Pathways of Arachidonic Acid Peroxyl Radical Reactions and Product Formation with Guanine Radicals

Peroxyl radicals were derived from the one-electron oxidation of polyunsaturated fatty acids by sulfate radicals that were generated by the photodissociation of peroxodisulfate anions in air-equilibrated aqueous solutions. Reactions of these peroxyl and neutral guanine radicals, also generated by oxidation with sulfate radicals, were investigated by laser kinetic spectroscopy, and the guanine oxidation products were identified by HPLC and mass spectrometry methods. Sulfate radicals rapidly oxidize arachidonic (ArAc), linoleic (LnAc), and palmitoleic (PmAc) acids with similar rate constants, (2−4) × 109 M−1 s−1. The C-centered radicals derived from the oxidation of ArAc and LnAc include nonconjugated Rn• (∼80%) and conjugated bis-allylic Rba• (∼20%) radicals. The latter were detectable in the absence of oxygen by their prominent, narrow absorption band at 280 nm. The Rn• radicals of ArAc (containing three bis-allylic sites) transform to the Rba• radicals via an intramolecular H-atom abstraction [rate constant (7.5 ± 0.7) × 104 s−1]. In contrast, the Rn• radicals of LnAc that contain only one bis-allylic site do not transform intramolecularly to the Rba• radicals. In the case of PmAc, which contains only one double bond, the Rba• radicals are not observed. The Rn• radicals of PmAc rapidly combine with oxygen with a rate constant of (3.8 ± 0.4) × 109 M−1 s−1. The Rba• radicals of ArAc are less reactive and react with oxygen with a rate constant of (2.2 ± 0.2) × 108 M−1 s−1. The ArAc peroxyl radicals formed spontaneously eliminate superoxide radical anions [rate constant = (3.4 ± 0.3) × 104 M−1 s−1]. The stable oxidative lesions derived from the 2′,3′,5′-tri-O-acetylguanosine or 2′,3′,5′-tri-O-acetyl-8-oxo-7,8-dihydroguanosine radicals and their subsequent reactions with ArAc peroxyl radicals were also investigated. The major products found were the 2,5-diamino-4H-imidazolone (Iz), dehydroguanidinohydantoin (Ghox), and diastereomeric spiroiminodihydantoin (Sp) nucleosides from 2′,3′,5′-tri-O-acetylguanosine and the Ghox and Sp nucleosides from 2′,3′,5′-tri-O-acetyl-8-oxo-7,8-dihydroguanosine. In air-saturated aqueous solutions, covalent alkylated guanine adducts were not detected

DNA Lesions Derived from the Site Selective Oxidation of Guanine by Carbonate Radical Anions

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order:  5‘-d(CCATCCTACC) [(5.7 ± 0.6) × 106 M-1 s-1] < 5‘-d(TATAACGTTATA), self-complementary duplex [(1.4 ± 0.2) × 107 M-1 s-1] < 5‘-d(CCATCGCTACC [(2.4 ± 0.3) × 107 M-1 s-1] 8 M-1 s-1], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of ∼2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40−60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed

Origins of Conformational Differences between <i>Cis</i> and <i>Trans</i> DNA Adducts Derived from Enantiomeric <i>anti</i>-Benzo[<i>a</i>]Pyrene Diol Epoxides

