The degree of alkylation of proteins was used to determined tissue doses Dt i.e. the concentration of free alkylating agent, integrated over time, in resting male mice exposed for 1–2 h to air containing 1–35 ppm ethylene oxide (EO). The exposure doses were thus 0.03–2% of LD50. The results agree with an absorption of all EO in alveolar ventilation, a rapid distribution to all organs, and a rapid detoxication and excertion (biological half-life about 9 min). Dt is proportional to the exposure dose within the range studied. In most organs, including the testes, the Dt is about 0.5 μM · h per ppm · h of expsoure. The degree of alkylation of DNA agreed with expectation from the doses determined. Expressing genetic risks of environmental chemicals in the frame of reference of radiation hazard will facilitate comparison and summations of risks of various origins. On the basis of dose-effect curves of EO and X-rays in barely, a tissue dose of EO in man of i m M · h may be provisionally set equal to 80 rad of low LET radiation. Allowing for the difference in alveolar ventilation between mouse and man, this would mean that epoxide operators working at 5 ppm EO 40 h/week receive a weeky gonad dose of EO amounting to about 4 “rad-equivalenst”. Various data show that this risk estimate is realistic.

Evaluation of genetic risks of alkylating agents: tissue doses in the mouse from air contaminated with ethylene oxide.

Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications.

In various biological systems, 1,2-epoxides are able to cause biological effects with genetic mechanisms: point mutations, deletions, chromosomal aberrations, gene conversion, crossing-over, cancer and virus (prophage) induction. Dose—response curves are linear, or should be interpreted to have a linear component that predominates at low doses or concentrations. In forward mutation systems the effectiveness is related to the dose in the target cells and to the chemical reactivity. By applying experimentally determined correction factors for difunctionality (in the case of diepoxides or monoepoxides with a reactive substituent such as a carbonyl group), genetic risks may be estimated by a unitary approach, valid for epoxides in general, if not for all alkylating agents. The use of this approach, to calculate type-II errors in negative carcinogenicity tests, indicates that no data presently available support a conclusion that certain epoxides are non-mutagenic or non-carcinogenic, i.e. are less effective than expected from reactivity data. Determination of the rad-equivalence of genetic risks, i.e. the use of corresponding risks from a unit dose of ionizing radiation as a standard, indicates that the risks associated with Threshold Limit Values for epoxides in work environments in Western countries are 1–2 orders of magnitude higher than permissible risks for radiological workers.

Genetic toxicity of some important epoxides

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

The relationship in Drosophila males between chemical reaction pattern of mono-functional alkylating agents (AA), described in terms of primary alkylation pattern with DNA and proteins as well as the Swain--Scott s factor, and their biological effectiveness were investigated. The agents chosen for comparative analysis were the nitrosamides ENU and MNU, the methanesulfonic esters iPMS, EMS and MMS, the dialkylsulfate DMS, and the nitrosamines DEN and DMN. Parameters of their biological activity were mortality (LC50) of treated adult males, induction in post-meiotic stages of X-chromosomal recessive lethal mutations and 2--3 translocations after either adult feeding or injection. Induced frequencies of recessive lethals, determined for each AA with a range of concentrations, served as biological dosimeter for interaction with target DNA in the germ line. The results are interpreted as indicating for these AA a causal connection between the pattern of primary alkylation of DNA and the quality of genetic damage observed. 1. The agent with the lowest s value, ENU, and its pendant DEN, failed to produce translocations at mutation frequencies that reached 44% for ENU. The highest chromosome-breaking activity was attributed to AA with high s, MMS and DMS. For MMS, the proportions of translocations (T) to mutations (M) approximately reached a 1 : 1 ratio in stored spermatozoa, at a recessive-lethal frequency of 14%. Ability to break chromosomes, as indicated by the T : M ratios, decreased in the sequence MMS greater than or equal to DMS, MNU greater than DMN greater than EMS greater than iPMS greater than ENU = DEN. 2. Nearly the reversed sequence in relative mutagenci effectivenss was obtained when the (directly acting) AA were arranged on the basis of their CM4/LC50 ratios (CM4, the exposure condition producing 4% recessive lethals after injection): ENU greater than EMS greater than iPMS, MNU greater than MMS = DMS. 3. Among the AA, EMS had a somewhat unique position, in that it was slightly less effective in the translocation test, and also less cytotoxic but more mutagenic in the recessive-lethal test than one would expect from its s value. This is taken as an indication of the influence on biological effectiveness of factors other than the s value, e.g. methylation versus ethylation and the lipid/water partition ratio. An example of the latter was also provided by DMS which, although having the same s as MMS, with its 5-fold higher lipid/water partition ratio, was more toxic than MMS. 4. For those AA that were clearly active in the translocation tests--MMS, DMS, MNU, DMN and EMS--delayed formation of exchanges was observed. Only in 17 out of 555 translocation tests with positive response translocations were already found in progeny from unstored spermatozoa. Consequently, it was concluded that performance of storage experiments in Drosophila is an absolute necessity for the detection of this type of rearrangement by AA. 5...

The relation between reaction kinetics and mutagenic action of mono-functional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila.

The bactericidal and mutagenic effectiveness in E. coli of a few unsubstituted and substituted methanesulfonic esters and diethyl sulfate was related to the substrate constants, s , of the alkylating agents. The relative LD 50 values of the compounds except isopropyl methanesulfonate (iPMS) were found to agree with the relative reaction rates at a nucleophilic constant n = 5.1–5.5; it is, hence, indicated that alkylation of protein SH plays a predominant role in cell killing. iPMS which has the lowest s of the investigated compounds seems to kill cells mainly by a genetic mechanism. In contrast to the sigmoid survival-dose curves obtained with the other substances, iPMS gives a nearly one-hit exponential survival curve. iPMS was also demonstrated to differ from the other compounds by the absence of after-effects in treated cells, such as the enhanced killing during a post-treatment incubation in the absence of Mg 2+ . The relative mutagenic effectiveness (mutation to streptomycin independence in the strain Sd- 4 ) of the compounds studied, including iPMS, agrees with relative alkylation rates at n = ca . 2,5, i . e . close to values found for DNA. Also with respect to mutation iPMS differs from other compounds, giving a practically linear dose-response curve. Ethyl methanesulfonate (EMS) and other compounds with intermidiate s values give, superimposed on a linear component distinguishable at low doses, exponential (“multi-hit”) mutation curves leading to high mutation frequencies around LD 50 .

I. Turtóczky

Papers

Reaction rates and biological action of alkylating agents. Preliminary report on bactericidal and mutagenic action in E. coli.