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

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 LC-MS based detection of BPDE-N2-dG in BaP-treated rodents, and indirectly through 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 an LC-ESI-MS/MS methodology employing high resolution/accurate mass analysis for detecting ultra-trace 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 an LOD of 1 amol of BPDE-N2-dG on-column, corresponding to 1 BPDE-N2-dG adduct/1011 nucleotides (1 adduct/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/1010 nucleotides). 29 human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and non-smoker DNA containing 3.1 and 1.3 BPDE-N2-dG adducts/1011 nucleotides, respectively.

The Future of DNA Adductomic Analysis

“…The complexity of the DNA samples should consist of the unmodified bases, which are very hydrophilic and therefore elute much earlier than many adducts, hydrolysis enzymes, and any impurities in the enzymes and unpolymerized constituents entering the sample solution when using plastic components, such as solid-phase extraction cartridges and molecular weight filters for sample preparation [ 29 , 36 , 72 ]. The impurities due to the use of hydrolysis enzymes can be nearly eliminated, or at least greatly reduced, by careful enzyme source and vendor selection, cleaning of the enzymes prior to use, and determining the minimal enzyme necessary for the analysis [ 72 , 73 ]. Impurities due to the use of plastics can be greatly reduced or eliminated by avoiding the use plastics either entirely or as much is practically possible and by careful type/vendor selection of consumables used for the experiments.…”

Analysis and Identification of 2′-Deoxyadenosine-Derived Adducts in Lung and Liver DNA of F-344 Rats Treated with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

“…32 With analytical power continuing to increase, measurement of low levels of DNA adducts in humans is starting to become an achievable feat. 54 …”

The safety evaluation of food flavoring substances: the role of genotoxicity studies

“…DNA adducts can be detected at extremely low levels, e.g. the reported limit of detection (LOD) is as low as 1 adduct per 10 11 nucleotides and the limit of quantitation (LOQ) is as low as 5 adducts per 10 11 nucleotides by LC/MS (Zhang et al 2006;Monien et al 2015;Villalta et al 2017;Yang et al 2019) and is even lower by accelerator MS (Hummel et al 2018;Madeen et al 2019). Therefore, it is critical to evaluate whether the level of DNA adducts detected is biologically significant.…”