Searching for assay controls for the Fpg- and hOGG1-modified comet assay

The formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine DNA glycosylase (hOGG1)-modified comet assays have been widely used in human biomonitoring studies. The purpose of this article is to assess differences in reported levels of Fpg- and hOGG1-sensitive sites in leukocytes and suggest suitable assay controls for the measurement of oxidatively damaged DNA. An assessment of the literature showed a large variation in the reported levels of Fpg-sensitive sites (range 0.05-1.31 lesions/106 bp). The levels of Fpg-sensitive sites are lower in studies where Fpg has been obtained from commercial suppliers or unknown sources as compared to Fpg from one particular non-commercial source (χ2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04-0.18 lesions/106 bp in leukocytes) compared to the Fpg-sensitive sites. Surprisingly, few publications have reported the use of oxidising agents as assay controls, with the exception of hydrogen peroxide. This may be due to a lack of consensus about suitable controls for the Fpg- and hOGG1-modified comet assay. A major challenge is to find an oxidising agent that only oxidises nucleobases and does not generate DNA strand breaks because this reduces the dynamic range of Fpg- and hOGG1-sensitive sites in the comet assay. Based on a literature search we selected the photosensitiser Ro19-8022 plus light, KBrO3, 4-nitroquinoline-1-oxide, Na2Cr2O7 and ferric nitrilotriacetate as possible assay controls. A subsequent assessment of these compounds for generating cryopreserved assay controls in mononuclear blood cells showed that Ro19-8022 plus light, KBrO3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA.

[1]  Yoshiki Miyachi,et al.  4-Nitroquinoline 1-oxide forms 8-hydroxydeoxyguanosine in human fibroblasts through reactive oxygen species. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[2]  A. Collins,et al.  Oxidative stress in humans: validation of biomarkers of DNA damage. , 2002, Carcinogenesis.

[3]  Steffen Loft,et al.  Intra-laboratory Comet Assay Sample Scoring Exercise for Determination of Formamidopyrimidine DNA Glycosylase Sites in Human Mononuclear Blood Cell DNA , 2004, Free radical research.

[4]  T. Noda,et al.  High accumulation of oxidative DNA damage, 8-hydroxyguanine, in Mmh/Ogg1 deficient mice by chronic oxidative stress. , 2002, Carcinogenesis.

[5]  Günter Speit,et al.  Sensitivity of the FPG protein towards alkylation damage in the comet assay. , 2004, Toxicology letters.

[6]  R. Marcos,et al.  Chromium-Induced Genotoxicity and Interference in Human Lymphoblastoid Cell (TK6) Repair Processes , 2011, Journal of toxicology and environmental health. Part A.

[7]  M. Chung,et al.  Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate. , 1996, Carcinogenesis.

[8]  P. Møller,et al.  No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly , 2015, Mutagenesis.

[9]  P. Møller,et al.  Oxidative DNA damage in human white blood cells in dietary antioxidant intervention studies. , 2002, The American journal of clinical nutrition.

[10]  J. Chipman,et al.  Modified comet assay as a biomarker of sodium dichromate-induced oxidative DNA damage: Optimization and reproducibility , 2004, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals.

[11]  Stefano Bonassi,et al.  Worldwide interest in the comet assay: a bibliometric study. , 2015, Mutagenesis.

[12]  A. Collins,et al.  Enhancing the sensitivity of the comet assay as a genotoxicity test, by combining it with bacterial repair enzyme FPG. , 2013, Mutagenesis.

[13]  M. Glasius,et al.  Biomarkers of oxidative stress and inflammation after wood smoke exposure in a reconstructed Viking Age house , 2014, Environmental and molecular mutagenesis.

[14]  Maria Dusinska,et al.  On the search for an intelligible comet assay descriptor , 2014, Front. Genet..

[15]  David H Phillips,et al.  Inter-laboratory variation in DNA damage using a standard comet assay protocol. , 2012, Mutagenesis.

[16]  T. Noda,et al.  Cell proliferation in liver of Mmh/Ogg1-deficient mice enhances mutation frequency because of the presence of 8-hydroxyguanine in DNA. , 2003, Cancer research.

[17]  A. Collins,et al.  Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors , 2011, Cell biochemistry and function.

[18]  A. Doherty,et al.  Anomalous genotoxic responses induced in mouse lymphoma L5178Y cells by potassium bromate. , 2010, Toxicology.

