to 2.1-fold higher than in incubations with cytosols from control animals. This increase corresponded to an increase in protein level and enzymatic activity of NQO1. Incubations of 3-ABA with microsomes of 3-NBA-treated rats led to up to a fivefold increase in DNA adduct formation relative to controls. The stimulation of DNA adduct formation correlated with the potential of 3-NBA to induce protein expression and activity of CYP1A1. These results demonstrate that 3-NBA is capable to induce NQO1 and CYP1A1 in lungs and kidney of rats thereby enhancing its own genotoxic and carcinogenic potential.
    • ERCC1 haplotypes modify bladder cancer risk: a case-control study.

      Ricceri, Fulvio; Guarrera, Simonetta; Sacerdote, Carlotta; Polidoro, Silvia; Allione, Alessandra; Fontana, Dario; Destefanis, Paolo; Tizzani, Alessandro; Casetta, Giovanni; Cucchiarale, Giuseppina; et al. (2010-02-04)
      Bladder cancer risk is highly influenced by environmental and/or predisposing genetic factors. In the last decades growing evidence of the major role played by DNA repair systems in the developing of bladder cancer has been provided. To better investigate the involvement of DNA repair genes previously reported to be significantly associated with bladder cancer risk, we examined in a case-control study (456 cases and 376 hospital controls) 36 single nucleotide polymorphisms (SNPs) in 10 DNA repair genes, through a better gene coverage and a deep investigation of the haplotype role. A single SNP analysis showed a significantly increased risk given by XRCC1-rs915927 G allele (OR=1.55, CI 95% 1.02-2.37 for dominant model) and a protective effect of the rare alleles of 3 ERCC1 SNPs: rs967591 (OR=0.66, CI 95% 0.46-0.95), rs735482 (OR=0.62, CI 95% 0.42-0.90) and rs2336219 (OR=0.63, CI 95% 0.43-0.93). Haplotype analysis revealed that cases had a statistically significant excess of XRCC3-TAGT and ERCC1-GAT haplotypes, whereas ERCC1-AAC, MGMT-TA, XRCC1-TGCC and ERCC2-TGAA haplotypes were significantly underrepresented. Together with other published data on large case-control studies, our findings provide epidemiological evidence supporting a link between DNA repair gene variants and bladder cancer development, and suggest that the effects of high-order interactions should be taken into account as modulating factors affecting bladder cancer risk. A detailed characterization of DNA repair genetic variation is warranted and might ultimately help to identify multiple susceptibility variants that could be responsible for joint effects on the risk.
    • Estimation of dietary sources and flavonoid intake in a Spanish adult population (EPIC-Spain).

      Zamora-Ros, Raul; Andres-Lacueva, Cristina; Lamuela-Raventos, Rosa M.; Berenguer, Toni; Jakszyn, Paula; Barricarte, Aurelio; Ardanaz, Eva; Amiano, Pilar; Dorronsoro, Miren; Larranaga, Nerea; et al. (2010-03)
      BACKGROUND: Epidemiologic studies have suggested associations between flavonoid intake and health benefits. Traditional Mediterranean diets consist of a high consumption of plant products rich in flavonoids. OBJECTIVE: This study estimates dietary flavonoid intake and main food sources in a Mediterranean population (Spanish adults). DESIGN: The study included 40,683 subjects aged 35 to 64 years from northern and southern regions of Spain who were included in the European Prospective Investigation into Cancer and Nutrition study Spanish cohort. Usual food intake was assessed by personal interviews using a computerized version of a validated diet history method. Expanded US Department of Agriculture databases for the flavonoid, isoflavone, and proanthocyanidin content were used. RESULTS: The median and mean of total flavonoids were 269.17 and 313.26 mg/day, respectively. The most abundant flavonoid subgroup was proanthocyanidins (60.1%), followed by flavanones (16.9%), flavan-3-ols (10.3%), flavonols (5.9%), anthocyanidins (5.8%), flavones (1.1%), and isoflavones (<0.01%). The main sources of total flavonoid intake were apples (23%), red wine (21%), unspecified fruit (12.8%), and oranges (9.3%). CONCLUSIONS: These results should be very useful for evaluating the relationships between flavonoid intake and several diseases.
    • 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.
    • European genome-wide association study identifies SLC14A1 as a new urinary bladder cancer susceptibility gene.

      Rafnar, Thorunn; Vermeulen, Sita H.; Sulem, Patrick; Thorleifsson, Gudmar; Aben, Katja K.H.; Witjes, J. Alfred; Grotenhuis, Anne J.; Verhaegh, Gerald W.; Hulsbergen-van de Kaa, Christina A.; Besenbacher, Soren; et al. (2011-11-01)
      Three genome-wide association studies in Europe and the USA have reported eight urinary bladder cancer (UBC) susceptibility loci. Using extended case and control series and 1000 Genomes imputations of 5 340 737 single-nucleotide polymorphisms (SNPs), we searched for additional loci in the European GWAS. The discovery sample set consisted of 1631 cases and 3822 controls from the Netherlands and 603 cases and 37 781 controls from Iceland. For follow-up, we used 3790 cases and 7507 controls from 13 sample sets of European and Iranian ancestry. Based on the discovery analysis, we followed up signals in the urea transporter (UT) gene SLC14A. The strongest signal at this locus was represented by a SNP in intron 3, rs17674580, that reached genome-wide significance in the overall analysis of the discovery and follow-up groups: odds ratio = 1.17, P = 7.6 × 10(-11). SLC14A1 codes for UTs that define the Kidd blood group and are crucial for the maintenance of a constant urea concentration gradient in the renal medulla and, through this, the kidney's ability to concentrate urine. It is speculated that rs17674580, or other sequence variants in LD with it, indirectly modifies UBC risk by affecting urine production. If confirmed, this would support the 'urogenous contact hypothesis' that urine production and voiding frequency modify the risk of UBC.
    • Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine.

      Cooke, Marcus S.; Singh, Rajinder; Hall, Georgina K.; Mistry, Vilas; Duarte, Tiago L.; Farmer, Peter B.; Evans, Mark D. (2006-12-15)
      While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 +/- 0.53 pmol/mumol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 +/- 0.39 pmol/mumol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.