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).
