TY - JOUR
T1 - Induction of A·T to G·C mutations by erroneous repair of depurinated DNA following estrogen treatment of the mammary gland of ACI rats
AU - Mailander, Paula C.
AU - Meza, Jane L.
AU - Higginbotham, Sheila
AU - Chakravarti, Dhrubajyoti
N1 - Funding Information:
We thank Drs. E.L. Cavalieri and E.G. Rogan for support, and Dr. M. Zahid for synthesizing the estradiol-3,4-quinone used in this study. This work was supported by NIH grant P20RR17675 from the National Center for Research Resources. PCM and DC were supported in part by a US Public Health Service grant P01 CA49210 from the National Cancer Institute. Core support at the Eppley Institute was provided by the grant P30 CA36727 from the National Cancer Institute.
PY - 2006/11
Y1 - 2006/11
N2 - Evidence suggests that the genotoxic mechanism of estrogens (estrone/estradiol) in breast cancer involves their oxidation to 3,4-quinones and reaction with DNA to form depurinating N3Ade and N7Gua adducts. We examined whether estrogen genotoxicity is mutagenic in the mammary gland of the female ACI rat, a model for estrogen-dependent breast cancer. Mutagenesis was studied by PCR amplification of the H-ras1 gene (exons 1-2), cloning in pUC18, transforming Escherichia coli, and sequencing the inserts in plasmids from individual colonies. Mammary glands of both estrogen-responsive (ACI and DA) and resistant (Sprague-Dawley) rats contained pre-existing mutations at frequencies of (39.8-58.8) × 10-5, the majority (62.5-100%) of which were A·T to G·C transitions. Estradiol-3,4-quinone (200 nmol) treatment of ACI rats caused rapid (6 h to 1 day) mutagenesis (frequency (83.3-156.1) × 10-5; A·T to G·C 70-73.3%). The estrogen-induced A·T to G·C mutations were detected as G·T heteroduplexes, as would be expected if N3Ade depurinations caused Gua misincorporations by erroneous repair. These heteroduplexes were identified by the T·G-DNA glycosylase (TDG) assay. TDG converts G·T heteroduplexes to G.abasic sites, rendering DNA templates refractory to PCR amplification. Consequently, A·T to G·C mutations present as G·T heteroduplexes in the DNA are eliminated from the spectra. TDG treatment of mammary DNA from estradiol-3,4-quinone-treated ACI rats brought A·T to G·C mutations down to pre-existing frequencies. Our results demonstrate that treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A·T to G·C mutations in the DNA of the mammary gland of ACI rats.
AB - Evidence suggests that the genotoxic mechanism of estrogens (estrone/estradiol) in breast cancer involves their oxidation to 3,4-quinones and reaction with DNA to form depurinating N3Ade and N7Gua adducts. We examined whether estrogen genotoxicity is mutagenic in the mammary gland of the female ACI rat, a model for estrogen-dependent breast cancer. Mutagenesis was studied by PCR amplification of the H-ras1 gene (exons 1-2), cloning in pUC18, transforming Escherichia coli, and sequencing the inserts in plasmids from individual colonies. Mammary glands of both estrogen-responsive (ACI and DA) and resistant (Sprague-Dawley) rats contained pre-existing mutations at frequencies of (39.8-58.8) × 10-5, the majority (62.5-100%) of which were A·T to G·C transitions. Estradiol-3,4-quinone (200 nmol) treatment of ACI rats caused rapid (6 h to 1 day) mutagenesis (frequency (83.3-156.1) × 10-5; A·T to G·C 70-73.3%). The estrogen-induced A·T to G·C mutations were detected as G·T heteroduplexes, as would be expected if N3Ade depurinations caused Gua misincorporations by erroneous repair. These heteroduplexes were identified by the T·G-DNA glycosylase (TDG) assay. TDG converts G·T heteroduplexes to G.abasic sites, rendering DNA templates refractory to PCR amplification. Consequently, A·T to G·C mutations present as G·T heteroduplexes in the DNA are eliminated from the spectra. TDG treatment of mammary DNA from estradiol-3,4-quinone-treated ACI rats brought A·T to G·C mutations down to pre-existing frequencies. Our results demonstrate that treatment with estradiol-3,4-quinone, an important metabolite of estrogens, produced A·T to G·C mutations in the DNA of the mammary gland of ACI rats.
KW - ACI rat
KW - Erroneous repair
KW - Estrogen mutagenesis
KW - Mammary gland
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U2 - 10.1016/j.jsbmb.2006.06.019
DO - 10.1016/j.jsbmb.2006.06.019
M3 - Article
C2 - 16982187
AN - SCOPUS:33750009769
SN - 0960-0760
VL - 101
SP - 204
EP - 215
JO - Journal of Steroid Biochemistry and Molecular Biology
JF - Journal of Steroid Biochemistry and Molecular Biology
IS - 4-5
ER -