Depurinating acylfulvene-DNA adducts: characterizing cellular chemical reactions of a selective antitumor agent

Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF-DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed.     

Qualitative study of in vivo melphalan adduct formation in the rat by miniaturized column-switching liquid chromatography coupled with electrospray mass spectrometry

In a general study of DNA adduct formation with melphalan, rats were intravenously injected with a single high dose (10 mg kg(-1)). Adduct formation was studied at the nucleoside level in the target organs liver, bone marrow, kidney and blood with the use of 2D liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts of dGuo and dAdo were detected under selected reaction monitoring in liver and bone marrow 10 h after administration of melphalan. In the DNA hydrolysates from kidney and blood a Gua-melphalan adduct was found, although in very low abundance. These first results of the search for in vivo-formed melphalan adducts in the rat showed that our miniaturized LC/MS technique is useful for investigating this type of compound. More experiments will be performed in this area to gather more information about the pharmacokinetics and the quantity of adducts formed.     

DNA-protein cross-links between guanine and lysine depend on the mechanism of oxidation for formation of C5 vs C8 guanosine adducts

The reaction between N(alpha)-acetyllysine methyl ester (Lys) and 2'-deoxyguanosine (dGuo) was used to study structural aspects of DNA-protein cross-link (DPC) formation. The precise structure of DPCs depended on the nature of the oxidant and cross-linking reactions in which a series of different oxidation conditions generated a distribution of adducts, principally spirodiiminodihydantoins with lysine appended at the purine position of C5 (5-Lys-Sp), C8 (8-Lys-Sp), or both C5 and C8 (5,8-diLys-Sp). Singlet oxygen oxidation of dGuo produced 5-Lys-Sp exclusively when Rose Bengal or methylene blue was used to photochemically generate (1)O2 in the presence of Lys, whereas riboflavin or benzophenone-mediated photochemistry generated lysine radicals and led to C8 adduct formation, yielding 8-Lys-Sp and 5,8-diLys-Sp. Notably, the yield of dGuo modifications from riboflavin photooxidation increased dramatically in the presence of lysine. Oxidation of deoxyguanosine/lysine mixtures with Na2IrCl6 or sulfate radicals produced both 5-Lys-Sp and 8-Lys-Sp. The same adducts were formed in single and double-stranded oligodeoxynucleotides, and these could be analyzed after nuclease digestion. Adduct formation in duplex DNA was somewhat dependent on the accessibility of lysine to C5 vs C8 of the purine. No adduct formation was detected between lysine and the other nucleobases T, C, or A. Overall, the precise location of adduct formation at C5 vs C8 of guanine appears to be diagnostic of the oxidation pathway.     

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

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.     

DNA Interaction with 17α‐Ethinylestradiol Studied Using Electrochemical Biosensors and Biosensing in Solution

The synthetic estrogen 17α‐ethinylestradiol (EE2) is an active component of oral contraceptives. It is considered as an endocrine disrupting compound that, once incorporated into an organism, affects the hormonal balance of animals and humans. In this study we characterized the DNA‐EE2 interaction using an electrochemical biosensor and biosensing in solution phase with the double stranded DNA from salmon sperm and deoxyguanosine monophosphate (dGMP). Differential pulse voltammetry method has been applied based on voltammetric anodic responses of the deoxyguanine (dGuo) and deoxyadenine (dAdo) as well as EE2 in the medium of phosphate buffer solution pH 7.0. Binding of EE2 to the nucleobases leads to a decrease of their anodic signals. Association constant for DNA‐EE2 interaction has been estimated to be about 1.1 ? 10³ L mol^?1 and 1.4 ? 10³ L mol^?1 for dGuo and dAdo responses, respectively. The association is reversible as indicated by decrease of the EE2 response in pure buffer solution due to leaching of EE2 from the surface attached DNA. The DNA‐EE2 association has been confirmed also by UV‐vis spectrometric experiments.     

