The analysis of DNA adducts: The transition from 32P-postlabeling to mass spectrometry

Abstract

The technique of 32P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10 μg DNA to achieve the detection of 1 adduct in 1010 normal bases. 32P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques 32P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field. Structural methods have since been developed to allow for positive identification of DNA adducts, but to this day, the same level of sensitivity and low sample requirements provided by 32P-postlabeling have not been matched. In this mini review we will discuss the 32P-postlabeling method and chronicle the transition to mass spectrometry via the hyphenation of gas chromatography, capillary electrophoresis, and ultimately liquid chromatography which, some 30 years later, is only just starting to approach the sensitivity and low sample requirements of 32P-postlabeling. This paper focuses on the detection of bulky carcinogen-DNA adducts, with no mention of oxidative damage or small alkylating agents. This is because the 32P-postlabeling assay is most compatible with bulky DNA adducts. This will also allow a more comprehensive focus on a subject that has been our particular interest since 1990.

Mechanistic relationships between hepatic genotoxicity and carcinogenicity in male B6C3F1 mice treated with polycyclic aromatic hydrocarbon mixtures

“…Our results showed that the BPDE-dG adduct has very similar trends with total DNA adducts. In future studies, high resolution mass spectrometry (Klaene et al 2012) or a combination of 32 P-postlabeling assay and mass spectrometry should be considered in detection and structure identification of DNA adducts (Farmer and Singh 2008). …”

Mass Spectrometric Mapping of the DNA Adductome as a Means to Study Genotoxin Exposure, Metabolism, and Effect

“…28 Over the years, sensitivity has continued to improve 6,14 as different research groups have focused on optimization of DNA adduct detection methods with MS [30][31][32][33][34][35][36][37] , including research into non-manual data mining and sequencing to locate DNA adduction sites. 38,39 Due to ongoing technical advancements and the use of stable isotope labeled internal standards, MS currently offers a reliable tool to measure low DNA adduct levels with the highest specificity. 12,14,27,28 Coupling of MS with LC by means of the electrospray interface (ESI) has enabled analysis of DNA adducts in very complex biological matrices 40 , whilst avoiding complex and labour-intensive sample preparation with derivatization for the initially envisioned use of GCcouplings.…”

A nontargeted screening method for covalent DNA adducts and DNA modification selectivity using liquid chromatography-tandem mass spectrometry

“…However, those methods are unable to provide information on the structure of DNA adducts, leading to some degree of ambiguity in the identities of unknown DNA adducts [6,7]. In addition, because of exposure to complicated mixtures of environmental contaminants in human exposure scenario, DNA adducts in human samples are often unknowns [8].…”