DNA adduct of the mitomycin C metabolite 2,7-diaminomitosene is a nontoxic and nonmutagenic DNA lesion in vitro and in vivo.

Abstract

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.

• Authors: Christopher D Utzat, Cristina C Clement, Leilani A Ramos, Arunangshu Das, Maria Tomasz, Ashis K Basu
• Journal: Chemical research in toxicology (Chem Res Toxicol) Vol. 18 Issue 2 Pg. 213-23 (Feb 2005) ISSN: 0893-228X [Print] United States
• PMID: 15720125 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA Adducts
• Mitomycins
• Oligonucleotides
• Mitomycin
• 2,7-diaminomitosene
• DNA

Topics

• Animals
• COS Cells
• DNA (drug effects)
• DNA Adducts (chemistry)
• DNA Damage
• Escherichia coli (chemistry, drug effects)
• Mitomycin (chemistry)
• Mitomycins (chemical synthesis, chemistry, pharmacology)
• Molecular Structure
• Mutagenicity Tests
• Oligonucleotides (chemical synthesis, chemistry, pharmacology)