13C2]-Acetaldehyde promotes unequivocal formation of 1,N2-propano-2'-deoxyguanosine in human cells

Acetaldehyde is an environmentally widespread genotoxic aldehyde present in tobacco smoke, vehicle exhaust and several food products. Endogenously, acetaldehyde is produced by the metabolic oxidation of ethanol by hepatic NAD-dependent alcohol dehydrogenase and during threonine catabolism. The formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2'-deoxyguanosine in DNA to form primarily N(2)-ethylidene-2'-deoxyguanosine. The subsequent reaction of N(2)-ethylidenedGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo), an adduct also found as a product of the crotonaldehyde reaction with dGuo. However, adducts resulting from the reaction of more than one molecule of acetaldehyde in vivo are still controversial. In this study, the unequivocal formation of 1,N(2)-propanodGuo by acetaldehyde was assessed in human cells via treatment with [(13)C(2)]-acetaldehyde. Detection of labeled 1,N(2)-propanodGuo was performed by HPLC/MS/MS. Upon acetaldehyde exposure (703 μM), increased levels of both 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdGuo), which is produced from α,β-unsaturated aldehydes formed during the lipid peroxidation process, and 1,N(2)-propanodGuo were observed. The unequivocal formation of 1,N(2)-propanodGuo in cells exposed to this aldehyde can be used to elucidate the mechanisms associated with acetaldehyde exposure and cancer risk.     

Phenylglycidyl ether adducts of 2′-deoxycytidine and 2′-deoxyadenosine: Stability in solution and structure analysis by electrospray tandem mass spectrometry

The adducts of phenylglycidyl ether with 2′-deoxyadenosine (dAdo) and 2′-deoxycytidine (dCyd) exhibit structural modifications. The N-1 adduct of dAdo underwent rearrangement to the N-6 adduct; the N-3 adduct of dCyd was deaminated to the corresponding 2′-deoxyuridine adduct. These structural modifications were studied by using liquid chromatography-electrospray tandem mass spectrometry, and kinetic data for both reactions are presented. The low energy (+) collision-activated dissociation spectra of the dAdo adducts allow the two positional isomers N-1 versus N-6 to be distinguished. The structure of the latter is independently proven by an extended NMR study. For the dCyd and 2′-deoxyuridine adducts, information about the alkylation site is found in the (?) collision-activated dissociation spectra. These spectra show the presence of an unexpected N-4-alkylated dCyd in addition to the two epimeric N-3 adducts.     

Comparison of the isolation of adducts of 2'-deoxycytidine and 2'-deoxyguanosine with phenylglycidyl ether by high-performance liquid chromatography on a reversed-phase column and a polystyrene-divinylbenzene column

2'-Deoxycitidine (dCyd) and 2'-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2'-deoxynucleoside adducts. Separation methods were developed on analytical and semi-preparative scales using high-performance liquid chromatography with photodiode-array detection on a reversed-phase column and on a polystyrene-divinylbenzene column. The use of the latter column was prompted by decomposition of the preparatively isolated dGuo-PGE adducts on the reversed-phase column. The use of a polystyrene-divinylbenzene column solved this problem and also revealed the presence of one more peak in both the dCyd- and dGuo-PGE reaction mixtures. The adducts of dCyd and dGuo were isolated on preparative reversed-phase and polystyrene-divinylbenzene columns and characterized by UV, fast atom bombardment mass and 360 MHz 1H NMR spectrometry. The adducts of dCyd were the diastereomers of N-3-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine and N4-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine whereas those of dGuo were the two diastereomers of N-7-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine and a third peak which appeared to be mainly N2-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine.     