The two enantiomeric metabolites of the carcinogen precursor benzo[a]pyrene, (+)- and (−)-anti-BPDE [(7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the corresponding 7S,8R,9R,10S enantiomer, respectively], bind predominantly to the exocyclic amino groups of dG residues in double-stranded DNA by either cis or trans addition to yield four stereoisomerically distinct [BP]-N2-dG adducts. Both the 10S (+)-trans and 10R (−)-trans adducts assume minor groove conformations in normal, full duplexes, but with opposite 5‘ or 3‘ orientations, respectively, relative to the modified strand. In contrast, the 10R (+)-cis and 10S (−)-cis adducts assume oppositely oriented base-displaced intercalative conformations in normal duplexes, with the inserted pyrenyl residues pointing toward the major groove in the (+)-cis isomer and toward the minor groove in the (−)-cis isomer. A BPDE-modified nucleoside is a small system which can be studied by computational methods with a very thorough survey of the potential energy surface. To investigate conformational differences between cis and trans adducts, and to elucidate origins governing the opposite orientations of these (+)- and (−)-diol epoxide adducts, we have carried out extensive investigations of the (+)- and (−)-trans-anti- and (+)- and (−)-cis-anti-[BP]-N2-dG deoxynucleoside adduct pairs. We report results for the (+)- and (−)-cis-anti pair, and compare them with the (+)- and (−)-trans-anti adducts. We created 373 248 different conformers for each adduct, which uniformly sampled at 5° intervals the possible rotamers about three flexible torsion angles governing base (χ) and carcinogen (α‘ and β‘) orientations, and computed each of their energies. The potential energy surface of the molecule was then mapped from these results. While four potential energy wells or structural domains are found for the (+)-trans adduct and four for the (−)-trans adduct, only two of these four domains are favored for each of the two cis adducts. In both cis and trans adducts, the (+)/(−) pairs of each structural domain are nearly mirror images. The most favored of the domains in both cis and trans adducts is observed experimentally in the duplexes containing each of these [BP]-N2-dG lesions. The opposite orientations in both cis and trans adducts stem from steric crowding at the benzylic ring, engendered when a (+) stereoisomer is rotated into the analogous conformation of its (−) partner, and vice versa. Furthermore, the key role of the difference in absolute configuration between trans and cis adducts at the hydroxyls of C9 and C8 in governing conformational preferences and flexibility is delineated. Cis adducts are less conformationally flexible than trans adducts because they are inherently more sterically crowded, with C9-OH and C8-OH on the same side of the benzylic ring as guanine and sugar, while they are on the opposite side of the benzylic ring in the trans adducts. Consequently, the cis adducts inherently favor less the minor groove position adopted by trans adducts in DNA duplexes because the C9-OH and C8-OH are directed inward into the minor groove in the cis adducts. In the trans adducts, the C9-OH and C8-OH are directed outward, away from the interior of the minor groove. Observed differential processing of these four adducts by replication, repair, and transcription enzymes may well stem from their differing conformational preferences

Lifetimes and Reaction Pathways of Guanine Radical Cations and Neutral Guanine Radicals in an Oligonucleotide in Aqueous Solutions

The exposure of guanine in the oligonucleotide 5′-d­(TCGCT) to one-electron oxidants leads initially to the formation of the guanine radical cation G^•+, its deptotonation product G­(-H)^•, and, ultimately, various two- and four-electron oxidation products via pathways that depend on the oxidants and reaction conditions. We utilized single or successive multiple laser pulses (308 nm, 1 Hz rate) to generate the oxidants CO₃^•– and SO₄^•– (via the photolysis of S₂O₈^2– in aqueous solutions in the presence and absence of bicarbonate, respectively) at concentrations/pulse that were ∼20-fold lower than the concentration of 5′-d­(TCGCT). Time-resolved absorption spectroscopy measurements following single-pulse excitation show that the G^•+ radical (p<i>K</i>_a = 3.9) can be observed only at low pH and is hydrated within 3 ms at pH 2.5, thus forming the two-electron oxidation product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single pulse excitation, the principal reactive intermediate is G­(-H)^•, which, at best, reacts only slowly with H₂O and lives for ∼70 ms in the absence of oxidants/other radicals to form base sequence-dependent intrastrand cross-links via the nucleophilic addition of N3-thymidine to C8-guanine (5′-G*CT* and 5′-T*CG*). Alternatively, G­(-H)^• can be oxidized further by reaction with CO₃^•–, generating the two-electron oxidation products 8-oxoG (C8 addition) and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), appear only after a second (or more) laser pulse. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses

