Increasing the sensitivity of an immunohistochemical method for detecting carcinogen adducts with DNA]

Application of polyclonal antibodies against N7-methylguanine (N7-MG) for immunohistochemical detection of the DNA damage following exposure to methylnitrosourea in cultured cells (CHO and IAR-27) has been shown. The approaches were applied: a) an extraction of nuclei, and b) a silver intensification of the end product of peroxidase reaction. Both the approaches were effective in increasing the sensitivity of immunohistochemical detection of N7-MG, the latter technique being most sensitive. It is proposed that the silver intensification technique is very perspective to have a high levels of sensitivity and specificity, a sufficient contrast and preservation of the cell morphology. The efficiency of the new approach in application to the purposes of molecular epidemiology is discussed.     

Monitoring of dimethyl sulphate-induced N3-methyladenine, N7-methylguanine and O(6)-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry

This work describes the determination of N3-methyladenine, N7-methylguanine and O(6)-methylguanine adducts in dimethyl sulphate-treated salmon-testes DNA employing reversed-phase high performance liquid chromatography (RP-HPLC) with UV-vis detection, followed by mass-spectrometric verification using electrospray ionisation in positive mode ESI(+). Within validation parameters, accuracy, precision, calibration parameters, limit of detection (LOD) and quantitation (LOQ) as well as stability of standard stock solutions were tested and presented for UV/vis detection. The limit of detection (LOD) was found to be 0.1 ng/mL for N3-methyladenine and 0.2 ng/mL for both N7-methylguanine and O(6)-methylguanine (S/N=3). The limit of quantitation (LOQ) was found to be 0.5 ng/mL for all measured compounds, (S/N=10). Quantitative results were obtained for each substance based on eight-point calibration. Intra- and inter-day precisions were within 1.73-6.96 and 2.26-7.58%, respectively, and correlation coefficients of calibration curves (R(2)) ranged from 0.9992 to 0.9997. Relative proportion of N7-methylguanine was accounted for 61.53+/-2.97% (R.S.D.=4.8), N3-methyladenine for 38.19+/-2.99% (R.S.D.=9.6) and O(6)-methylguanine for 0.29+/-0.02% (R.S.D.=5.1), respectively. The application of the above-mentioned techniques provides a valuable contribution for simultaneous determination of methylated DNA adducts, and may represent a suitable approach for similar monitoring/screening studies.     

Simultaneous quantitation of 7-methyl- and O6-methylguanine adducts in DNA by liquid chromatography-positive electrospray tandem mass spectrometry

A methodology has been developed and validated for the simultaneous quantitation of O6-methyl- and 7-methylguanine in DNA isolated from in vitro exposure to the model alkylating agents: N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS). After exposure, DNA was isolated and directly hydrolyzed under acid conditions to hydrolytes containing DNA bases (modified and unmodified). The hydrolytes were used for direct O6- and 7-methylguanine quantitation using a rapid and selective liquid chromatography-electrospray tandem mass spectrometry (LC/ESI-MS/MS). The lower limits of quantitation for O6-methyl- and 7-methylguanine were 75.8 and 151.5 fmol, respectively. Linearity of the calibration curve was greater than 0.999 from 75.8 to 151,600.0 fmol for O6-methylguanine and 0.999 from 151.5 to 303,200.0 fmol for 7-methylguanine. The intra-day assay precision relative standard deviation (R.S.D.) values for O6-methylguanine for quality control (QC) samples were < or =9.2% with accuracy values ranging from 90.8 to 118%, and for 7-methylguanine the R.S.D. values for QC samples were < or =11%, with accuracy values ranging from 92.9 to 119%. The inter-day assay precision (R.S.D.) values for O6-methylguanine QC samples were < or =7.9% with accuracy values ranging from 94.5 to 116%, and for 7-methylguanine QC samples were < or =7.1% with accuracy values ranging from 95.2 to 110.2%. This method was used for simultaneous determination of the levels of 7-methyl- and O6-methylguanine in DNA acidic hydrolytes present in a series of incubations from salmon testis DNA treated with either MNU or MMS.     

