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Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of λlacZ transgenic mice treated with N-ethyl-N-nitrosourea

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Author: Mientjes, E.J. · Hochleitner, K. · Luiten-Schuite, A. · Delft, J.H.M. van · Thomale, J. · Berends, F. · Rajewsky, M.F. · Lohman, P.H.M. · Baan, R.A.
Type:article
Date:1996
Institution: TNO Voeding
Source:Carcinogenesis, 11, 17, 2449-2454
Identifier: 233537
Keywords: Toxicology · 6 o alkylguanine dna alkyltransferase · dna · ethylnitrosourea · guanine derivative · 6 ethylguanine · 6-ethylguanine · 7 ethylguanine · 7-ethylguanine · DNA · drug derivative · guanine · mutagenic agent · animal experiment · animal tissue · article · brain · controlled study · dna adduct · dna repair · enzyme inhibition · female · immunoblotting · liver · mouse · mutagenicity · nonhuman · priority journal · transgene · transgenic mouse · animal · bacteriophage lambda · biosynthesis · DNA repair · drug effect · genetics · genome · lactose operon · metabolism · mutagen testing · physiology · polymerase chain reaction · sensitivity and specificity · Animals · Bacteriophage lambda · Brain · DNA · DNA Repair · Ethylnitrosourea · Female · Genome · Guanine · Immunoblotting · Lac Operon · Liver · Mice · Mice, Transgenic · Mutagenicity Tests · Mutagens · Polymerase Chain Reaction · Sensitivity and Specificity

Abstract

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.