The Mechanism of Action of Methyl Xanthines in Mutagenesis 



137 



II. TRACER STUDIES 



The first possibility to test was that these compounds, or products derived 

 from them, are utihzed for the synthesis of the nucleic acid of the host (3). 

 To do this we prepared these substances as well as some others, labeled with 

 carbon 14 in the 8-position of the heterocyclic nucleus. These were then added 

 to growing cultures o^ Escherichia co// under conditions similar to those employed 

 by NoviCK and Szilard (I, 2) in their studies. 



In Table I the data so obtained are presented. Adenine and guanine as 

 well as the deaminated derivatives are very well incorporated into the nucleic 



Table I. Incorporation and Mutagenicity 

 oj Various Purines 



*RSA = relative specific activity = ratio of the specific activity of the 

 purine isolated from the bacteria to that of the growth medium. 



acids of both the RNA and DNA type, whereas all methylated substances 

 are incorporated only to a very small extent, if at all. On the other hand, 

 the correlation of mutagenesis is the reverse. 



A mutation is a very rare event, and though these agents, when present 

 in quite high concentration, may raise the mutation rate by a factor of fifteen 

 or so, this still only corresponds to one event in 10'^ duplications. 



The small amount of radioactivity that is found associated with the DNA 

 from cells grown in the presence of radioactive mutagens is probably experi- 

 mental contamination. However, although these experiments are technically 

 excellent, they cannot begin to exclude the possibility that a methylxanthine 

 molecule is incorporated into the DNA molecule in the process of the rare 

 mutational event itself, since the resultant incorporation for one locus would 

 be many orders of magnitude below the trace amount observed here. Considera- 

 tion of the structures of these substances, however, makes this possibility 

 rather unlikely. 



In the formation of the normal 9-A^-riboside or 9-A^-deoxyriboside linkages, 

 the single replaceable hydrogen which may be in either the 7- or 9-position is 

 replaced by the glycosyl residue. In the case of caffeine or theobromine, which 

 are 7-methyl derivatives, this is not possible because of the prior replacement 

 of the hydrogen by the methyl group. Thus even though the methyl group is 



