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

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 liquid chromatography-mass spectrometry (LC-MS)-based detection of BPDE-N2-dG in BaP-treated rodents, and indirectly through high-performance liquid chromatography (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 a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodology employing high-resolution/accurate mass analysis for detecting ultratrace 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 a limit of detection (LOD) of 1 amol of BPDE-N2-dG on-column, corresponding to 1 BPDE-N2-dG adduct per 1011 nucleotides (1 adduct per 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 per 1010 nucleotides). Twenty-nine human lung DNA samples resulted in 20 positive measurements above the LOD, with smoker and nonsmoker DNA containing 3.1 and 1.3 BPDE-N2-dG adducts per 1011 nucleotides, respectively.

Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons.

LC-MS using electrospray ionization is currently the method of choice in bio-organic analysis covering a wide range of applications in a broad spectrum of biological media. The technique is noted for its high sensitivity but one major limitation that hinders achievement of its optimal sensitivity is the signal suppression due to matrix inferences introduced by the presence of co-extracted compounds during the sample preparation procedure. The analysis of DNA adducts of common environmental carcinogens is particularly sensitive to such matrix effects as sample preparation is a multistep process which involves "contamination" of the sample due to the addition of enzymes and other reagents for digestion of the DNA in order to isolate the analyte(s). This problem is further exacerbated by the need to reach low levels of quantitation (LOQ in the ppb level) while also working with limited (2-5 µg) quantities of sample. We report here on the systematic investigation of ion signal suppression contributed by each individual step involved in the sample preparation associated with the analysis of DNA adducts of polycyclic aromatic hydrocarbon (PAH) using as model analyte BaP-dG, the deoxyguanosine (dG) adduct of benzo[a]pyrene (BaP). The individual matrix contribution of each one of these sources to analyte signal was systematically addressed as were any interactive effects. The information was used to develop a validated analytical protocol for the target biomarker at levels typically encountered in vivo using as little as 2 µg of DNA and applied to a dose response study using a metabolically competent cell line.

Effects of the N terminus of mouse DNA polymerase κ on the bypass of a guanine-benzo[a]pyrenyl adduct.

DNA polymerase κ (Polκ), one of the typical member of the Y-family DNA polymerases, has been demonstrated to bypass the 10S(+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-deoxyguanine adducts (BPDE-dG) efficiently and accurately. A large structural gap between the core and little finger as well as an N-clasp domain are essential to its unique translesion capability. However, whether the extreme N-terminus of Polκ is required for its activity is unclear. In this work, we constructed two mouse Polκ deletions, which have either a catalytic core (mPolκ1-516) or a core without the first 21-residues (mPolκ22-516), and tested their activities in the replication of normal and BPDE-DNA. These two Polκ deletions are nearly as efficient as the full length protein (Polκ1-852) in normal DNA synthesis. However, steady-state kinetics reveals a significant reduction in efficiency of dCTP incorporation opposite the lesion by Polκ22-516, along with increased frequencies for misinsertion compared with Polκ1-852 The next nucleotide insertion opposite the template C immediately following the BPDE-dG was also examined, and the bypass differences induced by deletions were highlighted in both insertion and extension step. We conclude that the extreme N-terminal part of Polκ is required for the processivity and fidelity of Polκ during translesion synthesis of BPDE-dG lesions.

Transient isotachophoresis focusing of DNA and DNA-protein complexes is essentially enhanced by spontaneously dissolved aerial carbon dioxide in electrolytes.

The formation of a highly adapted high-E zone is critical to isotachophoresis separation and focusing. Recently, we discovered that the high-E zone is present only in a small portion of electrophoresis channel in the presence of EOF (Liu, S. Q. et al. J. Am. Chem. Soc. 2013, 135, 4644-4647). Accordingly, a much narrower high-E zone is presumably present in t-ITP. If so, it is hard to achieve efficient t-ITP focusing. Indeed, by online coupling t-ITP with CE-LIF immunoassay, the immunocomplexes of carcinogenic BPDE-dG adducts are not efficiently focused using a freshly prepared background electrolyte. Intriguingly, we observed that 20-day stored background electrolyte displays a 10-fold better focusing efficiency. We hypothesize that the unexpected phenomenon is associated with the dissolution of aerial carbon dioxide, which is mainly converted to ionic HCO3(-) in the weak alkaline background electrolyte. Consequently, HCO3(-) of high electrophoretic mobility will be continuously injected into the capillary along with the background electrolyte and act as an alternative leading ion to improve the focusing. By addition of dry ice (without causing significant pH decrease, ΔpH < 0.4) to freshly prepared background electrolytes, we immediately observed the enhanced focusing of immunocomplexes of the DNA adducts. NH4HCO3 and Na2CO3, included in the background electrolyte, also improve the focusing efficiency and reproducibility. All these consistently support our hypothesis. To understand the underlying mechanism, an advanced CE-SMFI was exploited to monitor in real time the motion of single DNA molecules and the E change throughout t-ITP. We uncovered that t-ITP can induce a local high-E zone, but the presence of HCO3(-) in the background electrolyte could greatly increase the E value in the high-E zone, which allows more DNA molecules to rapidly move backward and to be efficiently stacked at LE/TE boundary. This study provides new insight into nonuniform electric field-induced electrophoresis focusing.

