THE SUBTLETY OF ALKYLATING-AGENTS IN REACTIONS WITH BIOLOGICAL MACROMOLECULES

Genotoxic agents known to modify DNA by alkylation reactions (alkylating agents, AAs), either directly or after metabolic conversion to ultimately reactive intermediates, by no means represent a homogeneous class. For instance, their effectiveness for genotoxic damage, when expressed as the number of events (e.g., mutations) per unit exposure dose, varies over a more than 1-million-fold range in dose. Despite the multiplicity of chemical and biological processes involved between DNA adduct formation and expression of genotoxic damage, the principal aims of studies on structure-activity relationships (SARs) are to (a) dissect the multi-step process of genetic damage formation into its most essential components, (b) use SARs for making predictions and, at a later step (c) as a basis for regulatory measures. The analytical tools available for such a comprehensive analysis in eukaryotic systems include determination of multiple genetic endpoints: molecular mutation spectra, relative clastogenicity (clastogenic events in relation to forward mutation induction) and the quantitative measure of enhanced mutagenicity in repair-deficient conditions. The genetic activity profiles obtained in this way can then be compared with fundamental physico-chemical properties of the AAs under consideration (such as Swain-Scott's s value, a useful indicator of the selectivity of an AA in its reactions with nucleophiles of distinct nucleophilic strength n in DNA, RNA and proteins), their functionality (monofunctional versus cross-linking) and their tumorigenic potency (TD(50)s compared with measures of initial DNA interaction, i.e., O-6-/N7-alkylguanine ratios, s values or the covalent binding index determined in the liver in vivo). The combination of these different methods revealed that carcinogenic potencies of AAs in rodents vary over a 10,000-fold range in dose, with the extremes having the following characteristics: (i) Chemicals of a relatively ''high carcinogenic potency'', as indicated by a low TD50 in rodents, either have low nucleophilic selectivity (and therefore mainly act through O-alkylation in DNA) or are capable of cross-linking DNA. The monofunctional members of this group, typified by N-ethyl-N-nitrosourea, are active in both spermatogonia and post-spermatogonial stages in the mouse and in Drosophila. Cross-linking agents also have a low TD50 value in rodents but are expected generally not to display genetic action in premeiotic stages (exceptions mitomycin C and chlorambucil). (ii) A relatively low carcinogenic potential is associated with AAs of high Swain-Scott s values, typified by trimethyl phosphate, epichlorohydrin or methyl methanesulphonate. Efficient error-free repair of N-alkylation damage appears the responsible mechanism for their high TD,, in rodents and why they tend to be inactive in repair-competent germ cells of the mouse. Since AAs of high s values give relatively high degrees of alkylation of proteins (e.g., with the -SH group of esterified cysteine, n = 5.1) reaction products with strong nucleophiles are often formed in amounts orders of magnitude larger than the products at n = 2 (DNA). As a consequence, the ''window'' of the dose range not causing cell lethality but, at the same time, still producing a significant amount of damage (e.g., mutations) will be very small. These type of agents are expected to be ''trouble-makers'' particularly in the in vivo mutagenicity assays. Examples are acrolein, chloroethyl isocyanate, chloroethylene oxide (s = 0.71), epichlorohydrin (s = 0.93), 1,2-epoxybutane, methyl bromide (s = 1.0), methyl iodide (s = 1.20), methyl vinylsulphone and 2-oxopropyl methanesulphonate (s = 2).