Guanine Adducts Research Articles

Unraveling the Aflatoxin−FAPY Conundrum: Structural Basis for Differential Replicative Processing of Isomeric Forms of the Formamidopyrimidine-Type DNA Adduct of Aflatoxin B1

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.

In vitro nonenzymatic glycation of DNA nucleobases: an evaluation of advanced glycation end products under alkaline pH

The advanced glycation end products (AGEs) of DNA nucleobases have received little attention, perhaps due to the fact that adenine, guanine, cytosine and thymine do not dissolve under mild pH conditions. To maintain nucleobases in solution, alkaline pH conditions are typically required. The objectives of this investigation were twofold: to study the susceptibility of DNA nucleobases to nonenzymatic attack by different sugars, and to evaluate the factors that influence the formation of nucleobase AGEs at pH 12, i.e., in an alkaline environment that promotes the aldo-keto isomerization and epimerization of sugars. Varying concentrations of adenine, guanine, thymine and cytosine were incubated over time with constant concentrations of D-glucose, D-galactose or D/L-glyceraldehyde under different conditions of temperature and ionic strength. Incubation of the nucleobases with the sugars resulted in a heterogeneous assembly of AGEs whose formation was monitored by UV/fluorescence spectroscopy. Capillary electrophoresis and HPLC were used to resolve the AGEs of the DNA adducts and provided a powerful tool for following the extent of glycation in each of the DNA nucleobases. Mass spectrometry studies of DNA adducts of guanine established that glycation at pH 12 proceeded through an Amadori intermediate.

Conformations of adducts formed between the genotoxic benzo[ a]pyrene-7,8-dione and nucleosides studied by density functional theory

Benzo[ a]pyrene (BP) is a widely distributed environmental pollutant that is metabolized by mammalian cells to a variety of genotoxic and carcinogenic intermediates that form covalent adducts with cellular DNA. One such pathway involves the metabolic activation of BP by members of the aldo-keto-reductase (AKR) family of enzymes to the highly reactive ortho-quinone, benzo[ a]pyrene-7,8-dione (BPQ). This compound has been reported to react with the 2′-deoxynucleosides, dA and dG, under physiological conditions. Four BPQ–dG adducts and two –dA adducts were identified by mass spectrometry and NMR methods [Balu et al. (2004) Chem. Res. Toxicol. 17, 827–838]. However, the detailed conformations and absolute configurations around the linkage site have not been resolved. In order to determine the full conformations of these purine adducts, we carried out quantum mechanical geometry optimization using density functional theory. In the case of the BPQ–guanine adducts, six possible structures, each of which consists of two isomers, were identified. However, in the case of the adenine adducts, only four isomers were identified. The results suggest that stereoisomeric adduct pairs are expected to adopt opposite orientations with respect to the 5′→3′ direction of the modified DNA strands. The stereochemistry-dependent variations in adduct orientation may produce different biological effects, as has been observed in the case of DNA adducts derived from other metabolites of polycyclic aromatic hydrocarbons.

Base-displaced intercalated structure of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in the recognition sequence of the NarI restriction enzyme, a hotspot for -2 bp deletions.

The solution structure of the oligodeoxynucleotide 5'-d(CTCGGCXCCATC)-3'.5'-d(GATGGCGCCGAG)-3' containing the heterocyclic amine 8-[(3-methyl-3H-imidazo[4,5-f]quinolin-2-yl)amino]-2'-deoxyguanosine adduct (IQ) at the third guanine in the NarI restriction sequence, a hot spot for -2 bp frameshifts, is reported. Molecular dynamics calculations restrained by distances derived from 24 (1)H NOEs between IQ and DNA, and torsion angles derived from (3)J couplings, yielded ensembles of structures in which the adducted guanine was displaced into the major groove with its glycosyl torsion angle in the syn conformation. One proton of its exocyclic amine was approximately 2.8 A from an oxygen of the 5' phosphodiester linkage, suggesting formation of a hydrogen bond. The carcinogen-guanine linkage was defined by torsion angles alpha' [N9-C8-N(IQ)-C2(IQ)] of 159 +/- 7 degrees and beta' [C8-N(IQ)-C2(IQ)-N3(IQ)] of -23 +/- 8 degrees . The complementary cytosine was also displaced into the major groove. This allowed IQ to intercalate between the flanking C.G base pairs. The disruption of Watson-Crick hydrogen bonding was corroborated by chemical-shift perturbations for base aromatic protons in the complementary strand opposite to the modified guanine. Chemical-shift perturbations were also observed for (31)P resonances corresponding to phosphodiester linkages flanking the adduct. The results confirmed that IQ adopted a base-displaced intercalated conformation in this sequence context but did not corroborate the formation of a hydrogen bond between the IQ quinoline nitrogen and the complementary dC [Elmquist, C. E.; Stover, J. S.; Wang, Z.; Rizzo, C. J. J. Am. Chem. Soc. 2004, 126, 11189-11201].

