Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MS/MS

Abstract

Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using LC-MS/MS. However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from sixteen ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by UPLC-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylme-thylglucosinolate, 5-hydroxymethylfurfural and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02 – 7.1 adducts/108 nucleosides). 3,N4-etheno-2′-deoxycytidine and 1,N6-etheno-2′-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9 – 115.3 and 27.2 – 179/108 nucleosides, respectively). The same was true for N2-(trans-methylisoeugenol-3′-yl)-2′-deoxyguanosine, the major adduct of methyleugenol (1.7 – 23.7/108 nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed detecting adducts of furfuryl alcohol and methyleugenol in samples of human lung.

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

“…Monien et al used an optimized extraction and enrichment technique coupled with a UPLC-MS/MS method to analyze 16 DNA adducts from various sources in 10 non-tumor human lung tissue samples from cancer patients. 18 Several adducts were detected and quantified, but not BPDE- N 2 -dG, which was negative in all samples with detection limits of 1.0–3.0 adducts per 10 8 nucleotides. As mentioned earlier, 3 additional studies failed to detect BPDE- N 2 -dG in salivary, oral cell, or prostate DNA.…”

“…The levels of BPDE- N 2 -dG adducts in human samples, when analyzed using conventional mass spectrometry, 18–22 have been typically below the limits of detection. We developed an ultra-sensitive high resolution/accurate mass (HRAM) parallel reaction monitoring (PRM) method optimizing parameters of the mass spectrometric detection, liquid chromatography, and sample preparation which when combined allowed for a dramatic improvement in detection limits of our previous HRAM MS 2 quantitation methodologies.…”

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

“…In this respect, Monien et al 141 recently employed enrichment followed by UPLC-MS/MS analysis for monitoring simultaneously multiple DNA adducts derived from different genotoxic agents in DNA isolated from human lung biopsy specimens. Adducts derived from by-products of lipid peroxidation, furfuryl alcohol, and methyleugenol could be detected.…”

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts

“…This “pseudo-CNL” methodology was used to characterize lipid peroxidation DNA adducts in human tissues. 27–29 Numerous signals were detected; however, it is not known whether these analytes are DNA adducts or simply other compounds in the DNA digest matrix that lose 116 Da upon CID. The Turesky laboratory laid the groundwork for the linear ion trap (LIT)-MS based adductomics method.…”