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The origin of DNA single strand breaks induced by ethylating agents in mammalian cells
Abstract:
Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.
Insights
Diethylsulfate (DES) causes slow DNA repair, while N-ethyl-N-nitrosourea (ENU) induces rapidly repaired DNA single-strand breaks (ssb) in Chinese hamster ovary cells. The mechanisms for ENU-induced ssb repair remain unclear.
Area of Science:
- Molecular Biology
- Genotoxicology
- Biochemistry
Background:
- Ethylating agents like N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES) are used to study DNA damage and repair mechanisms.
- Understanding DNA repair kinetics is crucial for assessing the genotoxic potential of chemical exposures.
Purpose of the Study:
- To investigate and compare the kinetics of DNA single-strand break (ssb) induction and repair following treatment with ENU and DES in Chinese hamster ovary (CHO) cells.
- To correlate DNA adduct formation and removal with ssb repair rates.
- To elucidate the mechanisms underlying differential DNA repair responses to ethylating agents.
Main Methods:
- Treatment of CHO cells with ENU and DES.
- Quantification of DNA single-strand breaks using alkaline elution (AE).
- Measurement of DNA adducts and their repair kinetics using high-performance liquid chromatography (HPLC).
Main Results:
- DES-induced DNA ssb were repaired slowly, with over 50% remaining after 3 hours.
- In contrast, 45% of ENU-induced ssb were repaired within 10 minutes.
- HPLC analysis identified ethylated purines (3-ethyladenine and 7-ethylguanine) as likely contributors to DES-induced ssb, but the rapid repair of ENU-induced ssb could not be explained by these adducts alone.
Conclusions:
- Differential DNA repair kinetics exist for ssb induced by ENU and DES in CHO cells.
- Excision repair of ethylated purines explains DES-induced ssb, but not the rapid repair of ENU-induced ssb.
- The study highlights potential differences in sensitivity between AE and HPLC for detecting DNA damage and repair.