Methylation of 2′-Deoxyguanosine by a Free Radical Mechanism

The mechanistic aspects of the methylation of guanine in DNA initiated by methyl radicals that are derived from the metabolic oxidation of some chemical carcinogens remain poorly understood. In this work, we investigated the kinetics and the formation of methylated guanine products by two methods: (i) the combination of •CH3 radicals and guanine neutral radicals, G(−H)•, and (ii) the direct addition of •CH3 radicals to guanine bases. The simultaneous generation of •CH3 and dG(−H)• radicals was triggered by the competitive one-electron oxidation of dimethyl sulfoxide (DMSO) and 2′-deoxyguanosine (dG) by photochemically generated sulfate radicals in deoxygenated aqueous buffer solutions (pH 7.5). The photolysis of methylcob(III)alamin to form •CH3 radicals was used to investigate the direct addition of these radicals to guanine bases. The major end products of the radical combination reactions are the 8-methyl-dG and N2-methyl-dG products formed in a ratio of 1:0.7. In contrast, the methylation of dG by •CH3 radicals generates mostly the 8-methyl-dG adduct and only minor quantities of N2-methyl-dG (1:0.13 ratio). The methylation of the self-complementary 5′-d(AACGCGAATTCGCGTT) duplexes was achieved by the selective oxidation of the guanines with carbonate radical anions in the presence of DMSO as the precursor of •CH3 radicals. The methyl-G lesions formed were excised by the enzymatic digestion and identified by LC-MS/MS methods using uniformly 15N-labeled 8-methyl-dG and N2-methyl-dG adducts as internal standards. The ratios of 8-methyl-G/N2-methyl-G lesions derived from the combination of methyl radicals with G(−H)• radicals positioned in double-stranded DNA or that with the free nucleoside dG(−H)• radicals were found to be similar. Utilizing the photochemical method and dipropyl or dibutyl sulfoxides as sources of alkyl radicals, the corresponding 8-alkyl-dG and N2-alkyl-dG adducts were also generated in ratios similar to those obtained with DMSO

Principles Governing Conformations in Stereoisomeric Adducts of Bay Region Benzo[<i>a</i>]pyrene Diol Epoxides to Adenine in DNA: Steric and Hydrophobic Effects Are Dominant

The stereochemical properties of ligands that form covalent adducts with DNA have profound effects on their biochemical functions. Differing absolute configurations of substituents about chiral carbon atoms can lead to strikingly different conformations when such stereoisomeric compounds bind to DNA. The environmental chemical carcinogen, benzo[a]pyrene (BP), provides a remarkable example of such stereochemical effects. Metabolic activation of benzo[a]pyrene leads to a pair of enantiomers, (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and its (−)-(7S,8R,9R,10S) mirror-image, known as (+)- and (−)-anti-BPDE. Both (+)- and (−)-anti-BPDE react with the amino group of adenine in DNA via trans epoxide opening, yielding a pair of stereochemically distinct trans-anti-benzo[a]pyrenyl adducts, whose possible role in chemical carcinogenesis is of great interest. High-resolution NMR solution studies (Schurter et al. Biochemistry 1995, 34, 1364−75; Yeh et al. Biochemistry 1995, 34, 13570−81; Zegar et al. Biochemistry 1996, 35, 6212−24; Schwartz et al., Biochemistry 1997, 36, 11069−76) have revealed that in the 10S (+)- and 10R (−)-trans-anti adducts, the BP is classically intercalated, residing on the 3‘-side of the modified adenine in the (+)-trans-anti adduct and on the 5‘-side in the (−) stereoisomer. To elucidate the stereochemical principles underlying these conformational preferences, an extensive survey of the potential energy surface of each modified nucleoside was carried out, in which the energy of 373,248 structures for each adduct was computed using AMBER 5.0. Our results reveal near mirror image symmetries in the four pairs of low-energy structural domains of 10S (+)- and 10R (−)-trans-anti-[BP]−N6-dA adducts, which is rooted in the exact enantiomeric relationship of the BPDE precursors, and accounts for the opposite orientations observed in solution. Steric hindrance prevents an R isomer from assuming the orientation favored by the S isomer, and vice versa. The NMR solution structures of 10S (+)- and 10R (−)-trans-anti-[BP]−N6-dA adducts in DNA adopt conformations which are in the low-energy domains computed for the nucleoside adducts. In addition, we find that the preference for classical intercalation over a major groove position for the pyrenyl ring system in the [BP]−N6-dA adducts stems from the advantage of burying the hydrophobic pyrenyl moiety within the helix rather than having it exposed in the large major groove

