Effects of Polycyclic Aromatic Hydrocarbon Adducts with Deoxyguanosine and Deoxyadenosine in vivo and in vitro

Reactive metabolites from non-planar polycyclic aromatic hydrocarbon carcinogens react extensively with both deoxyadenosine and deoxyguanosine in DNA whereas those from planar molecules react predominantly only with deoxyguanosine. In vitro studies with single adducts in oligonucleotides showed that both types of adduct blocked primer extension and that the limited amount of nucleotide addition opposite the adduct varied with the polymerase, the sequence context of the adduct and the chemical structure of the adduct. When these same single adduct containing-oligonucleotides were introduced into a single-stranded vector that was allowed to replicate in Escherichia coli, the major events observed were blockage of replication, insertion of the correct nucleotide (i.e. T opposite an A adduct and C opposite a G adduct), and insertion of A opposite the adduct. In mouse skin, benzo[c]phenanthrene 4S,3R-dihydrodiol 2S,1R-epoxide initiated substantially more tumors per DNA adduct formed than did the other three isomers. This isomer generates mostly adenine adducts in DNA, suggesting that, in this case, adenine adducts are intrinsically more tumorigenic than guanine adducts.     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

Synthesis and Biochemical Characterization of Tricyclothymidine Triphosphate (tc‐TTP)

Tricyclo‐DNA (tc‐DNA) is a conformationally restricted oligonucleotide analogue that exhibits promising properties as a robust antisense agent. Here we report on the synthesis and biochemical characterization of tc‐TTP, the triphosphate of a tc‐DNA nucleoside containing the base thymine. Tc‐TTP turned out to be a substrate for the Vent (exo^?) DNA polymerase, a polymerase that allows for multiple incorporations of tc‐T nucleotides under primer extension reaction conditions. However, the substrate acceptance is rather low, as also observed for other sugar‐modified analogues. Tc‐TTP and tc‐nucleotide‐containing templates do not sustain enzymatic polymerization under physiological conditions; this indicates that tc‐DNA‐based antisense agents will not enter natural metabolic pathways that lead to long‐term toxicity.     

Incorporation of Nucleoside Probes Opposite O6‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding

O⁶‐Methylguanine (O⁶‐MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low‐fidelity Y‐family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y‐family polymerases bypass DNA adducts. Previous work showed that Dpo4‐mediated dTTP incorporation is favored opposite O⁶‐MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶‐MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶‐MeG in a DNA template. To this end, we utilized a new fluorescence‐based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶‐MeG by Dpo4. In single‐dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶‐MeG. Moreover, pyrimidine‐based dNTPs were generally better incorporated than purine‐based syn‐conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2‐position of dCTP increase the selectivity for incorporation opposite O⁶‐MeG without a significant loss of efficiency.     

Enzymatic Polymerization of Phosphonate Nucleosides

5′‐O‐Phosphonomethyl‐2′‐deoxyadenosine (PMdA) proved to be a good substrate of the Therminator polymerase. In this article, we investigated whether the A, C, T and U analogues of this phosphonate nucleoside (PMdN) series can function as substrates of natural DNA polymerases. PMdT and PMdU could only be polymerized enzymatically to a limited extent. Nevertheless, PMdA and PMdC could be incorporated into a DNA duplex with complete chain elongation by all the DNA polymerases tested. A mixed sequence of four nucleotides containing modified C, T and A residues could be obtained with the Vent(exo^?) and Therminator polymerases. The kinetic values for the incorporation of PMdA by Vent(exo^?) polymerase were determined; a reduced K_M value was found for the incorporation of PMdA compared to the natural substrate. Future polymerase directed evolution studies will allow us to select an enzyme with a heightened capacity to process these modified DNA building blocks into modified strands.     