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.     

Charge distribution in 7-methylguanine regarding cation-pi interaction with protein factor eIF4E

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.     

ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N-METHYL-N-NITROSOUREA AND METHYL METHANESULPHONATE

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N -[¹⁴C]methyl- N -nitrosourea and [¹⁴C]methyl methanesulphonate. The main product with both compounds was 7-methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7-methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O ⁶-methylguanine, accounting for about 12 per cent of the radioactivity present as 7-methylguanine and cytoplasmic RNA contained about half this amount of O ⁶-methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O ⁶-methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1-methyladenine, 3-methyladenine and 3-methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.     

Immunochemical detection of 7-methylguanine residues in nucleic acids

The ability of specific antibodies to react with 7-methylguanine residues in nucleic acids was investigated. Anti-7-methylguanine specific antibodies precipitated polymers of poly-guanylic acid which were methylated to an extent of 35 or 70% at the N-7 position of guanine, indicating that these antibodies could readily detect 7-methylguanine residues in a polynucleotide. This reaction was proportional to the total amount of 7-methylguanine present, suggesting further that quantitation of these residues is possible. To determine the minimal amount required for detection, varying amounts of 7-methylguanine were introduced into calf thymus DNA by alkylation with dimethyl sulfate. While showing no reaction with denatured nonalkylated DNA, the reaction of antibodies with alkylated DNA was proportional to the amount of 7-methylguanine in the preparations. Moreover, the antibodies appeared to detect differences in the distribution of 7-methylguanine residues in extensively methylated DNA. Precipitation was observed with DNA containing as little as one 7-methylguanine residue per 300 nucleotides, suggesting that these antibodies can be used to detect biologically significant levels of 7-methylguanine in viral and cellular nucleic acids.     

Base stacking of simple mRNA cap analogues. Association of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole and purine derivatives in aqueous solution

Equilibrium constants for the association of different ionic forms of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole, caffeine and various methylated adenines have been determined by distributing the latter compounds between an organic solvent and aqueous solutions of the 7-methylguanine derivatives. The data are compared to those obtained for the association of unsubstituted purine with the same cosolutes. The stacking affinity of both cationic and zwitterionic forms of the 7-methylguanine ring correlates with the ring polarizability rather than the polarizing power of the cosolute. The cationic species stacks usually more efficiently. The chemical nature of the N9-substituent has only a moderate influence on the base-stacking properties.     

Identification of C8-methylguanine in the hydrolysates of DNA from rats administered 1,2-dimethylhydrazine. Evidence for in vivo DNA alkylation by methyl radicals.

C8-Methylguanine was identified in the neutral hydrolysates of DNA isolated from the liver or colon tissue of rats administered 1,2-dimethylhydrazine. In all the samples examined, the biologically isolated adducts were characterized by co-elution with synthetic C8-methylguanine under different high pressure liquid chromatography conditions. The sample isolated from liver DNA was also identified by UV spectroscopy at different pH values and by mass spectrometry. The estimated yields of C8-methylguanine obtained in hydrolysates of DNA from the liver or colon tissue were comparable to those of O6-methylguanine. C8-Methylguanine was not detected when the spin trap alpha-(4-pyridyl-1-oxide)-N-tert- butylnitrone was administered together with 1,2-dimethylhydrazine. The spin trap also inhibited N7-methylguanine and O6-methylguanine yields, although to a lesser extent. These results constitute the first evidence that DNA alkylation by carbon-centered radicals can occur in vivo.     

Cleavage of tRNA and rRNA at 7-methylguanine in the presence of methylated carrier RNA

A method of site-specific cleavage of some tRNAs and rRNAs at the 7-methylguanine residue is described. After reduction of 7-methylguanine by sodium borohydride treatment in the presence of the exogenous carrier RNA methylated statistically by dimethylsulfate the polynucleotide chain is cut at the modified residue by aniline. It was shown that the previously used procedure which did not involve a methylated carrier RNA is not applicable for splitting rRNAs or low concentrations of tRNAs. The method described was successfully used for site-specific cleavage of 32P-terminally labelled yeast tRNAPhe and unlabelled E. coli 16S rRNA at 7-methylguanine position.     

