Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.
We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O(6)-(2-hydroxyethyl) 2'-deoxyguanosine (O(6)-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O(6)-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5mM HENU resulted in mutation frequencies of 7.2+/-2.2x10(-5), 45.2+/-2.9x10(-5) and 120.3+/-24.4x10(-5), respectively. Comparison of the mutation frequencies demonstrates that 1 and 5mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C-->A:T transitions (52%) and a significant number of A:T-->T:A transversions (16%). We propose that the observed G:C-->A:T transitions are produced by the DNA alkylation product O(6)-HOEtdG. These results suggest that the formation of O(6)-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.
Sprung
C N
CN
Brain Tumor Research Center of the Department of Neurological Surgery, University of California, Box-0555, San Francisco, CA 94143-0555, USA.
Wang
Y P
YP
Miller
D L
DL
Giannini
D D
DD
Dhananjaya
N
N
Bodell
W J
WJ
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002330:Carmustine / Q000633:toxicity*; D002460:Cell Line; D018736:DNA Adducts / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000633:toxicity*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D056968:Lac Repressors; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D009153:Mutagens; D012097:Repressor Proteins; C054529:O(6)-(2-hydroxyethyl)-2'-deoxyguanosine; C022146:1-(2-hydroxyethyl)-1-nitrosourea; C035018:N(7)-hydroxyethylguanine; D006147:Guanine; D003849:Deoxyguanosine; D005038:Ethylnitrosourea; D002330:Carmustine