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Tandem lesions, which are defined by two or more contiguously damaged nucleotides, are a hallmark of ionizing radiation. Recently, tandem lesions containing 5-formyl-2'-deoxyuridine (5-fdU) flanked by a 5'-8-OxodGuo or Fapy•dG were discovered, and they are more mutagenic in human cells than the isolated lesions. In the current study, we examined replication of these tandem lesions in Escherichia coli . Bypass efficiency of both tandem lesions was reduced by 30-40% compared to the isolated lesions. Mutation frequencies (MFs) of isolated 8-OxodGuo and Fapy•dG were low, and no mutants were isolated from replication of a 5-fdU construct. The types of mutations from 8-OxodGuo were targeted G → T transversion, whereas Fapy•dG predominantly gave G → T and G deletion. 5'-8-OxodGuo-5-fdU also gave exclusively G → T mutation, which was 3-fold and 11-fold greater, without and with SOS induction, respectively, compared to that of an isolated 8-OxodGuo. In mutY/mutM cells, the MF of 8-OxodGuo and 5'-8-OxodGuo-5-fdU increased 13-fold and 7-fold, respectively. The MF of 5'-8-OxodGuo-5-fdU increased 2-fold and 3-fold in Pol II- and Pol IV-deficient cells, respectively, suggesting that these polymerases carry out largely error-free bypass. The MF of 5'- Fapy•dG-5-fdU was similar without (13 ± 1%) and with (16 ± 2%) SOS induction. Unlike the complex mutation spectrum reported earlier in human cells for 5'- Fapy•dG-5-fdU, with G → T as the major type of errors, in E. coli , the mutations were predominantly from deletion of 5-fdU. We postulate that removal of adenine-incorporated opposite 8-OxodGuo by Fpg and MutY repair proteins is partially impaired in the tandem 5'-8-OxodGuo-5-fdU, resulting in an increase in the G → T mutations, whereas a slippage mechanism may be operating in the 5'- Fapy•dG-5-fdU mutagenesis. This study showed that not only are these tandem lesions more mutagenic than the isolated lesions but they may also exhibit different types of mutations in different organisms.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo , but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPC MdG ), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPC MdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPC MdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPC MdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N 2 -methyl-dG ( N 2 -MedG) and N 2 - n -butyl-dG ( N 2 - n BudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N 2 -MedG and N 2 - n BudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N 2 -MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N 2 -MedG and N 2 - n BuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N 2 -alkyl-dG lesions in mammalian cells.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M 1 dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M 1 dG in genomic DNA is enzymatically oxidized to 6-oxo-M 1 dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M 1 dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M 1 dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M 1 dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M 1 dG blocks further replication by the enzyme.

Alkylation represents a main form of DNA damage. The N 2 position of guanine is frequently alkylated in DNA. The SOS-induced polymerases have been shown to be capable of bypassing various DNA damage products in Escherichia coli . Herein, we explored the influences of four N 2 -alkyl-dG lesions (alkyl = ethyl, n -butyl, isobutyl, or sec -butyl) on DNA replication in AB1157 E. coli cells and the corresponding strains with polymerases (Pol) II, IV, and V being individually or simultaneously knocked out. We found that N 2 -Et-dG is slightly less blocking to DNA replication than the N 2 -Bu-dG lesions, which display very similar replication bypass efficiencies. Additionally, Pol II and, to a lesser degree, Pol IV and Pol V are required for the efficient bypass of the N 2 -alkyl-dG adducts, and none of these lesions was mutagenic. Together, our results support that the efficient replication across small N 2 -alkyl-dG DNA adducts in E. coli depends mainly on Pol II.

Polybrominated diphenyl ethers (PBDEs) are emerging organic environmental pollutants, which were accused of various toxic effects. Here, we studied the role of a potential PBDEs quinone metabolite, PBDEQ, on cytotoxicity, oxidative DNA damage, and the alterations of signal cascade in HeLa cells. PBDEQ exposure leads to reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) loss, lactate dehydrogenase (LDH) release, increasing terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) positive foci, and the elevation of apoptosis rate. Furthermore, we showed PBDEQ exposure result in increased DNA migration, micronucleus frequency, and the promotion of 8-OHdG and phosphorylation of histone H2AX (γ-H2AX) levels. Mechanism study indicated that PBDEQ caused poly(ADP-ribose) polymerase 1 (PARP-1) activation and apoptosis-inducing factor (AIF) nuclear translocation. All together, these results confirmed the occurrence of parthanatos-like cell death upon PBDEQ exposure.

Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N 2 -position of guanine to produce N 2 -BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2'-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine and 2'-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2'-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson-Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N 2 -BP-dG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N 2 -BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.

With the discovery of translesion synthesis DNA polymerases, great strides have been made in the last two decades in understanding the mode of replication of various DNA lesions in prokaryotes and eukaryotes. A database search indicated that approximately 2000 articles on this topic have been published in this period. This includes research involving genetic and structural studies as well as in vitro experiments using purified DNA polymerases and accessory proteins. It is a daunting task to comprehend this exciting and rapidly emerging area of research. Even so, as the majority of DNA damage occurs at 2'-deoxyguanosine residues, this perspective attempts to summarize a subset of this field, focusing on the most relevant eukaryotic DNA polymerases responsible for their bypass.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O 6 -methyl-2'-deoxyguanosine (O 6 -Me-dG), O 6 -[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O 6 -POB-dG), O 2 -methylthymidine (O 2 -Me-dT), and O 2 -POB-dT. We evaluated the ability of human DNA polymerase ν to bypass this damage to evaluate the structural constraints on substrates for pol ν and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ν on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O 6 -Me-dG is a good substrate for pol ν, while O 6 -POB-dG and the O 2 -alkyl-dT adducts are poor substrates for pol ν. The reactivity with O 6 -Me-dG is high with a preference for dCTP > dGTP > dATP > dTTP. The catalytic activity of pol ν toward O 6 -Me-dG is high and can potentially be involved in its bypass in vivo. In contrast, pol ν is unlikely to bypass O 6 -POB-dG or the O 2 -alkyl-dTs in vivo.

Ultraviolet A not only plays a major part in photoaging and skin tanning but also induces genetic damage and mutation in the epidermal basal layer of human skin. The photoprotective effect of oxyresveratrol and kuwanon O, two phenolic compounds from the root extract of Morus australis, in human primary epidermal keratinocytes was investigated in this study. Both of them were nontoxic to cells at a concentration less than 10 and 0.5 μM, respectively. After pretreatment at the concentrations of 5 and 10 μM, oxyresveratrol increased cell viability, exhibited significant suppressions on UVA- or H2O2-induced cellular ROS. UVA-enhanced nitrotyrosine was also reduced by post-treatment with oxyresveratrol at theses concentrations. Kuwanon O presented similar inhibitions on cellular ROS and nitrotyrosine with lower concentrations (0.25 and 0.5 μM), but there is no significant protection on cell survival after UVA irradiation. Their photoprotective effects also involved the enhanced repair of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and cyclobutane pyrimidine dimers (CPDs) as mediated by the augment of p53 expression after UVA radiation.

