Formation and persistence of O-6-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 O-6-benzylguanine to inactivate the repair protein O-6-alkylguanine-DNA alkyltransferase (AGT), O-6-ethylguanine (O-6-EtG) was determined in lacZ in liver and brain by means of a monoclonal antibody-based immunoaffinity assay, In addition, O-6-EtG and N-7-ethylguanine (N7-EtG) were assayed in total genomic DNA of liver and brain with an immunoslotblot procedure, In liver, the initial O-6-EtG level in total genomic DNA was 1.6 times that in lacZ, The extent of repair of O-6-EtG during the first 1.5 h after treatment was 2.1 times that in lacZ, At later time points, O-6-EtG repair was the same. N7-EtG repair in genomic DNA was evident. In contrast to the liver, little repair of O-6-EtG in total genomic and lacZ DNA occurred in the brain while N7-EtG was repaired, No initial difference in O-6-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 O-6-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, O-6-EtG-induced mutagenesis most likely is also very similar in both types of DNA.