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Discrimination of mutagenic intermediates derived from alkylating agents by mutational patterns generated in
R K Elespuru1, L L Stupar, J A Gordon
1Laboratory of Chemical and Physical Carcinogenesis, NCI-Frederick Cancer Research and Development Facility, MD 21702-1201.
Abstract:
Reactive intermediates (ultimate mutagens/carcinogens) generated by alkylating agents are unstable and difficult to characterize by chemical means. We have used a genetic system to distinguish the in vivo interactions of eight carcinogenic methylating agents and five ethylating agents by the patterns of induced mutations at different target sites in Escherichia coli WU3610. For this multiple locus assay, target sites were an amber (TAG) and an ochre (TAA) triplet, DNA encoding five suppressor tRNA anticodons, and one unidentified locus. Most of the mutations could be classified as specific sequence changes at the target loci by suppressor analysis using T4 bacteriophage. Ratios of the slopes of dose-response curves for induced mutations were used to generate a profile of preferred sites for mutagenesis independent of mutagen potency. 'Mutational fingerprints' derived from different methylating and ethylating agents were compared, as evidence for the existence of common intermediates responsible for their biological effects. Six methylating agents thought to act via SN1 mechanisms were found to generate similar mutational patterns, indicative of a common mechanism, while two methylating agents reacting via SN2 mechanisms gave different patterns. The mutational fingerprints of SN1- and SN-type ethylating agents were also distinct. Mutational fingerprints may be useful in distinguishing the interactions of different ultimate mutagens.
Insights
This study uses a genetic system to create "mutational fingerprints" for alkylating agents. Different fingerprints reveal distinct DNA interaction mechanisms, aiding in the identification of mutagens.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Reactive intermediates from alkylating agents are unstable and hard to characterize chemically.
- Understanding these intermediates is crucial for identifying mutagens and carcinogens.
Purpose of the Study:
- To develop a genetic method for distinguishing in vivo interactions of methylating and ethylating agents.
- To create "mutational fingerprints" to classify mutagen mechanisms based on induced mutation patterns.
Main Methods:
- Utilized a multiple locus genetic assay in Escherichia coli WU3610 targeting specific DNA sequences.
- Analyzed induced mutations using suppressor analysis with T4 bacteriophage to classify sequence changes.
- Generated dose-response curves to derive site-specific mutagenesis profiles, independent of mutagen potency.
Main Results:
- Mutational fingerprints differentiated between SN1 and SN2 mechanisms for methylating agents.
- Distinct mutational patterns were observed for SN1- and SN-type ethylating agents.
- Six methylating agents acting via SN1 mechanisms showed similar mutational patterns, suggesting common intermediates.
Conclusions:
- Mutational fingerprinting provides a viable method for distinguishing the biological interactions of different ultimate mutagens.
- The genetic approach effectively characterizes the in vivo behavior of carcinogenic alkylating agents.
- This technique aids in understanding the mechanisms of mutagenesis and chemical carcinogenesis.