Chloroethylnitrosoureas (CENUs) are bifunctional alkylating agents widely used for the clinical treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) within GC base pairs to form dG-dC cross-links. This lesion inhibits DNA double strand separation during replication and transcription and results in the apoptosis of cancer cells. However, O(6)-alkylguanine DNA alkyltransferase (AGT) repairs the DNA ICLs by removing the alkyl group at the O(6) position of either O(6)-(2-chloroethyl)deoxyguanosine (O(6)-ClEtdGuo) or N1,O(6)-ethanodeoxyguanosine (N1,O(6)-EtdGuo), which are intermediates in the formation of dG-dC cross-links. The action of AGT leads to drug resistance against CENUs. O(6)-Benzylguanine (O(6)-BG) was identified as an effective AGT inhibitor that enhances the antitumor effects of CENUs. In this study, the effect of O(6)-BG on the formation of dG-dC cross-links was investigated by treating human brain glioma SF767 cells with 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea (ACNU). The levels of dG-dC cross-link were determined using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results indicated that ACNU induced higher levels of dG-dC cross-link in SF767 cells pretreated with O(6)-BG compared to cells without O(6)-BG pretreatment. The highest dG-dC cross-linking levels were generally observed at 12 h for all drug concentration groups, a result which was consistent with cytotoxicity assay. These results provided direct evidence for the enhancement of dG-dC cross-linking levels caused by the inhibition of AGT by O(6)-BG. These data indicate that dG-dC cross-links may be developed as a biomarker for evaluating the activity of novel O(6)-BG analogues as AGT inhibitors for combination therapy with CENUs.

We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in vivo. In this study, we initially focused on DB[a,l]P-induced genotoxic effects in both oral and tongue tissues. Therefore, to fully assess the contribution of these DNA adducts in the initiation stage of carcinogenesis induced by DB[a,l]P, an LC-MS/MS method to simultaneously detect and quantify DB[a,l]PDE-dG and -dA adducts was developed. Mice were orally administered with DB[a,l]P (24 nmole, 3 times per week for 5 weeks) or its fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE, 12 nmole, single application); animals were sacrificed at 2, 7, 14, and 28 days after the last dose of carcinogen administration. Oral and tongue tissues were obtained and DNA were isolated followed by enzymatic hydrolysis. Following the development of an isotope dilution LC-MS/MS method, we successfully detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(2)-dG, as well as (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(6)-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (-)-anti-trans-DB[a,l]PDE-N(6)-dA were ≥2 folds higher than (-)-anti-cis-DB[a,l]PDE-N(6)-dA adduct and those of dG adducts in the oral tissues and tongue at all time points selected after the cessation of DB[a,l]P treatment. Levels of dG adducts were comparable in both tissues. Collectively, our results support that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE, and the levels and persistence of (-)-anti-trans-DB[a,l]PDE-N(6)-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPolη), iota (hPolι), kappa (hPolκ), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPolη, hRev1, hPolκ, and hPolι to bypass 50% of the 8-oxodG lesions encountered (t50(bypass)) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPolη bypassed 8-oxodG most efficiently, hPolη correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPolι bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPolκ was most accurate opposite 8-oxodG (92.3%), whereas hPolκ alone was the least accurate (18.5%). The t50(bypass) value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPolη alone, suggesting that hPolη outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.

For DNA-reactive chemicals, a low dose linear assessment of cancer risk is the science policy default. In the present study, we quantitated the endogenous and exogenous N7-methyl-G and O(6)-methyl-dG adducts in human lymphoblastoid cells exposed to low dose [D3]-methylnitrosourea. Endogenous amounts of both adducts remained nearly constant, while the exogenous adducts showed linear dose-responses. The data show that O(6)-methyl-dG adducts ≥1.8/10(8) dG correlated with published studies that demonstrated significant increases of mutations under these conditions. The combined results do not support linear extrapolations to zero when data are available for science-based regulations.

Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2'-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N(2)-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus Dpo4, the prototypic Y-family DNA polymerase, using templates that contained the adduct in either the 5'-CXG-3' or the 5'-TXG-3' sequence context. Although γ-OH-PdG partially blocked Dpo4-catalyzed DNA synthesis, full primer extension was observed, and the majority of bypass products were error-free. Conversion of the adduct into an irreversibly ring-opened derivative prior to reaction facilitated bypass and further improved the fidelity. Structures of ternary Dpo4·DNA·dNTP complexes were determined with primers that either were positioned immediately upstream of the lesion (preinsertion complexes) or had a 3'-terminal dC opposite the lesion (postinsertion complexes); the incoming nucleotides, either dGTP or dATP, were complementary to the template 5'-neighbor nucleotide. In both postinsertion complexes, the adduct existed as ring-opened species, and the resulting base-pair featured Watson-Crick hydrogen bonding. The incoming nucleotide paired with the 5'-neighbor template, while the primer 3'-hydroxyl was positioned to facilitate extension. In contrast, γ-OH-PdG was in the ring-closed form in both preinsertion complexes, and the overall structure did not favor catalysis. These data provide insights into γ-OH-PdG chemistry during replication bypass by the Dpo4 DNA polymerase and may explain why γ-OH-PdG-induced mutations due to primer-template misalignment are uncommon.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a known human carcinogen. It generates methyl and pyridyloxobutyl DNA adducts. The role of the methyl DNA adducts has been well-established in the tumorigenic properties of NNK. However, the role of the pyridyloxobutyl DNA adducts is unclear. Four pyridyloxobutyl DNA adducts have been characterized: 7-[4-3-(pyridyl)-4-oxobut-1-yl]guanine (7-pobG), O²-[4-3-(pyridyl)-4-oxobut-1-yl]-cytodine (O²-pobC), O²-[4-3-(pyridyl)-4-oxobut-1yl]thymidine (O²-pobdT), and O⁶-[4-3-(pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O⁶-pobdG). Mutagenic O⁶-pobdG is thought to contribute to the tumorigenic properties of the pyridyloxobutylation pathway. It is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT). To explore the role of O⁶-pobdG formation and repair in the tumorigenic properties of NNK, A/J mice were given single or multiple doses of the model pyridyloxobutylating agent 4-(acetoxymethyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) in the presence or absence of the AGT depletor, O⁶-benzylguanine. Levels of the four pyridyloxobutyl DNA adducts were measured in the lung at 8, 48, or 96 h following treatment and compared to the lung tumorigenic activity of these treatments. AGT depletion had only a modest effect on the levels of O⁶-pobdG and did not increase tumor formation. Three pyridyloxobutyl DNA adducts, 7-pobG, O²-pobdT, and O⁶-pobdG, persisted in lung DNA at significant levels for up to 96 h post-treatment, suggesting that all three adducts may contribute to the tumorigenic properties of NNK.

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5' nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.

