Synthesis of N2-trans-isosafrole-dG-adduct Bearing DNAs and the Bypass Studies with Human TLS Polymerases κ and η.

Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N2-SF-dG and N6-SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N2-SF-dG modified DNAs using phosphoramidite chemistry. The N2-SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N2-SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N2-SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.

Genotoxic C8-Arylamino-2'-deoxyadenosines Act as Latent Alkylating Agents to Induce DNA Interstrand Cross-Links.

DNA interstrand cross-links (ICLs) are extremely deleterious and structurally diverse, driving the evolution of ICL repair pathways. Discovering ICL-inducing agents is, thus, crucial for the characterization of ICL repair pathways and Fanconi anemia, a genetic disease caused by mutations in ICL repair genes. Although several studies point to oxidative stress as a cause of ICLs, oxidative stress-induced cross-linking events remain poorly characterized. Also, polycyclic aromatic amines, potent environmental carcinogens, have been implicated in producing ICLs, but their identities and sequences are unknown. To close this knowledge gap, we tested whether ICLs arise by the oxidation of 8-arylamino-2'-deoxyadenosine (ArNHdA) lesions, adducts produced by arylamino carcinogens. Herein, we report that ArNHdA acts as a latent cross-linking agent to generate ICLs under oxidative conditions. The formation of an ICL from 8-aminoadenine, but not from 8-aminoguanine, highlights the specificity of 8-aminopurine-mediated ICL production. Under the influence of the reactive oxygen species (ROS) nitrosoperoxycarbonate, ArNHdA (Ar = biphenyl, fluorenyl) lesions were selectively oxidized to generate ICLs. The cross-linking reaction may occur between the C2-ArNHdA and N2-dG, presumably via oxidation of ArNHdA into a reactive diiminoadenine intermediate followed by the nucleophilic attack of the N2-dG on the diiminoadenine. Overall, ArNHdA-mediated ICLs represent rare examples of ROS-induced ICLs and polycyclic aromatic amine-mediated ICLs. These results reveal novel cross-linking chemistry and the genotoxic effects of arylamino carcinogens and support the hypothesis that C8-modified adenines with low redox potential can cause ICLs in oxidative stress.

Mitochondria-targeted tetrahedral DNA nanostructures for doxorubicin delivery and enhancement of apoptosis.

Mitochondria-targeted nanoparticles, such as liposomes, polymers and inorganic particles, suffer from heterogeneity, low biocompatibility and low drug loading efficiency. Here, we present a novel delivery platform based on tetrahedral DNA nanostructures (TDNs) that enable the mitochondrial transportation of the anticancer drug doxorubicin (DOX) for cancer therapy. In our design, DOX was intercalated into TDNs, which executed the cell-killing function inside the tumor cells. Various numbers of d-(KLAKLAK)2 (KLA) were conjugated to TDNs to achieve the mitochondria targeting effect. The mean size of the KLA-modified TDNs was about 15 nm, and the TDNs were stable in FBS. The DOX loading efficiency of the TDNs was up to around 77%. The 3KLA-modified TDNs exhibited the most efficient DOX accumulation in mitochondria, leading to an effective release of cytochrome c, and the upregulated expression levels of caspase-9, caspase-3, p21 and p53. Meanwhile, 3KLA-TDNs/DOX elevated the pro-apoptotic Bax, reduced the anti-apoptotic Bcl-2 protein expression and increased the Bax/Bcl-2 ratio, which finally activated the mitochondria-mediated, programmed apoptosis pathway to enhance the anticancer efficacy in vitro. This 3KLA-TDN and DOX co-assembling strategy can be further developed to transport other anthracyclines and chemotherapeutic agents for enhanced apoptosis effects.

Formation of Bulky DNA Adducts by Non-Enzymatic Production of 1,2-Naphthoquinone-Epoxide from 1,2-Naphthoquinone under Physiological Conditions.

Quinones may be formed metabolically or abiotically from environmental pollutants and polycyclic aromatic hydrocarbons (PAHs); many are recognized as toxicological intermediates that cause a variety of deleterious cellular effects including mutagenicity. The PAH-o-quinone, 1,2-naphthoquinone (1,2-NQ), may exert its genotoxic effects through interactions with cellular nucleophiles such as DNA, however, the mechanisms of 1,2-NQ adduct formation are still under investigation. With the aim to further understand these mechanisms, the chemical structures of adducts formed from the reaction of 2'-deoxyguanosine (dG) with 1,2-NQ under physiological conditions were investigated by liquid chromatography electrospray ionization tandem mass spectrometry and 1H NMR analyses. Results showed that 1,2-NQ underwent non-enzymatic oxidation to form a 1,2-NQ-epoxide which in turn formed at least four bulky adducts with dG, and these adducts were more likely to be formed under physiological conditions. A mechanism was proposed whereby hydration of 1,2-NQ to form unstable naphthohydroquinones and 2-hydroxy-1,4-naphthoquinone resulted in formation of hydrogen peroxide that oxidized 1,2-NQ. These results suggest that the genotoxicity of 1,2-NQ may not only be caused through oxidative DNA damage and adduct formation through Michael addition but also through non-enzymatic oxidative transformation of 1,2-NQ itself to form an intermediate PAH-epoxide which covalently binds to DNA.