Unprecedented monofunctional metalation of adenine nucleobase in guanine- and thymine-containing dinucleotide sequences by a cytotoxic platinum-acridine hybrid agent

We have investigated the reactions of [PtCl(en)(ACRAMTU-S)](NO(3))(2) (2) (en = ethane-1,2-diamine; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, acridinium cation, 1), the prototype of a new class of cytotoxic DNA-targeted agents, with 2'-deoxyguanosine (dGuo) and random-sequence native DNA by in-line liquid chromatography/mass spectrometry (LC/MS) and NMR spectroscopy ((1)H, (195)Pt) to identify the covalent adducts formed by platinum. In the mononucleoside model system, two adducts are observed, [Pt(en)(ACRAMTU)(dGuo)](3+) (P1, major) and [Pt(en)(dGuo)(2)](2+) (P2, minor). The reaction, which proceeds significantly slower (half-life 11-12 h at 37 degrees C, pH 6.5) than analogous reactions with cisplatin and reactions of 2 with double-stranded DNA, results in the unexpected displacement of the sulfur-bound acridine ligand in approximately 15% of the adducts. This reactivity is not observed in double-stranded DNA, rendering 1 a typical nonleaving group in reactions with this potential biological target. In enzymatic digests of calf thymus DNA treated with 2, three adducts were identified: [Pt(en)(ACRAMTU)(dGuo)](3+) (A1, approximately 80%), [Pt(en)(ACRAMTU)[d(GpA)]](2+) (A2, approximately 12%), and [Pt(en)(ACRAMTU)[d(TpA)]](2+) (A3, approximately 8%). A1 and P1 proved to be identical species. In the dinucleotide adducts A2 and A3, complex 2 covalently modifies adenine at GA and TA base steps, which are high-affinity intercalation sites of the acridine derivative 1. A2 and A3, which may be formed in the minor groove of DNA, are the first examples of monofunctional adenine adducts of divalent platinum formed in double-stranded DNA. The analysis of the adduct profile indicates that the sequence specificity of 1 plays an important role in the molecular recognition between DNA and the corresponding conjugate, 2. Possible biological consequences of the unusual adduct profile are discussed.     

Separation of N2-ethyl-2'-deoxyguanosine-5'-monophosphate and four native deoxyribonucleoside monophosphates using capillary zone electrophoresis with polyethylene glycol as buffer additive

We investigated the separation of five deoxyribonucleoside monophosphates: 2'-deoxyguanosine-5'-monophosphate (dGMP), 2'-deoxyadenosine-5'-monophosphate (dAMP), 2'-deoxycytosine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dTMP) and a dGMP adduct possessing N2-ethyl-guanine, which has been noted in relation to mutagenesis of alcohol, using capillary zone electrophoresis (CZE). The concentration of polyethylene glycol (PEG) as a modifier and the pH of the running solutions can efficiently control the observed separation. Interaction of PEG with analytes was quantitatively evaluated. PEG worked effectively as a hydrophobic selector in these separations. The values of pKa of the acidic-NH-groups in the base moieties of dGMP, dTMP, and the dGMP adduct are close to that of boric acid used as buffer of the running solutions. The control of their charge was facilitated, enabling improved separations. A more sufficient and fast separation was achieved by both optimization of pH of the running solutions and PEG concentration compared with that obtained by pH control alone. On-line concentration using a stacking method followed by the PEG-assisted CZE was briefly studied.     

Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.     

Quantitative determination of melphalan DNA adducts using HPLC - inductively coupled mass spectrometry

For the quantification of Melphalan DNA adducts, an analytical approach based on the detection of phosphorus using liquid chromatography combined with inductively-coupled-plasma mass spectrometry (ICP-MS) was developed. In reaction mixtures of native 2'-deoxynucleotides-5'-monophosphates and Melphalan, which were separated using reversed phase chromatography, phosphate adducts were found as the most abundant modifications. Besides the phosphate adducts, several base alkylated adducts were observed. In calf thymus DNA incubated with Melphalan and enzymatically digested using Nuclease P1, the phosphate adducts as well as monoalkylated dinucleotides were found. The most abundant single Melphalan adduct observed in DNA was a ring-opened adenosine monophosphate. Some dinucleotide adducts and the adenosine adduct were identified using electrospray ionization mass spectrometry (ESI-MS).     

