Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage

Abstract

The formation of deoxyribonucleic acid (DNA) adducts can have important and adverse consequences for cellular and whole organism function. Available methods for identification of DNA damage and quantification of adducts are reviewed. Analyses can be performed on various samples including tissues, isolated cells, and intact or hydrolyzed (digested) DNA from a variety of biological samples of interest for monitoring in humans. Sensitivity and specificity are considered key factors for selecting the type of method for assessing DNA perturbation. The amount of DNA needed for analysis is dependent upon the method and ranges widely, from <1 μg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled (¹⁴C- and ³H-) binding, ³²P-postlabeling, and methods dependent on gas chromatography (GC) or high-performance liquid chromatography (HPLC) with detection by electron capture, electrochemical detection, single or tandem mass spectrometry, or accelerator mass spectrometry. Sensitivity is ranked, and ranges from ∼1 adduct in 10⁴ to 10¹² nucleotides. A brief overview of oxidatively generated DNA damage is also presented. Assay limitations are discussed along with issues that may have impact on the reliability of results, such as sample collection, processing, and storage. Although certain methodologies are mature, improving technology will continue to enhance the specificity and sensitivity of adduct analysis. Because limited guidance and recommendations exist for adduct analysis, this effort supports the HESI Committee goal of developing a framework for use of DNA adduct data in risk assessment.

Keywords::

• Biomarkers
• DNA adducts
• oxidative damage
• risk assessment

Acknowledgements

The authors thank the ILSI HESI staff, especially Dr. Michelle Embry and Ms. Syril Petit, for administrative and technical support. Valuable discussions with Dr. Lynn Pottenger, the authors of the companion framework report, and Dr. G. Don Jones are greatly appreciated.

Disclaimer: This document represents the consensus of the participants’ views expressed as individual scientists and does not necessarily represent the policies and procedures of their respective institutions.

Declaration of interest: The author have no commercial conflict of interest to declare. This publication stems from a subgroup of the HESI Biological Significance of DNA Adducts Project Committee, whose work is funded through ILSI/HESI.

Abbreviations

2D TLC    two-dimensional thin-layer chromatography

3-MeAde    3-methyladenine

8-oxoGua    8-oxo-7,8-dihydroguanine

8-oxodGuo    8-oxo-7,8-dihydro-2’-deoxyguanosine

AMS    accelerator mass spectrometry

B[a]P    benzo[a]pyrene

¹⁴C    carbon-14

CO₂    carbon dioxide

DNA    deoxyribonucleic acid

DMSO    dimethyl sulfoxide

EC    electrochemical detection

ECD    electron capture detection

ELISA    enzyme-linked immunosorbent assay

Endo III    endonuclease III

ESCODD    European Standards Committee on Oxidative DNA Damage

FL    fluorescence detection

Fpg    formamidopyrimidine-DNA glycosylase

GC    gas chromatography

GC-ECD    gas chromatography-electron capture detection

GC-MS    gas chromatography mass spectrometry

³H    tritium

hOGG1    human 8-oxoguanine-DNA glycosylase

HPLC    high-performance liquid chromatography

HPLC-EC    HPLC–electrochemical detection

ISB    immunoslot blot

LC-MS/MS    liquid chromatography-tandem mass spectrometry

LC-NMR    liquid chromatography-nuclear magnetic resonance

LM-PCR    ligation-mediated polymerase chain reaction

M₁-dG    malondialdehyde-2′-deoxyguanosine

MRM    multiple reaction monitoring

MS    mass spectrometry

MS/MS    tandem mass spectrometry

NMR    nuclear magnetic resonance

PAH    polycyclic aromatic hydrocarbons

PCR    polymerase chain reaction

³²P-ATP    phosphorus–32 adenosine triphosphate

RIA    radioimmunoassay

RNA    ribonucleic acid

SIM    selected ion monitoring

SRM    selected reaction monitoring

TLC    thin-layer chromatography

UV    ultraviolet