Quantification of benzo[a]pyrene diol epoxide DNA‐adducts by stable isotope dilution liquid chromatography/tandem mass spectrometry

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants found in car exhausts, charbroiled food, and tobacco smoke. Three pathways for the metabolic activation of B[a]P to ultimate carcinogens have been proposed. The most widely accepted pathway involves cytochrome-P450 (CYP) 1A1- and/or 1B1-mediated formation of B[a]P-7,8-oxide, which undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further CYP1A1- and/or CYP1B1-mediated activation of the dihydrodiol results in the formation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. In previous studies, it was demonstrated that (+)-anti-B[a]PDE was the most potent tumorigen of the CYP-derived B[a]PDE diastereomers. We have developed a stable isotope dilution, liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay for all eight (+/-)-anti-B[a]PDE-derived dGuo and dAdo DNA-adducts. The LC-MRM/MS assay was rigorously validated and used to show that (+)-anti-trans-B[a]PDE-dGuo was the major adduct formed when naked DNA and human bronchoalveolar adenocarcinoma H358 cells were treated with (+/-)-anti-B[a]PDE. The preference for DNA-adducts derived from (+)-anti-B[a]PDE was even more apparent in cellular DNA. Thus, the increased potency of (+)-anti-B[a]PDE as a tumorigen is most likely due its ability to preferentially form DNA-adducts when compared with (-)-anti-B[a]PDE. Also, the adduct profile suggests that this occurs by binding of (+)-anti-B[a]PDE to DNA in a manner that facilitates covalent binding to dGuo rather than dAdo residues.

Preparation, identification and analysis of stereoisomeric anti-benzo[a]pyrene diol epoxide–deoxyguanosine adducts using phenyl liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection

“…The low yield of the cis-(+)-anti-BPDE-N 2 -dG was consistent with its chemical instability [27]. By the established synthesis method, enzymatic digestion process is no longer required, and carcinogenic anti-BPDE (∼0.1 mg) is 10-100 fold less consumed than reported methods [19,20,23].…”

“…A number of methods have been developed for characterization and/or detection of BPDE-DNA adducts, including capillary electrophoresis (CE)-laser-induced fluorescence (LIF) and CE-LIF immunoassays [10][11][12][13], accelerator mass spectrometry [14], liquid chromatography or capillary electrophoresis coupled to mass spectrometry [15][16][17][18][19][20][21], and 32 P-postlabelling [20,22] and spectroscopic analysis [23,24]. Among these methods, CE-LIF immunoassays display advantages of measuring trace levels of DNA adducts without digestion of DNA [10,11].…”

“…A large amount of pure single BPDE-adducted nucleosides, which are not commercially available, are also required to be prepared in individual laboratories to characterize the stereochemistry of BPDE-DNA adducts generated in vitro or in vivo. Previously, single adducted nucleosides were obtained by reaction of anti-BPDE with DNA, followed by enzymatic digestion, liquid-liquid or solid-phase extraction, and HPLC purification [19,20,23]. In these procedures, multiple enzymatic digestions involved are time-consuming (∼24-72 h), which may cause excess hydrolysis of the BPDE-nucleosides into BPDE tetrols.…”

“…Another disadvantage is the low yield of anti-BPDEdG in DNA except (+)-trans-anti-BPDE-dG [24]. To produce enough anti-BPDE-dG standards, a large amount of highly carcinogenic anti-BPDE is often required (1-10 mg) [19,20,23]. An alternative is to use the reaction of dGMP and anti-BPDE, which only requires one-step enzymatic digestion for preparation of anti-BPDE-dGs [25].…”

Synthesis of 13C2-benzo[a]pyrene and its 7,8-dihydrodiol and 7,8-dione implicated as carcinogenic metabolites

“…17,18 In this connection, development of a stable isotope dilution liquid chromatography tandem mass spectrometric method for analysis of the metabolites of BP and its nucleoside adducts was also described. 19 In order to improve the scope and sensitivity of this methodology, 13 C-labelled analogues of BP and its active metabolites with at least two 13 C-atoms in the aromatic ring system are needed as authentic standards.…”