Carbocations from Oxidized Metabolites of Benzo[a]anthracene: A Computational Study of Their Methylated and Fluorinated Derivatives and Guanine Adducts

Structure-reactivity relationships and substituent effects on carbocation stability in benzo[a]anthracene (BA) derivatives have been studied computationally at the B3LYP/6-31G and MP2/6-31G levels. Bay-region carbocations are formed by O-protonation of the 1,2-epoxides in barrierless processes. This process is energetically more favored as compared to carbocation generation via zwitterion formation/O-protonation, via single electron oxidation to generate a radical cation, or via benzylic hydroxylation. Relative carbocation stabilities were determined in the gas phase and in water as solvent (PCM method). Charge delocalization mode in the BA carbocation framework was deduced from NPA-derived changes in charges, and substitution by methyl or fluorine was studied at different positions selected on basis of the carbocation charge density. A bay-region methyl group produces structural distortion with consequent deviation from planarity of the aromatic system, which destabilizes the epoxide, favoring ring opening. Whereas fluorine substitution at sites bearing significant positive charge leads to carbocation stabilization by fluorine p-pi back-bonding, a fluorine atom at a ring position which presented negative charge density leads to inductive destabilization. Methylated derivatives are less sensitive to substituent effects as compared to the fluorinated analogues. Although the solvent decreases the exothermicity of the epoxide ring-opening reactions due to greater stabilization of the reactants, it provokes no changes in relative reactivities. Relative energies in the resulting bay-region carbocations are examined taking into account the available biological activity data on these compounds. In selected cases, quenching of bay-region carbocations was investigated by analyzing relative energies (in the gas phase and in water) and geometries of their guanine adducts formed via covalent bond formation with the exocyclic amino group and with the N-7.

Chemical Reactivity as a Tool to Study Carcinogenicity: Reaction between Chloroethylene Oxide and Guanine

Chloroethylene oxide, an ultimate carcinogen of vinyl chloride, reacts with DNA giving rise to 7-(2-oxyethyl)guanine adduct in a nearly quantitative yield. This reaction represents an initial step of carcinogenesis associated with vinyl chloride. From experimental data for this reaction we calculated the second-order rate constant of 0.049 s(-1) M(-1), which corresponds to the activation free energy of 19.5 kcal/mol. We also performed a series of medium high ab initio and density functional theory simulations. Effects of hydration were considered in the framework of the Langevine dipoles solvation model and the solvent reaction field method of Tomasi and co-workers. In silico calculated activation free energies are in a good agreement with the experimental value. This fact presents strong evidence in favor of the validity of the proposed reaction mechanism and points to the applicability of quantum-chemical methods to studies of other reactions associated with carcinogenesis. Insignificant stereoselectivity of the studied reaction was also predicted.

Inhibition of Werner Syndrome Helicase Activity by Benzo[a]pyrene Diol Epoxide Adducts Can Be Overcome by Replication Protein A

RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N(6) of deoxyadenosine (dA) or N(2) of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.

