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.     

Intermolecular transacylation of phosphatidylethanolamine by a Butyrivibrio sp.

The alkylation of purine bases in DNA of several rat tissues was determined during weekly injections (10 mg/kg) of N-[3H]methyl-N-nitrosourea, a dose schedule known to selectively induce tumours of the nervous system. Each group of animals was killed 1 week after the final injection, and the DNA hydrolysates were analysed by chromatography on Sephadex G-10. After five weekly applications, O6-methylguanine had accumulated in brain DNA to an extent which greatly exceeded that in kidney, spleen and intestine. In the liver, the final O6-methylguanine concentration was less than 1% of that in brain. Between the first and the fifth injection, the O6-methylguanine/7-methylguanine ratio in cerebral DNA increased from 0.28 to 0.68. In addition, 3-methylguanine was found to accumulate in brain DNA whereas in the other organs no significant quantities of this base were detectable. The results are compatible with the hypothesis that O6-alkylation of guanine in DNA plays a major role in the induction of tumours by N-methyl-N-nitrosourea and related carcinogens. The kinetics of the increase of O6-methylguanine in cerebral DNA suggest that there is no major cell fraction in the brain which is capable of excising chemically methylated bases from DNA. This repair deficiency could be a determining factor in the selective induction of nervous-system tumours by N-methyl-N-nitrosourea and other neuro-oncogenic compounds.     

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.     

Nitrosamine-induced carcinogenesis. The alkylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate

1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.     

Analysis of bases of rat-liver nucleic acids after administration of the carcinogen dimethylnitrosamine

Dimethylnitrosamine is metabolized to form an alkylating intermediate, which may have significance for its carcinogenic action. However, certain other compounds that are known to be highly mutagenic, including nitrous acid and hydroxylamine, might also be formed. Owing to the general reactivity of these compounds, it would be difficult to detect their formation in the intact animal. Instead, the nucleic acids of carcinogen-treated animals were examined for products of reaction with nitrous acid and hydroxylamine, i.e. for deamination of adenine and guanine, and formation of N(6)-hydroxycytosine, respectively. A double-labelling technique was used to detect very small amounts of the abnormal bases. The purine moieties of DNA in adult rat liver were labelled either with (14)C or with (3)H, by treating the neonatal animals with [(14)C]formate or with [(3)H]formate, and then allowing a period for normal growth. During this time, in liver, the labels were largely lost by metabolic turnover from cell components other than DNA. The pyrimidine moieties in DNA were labelled by treating the neonatal animals with [(14)C]orotate. The purine constituents of RNA of adult rat liver were labelled by repeated administration of [(14)C]- or [(3)H]-formate to the adult rats. The [(14)C]nucleic acid-labelled rat could then be treated with the carcinogen, and the [(3)H]nucleic acid-labelled animal could be used as a control. By this means the experimental and control tissues could be homogenized together in a single preparation, and the nucleic acids from the two tissues could be isolated, hydrolysed and analysed in a single sample. It was therefore possible to have an internal control for artifacts due to changes taking place in the nucleic acid bases during the experimental procedures. With this technique, the formation in vivo of 7-methylguanine in rat-liver DNA and RNA after administration of dimethylnitrosamine was confirmed, and no evidence was found for the formation of xanthine, hypoxanthine, N(6)-hydroxycytosine, or any other abnormal base.     

Analysis of products of DNA modification by methylases: a procedure for the determination of 5- and N4-methylcytosines in DNA

Although many different methods are used for the identification of methylated heterocyclic bases in DNA not all of them possess the ability to discriminate N4-methylcytosine (m4C) and 5-methylcytosine (m5C). Therefore, some of the methods need additional reexamination. This paper reinvestigates some chromatographic systems (thin-layer chromatography, paper chromatography, electrophoresis) most widely used in the analysis of minor bases occurring in nucleic acids according to their ability to separate m4C and m5C. A simple procedure for the preparation of the sample and a chromatographic system for its analysis was developed. The recommended chromatographic systems may be used for the simultaneous separation of not only of m4C and m5C but also both methylated cytosines together with N6-methyladenine and 7-methylguanine.     

