Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells.

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed.

Spectroscopic studies of the interaction of aspirin and its important metabolite, salicylate ion, with DNA, A·T and G·C rich sequences.

Among different biological effects of acetylsalicylic acid (ASA), its anticancer property is controversial. Since ASA hydrolyzes rapidly to salicylic acid (SA), especially in the blood, interaction of both ASA and SA (as the small molecules) with ctDNA, oligo(dA·dT)15 and oligo(dG·dC)15, as a possible mechanism of their action, is investigated here. The results show that the rate of ASA hydrolysis in the absence and presence of ctDNA is similar. The spectrophotometric results indicate that both ASA and SA cooperatively bind to ctDNA. The binding constants (K) are (1.7±0.7)×10(3) M(-1) and (6.7±0.2)×10(3) M(-1) for ASA and SA, respectively. Both ligands quench the fluorescence emission of ethidium bromide (Et)-ctDNA complex. The Scatchard plots indicate the non-displacement based quenching (non-intercalative binding). The circular dichroism (CD) spectra of ASA- or SA-ctDsNA complexes show the minor distortion of ctDNA structure, with no characteristic peaks for intercalation of ligands. Tm of ctDNA is decreased up to 3°C upon ASA binding. The CD results also indicate more distortions on oligo(dG·dC)15 structure due to the binding of both ASA and SA in comparison with oligo(dA·dT)15. All data indicate the more affinity for SA binding with DNA minor groove in comparison with ASA which has more hydrophobic character.

A contact photo-cross-linking investigation of the active site of the 8-17 deoxyribozyme.

The small RNA-cleaving 8-17 deoxyribozyme (DNAzyme) has been the subject of extensive mechanistic and structural investigation, including a number of recent single-molecule studies of its global folding. Little detailed insight exists, however, into this DNAzyme's active site; for instance, the identity of specific nucleotides that are proximal to or in contact with the scissile site in the substrate. Here, we report a systematic replacement of a number of bases within the magnesium-folded DNAzyme-substrate complex with thio- and halogen-substituted base analogues, which were then photochemically activated to generate contact cross-links within the complex. Mapping of the cross-links revealed a striking pattern of DNAzyme-substrate cross-links but an absence of significant intra-DNAzyme cross-links. Notably, the two nucleotides directly flanking the scissile phosphodiester cross-linked strongly with functionally important elements within the DNAzyme, the thymine of a G.T wobble base pair, a WCGR bulge loop, and a terminal AGC loop. Mutation of the wobble base pair to a G-C pair led to a significant folding instability of the DNAzyme-substrate complex. The cross-linking patterns obtained were used to generate a model for the DNAzyme's active site that had the substrate's scissile phosphodiester sandwiched between the DNAzyme's wobble thymine and its AGC and WCGR loops.

Mutagenic mechanism of the A-T to G-C transition induced by 5-bromouracil: an ab Initio Study.

The tautomerisms of uracil, 5-bromouracil (BrU), G-U, G-BrU, A-U, and A-BrU have been studied theoretically in an effort to investigate the mutagenicity of BrU. The ab initio calculations have been performed using HF and B3LYP methods with various basis sets. The relative stability of all tautomers was established. The intermolecular interactions between U, BrU, U*, BrU* (asterisks denote enol forms), and water have been studied. It shows that the possibility of tautomerism from BrU to BrU* is much more likely than that from U to U*. Further research indicates that BrU* tends to pair with guanine more easily than U*. The proton transfer process has been investigated by potential energy surface (PES) scan and transition state analysis. The results show that the proton transfer between G-U* and G*-U is monodirectional barrier-free proton transfer (BFPT), which terminates the base mismatch induced by U*. On the other hand, the proton transfer between G-BrU* and G*-BrU is bidirectional BFPT, which makes the base mismatch induced by BrU* sustained. On the basis of all of these calculated results, a new mutagenic mechanism for the A-T to G-C transition induced by 5-bromouracil is described in detail for the first time. It might give a new insight into the origin of the mutagenicity of the 5-Br derivative.

The value of DNA methylation analysis in basic, initial toxicity assessments.

