High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantitation of Benzo[a]pyrene−DNA Adducts

Abstract

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Ultra-performance liquid chromatography–tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide–DNA adducts

“…The method development for stereoisomeric analysis of BPDE-DNA adducts takes multiple steps and labor-intensive. It often involves with the preparation of single stereoisomers of adducted nucleosides or nucleotides (which are not commercially available), and the structural and stereochemical characterization, enzymatic digestion of genomic DNA, and highly efficient and stereoselective separation and coupled sensitive detection [10,21,28]. Among these steps, the stereoselective separation of the adduct stereoisomers is most critical and tunable factor for achieving high sensitivity of detection.…”

Integrating Field Analyses with Laboratory Exposures to Assess Ecosystems Health

“…In lung DNA, one adduct was tentatively identified as the anti-benzo [a]pyrene -7,8-diol-9,10-oxide-deoxyguanosine adduct (Culp & Beland, 1994). The identity of this adduct was confirmed by analysis of the lung DNA of female B6C3F1 mice fed a diet of coal tar from manufactured gas plant residue; however, based on the levels of this adduct, it was suggested that benzo [a]pyrene may contribute only a small fraction to the DNA adducts formed in the lung tissue of mice administered coal tar (Beland et al, 2005). Female B6C3F1 mice were fed diets of coal tar, and DNA adduct formation, cell proliferation and mutations in tumours in the K-ras, H-ras and p53 genes were determined.…”

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: The increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

“…[26,89,90]. In all of these studies there were inter-laboratory comparison of DNA samples that were modified in vitro, extracted from exposed animals and, in the case of B[a]P extracted from human tissue.…”