Nanotoxicology test of gold nanoparticles (Au NPs) and gold-cobalt (Au-Co) nanoalloy is an important step in their safety evaluation for biomedical applications. The Au and Au-Co NPs were prepared by reducing the metal ions using sodium borohydride (NaBH(4)) in the presence of polyvinyl pyrrolidone (PVP) as a capping material. The average size and shape of the nanoparticles (NPs) were characterized using high resolution transmission electron microscopy (HRTEM). Cobalt presence in the nanoalloy was confirmed by energy dispersive X-ray spectroscopy (EDX) analysis, and the magnetic properties of these particles were determined using a vibrating sample magnetometer (VSM). The Gold and gold-cobalt NPs of average size 15 ± 1.5 nm were administered orally to mice with a dose of 80, 160, and 320 mg/kg per body weight (bw) using gavages. Samples were collected after 7 and 14 days of the treatment. The results indicated that the Au-Co NPs were able to induce significant alteration in the tumor-initiating genes associated with an increase of micronuclei (MNs) formation and generation of DNA adduct (8-hydroxy-2-deoxyguanosine, 8-OHdG) as well as a reduction in the glutathione peroxidase activity. This action of Au-Co NPs was observed using 160 and 320 mg/kg bw at both time intervals. However, Au NPs had much lower effects than Au-Co NPs on alteration in the tumor-initiating genes, frequency of MNs, and generation of 8-OHdG as well as glutathione peroxidase activity except with the highest dose of Au NPs. This study suggests that the potential to cause in vivo genetic and antioxidant enzyme alterations due to the treatment by Au-Co nanoalloy may be attributed to the increase in oxidative stress in mice.

DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N(2)-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N(2)-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3' termini, and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers, and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ~500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N(2)-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension., (© 2012 American Chemical Society)

trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N(2)-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N(2)-dGuo (1,N(2)-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N(2)-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N(2)-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, the (6S,8R,11S)-1,N(2)-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, ring opening of the adduct to the corresponding N(2)-dGuo aldehyde species occurred. This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair.

Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.

Experimental and epidemiological data associate the exposure of estrogens to cancer development in several tissues, particularly, the breast, endometrium, liver, and kidney. One plausible mechanism of estrogen-mediated carcinogenicity is DNA damage by redox cycling of estrogen catechols. Reports have shown that metabolism of estrogens results in 2- and 4-hydroxylation to catechol metabolites which can then redox cycle. We examined the capacity of the endogenous estrogen, 17β-estradiol, and two equine estrogens which formulate a significant proportion of hormone replacement drugs, equilenin and equilin, to induce oxidatively generated DNA damage. Microsome/Cu(II)-mediated activation of all three estrogens resulted in numerous oxidation DNA adducts, as detected by (32)P-postlabeling/TLC. Essentially the same DNA oxidation pattern was also found when catechol estrogens were incubated with DNA in the presence of Cu(II) suggesting that redox cycling of catechol estrogens mediates the formation of these DNA adducts. Since the oxidation patterns induced by estrogen catechols and other chemically diverse catechols were chromatographically identical to those generated by Fenton-type chemistry and these adducts were inhibited by known ROS modifiers (up to 96%), this oxidatively generated DNA damage is believed to be the product of the attack of free radicals on DNA, rather than direct addition of the estrogen quinones. These data support a mechanistic role by endogenous and synthetic estrogens to induce oxidative DNA damage in addition to specific DNA adducts.

Ochratoxin A (OTA) is a fungal toxin that is classified as a possible human carcinogen based on sufficient evidence for carcinogenicity in animal studies. The toxin is known to promote oxidative DNA damage through production of reactive oxygen species (ROS). The toxin also generates covalent DNA adducts, and it has been difficult to separate the biological effects caused by DNA adduction from that of ROS generation. In the current study, we have derived structure-activity relationships (SAR) for the role of the C5 substituent of OTA (C5-X = Cl) by first comparing the ability of OTA, OTBr (C5-X = Br), OTB (C5-X = H), and OTHQ (C5-X = OH) to photochemically react with GSH and 2'-deoxyguanosine (dG). OTA, OTBr, and OTHQ react covalently with GSH and dG following photoirradiation, while the nonchlorinated OTB does not react photochemically with GSH and dG. These findings correlate with their ability to generate covalent DNA adducts (direct genotoxicity) in human bronchial epithelial cells (WI26) and human kidney (HK2) cells, as evidenced by the (32)P-postlabeling technique. OTB lacks direct genotoxicity, while OTA, OTBr, and OTHQ act as direct genotoxins. In contrast, their cytotoxicity in opossum kidney epithelial cells (OK) and WI26 cells did not show a correlation with photoreactivity. In OK and WI26 cells, OTA, OTBr, and OTB are cytotoxic, while the hydroquinone OTHQ failed to exhibit cytotoxicity. Overall, our data show that the C5-Cl atom of OTA is critical for direct genotoxicity but plays a lesser role in OTA-mediated cytotoxicity. These SARs suggest different mechanisms of action (MOA) for OTA genotoxicity and cytotoxicity and are consistent with recent findings showing OTA mutagenicity to stem from direct genotoxicity, while cytotoxicity is derived from oxidative DNA damage.

Benzene is employed in large quantities in the chemical industry and is an ubiquitous contaminant in the environment. There is strong epidemiological evidence that benzene exposure induces hematopoietic malignancies, especially acute myeloid leukemia, in humans, but the chemical mechanisms remain obscure. E,E-Muconaldehyde is one of the products of metabolic oxidation of benzene. This paper explores the proposition that E,E-muconaldehyde is capable of forming Gua-Gua cross-links. If formed in DNA, the replication and repair of such cross-links might introduce structural defects that could be the origin of the carcinogenicity. We have investigated the reaction of E,E-muconaldehyde with dGuo and found that the reaction yields two pairs of interconverting diastereomers of a novel heptacyclic bis-adduct having a spiro ring system linking the two Gua residues. The structures of the four diastereomers have been established by NMR spectroscopy and their absolute configurations by comparison of CD spectra with those of model compounds having known configurations. The final two steps in the formation of the bis-nucleoside (5-ring → 6-ring → 7-ring) have significant reversibility, which is the basis for the observed epimerization. The 6-ring precursor was trapped from the equilibrating mixture by reduction with NaBH(4). The anti relationship of the two Gua residues in the heptacyclic bis-adduct precludes it from being formed in B DNA, but the 6-ring precursor could readily be accommodated as an interchain or intrachain cross-link. It should be possible to form similar cross-links of dCyt, dAdo, the ε-amino group of lysine, the imidazole NH of histidine, and N termini of peptides with the dGuo-muconaldehyde monoadduct.

O(6)-POB-dG (O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O(6)-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI(+)-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O(6)-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O(6)-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI(+)-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5'-AATAGTATCT[O(6)-POB-G]GAGCC-3') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).