Mutation and DNA modification in Salmonella exposed to N-nitrosodimethylamine under UVA- and sunlight-irradiation.

Previously, we reported that when Salmonella typhimurium and Escherichia coli were treated with N-nitrosodimethylamine (NDMA) under irradiation with ultraviolet-A (UVA), mutagenesis of the bacteria took place without externally added activation enzymes. We also observed the formation of O(6)-methylguanine (O(6)-meG), N(7)-methylguanine (N(7)-meG) and 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) in calf thymus DNA treated with NDMA plus UVA. In this study, we observed the mutagenicity of NDMA under irradiation of natural sunlight in S. typhimurium. Furthermore, we detected the formation of O(6)-meG, N(7)-meG and 8-oxodG in calf thymus DNA treated with NDMA plus simulated sunlight. Regarding the mutagenesis of S. typhimurium by NDMA plus UVA, we have now identified and quantified O(6)-meG formed in the genomic DNA of the bacteria under conditions of the mutagenesis.     

Relationship of methyl purines produced by MNNG in adenovirus 5 DNA to viral inactivation in repair-deficient (Mer-) human tumor cell strains

Adenovirus 5 treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) has greater plaque-forming ability in cell strains having the Mer+ phenotype than in strains having the Mer- phenotype. MNNG-treated Mer- strains repair the N3-methyladenine (N3MeA) but not the O6-methylguanine (O6MeG) produced in their DNA, while MNNG-treated Mer+ strains repair both of these adducts. The fate of N7-methylguanine (another DNA adduct produced by MNNG) is similar in Mer+ and Mer- strains. We show in this paper that 2.3 +/- 0.4 O6MeG and 1.4 N3MeA per adenovirus genome correlate with one lethal hit when the survival assay is done using Mer- strains as viral hosts. We suggest that O6MeG is the lesion lethal to the virus.     

Simultaneous determination of N7-alkylguanines in DNA by isotope-dilution LC-tandem MS coupled with automated solid-phase extraction and its application to a small fish model

In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.     

Quantitative high-pressure liquid chromatographic analysis of methylated purines in DNA of rats treated with chemical carcinogens

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O⁶-methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.     

DNA methylation by tert-butyl hydroperoxide-iron (II): a role for the transition metal ion in the production of DNA base adducts.

Metabolic degradation of both endogenous and exogenous peroxides is associated with the etiology of several diseases including cancer. Tert-butyl hydroperoxide (TBHP) has been widely employed as a model compound to study the cytotoxicity and promoting effects of organic peroxides. Recently, we reported that incubations of TBHP with iron (II) and calf thymus DNA led to generation of high yields of methyl radicals and to DNA methylation. Interestingly, DNA was methylated to products expected from both free radical and ionic mechanisms such as 8-methylguanine (C8-MeGua) and 7-methylguanine (N7-MeGua), respectively. To elucidate the mechanisms by which methyl radicals can produce different types of DNA adducts, we examined the effects of transition metal ions (iron (II), iron (III) and copper (I)) and metal ion chelators (ethylenediamine-N,N,N",N"-tetraacetate (EDTA) and desferal) on the nature and the yields of the DNA adducts produced during TBHP decomposition. The results led us to propose that a direct methyl radical attack on DNA guanine residues produces C8-MeGua whereas N7-MeGua and 3-methyladenine (N3-MeAde) are likely to be produced by attack of nucleophilic DNA centers on methyl radical generated in situ by the assistance of transition metal ions bound to DNA.     

Selective changes in tRNA methyltransferase activity in confluent monolayers of WI-38 cells stimulated to proliferate.

In quiescent confluent monolayers of WI-38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzyme show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2 microgram/ml). The increases in activity were not equivalent for the different base-specific enzymes. The contribution of the N2-methylguanine specific enzyme remained relatively constant, while that of the N2,N2-dimethyl-guanine specific and 1-methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1-methylguanine and the 7-methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non-coordinate variations in the expression of the different base-specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.     

