Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column-switching liquid chromatography/tandem mass spectrometry

Rajinder Singh,William P Steward,Heinrich Frank,Friederike Teichert,Rebecca Cordell,Albrecht Seidel,Jonathan Roach,Peter B Farmer,Margaret M Manson,Mai-Kim Cheng

doi:10.1002/rcm.4645

Abstract

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software-based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH-modified DNA samples were obtained by reaction of the anti-dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2'-deoxynucleosides. Positive LC/electrospray ionisation (ESI)-MS/MS collision-induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2'-deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2'-deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH-adducted 2'-deoxynucleosides was achieved using online column-switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H](+) to [M+H-116](+) transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H-116](+) transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods.

Highlights

Polycyclic aromatic hydrocarbons (PAHs) represent a group of structurally related organic compounds, formed primarily by the incomplete combustion of organic matter, which are ubiquitously occuring environmental contaminants 1-6
No product ions resulting from the neutral loss of 17 u corresponding to –NH3 were observed, confirming that polycyclic aromatic hydrocarbon (PAH)-dihydrodiol epoxide adduct formation for each 2′-deoxynucleoside was by reaction with the exocyclic – NH2 group at position N2 for guanine, N6 for adenine and N4 for cytosine
The importance of determining multiple PAH DNA adducts rather than single adducts was highlighted by Beland et al who reported that for mice fed a diet containing coal tar, the B[a]PDE-N2-dG adduct determined by liquid chromatography (LC)-M/mass spectrometry (MS) only represented a small proportion of the total adduct burden in lung DNA as determined by 32P postlabelling

Summary

Introduction

Polycyclic aromatic hydrocarbons (PAHs) represent a group of structurally related organic compounds, formed primarily by the incomplete combustion of organic matter, which are ubiquitously occuring environmental contaminants 1-6. Sample loading: An aliquot of the PAH-dihydrodiol epoxide modified calf thymus DNA mixture (1 μg/μL of hydrolysed DNA) containing 1 pmol of the [15N5]B[a]PDE-N2-dG stable isotope internal standard was injected onto the trap column using pump A with the switching valve in position 1.

Results

Conclusion