Human DNA polymerases catalyze lesion bypass across benzo[a]pyrene-derived DNA adduct clustered with an abasic site.

The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residue in the certain position were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. Digestion with uracil DNA glycosylase was utilized to generate an AP site which was then hydrolyzed by APE1, and the resulting gap was processed by X-family DNA polymerases ß (Polß) and λ (Polλ), or Y-family polymerase ι (Polι). By varying reaction conditions, namely, Mg2+/Mn2+ replacement/combination and ionic strength decrease, we found that under certain conditions both Polß and Polι can catalyze lesion bypass across both cis- and trans-BP adducts in the presence of physiological dNTP concentrations. Polß and Polι catalyze gap filling trans-lesion synthesis in an error prone manner. By contrast, Polλ selectively introduced the correct dCTP opposite the modified dG in the case of cis-BP-dG adduct only, and did not bypass the stereoisomeric trans-adduct under any of the conditions examined. The results suggest that Polλ is a specialized polymerase that can process these kinds of lesions.

In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ.

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N(2)-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N(2)-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

Plastic antibody for DNA damage: fluorescent imaging of BPDE-dG adducts in genomic DNA.

Exogenous and endogenous genotoxic carcinogens and their in vivo metabolites may result in DNA damage and cause adverse health effects. It is very difficult to specifically detect trace DNA damage in the presence of a large excess of unmodified nucleosides. Here we report a molecularly imprinted polymer (MIP) nanocomposite, namely nanoMIP, which can be used as a "plastic antibody" for specific recognition of benzo[a]pyrene diol epoxide (BPDE)-DNA adduct. Enhanced binding affinity (880 nM) of nanoMIPs was achieved by using two tailor-made functional monomers. The property of binding kinetics was greatly improved in virtue of the well-defined nanostructure, which was fabricated by initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP). The BPDE-adducted DNA can be specifically captured by synthetic nanoMIP. By taking advantage of this antibody-mimicking behavior, we further developed a fluorescently imaged particle counting immunoassay (FIPCIA) method for ultrasensitive detection of BPDE-ssDNA adducts using a laser scanning confocal microscope (LSCM). The number of countable fluorescent dots is proportional to the content of BPDE-ssDNA adducts in the DNA sample. The proposed plastic antibody-based FIPCIA method can detect traces of BPDE-DNA adducts as low as 18 pM in human lung carcinoma A549 cells. This highly-sensitive detection of DNA lesions offers a promising alternative to immunogenic antibody-based immunoassays for genomics and DNA modification analysis.

The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site.

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.

Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa.

Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.

Inhibitory effects of chrysoeriol on DNA adduct formation with benzo[a]pyrene in MCF-7 breast cancer cells.

