Mutation Research/Genetic Toxicology and Environmental Mutagenesis
MinireviewDNA oxidation: Investigating its key role in environmental mutagenesis with the comet assay
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)
Oxidative stress: introductory remarks
- et al.
Molecular mechanism of oxidative stress perception by the Orp1 protein
J. Biol. Chem.
(2007) - et al.
Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome
Free Rad. Biol. Med.
(2002) - et al.
Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility
Free Rad. Biol. Med.
(2000) - et al.
Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair
Mol. Cell
(1998) - et al.
Functional characterization of human MutY homolog (hMYH) missense mutation (R231L) that is linked with hMYH-associated polyposis
Cancer Lett.
(2007) Molecular genetics and structural biology of human MutT homolog, MTH1
Mutat. Res.
(2001)- et al.
Oxidative DNA damage repair in mammalian cells: a new perspective
DNA Repair
(2007) - et al.
The single cell gel electrophoresis assay (comet assay): a European review
Mutat. Res.
(1993) - et al.
Impaired base excision repair and accumulation of oxidative base lesions in CD4+ T cells of HIV-infected patients
Blood
(2005)
Metallothionein-III prevents γ-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells
J. Biol. Chem.
Increased oxidative damage and mitochondrial abnormalities in the peripheral blood of Huntington's disease patients
Biochem. Biophys. Res. Commun.
Prognostic significance of oxidative DNA damage evaluated by 8-hydroxy-2′-deoxyguanosine in patients undergoing radical nephrectomy for renal cell carcinoma
Urology
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.
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.
Macromolecule oxidation and DNA repair in mussel (Mytilus edulis L.) gill following exposure to Cd and Cr(VI)
Aquat. Toxicol.
Investigating the relationship between embryotoxic and genotoxic effects of benzo[alpha]pyrene, 17 alpha-ethinylestradiol and endosulfan on Crassostrea gigas embryos
Aquat. Toxicol.
Hydroxyl radicals (OH) are associated with titanium dioxide (TiO(2)) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells
Mutat. Res.
In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A
Toxicol. Appl. Pharmacol.
Toxicological potential of 2-alkylcyclobutanones – specific radiolytic products in irradiated fat-containing food – in bacteria and human cell lines
Food Chem. Toxicol.
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.
DNA-damage in human lung epithelial cells exposed to respirable alpha-quartz
Toxicol. In Vitro
Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay
Toxicol. In Vitro
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
Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay
J. Hazard. Mater.
Humic acid induced genotoxicity in human peripheral blood lymphocytes using comet and sister chromatid exchange assay
J. Hazard. Mater.
Dietary exposure to diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase 1 deficient mice
Toxicol. Lett.
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.
Oxidatively damaged DNA and its repair after experimental exposure to wood smoke in healthy humans
Mutat. Res.
Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology
Cancer Metast. Rev.
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