Minireview
DNA oxidation: Investigating its key role in environmental mutagenesis with the comet assay

https://doi.org/10.1016/j.mrgentox.2008.10.013Get rights and content

Abstract

DNA oxidation, which can have potentially serious mutagenic consequences, commonly accompanies exposure to environmental mutagens. Oxidised bases can be measured chromatographically, but spurious oxidation during sample preparation leads to serious over-estimation of low levels of damage. A more reliable approach is to employ endonucleases specific for oxidised bases, to introduce breaks in cellular DNA that are then most commonly measured using the comet assay (alkaline single cell gel electrophoresis). The two enzymes in general use are formamidopyrimidine DNA glycosylase, which detects primarily 8-oxo-7,8-dihydroguanine (8-oxoGua), and endonuclease III which recognises oxidised pyrimidines. We give a brief account of the recommended experimental procedures, and then describe applications in various areas of environmental research. Cultured cell lines or white blood cells have been exposed to a range of environmental mutagens, including natural products, industrial chemicals, radiation and nanoparticles. In vivo exposure of animals and humans to pollutants is more challenging but can give particularly valuable information in relation to real life exposure. Possibly the most useful application is in biomonitoring of human population groups suffering actual exposure to environmental or occupational mutagens. Finally, the potential use of this technique to monitor effects of contaminants in the natural environment has yet to be fully exploited.

Section snippets

Introduction: the origin and significance of DNA oxidation damage

DNA oxidation is known to be one of the most common kinds of damage to human DNA. It is mainly induced by reactive oxygen species (ROS), which include free radicals and other highly reactive forms of oxygen (e.g. hydrogen peroxide, superoxide anion radical, singlet oxygen, hydroxyl radical, nitric oxide and peroxynitrite). ROS are produced in cells during normal metabolic processes involving oxygen. They are released during cellular respiration, processes of biosynthesis and biodegradation,

Methodology: how is DNA oxidation best measured?

The question how best to measure DNA oxidation as a biomarker in population studies is still a vexed one. The traditional methods, GC–MS and HPLC with electrochemical detection (HPLC-ECD) have been widely used to measure 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) (or the base 8-oxoGua), but it was recognised in the mid-1990s that DNA is prone to oxidation during sample preparation, especially for GC–MS [16], and this can lead to a serious artefact when attempting to measure low background

Enzymes used to detect DNA oxidation damage

The information about DNA damage given by the comet assay reflects the number of single or double strand breaks formed in the cellular DNA before or during the process of electrophoresis. Combining the comet assay with bacterial repair enzymes recognising specific DNA damage is therefore a necessary step to allow detection of lesions that are not frank breaks. The use of enzymes increases both the sensitivity of the assay (in terms of the ability to detect a wider range of damage overall), and

Outline protocol for measuring oxidised bases with the comet assay

Thirty microliters of cell suspension (approximately 106 cells/ml in PBS) are mixed with 140 μl of 1% low melting point agarose (37 °C). Two drops of 70 μl are placed on a glass microscope slide (precoated with 1% normal agarose and dried), covered with two coverslips and left in the fridge for 5 min to allow the agarose to set. After this time the coverslips are removed and the slides are placed in lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris base, adjusted to pH 10 with NaOH, plus 1% Triton

Application to ecotoxicology: effects on natural habitats

Monitoring the environment for genotoxic contaminants can have two purposes; either assessing the risk of those contaminants for the animals and plants normally inhabiting the environment and for the biological integrity of the environment, or assessing the risk to humans, for example from food crops grown on contaminated soil, or from consumption of contaminated water. Essentially different criteria apply. When discussing the environment and its inhabitants, we are interested in populations,

Application to environmental mutagens relevant to human health

During the last two years, numerous studies of environmental mutagens relevant to human health using the comet assay in combination with EndoIII and/or FPG have been reported. They can be classified in 4 groups: cell studies, animal experiments, human biomonitoring and human experimental exposure. We summarize them in Table 2, Table 3, Table 4, Table 5.

