EFFECTS ON PROTOZOA 753 



hypothesis that arsenicals act by the usually accepted mechanism. Protec- 

 tion data have little or no bearing on how the arsenicals, or other inhibitors, 

 act, as has been clearly stated by Eagle and Doak (1951) and emphasized 

 repeatedly in the present work. Let us see if any useful information can be 

 obtained from the studies on recovery. The pertinent results may be sum- 

 marized as follows: (1) cysteine added to a suspension of spirochaetes being 

 progressively inactivated by phenylarsenoxide immediately stops the 

 action of the arsenical, and the number of motile organisms remains rela- 

 tively constant (Eagle, 1940); (2) trypanosomes immobilized by phenylar- 

 senoxide can be resuscitated by cysteine and dimercaprol, and simulta- 

 neously the arsenical is partially withdrawn from the cells (Eagle et al., 

 1946); and (3) dimercaprol is able to halt and reverse the trypanocidal 

 action of various arsenicals in vivo. The results with dimercaprol indicate 

 that it binds arsenicals more tightly than the trypanosomal groups, since 

 essentially complete reactivation can be obtained if the dimercaprol is 

 added within 5-10 min (phenylarsenoxide was used at 0.013 raM and this 

 completely immobilizes within 1 min and begins to cause degenerative 

 changes by 5 min). If dimercaprol is added 15 min or longer after the arsen- 

 ical, progressively less reversal is observed, which is not surprising since a 

 good deal of lysis takes place after 15 min. It may seem rather remarkable 

 that cysteine is effective, but very high molar ratios with respect to the 

 arsenical must be used; e. g., ratios of cysteine/phenylarsenoxide greater 

 than 625 were quite effective in an experiment of Eagle et al. (1946), but 

 at a ratio of 312 there is no reversal at all, even if the cysteine is added 

 immediately after the arsenical. Dimercaprol is effective at a molar ratio 

 of 3 or above. Nevertheless, the fact that cysteine can reverse the immobili- 

 zation and withdraw the arsenicals from the trypanosomes may possibly 

 indicate that the arsenicals do not inhibit here by reaction with lipoate 

 since, in systems in which lipoate is the site of attack, cysteine is generally 

 unable to reverse. It is not so much a question of whether the arsenicals 

 combine with SH groups to produce their trypanocidal effects — actually 

 from what we know there is no other possibility — but of the exact nature 

 of the cellular thiols reacted. Reversal data are frequently suggestive but 

 unless obtained from experiments carefully designed to provide relative 

 binding affinities, they must be interpreted cautiously. For example, in 

 the work of Eagle et al. (1946), although the trypanosome/medium ratio 

 of arsenical was reduced from 154 to 33 by cysteine (molar ratio of 455), 

 we note that there is still much arsenical in the cells, and the experiments 

 with dimercaprol are similar in that the trypanosomes after reversal con- 

 tain 10-30 times as much arsenical as the supernate. Does this mean that 

 the more weakly bound fraction of the cellular arsenical is responsible for 

 the immobilization? It seems that resuscitated trypanosomes can still 

 contain a good deal of arsenical, although the experiments on reactivation 