Quantum dots (QDs) are a novel class of inorganic fluorophores which are gaining widespread recognition as a result of their exceptional photophysical properties and their applications as a biomarker and in molecular biomedical imaging. The aim of this study was to evaluate the in vivo genotoxicity in mice exposed to CdSe quantum dots of average size 5.0 ± 0.2 nm and CdSe doped with 1% cobalt ions of similar size. The quantum dots are surface modified using mercaptoacetic acid (MAA) in order to be biocompatible and water-soluble. The MAA-QDs were given to the mice orally at doses of 500, 1000, and 2000 mg/kg by weight of MAA-QDs. Bone marrow and liver samples were collected after two and seven days of treatment. The results indicated that after two days of treatment, the high dose of doped MAA-QDs was significantly able to induce DNA damage, formation of micronuclei (MNs), and generation of DNA adduct (8-hydroxy-2-deoxyguanosine, 8-OHdG). However, increasing DNA damage and the frequency of MNs formation as well as the generation of DNA adducts were observed with both the undoped MAA-QDs (2000 mg/kg) and doped MAA-QDs (1000 and 2000 mg/kg) after seven days of treatment. The results of our study indicate that exposure to high doses of pure MAA-QDs or MAA-QDs doped with cobalt has the potential to cause indirect in vivo genetic damage, which may be attributed to free radical-induced oxidative stress in mice.

Benzetheno adducts derived from p-benzoquinone (p-BQ), a reactive metabolite of benzene, were reported to be formed by the reaction of p-BQ with DNA in vitro but have never been detected either in vivo or in experiments with living cells. Two of them, 3-hydroxy-3,N(4)-benzetheno-2'-deoxycytidine (DCBQ) and 7-hydroxy-1,N(2)-benzetheno-2'-deoxyguanosine (DGBQ), were administered to rats by single ip injections at the doses of 2 mg/kg each. The excretion of unchanged compounds DCBQ and DGBQ within 2 days amounted to 8.2 ± 1.9 and 4.5 ± 1.2% (mean ± SE) of the dose, respectively. Additionally, deribosylated metabolites of DCBQ and DGBQ, 3-hydroxy-3,N(4)-benzethenocytosine (CBQ) and 7-hydroxy-1,N(2)-benzethenoguanine (GBQ), were found amounting to 45.7 ± 10.2 and 2.9 ± 2.1% of the dose, respectively. An additional portion of CBQ and GBQ was liberated from their corresponding conjugates by acidic hydrolysis. Therefore, total recoveries of CBQ and GBQ in urine were 82.1 ± 13.5 and 11.6 ± 5.1% of the dose. To identify conjugated metabolites, DCBQ and DGBQ were administered intraperitoneally at the doses 10.5 and 11.0 mg/kg, respectively, to one animal each. Glucuronides of DCBQ, DGBQ, and GBQ as well as sulfates of DGBQ, CBQ, and GBQ were identified by ESI-LC-MS according to (M - H)(-) ions and their fragmentation. In addition, two oxygenated metabolites and their corresponding conjugates were detected for DGBQ and GBQ. One of these metabolites was identified as 2,7-dihydroxy-1,N(2)-benzethenoguanine OGBQ1. It coeluted with the product obtained by the reaction of HQ and p-BQ mixture with 8-hydroxy-2'-deoxyguanosine followed by acid hydrolysis. These findings suggest that both DCBQ and DGBQ undergo extensive biotransformation in vivo. CBQ appears to be the only p-BQ derived DNA adduct, which can be efficiently recovered from its conjugates and might be therefore useful in molecular dosimetry of benzene.

Metabolic activation of estrogens to catechols and further oxidation to highly reactive o-quinones generates DNA damage including apurinic/apyrimidinic (AP) sites. 4-Hydroxyequilenin (4-OHEN) is the major catechol metabolite of equine estrogens present in estrogen replacement formulations, known to cause DNA strand breaks, oxidized bases, and stable and depurinating adducts. However, the direct formation of AP sites by 4-OHEN has not been characterized. In the present study, the induction of AP sites in vitro by 4-OHEN and the endogenous catechol estrogen metabolite, 4-hydroxyestrone (4-OHE), was examined by an aldehyde reactive probe assay. Both 4-OHEN and 4-OHE can significantly enhance the levels of AP sites in calf thymus DNA in the presence of the redox cycling agents, copper ion and NADPH. The B-ring unsaturated catechol 4-OHEN induced AP sites without added copper, whereas 4-OHE required copper. AP sites were also generated much more rapidly by 4-OHEN. For both catechol estrogens, the levels of AP sites correlated linearly with 8-oxo-dG levels, implying that depuriniation resulted from reactive oxygen species (ROS) rather than depurination of estrogen-DNA adducts. ROS modulators such as catalase, which scavenges hydrogen peroxide and a Cu(I) chelator, blocked the formation of AP sites. In MCF-7 breast cancer cells, 4-OHEN significantly enhanced the formation of AP sites with added NADH. In contrast, no significant induction of AP sites was detected in 4-OHE-treated cells. The greater redox activity of the equine catechol estrogen produces rapid oxidative DNA damage via ROS, which is enhanced by redox cycling agents and interestingly by NADPH-dependent quinone oxidoreductase.

Reactive oxygen species can induce the formation of not only single-nucleobase lesions, which have been extensively studied, but also tandem lesions. Herein, we report a high frequency of formation of a type of tandem lesion, where two commonly observed oxidatively induced single-nucleobase lesions, that is, thymidine glycol (Tg) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), are vicinal to each other in calf thymus DNA upon exposure to Cu(II)/ascorbate along with H(2)O(2) or gamma-rays. We further explored how the tandem lesions perturb the efficiency and fidelity of DNA replication by assessing the replication products formed from the propagation, in Escherichia coli cells, of the single-stranded pYMV1 shuttle vectors containing two tandem lesions [5'-(8-oxodG)-Tg-3' and 5'-Tg-(8-oxodG)-3'] or an isolated Tg or 8-oxodG. The bypass efficiencies for the two tandem lesions were approximately one-half of those for the two isolated single-nucleobase lesions. The presence of an adjacent Tg could lead to significant increases in G-->T transversion at the 8-oxodG site as compared to that of a single 8-oxodG lesion; the frequencies of G-->T mutation were approximately 18, 32, and 28% for 8-oxodG that is isolated, in 5'-(8-oxodG)-Tg-3' and in 5'-Tg-(8-oxodG)-3', respectively. Moreover, both pol IV and pol V are involved, in part, in bypassing the Tg, either present alone or as part of the tandem lesions, in E. coli cells. Together, our results support that complex lesions could exert greater cytotoxic and mutagenic effects than when the composing individual lesions are present alone.