Ru(TAP)32+ uses multivalent binding to accelerate and constrain photo-adduct formation on DNA.

Ru(ii)-complexes with polyazaaromatic ligands can undergo direct electron transfer with guanine nucleobases on blue light excitation that results in DNA lesions with phototherapeutic potential. Here we use single molecule approaches to demonstrate DNA binding mode heterogeneity and evaluate how multivalent binding governs the photochemistry of [Ru(TAP)3]2+ (TAP = 1,4,5,8-tetraazaphenanthrene).

A computational foray into the mechanism and catalysis of the adduct formation reaction of guanine with crotonaldehyde.

Crotonaldehyde, a common environmental pollutant and product of endogenous lipid peroxidation, reacts with guanine to form DNA adducts with pronounced genotoxicity and mutagenicity. Here, we explore the molecular mechanism of this adduct formation using double-hybrid density functional theory methods. The reaction can be envisaged to occur in a two-step fashion via an aza-Michael addition leading to an intermediate ring-open adduct followed by a cyclization reaction giving the mutagenic ring-closed adduct. We find that (i) a 1,2-type addition is favored over a 1,4-type addition for the aza-Michael addition, and (ii) an initial tautomerization of the guanine moiety in the resulting ring-open adduct significantly reduces the barrier toward cyclization compared to the direct cyclization of the ring-open adduct in its keto-form. Overall, the aza-Michael addition is found to be rate-determining. We further find that participation of a catalytic water molecule significantly reduces the energy barriers of both the addition and cyclization reaction. © 2018 Wiley Periodicals, Inc.

In Vitro DNA Adduction Resulting from Metabolic Activation of Diosbulbin B and 8-Epidiosbulbin E Acetate.

Diosbulbin B (DBB) and 8-epidiosbulbin E acetate (EEA), belonging to furan-containing diterpenoid lactones, are the primary components of Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Our earlier studies indicated that consumption of DBB or EEA induced acute hepatotoxicities. Both DBB and EEA were bioactivated by P450 3A4 to generate the corresponding cis-enedial reactive metabolites which are associated with the hepatotoxicities. It has been proposed that the electrophilic intermediates attack cellular nucleophiles such as protein or DNA, thought to be a mechanism of triggering toxicities. The purposes of our present study were to define the interaction of the electrophilic reactive metabolites originating from DBB and EEA with 2'-deoxyguanosine (dGuo), 2'-deoxycytidine (dCyd), and 2'-deoxyadenosine (dAdo) and to characterize DNA adducts arising from the reactive metabolites of DBB and EEA. The reactive metabolites of DBB and EEA were found to covalently bind to the exocyclic and endocyclic nitrogens of dCyd, dGuo, and dAdo to generate oxadiazabicyclo[3.3.0]octaimine adducts. The reactive metabolites of DBB and EEA also attacked dGuo, dAdo, and dCyd of calf thymus DNA. The DNA adducts possibly contribute to the toxicologies of DBB and EEA.

Site-specific cross-linking of proteins to DNA via a new bioorthogonal approach employing oxime ligation.

DNA-protein cross-links (DPCs) are super-bulky DNA adducts induced by common chemotherapeutic agents, reactive oxygen species, and aldehydes, and also formed endogenously as part of epigenetic regulation. Despite their presence in most cells and tissues, the biological effects of DPCs are poorly understood due to the difficulty of constructing site-specific DNA-protein conjugates. In the present work, a new approach of conjugating proteins to DNA using oxime ligation was used to generate model DPCs structurally analogous to lesions formed in cells. In our approach, proteins and peptides containing an unnatural oxy-Lys amino acid were cross-linked to DNA strands functionalized with 5-formyl-dC or 7-(2-oxoethyl)-7-deaza-dG residues using oxime ligation. The conjugation reaction was site-specific with respect to both protein and DNA, provided excellent reaction yields, and formed stable DPCs amenable to biological evaluation.

Quantitative synthesis of protein-DNA conjugates with 1 : 1 stoichiometry.

We describe here a binding-facilitated reaction strategy, enabling quantitative conjugation of DNA to native proteins with a desirable 1 : 1 stoichiometry. The technique takes advantage of the iterative affinity interaction and covalent binding processes to achieve complete conjugation. The complete conjugation obviates the need for separation of the protein-DNA conjugates as required by other DNA-protein conjugation methods.