[19]  Maria Dusinska,et al.  Can the comet assay be used reliably to detect nanoparticle‐induced genotoxicity? , 2015, Environmental and molecular mutagenesis.

[20]  B. Epe,et al.  Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022. , 1999, Mutation research.

[21]  L. Rasmussen,et al.  Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages. , 2012, Mutagenesis.

[22]  Andrew Collins,et al.  Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  A. Collins,et al.  Supplementation of a western diet with golden kiwifruits (Actinidia chinensis var.'Hort 16A':) effects on biomarkers of oxidation damage and antioxidant protection , 2011, Nutrition journal.

[24]  G. Wörtwein,et al.  Dynamic regulation of cerebral DNA repair genes by psychological stress. , 2015, Mutation research. Genetic toxicology and environmental mutagenesis.

[25]  J. Chipman,et al.  Induction of DNA-strand breaks in human peripheral blood lymphocytes and A549 lung cells by sodium dichromate: association with 8-oxo-2-deoxyguanosine formation and inter-individual variability. , 2001, Mutagenesis.

[26]  David H Phillips,et al.  Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories. , 2014, Mutagenesis.

[27]  A. Collins,et al.  DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay. , 2012, Mutation research.

[28]  T. Becker,et al.  Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A. , 2002, Mutation research.

[29]  Mark R. Miller,et al.  Cardiovascular health effects of oral and pulmonary exposure to multi-walled carbon nanotubes in ApoE-deficient mice. , 2016, Toxicology.

[30]  P. Møller,et al.  Aging and oxidatively damaged nuclear DNA in animal organs. , 2010, Free radical biology & medicine.

[31]  K. Kanki,et al.  In vivo mutagenicity and initiation following oxidative DNA lesion in the kidneys of rats given potassium bromate , 2006, Cancer science.

[32]  Jacob S. Lamson,et al.  Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver , 2012, Particle and Fibre Toxicology.

[33]  P. Møller,et al.  Controlled exposure to diesel exhaust and traffic noise--Effects on oxidative stress and activation in mononuclear blood cells. , 2015, Mutation research.

[34]  Andrew R Collins,et al.  Measuring oxidative damage to DNA and its repair with the comet assay. , 2014, Biochimica et biophysica acta.

[35]  S. Bonassi,et al.  The comet assay as a tool for human biomonitoring studies: the ComNet project. , 2014, Mutation research. Reviews in mutation research.

[36]  Yi Cao,et al.  Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals , 2015, Environmental and molecular mutagenesis.

[37]  P. Møller,et al.  Oxidative stress and inflammation generated DNA damage by exposure to air pollution particles. , 2014, Mutation research. Reviews in mutation research.

[38]  Steffen Loft,et al.  An ECVAG† trial on assessment of oxidative damage to DNA measured by the comet assay , 2009, Mutagenesis.

[39]  L. Jensen,et al.  Hepatic Oxidative Stress, Genotoxicity and Vascular Dysfunction in Lean or Obese Zucker Rats , 2015, PloS one.

[40]  Steffen Loft,et al.  Dietary antioxidants and beneficial effect on oxidatively damaged DNA. , 2006, Free radical biology & medicine.

[41]  G. Speit,et al.  Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting. , 2014, Mutation research.

[42]  A. Zhitkovich,et al.  Genetic and epigenetic mechanisms in metal carcinogenesis and cocarcinogenesis: nickel, arsenic, and chromium. , 2008, Chemical research in toxicology.

[43]  Escodd.  Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus. , 2002, Carcinogenesis.

[44]  O. Raaschou-Nielsen,et al.  Genotoxic potential of the perfluorinated chemicals PFOA, PFOS, PFBS, PFNA and PFHxA in human HepG2 cells. , 2010, Mutation research.

[45]  Steffen Loft,et al.  Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring. , 2008, Mutagenesis.

[46]  M. Bucciolini,et al.  Calibration of the comet assay for the measurement of DNA damage in mammalian cells , 2006, Free radical research.

[47]  T. Downs,et al.  Weak silica nanomaterial-induced genotoxicity can be explained by indirect DNA damage as shown by the OGG1-modified comet assay and genomic analysis , 2017, Mutagenesis.

[48]  A. Mally,et al.  Are 4-hydroxy-2(E)-nonenal derived mercapturic acids and (1)H NMR metabonomics potential biomarkers of chemically induced oxidative stress in the kidney? , 2007, Toxicology.

