Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon-DNA adducts generate oncogenic mutations found in mouse skin papillomas (Proc. Natl. Acad. Sci. USA 92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E 1 ) and estradiol (E 2 ) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA in vitro to form the depurinating adduct 4-OHE 1 (E 2 )-1(␣,␤)-N7Gua at 59-213 mol͞mol DNA-phosphate whereas the level of stable adducts was 0.1 mol͞mol DNA-phosphate. In female Sprague-Dawley rats treated by intramammillary injection of E 2 -3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 mol 4-OHE 2 -1(␣,␤)-N7Gua͞molDNA-phosphate. When 4-OHE 1 (E 2 ) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87-440 mol of 4-OHE 1 (E 2 )-1(␣, ␤)-N7Gua was formed. After treatment with 4-OHE 2 , rat mammary tissue contained 1.4 mol of adduct͞mol DNA-phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.

Catechol estrogens (CE) are among the major metabolites of estrone (E1) and 17 beta-estradiol (E2). Oxidation of these metabolites to semiquinones and quinones could generate ultimate carcinogenic forms of E1 and E2. The 2,3- and 3,4-quinones of E1 and E2 were synthesized by MnO2 oxidation of the corresponding CE, following the method for synthesizing E1-3,4-quinone [Abul-Hajj (1984) J. Steroid Biochem. 21, 621-622]. Characterization of these compounds was accomplished by UV, nuclear magnetic resonance, and mass spectrometry. The relative stability of these compounds was determined in DMSO/H2O (2:1) at room temperature, and the 3,4-quinones were more stable than the 2,3-quinones. The four quinones directly reacted with calf thymus DNA to form DNA adducts analyzed by the 32P-postlabeling method. The adducts were compared to those formed when the corresponding CE were activated by horseradish peroxidase (HRP) to bind to DNA. The E1- and E2-2,3-quinones formed much higher levels of DNA adducts than the corresponding 3,4-quinones. In addition, many of the adducts (70-90%) formed by the E1- and E2-2,3-quinones appeared to be the same as those formed by activation of 2-OHE1 or 2-OHE2 by HRP to bind to DNA. Little overlap was observed between the adducts formed by E1- and E2-3,4-quinones and HRP-activated 4-OHE1 and 4-OHE2. These results suggest that semiquinones and/or quinones are ultimate reactive intermediates in the peroxidatic activation of catechol estrogens.

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We have determined an X-ray crystal structure for the N-methyl iodide derivative of the nonsteroidal contraceptive centchroman. The pendant aromatic substituents on C-3 and C-4 of the chroman system are nearly perpendicular to the plane of the chroman system, an orientation expected in such a chroman, but perturbed to some degree by the gem dimethyl substituents at C-2. Structural superposition with other nonsteroidal antiestrogens, tamoxifen and nafoxidine, shows a similar disposition of the tertiary amine side chains responsible for antagonist activity. The aryl rings also show good superposition, but in contrast to tamoxifen and nafoxidine, which have the potential for ring double bond conjugation, the centchroman aryl rings show a larger dihedral twist. While different superpositions between the enantiomers of centchroman and the bioactive enantiomer of estradiol (d-estradiol, 8 beta,9 alpha,13 beta,14 alpha,17 beta) are possible, when the chroman ring system is positioned over the AB rings of estradiol, then (3R,4R)-centchroman makes the best fit. The aryl substituents in both enantiomers make comparable overlays with the steroidal skeleton, but the axial methyl group at C-2 in (3R,4R)-centchroman is directed downward along the C-7 alpha axis of estradiol, a site where many substituents are known to be well tolerated by the estrogen receptor, while in the 3S,4S-enantiomer, this methyl group is projected upward. Thus, we suggest that the bioactive l-enantiomer of centchroman will have the 3R,4R absolute configuration.