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Mutagenic specificity of model estrogen-DNA adducts in mammalian cells
I Terashima1, N Suzuki, S Itoh
1Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA.
Abstract:
Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic properties of the model estrogen-DNA adducts N2-[3-methoxyestra-1,3,5(10)-trien-6(alpha, beta)-yl]-2'-deoxyguanosine (dG-N2-3MeE) and N6-[3-methoxyestra-1,3, 5(10)-trien-6(alpha,beta)-yl]-2'-deoxyadenosine (dA-N6-3MeE) in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'TCCTCCTCXCCTCTC; X = dG, dA, dG-N2-3MeE, or dA-N6-3MeE) containing an unmodified or model estrogen lesion were inserted into single-stranded (ss) phagemid vectors. These ss vectors were transfected into COS-7 cells. The progeny plasmid obtained were used to transform Escherichia coli DH10B. The transformants were analyzed by oligodeoxynucleotide hybridization and sequencing to determine the mutation frequency and spectrum. Preferential incorporation of dCMP, the correct base, was observed opposite the dG-N2-3MeE lesion. Targeted mutations showing G --> T transversions were detected, along with a small number of G --> C transversions. When a dA-N6-3MeE-modified oligodeoxynucleotide was used, preferential incorporation of dTMP, the correct base, was also observed. Targeted mutations representing A --> T transversions were detected, accompanied by a small amount of A --> G transitions. The frequency of mutation observed opposite dA-N6-3MeE (17.5%) was 2.3 times higher than that observed opposite dG-N2-3MeE (7.5%). These results indicate that estrogen DNA adducts have mutagenic potential in mammalian cells.
Insights
Estrogen DNA adducts, N2-[3-methoxyestra-1,3,5(10)-trien-6(alpha, beta)-yl]-2'-deoxyguanosine (dG-N2-3MeE) and N6-[3-methoxyestra-1,3,5(10)-trien-6(alpha,beta)-yl]-2'-deoxyadenosine (dA-N6-3MeE), show mutagenic potential in mammalian cells. These adducts induce specific mutations, with dA-N6-3MeE being more mutagenic than dG-N2-3MeE.
Area of Science:
- Molecular biology
- Toxicology
- Genetics
Background:
- Estrogen exposure is linked to various cancers, suggesting a role for estrogen-DNA adducts in mutagenesis.
- Understanding the mutagenic mechanisms of these adducts is crucial for assessing their carcinogenic potential.
Purpose of the Study:
- To investigate the mutagenic properties of specific estrogen-DNA adducts, dG-N2-3MeE and dA-N6-3MeE, in mammalian cells.
- To determine the mutation frequency and spectrum induced by these adducts.
Main Methods:
- Site-specifically modified oligodeoxynucleotides containing estrogen adducts were synthesized.
- These modified oligonucleotides were inserted into single-stranded phagemid vectors and transfected into simian kidney (COS-7) cells.
- Progeny plasmids were analyzed for mutation frequency and spectrum using hybridization and sequencing.
Main Results:
- The dG-N2-3MeE adduct preferentially led to dCMP incorporation and induced G --> T transversions and some G --> C transversions.
- The dA-N6-3MeE adduct preferentially led to dTMP incorporation and induced A --> T transversions and some A --> G transitions.
- The mutation frequency opposite dA-N6-3MeE (17.5%) was significantly higher (2.3-fold) than that opposite dG-N2-3MeE (7.5%).
Conclusions:
- Estrogen DNA adducts possess mutagenic potential in mammalian cells.
- The specific type of estrogen adduct influences the mutation frequency and spectrum.
- These findings contribute to understanding the genotoxic mechanisms of estrogen-related carcinogenesis.