Chloroethylating anticancer drug-induced mutagenesis and its repair in Escherichia coli

Chloroethylnitrosourea (CENU) derivatives, such as nimustine (ACNU) and carmustine (BCNU), are employed in brain tumor chemotherapy due to their ability to cross the blood-brain barrier. They are thought to suppress tumor development through DNA chloroethylation, followed by the formation of interstrand cross-links (ICLs) that efficiently block replication and transcription. However, the alkylation of DNA and ICLs may trigger genotoxicity, leading to tumor formation as a side effect of the chemotherapeutic treatment. Although the involvement of -alkylguanine-DNA alkyltransferase (AGT) in repairing chloroethylated guanine ( -chloroethylguanine) has been reported, the exact lesion responsible for the genotoxicity and the pathway responsible for repairing it remains unclear. We examined the mutations induced by ACNU and BCNU using a series of strains CC101 to CC111, in which reverse mutations due to each episome from F'101 to F'106 and frameshift mutations due to each episome from F'107 to F'111 could be detected. The mutant frequency increased in CC102, which can detect a GC to AT mutation. To determine the pathway responsible for repairing the CENU-induced lesions, we compared the frequency of mutations induced by CENU in the wild-type strain to those in the (AGT-deficient) strain, (nucleotide excision repair (NER)-deficient) strain, mismatch repair (MMR)-deficient strains, and (recombination deficient) strain of CC102. The frequencies of mutations induced by ACNU and BCNU increased in the strain, demonstrating that -chloroethylguanines were formed, and that a portion was repaired by AGT.Mutation induced by ACNU in NER-deficient strain showed a similar profile to that in AGT-deficient strain, suggesting that an NER and AGT play at the similar efficacy to protect from mutation induced by ACNU. -Chloroethylguanine is reported to form ICLs if it is not repaired. We examined the survival rates and the frequencies of mutations induced by ACNU and BCNU in the strain, the strain, as well as a double-deficient strain of CC102. The mutation profile of the double-deficient strain was similar to that of the NER-deficient strain, suggesting that an NER protects from mutations but not recombination. In addition, cell death was more pronounced in the double-deficient strain than in the single-deficient strains. These results suggest that the toxic lesions induced by CENU were repaired additively or synergistically by NER and recombination. In other words, lesions, such a