Reduction of estrogen-induced transformation of mouse mammary epithelial cells by N-acetylcysteine

Abstract

A growing number of studies indicate that breast cancer initiation is related to abnormal estrogen oxidation to form an excess of estrogen-3,4-quinones, which react with DNA to form depurinating adducts and induce mutations. This mechanism is often called estrogen genotoxicity. 4-Catechol estrogens, precursors of the estrogen-3,4-quinones, were previously shown to account for most of the transforming and tumorigenic activity. We examined whether estrogen-induced transformation can be reduced by inhibiting the oxidation of a 4-catechol estrogen to its quinone. We demonstrate that E6 cells (a normal mouse epithelial cell line) can be transformed by a single treatment with a catechol estrogen or its quinone. The transforming activities of 4-hydroxyestradiol and estradiol-3,4-quinone were comparable. N-Acetylcysteine, a common antioxidant, inhibited the oxidation of 4-hydroxyestradiol to the quinone and consequent formation of DNA adducts. It also drastically reduced estrogen-induced transformation of E6 cells. These results strongly implicate estrogen genotoxicity in mammary cell transformation. Since N-acetylcysteine is well tolerated in clinical studies, it may be a promising candidate for breast cancer prevention.

Figure 1. Cytotoxicity of 4-OHE₂ and E₂-3,4-Q in normal mouse mammary epithelial cells (E6)

Cells were treated with the estrogens at Day 0. Cell numbers were determined by the MTT assay using a standard curve.

Figure 2. N-acetylcysteine rescues E6 cells from 4-OHE₂ and E₂-3,4-Q cytotoxicity

Equimolar amounts of N-acetylcysteine were added to the experiments at Day 0. Cell numbers were determined by the MTT assay using a standard curve.

Figure 3. N-acetylcysteine inhibits estrogen-DNA adduct formation in E6 cells

E₂ and E₁ are interconverted in the cell; therefore, we combined the conjugates and adducts from E₁ and E₂ together. The quinone produced less conjugates and DNA adducts than the catechol (note the difference in the Y-axes of B & C).

Figure 3. N-acetylcysteine inhibits estrogen-DNA adduct formation in E6 cells

E₂ and E₁ are interconverted in the cell; therefore, we combined the conjugates and adducts from E₁ and E₂ together. The quinone produced less conjugates and DNA adducts than the catechol (note the difference in the Y-axes of B & C).

Figure 4. Colony morphology of estrogen-transformed E6 cells

Figure 5. Spectrum of pre-existing H-ras mutations in E6 cells

Exon 1-2 region (GenBank Accession No. U89950) was analyzed for mutations. The mutations are shown over the top line of the cartoon, and the wild-type nucleotides are shown below.