An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.     

Studies on the stability of trialkyl phosphates and di-(2'deoxythymidine) phosphotriesters in alkaline and neutral solution. A model study for hydrolysis of phosphotriesters in DNA and on the influence of a beta hydroxyethyl ester group

Various trialkyl phosphates were investigated as model compounds for DNA-phosphotriesters for their stability in neutral or alkaline conditions. The results show that phosphotriesters were highly stable even at strongly alkaline pH, with the exception of diethyl 2-hydroxyethyl phosphate (DHP). The extreme instability of the latter was found to be due to the 2-hydroxy function. In accordance with earlier interpretations the 2-hydroxyethyl group is proposed to participate in the formation of a highly reactive dioxaphospholane ring intermediate which decays rapidly by hydrolysis. Alkylation of 3'- and 5'-deoxythymidine monophosphates with methyl- or hydroxyethylnitrosourea (MNU, HENU) results in practically exclusive phosphate alkylation. In analogy with the model phosphotriesters, di(2'-deoxythymidine) phosphotriesters generated after reaction with MNU or HENU showed extreme dependence of their stabilities on the nature of the alkyl group transferred to phosphate. Whereas the methyl phosphotriester was highly stable, the corresponding hydroxyethyl analogue showed half lives of decay of less than 1 min (pH 12.5), 27 min (pH 9.1) and 60 min (pH 7). Thus the introduction of a 2-hydroxyethyl function into phosphate strongly decreases the stability of the phosphate link of DNA, resulting in DNA single strand breaks, in analogy to RNA phosphotriesters which have been found earlier to be highly unstable because of the presence of the ribose 2'-OH-group.     

Assays for phosphotriester formation in the reaction of bacteriophage R17 with a group of alkylating agents.

The interaction of bacteriophage R17 with 8 compounds has been studied, comparing the contribution of degradation of ribonucleic acid to the total toxicity. Breaks in the RNA chain result from the hydrolysis of phosphotriesters and thus are a measure of the extent of O-alkylation and of the SN1-type mechanism of the reaction. With many alkylating agents mutagenicity and carcinogenicity increase with increasing SN1 character of the reaction. In experiments with methyl methanesulphonate no evidence of degradation was observed at up to 19 times the mean lethal dose (620 methylations/RNA molecule). Breaks in the RNA chain accounted for 1 in 10 of the lethal lesions with beta-hydroxyethyl methanesulphonate, 1 in 60 with bis-(2-chloromethyl)methylamine (nitrogen mustard, HN2), less than 1 in 125 with 2,2-dichlorvinyl dimethyl phosphate (dichlorovos, DDVP), and 1 in 200 with propylene oxide. The hydrolysis rate of bis-(2 chloroethyl)ether was too slow for any reaction to be detected. In reactions with the carcinogen bis-(2-chloromethyl)ether the toxicity observed could be accounted for by the formaldehyde produced on hydrolysis. Cross-linking of the bacteriophage components by formaldehyde reduced the survival range over which the physical state of the RNA could be studied. No evidence of RNA degradation was observed. Reaction of the formaldehyde led to a progressive loss of biological activity over 24 h, a loss which was partially reversed by dialysis.     

Inactivation and mutation of coliphage T4 by aliphatic nitrosamides and methanesulphonates: in vitro recovery of infectivity of T4 inactivated by isopropyl methanesulphonate.

The inactivation and mutation (to r phenotype) of extracellular coliphage T4 wild-type by the monofunctional alkylating agents N-methyl- and N-ethyl-N-nitrosourea and isopropyl methanesulphonate were investigated. The rate and extent of change in phage infectivity observed during the post-treatment period were found to correlate with what is known of the mechanisms by which these agents react in vitro. Loss of phage infectivity was found to occur during the period following treatment with these agents, but that resulting from treatment with isopropyl methanesulphonate was preceded, in the first 24 to 48 h, by a recovery of infectivity. This suggested that changes in phage infectivity occurring after treatment with monofunctional alkylating agents are resultant of various processes which diversely promote loss and recovery of infectivity. The mutagenicity of N-methyl-N-nitrosourea was similar to that of its ethyl homologue at a level of phage survival of 4 x 10-3, but less than that of isopropyl methanesulphonate. At a level of survival of 3 x 10-2 ethyl methanesulphonate was a mutagenic as its isopropyl homologue, but methyl methanesulphonate was only slightly if at all mutagenic. These results could not be correlated with the compounds' reaction mechanisms. The efficiency of isopropyl methanesulphonate (compared with its toxicity to phage) was found to decrease as the severity of the dose was increased.     

Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I

A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I). Escherichia coli DNA pol I bypassed the lesion efficiently, but partial inhibition was observed for T4 DNA polymerase. The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center. These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase–DNA–dNTP complex. When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion. The replication block was overcome by higher dNTP concentrations. Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks.     

The inactivation of bacteriophage R17 by ethylating agents: the lethal lesions.

The biological inactivation of bacteriophage R17 by ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENUA) has been studied. At the mean lethal dose for the first compound 8 moles ethyl are bound/mole RNA and with the nitroso compound 3.5 moles ethyl are bound. Analysis of the amounts of the different ethylated derivatives formed shows that the toxicity of the sulphonate can be accounted for by the formation of 3-ethylcytosine, O6-ethylguanine, 1-ethyladenine and chain breaks produced on the hydrolysis of ethyl phosphotriesters. With the nitroso derivative on the other hand, the sum of chain breaks and of bases alkylated on a position involved in specific hydrogen bonding between base pairs only accounts for 65% of the observed toxicity. The possibility that 3-ethyladenine may constitute a lethal lesion is discussed.     

Synthesis and properties of oligodeoxyribonucleotides containing an ethylated internucleotide phosphate

Internucleotide phosphotriesters comprise an important class of DNA lesions produced by carcinogenic alkylating agents. To avoid confusion resulting from the presence of other DNA lesions, synthetically prepared oligonucleotides containing ethylated internucleotide phosphates as the sole form of damage were employed to investigate several chemical and biochemical properties of DNA alkyl phosphotriesters. A total of four oligonucleotides were synthesised for this study, the dimers Tp(Et)T and pTp(Et)T and the decamer d-TpTpTp(Et)TpCpTpApTpTpT together with its unmodified analogue. The dimers were characterized by UV and phosphorus NMR spectroscopy and the decamers by two-dimensional homochromatography, alkali hydrolysis, and variable-temperature circular dichroism (CD). Alkali hydrolysis of the ethylated decamer produced strand breaks in approximately 75% of the molecules. This is in close agreement with data previously obtained for dinucleoside ethyl phosphotriesters and triesters in alkylated cellular DNA. Results from the CD study suggest that the ethyl substituent does not disrupt base stacking within the oligomer. The interactions of two enzymes with the alkylated oligonucleotides were examined. First, it was found that ethylation of the internucleotide phosphate renders TpT inactive as a substrate for T4 polynucleotide kinase, implying that a negative charge is required on the 3'-phosphate group of the nucleotide to be phosphorylated. Hence, postlabeling assays of DNA damage that depend upon enzymatic phosphorylation of modified 3'-nucleotides cannot be applied to dinucleoside alkyl phosphotriesters. Second, both decamers, when annealed to a single-stranded plasmid template, were able to prime DNA synthesis, catalyzed by Escherichia coli DNA polymerase I, with equal effectiveness. The use of this reaction as a means of site-specifically incorporating phosphotriesters into viral vectors is recognized.     

Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3''-5'')thymidine.

1. The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates.     

DFT investigations of phosphotriesters hydrolysis in aqueous solution: a model for DNA single strand scission induced by N-nitrosoureas

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

The molecular basis for biological inactivation of nucleic acids. The action of methylating agents on the ribonucleic acid-containing bacteriophage R17

1. The inactivation of an RNA-containing bacteriophage after reaction with four methylating agents was studied. Measurements of the extent of methylation of the RNA and of the nature and amounts of the various reaction products were made. In experiments with dimethyl sulphate and methyl methanesulphonate inactivation can be quantitatively accounted for by methylation at two of the positions involved in hydrogen bonding: N-1 of adenine and N-3 of cytosine. In experiments with N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine methylation at N-1 of adenine and N-3 of cytosine accounts for only about one-half of the observed inactivation. Scission of the RNA chain during reaction accounts for a further 20% of the inactivation. To account for the remainder it seems necessary to postulate that formation of O(6)-methylguanine constitutes a lethal lesion. 2. Breaks in the RNA chain formed on reaction with the nitroso derivatives presumably result from methylation of the phosphate diester group followed by hydrolysis of the unstable triester thus formed.     