Fast repair activities of quercetin and rutin toward dGMP hydroxyl radical adducts

The repair activities and mechanisms of both quercetin and rutin towards the oxidizing deoxyguanosine monophosphate (dGMP) hydroxyl radical adduct were investigated with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP aqueous solution containing 0.1 mM quercetin, the transient absorption spectrum of the dGMP hydroxyl radical adduct decays with the formation of phenoxyl radical of quercetin within tens of microseconds. It indicates that there is a repair reaction between dGMP hydroxyl radical adduct and quercetin. The repair activity of rutin towards hydroxyl radical adducts of dGMP was also observed. The rate constants of the repair reactions were calculated to be 3.05×10⁸ and 1.31×10⁸ M⁻¹ s⁻¹ for quercetin and rutin, respectively. This result together with our previous studies demonstrated that non-enzymatic, fast repair is a universal repair mechanism of phenolic antioxidants.     

Isomeric equilibria in aqueous solution involving aromatic ring stacking in the sexternary complexes formed by the quaternary cis-(NH3)2Pt(2'-deoxyguanosine-N7)(dGMP-N7) complex and the binary Cu(2,2'-bipyridine)2+ or Cu(1,10-phenanthroline)2+ complexes (dGMP2- = 2'-deoxyguanosine 5'-monophosphate)

To the best of our knowledge, for the first time the stabilities of sexternary complexes are determined by potentiometric pH titrations in aqueous solution at 25 degrees C and I = 0.1 M (NaNO3). The sexternary complexes form by binding of the binary Cu(Arm)2+ complexes, where Arm = 2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), to the -PO3(2-) group present in the quaternary cis-(NH3)2Pt(dGuo)(dGMP) complex. It is shown by stability constant comparisons and spectrophotometric measurements (observation of charge-transfer bands for the Phen system) that the [cis-(NH3)2Pt(dGuo)(dGMP).Cu(Arm)]2+ complexes can fold in such a way that aromatic ring stacking between the aromatic rings of Bpy or Phen and a guanine residue (most probably the one of dGMP2-) becomes possible. The formation degree of the stacks reaches approximately 25 and 50% for the [cis-(NH3)2Pt-(dGuo)(dGMP).Cu(Bpy)]2+ and [cis-(NH3)2Pt(dGuo)(dGMP).Cu(Phen)]2+ species, respectively. By comparisons with Cu(Arm)(dGMP) complexes, it is shown that the cis-(NH3)2Pt2+ unit coordinated to N7 of the guanine residues in the sexternary complexes inhibits stacking but does not prevent it. This result is of general importance because it demonstrates that in aqueous solution purine residues of nucleotides or nucleic acids that carry a metal ion at N7 can still undergo stacking interactions with other suitable aromatic ring systems.     

Fluorinated alcohol mediated control over cis vs trans opening of benzo[a]pyrene-7,8-diol 9,10-epoxides at C-10 by the exocyclic amino groups of O6-allyl protected deoxyguanosine and of deoxyadenosine

A detailed study was carried out on the stereoselective control of cis- vs trans-opening of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene {B[a]P DE-1 (1)} and (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene {B[a]P DE-2 (2)} at C-10 by the exocyclic amino groups of protected purine nucleosides in the fluorinated alcohols trifluoroethanol (TFE), hexafluoropropan-2-ol (HFP), and perfluoro-tert-butanol (PFTB). Addition of the 2-amino group of O6-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (3) and of the 6-amino group of 3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (4) occurs at C-10 of the epoxides. The observed cis:trans ratio for the reaction of DE-1 (1) in the presence of 5 equiv of 3 over the range of 10-250 equiv of fluorinated alcohol varied from 53:47 to 87:13 for TFE, 60:40 to 92:8 for HFP, and 52:48 to 73:27 for PFTB. The corresponding ratios for DE-2 (2) varied from 22:78 to 72:28 for HFP under the same set of conditions. In contrast, the corresponding ratios for DE-2 (2) remained unchanged ( approximately 40:60) for TFE and for PFTB over the range of 25-250 molar equiv. Unlike the addition of the dGuo reactant 3, the corresponding addition of the dAdo reactant (4) to the DEs (1 or 2) in over 25 molar equiv of TFE occurred highly stereoselectively to afford only cis adducts for both DEs. A highly efficient HPLC separation of dGuo adduct diastereomers derived from DE-2 (2) was developed using acetone as a modifier in CH2Cl2 or in n-hexane. Through the use of varying molar ratios of the different fluorinated alcohols described above and the newly developed HPLC separation method, the four possible phosphoramidites (cis/trans, R/S) of the B[a]P DE-2 N2-dGuo adducts can be prepared in an efficient fashion on gram scale for use in oligonucleotide synthesis.     