The Carcinogenic Significance of Reactive Intermediates Derived from 3-Acetoxy- and 5-Acetoxy-2-hydroxy-N-nitrosomorpholine

N-Nitroso-2-hydroxymorpholine (NHMOR), a relatively reactive metabolite of two potent carcinogens, N-nitrosodiethanolamine (NDELA) and N-nitrosomorpholine (NMOR), has been reported to not be carcinogenic. Two isomeric acetate esters of the alpha-hydroxynitrosamines expected to be produced from the cytochrome P450-mediated metabolism of NHMOR have been synthesized, and their hydrolytic decomposition products, hydrolysis rates, and deoxyguanosine (dG) reaction adducts have been determined. N-Nitroso-3-acetoxy-2-hydroxymorpholine was prepared in high yield from the reaction of N-nitroso-2,3-dehydromorpholine with dry peracetic acid in glacial acetic acid or by the reaction of its dimethyldioxirane-produced epoxide with glacial acetic acid. The hydrolysis of this alpha-acetoxynitrosamine gave acetaldehyde (10%), ethylene glycol (55%), glyoxal (95%), and acetic acid. The pH rate profile for the hydrolysis of this nitrosamine was abnormal in that it exhibited pronounced base-catalyzed hydrolysis beginning at pH 5. The mechanism of hydrolytic decomposition is proposed to involve neighboring group participation with the formation of a reactive epoxide intermediate. N-Nitroso-3-acetoxy-2-hydroxymorpholine reacted with dG to give these guanine adducts after acidic deglycosylation: 1,N2-glyoxal (65%), 7-(2-hydroxyethyl)guanine (9%), and O6-hydroxyethylguanine (3%). N-Nitroso-5-acetoxy-2-hydroxymorpholine was synthesized from 2-hydroxyethylvinylnitrosamine by its oxidative conversion to the corresponding aldehyde followed by reaction with dry peracetic acid in glacial acetic. The hydrolytic decomposition products of this nitrosamine were 2-acetoxyacetaldehyde (65%), a rearrangement product, glycol aldehyde (15%), a trace of glyoxal, and acetic acid. The pH rate profile for the hydrolysis of this acetate is similar to other alpha-acetoxynitrosamines in that it exhibits a pH-independent region which gives way to base-catalyzed ester hydrolysis beginning at pH 7. The lower pH ( approximately 7 < 9) onset of base catalysis is proposed to involve base-catalyzed opening of the hemiacetal and intramolecular acyl transfer to give an unstable alpha-hydroxynitrosamine. N-Nitroso-5-acetoxy-2-hydroxymorpholine was less reactive toward dG and gave the 1,N2-etheno-dG adduct (44%). The products from both of the isomeric alpha-acetoxy nitrosamines were judged to arise from diazonium ions produced from unstable alpha-hydroxynitrosamine intermediates. The high yield of the rearrangement product 2-acetoxyacetaldehyde could explain the low carcinogenic potential of NHMOR if it is mainly alpha-hydroxylated at the 5 carbon. Hydroxylation of NHMOR at carbon 3 is expected to yield a carcinogenic outcome.

Formation of Inter- and Intrastrand Imine Type DNA−DNA Cross-Links through Secondary Reactions of Aldehydic Adducts

Acrolein-derived DNA adducts of guanine have previously been detected in tissues of several species, including humans, and have been shown to be mutagenic in mammalian cells and potentially carcinogenic in higher organisms. In duplex DNA, the predominant acrolein-derived lesion, gamma-hydroxy-1,N2-propanodeoxyguanosine (gamma-HOPdG), exists in an equilibrated mixture of ring-opened and ring-closed forms. We have previously shown that the acyclic form can undergo secondary chemical reactions to form both interstrand DNA-DNA cross-links in a CpG sequence context and DNA-protein and DNA-peptide cross-links. Investigations using duplex DNAs containing gamma-HOPdG in a CpG sequence context reversibly created at least two cross-linked species: an imine, which is a minor species but could be readily reduced by NaBH4, and a major heat labile, nonreducible species that we formulate as a carbinolamine cross-link. The DNA came to equilibrium over several days with the carbinolamine species being significantly more abundant than the reducible imine. In an effort to find other types of DNA-DNA cross-links, we have developed a high throughput screen to evaluate the effects of DNA sequence and lesion structure on the formation of reducible interstrand and intrastrand cross-links. These data reveal that four different lesions, two involving deoxyguanosine and two involving deoxyadenosine, can react with nearby bases to form inter- and intrastrand DNA cross-links.