Synthesis and properties of some cyclic AMP alkyl phosphotriesters

Cyclic AMP was converted to its phosphotriesters according to the classical approach of phosphate activation with a sulfonyl chloride, followed by esterification with an alcohol. The methyl, ethyl, propyl, butyl and cetyl triesters were prepared, and some of their physical-chemical properties determined. Alkaline hydrolysis of these alkyl phosphotriesters resulted predominantly in ring opening. On the other hand, nucleophilic attack by thiourea led to the formation of cAMP as the main product. The conclusion can be drawn from these results that cAMP phosphotriesters could serve as suitable storage forms of cAMP, and cyclic triesters may be the best vehicle of transporting nucleotides through biological membranes.     

The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.     

Phosphotriester formation by the haloethylnitrosoureas and repair of these lesions by E. coli BS21 extracts.

The alkylation of phosphates in DNA by therapeutically active haloethylnitrosoureas was studied by reacting N-chloroethyl-N-nitrosourea (CNU) with dTpdT, separating the products by HPLC, and identifying them by co-chromatography with authentic markers. Both hydroxyethyl and chloroethyl phosphotriesters of dTpdT were identified; a similar reaction between CNU and dTR yielded 3-hydroxyethyl and 3-chloroethyl dTR as the major products of ring alkylation. A DNA-like substrate for repair studies was synthesized by reacting 14C-labelled N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (14C-CCNU) with poly dT and annealing the product to poly dA. An extract of E. coli strain BS21 selectively transferred a chloroethyl group from one of the chloroethyl phosphotriester isomers in this substrate to the bacterial protein; chemical instability of the hydroxyethyl phosphotriesters precluded definite conclusions about the repair of this product.     

The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo.

The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.     

Structural and conformational studies on deoxyguanosyl-3',5'-deoxyadenosine monophosphate and its ethyl phosphotriester analogs--left-handed dimers

The mode of base-base stacking, the handedness and the sugar(dGpA)phosphate backbone conformation of deoxyguanosyl 3'-5' deoxyadenosine and its diastereomeric ethyl phosphotriester analogs were studied by 1H NMR, UV and CD spectroscopy. The results indicate the three dimers are left-handed, while the sugar phosphate backbone is comprised predominantly of C2-endo,gg(C4-C5) and g'g (C5-O) conformers. The two bases are extensively stacked and interact about 90 degrees along the dyad axes. The extent of base overlap in dGpA is slightly greater than in either ethyl phosphotriester analog. The absolute configurations of the two ethyl phosphotriester diastereoisomers of dGpA can be assigned by one-dimensional and two-dimensional 1H NMR nuclear Overhauser enhancement experiments.     

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

1. The following methods for hydrolysis of methyl-(14)C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH(4) (+) form) at pH6 or 8.9, or on Dowex 50 (H(+) form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O(6)-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose-phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson-Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson-Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.     

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.     

Sequence specific block of in vitro DNA synthesis with isopropyl phosphotriesters in template oligodeoxyribonucleotides.

We have synthesized four oligodeoxyribonucleotides each bearing an isopropyl phosphotriester at a defined position. These oligomers were used as templates for in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I large fragment. Results showed that the phosphotriester inhibits the DNA chain elongation and the level of the inhibition is dependent on the base 5' to the phosphotriester.     

31P NMR Studies on Oxidative Transformations of Aryl Nucleoside H-Phosphonate Diesters

Abstract

Various aspects of oxidative coupling of nucleoside aryl H-phosphonate diesters with nucleosides to produce important synthetic intermediates in the phosphotriester approach to oligonucleotide synthesis, dinucleoside aryl phosphotriesters, was investigated.     

Nitrosamine-induced carcinogenesis. The alkylation of N-7 of guanine of nucleic acids of the rat by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate

1. The extent of ethylation of N-7 of guanine in the nucleic acids of rat tissue in vivo by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate was measured. 2. All compounds produced measurable amounts of 7-ethyl-guanine. 3. A single dose of diethylnitrosamine or N-ethyl-N-nitrosourea produced tumours of the kidney in the rat. Three doses of ethyl methanesulphonate produced kidney tumours, but a single dose did not. 4. A single dose of diethylnitrosamine produced twice as much ethylation of N-7 of guanine in DNA of kidney as did N-ethyl-N-nitrosourea. A single dose of both compounds induced kidney tumours, although of a different histological type. 5. A single dose of ethyl methanesulphonate produced ten times as much ethylation of N-7 of guanine in kidney DNA as did N-ethyl-N-nitrosourea without producing tumours. 6. The relevance of these findings to the hypothesis that alkylation of a cellular component is the mechanism of induction of tumours by nitroso compounds is discussed.