Effect of Dietary Restriction on DNA-Adduct Formation of Benzo[a]pyrene and 2-Acetylaminofluorene in Mouse Liver

Dietary restriction (DR) alters hepatic drug metabolizing enzyme activities that affect the metabolic activation of xenobiotics. Previously, we have studied the effect of DR on the formation of benzo[a]pyrene (BaP)-DNA adducts in male Fischer 344 rats. In this study, the effects of DR on the formation of specific BaP-DNA adducts in mouse livers were investigated. In addition, 2-acetylaminofluorene (2-AAF) modified DNA adducts in livers of mice and rats were also examined. DR reduced the body and liver weights of male B6C3F₁ mice. Both the total [³H]BaP-DNA binding and the specific BaP-DNA adduct, N²-dG-BaP, detected by ³²P-postlabeling technique, were found to be greater in DR mice than in animals fed al libitum (AL). The formation of the 2-AAF-DNA adduct, AF-C8-dG, was not affected by DR. Similar results were obtained from the in vitro DNA adduct formation of these two carcinogens mediated by rat and mouse liver microsomes. The effect of DR on the formation of BaP- and 2-AAF-modified DNA adducts is discussed.     

DNA Adducts Formed in vitro from Fractionated Extract of Urban Air Particles

This study is a part of an ongoing interdisciplinary project on the health effects of air pollution (Teplice Program) in the highly polluted district of Teplice, Northern Bohemia. In our previous studies we found the relationship between DNA adduct levels detected in white blood cells of selected Teplice population and personal exposure to PAH associated with respirable particles. In this pilot study we used ³²P-postlabeling assay for DNA adduct detection in an in vitro model system for evaluation of genotoxic activity of fractionated urban air extractable organic matter (EOM). To identify some of the specific DNA adducts formed we coupled TLC with HPLC analysis of labeled adducts. The urban air particles were collected by high-volume sampler during January-March 1992 in Teplice. EOM was extracted by dichlormethane (DCM) and crude extract was fractionated into five fractions to obtain a gross partition of different chemical classes. Fractions were incubated with calf thymus DNA (dose 100 μg/ml incubate) under oxidative and reductive conditions using two metabolic activation system: 1) an oxidative rat liver S9 system (S9) and 2) a reductive xanthine oxidase catalyzed system (XO). The different DNA adduct patterns and levels were determined using S9 and XO-mediated metabolism followed by postlabeling with both nuclease P1 and butanol extraction enrichment procedures for all fractions examined. The moderately polar fraction (DCM) contained over 50% of the total DNA adduct forming activity both without and with S9 activation. The highly polar fraction (methanol) contained about 60 % of the DNA adduct forming activity under reductive conditions. Some of the main distinct DNA adducts obtained with S9 and XO mediated metabolism were tentatively identified by HPLC comparing with standards of PAH- and nitro-PAH DNA adducts.     

Relationship between Mutagenicity of Heterocyclic Aromatic Amines and Stability of Their DNA Adducts: A Study by Semi-empirical Molecular Orbital Method

Quantitative structure-mutagenicity correlations were investigated for heterocyclic aromatic amines (HCAs) by use of a DNA model with three-base pairs. DNA adducts of thirteen HCAs were optimized by the PM3 method and energy decrease, ΔE, of each HCA due to formation of a DNA adduct was obtained as the stability of the adduct. The calculations for the HCA-DNA adducts revealed the interaction between HCA's methyl group and DNA's phosphate, which plays an important role in the stabilization of the adducts. The ΔE values plotted against the logarithm of HCA's mutagenicity, M, provided an almost straight line with the regression coefficient (R) of ?0.89 (R ²= 0.79). This good correlation suggests that binding reaction between HCA's nitrenium ion and DNA is an important rate-determining step in the metabolic transformation of HCAs.     

DNA Adducts Derived from Diesel Emissions - Assessment of Genotoxic Potency in Vitro and Human Exposure Monitoring

Diesel particle extracts, which originated from three different diesel fuels, were used to study the activation of polycyclic aromatic hydrocarbons (PAHs). DNA adducts were analyzed in vitro calf thymus, human skin tissue culture and in lymphocytes isolated from diesel exposed workers. Direct-acting mutagens (e.g. nitro-PAHs) measured by Ames test were compared with DNA adducts formed in vitro by nitroreductive xanthine oxide enzyme. PAH-DNA adducts were analyzed by ³²P-postlabeling, and when characterizing adducts from skin DNA, a solid-phase micro extraction (SPME) method was developed for sample preparation before HPLC analysis. A good accordance between mutagenicity and DNA adducts showed that the three extracts contain higher amounts of direct-acting PAHs than the PAHs needing S9 activation. Skin DNA adducts demonstrated two-fold differences between the tissue cultures. ³²P-postlabeling and HPLC analysis did not confirm the identity of skin DNA adducts with the BPDE-DNA standard. The pilot study on 13 diesd exposed bus garage and waste collection workers showed low levels of PAH exposure (<50 ng/m³) and lymphocyte PAH-DNA adducts less than 2 adducts/10⁸ nucleotides.     