Base Selectivity and Effects of Sequence and DNA Secondary Structure on the Formation of Covalent Adducts Derived from the Equine Estrogen Metabolite 4-Hydroxyequilenin

Equilenin, an important component of a widely prescribed hormone replacement formulation for postmenopausal women, is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN). The oxidized o-quinone derivative of 4-OHEN is known to form cyclic covalent adducts with DNA [Bolton, J. (1998) Chem. Res. Toxicol. 11, 1113] in vitro and in vivo. The characteristics of 4-OHEN-DNA adduct formation were investigated with the oligonucleotides 5'-d(CCATCGCTACC) (I), its complementary strand 5'-d(GGTAGCGATGG) (II), one rich in C and the other in G, and the duplexes I.II. The identities of the modified bases were elucidated in terms of four stereoisomeric 4-OHEN-2'-deoxynucleoside standards described earlier [Shen et al. (2001) Chem. Res. Toxicol. 11, 94; Embrechts et al. J. Mass Spectrom. 36, 317). The reactions of 4-OHEN with C are favored overwhelmingly in both single-stranded I and II with no guanine adducts observed in either case, and only minor proportions of A adducts were detected in sequence II. However, guanine adducts are observed in oligonucleotides that contain only G and unreactive T residues. The relative levels of cyclic covalent adducts observed in single-stranded I, II, and duplex I.II are approximately 54:21:5, with only the end C groups in I modified in the I.II duplex. When 4-OHEN is reacted with calf thymus DNA, the reaction yield of cyclic adducts is more than approximately 10(3)-fold lower than in I. The cyclic 4-OHEN adducts lead to a pronounced thermal destabilization of duplexes I.II. Overall, cyclic adduct formation is markedly dependent on the sequence context and secondary structure of the DNA. The latter effect is attributed to the poor accessibilities of 4-OHEN to the reactive nucleotide Watson-Crick hydrogen-bonding interface in the interior of the duplex. In the single-stranded oligonucleotides I and II, the strikingly different selectivities of adduct formation are attributed to the formation of noncovalent preassociation complexes that favor reaction geometries with C, rather than with A or G. Finally, the levels of several typical biomarkers of oxidative DNA damage (including 8-oxo-2'-deoxyguanosine) are formed in I in aqueous solutions with a yield at least 10 times smaller than the yield of cyclic 4-OHEN-dC adducts under identical reaction conditions.

Molecular Dynamics and Free Energy Calculations of the B and Z Forms of C8-Arylguanine Modified Oligonucleotides

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8- tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6–31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5′-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.

Cisplatin interaction with cysteine and methionine, a theoretical DFT study

Interactions of hydrated cisplatin complexes with sulphur-containing amino acids cysteine and methionine were explored. The square-planar cis-[Pt(NH 3) 2(H 2O)X] + complexes (where X = Cl − and OH −) were chosen as mono- and dihydrated reactants. Calculations using density functional theory (DFT) techniques with B3LYP functional were performed. The isolated molecules and the supermolecular approaches were employed for the determination of the reaction energies. Bond dissociation energies (BDE) were estimated in the model of isolated molecules and supermolecules were used for the determination of the association energies between the two interacting parts. Formation of monodentate complexes by replacing the aqua-ligand with the S, N, and O-sites of both amino acids represents an exothermic process. The highest BDE was found in cysteine structures for the Pt–S coordination. The bonding energy is about 114 kcal/mol, which is comparable with cisplatin–guanine adducts. Analogous BDE for methionine complexes is smaller by about 40 kcal/mol. This correlates well with the known fact that cysteine forms irreversible cisplatin adducts while similar adducts in the methionine case are reversible. The formation of chelate structures is an exothermic reaction only for the hydroxo-form of reactants in the supermolecular approach where additional association interactions between the released water and chelate molecules sufficiently stabilize the final product.