Genetic control of predominantly error-free replication through an acrolein-derived minor-groove DNA adduct.

Acrolein, an α,ß-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N2-propano-2' deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.

Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B2a and Biological Amines.

Aflatoxin B2a has been shown to bind to proteins through a dialdehyde intermediate under physiological conditions. The proposed structure of this adduct has been published showing a Schiff base interaction, but adequate verification using structural elucidation instrumental techniques has not been performed. In this work, we synthesized the aflatoxin B2a amino acid adduct under alkaline conditions, and the formation of a new product was determined using high performance liquid chromatography-time-of-flight mass spectrometry. The resulting accurate mass was used to generate a novel proposed chemical structure of the adduct in which the dialdehyde forms a pyrrole ring with primary amines rather than the previously proposed Schiff base interaction. The pyrrole structure was confirmed using 1H, 13C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation NMR and tandem mass spectrometry. Reaction kinetics show that the reaction is overall second order and that the rate increases as pH increases. Additionally, this study shows for the first time that aflatoxin B2a dialdehyde forms adducts with phosphatidylethanolamines and does so through pyrrole ring formation, which makes it the first aflatoxin-lipid adduct to be structurally identified. Furthermore, oxidation of the pyrrole adduct produced a product that was 16 m/z heavier. When the aflatoxin B2a-lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and 1H NMR spectrum that match aflatoxin B1-lysine, which suggest the transformation of the pyrrole ring to a pyrrolin-2-one ring. These data give new insight into the fate and chemical properties of biological adducts formed from aflatoxin B2a as well as possible interferences with known aflatoxin B1 exposure biomarkers.

Epoxyeicosatrienoic acids (EETs) form adducts with DNA in vitro.

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.

Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.

Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O(2)-methylthymidine (O(2)-Me-dT) and O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT). To investigate the biological effects of these O(2)-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O(2)-Me-dT or O(2)-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O(2)-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O(2)-Me-dT was less genotoxic and allowed 55% TLS. However, O(2)-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O(2)-POB-dT (MF 53%) in HEK293T cells. The major type of mutations in each case was targeted T → A transversions (56% and 47%, respectively, for O(2)-Me-dT and O(2)-POB-dT). Both lesions induced a much lower frequency of T → G, the dominant mutation in bacteria. siRNA knockdown of the TLS polymerases (pols) indicated that pol η, pol ζ, and Rev1 are involved in the lesion bypass of O(2)-Me-dT and O(2)-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O(2)-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O(2)-POB-dT, it was decreased by 44% in pol ζ knockdown cells, indicating that these TLS pols are critical for mutagenesis. Additional decrease in both TLS efficiency and MF was observed in cells deficient in pol ζ plus other Y-family pols. This study provided important mechanistic details on how these lesions are bypassed in human cells in both error-free and error-prone manner.

Formation of DNA-copolymer fibrils through an amyloid-like nucleation polymerization mechanism.

Conjugation of a hydrophobic poly(2-oxazoline) bearing tertiary amide groups along its backbone with a short single stranded nucleotide sequence results in an amphiphilic comb/graft copolymer, which organizes in fibrils upon direct dissolution in water. Supported by circular dichroism, atomic force microscopy, transmission electron microscopy, and scattering data, fibrils are formed through inter- and intramolecular hydrogen bonding between hydrogen accepting amide groups along the polymer backbone and hydrogen donating nucleic acid grafts leading to the formation of hollow tubes.

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 µM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage and the risk of hepatocellular cancer in smokers.

Capturing labile sulfenamide and sulfinamide serum albumin adducts of carcinogenic arylamines by chemical oxidation.

Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by N-oxidation of the exocyclic amine group to produce N-hydroxylated metabolites, which are critical intermediates implicated in toxicity and DNA damage. The arylhydroxylamines and their oxidized arylnitroso derivatives can also react with cysteine (Cys) residues of glutathione or proteins to form, respectively, sulfenamide and sulfinamide adducts. However, sulfur-nitrogen linked adducted proteins are often difficult to detect because they are unstable and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine present in tobacco smoke, were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts at the Cys(34) residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve as biomarkers of these tobacco and dietary carcinogens.

Structural probes in quadruplex nucleic acid structure determination by NMR.

Traditionally, isotope-labelled DNA and RNA have been fundamental to nucleic acid structural studies by NMR. Four-stranded nucleic acid architectures studies increasingly benefit from a plethora of nucleotide conjugates for resonance assignments, the identification of hydrogen bond alignments, and improving the population of preferred species within equilibria. In this paper, we review their use for these purposes. Most importantly we identify reasons for the failure of some modifications to result in quadruplex formation.

Nucleoside triphosphates--building blocks for the modification of nucleic acids.

Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.