DEB-FAPy-dG Adducts of 1,3-Butadiene: Synthesis,Structural Characterization,and Formation in 1,2,3,4-Diepoxybutane Treated DNA**

Metabolic activation of the human carcinogen 1,3-butadiene (BD) by cytochrome 450 monooxygenases gives rise to a genotoxic diepoxide, 1,2,3,4-diepoxybutane (DEB). This reactive electrophile alkylates guanine bases in DNA to produce N7-(2-hydroxy-3,4-epoxy-1-yl)-dG (N7-DE-dG) adducts. Because of the positive charge at the N7 position of the purine heterocycle, N7-DEB-dG adducts are inherently unstable and can undergo spontaneous depurination or base-catalyzed imidazole ring opening to give N⁶-[2-deoxy-D-erythro-pentofuranosyl]-2,6-diamino-3,4-dihydro-4-oxo-5-N-1-(oxiran-2-yl)propan-1-ol-formamidopyrimidine (DEB-FAPy-dG) adducts. Here we report the first synthesis and structural characterization of DEB-FAPy-dG adducts. Authentic standards of DEB-FAPy-dG and its ¹⁵N₃-labeled analogue were used for the development of a quantitative nanoLC-ESI⁺-HRMS/MS method, allowing for adduct detection in DEB-treated calf thymus DNA. DEB-FAPy-dG formation in DNA was dependent on DEB concentration and pH, with higher numbers observed under alkaline conditions.     

STUDIES ON THYMINE-DERIVED UV PHOTOPRODUCTS IN DNA—I. FORMATION AND BIOLOGICAL ROLE OF PYRIMIDINE ADDUCTS IN DNA

Abstract— Pyrimidine (Pyr) adducts constitute a significant fraction of the photoproducts formed in DNA exposed to far UV light. The primary and secondary DNA structure affects the rate of Pyr adduct formation; for example, it increases with decreasing (Ade + Thy)/(Gua + Cyt) of the DNA and with increasing dehydration, and it is greater in double-stranded than in single-stranded DNA. Pyrimidine adducts do not appear to be involved in inter-strand cross-links, and 313 nm-induced photolysis of Pyr adducts does not cause strand breakage. The action spectrum for Pyr adduct formation is qualitatively similar to that for Pyr < > Pyr formation; the calculated quantum yields for its formation is essentially wavelength independent over the range 254 nm to 280 nm, but decreased somewhat at shorter wavelengths (240 nm). The biological role of Pyr adducts is still not clear. The data suggest that either Pyr adducts and their photolysis products are not lethal, or that both are lethal but can be repaired under certain conditions.     

New insights in the formation of deoxynucleoside adducts with the heterocyclic aromatic amines PhIP and IQ by means of ion trap MS<Superscript>n</Superscript> and accurate mass measurement of fragment ions

The formation of adducts by reaction of active metabolites of two heterocyclic aromatic amines (NHOH-PhIP and NHOH-IQ) at nucleophilic sites of deoxynucleosides has been studied by LC-MS(n) analyses of the obtained reaction mixtures. Sequential MS(3) experiments were carried out on an ion trap mass spectrometer to gain extensive structural information on each adduct detected in the first MS step. Attribution of ions was supported by accurate mass measurements performed on an Orbitrap mass analyzer. Particular attention was given to ions diagnostic of the linking between the heterocyclic aromatic amine (HAA) and the deoxynucleoside. By this way, the structures of five adducts have been characterized in this study, among which two are new compounds: dG-N7-IQ and dA-N(6)-IQ. No depurinating adduct was found in the reactions investigated therein. As expected, the C8 and N(2) atoms of dG were found as the most reactive sites of deoxynucleosides, resulting in the formation of two different adducts with IQ and one adduct with PhIP. An unusual non-depurinating dG-N7-IQ adduct has been characterized and a mechanism is proposed for its formation on the basis of the reactivity of arylamines. A dA-N(6)-IQ adduct has been identified for the first time in this work, showing that HAAs can generate DNA adducts with bases other than dG.     