Interstrand and Intrastrand DNA−DNA Cross-Linking by 1,2,3,4-Diepoxybutane: Role of Stereochemistry

1,2,3,4-Diepoxybutane (DEB) is a bifunctional electrophile capable of forming DNA-DNA and DNA-protein cross-links. DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then form 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) lesions. All three optical isomers of DEB are produced metabolically from 1,3-butadiene, but S,S-DEB is the most cytotoxic and genotoxic. In the present work, interstrand and intrastrand DNA-DNA cross-linking by individual DEB stereoisomers was investigated by PAGE, mass spectrometry, and stable isotope labeling. S,S-, R,R-, and meso-diepoxides were synthesized from l-dimethyl-2,3-O-isopropylidene-tartrate, d-dimethyl-2,3-O-isopropylidene-tartrate, and meso-erythritol, respectively. Total numbers of bis-N7G-BD lesions (intrastrand and interstrand) in calf thymus DNA treated separately with S,S-, R,R-, or meso-DEB (0.01-0.5 mM) were similar as determined by capillary HPLC-ESI(+)-MS/MS of DNA hydrolysates. However, denaturing PAGE has revealed that S,S-DEB produced the highest number of interchain cross-links in 5'-GGC-3'/3'-CCG-5' sequences. Intrastrand adduct formation by DEB was investigated by a novel methodology based on stable isotope labeling HPLC-ESI(+)-MS/MS. Meso DEB treatment of DNA duplexes containing 5'-[1,7, NH(2)-(15)N(3),2-(13)C-G]GC-3'/3'-CCG-5' and 5'-GGC-3'/3'-CC[(15)N(3),2-(13)C-G]-5' trinucleotides gave rise to comparable numbers of 1,2-intrastrand and 1,3-interstrand bis-N7G-BD cross-links, while S,S DEB produced few intrastrand lesions. R,R-DEB treated DNA contained mostly 1,3-interstrand bis-N7G-BD, along with smaller amounts of 1,2-interstrand and 1,2-intrastrand adducts. The effects of DEB stereochemistry on its ability to form DNA-DNA cross-links may be rationalized by the spatial relationships between the epoxy alcohol side chains in stereoisomeric N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine adducts and their DNA environment. Different cross-linking specificities of DEB stereoisomers provide a likely structural basis for their distinct biological activities.

Kinetic Analysis of Steps in the Repair of Damaged DNA by Human O6-Alkylguanine-DNA Alkyltransferase

Rates of individual steps in the removal of alkyl groups from O6-methyl (Me) and -benzyl (Bz) guanine in oligonucleotides by human O6-alkylguanine DNA alkyltransferase (AGT) were estimated using rapid reaction kinetic methods. The overall reaction yields hyperbolic plots of rate versus AGT concentration for O6-MeG but linear plots for the O6-BzG reaction, which is approximately 100-fold faster. The binding of AGT and DNA (double-stranded 30-mer/36-mer complex) appears to be diffusion-limited. The rate of dissociation of the complex is approximately 25-fold slower (approximately 1 s(-1)) for DNA containing O6-MeG or O6-BzG than unmodified DNA. The fluorescent dC-analog 6-methylpyrrolo[2,3-d]pyrimidine-2(3H) one deoxyribonucleoside (pyrrolo dC), which pairs with G, was positioned opposite G, O6-MeG, or O6-BzG and used as a probe of the rate of base flipping. A rapid increase of fluorescence (k approximately 200 s(-1)) was observed with O6-MeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Only weak and slower fluorescence changes were observed with G:pyrrolo dC or T:2-aminopurine pairs. These rate estimates were used in a kinetic model in which AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is rate-limiting in the case of O6-MeG but not O6-BzG, and releases the dealkylated DNA. The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions.

Recognition and incision of gamma-radiation-induced cross-linked guanine-thymine tandem lesion G[8,5-Me]T by UvrABC nuclease.

