BIOLOGICAL alkylating agents—“mustards”, ethylenimines, epoxides and alkyl alkanesulphonates—have been believed to induce mutations by causing atypical base pairing during DNA replication at sites bearing a guanine residue which has suffered alkylation at the 7(N) position1,2. The most abundant reaction product found in acid hydrolysates of treated DNA is 7-alkyl guanine3,4, although alkylated adenines and cytosine, notably 3-alkyl adenine, are recovered as minor products. Furthermore, genetic studies of alkylation mutants in bacteria and bacteriophages have shown that many point mutational events involve guanine to adenine transitions5,6.

Possible Relevance of O–6 Alkylation of Deoxyguanosine to the Mutagenicity and Carcinogenicity of Nitrosamines and Nitrosamides

INTRODUCTION . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 201 S OURCES OF AP SITES . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 202 Spontaneous DepurinationlDepyrimidination . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 Chemically Induced DepurinationlDepyrimidination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 203 DepurinationlDepyrimidination by DNA Glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 207 REPAIR OF AP SITES . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 209 MUTAGENESIS BY AP SITES . . . .... . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Misincorporation by DNA Polymerases in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 210 Mutagenesis in E. coli... . . . . . . . . . ..... . . . . . . . . . 2 1 2 Mutagenesis in Animal Cells . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 3 A P Sites a s Intermediates in Spontaneous Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 AP Sites as Intermediates in Chemical Mutagenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 MOLECULAR MECHANISMS FOR MUTAGENESIS BY AP S ITES . . . . . . . . .. . . . . . . . . . 219

Mutagenesis by Apurinic/Apyrimidinic Sites

Four different base-specific chemical reactions generate a means of directly sequencing RNA terminally labeled with 32P. After a partial, specific modification of each kind of RNA base, an amine-catalyzed strand scission generates labeled fragments whose lengths determine the position of each nucleotide in the sequence. Dimethyl sulfate modifies guanosine. Diethyl pyrocarbonate attacks primarily adenosine. Hydrazine attacks uridine and cytidine, but salt suppresses the reaction with uridine. In all cases, aniline induces a subsequent strand scission. The electrophoretic fractionation of the labeled fragments on a polyacrylamide gel, followed by autoradiography, determines the RNA sequence. RNA labeled at the 3' end yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.

https://www.pnas.org/content/pnas/76/4/1760.full.pdf

Direct chemical method for sequencing RNA.

During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

Probing the structure of RNAs in solution

We include in this review an assessment of the formation, environmental fate, and mammalian and ecotoxicity of CW agent degradation products relevant to environmental and occupational health. These parent CW agents include several vesicants: sulfur mustards [undistilled sulfur mustard (H), sulfur mustard (HD), and an HD/agent T mixture (HT)]; nitrogen mustards [ethylbis(2-chloroethyl)amine (HN1), methylbis(2-chloroethyl)amine (HN2), tris(2-chloroethyl)amine (HN3)], and Lewisite; four nerve agents (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX), tabun (GA), sarin (GB), and soman (GD)); and the blood agent cyanogen chloride. The degradation processes considered here include hydrolysis, microbial degradation, oxidation, and photolysis. We also briefly address decontamination but not combustion processes. Because CW agents are generally not considered very persistent, certain degradation products of significant persistence, even those that are not particularly toxic, may indicate previous CW agent presence or that degradation has occurred. Of those products for which there are data on both environmental fate and toxicity, only a few are both environmentally persistent and highly toxic. Major degradation products estimated to be of significant persistence (weeks to years) include thiodiglycol for HD; Lewisite oxide for Lewisite; and ethyl methyl phosphonic acid, methyl phosphonic acid, and possibly S-(2-diisopropylaminoethyl) methylphosphonothioic acid (EA 2192) for VX. Methyl phosphonic acid is also the ultimate hydrolysis product of both GB and GD. The GB product, isopropyl methylphosphonic acid, and a closely related contaminant of GB, diisopropyl methylphosphonate, are also persistent. Of all of these compounds, only Lewisite oxide and EA 2192 possess high mammalian toxicity. Unlike other CW agents, sulfur mustard agents (e.g., HD) are somewhat persistent; therefore, sites or conditions involving potential HD contamination should include an evaluation of both the agent and thiodiglycol.

The sources, fate, and toxicity of chemical warfare agent degradation products.

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The reaction of mono- and di-functional alkylating agents with nucleic acids