DNA methylation is an epigenetic mechanism regulating patterns of gene expression. Our goal was to see if the assessment of DNA methylation might be a useful tool, when used in conjunction with initial, basic in vitro tests, to provide a more informative preliminary appraisal of the toxic potential of chemicals to prioritize them for further evaluation. We sought to give better indications of a compound's toxic potential and its possible mechanism of action at an earlier time and, thereby, contribute to a rational approach of an overall reduction in testing by making improved early decisions. Global and GC-rich patterns of DNA methylation were evaluated along with more traditional cytolethality measurements, e.g., cytolethality and genotoxicity assessments, on rat hepatoma (H4IIE) cells. The relative toxic potential of model compounds camptothecin, 5-fluorouracil, rotenone, and staurosporine was estimated by employing DNA methylation assessments combined with our cytolethality data plus genotoxicity information gleaned from the literature. The overall contribution of the methylation assessment was threefold; it (1) strengthened a ranking based on genotoxicity; (2) provided an indication that a compound might be more potentially problematic than what cytolethality and genotoxicity assessments alone would indicate; and (3) suggested that compounds, particularly nongenotoxins, that are more potent regarding their ability to alter methylation, especially at noncytolethal concentrations, may be more potentially toxic. Altered methylation per se is not proof of toxicity; this needs to be viewed as a component of an evaluation.

Pentamidine-induced alteration in restriction endonuclease cleavage of plasmid DNA.

We have used restriction enzymes and DNaseI as probes to determine the specificity of pentamidine binding to plasmid DNA. Cleavage of plasmid pAZ130 by EcoRI, EcoRV and ApaI is inhibited by pentamidine, cleavage by XbaI, NotI and AvaI is unaffected, while cleavage by XhoI, which recognizes the same sequence as AvaI, is stimulated. DNaseI footprinting of DNA containing these restriction sites revealed that pentamidine protection is not strictly limited to AT-rich regions. We suggest that perturbation of the DNA micro- environment by pentamidine binding is responsible for its effect on nucleases.

TectoRNA: modular assembly units for the construction of RNA nano-objects.

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.

Hydroxyl radicals are involved in the oxidation of isolated and cellular DNA bases by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA) is a heme precursor, pathological accumulation of which is associated with liver cancer. We show that the reactive oxygen species produced upon ALA metal-catalyzed oxidation promote the formation of several radical-induced base degradation products in isolated DNA. The distribution of modified bases is similar to that obtained upon gamma irradiation. This observation strongly suggests the involvement of hydroxyl radicals in the ALA-mediated DNA damage. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-hydroxy-2'-deoxycytidine in organ DNA of rats chronically treated with ALA were observed. This is strongly suggestive of the implication of hydroxyl radicals in the ALA-induced degradation of cellular DNA.

Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG1 and/or abasic site formation.

Oxidative damage of DNA by endogenously generated oxygen radicals contributes to the mutagenic process. Hydroxy, alkoxy, and peroxy radicals all have the potential to react with DNA, giving rise to strand breaks and potentially mutagenic oxidative base damage. Although reactions of the hydroxy radical with DNA have been well studied, far less is known about the reactivities of these other radicals with DNA and their mutation-inducing potential. Frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation were measured by glyoxal gel electrophoretic analysis. We report the spectrum of mutations induced in Escherichia coli upon transfection with peroxy radical treated DNA carrying the lacZ alpha gene as a reporter. Transfection of DNA exposed to micromolar amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS-inducible cells. Sequencing analysis of DNA isolated from mutants showed that among base substitution mutants 88% consisted of transversions at G, with a nearly equal number of G --> C and G --> T mutants. Transition mutations were rarely detected, in contrast to control experiments. Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed to any detectable degree. The oxidative G lesions are sensitive to digestion by the Fpg protein. We were unable to detect the formation of 8-oxo-dG by HPLC/electrochemical analysis of peroxy radical oxidation of dG, suggesting that the G --> T transversions were not caused by this base lesion.

Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.

In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924].

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene.

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the supF gene of Escherichia coli were recovered from bacteria deficient in nucleotide excision repair and in DNA-alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7-fold above the spontaneous value. A total of 73 independent supF- mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle-vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C-->A:T transitions. A substantial proportion (6/18) of CCNU-induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C-->A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O6-chloroethylguanine lesion with thymine. Most G:C-->A:T transitions (34/39) were located at three 5'-GG-3' hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C-->A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6-chloroethylguanine formation, reducing its incidence in the transcribed DNA strand.

Base sequence selectivity in the alkylation of DNA by 1,3-dialkyl-3-acyltriazenes.

