N5-Substituted formamidopyrimidine adducts have been observed from the reaction of dGuo or DNA with aziridine containing electrophiles, including nitrogen mustards. However, the role of substituted Fapy-dGuo adducts in the biological response to nitrogen mustards and related species has not been extensively explored. We have developed chemistry for the site-specific synthesis of oligonucleotides containing an N5-nitrogen mustard Fapy-dGuo using the phosphoramidite approach. The lesion was found to be a good substrate for Escherichia coli endonuclease IV and formamidopyrimidine glycosylase.

Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA–protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and Nα-acetyllysine (AcLys) and determined the structure of the cross-link to be the N2-ethyl-1-carboxamide with the lysine side chain amino group (1). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement.

N6-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) has been identified as a stable DNA adduct that arises from the reaction of DNA with a variety of methylating agents. Since this lesion persists in DNA and may contribute to the overall mutagenesis from electrophilic methylating agents, the MeFapy-dGuo lesion was incorporated into oligonucleotides, and its replication bypass was examined in vitro with a panel of eukaryotic high fidelity (hPols α, β, and δ/PCNA) and translesion (hPols η, κ, ι, Rev1, ν, and yPol ζ) polymerases to address its miscoding potential. The MeFapy-dGuo was found to be a strong block to the high fidelity polymerases at either the insertion or the extension step. Efficient translesion synthesis was observed for hPols η and κ, and the combined activities of hRev1 and yPol ζ. The nucleotide sequences of the extension products were determined by mass spectrometry. The error-free extension product was the most abundant product observed for each polymerase. Misreplication products, which included misinsertion of Thy, Gua, and Ade opposite the MeFapy-dGuo lesion, as well as an interesting one-nucleotide deletion product, were observed when hPols η and κ were employed; these events accounted for 8–29% of the total extension products observed. The distribution and abundance of the misreplication products were dependent on the polymerases and local sequence context of the lesion. Collectively, these data suggest that although MeFapy-dGuo adducts represent a relatively minor proportion of the total alkylated lesions, their miscoding potentials could significantly contribute to genomic instability.

2,3-Epoxy-4-hydroxynonanal (EHN) is a potential product of lipid peroxidation that gives rise to genotoxic etheno adducts. We have synthesized all four stereoisomers of EHN and individually reacted them with 2′-deoxyguanosine. In addition to 1,N2-etheno-2′-deoxyguanosine, 12 stereoisomeric products were isolated and characterized by 1H NMR and circular dichroism spectroscopy. The stereochemical assignments were consistent with selective NOE spectra, vicinal coupling constants, and molecular mechanics calculations. Reversed-phase HPLC conditions were developed that could separate most of the adduct mixture.

2-Amino-3-methylimidazo[1,2-d]naphthalene (cIQ) is a carbocyclic analogue of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in which a naphthalene ring system replaces the quinoline unit of IQ. The activity of cIQ in Ames Salmonella typhimurium tester strain TA98 is known to be 4−5 orders of magnitude lower than IQ. cIQ undergoes efficient bioactivation with rat liver microsomes. The C8-dGuo adduct was formed when calf thymus DNA was treated with the N-hydroxy-cIQ metabolite and either acetic anhydride or extracts from cells that overexpress N-acetyl transferase (NAT). These studies indicate that bioactivation, the stability of the N-hydroxylamine ester, and the reactivity of the nitrenium ion with DNA of cIQ are similar to IQ and that none of these factors account for the differences in mutagenic potency of these analogues in Ames assays. Oligonucleotides were synthesized that contain the C8-dGuo adduct of cIQ in the frameshift-prone CG-dinucleotide repeat unit of the NarI recognition sequence. We have examined the in vitro translesion synthesis of this adduct and have found it to be a strong replication block to Escherichia coli DNA polymerase I, Klenow fragment exo− (Kf−), E. coli DNA polymerase II exo− (pol II−), and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Previous studies by Fuchs and co-workers identified E. coli pol II as the polymerase responsible for two-base deletions of the C8-dGuo adduct of N-acetyl-2-aminofluorene in the NarI sequence. Our observation that pol II is strongly inhibited by the C8-dGuo adduct of cIQ suggests that one of the other SOS inducible polymerases (E. coli pol IV or pol V) is required for its bypass, and this accounts for the greatly attenuated mutagenicity in the Ames assays as compared with IQ.

Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories, and the results were compared to that of the 1,N2-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in 5′-TXG-3′ local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5′-CXG-3′ local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N2-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5′-TXG-3′ sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pKa for this transition was lower than that observed for the 1,N2-ε-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pKa value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N2-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N2 positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS3 or MS4 scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 108 DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1−2% of the levels of the monomeric 1,N2-dG adducts in CT DNA treated with either enal.

Oligonucleotides containing a site-specific N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesion were synthesized, and their in vitro replication by Escherichia coli DNA polymerase I Klenow fragment (exo−) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) resulted in the misincorporation of Ade, Gua, and Thy opposite the MeFapy-dGuo lesion in addition to the correct insertion of Cyt. However, sequencing of the full-length extension products revealed that the initial insertion of Cyt opposite the lesion was extended most efficiently. Two sequences were examined, and the misincorporation was sequence-dependent. Improvements in the method for the mass spectrometric sequencing of the extension products were developed; a 5′-biotinylated primer strand was used that contained a dUrd near the template-primer junction. The extended primer was immobilized with streptavidin-coated beads, allowing it to be washed free of polymerase, the template strand, and other reagents. The extended primer was cleaved from the solid support with uridine DNA deglycosylase and piperidine treatment, and the extension products were sequenced by LC-ESI-MS-MS. The purification steps afforded by the biotinylated primer resulted in improved sensitivity for the MS analysis. Translesion synthesis of a template with a local 5′-T-(MeFapy-dGuo)-G-3′ sequence resulted in only error-free bypass and extension, whereas a template with a local 5′-T-(MeFapy-dGuo)-T-3′ sequence also resulted in an interesting deletion product and the misincorporation of Ade opposite the MeFapy-dGuo lesion.

