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DNA adducts, mutant frequencies, and mutation spectra in various organs of lambda lacZ mice exposed to ethylating
E J Mientjes1, A Luiten-Schuite, E van der Wolf
1Department of Molecular Toxicology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands.
Abstract:
To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.
Insights
This study examined DNA adducts and mutations in mouse tissues after exposure to N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). O6-ethylguanine (O6-EtG) was not the primary mutagenic lesion in liver, but may be in bone marrow.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- DNA adducts are chemical modifications to DNA that can lead to mutations.
- Understanding the relationship between specific DNA adducts and mutagenesis is crucial for assessing chemical toxicity.
- Tissue-specific differences in DNA repair and mutagenesis can influence toxicological outcomes.
Purpose of the Study:
- To investigate the tissue-specific relationship between DNA adduct formation and mutagenesis in vivo.
- To determine if O6-ethylguanine (O6-EtG) is a major premutagenic lesion in different mouse tissues after exposure to ethylating agents.
- To compare the mutagenic potential of N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS) in liver, bone marrow, and brain.
Main Methods:
- Lambda lacZ transgenic mice were treated with ENU, DEN, and EMS.
- DNA adduct levels (O6-EtG, N7-EtG) and mutant frequency (MF) in the lacZ gene were measured in liver, bone marrow, and brain at various time points.
- Mutant DNA sequences were analyzed to identify mutation types.
Main Results:
- In mouse liver, O6-EtG was rapidly removed, and mutant frequency continued to rise, suggesting O6-EtG is not the main premutagenic lesion.
- Sequence analysis revealed GC-->AT transitions and TA-->AT transversions in liver mutants, with the latter possibly arising from O2-ethylthymine.
- In bone marrow after ENU treatment, O6-EtG may be a major premutagenic lesion at 3 days, correlating with mutation induction and GC-->AT mutations.
- EMS treatment resulted in high N7-EtG but low O6-EtG and no increased MF in liver and bone marrow.
- No adducts or mutations were detected in bone marrow after DEN treatment or in the brain after ENU or EMS treatment.
Conclusions:
- The role of O6-EtG as a premutagenic lesion is tissue-specific, being potentially significant in bone marrow but not in liver.
- Other DNA adducts, such as O2-ethylthymine, may contribute to mutagenesis.
- The brain appears resistant to the mutagenic effects of these ethylating agents, despite adduct formation.