[49]  Maria Dusinska,et al.  Measurement of DNA oxidation in human cells by chromatographic and enzymic methods. , 2003, Free radical biology & medicine.

[50]  Yi Cao,et al.  Applications of the comet assay in particle toxicology: air pollution and engineered nanomaterials exposure. , 2015, Mutagenesis.

[51]  Peter Møller,et al.  Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group. , 2010, Mutagenesis.

[52]  B. Epe,et al.  Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases. , 1997, Carcinogenesis.

[53]  B. Epe,et al.  Oxidative DNA damage induced by potassium bromate under cell-free conditions and in mammalian cells. , 1995, Carcinogenesis.

[54]  P. Møller,et al.  Controlled human wood smoke exposure: oxidative stress, inflammation and microvascular function , 2012, Particle and Fibre Toxicology.

[55]  David H Phillips,et al.  Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial. , 2010, Mutagenesis.

[56]  P. Møller,et al.  Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells , 2014, Oncotarget.

[57]  Steffen Loft,et al.  Hepatic toxicology following single and multiple exposure of engineered nanomaterials utilising a novel primary human 3D liver microtissue model , 2014, Particle and Fibre Toxicology.

[58]  Catherine C. Smith,et al.  hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII. , 2006, Mutagenesis.

[59]  Maria Dusinska,et al.  Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles , 2014, Nanotoxicology.

[60]  F. Nesslany,et al.  Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: determination of No-Observed-Genotoxic-Effect-Levels. , 2011, Mutation research.

[61]  Peter Møller,et al.  Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA. , 2006, Mutation research.

[62]  H. Karlsson,et al.  The comet assay in nanotoxicology research , 2010, Analytical and bioanalytical chemistry.

[63]  Amaya Azqueta,et al.  The essential comet assay: a comprehensive guide to measuring DNA damage and repair , 2013, Archives of Toxicology.

[64]  Antonio Marcomini,et al.  Genotoxicity, cytotoxicity, and reactive oxygen species induced by single‐walled carbon nanotubes and C60 fullerenes in the FE1‐Muta™Mouse lung epithelial cells , 2008, Environmental and molecular mutagenesis.

[65]  H. Karlsson,et al.  DNA damage induced by micro- and nanoparticles--interaction with FPG influences the detection of DNA oxidation in the comet assay. , 2012, Mutagenesis.

[66]  Steffen Loft,et al.  DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG). , 2013, Mutation research.

[67]  G. Speit,et al.  Comparative evaluation of the genotoxic properties of potassium bromate and potassium superoxide in V79 Chinese hamster cells. , 1999, Mutation research.

[68]  C. Del Bo’,et al.  Comparison of DNA damage by the comet assay in fresh versus cryopreserved peripheral blood mononuclear cells obtained following dietary intervention. , 2015, Mutagenesis.

[69]  Trevor M. Penning,et al.  Analysis of 7,8-Dihydro-8-oxo-2′-deoxyguanosine in Cellular DNA during Oxidative Stress , 2009, Chemical research in toxicology.

[70]  A. Collins,et al.  Oxidative damage to DNA: do we have a reliable biomarker? , 1996, Environmental health perspectives.

[71]  G. Speit,et al.  The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay. , 2016, Mutagenesis.

[72]  Amaya Azqueta,et al.  Controlling variation in the comet assay , 2014, Front. Genet..

[73]  Amaya Azqueta,et al.  Comet assay to measure DNA repair: approach and applications , 2014, Front. Genet..

[74]  A. Collins,et al.  European Standards Committee on Oxidative DNA Damage (ESCODD). Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage , 2000 .

[75]  Hajime Kojima,et al.  Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT). , 2015, Mutation research. Genetic toxicology and environmental mutagenesis.

[76]  V. D’Agata,et al.  Direct‐oxidative DNA damage and apoptosis induction in different human respiratory cells exposed to low concentrations of sodium chromate , 2009, Journal of applied toxicology : JAT.

[77]  E. Park,et al.  Influence of iron-overload on DNA damage and its repair in human leukocytes in vitro. , 2011, Mutation research.

[78]  P. Møller,et al.  Interventions with antioxidants and nutrients in relation to oxidative DNA damage and repair. , 2004, Mutation research.

[79]  David H Phillips,et al.  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. , 2013, Mutagenesis.

[80]  P. Møller,et al.  Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells. , 2015, Mutagenesis.

[81]  Steffen Loft,et al.  Harmonising measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and urine , 2012, Free radical research.