The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses indicate that the DNA interstand cross-links contain a mixture of carbinolamine and Schiff base, with the carbinolamine forms of the linkages predominating in duplex DNA. The reduced derivatives of the enal-mediated N2-dG:N2-dG interstrand cross-links can be processed in mammalian cells by a mechanism not requiring homologous recombination. Mutations are rarely generated during processing of these cross-links. In contrast, the reduced acrolein-mediated N2-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol κ in human, are implicated in error-free bypass of model acrolein-mediated N2-dG secondary adducts, the interstrand cross-links, and the peptide conjugates.

Vinyl chloride induces hepatic angiosarcomas, which are otherwise rare malignancies. The biochemical basis involves the formation of the epoxide, which reacts with DNA to give ∼98% of the 7-(2-oxoethyl) adduct (4) of dGuo plus small amounts of the etheno derivatives of dGuo, dCyd, and dAdo. The carcinogenicity is generally ascribed to the etheno adducts, not 4, because 4 has been shown to disappear from cells rapidly and to have negligible mutagenicity, which argues against its biological importance, whereas etheno adducts are both persistent and mutagenic. It has also been shown that apurinic sites derived from 4 are unlikely to be crucial lesions. A confounding factor with regard to the etheno hypothesis is that etheno adducts arise in unexposed cells by reactions of various lipid peroxidation products. The present study explores the possibility that a major contributor to the carcinogenicity of vinyl chloride may be formamidopyrimidine (FAPy) 12, N-[2-amino-6-[(2-deoxy-β-d-erythro-pentofuranosyl)amino]-3,4-dihydro-4-oxo-5-pyrimidinyl]-N-(2-oxoethyl)-formamide, which can arise by ring opening of 4, although its formation has not been observed until the present study. N7 adduct 4 undergoes deglycosylation to give 7-(2-oxoethyl)-Gua (13) in acid and imidazolium ring-opening to 12 in base. At pH 7.4, both processes occur with the formation of 12 representing ∼10% of the product mixture. FAPy 12 spontaneously cyclizes to 22, which upon mild acid treatment yields the deglycosylation product 2-amino-3,4,7,8-tetrahydro-7-hydroxy-4-oxopteridine-5(6H)-carbaldehyde (14). The structure of 14 has been established by NMR and mass spectroscopy and by independent synthesis. Reaction of the epoxide of crotonaldehyde with dGuo failed to give either 13 or 14, indicating that both compounds are unique products of the reactions of dGuo with the epoxides of vinyl monomers. Although FAPy 12 was found to be unstable, carbinolamine 22 arising from cyclization of 12 may be an important contributor to the carcinogenicity of vinyl chloride.

A phosphoramidite reagent of N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-1,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesions was synthesized in four steps from 2′-deoxyguanosine. Fapy nucleosides can rearrange to the pyranose form when the 5′-hydroxyl group is unprotected. The phosphoramidite was incorporated into oligonucleotides using solid-phase synthesis by adjusting the deprotection time for removal of the 5′-dimethoxytrityl group of the MeFapy-dGuo nucleotide, thereby minimizing its rearrangement to the ribopyranose. The furanose and pyranose forms were differentiated by a series of two-dimensional NMR experiments.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine formed in all cooked meats. IQ has been found to be a potent inducer of frameshift mutations in bacteria and carcinogenic in laboratory animals. Upon metabolic activation, IQ forms covalent adducts at the C8- and N2-positions of deoxyguanosine with a relative ratio of up to ∼4:1. We have previously incorporated the major dGuo-C8-IQ adduct into oligonucleotides through the corresponding phosphoramidite reagent. We report here the sequence-specific synthesis of oligonucleotides containing the minor dGuo-N2-IQ adduct. Thermal melting analysis revealed that the dGuo-N2-IQ adduct significantly destabilizes duplex DNA.

Background levels of etheno adducts have been attributed to the reaction of DNA with 2,3-epoxyaldehydes, a proposed product of lipid peroxidation. We have examined the reaction of (2R,3S)-epoxyhexanal with dGuo to give 7-(1S-hydroxybutyl)-1,N2-etheno-dGuo. We observed that the stereochemistry of the side chain scrambled over time. This process provided insight into the mechanism for the formation of 1,N2-etheno-dGuo from 4,5-epoxy-2-decenal [Lee, S. H., et al.(2002) Chem. Res. Toxicol.15, 300–304]. The mechanistic proposal predicts that 2-octenal is a by-product of the reaction. The reaction of 4,5-epoxy-2-decenal was reinvestigated, and the 2-octenal adduct of dGuo was identified as a product of this reaction in support of the mechanistic proposal. Also observed are products that appear to be derived from 2,3-epoxyoctanal, which can be formed through Schiff base formation of 4,5-epoxy-2-decenal with the dGuo followed by hydration of the double bond and retro-aldol reaction.

2,3-Epoxy-4-hydroxynonanal (EHN) is a potential product of lipid peroxidation that gives rise to genotoxic etheno adducts. We have synthesized all four stereoisomers of EHN and individually reacted them with 2′-deoxyguanosine. In addition to 1,N2-etheno-2′-deoxyguanosine, 12 stereoisomeric products were isolated and characterized by 1H NMR and circular dichroism spectroscopy. The stereochemical assignments were consistent with selective NOE spectra, vicinal coupling constants, and molecular mechanics calculations. Reversed-phase HPLC conditions were developed that could separate most of the adduct mixture.