Primer Length Dependence of Binding of DNA Polymerase I Klenow Fragment to Template−Primer Complexes Containing Site-Specific Bulky Lesions^†

The binding of the benzo[a]pyrene metabolite anti-BPDE (r7,t8-dihydroxy-t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene) to the N2 group of 2‘-deoxyguanosine residues (dG*) is known to adversely affect the Michaelis−Menten primer extension kinetics catalyzed by DNA Pol I and other polymerases. In this work, the impact of site-specific, anti-BPDE-modified DNA template strands on the formation of Pol I (Klenow fragment, KF)/template−primer complexes has been investigated. The 23-mer template strand 5‘-d(AAC G*C-1 T-2 ACC ATC CGA ATT CGC CC), I (dG* = (+)-trans- and (−)-trans-anti-BPDE-N2-dG), was annealed with primer strands 18, 19, or 20 bases long. Complex formation of these template−primer strands with KF- (exonuclease-free) at different enzyme concentrations was determined using polyacrylamide gel mobility shift assays in the absence of dNTPs. The lesion dG* causes an increase in the dissociation constants, Kd, of the monomeric, 1:1 KF-/DNA template−primer complexes by factors of 10−15 when the 3‘-end base of the primer strand is positioned either opposite dG*, or opposite dC-1 in I, and the shapes of the binding isotherms are sigmoidal. The sigmoidal shapes are attributed to the formation of dimeric 2:1 KF-/DNA template−primer complexes. In contrast, when the 3‘-end of the primer strand extends only to dT-2 in I, the Kd of 1:1 complexes is increased by factors of only 2−3, the shapes of the binding isotherms are hyperbolic and nonsigmoidal and are similar to those observed with the unmodified control, and monomeric KF-/DNA complexes are dominant. The impact of bulky lesions on polymerase/DNA complex formation in polymerase-catalyzed primer extension reactions needs to be taken into account in interpreting the site-specific Michaelis−Menten kinetics of these reactions

Fluorescence Characteristics of Site-Specific and Stereochemically Distinct Benzo[<i>a</i>]pyrene Diol Epoxide−DNA Adducts as Probes of Adduct Conformation

Spectroscopic fluorescence quenching techniques are described for distinguishing the conformational characteristics of adducts derived from the binding of the benzo[a]pyrene metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz[a]pyrene) to the exocyclic amino groups of guanine ([BP]-N 2-dG) and adenine ([BP]-N 6-dA) in double stranded oligonucleotides. These methods are calibrated by comparing the fluorescence quenching and UV absorbance characteristics of different, stereoisomeric anti-[BP]-N 2-dG adducts of known adduct conformations, previously established by high-resolution NMR techniques. It is shown that intercalative adduct conformations can be distinguished from solvent-exposed adduct conformations, e.g., adducts in which the pyrenyl residues are positioned in the minor groove. These low resolution fluorescence methods are at least 4 orders of magnitude more sensitive than the high-resolution NMR techniques; the fluorescence methods are useful for distinguishing adduct conformations when either small amounts of material are available or the NMR signals are of such poor quality that high-resolution structures cannot be determined. This methodology is illustrated using a variety of anti-BPDE-modified oligonucleotides of varying adduct conformations. It is shown that the 10S (+)-trans-anti-[BP]-N 6-dA adduct in an oligonucleotide duplex containing an N-ras protooncogene sequence, believed to be conformationally heterogeneous and disordered, is significantly more exposed to the solvent environment than the stereoisomeric, intercalated 10R adduct [Zegar et al. (1996) Biochemistry 35, 6212]. These differences suggest an explanation for the greater efficiencies of excision of the 10S adduct (relative to the 10R adduct) by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849]

Conformational Determinants of Structures in Stereoisomeric <i>Cis</i>-Opened <i>anti</i>-Benzo[<i>a</i>]pyrene Diol Epoxide Adducts to Adenine in DNA

