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The relationship between DNA damage and mutation frequency in mammalian cell lines treated with
1Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Republic of China.
Abstract:
The induction of DNA single-strand breaks in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells by N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) was demonstrated by the alkaline elution technique. Without metabolic activating system (i.e., rat liver S9 fraction), N-NO-2-FAA exhibits more direct and strong damaging effects on DNA than its parent compound, 2-FAA, at equal concentration in both cell lines. To compare the DNA-damaging potency of N-NO-2-FAA with other well-known carcinogens, such as benzo[a]pyrene, 2-nitrofluorene, and N-methyl-N'-nitrosoguanidine (MNNG), the order of potency is as follows: MNNG (5 microM) greater than N-NO-2-FAA (150 microM) greater than benzo[a]pyrene (20 microM) at equitoxic concentrations, LD37, in the same cell system. Another parallel experiment indicated that N-NO-2-FAA could disrupt the superhelicity of circular plasmid DNA (pBR 322) at a dose range of 0.1-50 mM; however, a complete conversion to form III linear DNA was found at the highest concentration (50 mM). After treatment with various concentrations of N-NO-2-FAA, ouabain resistance (ouar) was induced in C3H10T1/2 cells, while both ouar and 6-thioguanine resistance (6-TGr) were induced in CHO cells. The mutation frequency in the Na+/K+-ATPase locus in CHO cells (1.5 X 10(-6) mutants/microM) is higher than that in C3H10T1/2 cells (1.0 X 10(-6) mutants/microM). The maximal mutation frequency at the Na+/K+-ATPase gene locus was attained with 30 min of exposure in C3H10T1/2 cells, whereas the mutation frequency in CHO cells continued to increase up to 80 min of treatment. Similarly, the maximal mutation frequency at the HPRT locus also continued to increase up to 80 min of treatment. Finally, a linear plot of alkali-labile lesions versus 6-TGr mutations was obtained; but the same relationship was not observed in the case of ouar mutation.
Insights
N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) directly damages DNA and induces mutations in mouse and hamster cells. Its DNA-damaging potency is significant, ranking below MNNG but above benzo[a]pyrene.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) is a chemical compound with potential genotoxic effects.
- Understanding the DNA-damaging and mutagenic potential of N-NO-2-FAA is crucial for assessing its carcinogenic risk.
Purpose of the Study:
- To investigate the DNA-damaging effects of N-NO-2-FAA in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells.
- To compare the DNA-damaging potency of N-NO-2-FAA with known carcinogens.
- To evaluate the mutagenic potential of N-NO-2-FAA in mammalian cells.
Main Methods:
- Alkaline elution technique to detect DNA single-strand breaks.
- Treatment of cells with N-NO-2-FAA with and without metabolic activation (rat liver S9 fraction).
- Assessment of DNA superhelicity disruption in plasmid DNA.
- Induction of ouabain resistance (ouar) and 6-thioguanine resistance (6-TGr) mutations.
Main Results:
- N-NO-2-FAA induced significant DNA single-strand breaks in both cell lines, more potently than its parent compound, 2-FAA, without metabolic activation.
- The DNA-damaging potency order was MNNG > N-NO-2-FAA > benzo[a]pyrene at equitoxic concentrations.
- N-NO-2-FAA disrupted plasmid DNA superhelicity and induced mutations (ouar and 6-TGr) in a dose- and time-dependent manner.
- Mutation frequency was higher in CHO cells than in C3H10T1/2 cells, and mutation induction kinetics differed between cell types and loci.
Conclusions:
- N-NO-2-FAA is a direct-acting genotoxic agent capable of inducing DNA damage and mutations in mammalian cells.
- The study provides quantitative data on the DNA-damaging potency and mutagenicity of N-NO-2-FAA.
- Findings contribute to the understanding of the mechanisms of chemical carcinogenesis and genotoxicity testing.