The comet assay in combination with EndoIII or FPG can be used to study the capacity of cells for DNA repair. Briefly, cells treated with an

Conclusions

The comet assay is widely considered to be a powerful technique for investigating effects of environmental mutagens on cells, animals or humans. The inclusion of lesion-specific enzymes to introduce breaks at sites of oxidised bases increases its sensitivity and specificity. Most recent studies have involved experimental systems with cultured cells: applications in human biomonitoring or ecotoxicology are relatively few in number. While the comet assay is simple and reliable, it remains

Conflict of interest statement

None.

Acknowledgement

We acknowledge the support of EC contract FOOD-CT-2005-016320 (NewGeneris) in preparing this review.

References (61)

  • H.G. Jeong et al.

    Metallothionein-III prevents γ-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

    J. Biol. Chem.

    (2004)
  • C.M. Chen et al.

    Increased oxidative damage and mitochondrial abnormalities in the peripheral blood of Huntington's disease patients

    Biochem. Biophys. Res. Commun.

    (2007)
  • H. Miyake et al.

    Prognostic significance of oxidative DNA damage evaluated by 8-hydroxy-2′-deoxyguanosine in patients undergoing radical nephrectomy for renal cell carcinoma

    Urology

    (2004)
  • S. Hoffmann et al.

    Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of mammalian cells

    Free Rad. Biol. Med.

    (2004)
  • J. Rank et al.

    DNA damage, acetylcholinesterase activity and lysosomal stability in native and transplanted mussels (Mytilus edulis) in areas close to coastal chemical dumping sites in Denmark

    Aquat. Toxicol.

    (2007)
  • C. Emmanouil et al.

    Macromolecule oxidation and DNA repair in mussel (Mytilus edulis L.) gill following exposure to Cd and Cr(VI)

    Aquat. Toxicol.

    (2007)
  • N. Wessel et al.

    Investigating the relationship between embryotoxic and genotoxic effects of benzo[alpha]pyrene, 17 alpha-ethinylestradiol and endosulfan on Crassostrea gigas embryos

    Aquat. Toxicol.

    (2007)
  • J.F. Reeves et al.

    Hydroxyl radicals (OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells

    Mutat. Res.

    (2008)
  • L. Arbillaga et al.

    In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A

    Toxicol. Appl. Pharmacol.

    (2007)
  • A. Hartwig et al.

    Toxicological potential of 2-alkylcyclobutanones – specific radiolytic products in irradiated fat-containing food – in bacteria and human cell lines

    Food Chem. Toxicol.

    (2007)
  • H. Li et al.

    Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line

    Toxicol. Appl. Pharmacol.

    (2008)
  • C. Fanizza et al.

    DNA-damage in human lung epithelial cells exposed to respirable alpha-quartz

    Toxicol. In Vitro

    (2007)
  • N. Arranz et al.

    Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay

    Toxicol. In Vitro

    (2007)
  • W. Sudprasert et al.

    Effects of low-dose gamma radiation on DNA damage, chromosomal aberration and expression of repair genes in human blood cells

    Int. J. Hyg. Env. Health

    (2006)
  • C.S. Chen et al.

    Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay

    J. Hazard. Mater.

    (2008)
  • Y.C. Hseu et al.

    Humic acid induced genotoxicity in human peripheral blood lymphocytes using comet and sister chromatid exchange assay

    J. Hazard. Mater.

    (2008)
  • L. Risom et al.

    Dietary exposure to diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase 1 deficient mice

    Toxicol. Lett.

    (2007)
  • M.E. Fracasso et al.

    DNA damage and repair capacity by comet assay in lymphocytes of white-collar active smokers and passive smokers (non- and ex-smokers) at workplace

    Toxicol. Lett.

    (2006)
  • P.H. Danielsen et al.

    Oxidatively damaged DNA and its repair after experimental exposure to wood smoke in healthy humans

    Mutat. Res.

    (2008)
  • D.R. Spitz et al.

    Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology

    Cancer Metast. Rev.

    (2004)
  • Cited by (160)

    View all citing articles on Scopus
    View full text