The base sequence selectivity of DNA alkylation for a series of structurally related 1,3-dialkyl-3-acyltriazenes was examined with calf thymus DNA or polymers containing the sequences GGG, CGC, TGT, and AGA. The reaction products at the N7 and the O6 positions of guanine were identified, quantitated, and then correlated with the decomposition rates of the triazenes, 1-(2-chloroethyl)-3-methyl-3-carbethoxy- (CMC), 1-(2-chloroethyl)-3-methyl-3-acetyl- (CMA), 1-(2-hydroxyethyl)-3-methyl-3-carbethoxy- (HMC), 1-(2-hydroxyethyl)-3-methyl-3-acetyl- (HMA), and 1,3-dimethyl-3-acetyl- (DMA). The results of these studies revealed that DNA sequences with runs of purines were more reactive toward alkylation by all of the triazenes tested, irrespective of whether the alkylation was measured by N7, O6, or total guanine adducts. Within this generalization, the (hydroxyethyl)triazenes showed a preference for the AGA sequence, while the (chloroethyl)triazenes favored the GGG sequence. The structure of the 3-acyl group of the triazene also played a role in the extent of alkylation of a particular sequence of DNA. Both the (chloroethyl)- and the (hydroxyethyl)triazenes produced higher alkylation product yields for the 3-carbethoxytriazenes as compared with the 3-acetyl derivatives for most of the sequences examined. These overall patterns correlated well with the order of decomposition of the triazenes at 37 degrees C: HMC > DMA > HMA > CMC > CMA. This study has demonstrated how varying the structure of 1,3-dialkyl-3-acyltriazenes can modulate DNA alkylation, a finding which may be important in the design of new triazene antitumor agents.

The effect of AT and GC sequence specific minor groove-binding agents on restriction endonuclease activity.

The ability of the naturally occurring A/T specific DNA minor groove binders netropsin and diastamycin A and two synthetic G/C selective oligopeptide analogues (1 and 2), to interfere with the catalytic activity of restriction endonucleases has been investigated. Enzymes were chosen to have A/T rich (EcoRI, EcoRV) or G/C rich (BalI, NruI) recognition sequences. An agarose gel assay was used to measure the cleavage of 32P-labelled DNA and ligand-DNA binding data was obtained using methidium-propyl EDTA footprinting. Netropsin and distamycin bind at the recognition sites, and dose-dependently inhibited cleavage by, EcoRI and EcoRV, (EcoRI > EcoRV). They were also more effective at inhibiting the catalytic activity of BalI than either 1 or 2. NruI was inhibited by distamycin and 2, but not by netropsin or 1. DNA footprinting revealed that neither 1 or 2 bound to the BalI or NruI recognition sequences under the conditions used whereas netropsin and distamycin footprint at adjacent sites. 1 binds to two of the three recognition sequences for the enzyme Fnu4HI (GCNGC) in the fragment studied and was shown to inhibit DNA cleavage only at these two sites. 2 binds strongly to two GGGCTC sequences which are recognition sites for the enzyme BanII. In this case a pronounced stimulation of cleavage was observed in the presence of 2 over a wide dose range. The results indicate that enzyme inhibition does not necessarily result from simultaneous occupancy of a common site, or at nearby flanking sequences, and in some circumstances, a pronounced stimulation of enzyme cleavage can occur.

DNA base and deoxyribose modification by the carbon-centered radical generated from 4-(hydroxymethyl)benzenediazonium salt, a carcinogen in mushroom.

Modification of the base and the sugar moieties of DNA with 4-(hydroxymethyl)benzenediazonium salt (HMBD), a carcinogen in the mushroom Agaricus bisporus, was investigated. When deoxyribonucleosides dGuo, dAdo, dThd, and dCyd were incubated with HMBD at pH 7.4 and 37 degrees C, the levels of all the nucleosides were decreased. The decrease was inhibited by ethanol or Cys. When deoxyribose was incubated with HMBD, malonaldehyde was released as assessed by the thiobarbituric acid reactivity. The release was inhibited by ethanol. Major products of the reaction of dGuo and dAdo with HMBD were isolated, and their structures were established to be 8-[4-(hydroxymethyl)phenyl]dGuo (8-HMP-dGuo) and 8-[4-(hydroxymethyl)phenyl]dAdo), respectively. Calf thymus DNA treated with HMBD was enzymatically digested into nucleosides, in which 8-HMP-dGuo and 8-HMP-dAdo were detected. Formation of the modified nucleosides in DNA was inhibited by ethanol or 2-mercaptoethanol. Malonaldehyde was released from DNA treated with HMBD, which indicated that the deoxyribose moiety of DNA had been damaged. The results indicate that the 4-(hydroxymethyl)phenyl radical generated from HMBD can directly modify the base and the sugar moieties of DNA under the mild conditions. Inhibitory effect of ethanol was ascribable to its scavenging activity for the carbon-centered radical. The inhibitory effect of Cys and 2-mercaptoethanol was found to be due to the formation of the reversible adducts between HMBD and the SH compounds.

