Cancer a Comprehensive Treatise 2

Publisher Summary This chapter discusses the three main reasons for the chemical modification of nucleic acids that are available on modifying agents. These agents are used for inactivations of various kinds, for directed functional changes of nucleic acids in vivo and for the elucidation, through resulting modification, of structural and functional characteristics peculiar to synthetic or native polynucleotides. Some of these agents are useful for one, two, or all three of these purposes. One of the few agents that serve all three of them is formaldehyde. It is widely used as an inactivator of viruses to obtain vaccines and is reported to exert a cytostatic (carcinostatic) effect. It is also one of the most promising mutagenic agents affecting multicellular organisms. Formaldehyde is used extensively in structural and functional studies of nucleic acids as an agent not so much for causing denaturation as for preventing renaturation, and as a fixator of nucleic acids and nucleoproteins in electron microscope and sedimentation investigations. Recently, some progress in the structural study of formaldehyde interaction products with nucleotides and nucleic acids has been made. The chapter emphasizes on the evidence for the formation of various structures and the molecular mechanisms of biological and other effects of formaldehyde.

Reactions of nucleic acids and nucleoproteins with formaldehyde.

Alkylation of DNA can lead to induction of potentially miscoding groups (promutagenic) or potentially template-inactivating groups (lethal). The proportions of these are found to vary with the chemical nature of the alkylating agent. Agents of low Swain and Scott s factor (or those tending to Ingold's SN i type) react relatively more extensively at O-atom sites in DNA, and yield relatively more of the miscoding O6-alkylguanine residues. Phosphotriester formation is also relatively more extensive with SN i agents. Inactivation of DNA can result from depurinations, strand breakage, and cross-linkage. Both promutagenic and lethal lesions are subject to repair; 3 principal enzymatic systems appear to exist; one for excision and repair of cross-links or aralkyl groups resembles the uvr system; others for repair of single-strand breaks parallel repair of X-ray-induced breaks (exr, rec systems); another, less well defined at present, recognizes certain methylated bases, and depurinated sites (probably Goldthwait 's endonuclease II). These factors can be shown to influence dose-response in alkylation mutagenesis. This, broadly, can be classified as linear with the promutagenic group-inducing or directly miscoding agents, and is independent of cytotoxicity; whereas with other agents non-linear response parallels the occurrence of “shouldered” survival curves, and reflects mutation induction by “repairs errors”. Additionally, alkylation of cellular constituents other than DNA, e.g. repair enzymes, may influence dose response, and will again depend on chemical reactivity of the agent.

Some chemical aspects of dose-response relationships in alkylation mutagenesis

Publisher Summary This chapter discusses some studies on the interaction of various compounds with cellular DNA and the biochemical and biological consequences of these reactions in a variety of cell systems and it focuses attention on DNA as the principal target for a number of chemotherapeutically useful cytotoxic agents and for numerous known carcinogens. The chapter further discusses some observations establishing that some DNA reactions are recognized and modified by various enzyme systems. These DNA repair processes clearly protect cells from lethal effects of DNA reactions in both bacterial and mammalian cells. Decreased sensitivity of sublines of cells in culture have been shown in some instances to be related either to their increased capacity for excision repair of damage to their DNA or to their greater capacity to carry out postreplication DNA repair. However, it is by no means established, although quite likely, that clinical resistance to cytotoxic agents in cancer chemotherapy is similarly characterized by the emergence of, or conversion of a tumor cell to, a cell with altered DNA repair capacity that confers decreased sensitivity to cytotoxic agents as compared with that of the original tumor cell.

The Repair of DNA Modified by Cytotoxic, Mutagenic, and Carcinogenic Chemicals

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

P. Brookes

Papers

Effects of alkylating agents on the induced synthesis of β-galactosidase by Escherichia coli Bs−1

Nature of Alkylation Lesions and Their Repair: Significance for Ideas on Mutagenesis