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Deoxyribonucleotide pools as targets for mutagenesis by N-methyl-N-nitrosourea
A Arecco1, B J Mun, C K Mathews
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Abstract:
This paper describes a biological test of the hypothesis that one or more components of the intracellular nucleotide pool represent a significant target for the mutagenic effects of alkylating agents. In other words, we ask whether mutagenesis can occur either through alkylation of susceptible nucleotide residues in DNA, or through alkylation of a free nucleotide, followed by its incorporation into DNA. Our approach is based upon the premise that if a nucleotide pool is a mutagenic target, then transient expansion of that pool should increase the target size and enhance mutagenesis following subsequent treatment with an alkylating agent. Working either with V79 hamster lung fibroblasts or Chinese hamster embryo fibroblasts (CHEF/18), we treated cells for 30 min, under conditions that expanded one or more pools of deoxyribonucleoside triphosphates. This was followed immediately by a 30-min treatment with 0.5 mM N-methyl-N-nitrosourea. After 8 days of additional culture for recovery of cells and expression of mutations, we plated in selective media to determine the abundance of 6-thioguanine-resistant mutants in each culture. We found that conditions which expand pools of either dATP or dTTP and dGTP stimulate mutagenesis by MNU, with the degree of stimulation varying in different experiments from 2- to 6-fold. Although alternate interpretations can be entertained, the data are consistent with the hypothesis that nucleotide pools represent alkylation targets. A biochemical test of the hypothesis is warranted. During our studies we made several other noteworthy observations: (1) treatment of V79 cells with mutagen alone does not significantly affect dNTP pools; (2) deoxynucleotide pool perturbations are quite short-lived following transfer of cells to normal medium; (3) deoxyuridine is significantly more effective than thymidine in expanding dTTP pools; (4) deoxyuridine by itself is significantly mutagenic, particularly to CHEF/18 cells.
Insights
Expanding intracellular nucleotide pools, like deoxyadenosine triphosphate (dATP) and deoxythymidine triphosphate (dTTP) and deoxyguanosine triphosphate (dGTP), enhances mutagenesis from alkylating agents. This suggests nucleotide pools are targets for mutagenic effects.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Alkylation of DNA is a known mutagenic mechanism.
- The role of intracellular nucleotide pools as direct targets for alkylating agents remains unclear.
- Understanding these targets is crucial for assessing mutagenicity risks.
Purpose of the Study:
- To biologically test the hypothesis that intracellular nucleotide pools are targets for alkylating agent mutagenesis.
- To determine if expanding nucleotide pools increases susceptibility to mutagenesis.
- To investigate the mutagenic potential of altered nucleotide pools.
Main Methods:
- Cultured V79 hamster lung fibroblasts and Chinese hamster embryo fibroblasts (CHEF/18).
- Transiently expanded deoxyribonucleoside triphosphate pools using specific conditions.
- Treated cells with N-methyl-N-nitrosourea (MNU), an alkylating agent.
- Assessed mutagenesis by quantifying 6-thioguanine-resistant mutants after recovery.
Main Results:
- Expanding pools of dATP, or dTTP and dGTP, significantly stimulated MNU-induced mutagenesis (2- to 6-fold).
- Mutagen treatment alone did not significantly alter deoxynucleotide triphosphate (dNTP) pools.
- Deoxynucleotide pool perturbations were transient.
- Deoxyuridine was more effective than thymidine in expanding dTTP pools and was itself mutagenic.
Conclusions:
- The results support the hypothesis that intracellular nucleotide pools are alkylation targets.
- Further biochemical investigation is warranted to confirm these findings.
- Deoxyuridine exhibits mutagenic properties and influences dTTP pool expansion.