Metabolic activation of the carcinogenic tobacco-specific N-nitrosamines leads to the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts. We recently developed a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method for the analysis of HPB-releasing DNA adducts in human oral cells. However, given the limited amounts of DNA that can be extracted from oral cells, higher sensitivity and selectivity are required for the reliable analysis of these adducts in future studies. We have developed a new sensitive LC-nanoelectrospray ionization-high-resolution MS/MS method for the analysis of HPB-releasing DNA adducts in oral cells. A new procedure was also developed for guanine analysis by LC-MS/MS. The detection limit of the developed assay is 5 amol, and the limit of quantitation is 0.35 fmol HPB on-column, starting with 50 pg of DNA. The method was tested by analyzing oral samples from 65 smokers, including 30 head and neck squamous cell carcinoma (HNSCC) patients and 35 cancer-free controls. In all smokers, the levels of HPB-releasing DNA adducts averaged 6.22 ± 16.18 pmol/mg DNA, with significant interindividual variation being consistent with previous reports. The median HPB-releasing DNA adduct level was 6.6 times greater for those with HNSCC than for smokers without HNSCC (p = 0.002). The developed highly sensitive and selective method is a valuable tool for future measurement of HPB-releasing DNA adducts in tobacco users, which can potentially provide critical insights for the identification of individuals at risk for cancer.
Bibliographical noteFunding Information:
This study was supported by Grant Nos. P01-CA138338 and R01-CA179246 from the National Cancer Institute, Grant No. K23-DE023572 from the National Institute of Dental and Craniofacial Research, and by startup funds to IS from the Masonic Cancer Center. Mass spectrometry analysis was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by Grant No. CA-77598 from the National Cancer Institute.