• Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity.

      Zijno, Andrea; Porcedda, Paola; Saini, Francesca; Allione, Alessandra; Garofalo, Bruno; Marcon, Francesca; Guarrera, Simonetta; Turinetto, Valentina; Minieri, Valentina; Funaro, Ada; et al. (2010-02-03)
      As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved.
    • Up-regulation of 8-oxo-dGTPase activity of MTH1 protein in the brain, testes and kidneys of mice exposed to (137)Cs gamma radiation.

      Bialkowski, Karol; Szpila, Anna; Kasprzak, Kazimierz S. (2009-08)
      Abstract Mammalian MTH1 protein is an antimutagenic (2'-deoxy)ribonucleoside 5'-triphosphate pyrophosphohydrolase that prevents the incorporation of oxidatively modified nucleotides into nucleic acids. It decomposes most specifically the miscoding products of oxidative damage to purine nucleic acid precursors (e.g. 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP, 8-oxo-GTP) that may cause point mutations or transcription errors when incorporated into DNA and RNA, respectively. The increased expression of MTH1 mRNA and MTH1 protein was previously proposed as a molecular marker of oxidative stress. Therefore, we hypothesized that increased 8-oxo-dGTPase activity of MTH1 protein in mouse organs could serve as a dose-dependent marker of exposure to ionizing radiation, which is known to induce oxidative stress. To test our hypothesis, we measured 8-oxo-dGTPase activity in six organs of male BL6 mice after exposure to 0, 10, 25 and 50 cGy and 1 Gy of (137)Cs gamma radiation given as a single whole-body dose (1 Gy/min). The mice were killed 4, 8 and 24 h after irradiation. A statistically significant induction of 8-oxo-dGTPase was found in brains, testes and kidneys but not in lungs, hearts or livers. Brains, which demonstrated the highest (4.3-fold) increase of 8-oxo-dGTPase activity, were shown to express approximately 50% higher levels of MTH1 protein. However, due to the lack of a simple positive correlation between the dose and the observed 8-oxo-dGTPase activity in brain, testes and kidneys, we conclude that measurements of 8-oxo-dGTPase activity in these organs may serve as a rough indicator rather than a quantifiable marker of radiation-induced oxidative stress.
    • 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.
    • Urinary excretion rates of 8-oxoGua and 8-oxodG and antioxidant vitamins level as a measure of oxidative status in healthy, full-term newborns.

      Dziaman, Tomasz; Gackowski, Daniel; Rozalski, Rafal; Siomek, Agnieszka; Szulczynski, Jaroslaw; Zabielski, Romuald; Olinski, Ryszard (2007-09)
      The aim of the present study was to evaluate the oxidative status in healthy full-term children and piglets. Urinary excretion of 8-oxoGua (8-oxoguanine) and 8-oxodG (8-oxo-2'-deoxyguanosine) were determined using HPLC/GS/MS methodology and concentrations of vitamins A, C and E with HPLC technique. The levels of 8-oxoGua in urine samples were about 7-8 times higher in newborn children and piglets when compared with the level of adult subjects, while in the case of 8-oxodG the difference was about 2.5 times. The levels of vitamin C and E in umbilical cord blood of newborn children significantly depend on the concentration of these compounds in their mother's blood. However, the values of vitamin C in human's cord blood were about 2-times higher than in respective mother blood, while the level of vitamin E showed an opposite trend. The results suggest that: (i) healthy, full-term newborns are under potential oxidative stress; (ii) urinary excretion of 8-oxoGua and 8-oxodG may be a good marker of oxidative stress in newborns; and (iii) antioxidant vitamins, especially vitamin C, play an important role in protecting newborns against oxidative stress.
    • Urinary measurement of 8-OxodG, 8-OxoGua, and 5HMUra: a noninvasive assessment of oxidative damage to DNA.

      Olinski, Ryszard; Rozalski, Rafal; Gackowski, Daniel; Foksinski, Marek; Siomek, Agnieszka; Cooke, Marcus S. (2008-05-26)
      Numerous DNA repair pathways exist to prevent the persistence of damage, and are integral to the maintenance of genome stability, and hence prevention of disease. Excised lesions arising from repair may ultimately appear in the urine where their measurement has been acknowledged to be reflective of overall oxidative stress. The development of reliable assays to measure urinary DNA lesions, such as HPLC prepurification followed by gas chromatography/mass spectrometry, offers the potential to assess whole body oxidative DNA damage. However, some studies suggest a possibility that confounding factors may contribute to urinary levels of 7,8-dihydro-8-oxoguanine (8-oxoGua) and 7,8-dihydro-8-oxo-2 -deoxyguanosine (8-oxodG). This article considers several possible sources of urinary lesions: (a) the repair of oxidatively damaged DNA; (b) a possible dietary influence; and (c) cell death. The authors conclude that data from their laboratories, along with a number of literature reports, form an argument against a contribution from cell death and diet. In the absence of these confounding factors, urinary measurements may be attributed entirely to the repair of DNA damage and suggests their possible use in studying associations between DNA repair and disease.
    • Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: the increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens.

