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DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis
A A van Zeeland1, A de Groot, A Neuhäuser-Klaus
1Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratories, State University of Leiden, The Netherlands.
Abstract:
DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.
Insights
Ethylating agents like ENU, EMS, and DES cause DNA adducts. Their specific DNA reaction sites correlate with mutagenic potency in mouse germ cells, impacting mutation induction.
Area of Science:
- Toxicology
- Genetics
- Molecular Biology
Background:
- DNA adducts are key intermediates in chemical mutagenesis.
- Ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES) are known mutagens.
- Understanding the relationship between DNA adduct formation and mutagenicity is crucial for risk assessment.
Purpose of the Study:
- To investigate the relationship between DNA adduct formation and mutagenic potency of ENU, EMS, and DES in mice.
- To determine the specific DNA reaction sites (O6-guanine vs. N-7 guanine) of these agents.
- To correlate DNA alkylation patterns with mutation induction in different germ cell stages.
Main Methods:
- Mice were injected intraperitoneally with ENU, EMS, and DES.
- DNA adduct levels (O6-ethylguanine and 7-ethylguanine) were quantified in various organs.
- Mutagenicity was assessed using the mouse specific-locus assay.
- Dose-response relationships for DNA adducts and mutations were analyzed.
Main Results:
- ENU, favoring O6-alkylation, was primarily mutagenic in spermatogonia.
- EMS and DES, favoring N-alkylation, were more mutagenic in post-meiotic germ cells.
- The dose-response for ENU adducts was non-linear, but mutation induction in spermatogonia was linear.
- High mutagenic potency of EMS/DES in late spermatogenesis may involve apurinic site accumulation.
Conclusions:
- The type of DNA adduct formed by ethylating agents dictates their mutagenic target and potency in male germ cells.
- O6-ethylguanine is the likely mutagenic lesion for ENU in spermatogonia.
- Mutagenesis by EMS and DES in post-meiotic cells may involve indirect mechanisms.
- Approximately 10 O6-ethylguanine residues per gene are needed for mutation induction in mammalian cells.