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. 2017 Dec 5;89(23):12735-12742.
doi: 10.1021/acs.analchem.7b02856. Epub 2017 Nov 17.

Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[a]pyrene in Human Lung

Peter W Villalta  1 , J Bradley Hochalter  1 , Stephen S Hecht  1
Affiliations

Ultrasensitive High-Resolution Mass Spectrometric Analysis of a DNA Adduct of the Carcinogen Benzo[a]pyrene in Human Lung

Peter W Villalta et al. Anal Chem. .

Abstract

Benzo[a]pyrene (BaP), an archetypical polycyclic aromatic hydrocarbon, is classified as "carcinogenic to humans" and is ubiquitous in the environment, as evident by the measurable levels of BaP metabolites in virtually all human urine samples examined. BaP carcinogenicity is believed to occur mainly through its covalent modification of DNA, resulting in the formation of BPDE-N2-dG, an adduct formed between deoxyguanosine and a diol epoxide metabolite of BaP, with subsequent mutation of critical growth control genes. In spite of the liquid chromatography-mass spectrometry (LC-MS)-based detection of BPDE-N2-dG in BaP-treated rodents, and indirectly through high-performance liquid chromatography (HPLC)-fluorescence detection of BaP-7,8,9,10-tetraols released from human DNA upon acid hydrolysis, BPDE-N2-dG adducts have rarely if ever been observed directly in human samples using LC-MS techniques, even though sophisticated methodologies have been employed which should have had sufficient sensitivity. With this in mind, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodology employing high-resolution/accurate mass analysis for detecting ultratrace levels of these adducts. These efforts are directly translatable to the development of sensitive detection of other small molecules using trap-based LC-ESI-MS/MS detection. The developed methodology had a limit of detection (LOD) of 1 amol of BPDE-N2-dG on-column, corresponding to 1 BPDE-N2-dG adduct per 1011 nucleotides (1 adduct per 10 human lung cells) using 40 μg of human lung DNA. To our knowledge, this is the most sensitive DNA adduct quantitation method yet reported, exceeding the sensitivity of the 32P-postlabeling assay (∼1 adduct per 1010 nucleotides). Twenty-nine human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and nonsmoker DNA containing 3.1 and 1.3 BPDE-N2-dG adducts per 1011 nucleotides, respectively.

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Figures

Figure 1
Figure 1
Plots of the average (triplicate) ion signal response resulting from analysis of varying amounts of analyte (BPDE-N2-dG) with constant amounts of internal standards ([13C10]BPDE-N2-dG). Error bars represent one relative standard deviation of the triplicate measurements. Samples for (A) are neat standards in 90% H2O, 10% ACN. Samples for (B) are from spiked ct-DNA (40 μg) processed in the same fashion as used for samples derived from lung tissue.
Figure 2
Figure 2
Extracted ion chromatograms for the BPDE-N2-dG analyte and [13C10]BPDE-N2-dG internal standard ion signal with a mass tolerance of 5 ppm for a typical smoker sample (A) and no-smoker sample (B).
Scheme 1
Scheme 1
Metabolism of BaP to BaP-7,8-epoxide (2), BaP-7.8-diol (3), and BaP-7,8-diol-9,10-epoxide (BPDE, 4), and reaction with DNA to give BPDE-N2-dG (5).
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