Measurement of Oxidatively‐Induced Clustered DNA Lesions Using a Novel Adaptation of Single Cell Gel Electrophoresis (Comet Assay)

Georgakilas, Alexandros G.; Holt, Stewart M.; Hair, Jessica M.; Loftin, Charles W.

doi:10.1002/0471143030.cb0611s49

Cited by 18 publications

(18 citation statements)

References 40 publications

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“…One example is the inclusion of purified lesion-specific enzymes. These enzymes can convert otherwise undetectable base lesions into detectable strand breaks and abasic sites 1,[19][20][21] . A widely used example is the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg), which reveals oxidative DNA modifications 19,22 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…These enzymes can convert otherwise undetectable base lesions into detectable strand breaks and abasic sites 1,[19][20][21] . A widely used example is the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg), which reveals oxidative DNA modifications 19,22 . Because the enzyme digestion step is applied after cell lysis, the system can be treated the same way as a traditional agarose slide without alterations to the protocol.…”

Section: Representative Resultsmentioning

confidence: 99%

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CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Prasongtanakij

Wood

et al. 2014

JoVE

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DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The 'CometChip' is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

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Section: Representative Resultsmentioning

confidence: 99%

Section: Representative Resultsmentioning

confidence: 99%

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Prasongtanakij

Wood

et al. 2014

JoVE

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“…However, differences exist in microdistribution (single molecules and clusters of ROS produced by radiation vs. single molecules produced by endogenous processes), relative yield of specific products (mainly O 2 •− and H 2 O 2 produced by endogenous processes vs. • OH in highest yield in irradiated cells) and timing of production (chronic release of endogenous ROS versus instantaneous production during irradiation) [92], As a result, damage from metabolic ROS is randomly distributed in the DNA, radiation-induced DNA damage frequently occurs in clusters [93]. Whereas ∼ 1/3 of DNA damage from ionizing radiation (low LET type) emanates from direct interaction of DNA with the irradiating particle and 2/3 due to indirect effects (Fig.…”

Section: Primary Effects Of Ionizing Radiationmentioning

confidence: 99%

“…Techniques such as the ‘comet assay’ [93; 94] and newer techniques (e.g. gas chromatography, high pressure liquid chromatography, immunoassays) [4; 68] can distinguish gross DNA damage produced by ionizing radiation and damage from oxidative attacks.…”

Section: Primary Effects Of Ionizing Radiationmentioning

confidence: 99%

Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury

2012

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Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through direct interactions of radiation with target macromolecules or via products of water radiolysis. Further, the oxidative damage may spread from the targeted to neighboring, non-targeted bystander cells through redox-modulated intercellular communication mechanisms. To cope with the induced stress and the changes in the redox environment, organisms elicit transient responses at the molecular, cellular and tissue levels to counteract toxic effects of radiation. Metabolic pathways are induced during and shortly after the exposure. Depending on radiation dose, dose-rate and quality, these protective mechanisms may or may not be sufficient to cope with the stress. When the harmful effects exceed those of homeostatic biochemical processes, induced biological changes persist and may be propagated to progeny cells. Physiological levels of reactive oxygen and nitrogen species play critical roles in many cellular functions. In irradiated cells, levels of these reactive species may be increased due to perturbations in oxidative metabolism and chronic inflammatory responses, thereby contributing to the long-term effects of exposure to ionizing radiation on genomic stability. Here, in addition to immediate biological effects of water radiolysis on DNA damage, we also discuss the role of mitochondria in the delayed outcomes of ionization radiation. Defects in mitochondrial functions lead to accelerated aging and numerous pathological conditions. Different types of radiation vary in their linear energy transfer (LET) properties, and we discuss their effects on various aspects of mitochondrial physiology. These include short and long-term in vitro and in vivo effects on mitochondrial DNA, mitochondrial protein import and metabolic and antioxidant enzymes.

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“…The concepts in DNA cluster quantification are simple, but their execution is technically and analytically demanding. Dr. Georgakilas' group, using novel adaptations of DNA gel electrophoresis, has been able to provide to the scientific community relatively easy and reliable methodologies for the detection of OCDLs in a variety of cell and tissues [3,[9][10][11] . In early 2007, Gollapalle et al [4] showed for the first time in vivo that X-rays can induce a radiation-delayed effect in mice several weeks after the initial irradiation.…”

Section: Detection Of Ocdls In Vivo: Potential Clinical Applicationsmentioning

confidence: 99%

Detection of clustered DNA lesions: Biological and clinical applications

Georgakilas

2011

WJBC

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Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.

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Measurement of Oxidatively‐Induced Clustered DNA Lesions Using a Novel Adaptation of Single Cell Gel Electrophoresis (Comet Assay)

Cited by 18 publications

References 40 publications

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury

Detection of clustered DNA lesions: Biological and clinical applications

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