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.     

Methylated purines in the deoxyribonucleic acid of various Syrian-golden-hamster tissues after administration of a hepatocarcinogenic dose of dimethylnitrosamine.

1. DNA was extracted from livers, kidneys and lungs of Syrian golden hamsters at various times (up to 96h) after injection of a hepatocarcinogenic dose of [14C]dimethylnitrosamine. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. At 7h after dimethylnitrosamine administration liver DNA was alkylated to the greatest extent, followed by that of lung and kidney, the values for which were 8 and 3% respectively of those for liver. 3. The O6-methylguanine/7-methylguanine ratios were initially the same in all three organs and in the liver DNA of rats under similar conditions of dose. 4. O6-Methylguanine was the most persistent alkylated purine in all three hamster tissues. There was evidence for excision of 7-methyl-guanine, the highest activity for this being present in the liver. 5. Detectable amounts of the minor products 3-methyladenine, 1-methyladenine, 3-methylguanine and 7-methyladenine were present in most hamster tissues, and their individual rates of loss from liver DNA were determined. 6. Ring-labelling of the normal purines in DNA was highest in the liver, followed closely by the lung (80% of that in liver) whereas the kidney had very low incorporation (3% of that in liver). 7. The results are discussed with respect to the hepatotoxicity of dimethylnitrosamine, the miscoding potential of the various alkylation products and the induction of liver tumours in hamsters.     

A tracking marker for the second dimension of the two dimensional gel electrophoresis of ribosomal proteins.

Conditions for the analysis of 7-methylguanine (7-MG) in the presence of major nucleic acid bases and other minor bases by means of high-performance liquid chromatography (hplc) were established. Purine bases were obtained by Chargaff's method to yield apurinic acid and were analyzed by hplc with strong cation-exchange resin, Zipax SCX. The method was applied to the determination of 7-MG in the liver DNA from rats treated with DMN and it was revealed that the results were comparable to those obtained by radioisotope method in terms of sensitivity and reproducibility. The lower limits of quantitation and detection of 7-MG were determined to be 10 and 4 ng, respectively.     

Formation of 8-methylguanine as a result of DNA alkylation by methyl radicals generated during horseradish peroxidase-catalyzed oxidation of methylhydrazine

Methylhydrazine oxidation promoted by horseradish peroxidase-H2O2 or ferricyanide led to the generation of high yields of methyl radicals and to the formation of 7-methylguanine and 8-methylguanine upon interaction with calf thymus DNA. Methyl radicals were identified by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone and tert-nitrosobutane. The methylated guanine products were identified in the neutral hydrolysates of treated DNA by high pressure liquid chromatography (HPLC) analysis and spiking with authentic samples. The structure of 8-methylguanine, a product not previously reported in enzymatic systems, was confirmed by HPLC chromatography, UV absorbance, and mass spectrometry. The formation of 8-methylguanine suggests a possible role for carbon-centered radicals as DNA-alkylating agents.     

Improved automated solid-phase microsequencing of peptides using DABITC

The methylated purines O⁶-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H₃PO₄. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O⁶-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O⁶-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [¹⁴C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O⁶-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.     

The fate of 7[14C]-methylguanine after administration to the rat

1. To assess the significance of the methylation of nucleic acids known to be caused by certain carcinogens, the metabolic fate of 7[¹⁴C]-methylguanine was studied, with special reference to its possible incorporation into RNA and DNA. 2. The major part (approx. 95%) of the dose was excreted unchanged in the urine. A small amount of N-demethylation took place, as evidenced by the formation of radioactive adenine and guanine, and expiration of ¹⁴C-labelled carbon dioxide. No evidence was obtained for the direct incorporation of 7-methylguanine into systems synthesizing nucleic acids, i.e. RNA in liver, DNA in intestine or in the foetus.     

Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine.

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.     

Similarities between the biochemical actions of cycasin and dimethylnitrosamine

1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.     

Organ-specific effects of DNA methylation by alkylating agents in the inbred Swiss mouse.

Young adult inbred Swiss mice given single or repeated equitoxic doses of N-methyl-N-nitrosourea (MNUA) or methyl methanesulphonate (MMS) develop thymomas and pulmonary adenomas only following MNUA in spite of nearly identical overall alkylation of DNA of tumour target tissues by both agents due mainly to the biologically ineffective product 7-methylguanine. The main difference in DNA alkylation was the production of O6-methylguinine, a known pre-mutagenic product, by MNUA in amounts 10 or more times as large as following MMS. This supports the possibility that somatic mutations are a part of the process of carcinogenesis.     

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.     

Purification and characterization of human 3-methyladenine-DNA glycosylase.

A human cDNA coding sequence for a 3-methyladenine-DNA glycosylase was expressed in Escherichia coli. In addition to the full-length 3-methyladenine-DNA glycosylase coding sequence, two other sequences (resulting from differential RNA splicing and the truncated anpg cDNA) derived from that sequence were also expressed. All three proteins were purified to physical homogeneity and their N-terminal amino acid sequences are identical to those predicted by the nucleic acid sequences. The full-length protein has 293 amino acids coding for a protein with a molecular mass of 32 kDa. Polyclonal antibodies against one of the proteins react with the other two proteins, and a murine 3-methyladenine-DNA glycosylase, but not with several other E. coli DNA repair proteins. All three proteins excise 3-methyl-adenine, 7-methylguanine, and 3-methylguanine as well as ethylated bases from DNA. The activities of the proteins with respect to ionic strength (optimum 100 mM KCl), pH (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: Km 9 nM and kcat 10 min-1, 7-methylguanine: Km 29 nM and kcat 0.38 min-1) are comparable. In contrast to these results, however, the thermal stability of the full-length and splicing variant proteins at 50 degrees C is less than that of the truncated protein.     

Role of tautomerism in RNA biochemistry

Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.     

The tentative identification of DNA-adducts generated by trans-4-dimethylaminostilbene and trans-4-acetylaminostilbene in rats.

Tritium-labeled trans-4-dimethylaminostilbene (DAS) and trans-4-acetylaminostilbene (AAS) were administered orally to female Wistar rats. RNA and DNA were isolated from livers after 24 h and 28 days. Hydrolysates were analyzed by gel filtration and HPLC. Total binding to nucleic acids and elution profiles of hydrolysates were very similar for both aminostilbene derivatives. The large polar fraction eluting early in Sephadex LH20 chromatograms accounting for 60-80% of total DNA-bound radioactivity could not be assigned to individual base adducts and most likely is not due to incomplete hydrolysis but rather to cross-links between bases or proteins and bases. Of the total radioactivity bound to nucleic acids 6-7% in RNA and 3-5% in DNA could be tentatively identified as four isomeric, cyclic guanine adducts (predominantly (S,S)- and (R,R)-guanine-N2,beta-N3,alpha-N-acetylaminobibenzyl) by cochromatography with synthetic reference compounds. The most abundant single adduct accounting for 20-30% of RNA-bound radioactivity (fraction G in Sephadex LH20 chromatography) could not be identified. The long-term experiment revealed different persistence of DNA adducts: the polar material decreased to about 2/3, the cyclic guanine adducts (fraction d-B) to about 1/3 to 1/2 within 4 weeks, whereas one of the unidentified DNA-adducts (fraction d-E) persisted completely. AAS labeled in the acetyl group was administered in an additional experiment. The presence of the acetyl group could be demonstrated in most of the adducts, but non-acetylated adducts were found also. The ratio of non-acetylated:acetylated cyclic B-adducts in RNA was 1:2 from DAS and 1:13 from AAS, in DNA 1:3 and 1:10, respectively.     

Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain.

This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed.