Structural Alterations in Breast Stromal and Epithelial DNA: The Influence of 8,5-cyclo-2-Deoxyadenosine

Abstract

(5'S)-8,5'-Cyclo-2'-deoxyadenosine (S-cdA), which arises from the reaction of the hydroxyl radical (*OH) with 2'-deoxyadenosine in DNA, is a lesion comprising a base-sugar linkage that distorts the DNA backbone. This structure impedes transcription and blocks polymerase action. Further, a single S-cdA lesion in the TATA box reduces gene expression. Considering the ability of S-cdA to disrupt DNA structure, which is likely associated with increased cancer risk, we determined S-cdA concentrations in the DNA of stroma, epithelium, and myoepithelium from normal breast tissues using liquid chromatography/mass spectrometry (LC/MS). We also identified differences in the base and backbone structures using Fourier transform-infrared (FT-IR) spectroscopy. LC/MS revealed that the lowest concentration of S-cdA in the stroma (0.04 +/- 0.02 lesions/10(6) bases) occurred in women ages 17 to 30. The highest concentration (0.13 +/- 0.07 lesions/10(6) bases) was found in women 33 to 46. FT-IR spectroscopy showed significant base and backbone differences in the stromal DNA between the women under 30 and those over 50. These findings imply that distortions in the geometry of the helix increase with age, reaching significant proportions in older women. No differences were found in the S-cdA concentrations between the three cell types, suggesting that the *OH attack on the base structure may be essentially random. Initial insight is provided on changes in DNA structure that potentially affect gene expression and increase breast cancer risk.

Oxidative Changes in the DNA of Stroma and Epithelium from the Female Breast: Potential Implications for Breast Cancer

“…23 In an age-related study of the stroma, the highest mean concentration of this cyclopurine nucleoside was found in women 33 to 46 years old, an age corresponding to a rise in breast cancer incidence. 26 In the present blinded study, plots of mean 8-OH-dG and 8-OH-dA concentrations in stromal DNA vs. age revealed three discernable groups for each lesion.…”

“…Stromal, epithelial, and myoepithelial cells were isolated using StemCell Technologies (Vancouver, BC) protocol with minor modifications as described. 23 The purity (≥ 80%) of the three cell types was determined via FACS analysis. 23 DNA extraction and analysis by FT-IR spectroscopy.…”

“…23 The purity (≥ 80%) of the three cell types was determined via FACS analysis. 23 DNA extraction and analysis by FT-IR spectroscopy. DNA (~50 µg) was extracted from each sample (2-15 × 10 6 cells) using Qiagen 100/G Genomic-tips (Qiagen, Chatsworth, CA).…”

“…DNA (~50 µg) was extracted from each sample (2-15 × 10 6 cells) using Qiagen 100/G Genomic-tips (Qiagen, Chatsworth, CA). 23 Spectral analysis was performed using an FT-IR microscope spectrometer (System 2000, PerkinElmer). 24 Spectral measurements were converted (Fourier-transformed) into absorbance.…”

“…25 Statistical analyses were performed using SPSS for Microsoft Windows 11.0 statistical program (SPSS, Chicago, IL), as described. 23 …”