260 PETER ALEXANDER AND MISS D. I. CONNELL 



other than somatic mutations, which have been put forward for life-span 

 shortenmg by radiation, such as imperfect repair of radiosensitive organs. 



A more decisive test for the somatic mutation hypothesis would be whether 

 the monofunctional alkylating agent derived from myleran (colloquially 

 "half-myleran") ethyl methane sulphonate (EMS) CHg . SO2 . . CH2 . CH3 

 brings about life-span shortening. This substance has a very low toxicity yet it 

 has been shown to be a very powerful mutagen in all the systems in which it has 

 been tested {Droso'phila, Fahmy and Fahmy, 1956; Neurosjjora, Westergaard, 

 1957; barley, Heslot and Ferrary, 1958; bacteria. Loveless and Howarth, 

 1959; bacteriophage, Loveless, 1959). With EMS effects due to cell killing 

 and mutagenicity can be clearly separated since in bacteria even a 1% 

 solution does not kiU (Loveless and Howarth, 1959). We find that in mice the 

 LD5Q dose of EMS is eight times greater than that for myleran. The ratio of 

 the actual cell killing efficiency of myleran and EMS is, however, even greater 

 than 8 : 1 since mice given a lethal dose of EMS do not die from bone-marrow 

 depletion following destruction of dividing ceUs as is the case for myleran and 

 X-rays. With EMS death is due to acute kidney damage which is probably 

 brought about by the large amount of strong acid released when EMS 

 hydrolyzes in the body. Mustards and myleran also hydrolyze with production 

 of strong acid but at the dose levels used the amount of acid released is 

 physiologically neutralized. The mouse cannot, however, cope with acid 

 from 400 mg/kg of EMS (the LDgg dose) and kidney necrosis results. In 

 Droso]}hila also, myleran is much more toxic than EMS (Fahmy, private 

 communication) . 



EXPERIMENTAL 



Males from an mbred CBA strain were bred by brother-sister mating and 

 maintained on rat cake nuts. EMS obtained from Eastman Kodak was 

 suspended in arachis oil and injected intraperitoneaUy. The LDgg dose was 

 found to lie between 400 and 450 mg/kg and a dose corresponding to half the 

 LD5Q (i.e. 200 mg/kg) was given three times at 3-weekly intervals starting with 

 mice 11 to 14 weeks old. This regime produced no acute deaths. The procedure 

 used with myleran and chlorambucil was exactly comparable and has been 

 described (Alexander and Connell, 1960). The X-ray irradiation was carried 

 out with 250 kV X-rays at a dose-rate of 55 r/min and given in four doses of 

 300 r, 300 r, 300 r and 200 r at 3-week intervals, a procedure which killed 

 none of the animals. 



RESULTS AND DISCUSSION 



Table I shows that EMS has no significant effect on the average life-span 

 when defined as the time for half the animals in a given experiment to be dead. 