Nucleotide excision repair (NER) plays an important role in maintaining the integrity of DNA by removing various types of bulky or distorting DNA adducts in both prokaryotic and eukaryotic cells. In Escherichia coli, the excision repair proteins UvrA, UvrB, and UvrC recognize and incise the bulky DNA damages induced by UV light and chemical carcinogens. In this process, when a putative lesion in DNA is identified initially by UvrA, a subsequent strand opening is carried out by UvrB that not only ensures that the distortion is indeed due to a damaged nucleotide but also recognizes the chemical structure of the modified nucleotides with varying efficiencies. UvrB also recruits UvrC that catalyzes both the 3'- and the 5'-incisions. Herein, we examined the interaction of UvrABC with a DNA substrate containing a single G[8,5-Me]T cross-link and compared it with T[6,4]T (the 6-4 pyrimidine-pyrimidone photoproduct) and the C8 guanine adduct of N-acetyl-2-aminofluorene (AAF). The intrastrand vicinal cross-link G[8,5-Me]T containing a covalent bond between the C8 position of guanine and the 5-methyl carbon of the 3'-thymine is formed by X-radiation, while T[6,4]T is a vicinal cross-link induced by the UV light. We also selected the AAF adduct for comparison because it represents a highly distorting monoadduct containing a covalent linkage at the C8 position of guanine. The dissociation constants (K(d)) for UvrA protein binding to DNA substrates containing the G[8,5-Me]T, T[6,4]T, and AAF adducts, as determined by gel mobility shift assays, were 3.1 +/- 1.3, 2.8 +/- 0.9, and 8.2 +/- 1.9, respectively. Although UvrA had a considerably higher affinity for G[8,5-Me]T than for the AAF adduct, the G[8,5-Me]T intrastrand cross-link was incised by UvrABC much less efficiently than the T[6,4]T intrastrand cross-link and the AAF adduct. Similar incision results also were obtained with the DNA substrates containing the adducts in a six-nucleotide bubble, indicating that the inefficient incision of G[8,5-Me]T cross-link by UvrABC was probably due to the lack of efficient recognition of the adduct by UvrB at the second step of DNA damage recognition in the E. coli NER. Indeed, as compared to T[6,4]T and AAF substrates, which clearly showed UvrB-DNA complex formation, very little UvrB complex was detectable with the G[8,5-Me]T substrate. Our result suggests that G[8,5-Me]T intrastrand cross-link is more resistant to excision repair in comparison with the T[6,4]T and AAF adducts and thus will likely persist longer in E. coli cells.

Acrylamide and glycidamide adducts of Guanine

Acrylamide, a chemical of industrial use, which may be formed in thermally processed food, is known to produce various undesirable effects in animals and human beings. Some of these effects may be attributed to the adduct formation of acrylamide and its metabolite glycidamide with Guanine. We have modeled the adducts, making use of semi-empirical QM calculations to compare their stabilities. Our results support experimental findings reported in the literature.

Reaction of 1,2,3,4-diepoxybutane with 2'-deoxyguanosine: initial products and their stabilities and decomposition patterns under physiological conditions.

1,2,3,4-Diepoxybutane (DEB), an in vivo metabolite of 1,3-butadiene (BD), is a carcinogen and a potent mutagen. Previously, DEB was shown to react with 2'-deoxyguanosine (dG) under physiological conditions to produce seven major nucleoside adducts resulting from alkylation at the N1- (P8 and P9), N7- (P5 and P5'), and both the N1- and the N2-positions of dG to form six-membered (P4-1 and P4-2) and seven-membered fused ring systems (P6), respectively [Zhang, X.-Y., and Elfarra, A. A. (2003) Chem. Res. Toxicol. 16, 1606. Zhang, X.-Y., and Elfarra, A. A. (2004) Chem. Res. Toxicol. 17, 521]. In the present study, the stabilities and decomposition products of the seven adducts under in vitro physiological conditions (phosphate buffer containing KCl, pH 7.4, 37 degrees C) were investigated. The results showed that P4-1, P4-2, and P6 were stable, whereas P5, P5', P8, and P9 were labile with half-lives of 2.6, 2.7, 16, and 16 h, respectively. P5 and P5' decomposed initially by the loss of the deoxyribose moiety to yield the corresponding guanine adduct P5D, which exhibited a half-life of 33 h and decomposed through opening of the remaining oxirane ring by dihydrogen phosphate ion, water, or chloride ion. Decomposition of P8 yielded P4-1, P6, and nucleoside products resulting from opening of the oxirane ring by dihydrogen phosphate ion, water, or chloride ion. Similarly, decomposition of P9 led to the formation of P4-2, P6, and nucleoside products resulting from opening of the oxirane ring by dihydrogen phosphate ion, water, or chloride ion. These results indicate that the initial products of the reaction of DEB with dG are P5, P5', P8, and P9, whereas P4-1, P4-2, and P6 are secondary products. The results may also facilitate development of useful biomarkers of exposure to DEB.