We investigated peroxidase-mediated oxidation of and the formation of the (deoxy)guanosine adduct by 1-phenylazo-2-hydroxynaphthalene (Solvent Yellow 14, Sudan I), a liver and urinary bladder carcinogen for rodents and a potent contact allergen and sensitizer for humans. Using thin layer chromatography (TLC) and/or high performance liquid chromatography (HPLC) combined with mass and/or nuclear magnetic resonance (NMR) spectrometry, we characterized the structures of two major peroxidase-mediated Sudan I metabolites and those of the adducts of (deoxy)guanosine that are formed during Sudan I oxidation. Peroxidase oxidizes Sudan I to radical species that react with another Sudan I radical to form the Sudan I dimer, or in the presence of (deoxy)guanosine, the oxidized Sudan I can attack the exocyclic amino group of guanine, forming the 4-[(deoxy)guanosin-N(2)-yl]Sudan I adduct. The reaction product with a second Sudan I radical results in a dimer where the oxygen 2 radical of Sudan I reacted with carbon 1 in the second Sudan I skeleton. The Sudan I dimer is unstable and decomposes spontaneously to the second oxidation product. This compound consists of the 4-oxo-Sudan I skeleton connected via the oxygen of its 2-hydroxyl group and nitrogen of its azo group with carbon 1 of 2-oxonaphthalene, having a unique spironaphthooxadiazine structure. If (deoxy)guanosine is present during the formation of this Sudan I metabolite, an adduct, in which this Sudan I metabolite is bound to the exocyclic amino group of guanine, is generated. This (deoxy)guanosine adduct is again unstable and decomposes spontaneously to the same adduct that is formed by the direct reaction of oxidized Sudan I, the 4-[(deoxy)guanosin-N(2)-yl]Sudan I adduct. The results presented here are the first structural characterization of Sudan I-(deoxy)guanosine adducts formed during the oxidation of this carcinogen by peroxidase.

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N(2)-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH(3)CN) to the chemically stable N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [(15)N(5)]N(2)-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides with the incorporation of NaBH(3)CN in the DNA hydrolysis buffer. The method required 50 microg of hydrolyzed DNA on column for the analysis, and the limit of detection for N(2)-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N(2)-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 +/- 21.9 vs 348.4 +/- 19.1 adducts per 10(8) 2'-deoxynucleosides) of N(2)-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N(2)-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 +/- 0.7 adducts per 10(8) 2'-deoxynucleosides. No N(2)-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH(3)CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development.

The apoptotic effects of docosahexaenoic acid (DHA) and other omega-3 polyunsaturated fatty acids (PUFAs) have been documented in cell and animal studies. The molecular mechanism by which DHA induces apoptosis is unclear. Although there is no direct evidence, some studies have suggested that DNA damage generated through lipid peroxidation may be involved. Our previous studies showed that DHA, because it has a high degree of unsaturation, can give rise to the acrolein-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) as a major class of DNA adducts via lipid oxidation. As a first step to investigate the possible role of oxidative DNA damage in apoptosis induced by DHA, we examined the relationships between oxidative DNA damage and apoptosis caused by DHA in human colon cancer HT-29 cells. Apoptosis and oxidative DNA damage, including Acr-dG and 8-oxo-deoxyguanosine (8-oxo-dG) formation, in cells treated with DHA and omega-6 PUFAs, including arachidonic acid (AA) and linoleic acid (LA), were measured. DHA induced apoptosis in a dose- and time-dependent manner with a concentration range from 0 to 300 microM as indicated by increased caspase-3 activity and PARP cleavage. In contrast, AA and LA had little or no effect at these concentrations. The Acr-dG levels were increased in HT-29 cells treated with DHA at 240 and 300 microM, and the increases were correlated with the induction of apoptosis at these concentrations, while no significant changes were observed for 8-oxo-dG. Because proteins may compete with DNA to react with acrolein, we then examined the effects of BSA on DHA-induced apoptosis and oxidative DNA damage. The addition of BSA to HT-29 cell culture media significantly decreases Acr-dG levels with a concomitant decrease in the apoptosis induced by DHA. The reduced Acr-dG formation is attributed to the reaction of BSA with acrolein as indicated by increased levels of total protein carbonyls. Similar correlations between Acr-dG formation and apoptosis were observed in HT-29 cells directly incubated with 0-200 microM acrolein. Additionally, DHA treatment increased the level of DNA strand breaks and caused cell cycle arrested at G1 phase. Taken together, these results demonstrate the parallel relationships between Acr-dG level and apoptosis in HT-29 cells, suggesting that the formation of Acr-dG in cellular DNA may contribute to apoptosis induced by DHA.

Reactive oxygen species can induce the formation of tandem DNA lesions. We recently showed that the treatment of calf thymus DNA with Cu2+/H2O2/ascorbate could result in the efficient formation of a tandem lesion where a 5,6-dihydroxy-5,6-dihydrothymidine (or thymidine glycol) is situated on the 5' side of an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). In the present study, we assessed how the 5'-Tg-(8-oxodG)-3' and 5'-(8-oxodG)-Tg-3' tandem lesions are replicated by purified DNA polymerases and how they are recognized by base excision repair enzymes. Our results revealed that the tandem lesions blocked primer extension mediated by the Klenow fragment and yeast polymerase eta more readily than when the Tg or 8-oxodG was present alone. The mutagenic properties of Tg or 8-oxodG differed while they were present alone or in tandem. Moreover, the human 8-oxoguanine-DNA glycosylase (hOGG1)-mediated cleavage of 8-oxodG was compromised considerably by the presence of a neighboring 5' Tg, whereas the presence of Tg as the adjacent 3' nucleoside enhanced 8-oxodG cleavage by hOGG1. The efficiency for the cleavage of Tg by endonuclease III was not affected by the presence of an adjoining 8-oxodG. These results supported the notion that the replication and repair of tandem single-nucleobase lesions depend on the types of lesions involved and their spatial arrangement.

Acrolein (Acr) is a major toxicant in cigarette smoke (CS); it can interact with DNA forming two major adduct isomers: alpha-OH-Acr-dG and gamma-OH-Acr-dG. Previously, we found that the Acr-DNA binding pattern in the human p53 gene coincides with the p53 mutational pattern in CS-related lung cancer; hence, we proposed that Acr is a major lung cancer etiological agent [ Feng , Z. , Hu , W. , Hu , Y. , and Tang , M.-s. ( 2006 ) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair . Proc. Natl. Acad. Sci. U.S.A. 103 , 15404 - 15409 ]. This hypothesis has been brought into question with recent work that failed to detect Acr-induced mutations in the pSP189 system [ Kim , S. I. , Pfeifer , G. P. , and Besaratinia , A. ( 2007 ) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells . Cancer Res. 67 , 11640 - 116472 ]. To resolve this controversy, we determined the level and the type of Acr-dG formation, and the mutagenicity of Acr-dG adducts in the same pSP189 system. We also mapped the Acr-dG adduct distribution at the nucleotide level and the Acr-dG-induced mutational spectrum in this system. We found that (1) gamma-OH-Acr-dG is the major adduct formed in Acr-modified DNA based on the LC-ESI-MS/MS analysis; (2) the mutation frequency is proportional to the extent of Acr modifications, the majority of which are G:C to T:A and G:C to A:T mutations; and (3) sequences with a run of Gs are the mutational hotspots. Using the UvrABC nuclease incision method to map the Acr-dG distribution in the supF gene sequence, we confirmed that Acr-DNA adducts preferentially form in guanine-rich sequences that are also mutational hotspots. These results reaffirm that Acr-dG adducts are mutagenic and support our hypothesis that Acr is a major etiological agent for CS and cooking fume-related lung cancer.