O4‐Alkylated‐2‐Deoxyuridine Repair by O6‐Alkylguanine DNA Alkyltransferase is Augmented by a C5‐Fluorine Modification

Oligonucleotides containing various adducts, including ethyl, benzyl, 4‐hydroxybutyl and 7‐hydroxyheptyl groups, at the O⁴ atom of 5‐fluoro‐O⁴‐alkyl‐2′‐deoxyuridine were prepared by solid‐phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5‐fluoro‐2′‐deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B‐form DNA structure. O⁶‐Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5‐fluoro‐O⁴‐benzyl‐2′‐deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O⁴‐ethyl and benzyl adducts of 5‐fluoro‐2‐deoxyuridine. Computational assessment of N1‐methyl analogues of the O⁴‐alkylated nucleobases revealed that the C5‐fluorine modification had an influence on reducing the electron density of the O⁴?C_α bond, relative to thymine (C5‐methyl) and uracil (C5‐hydrogen). These results reveal the positive influence of the C5‐fluorine atom on the repair of larger O⁴‐alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT.     

Biotransformation and DNA Adduct Formation of Trans-8,9-Dihydroxy-8,9-Dihydrodibenzo[a,l]Pyrene by Induced Rat Liver and Human CYP1A1 Microsomes

In order to explain the adduct patterns observed from the human CYP1A1-mediated binding of dibenzo[a, l]pyrene (DB[a, l]P) to DNA, we have investigated the further metabolism and DNA adduct activity of trans-DB[a, l]P-8,9-diol by induced rat liver and human CYP1A1 microsomes. trans-DB[a, l]P-8,9-diol was synthesized and metabolic studies with β-naphthoflavone-induced rat liver microsomes indicated three major metabolites: 2 diastereomers of trans,trans-8,9,11,12-tetrahydro-8,9,11,12-tetrahydroxy-DB[a, l]P and 8,9,13,14-tetrahydro-8,9,13,14-tetrahydroxy-DB[a, l]P. DB[a, l]P when activated by CYP1A1/epoxide hydrase (EH) and calf thymus DNA gave a complex pattern of DNA adducts most of which cochromatograph with syn- and anti-DB[a, l]P fjord region diol epoxide-DNA standards. Two highly polar eluting adducts were also observed, one which cochromatographs with the single major DNA adduct obtained from the CYP1A1/EH activation of trans-DB[a, l]P-8,9-diol. The relative retention time of this adduct suggests either a bis-diol epoxide adduct or a more polar diol epoxide adduct.     

Synthesis and Structure Determination of the Adducts of Dibenzo[a,l]pyrene Diol Epoxides and Deoxyadenosine or Deoxyguanosine

Abstract

(±)?Syn?dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE) and (±)?anti?DB[a,l]PDE were reacted with deoxyadenosine (dA) or deoxyguanosine (dG) in dimethylformamide at 100 °C for 30 min. The crude products were purified by reverse phase HPLC under gradient and isocratic conditions. The structure of each adduct was assigned by 1D and 2D NMR spectra and by fast atom bombardment mass spectrometry. Five adducts were isolated from the reaction of (±)?syn?DB[a,l]PDE and dA: syn?DB[a,l]PDE?N⁶dA?1, syn?DB[a,l]PDE?N⁶dA?2, syn?DB[a,l]PDE?N⁶dA?3, syn?DB[a,l]PDE?N⁶dA?4 and syn?DB[a,l]PDE?N7Ade. Four adducts were isolated from the reaction of (±)?anti?DB[a,l]PDE and dA: anti?DB[a,l]PDE?N⁶dA?1, anti?DB[a,l]PDE?N⁶dA?2, anti?DB[a,l]PDE?N⁶dA?3 and anti?DB[a,l]PDE?N⁶dA?4. Two adducts were isolated from the reaction of (±)?syn?DB[a,l]PDE and dG: (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N²dG and (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N7Gua. Two adducts were isolated from the reaction of (±)?anti?DB[a,l]PDE and dG: (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N²dG and (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N7Gua.     

