Effect of cations on the formation of DNA alkylation products in DNA reacted with 1-(2-Chloroethyl)-1-nitrosourea.

Abstract

The purpose of this study was to examine the influence of cations on the formation of the individual DNA alkylation products derived from 1-(2-chloroethyl)-1-nitrosourea (CNU). Reaction of calf-thymus DNA with [(3)H]CNU in 10 mM triethanolamine buffer produced 13 DNA adducts. Seven of these adducts were identified as N7-(2-hydroxyethyl)guanine, N7-(2-chloroethyl)guanine, 1, 2-(diguan-7-yl)ethane, N1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N1-2-deoxyguanosinyl)-2-(N3-2-deoxycytidyl)ethane, O(6)-(2-hydroxyethyl)-2-deoxyguanosine, and phosphotriesters. The ratios of the individual products indicated that the chloroethyl and hydroxyethyl adducts are derived from different alkylating intermediates. The influence of cations on the formation of these DNA alkylation products was investigated by the addition of either NaCl, MgCl(2), or spermine. The results demonstrated that (1) the levels of DNA alkylation were inversely proportional to ionic strength, (2) the extent of inhibition was dependent on the alkylation product, and (3) the order of relative effectiveness of inhibition of DNA alkylation by these cations was as follows: spermine > Mg > Na. These results support a model whereby reactions which proceed via an S(N)2 mechanism are more sensitive to the effects of ionic strength than reactions which proceed via an S(N)1 mechanism. In 9L cells treated with CNU, the same alkylation products were formed as in purified DNA; however, the product distribution was different. We interpret this to indicate that within cells, cations modify the reaction of intermediates derived from CNU with DNA.