The role of mutagenic metal ions in mediating in vitro mispairing by alkylpyrimidines.

A variety of alkylating mutagens and carcinogens produce pyrimidine adducts in DNA that block DNA synthesis in vitro. Since DNA synthesis past the lesion is a necessary step to produce mutations, we investigated the role of the mutagenic metal ion Mn++ in facilitating DNA synthesis past alkylpyrimidines. In the presence of the natural metal activator Mg++, N3-ethyldeoxythymidine (N3-Et-dT) and O2-ethyldeoxythymidine (O2-Et-dT), present at a single site in DNA, blocked in vitro DNA synthesis 3' to the lesion and after incorporating dA opposite each lesion. The presence of Mn++ permitted postlesion synthesis with dT misincorporated opposite N3-Et-dT and O2-Et-dT, implicating these lesions in A.T-->T.A transversion mutagenesis. The DNA synthesis block by O4-ethyldeoxythymidine (O4-Et-dT) in the presence of Mg++ was partial and was also removed by Mn++. Consistent with in vivo studies, dG was incorporated opposite O4-Et-dT during postlesion synthesis, leading to A.T-->G.C transition mutagenesis. We also have discovered a new class of DNA adducts, N3-hydroxyalkyldeoxyuridine (3-HA-dU) lesions, which are produced by mutagenic and carcinogenic aliphatic epoxides. 3-HA-dU is formed after initial alkylation at the N3 position of dC followed by a rapid hydrolytic deamination. As observed with the analogous mutagenic N3-Et-dT, the ethylene oxide-induced 3-hydroxyethyldeoxyuridine (3-HE-dU) blocked in vitro DNA synthesis, which could be by-passed in the presence of Mn++. The nucleotide incorporated opposite 3-HE-dU during postlesion synthesis is being identified. These studies suggest a role for Mn++ in mediating mutagenic and carcinogenic effects of environmentally important ethylating agents and aliphatic epoxides.

Alkylation of guanine and cytosine in DNA by bizelesin. Evidence for a covalent immobilization leading to a proximity-driven alkylation of normally unreactive bases by a (+)-CC-1065 cross-linking compound.

Bizelesin, an intrahelical DNA-DNA interstrand cross-linker related to (+)-CC-1065, has been shown to alkylate DNA through guanine in restriction enzyme sequences in which there is a suitably positioned adenine contained in a highly reactive monoalkylation sequence on the opposite strand. Oligomers containing the sequence 5'-TTTTTN*, in which "N" was either G, C, or T, were synthesized to evaluate the cross-linking potential of bizelesin at nonadenine bases. Kinetic analysis of monoalkylation and cross-linking events demonstrates that it is the reaction at "N" (guanine or cytosine) that results in the cross-link which is the slow step. On the basis of this analysis and the normal unreactivity of guanine and cytosine to alkylation by the cyclopropapyrroloindole alkylating moiety of (+)-CC-1065, we propose that the molecular mechanism for this type of cross-linking reaction most likely involves a covalent immobilization of the second alkylating arm, resulting in a "proximity-driven" reaction.

Identification of hydroxyl radical-induced lesions in DNA base structure: biomarkers with a putative link to cancer development.

