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Mutation of potassium permanganate- and hydrogen peroxide-treated plasmid pZ189 replicating in CV-1 monkey kidney
S A Akman1, G P Forrest, J H Doroshow
1Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA 91010.
Abstract:
We examined the effect of the oxidation of plasmid pZ189 by KMnO4, which does not produce free radicals, and H2O2/Fe(2+)-diethylenetriaminepentaacetic acid (DTPA), which does, on the mutation frequency of pZ189 transfected into monkey kidney CV-1 cells. Treatment with 1.5 mM KMnO4 increased the content of certain modified bases, principally Thy and Cyt modified at C-5 and C-6, by up to 300-fold, as measured by GC/MS; however, the mutation frequency increased only 5-fold above background. 1.0 mM H2O2/0.1 mM Fe(2+)-DTPA treatment, which increased the mutation frequency 10-fold above background, increased the content of certain modified bases by up to 4-fold. Sequence analysis revealed both deletions and point mutations, with a predominance of C-G substitutions, among H2O2/Fe(2+)-DTPA-associated mutations. These data suggest that KMnO4-modified DNA is only weakly mutagenic in DNA replicating in mammalian nuclei, despite substantial production of Thy glycol and other base modifications, whereas H2O2/Fe(2+)-DTPA-modified DNA is more mutagenic. H2O2/Fe(2+)-DTPA generated mutations occur predominantly at C-G base pairs.
Insights
Potassium permanganate (KMnO4) DNA oxidation weakly increases mutations, while hydrogen peroxide (H2O2) with iron and DTPA is more mutagenic, causing primarily C-G substitutions.
Area of Science:
- Molecular Biology
- Toxicology
- Genetics
Background:
- DNA damage can arise from various oxidative agents.
- Understanding the mutagenicity of different DNA modifications is crucial for assessing risk.
Purpose of the Study:
- To compare the mutagenic potential of two distinct DNA oxidation methods.
- To investigate the types of mutations induced by these oxidative treatments.
Main Methods:
- Plasmid pZ189 was oxidized using potassium permanganate (KMnO4) or hydrogen peroxide/iron(II)-diethylenetriaminepentaacetic acid (H2O2/Fe(2+)-DTPA).
- Oxidized plasmids were transfected into monkey kidney CV-1 cells.
- Mutation frequencies were analyzed using sequence analysis and base modification levels were measured by gas chromatography/mass spectrometry (GC/MS).
Main Results:
- KMnO4 treatment significantly increased base modifications (up to 300-fold) but only caused a modest 5-fold increase in mutation frequency.
- H2O2/Fe(2+)-DTPA treatment increased mutation frequency 10-fold with only a 4-fold increase in base modifications.
- Mutations induced by H2O2/Fe(2+)-DTPA predominantly involved deletions and C-G substitutions.
Conclusions:
- DNA modified by KMnO4 is weakly mutagenic in mammalian cells, despite extensive base damage.
- Oxidative damage induced by H2O2/Fe(2+)-DTPA is more mutagenic and primarily targets C-G base pairs.