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Mutations induced by dacarbazine activated with cytochrome P-450
A Mudipalli1, S S Nadadur, A E Maccubbin
1Grace Cancer Drug Center, Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263.
Abstract:
The mutagenicity of the antitumor drug dacarbazine (DTIC) is due to alkylation of cellular DNA by metabolites resulting from the metabolism of this drug by the mixed function oxidase system. In the present study, we used an in vitro shuttle vector assay to study the base and sequence specificity of mutagenesis by DTIC. The shuttle vector plasmid pSP189 was treated with DTIC (1-2.5 mM) in vitro in a reconstituted cytochrome P-450 system at 37 degrees C for either 30 or 60 min. SupF tRNA gene insert contained in the plasmid was sequenced after replication of the drug-treated plasmid in human Ad 293 cells followed by amplification in indicator bacteria. Mutagenesis of DTIC in this system was dependent upon the presence of the cytochrome P-450 reconstituted system and NADPH. Mutations induced by DTIC included single base substitutions (35%), single base deletions (30.5%), single base insertions (19.4%) and large deletions (13.8%). Among the substitutions, transversions and transitions were in the ratio of 1:0.7. Base pairs 108 and 127 in the SupF tRNA of the pSP189 were identified as mutational hot spots.
Insights
The antitumor drug dacarbazine (DTIC) causes DNA mutations, including substitutions and deletions, through its metabolites. This study identified specific DNA sequences as mutational hotspots, crucial for understanding DTIC
Area of Science:
- Molecular Toxicology
- Genetics
- Cancer Research
Background:
- Antitumor drug dacarbazine (DTIC) is known to be mutagenic.
- DTIC's mutagenicity arises from DNA alkylation by its metabolites, processed by the mixed-function oxidase system.
Purpose of the Study:
- To investigate the base and sequence specificity of dacarbazine (DTIC)-induced mutagenesis.
- To characterize the types of mutations induced by DTIC in a specific DNA sequence.
Main Methods:
- An in vitro shuttle vector assay using the pSP189 plasmid.
- Treatment of the plasmid with DTIC in a reconstituted cytochrome P-450 system with NADPH.
- Sequencing of the SupF tRNA gene insert after replication in human Ad 293 cells and amplification in bacteria.
Main Results:
- DTIC-induced mutagenesis was dependent on the cytochrome P-450 system and NADPH.
- Mutations included single base substitutions (35%), deletions (30.5%), insertions (19.4%), and large deletions (13.8%).
- Specific base pairs (108 and 127) in the SupF tRNA were identified as mutational hotspots.
Conclusions:
- Dacarbazine (DTIC) induces a spectrum of mutations in a sequence-specific manner.
- The study highlights specific mutational hotspots, providing insights into DTIC's genotoxic mechanisms.
- Understanding these mechanisms is vital for cancer therapy and drug safety.