The alkylated polycyclic aromatic hydrocarbon 1-methylpyrene is a carcinogen in rodents and has been detected in various environmental matrices and foodstuffs. It is activated metabolically by benzylic hydroxylation to 1-hydroxymethylpyrene followed by sulfoconjugation to yield electrophilic 1-sulfooxymethylpyrene (1-SMP) that is prone to form DNA adducts. An LC-MS/MS method using multiple reaction monitoring (MRM) of fragment ions has been developed for specific detection and quantification of N (2)-(1-methylpyrenyl)-2'-deoxyguanosine (MP-dGuo) and N (6)-(1-methylpyrenyl)-2'-deoxyadenosine (MP-dAdo) formed in DNA in the presence of 1-SMP. DNA samples were spiked with stable isotope internal standards, [ (15)N 5, (13)C 10]MP-dGuo and [ (15)N 5]MP-dAdo, followed by enzymatic digestion to 2'-deoxynucleosides and solid-phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS. The limits of detection were 10 fmol of MP-dGuo and 2 fmol of MP-dAdo or three molecules of MP-dGuo and 0.6 molecules of MP-dAdo per 10 (8) 2'-deoxynucleosides using 100 mug of herring sperm DNA as the sample matrix. The method was validated with herring sperm DNA reacted with 1-SMP in vitro. Hepatic DNA was analyzed from rats that were dosed intraperitoneally with 9.3 mg 1-SMP per kg body weight and killed after various time periods. Levels of MP-dGuo and MP-dAdo in rat liver were found to increase, reaching their maxima at approximately 3 h, and then decrease over time. A good correlation was observed between the results obtained using LC-MS/MS and MRM and those from (32)P-postlabeling. MRM allowed the more precise quantification of specific 1-MP adducts, in addition to a time reduction of the analysis when compared with (32)P-postlabeling.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of nevirapine use is severe hepatotoxicity, which raises concerns about its administration, particularly in the perinatal and pediatric settings. Nevirapine metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP and the formation of phenolic derivatives. Further metabolism of 12-hydroxy-NVP by phase II esterification may produce electrophilic derivatives capable of reacting with DNA to yield covalent adducts, which could potentially be involved in the initiation of mutagenic and carcinogenic events. In the present study, we have investigated the reactivity of the synthetic model electrophile, 12-mesyloxy-NVP, toward 2'-deoxynucleosides and DNA. Parallel synthetic studies were conducted with 12-bromo-NVP and 3',5'- O-bis( tert-butyldimethylsilyl)-2'-deoxynucleosides, using palladium(0) catalysis. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in the reactions with 12-mesyloxy-NVP and characterized by NMR and MS. The adduct structures consistently involved binding through C12 of NVP and N7 or N9 of deoxyguanosine; N1, N3, or N9 of deoxyadenosine; and N3 of deoxycytidine. Reactions conducted under palladium(0) catalysis yielded adducts through O (6) and N1 of deoxyguanosine, N1 of deoxyadenosine, and N3 of deoxycytidine. At least seven deoxynucleoside-NVP adducts were present in DNA reacted with 12-mesyloxy-NVP and enzymatically hydrolyzed. Four of these adducts were identified as 12-(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, 12-(deoxyguanosin- O(6)-yl)nevirapine, and 12-(deoxyadenosin- N(6)-yl)nevirapine. One depurinating adduct, 12-(guanin-N7-yl)nevirapine, was identified in the thermal neutral DNA hydrolysate. If formed in vivo, some of these adducts would have considerable mutagenic potential. Our results thus suggest that NVP metabolism to 12-hydroxy-NVP may be a factor in NVP hepatocarcinogenicity.

The reaction of catechol estrogen quinones with DNA to produce the depurinating adducts, 4-OHE 2(E 1)-1-N7Gua and 4-OHE 2(E 1)-1-N3Ade, has been linked to the initiation of breast and other human cancers. A better understanding into the mechanism of how these adducts are formed would be useful to studies aimed at correlating adduct formation to DNA damage. Possible reaction intermediates, produced as a result of Michael addition of deoxyguanosine (dG) to catechol estrogen-3,4-quninones, have been modeled using density functional theory to determine likely intermediates on the potential energy surface (PES) of this reaction. Specifically, the sequence of elimination events, glycosidic bond cleavage and rearomatization of the estrogen A ring, was explored. Consistent with known experimental procedures, B3LYP calculations indicate that a proton source is needed to effect the Michael addition. Calculations also indicate that a catalytic mechanism, where one catechol estrogen quinone could adduct multiple purine bases, is unlikely. Experimental investigation toward an observed cationic reaction intermediate was also consistent with a stoichiometric reaction between estrone-3,4-quinone (E 1-3,4-Q) and dG. HPLC-MS analysis indicates that the cationic reaction intermediate contains the 2'-deoxyribose moiety. Assay of 4-OHE 1-1-N7Gua adduct formation and 2'-deoxyribose formation at different times during the reaction of E 1-3,4-Q with dG indicates that equimolar amounts of each are produced, further supporting a stoichiometric process with respect the catechol estrogen quinone. Differences in the UV spectroscopy of cationic reaction intermediate and the 4-OHE 1-1-N7Gua adduct allowed for kinetic analysis of the glycosidic bond cleavage process. Kinetic scanning analysis indicates that the decomposition of the cationic reaction intermediate is a first-order process with a t 1/2 of 40 min at 30 degrees C. Measurement of the unimolecular rate constant k at different temperatures afforded an Arrhenius plot, which provided values for Delta H, Delta S, and Delta G of 24.7 kcal/mol, 7.2 eu, and 26.8 kcal/mol, respectively. The computational data in conjunction with experimental results are consistent with a mechanism that involves a proton-assisted Michael addition to form an alpha-ketoenol ring system, followed by slow loss of the proton at C1 to restore the aromatic A ring, then fast cleavage of the glycosidic bond to form the 4-OHE 1-1-N7Gua adduct.

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.

The reaction of N-nitrosotolazoline, the nitrosation product of a representative imidazoline receptor drug tolazoline, with DNA, deoxyguanosine (dG), or deoxyadenosine (dA) produces adducts containing the 2-phenylacetamidoethyl group. The synthesis and characterization of 2-phenylacetamidoethyl-guanine derivatives (O6-dG, O6-Gua, N2-Gua, and 7-Gua) and 2-phenylacetamidoethyladenine derivatives (1-Ade, 3-Ade, 7-Ade, and N6-Ade) are described. In addition to the use of an established UV spectral method for confirming the structure of the alkyl adenines, a new 13C NMR method for determining the N-alkylation site is presented. In combination with the synthesized standards, HPLC MS/MS methods were used to determine the nature and the quantity of adducts produced. N-Nitrosotolazoline reacted with dG to give 7-(2-phenylacetamidoethyl)deoxyguanosine (major), O6-(2-phenylacetamidoethyl)deoxyguanosine, and 5'-O-phenyacetyldeoxyguanosine. The reaction of N-nitrosotolazoline with dA produced the 1-, 3-, 7-, N6, and 5-O'-2-phenylacetamidoethyl adenine and dA derivatives as well as several phenylacetyl adducts. Reaction of N-nitrosotolazoline with DNA in vitro resulted in the detection of 2-phenylacetamidoethyl adducts (adduct, relative %): 7-Gua, 60%; 3-Ade, 30%; O6-Gua, 8%; and 7-Ade, 2%. Comparison of these data with appropriate literature data, as well as our work on the mechanism of N-nitrosotolazoline hydrolytic decomposition, is consistent with the adducts being produced from a 2-phenylacetamidoethyldiazonium intermediate. The results show that N-nitrosotolazoline, and presumably other N-nitrosoimidazolines, if produced by endogenous nitrosation pathways, are capable of alkylating DNA without additional metabolic transformation and are probable carcinogens.