Mustard gas [di-(2-chloroethyl) sulphide] and analogous alkylating agents produce a wide range of biological effects (cf. review by Boyland, 1948). Some ofthese effects, such as induction ofmutations (Auerbach & Robson, 1946) and interference with mitosis and chromosome breakage (Darlington & Koller, 1947), stimulated interest in the reactions of alkylating agents with deoxyribonucleic acid. Previous studies of such reactions, mainly by physicochemical methods, have led to various suggestions regarding the chemical group or groups in nucleic acids involved. Chanutin & Gjessing (1946) showed by ultraviolet spectroscopy that the nitrogen mustards di-(2-chloroethyl)methylamine and tri-(2-chloroethyl)amine reacted with the bases of deoxyribonucleic acid, but the groups reacting were not specified. Elmore, Gulland, Jordan & Taylor (1948), on the basis ofelectrometric titration experiments, suggested that both basic and phosphate groups of deoxyribonucleic acid reacted with mustard gas. Press & Butler (1952) found that alkylation of deoxyribonucleic acid by di-(2chloroethyl)methylamine in sodium bicarbonate solution resulted in a decrease of primary amino nitrogen and of purine nitrogen precipitable as purine silver salt, but no specific sites of reaction were established. Wheeler, Morrow & Skipper (1955) concluded from experiments with methylated xanthines and mustard gas that alkylation of ring nitrogen atoms of purines could occur. Alexander, Cousens & Stacey (1957) postulated that phosphotriesters were produced by alkylation of deoxyribonucleic acid in neutral aqueous solution by a variety of agents. This was based on observations that alkylation was not accompanied by liberation of acid. Reiner & Zamenhof (1957), from experiments on the binding of protamine by deoxyribonucleic acid and paper chromatography of hydrolysed alkylated deoxyribonucleic acid, claimed that whereas dimethyl sulphate reacted exclusively with the purine moieties at N-7, diethyl sulphate reacted exclusively with primary phosphate groups. The only study of products formed by alkylation in vivo of nucleic acid is that by Wheeler & Skipper (1957). After administration of di-(2-chloroethyl)[14C]methylamine to rats and mice isotopic labelling of both ribonucleic acid and deoxyribonucleic acid was found, and it was shown that this was associated with the purine fraction. In most of the earlier chemical studies of alkylation of nucleic acids the conditions were very different from those which would occur in vivo, e.g. large concentrations of alkylating agents, and sometimes alkaline pH, were used. Further, the methods used were not such as to enable the sites of reaction to be specified. In view of this uncertainty and the fact that preliminary experiments (Lawley & Wallick, 1957; Lawley, 1957a, b) had indicated N-7 of guanine moieties to be the most reactive centre towards alkylation in nucleic acid, it was decided to reinvestigate this reaction with a typical alkylating agent. Mustard gas, di-(2-chloroethyl) [35S]sulphide, was chosen since it is known to be one of the most reactive of the alkylating agents and also is obtainable at a high specific radioactivity. The reaction of mustard gas with nucleic acid under mild conditions has therefore been studied chemically and compared with that resulting when tobacco mosaic virus, protoplasts of Bacillus megaterium, and the Ehrlich ascites tumour in the mouse are treated with this reagent.

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The reaction of mustard gas with nucleic acids in vitro and in vivo

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8. Effects of the sulphur mustards on nucleic acid synthesis in sensitive and resistant strains were studied. DNA synthesis was inhibited in both strains at low doses in a dose-dependent manner, but RNA and protein synthesis were not affected in this way. 9. DNA synthesis in E. coli B(s-1) was permanently inhibited by low doses of mustards. In the resistant strains 15T(-) and B/r a characteristic recovery in DNA synthesis was observed after a dose-dependent time-lag. This effect could be shown at low doses in the region of the mean lethal dose. 10. Cellular DNA was isotopically prelabelled and the effect of mustards on stability of DNA was investigated. With resistant strains a dose-dependent release of DNA nucleotide material into acid-soluble form was found; this was much more extensive with the difunctional mustard (about 400 nucleotides released per DNA alkylation) than with the monofunctional mustard (about 10 nucleotides per alkylation). With the sensitive strain no dose-dependent release was found, though the DNA was less stable independent of cellular alkylation. 11. The results are discussed in terms of the concepts that alkylation of cellular DNA induces lesions which interfere with DNA replication, but which can be enzymically ;repaired'. The possible nature of these lesions is discussed in terms of the known reactions of the alkylating agents with DNA.

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Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids

Barnum, C. P., Huseby, R. A. & Vermund, H. (1953). Cancer Res. 13, 880. Dounce, A. L. (1955). In The Nucleic Acids, p. 147. Ed. by Chargaff, E. & Davidson, J. N. New York: Academic Press Inc. Eagle, H., Oyama, V. I., Levy, M., Horton, C. L. & Fleischman, R. (1956). J. biol. Chem. 218, 607. Ficq, A. (1953). Experientia, 9, 377. Ficq, A. (1955). Exp. Cell Bes. 9, 286. Harris, H. (1955). Brit. J. exp. Path. 38, 115. Harris, H. (1959). Biochem. J. 72, 54. Luck6, B., Strumia, M., Mudd, S., McCutcheon, M. & Mudd, E. B. H. (1933). J. Immunol. 24, 455. McClintock, B. (1934). Z. Zellfor8ch. 21, 294. Pelc, S. R. (1956). Int. J. appl. Radiat. I8otope8, i, 172. Slider, E. M. & Downey, H. (1950). In MWClung'8 Handbook of Micro8copcal Technique, p. 226. Ed. by Jones, R. McC. London: Cassell. Smellie, R. M. S. (1955). In The Nucleic Acid8, p. 408. Ed. by Chargaff, E. & Davidson, J. N. New York: Academic Press Inc. Smellie, R. M. S., McIndoe, W. M., Logan, R., Davidson, J. N. & Dawson, I. M. (1953). Biochem. J. 54, 280. Vincent, W. S. (1955). Int. Rev. Cytol. 4, 269. Watts, J. W. & Harris, H. (1959). Biochem. J. 72, 147.

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P. Brookes

Papers

The reaction of mono- and di-functional alkylating agents with nucleic acids

The reaction of mustard gas with nucleic acids in vitro and in vivo

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids

Intermediate reactions in protein synthesis by the isolated cytoplasmic-membrane fraction of Bacillus megaterium

Site of synthesis of the peptide component of the cell wall of Bacillus megaterium.