Estrogen-nucleic acid adducts: reaction of 3,4-estrone o-quinone with nucleic acid bases.

Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to be a prerequisite for its genotoxic effects. Previous studies have shown that 3,4-estrone quinone (3,4-EQ) can redox-cycle and is capable of inducing exclusively single-strand DNA breaks in MCF-7 breast cancer cells [Nutter et al. (1991) J. Biol. Chem. 226, 16380-16386]. These studies, however, could not provide conclusive evidence about the mechanism of estrogen carcinogenesis. In order to explore this in more detail, we have shown previously that 3,4-EQ can react with adenine under electrochemical reductive conditions to yield an estrogen-nucleic acid adduct [Abul-Hajj et al. (1995) J. Am. Chem. Soc. 117, 6144-6145]. In this paper, we report the synthesis and identification of seven estrogen-nucleic acid adducts obtained from reaction of 3,4-EQ with adenine, thymine, and cytosine. Initial purification of reaction mixtures using TLC followed by HPLC gave sufficient quantities of reaction products for identification using 1H-NMR and mass spectral determinations. Reaction of 3,4-EQ with adenine, thymine, and cytosine gave the following estrogen-nucleic acid adducts: 8-(4-hydoxyestrone-1-yl)adenine, 3-adenylimino-1,5(10)-estradiene-4,17-dione,4-adenylimino-1, 5(10)-estradiene-3,17-dione, N1- [4-hydroxyestrone-1(alpha,beta)-yl]thymine, N4-(4-hydroxyestrone-1- yl)cytosine, and N4-(4-hydroxy- estrone-2-yl)cytosine. No reaction products were obtained with guanine presumably due to poor solubility in DMF.

Oxidative transformation of 2-hydroxyestrone. Stability and reactivity of 2,3-estrone quinone and its relationship to estrogen carcinogenicity.

The carcinogenicity of estrogens in rodents and man has been attributed to either alkylation of cellular macromolecules and/or redox-cycling, generation of active radicals, and DNA damage. Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to be a prerequisite for its genotoxic effects. 4-Hydroxyestradiol, although not 2-hydroxyestradiol, is a potent inducer of tumors in hamsters. Previous studies have shown that 3,4-estrone quinone can redox-cycle and is capable of inducing exclusively single strand DNA breaks in MCF-7 breast cancer cells, as well as react with various nucleophiles (thiol, imidazole, amino, phenolate, and acetoxy) to give Michael addition products. These results support the possible involvement of 3,4-catechol/quinone estrogens in estrogen's carcinogenicity. To explain the decreased carcinogenicity of 2-hydroxyestrogens, the reactions of 2,3-estrone quinone (2,3-EQ) with nucleophiles were investigated. Reactions of 4-methylimidazole with 2,3-EQ gave a complex mixture of products leadng to the formation of the catechol, C-O dimerization product, and a 1,6-Michael addition product identified as the 1-(4-methylimidazolo)-2-hydroxyestrone. Reactions of 2,3-EQ under mildly basic conditions with either ethyl phenolate or acetate gave several products which were characterized as the C-O and C-C dimers, catechol, and 3,5-dihydroxy-1(10), 3-estradiene-2, 17-dione. No Michael addition products were detected under these experimental conditions. The same products were also observed during the synthesis of 2,3-EQ, which led us to postulate that the lack of carcinogenicity of 2-hydroxyestrogens may be related to the increased reactivity and decreased stability of the quinone under physiological conditions. These results are contrasted with those obtained with 3,4-EQ which is much more stable and therefore could diffuse from the site of formation to the target tissue. These results along with rapid methylation and clearance may be very likely explanations for the decreased carcinogenicity of 2-hydroxyestrogens.

Reactions of 3,4-estrone quinone with mimics of amino acid side chains.

Reaction of 3,4-estrone o-quinone (3,4-EQ) with several amino acid side chain mimics, including 4-ethylphenol, 4-methylimidazole, acetic acid, and propanethiol, gave a mixture of several products including the catechol, Michael addition products, and dimeric products of the catechol. On the other hand, several other amino acid side chain mimics, including ethanol, acetamide, 1-ethylguanidine, and 3-methylindole, did not result in any addition products or catechol formation. Michael addition to 3,4-EQ with 4-methylimidazole, acetate, and 4-ethyl phenoxide resulted in 1,4-addition, leading to C-1 adducts while reaction with propanethiol gave the C-2 addition product.

Reaction of lysine with estrone 3,4-o-quinone.

Reaction of lysine with estrone 3,4-o-quinone gave a complex mixture of products. Six compounds were isolated and identified using spectroscopic techniques. Among the reaction products isolated were 4-hydroxyestrone (2), 3-aminoisoestrone (3), 3-(N-pentyl-5-amino)-isoestrone (4), 1-lysylestrone 3,4-o-iminoquinone (5), and two dimeric products of 3,4-catechol estrone (6 and 7).