Cytochrome P450 (CYP) 1 families including CYP1A1, 1A2 and 1B1 are well known to be deeply involved in the initiation of several cancers, due to the fact that they activate environmental pro-carcinogens to form ultimate carcinogens. Benzo[a]pyrene (BaP) is one of the major classes of prototypical pro-carcinogen. It is activated by the CYP1 family to its ultimate carcinogenic forms, mainly BaP-7,8-diol-9,10-epoxide (BPDE), and it forms adducts with DNA. This has been recognized to be a major initiation pathway for cancer. Our previous study demonstrated that chrysoeriol, which is a dietary methoxyflavonoid, selectively inhibited CYP1B1 enzymatic activity and might protect the CYP1B1 related-diseases such as breast cancer. In the present study, we further examined the effects of chrysoeriol on the other initiation pathway of cancer relating to the CYP1 family with BaP in human breast cancer MCF-7 cells. The effects of chrysoeriol on the formation of BPDE-DNA adducts were analyzed specifically using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. When MCF-7 cells were incubated with 2 microM BaP for 24h, three types of BPDE-dG adducts, especially (+)-trans-BPDE-dG as the dominant adduct, were detected. Co-treatment of MCF-7 cells with 10 microM chrysoeriol and BaP remarkably reduced (+)-trans-BPDE-dG formation. Chrysoeriol (1-10 microM) dose-dependently inhibited both EROD activity and the gene expressions of CYP1A1, 1B1 and 1A2 stimulated by treatment with BaP. In addition, the same amounts of chrysoeriol significantly inhibited the binding of BaP to the aryl hydrocarbon receptor (AhR), which is the key factor concerning the induction of the CYP1 families. In conclusion, our results clearly indicate that chrysoeriol inhibited the formation of BPDE-DNA adducts via regulation of the AhR pathway stimulated by BaP. As a consequence chrysoeriol may be involved in the chemoprevention of environmental pro-carcinogens such as BaP.

Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N(2)-dG adducts by capillary electrophoresis immunoassay.

The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90 mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5'-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K(b): 3.57 x 10(8) M(-1)) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(-)-anti-BPDE-dG displays lowest affinity (K(b): 1.14 x10(7) M(-1)). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90 mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90 mers (K(b): 6.36+/-0.54 x 10(8)M(-1)) than trans-(-)-anti-BPDE-90 mers (K(b): 4.52+/-0.52 x 10(8) M(-1)). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K(b): 3.74 x 10(8) M(-1)), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA.

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

An ultra-performance liquid chromatography tandem mass spectrometry with multiple reaction monitoring method (UPLC-MRM/MS) is developed for fast and sensitive analysis of four genotoxic stereoisomers of anti-benzo[a]pyrene diol epoxide (BPDE)-N(2)dG adducts (trans-(+), trans-(-), cis-(+) and cis-(-)), which result from environmental exposure to ubiquitous pollutant benzo[a]pyrene (B[a]P). The developed method displays a low limit of detection of <0.7 fmol (S/N=3) for the four stereoisomers of anti-BPDE-N(2)dG, a dynamic range of 2 orders of magnitude (2.3-630 fmol, R(2)> or =0.997), and one separation of 2-4 min. The developed method enables us to use the stereoisomers of anti-BPDE-N(2)dG as a biomarker and to study the stereoselectivity of metabolic activation of B[a]P in human lung A549 cells. The UPLC-MRM/MS analysis of cellular DNA exposed to B[a]P show that activation of B[a]P in A549 cells predominantly induces trans-(+)-anti-BPDE-N(2)dG with cis-(+)-anti-BPDE-N(2)dG and one syn-BPDE-N(2)dG as two minorities, while trans-(-)-anti-BPDE-N(2)dG and cis-(-)-anti-BPDE-N(2)dG are absent. The observed preferential formation of trans-(+)-anti-BPDE-N(2)dG in B[a]P treated A549 cells may result from combined stereoselectivity of the metabolic activation of B[a]P and the reaction of anti-BPDE with dsDNA. The results also suggest that a number of key optical intermediates are formed during activation of B[a]P in A549 cells, including trans-(+)-B[a]P-7,8-dihydrodiol and trans-(-)-B[a]P-7,8-dihydrodiol and their corresponding downstream metabolites (+)-anti-BPDE and (+)-syn-BPDE.

Synthesis and characterization of DNA fluorescent probes containing a single site-specific stereoisomer of anti-benzo[a]pyrene diol epoxide-N2-dG.

We present a comprehensive study of synthesis and characterization of DNA probes containing covalently bound benzo[a]pyrene diol epoxide (BPDE) isomers at a defined site. Short oligonucleotides of 16mers containing a single trans-(+)- or trans-(-)-anti-BPDE-N(2)-guanine adducts (BPDE-16mer) were first synthesized and then ligated with a fluorescently labeled single-stranded oligonucleotide. The ligation products (double-stranded or single-stranded 90mers) contained a single BPDE adduct of defined stereochemistry and a fluorescent label. The BPDE adduct could be recognized by a specific antibody, and the fluorescent label was useful for highly sensitive laser-induced fluorescence detection. The incorporation of single BPDE in the 16mers was validated by liquid chromatography, UV spectroscopy, and tandem mass spectrometry analysis. The stereochemistry of the single BPDE adducts in the 16mers was further identified by enzyme digestion-coupled stereoselective chromatography analysis. The ligation of BPDE-16mer with normal oligonucleotides for the synthesis of tetramethylrhodamine (TMR)-BPDE-90mers was evaluated. It was found that the modification of the 16mer by anti-BPDE could significantly reduce the ligation yield of ds90mer and lead to the formation of gapped DNA. The incorporation of a single anti-BPDE adduct in ligated ds90mers was confirmed using an antibody specific to the anti-BPDE-dG and affinity capillary electrophoresis. The detection limits of the TMR-BPDE-90mers by capillary electrophoresis coupled with laser-induced fluorescence are below 4 x 10(-19) mol.

Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts.

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine ((Me)C, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O(6)-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of (Me)C were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N(2)-BPDE-dG adducts.

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.

Benzo[a]pyrene, a common environmental pollutant, can be metabolized into reactive anti-benzo[a]pyrene diol epoxide (anti-BPDE), which predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. To characterize the stereochemistry of these adduct isomers, preparation of single adducted deoxyguanosine (dG) is required for efficient enantiomeric analysis. Here, we demonstrate an improved method for preparation, identification, and analysis of four BPDE-adducted dGs, including (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-BPDE-N(2)-dG. These stereoisomerically adducted nucleosides were first synthesized by a direct reaction of (+/-)-anti-BPDE with dG, followed by optimized solid-phase extraction (SPE) and HPLC purification. The reaction of (+/-)-anti-BPDE and dG displayed a yield as high as 45%. The developed preparation method does not require any enzymatic digestion. Based on highly efficient separation achieved by optimization of stationary phase and mobile phase, LC-UV-MS/MS and LC-diode array detection (DAD)-fluorescence detection (FL) methods were established for characterization and analysis of the four stereoisomeric anti-BPDE-dGs. The established LC-DAD-FL method may provide characterization and analysis of four stereoisomeric anti-BPDE-dGs and two interfering anti-BPDE tetrols by taking advantage of their distinct fluorescence quenching.

Spectral differentiation and immunoaffinity capillary electrophoresis separation of enantiomeric benzo(a)pyrene diol epoxide-derived DNA adducts.

Antibody cross-reactivity makes separation and differentiation of enantiomeric analytes one of the most challenging problems in immunoanalytical research, particularly for the analysis of structurally related biological molecules [such as benzo( a)pyrene (BP) metabolites and BP-derived DNA adducts]. It has recently been shown that the interaction of enantiomers of BP tetrols (BPT) with a promiscuous anti-polycyclic aromatic hydrocarbon ( anti-PAH) monoclonal antibody (mAb) allowed for separation of all four enantiomeric isomers using immunoaffinity capillary electrophoresis [ Grubor, N. M. , Armstrong, D. W. , and Jankowiak, R. ( 2006) Electrophoresis 27, 1078 ] and unambiguous spectral resolution using fluorescence line narrowing spectroscopy (FLNS) [ Grubor, N. M. , Liu, Y. , Han, X. , Armstrong, D.W. , and Jankowiak, R. ( 2006) J. Am.Chem. Soc. 128, 6409 ]. Here, we expand the use of the above two methodologies to the group of biologically important molecules that are products of BP diol epoxide (BPDE)-induced DNA damage. Four diastereomeric anti-BPDE-derived deoxyguanosine (dG) adducts, that is, (+)- and (-)- anti-trans-BPDE- N (2)-dG and (+)- and (-)- anti-cis-BPDE- N (2)-dG, were electrophoretically separated and spectroscopically differentiated using 8E11 mAb raised against BP-DNA conjugates. In fluorescence line narrowing spectroscopy (FLNS) experiments, complexes of BPDE-dG adducts with mAb revealed differences in fluorescence origin band positions, bandwidths, and vibrational patterns for all four BPDE- N (2)-dG adducts. Narrow fluorescence origin bands observed for (-)- trans-BPDE-dG (70 cm (-1)) and (+)- trans-BPDE- N (2)-dG (80 cm (-1)) suggest spatial constraint within the mAb binding pocket. Broader origin bands observed for cis type adducts ( approximately 120 cm (-1)) in 8E11 mAb suggest different binding geometries and/or conformational changes, as also indicated by changes in vibrational frequencies observed for the (+)- anti-cis and (-)- anti-cis adducts complexed with mAb. FLNS revealed that binding conformations and interactions within the mAb binding pocket are different for each adduct, enabling unambiguous positive identification. The methodologies described in this manuscript could also be used for analysis of DNA adducts following enzymatic hydrolysis of BPDE-adducted DNA to free nucleosides.