Hydroxyl radical-induced DNA base lesions of guanine and adenine were originally found in neoplastic and microscopically normal livers of fish exposed to environmental carcinogens. They were later identified in a mammalian tissue--the cancerous female breast. This evidence suggested that the base lesions are broadly present in the cancerous and microscopically normal tissues of a variety of eukaryotic organisms. The base lesion concentrations in both neoplastic tissues frequently exceeded 1 modified base in 1000 normal bases. By contrast, the base lesion:normal base ratios in healthy tissues were generally 10-100 times less. A greater variety of base lesions was found subsequently in the cancerous lung, brain, and other human tissues, although information relating to their biological significance is largely confined to the originally found purine derivatives. The biochemistry of the base lesions and the relationship of ring-opening (Fapy) derivatives to OH adducts in the DNA of normal and cancerous tissues is discussed with regard to the etiology of cancer and the potential use of the lesions as biomarkers for cancer risk assessment.

N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli: multiple roles for UmuC protein.

Backmutations of an ochre (UAA) nonsense defect in the tyrA gene of Escherichia coli were induced by N-ethyl-N-nitrosourea (ENU) in both UmuC+ and UmuC- strains. This site is particularly flexible to base substitution mutation and all but one (TAA-->TGA) substitution can be recovered using a reversion assay. Employing direct sequencing of polymerase chain reaction-amplified genomic DNA and/or colony-hybridization methods, the changes induced by several separate doses of ENU in a total of 587 independent backmutations were investigated. In the UmuC+ strain, all possible single-base substitutions were recovered. Different frequencies for individual single-base substitutions were obtained and correlations with the surrounding base sequence could be seen. Transitions occurred most frequently at thymine residues having a purine on the 5'-side while transversions occurred more frequently at thymine residues having a cytosine on the 5'-side. In the UmuC- strain, ENU failed to induce A:T-->T:A and A:T-->C:G transversions and the frequency of A:T-->G:C transitions was reduced. However, an unidentified class of extragenic mutations were induced to a greater extent. These results suggest distinct pathways for ENU-induced mutagenesis at ethylated thymine residues and delineate several separate functions for the UmuC protein.

8-Aminoguanine: a base modification produced in rat liver nucleic acids by the hepatocarcinogen 2-nitropropane.

2-Nitropropane (2-NP), an important industrial chemical and a hepatocarcinogen in rats, had previously been found to produce several modifications of nucleosides in rat liver RNA and DNA that are discernible using HPLC with electrochemical detection. While one of these modifications has been identified as an increase in the levels of 8-oxoguanosine and 8-oxo-2'-deoxyguanosine in RNA and DNA, respectively, the others had not been identified. We now present evidence that a major modification in rat liver nucleic acids due to the administration of 2-NP is the amination of guanine at C8, apparently a completely novel in vivo reaction. 8-Aminoguanosine, isolated from hydrolysates of liver RNA from 2-NP-treated rats, cochromatographed with synthetic or commercially-obtained standard on reverse-phase as well as cation-exchange HPLC, and its UV spectral characteristics at acidic, neutral, and basic pH were identical to those of the standard. Acid hydrolysis produced 8-aminoguanine, which had a retention time and fragmentation pattern identical to that of the standard on gas chromatography-mass spectrometry of the trimethylsilyl derivatives. Evidence for the presence of 8-aminodeoxyguanosine in liver DNA of rats treated with 2-NP was also obtained by cochromatography with synthetic standard on HPLC. Hydroxylamine-O-sulfonic acid was found to react with RNA and DNA to give 8-oxo- and 8-amino-substituted guanines. We propose, as a working hypothesis, that 2-NP may be metabolized to hydroxylamine-O-sulfonate or acetate, which yield the reactive nitrenium ion, NH2+, capable of aminating cellular macromolecules in vivo.

[Defects in the secondary structure of DNA recognizable by nuclease S1 and their possible role in the aging of mammalian somatic cells]. / Defekty vtorichnoi struktury DNK, opoznavaemye nukleazoi S1, i ikh vozmozhnanaia rol' v starenii somaticheskikh kletok mlekopitaiushchikh.

The amount of defects in DNA secondary structure recognized by nuclease S1 and their localization by sequences of various nucleotide composition were studied in relation to the age factor. It was established that a significant twofold increase in defects of DNA secondary structure was observed in hepatic cells of very old (30 months and older) intact mice only, as well as in radiation-induced acceleration of aging at the age 19 months. The prevailing localization of the defects of DNA secondary structure has been demonstrated in those sequences which were enriched with AT pairs by 3% (as compared with the average level). It was concluded that the mentioned defects of DNA secondary structure are not the cause of aging. Nevertheless they can play an essential role in the process of irreversible destruction of genome in the terminal phase of aging.