Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of lambda(lacZ) transgenic mice treated with N-ethyl-N-nitrosourea.
LacZ transgenic mice are suitable for short-term mutagenicity studies in vivo. Mutagenicity in these mice is determined in the lacZ transgene. Since the lacZ gene is of bacterial origin the question has been raised whether DNA-adduct formation and repair in the transgene are comparable to those in total genomic DNA. Mice were treated with N-ethyl-N-nitrosourea (ENU) and killed at several time points following treatment. Some mice were pretreated with O6-benzylguanine to inactivate the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). O6-ethylguanine (O6-EtG) was determined in lacZ in liver and brain by means of a monoclonal antibody-based immunoaffinity assay. In addition, O6-EtG and N7-ethylguanine (N7-EtG) were assayed in total genomic DNA of liver and brain with an immunoslotblot procedure. In liver, the initial O6-EtG level in total genomic DNA was 1.6 times that in lacZ. The extent of repair of O6-EtG during the first 1.5 h after treatment was 2.1 times that in lacZ. At later time points, O6-EtG repair was the same. N7-EtG repair in genomic DNA was evident. In contrast to the liver, little repair of O6-EtG in total genomic and lacZ DNA occurred in the brain while N7-EtG was repaired. No initial difference in O6-EtG levels were found in lacZ and genomic brain DNA. These findings indicate that in the liver, total genomic DNA is more accessible than lacZ to ENU and/or the AGT protein, during the first 1.5 h following treatment. Because the difference in O6-EtG levels in the transgene and genomic DNA in the liver is restricted to the first 1.5 h after treatment, while the fixation of mutations occurs at later time points, O6-EtG-induced mutagenesis most likely is also very similar in both types of DNA.
Mientjes
E J
EJ
Department of Molecular Toxicology, TNO Food and Nutrition Research Institute, Rijswijk, The Netherlands.
Hochleitner
K
K
Luiten-Schuite
A
A
van Delft
J H
JH
Thomale
J
J
Berends
F
F
Rajewsky
M F
MF
Lohman
P H
PH
Baan
R A
RA
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001921:Brain / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D005038:Ethylnitrosourea / Q000633:toxicity*; D005260:Female; D016678:Genome; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015151:Immunoblotting; D007763:Lac Operon; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D009153:Mutagens; C022007:7-ethylguanine; C007673:6-ethylguanine; D006147:Guanine; D004247:DNA; D005038:Ethylnitrosourea