An association between BPDE-like DNA adduct levels and P53 gene mutation in pterygium.

PURPOSE: A previous report of ours noted that not only p53 protein overexpression, but also p53 gene mutation. were indeed detected in pterygium. BaP 7,8-diol 9,10-epoxide (BPDE), an ultimate metabolite of BaP, attacks deoxyguanosine to form a BPDE-N2-dG adduct resulting in p53 mutations. The relationship between BPDE-like DNA adduct levels and abnormal p53 has not been clear in pterygium. Therefore, BPDE-like DNA adduct, p53 protein expression and p53 gene mutation were examined in this study to provide more molecular evidence to understand the cause of p53 gene mutation in pterygium. METHODS: In this study, immunohistochemical staining, using a monoclonal antibody (DO7) against p53 and a polyclonal antibody against BPDE-like DNA adducts, was performed on 73 pterygial specimens. DNA samples for p53 mutation analysis were extracted from epithelial cells and then subjected to DNA sequencing for the determination of mutations in exons 4, 5, 6, 7, and 8 of the p53 gene. RESULTS: BPDE-like DNA adducts were detected in 36.1% (26/73) pterygium samples. No correlation between adduct levels and p53 protein expression was found in these samples. Additionally, the p53 gene mutation and p53 mutation pattern also did not correlate with BPDE-like DNA adduct levels. CONCLUSIONS: Our data provides evidence that BPDE-like DNA adducts are indeed detected in pterygium samples, and they are only minor contributors to the abnormal p53 gene.

Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences.

G --> T transversion mutations in the p53 tumor suppressor gene are characteristic of smoking-related lung tumors, suggesting that these genetic changes may result from exposure to tobacco carcinogens. It has been previously demonstrated that the diol epoxide metabolites of bay region polycyclic aromatic hydrocarbons present in tobacco smoke, e.g., benzo[a]pyrene diol epoxide (BPDE), preferentially bind to the most frequently mutated guanine nucleotides within p53 codons 157, 158, 248, and 273 [Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Science 274, 430-432]. However, the methodology used in that work (ligation-mediated polymerase chain reaction in combination with the UvrABC endonuclease incision assay) cannot establish the chemical structures and stereochemical identities of BPDE-guanine lesions. In the present study, we employ a stable isotope-labeling HPLC-MS/MS approach [Tretyakova, N., Matter, B., Jones, R., and Shallop, A. (2002) Biochemistry 41, 9535-9544] to analyze the formation of diastereomeric N(2)-BPDE-dG lesions within double-stranded oligodeoxynucleotides representing p53 lung cancer mutational hotspots and their surrounding DNA sequences. (15)N-labeled dG was placed at defined positions within DNA duplexes containing 5-methylcytosine at all physiologically methylated sites, followed by (+/-)-anti-BPDE treatment and enzymatic hydrolysis of the adducted DNA to 2'-deoxynucleosides. Capillary HPLC-ESI(+)-MS/MS was used to establish the amounts of (-)-trans-N(2)-BPDE-dG, (+)-cis-N(2)-BPDE-dG, (-)-cis-N(2)-BPDE-dG, and (+)-trans-N(2)-BPDE-dG originating from the (15)N-labeled bases. We found that all four N(2)-BPDE-dG diastereomers were formed preferentially at the methylated CG dinucleotides, including the frequently mutated p53 codons 157, 158, 245, 248, and 273. The contributions of individual diastereomers to the total adducts number at a given site varied between 70.8 and 92.9% for (+)-trans-N(2)-BPDE-dG, 5.6 and 16.7% for (-)-trans-N(2)-BPDE-dG, 2.1 and 8.5% for (-)-cis-N(2)-BPDE-dG, and 0.5 and 8.3% for (+)-cis-N(2)-BPDE-dG. The relative yields of the minor N(2)-BPDE-dG stereoisomers were elevated at the sites of inefficient adduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequently adducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased the yields of N(2)-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactions between BPDE and 5-methylcytosine. The targeted formation of N(2)-BPDE-dG at (Me)CG dinucleotides within the p53 gene is consistent with the high prevalence of G --> T transversions at these sites in smoking-induced lung cancer.