Active and passive smoking are major risk factors for lung cancer. Pro-oxidants in tobacco smoke have been implicated in smoking-associated disease development due to their potential role in inducing oxidative stress. Previous studies have failed to associate increased levels of oxidative damage to DNA with the formation of the potentially mutagenic lesion, 8-oxo-2-deoxyguanosine (8-oxodG), probably due to repair of this lesion. However, no systematic studies have been performed to assess the dose- and time-dependent formation and removal of this lesion by cigarette smoke exposure. In the present study, female A/J mice were exposed to side-stream cigarette smoke in a whole body exposure chamber for 6 h a day, 5 days a week for up to 6 weeks. Age-matched controls were maintained in filtered ambient air. Lung tissues were harvested from 2, 4, and 6 weeks smoke-exposed mice after 1, 3, 6, and 20 h, following the cessation of smoking. A significant increase in the levels of 8-oxodG in lung DNA was observed in 10 day smoke-exposed mice at 1 (11.5+/-1.1/10(6) nucleotides), 3 (20.2+/-2.7/10(6) nucleotides; p=0.0008), and 6 h (17.2+/-1.0/10(6) nucleotides; p<0.005) postcessation, as compared with age-matched sham treatment (8.8+/-2.3/10(6) nucleotides) (mean+/-SD). The levels significantly declined 20 h after the cessation of smoke exposure (14.0+/-1.6/10(6) nucleotides), although they were still higher than the control. Our results strongly suggest that there is a significant increase in the 8-oxodG levels immediately after the cessation of smoking, which is repaired over time. This initial increase in 8-oxodG levels may lead to gene mutations, and accumulation of such mutations over time can eventually lead to malignant transformation of the cells.

Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence-selective formation of other tobacco carcinogen-DNA adducts along K-ras- and p53-derived duplexes and the preferential modification of endogenously methylated CG dinucleotides by benzo[a]pyrene diol epoxide and acrolein.

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

An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS(n)) technique has been developed for the characterization and quantification of 2'-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP-DNA adducts were analyzed by MS/MS and MS(n) scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 10(8) DNA bases, and the limit of quantification (LOQ) was 3 adducts per 10(8) DNA bases in both MS/MS and MS(3) scan modes, using 27 microg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-[2H3C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H - 116]+). The consecutive reaction monitoring (CRM) scan modes in MS(3) and MS(4) were used to measure and further characterize product ions of the aglycone ion (BH2+) (Guanyl-PhIP). The MS(3) scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MS(n) scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LC-ESI/MS/MS(n) method is the first reported application on the use of the MS(3) scan mode for the analysis of DNA adducts in vivo.

Crotonaldehyde, a mutagen and carcinogen, reacts with deoxyguanosine (dGuo) in DNA to generate a pair of diastereomeric 1,N(2)()-propanodeoxyguanosine adducts (Cro-dGuo, 2), which occur in (6S,8S) and (6R,8R) configurations. They can also be formed through the consecutive reaction of two acetaldehyde molecules with dGuo. Cro-dGuo adducts inhibit DNA synthesis and induce miscoding in human cells. Considering their potential role in carcinogenesis, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to explore the presence of Cro-dGuo adducts in DNA from various human tissues, such as liver, lung, and blood. DNA was isolated from human tissues and enzymatically hydrolyzed to deoxyribonucleosides. [(15)N(5)]Cro-dGuo was synthesized and used as an internal standard. The Cro-dGuo adducts were enriched from the hydrolysate by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring (SRM). This method allows the quantitation of the Cro-dGuo adducts at a concentration of 4 fmol/micromol dGuo, corresponding to about 1 adduct per 10(9) normal nucleosides starting with 1 mg of DNA, with high accuracy and precision. DNA from human liver, lung, and blood was analyzed. The Cro-dGuo adducts were detected more frequently in human lung DNA than in liver DNA but were not detected in DNA from blood. The results of this study provide quantified data on Cro-dGuo adducts in human tissues. The higher frequency of Cro-dGuo in lung DNA than in the other tissues investigated is potentially important and deserves further study.

Tetranitromethane (TNM) is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel. TNM was reported to induce pulmonary adenocarcinomas and squamous cell carcinomas in mice and rats. However, the mechanisms underlying carcinogenesis induced by TNM has not yet been clarified. We previously revealed that nitroTyr and nitroTyr-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Since TNM is a reagent for nitration of Tyr in proteins and peptides, we have hypothesized that TNM-treated Tyr and Tyr-containing peptides induce DNA damage by the modification of Tyr. We examined DNA damage induced by TNM-treated amino acids or peptides using (32)P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. TNM-treated Tyr and Lys-Tyr-Lys induced DNA damage including the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of Cu(II) and NADH. DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). The cytosine residue of the ACG sequence complementary to codon 273, well-known hotspots of the p53 gene, was cleaved with piperidine and Fpg treatments. On the other hand, nitroTyr and Lys-nitroTyr-Lys did not induce DNA damage in the presence of Cu(II) and NADH. Time-of-flight mass spectrometry confirmed that reactions between Lys-Tyr-Lys and TNM yielded not only Lys-nitroTyr-Lys but also Lys-nitrosoTyr-Lys. Therefore, it is speculated that the nitrosotyrosine residue can induce oxidative DNA damage in the presence of Cu(II) and NADH. It is concluded that Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM. TNM is a new type of carcinogen that induces DNA damage not by itself but via Tyr modification.

N(2)-Ethyldeoxyguanosine (N(2)-ethyldGuo) is a DNA adduct formed by reaction of the exocyclic amine of dGuo with the ethanol metabolite acetaldehyde. Because ethanol is a human carcinogen, we assessed the biological consequences of replication of template N(2)-ethyldGuo, in comparison to the well-studied adduct O(6)-ethyldeoxyguanosine (O(6)-ethyldGuo). Single chemically synthesized N(2)-ethyldGuo or O(6)-ethyldGuo adducts were placed site specifically in the suppressor tRNA gene of the mutation reporting shuttle plasmid pLSX. N(2)-EthyldGuo and O(6)-ethyldGuo were both minimally mutagenic in double-stranded pLSX replicated in human 293 cells; however, the placement of deoxyuridines on the complementary strand at 5'- and 3'-positions flanking the adduct resulted in 5- and 22-fold enhancements of the N(2)-ethyldGuo- and O(6)-ethyldGuo-induced mutant fractions, respectively. The fold increase in the N(2)-ethyldGuo-induced mutant fraction in deoxyuridine-containing plasmids was similar after replication in 293T cells, a mismatch repair deficient variant of 293 cells, indicating that postreplication mismatch repair has little role in modulating N(2)-ethyldGuo-mediated mutagenesis. The mutation spectrum generated by N(2)-ethyldGuo consisted primarily of single base deletions and adduct site-targeted transversions, in contrast to the exclusive production of adduct site-targeted transitions by O(6)-ethyldGuo. The yield of progeny plasmids after replication in 293 cells was reduced by the presence of N(2)-ethyldGuo in parental plasmids with or without deoxyuridine to 39 or 19%, respectively. Taken together, these data indicate that N(2)-ethyldGuo in DNA exerts its principal biological activity by blocking translesion DNA synthesis in human cells, resulting in either failure of replication or frameshift deletion mutations.