      Farmer, Peter B.; Singh, Rajinder (2009-08-07)
      The carcinogens to which humans are exposed are normally in the form of complex mixtures, and much effort has gone into determining the nature of the most significant carcinogenic components in these mixtures and their mechanisms of action. Essential to achieving this aim in exposed populations is the use of biomarkers, which can characterize the chemical nature of the carcinogens involved and identify key biological effects that result from the exposure. DNA adducts are particularly appropriate as biomarkers in the case of genotoxic carcinogens as they indicate the biologically effective dose of the genotoxin in the target tissue under study. This review considers in particular the use of mass spectrometry (MS), which is having an increasing role in the determination of DNA adducts. Compared to other existing DNA damage detection methods, such as 32P-postlabeling, HPLC-fluorescence or electrochemical detection, immunoassay-based techniques and modified Comet assays, MS provides improved structural characterization of adducts. Greater selectivity in the analyses is achieved by the use of tandem MS with selected reaction monitoring or constant neutral loss of ions. Use of capillary/nano liquid chromatography and micro/nano electrospray ionization improves the analytical sensitivity and higher throughput may be obtained by the use of online-column switching. The application of microfluidics technology offers exciting new possibilities for interfacing sample preparation to the mass spectrometer. Despite these improvements in the use of MS for adduct detection, the main current requirement is to validate these methods both analytically and in molecular epidemiology studies. More knowledge of the stability of stored samples is required. Development of sensitive mass spectrometric DNA adductomic screening systems, and of long-term biomarkers (e.g., phosphotriester adducts that are not repaired efficiently) seems important areas for the future assessment of the effects of human exposure to environmental genotoxins, together with studies of dose-response relationships at low doses.
    • Validation of biomarkers for the study of environmental carcinogens: a review.

      Gallo, Valentina; Khan, Aneire; Gonzales, Carlos; Phillips, David H.; Schoket, Bernadette; Gyorffy, Erika; Anna, Livia; Kovacs, Katalin; Moller, Peter; Loft, Steffen; et al. (2008-08)
      There is a need for validation of biomarkers. Our aim is to review published work on the validation of selected biomarkers: bulky DNA adducts, N-nitroso compounds, 1-hydroxypyrene, and oxidative damage to DNA. A systematic literature search in PubMed was performed. Information on the variability and reliability of the laboratory tests used for biomarkers measurements was collected. For the evaluation of the evidence on validation we referred to the ACCE criteria. Little is known about intraindividual variation of DNA adduct measurements, but measurements have a good repeatability irrespective of the technique used for their identification; reproducibility improved after the correction for a laboratory factor. A high-sensitivity method is available for the measurement of 1-hydroxypyrene in urine. There is consensus on validation of biomarkers of oxidative damage DNA based on the comet assay and chromatographic measurement in blood while urinary measurements by chromatographic assays are well validated, and ELISA-based assays appear to lack specificity. Immunoassays for the quantification of adducts of N-nitroso compounds are useful for large epidemiological studies, given their sensitivity, the small amount of DNA required and their potential for rapid and high-throughput analysis.
    • 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.
    • Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring.

      Forchhammer, Lykke; Brauner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard; Danielsen, Pernille Hogh; Nielsen, Claus; Jensen, Annie; Loft, Steffen; Friis, Gitte; Moller, Peter (2008-05)
      The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.
    • 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; et al. (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.
    • Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial.

      Forchhammer, Lykke; Johansson, Clara; Loft, Steffen; Moller, Lennart; Godschalk, Roger W. L.; Langie, Sabine A. S.; Jones, George D. D.; Kwok, Rachel W. L.; Collins, Andrew R.; Azqueta, Amaya; et al. (2010-03)
      The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.
    • Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine.

      Bergstrom, Therese; Bergman, Jan; Moller, Lennart (2011-11)
      In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. β-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.
    • Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--alone or in synergism with metals.

      Bergstrom, Therese; Ersson, Clara; Bergman, Jan; Moller, Lennart (2012-03-30)
      Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins' chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins' ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, β-carotene, folic acid, α-tocopherol, δ-tocopherol or γ-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose-response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.
    • Vitamins B2 and B6 and genetic polymorphisms related to one-carbon metabolism as risk factors for gastric adenocarcinoma in the European prospective investigation into cancer and nutrition.

      Eussen, Simone J. P. M.; Vollset, Stein Emil; Hustad, Steinar; Midttun, Oivind; Meyer, Klaus; Fredriksen, Ase; Ueland, Per Magne; Jenab, Mazda; Slimani, Nadia; Ferrari, Pietro; et al. (2010-01)
      B vitamins and polymorphisms in genes coding for enzymes involved in one-carbon metabolism may affect DNA synthesis and methylation and thereby be implicated in carcinogenesis. Previous data on vitamins B2 and B6 and genetic polymorphisms other than those involving MTHFR as risk factors for gastric cancer (GC) are sparse and inconsistent. In this case-control study nested within the European Prospective Investigation into Cancer and Nutrition cohort, cases (n = 235) and controls (n = 601) were matched for study center, age, sex, and time of blood sampling. B2 and B6 species were measured in plasma, and the sum of riboflavin and flavin mononucleotide was used as the main exposure variable for vitamin B2 status, whereas the sum of pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid was used to define vitamin B6 status. In addition, we determined eight polymorphisms related to one-carbon metabolism. Relative risks for GC risk were calculated with conditional logistic regression, adjusted for Helicobacter pylori infection status and smoking status. Adjusted relative risks per quartile (95% confidence interval, P(trend)) were 0.85 (0.72-1.01, 0.06) for vitamin B2 and 0.78 (0.65-0.93, <0.01) for vitamin B6. Both relations were stronger in individuals with severe chronic atrophic gastritis. The polymorphisms were not associated with GC risk and did not modify the observed vitamin-cancer associations. In summary, results from this large European cohort study showed an inverse association between vitamin B2 and GC risk, which is borderline significant, and a significant inverse association between vitamin B6 and GC risk.