High performance liquid chromatography studies on the interactions of cis-Pt(NH3)2−(H2O)22+ with guanine and methylated g

Time dependence studies, using high performance liquid chromatography (HPLC), on the reaction between cis-diamminediaquoplatinum and guanine, N1-methylguanine, N7-methylguanine, N9-methylguanine, and N1 ,N7-dimethylguanine are reported. Each reaction gave rise to eight or more compounds; the major components have been prepared and characterization by ¹H and ¹⁹⁵Pt nuclear magnetic resonance has been attempted. Species of the form ((NH₃)₂Pt(NO₃)-(G-H)-(NO₃)-Pt(NH₃)₂)⁺, (NH₃)₂,Pt(G-H)(NO₃) monomer and (NH₃)₂Pt(G-H)(NO₃) dimer, where G-H indicates the guanine monoanion, are postulated.     

Structural studies of O6-methyldeoxyguanosine and related compounds: a promutagenic DNA lesion by methylating carcinogens.

O6-Methylation of guanine residues in DNA can induce mutations by formation of base mispairing due to the deprotonation of N(1). The electronic, geometric and conformational properties of three N(9)-Substituted O6-methylguanine derivatives, O6-methyldeoxyguanosine (O6mdGuo), O6-methylguanosine (O6mGuo) and O6, 9-dimethylguanine (O6mdGua), were investigated by X-ray and/or NMR studies. O6mdGuo crystallizes in the monoclinic space group P2(1) with cell parameters a = 5.267(1), b = 19.109(2), c = 12.330(2) A, beta = 92.45(1) degrees, V = 1239.8(3) A3, z = 4 (two nucleosides per asymmetric unit), and O6mGua in the monoclinic space group P2(1)/n with cell parameters a = 10.729(2), b = 7.640(1) c = 10.216(1) A, beta = 92.17(2) degrees, V = 836.7(2) A3, z = 4. The geometry and conformation of O6-methylguanine moieties observed in both crystals and are very similar. Furthermore, the molecular dimensions of the O6methylguanine residue resemble more closely those of adenine than those of guanine. The methoxy group is coplanar with the purine ring, the methyl group being cis to N(1). The conformation of O6-methylguanine nucleosides is variable. The glycosidic conformation of O6mdGuo is anti for molecule (a) and high-anti for molecule (b) in the crystal, while that of O6mGuo is syn [Parthasarathy, R & Fridey, S. M. (1986) Carcinogenesis 7, 221-227]. The sugar ring pucker of O6mdGuo is C(2')-endo for molecule (a) and C(1')-exo for molecule (b). The C(4')-C(5') exocyclic bond conformation in O6mdGuo is gauche- for molecule (a) but trans for molecule (b), in contrast with gauche+ for O6mGuo. The hydrogen bonds exhibited by O6-methylguanine derivatives differ from those in guanine derivatives; the amino N(2) and ring N(3) and N(7) atoms of O6-methylguanine residues are involved in hydrogen bonding. 1H-NMR data for O6mdGuo and O6mdGuo reveal the predominance of a C(2')-endo type sugar puckering. In O6mdGuo, however, a contribution of a C(1')-exo sugar puckering is significant. The NOE data also indicate that O6mdGuo molecules exist with nearly equal population for anti (including high anti) and syn glycosidic conformations. These observations and their biological implications are discussed.     

Methylation of DNA guanine via the 1-carbon pool in dimethylnitrosamine-treated rats

Treatment of rats with radioactive methionine and nonradioactive dimethylnitrosamine resulted in the formation of radioactive 7-methylguanine in rat-liver DNA. By comparing the specific activity of administered [14C-Me]-dimethylnitrosamine to the specific activity of isolated 7-methylguanine it was determined that following 20 mg/kg dimethylnitrosamine DNA methylation via the 1-carbon pool may account for up to 30% of the total 7-methylguanine formed.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.