Reactive and redox-active polycyclic aromatic hydrocarbon (PAH) o-quinones produced by Aldo-Keto Reductases (AKRs) have the potential to cause depurinating adducts leading to the formation of abasic sites and oxidative base lesions. The aldehyde reactive probe (ARP) was used to detect these lesions in calf thymus DNA treated with three PAH o-quinones (BP-7,8-dione, 7,12-DMBA-3,4-dione, and BA-3,4-dione) in the absence and presence of redox-cycling conditions. In the absence of redox-cycling, a modest amount of abasic sites were detected indicating the formation of a low level of covalent o-quinone depurinating adducts (>3.2 x 10(6) dNs). In the presence of NADPH and CuCl2, the three PAH o-quinones increased the formation of abasic sites due to ROS-derived lesions destabilizing the N-glycosidic bond. The predominant source of AP sites, however, was revealed by coupling the assay with human 8-oxoguanine glycosylase (hOGG1) treatment, showing that 8-oxo-dGuo was the major lesion caused by PAH o-quinones. The levels of 8-oxo-dGuo formation were independently validated by HPLC-ECD analysis. Apyrimidinic sites were also revealed by coupling the assay with Escherichia coli (Endo III) treatment showing that oxidized pyrimidines were formed, but to a lesser extent. Different mechanisms were responsible for the formation of the oxidative lesions depending on whether Cu(II) or Fe(III) was used in the redox-cycling conditions. In the presence of Cu(II)-mediated PAH o-quinone redox-cycling, catalase completely suppressed the formation of the lesions, but mannitol and sodium benzoate were without effect. By contrast, sodium azide, which acts as a *OH and 1O2 scavenger, inhibited the formation of all oxidative lesions, suggesting that the ROS responsible was 1O2. However, in the presence of Fe(III)-mediated PAH o-quinone redox-cycling, the *OH radical scavengers and sodium azide consistently attenuated their formation, indicating that the ROS responsible was *OH.

A sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution mass spectrometric method was developed to identify and quantify the endogenous cyclic DNA adducts derived from trans-4-hydroxy-2-nonenal with 2'-deoxyguanosine (HNE-dG) in human brain tissues. Authentic and 13C and 15N stable isotope-labeled HNE-dG were synthesized to serve as standards. The in vitro HNE-modified calf-thymus DNA as well as the DNA samples isolated from human brain tissues of normal and Alzheimer's disease subjects were enzymatically digested to nucleosides in vitro with the presence of internal standard (HNE-dG-13C10, 15N5). The enzymatic digests were cleaned up by solid phase extraction. Only 1-2 microg of DNA digests was loaded on a laboratory-constructed reversed phase capillary chromatography column, and the HNE-dG adducts were separated from intact nucleosides and quantified by a high capacity ion trap mass spectrometer in the MS/MS mode. This method was able to quantify an adduct level of approximately 40 lesions/10(9) normal DNA nucleosides. The detected level of HNE-dG adducts in hippocampus/parahippocampal gyrus and inferior parietal regions of postmortem brains from AD subjects were 556 +/- 379 and 238 +/- 72 adducts per 10(9) normal nucleosides, respectively. These results were consistent with the 32P postlabeling results, which detected 400-600 adducts per 10(9) normal nucleotides in the hippocampus.

Pentachlorophenol (PCP), a widespread environmental pollutant that is possibly carcinogenic to humans, is metabolically oxidized to tetrachloroquinone. DNA adducts attributable to tetrachloroquinone have been observed previously in vitro and detected in vivo. In addition, an unidentified adduct in these studies coeluted with the product of the reaction of deoxyguanosine (dG) and tetrachlorobenzoquinone (Cl4BQ). We have synthesized, isolated, purified, and characterized the predominant adduct formed from the reaction of dG and Cl4BQ. The preparation of a 13C-labeled version of this adduct facilitated its structural characterization. On the basis of 1H NMR, 13C NMR, MS, IR, UV, and cyclic voltammetry, we propose that the adduct is a dichlorobenzoquinone nucleoside in which two chlorine atoms in Cl4BQ have been displaced by reaction at the 1- and N2-positions of dG. The 1H and 13C NMR chemical shifts are consistent with the dichlorobenzoquinone assignment. In contrast, under standard analytical conditions, LC-MS data are consistent with a reduced hydroquinone structure, similar to what may be expected based on results from other chloroquinones. Data from the present study indicate that this reduction could be occurring in the electrospray ionization source and that the initial product of the reaction of dG and Cl4BQ is a dichlorobenzoquinone. The results of this study contribute to the hypothesis that direct reactions between chlorophenols and DNA may play a role in the toxic effects of chlorophenols and indicate a potential difference in reactivity and biological influence between PCP and other less substituted chlorophenols or phenols.

7,8-dihydro-8-oxoguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at the nucleobase guanine. Recent studies have shown that 8-oxoG has a lower reduction potential than the parent guanine and is a hot spot for further oxidation. Spiroiminodihydantoin (Sp) has been identified as one of these further oxidation products. Chromium(VI) is a human carcinogen that, when reduced by a cellular reductant such as ascorbate, can oxidize DNA. In this study, duplex DNA was reacted with Cr(VI) and ascorbate to identify and quantify the base lesions formed. Guanine bases were observed to be preferentially oxidized with 5' guanines within purine repeats showing enhanced oxidation. Trapping of the guanine lesions by the base excision repair enzymes hOGG1 and mNEIL2 showed nearly exclusive trapping by mNEIL2, suggesting that 8-oxoG was not the major lesion but rather a lesion recognized by mNEIL2 such as Sp. Formation of the Sp lesion in the Cr(VI)/Asc oxidation reaction with DNA was confirmed by LC-ESI-MS detection. HPLC-ECD was used to identify and quantify any 8-oxoG arising from Cr(VI)/Asc oxidation of DNA. Concentrations of Cr(VI) (3.1-50 microM) with a corresponding 1:10 ratio of Asc oxidized between 0.3% and 1.5% of all guanines within the duplex DNA strand to Sp. 8-oxoG was also identified but with the highest Cr(VI) concentration converting approximately 0.1% of all guanines to 8-oxoG. These results show that Sp was present in concentrations approximately 20 times greater than that of 8-oxoG in this system. The results indicate that 8-oxoG, while present, was not the major product of Cr(VI)/Asc oxidation of DNA and that Sp predominates under these conditions. These results further imply that Sp may be the lesion that accounts for the carcinogenicity of this metal in cellular systems.