Synthesis and dissociation of amine‐blocked diisocyanates and polyurethane prepolymers

Substituted N‐methylanilines are shown to act as blocking agents for toluenediisocyanate. N‐methylaniline‐, N‐methyl‐p‐anisidine‐ and N‐methyl‐p‐nitroaniline‐blocked toluene diisocyanates have been prepared and characterized by FTIR, ¹H NMR and ¹³C NMR spectroscopies, and nitrogen content analysis. A new method for determining the minimum deblocking temperature of the blocked isocyanate is described. The method has advantages in that it can be used to find the minimum deblocking temperature of even non‐volatile blocking agents. The minimum deblocking temperature of the adducts is found to be in the following order: N‐methyl‐p‐anisidine–TDI adduct < N‐methyaniline–TDI adduct < N‐methyl‐p‐nitroaniline–TDI adduct. The anilines exhibit the same trend when they block a polyurethane prepolymer prepared using polypropylene glycol of molecular weight 2000 g mol^?1 and tolylene‐2,6‐diisocyanate. The deblocking temperatures are lower in the case of blocked prepolymers than in the blocked adducts. The blocked adducts and prepolymers are reacted with pyromellitic dianhydride (PMDA) in dimethylpropylene urea (DMPU) and the evolution of carbon dioxide is monitored to study the completion of imidization. The reaction time is in accordance with the deblocking ability of the adducts. The regeneration of the blocking agent is confirmed by gas chromatography. © 2002 Society of Chemical Industry     

Metabolism and DNA-Binding of 3-Nitrobenzanthrone in Primary Rat Alveolar Type II Cells,in Human Fetal Bronchial,Rat Epithelial and Mesenchymal Cell Lines

3-Nitrobenzanthrone (NBA) is a suspected human carcinogen and has been identified in diesel exhaust and in airborne particulates. Human exposure to NBA is thought to occur primarily via the respiratory tract and bronchial as well as alveolar epithelial cells are believed to be primary targets for lung carcinogenesis. Nitroarenes require metabolic activation to DNA binding metabolites to become genotoxic carcinogens. In this study the metabolism of NBA as well as its metabolic intermediate 3-nitrosobenzanthrone was investigated in cultures of rat lung alveolar type II cells, in human fetal bronchial (HFBE) and rat bronchial epithelial (R3/1) as well as mesenchymal Rwd009 cells. 3-Aminobenzanthrone (ABA) was identified as the major metabolite from both substrates, but also small amounts of 3-acetyl-ABA were observed during short term incubations (6 to 24 h) with NBA. Inhibition studies with allopurinol in alveolar type II cells indicate that the cytosolic enzyme xanthine oxidase contributes substantially to the biotransformation of NBA. ³²?Postlabeling analysis of DNA adducts in these cells demonstrates the formation of 5 and 6 different adducts after exposure of the cells with NBA and 3-nitrosobenzanthrone, respectively. Different oligonucleotides were modified with N?acetoxy-N?acetyl-3-ABA and used as reference materials for postlabeling analysis. Based on co-chromatography experiments, the presence of N?acetoxy-ABA-dA adducts in alveolar type II epithelial cells could be excluded. In conclusion, it was shown that metabolic conversion of NBA is associated with DNA adduct formation in rat alveolar type II epithelial cells. The formation and covalent DNA binding of reactive NBA metabolites may provide the rational for a mechanism of lung carcinogenesis based on direct genotoxicity.     

Transient N4-Acyl-DNA Protection against Cleavage by Restriction Endonucleases

A set of five N⁴-acyl-modified 2′-deoxycytidine 5′-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N⁴-acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N⁴-acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N⁴ modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N⁴-modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.     