Dissociation energies of deoxyribose nucleotide dimer anions measured using blackbody infrared radiative dissociation

The dissociation kinetics of deprotonated deoxyribose nucleotide dimers were measured using blackbody infrared radiative dissociation. Experiments were performed with noncovalently bound dimers of phosphate, adenosine (dAMP), cytosine (dCMP), guanosine (dGMP), thymidine (dTMP), and the mixed dimers dAMP.dTMP and dGMP.dCMP. The nucleotide dimers fragment through two parallel pathways, resulting in formation of the individual nucleotide or nucleotide + HPO3 ion. Master equation modeling of this kinetic data was used to determine threshold dissociation energies. The dissociation energy of (dGMP.dCMP-H)- is much higher than that for the other nucleotide dimers. This indicates that there is a strong interaction between the nucleobases in this dimer, consistent with the existence of Watson-Crick hydrogen bonding between the base pairs. Molecular mechanics simulations indicate that Watson-Crick hydrogen bonding occurs in the lowest energy structures of (dGMP.dCMP-H)-, but not in (dAMP.dTMP-H)-. The trend in gas phase dissociation energies is similar to the trend in binding energies measured in nonaqueous solutions within experimental error. Finally, the acidity ordering of the nucleotides is determined to be dTMP < dGMP < dCMP < dAMP, where dAMP has the highest acidity (largest delta Gacid).     

Separation and identification of DMPO adducts of oxygen-centered radicals formed from organic hydroperoxides by HPLC-ESR,ESI-MS and MS/MS

Many electron spin resonance (ESR) spectra of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) radical adducts from the reaction of organic hydroperoxides with heme proteins or Fe(2+) were assigned to the adducts of DMPO with peroxyl, alkoxyl, and alkyl radicals. In particular, the controversial assignment of DMPO/peroxyl radical adducts was based on the close similarity of their ESR spectra to that of the DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the peroxyl adducts from the DMPO/superoxide adduct. Although recent reports assigned the spectra suggested to be DMPO/peroxyl radical adducts to the DMPO/methoxyl adduct based on independent synthesis of the adduct and/or (17)O-labeling, (17)O-labeling is extremely expensive, and both of these assignments were still based on hyperfine coupling constants, which have not been confirmed by independent techniques. In this study, we have used online high performance liquid chromatography (HPLC or LC)/ESR, electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) to separate and directly characterize DMPO oxygen-centered radical adducts formed from the reaction of Fe(2+) with t-butyl or cumene hydroperoxide. In each reaction system, two DMPO oxygen-centered radical adducts were separated and detected by online LC/ESR. The first DMPO radical adduct from both systems showed identical chromatographic retention times (t(R) = 9.6 min) and hyperfine coupling constants (a(N) = 14.51 G, a(H)(beta) = 10.71 G, and a(H)(gamma) = 1.32 G). The ESI-MS and MS/MS spectra demonstrated that this radical was the DMPO/methoxyl radical adduct, not the peroxyl radical adduct as was thought at one time, although its ESR spectrum is nearly identical to that of the DMPO/superoxide radical adduct. Similarly, based on their MS/MS spectra, we verified that the second adducts (a(N) = 14.86 G and a(H)(beta) = 16.06 G in the reaction system containing t-butyl hydroperoxide and a(N) = 14.60 G and a(H)(beta) = 15.61 G in the reaction mixture containing cumene hydroperoxide), previously assigned as DMPO adducts of t-butyloxyl and cumyloxyl radical, were indeed from trapping t-butyloxyl and cumyloxyl radicals, respectively.