Identification and Quantification of Stable DNA Adducts Formed from Dibenzo[a,l]pyrene or Its Metabolites in Vitro and in Mouse Skin and Rat Mammary Gland

The stable adducts of dibenzo[a,l]pyrene (DB[a,l]P) formed by rat liver microsomes in vitro were previously quantified, whereas the depurinating adducts were both identified and quantified [Li, et al. (1995) Biochemistry 34, 8043]. In this article, we report the identification and quantification of the stable DNA adducts obtained from DB[a,l]P and DB[a,l]P-11,12-dihydrodiol activated by rat liver microsomes and from reaction of (+/-)-anti-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) and (+/-)-syn-DB[a,l]PDE with DNA in vitro. In addition, the stable DNA adducts were identified and quantified following treatment of mouse skin with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (+/-)-anti-DB[a,l]PDE, or (+/-)-syn-DB[a,l]PDE in vivo and treatment of rat mammary gland with DB[a,l]P in vivo. The DNA adducts were analyzed by the (32)P-postlabeling method, and the major adducts were identified by comparison with standards. The six stable adducts of DB[a,l]P formed by rat liver microsomes in vitro were either guanine or adenine adducts of anti-DB[a,l]PDE or syn-DB[a,l]PDE. About 43% of the detected stable adducts from microsomes were with guanine and 44% were with adenine. The pattern of adducts formed from DB[a,l]P-11,12-dihydrodiol with microsomes was very similar to that from DB[a,l]P. Reaction of (+/-)-anti-DB[a,l]PDE with DNA in vitro formed higher levels of stable adducts (55% from guanine and 39% from adenine) than (+/-)-syn-DB[a,l]PDE did (about 44% with guanine and 47% with adenine). In mouse skin treated with DB[a,l]P, 1% of the total adducts detected were stable adducts, comprised of 51% guanine adducts and 46% from adenine; with DB[a,l]P-11,12-dihydrodiol, 54% of the total were stable adducts, with a pattern of adducts similar to those formed from DB[a,l]P. Treatment of mouse skin with (+/-)-syn-DB[a,l]PDE formed 68% stable adducts, mostly at guanine. With (+/-)-anti-DB[a,l]PDE, mouse skin contained almost exclusively (97%) stable adducts: 61% guanine adducts and 33% adenine adducts. In rat mammary gland treated with DB[a,l]P, 2% of the total adducts were stable, with 42% guanine adducts and 55% adenine adducts. Approximately equal to or greater amounts of stable guanine adducts were formed in all systems, except for rat mammary gland. In contrast, the majority of depurinating adducts were adenine adducts. The carcinogenic potencies of these compounds in mouse skin, published earlier, do not qualitatively or quantitatively correlate with stable adducts, but rather with depurinating adducts.

Stable Isotope Labeling−Mass Spectrometry Analysis of Methyl- and Pyridyloxobutyl-Guanine Adducts of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone inp53-Derived DNA Sequences

The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.

DNA Adduct of the Mitomycin C Metabolite 2,7-Diaminomitosene Is a Nontoxic and Nonmutagenic DNA Lesion in Vitro and in Vivo

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.