Reaction of N-(2-chloroethyl)-N-nitrosoureas with DNA: effect of buffers on DNA adduction, cross-linking, and cytotoxicity.
N-(2-Chloroethyl)nitrosoureas (CNU) are clinically used anticancer drugs whose cytotoxicity is associated with the generation of DNA interstrand cross-links. While studying the sequence selectivity for a series of CNU, a dramatic increase in the formation of N7-alkyldeoxyguanosine was observed when Tris buffer was used rather than phosphate or cacodylate buffers. Moreover, the formation of N7-alkyldeoxyguanosine lesions continues in Tris long after all of the CNU has hydrolyzed. These effects are not seen with the monofunctional alkylating analogues, e.g., N-methyl- and N-(2-hydroxyethyl)-N-nitrosourea. In order to determine if the nature of the CNU-mediated DNA damage was altered by Tris, studies were initiated on the following: (1) alkylation of N7-G in end-labeled DNA restriction fragments; (2) covalent modification of DNA with [ethyl-3H]-N-(2-chloroethyl)-N-nitrosourea; and (3) cytotoxicity in L1210 cells. The data presented demonstrate that Tris increases the yield of the "normal" CNU monofunctional cross-linked adducts, i.e., N7-(2-hydroxyethyl)deoxyguanosine, N7-(2-chloroethyl)deoxyguanosine, O6-(2-chloroethyl)deoxyguanosine, and bifunctional adducts, i.e., 1-(deoxycytid-3-yl)-2-(deoxyguanosin-1-yl)ethane and 1,2-bis(deoxyguanosin-7-yl)ethane. In addition, CNU appears to react with Tris to give a long-lived alkylating intermediate that affords large amounts of DNA adducts not seen with CNU in the absence of Tris. However, in vivo toxicity of CNU in L1210 cells is not affected by the presence of Tris, indicating that the reaction pathway(s) responsible for cross-linking is not significantly sensitive to the nature of the buffer.
Chen
F X
FX
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Bodell
W J
WJ
Liang
G
G
Gold
B
B
D000478:Alkylation / Q000187:drug effects; D000818:Animals; D001483:Base Sequence / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000633:toxicity*; D018736:DNA Adducts / Q000737:chemistry / Q000187:drug effects* / Q000633:toxicity*; D004249:DNA Damage; D004347:Drug Interactions; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000737:chemistry / Q000633:toxicity; D007939:Leukemia L1210 / Q000235:genetics* / Q000473:pathology*; D051379:Mice; D014325:Tromethamine / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D014325:Tromethamine; C017139:1-(2-chloroethyl)-1-nitrosourea; D005038:Ethylnitrosourea