Synthesis of Stereoisomeric N 6-Deoxyadenosine Adducts of syn- and anti- Dihydrodiol Epoxides of Benzo[a]pyrene and Their Incorporation into 18-mer DNA Sequences from Human Ha-ras Protooncogene

Oligonucleotide sequences synthesized with specifically positioned and structurally defined adducts of dihydrodiol epoxides (DE) of polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) are useful tools to study in detail the solution structure of corresponding duplexes by NMR techniques as well as the interaction of a single adduct with DNA polymerases. Here we report the successful incorporation of trans-N ⁶-dA-B[a]PDE adducts derived from the syn- and anti-diastereomers of B[a]PDE into 18-mer oligonucleotides representing partial human Ha-ras sequences surrounding codon 61 (CAG). The key step in our approach is a nucleophilic displacement reaction of a deoxyinosine derivative activated at the 6-position by a sulfonate group with a racemic aminotriol providing a regio- and stereospecific synthesis of the N ⁶-dA adducts. The aminotriol precursors are prepared by direct aminolysis of the DE's or by a stereoselective opening of the oxirane ring with sodium azide followed by reduction. The fully protected diastereomeric trans-N ⁶-dA-B[a]PDE adducts were separated by preparative HPLC and subsequently transformed into the corresponding phosphoramidites. The synthesis of four 18-mers was performed on a DNA synthesizer incorporating in each sequence [d(5′-GGC·CA*G·GAG·GAG·TAC·AGC-3′)] a single dA adduct (A*) at Codon 61 using standard phosphoramidite chemistry. After extensive purification of the 18-mers by reverse phase HPLC and analysis by PAGE, the presence of the trans-N⁶ -dA-B[a]PDE adducts in the oligonucleotides was confirmed by fluorescence spectroscopy.     

Glutathione Conjugation and DANN Adduct Formation of Diol Epoxides in V79 Cells Expressing Human Glutathione Transferase P1-1

V79 cells and cells over-expressing glutathione transferase (GST) P1-1 have been incubated with the (+)- and (?)-anti?enantiomers of trans?7,8-dihydroxy-9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and trans?9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydrobenzo[c]chrysene (BCDE) and the formation of glutathione (GSH) conjugates and DNA adducts monitored. With (+)-anti?BPDE and (?)-anti?BCDE, the results demonstrate a several fold increase in conjugate formation concomitant with reduced levels of DNA adducts in GST expressing cells relative to control cells. Thus, the effects were restricted to the enantiomers with R?absolute configuration at the benzylic oxirane carbon. The rate of conjugate formation of BCDE relative to BPDE is significantly lower indicating reduced accessibility of the more lipophilic BCDE for GSTP1-1.     

Preparation of diastereomerically pure 9-carboxybicyclo[6.1.0]nonane derivatives

Reactions of cyclooctene ( 1 ) with dibromocyanoacetic esters and copper(I) bromide give (8–10) : 1 mixtures of isomers ( 2 , 3 ), not stereochemically pure compounds as reported by others. The stereochemistry is elucidated by independent synthesis of one diastereomer ( 3a ). Carbenoid addition of alkoxycarbonylmethylene to 1 and 1,5-cyclooctadiene leads also to exo/endo adduct mixtures. Methods are developed to generate diastereomerically pure compounds ( exo -7a , exo -8, endo -9 , exo -10 , endo -10 ) from these. Endo esters of this series undergo very facile base catalyzed epimerization.     

Production and Immunochemical Characterization of a High-Affinity Monoclonal Antibody Specific for 7-(Benzo[a]pyren-6-yl)guanine (Bp-6-N7GUA), a Depurinating DNA Adduct of Benzo[a]pyrene

Molecular dosimetry of depurinating DNA adducts of benzo[a]pyrene (BP) offers a promising new approach to determining risk of PAH-induced cancer. Depurinating adducts are the predominant form of BP-induced DNA damage, are excreted in urine and, importantly, are linked to cancer initiation. We have produced a monoclonal antibody (MAb) with high specific affinity for BP-6-N7Gua, a major depurinating DNA adduct of BP, and have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA). The I_50S (quantity producing 50% inhibition of MAb binding in the ELISA) of selected BP adducts and metabolites were determined. The results indicated 1) a high degree of discrimination between BP-6-N7Gua (I₅₀=750 fmol) and BP (I₅₀=900,000 fmol), 2) high affinity of the MAb for BP-6-N7Ade (I₅₀=1,500 fmol), another major depurinating DNA adduct, and 3) specific structural requirements for MAb-adduct binding. In addition, the competitive ELISA provided an accurate determination of BP-6-N7Gua added to normal human urine, at a sensitivity of 200 fmol.