As part of a comprehensive effort to understand the origins of the variety of structural motifs adopted by (+)- and (−)-cis- and trans-anti-[BP]-N2-dG and -N6-dA adducts, with the goal of contributing to the elucidation of the structure−function relationship, we present results of our comprehensive computational investigation of the C10R (+)-cis- and C10S (−)-cis-anti-[BP]-N6-dA adducts on the nucleoside level. We have surveyed the potential energy surface of these two adducts by varying systematically, at 5° intervals in combination, the three key torsion angle determinants of conformational flexibility (χ, α‘, and β‘) in each adduct, creating 373 248 structures, and evaluating each of their energies. This has permitted us to map the entire potential energy surface of each adduct and to delineate the low-energy regions. The energy maps possess a symmetric relationship in the (+)/(−) adduct pair. This symmetry in the maps stems from the mirror image configuration of the benzylic rings in the two adducts, which produces opposite orientations of the BP residues in the C10R and C10S adducts on the nucleoside level. These opposite orientations result from primary steric hindrance between the base and the BP moiety which ensues when a (+) stereoisomer is rotated to the conformation favored by the (−) stereoisomer, and vice versa. Moreover, this steric hindrance manifested on the nucleoside level governs the structure on the duplex DNA level, accounting for observed opposite orientations in high-resolution NMR studies of C10R/C10S adduct pairs

Stereochemical Origin of Opposite Orientations in DNA Adducts Derived from Enantiomeric <i>anti</i>-Benzo[a]pyrene Diol Epoxides with Different Tumorigenic Potentials^†

When covalently linked to DNA, enantiomeric pairs of mirror image aromatic diol epoxides with differing tumorigenic potencies adopt opposite orientations along the DNA helix. This phenomenon has been observed by high-resolution NMR solution studies in a number of systems. Preliminary modeling efforts [Geacintov et al. (1997) Chem. Res. Toxicol. 10, 111−146) had suggested that the origin of the opposite orientation effect may be manifested even at the level of the carcinogen-modified nucleoside due to primary steric hindrance effects between the aromatic moiety and the attached base and sugar. Such a small system can be computationally investigated extensively, since a very thorough survey of the potential energy surface is feasible. Consequently, in an effort to understand the underlying origins of the opposite orientations in (+)-trans and (−)-trans-anti adduct pairs, we have undertaken an extensive investigation of the paradigm 10S (+) and 10R (−)-trans-anti-[BP]-N2-dG mononucleoside adduct pair, derived from the binding of the (+)-7R,8S,9S,10R and (−)-7S,8R,9R,10S enantiomers of 7,8-dihydro-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP) to the exocyclic amino group of 2‘-deoxyguanosine. In the present work we created 373248 different conformers for each adduct, which uniformly sampled the possible rotamers about the three flexible torsion angles governing the orientation of the base (χ) and its covalently linked BP residue (α‘, β‘) at 5° intervals, and computed each of their energies with AMBER 4.0. The extensive results permitted us to map the potential energy surface of the molecule. Only four low-energy structural domains are found for the (+)-trans adduct and four for the (−)-trans adduct; the (+)/(−) pairs of each structural domain are mirror images, with the mirror image symmetry broken by the sugar and its attached C4‘−C5‘ group. The most favored of these four is observed experimentally in the duplexes containing the same (+) and (−)-trans-anti-[BP]-N2-dG adducts (Cosman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914−1918; de los Santos et al. (1992) Biochemistry 31, 5245−5252). The origin of the opposite orientations resides in steric hindrance effects resulting from the mirror image relationship of the BP benzylic rings in the adduct pair, such that rotation of one stereoisomer into the conformational domain preferred by the other causes crowding between the base and the BP benzylic ring. Limited conformational flexibility in the torsion angle β‘, the one closest to the bulky BP moiety at the linkage site to guanine, plays a key role in governing the orientations in each adduct. The opposite orientation phenomenon is likely to manifest itself when the adducts are processed by cellular enzymes involved in replication, repair, and transcription and thus play a role in the differing biological outcomes stemming from the (+) and (−)-trans-anti adducts