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(α,β)-N7Gua at 59–213 μmol/mol DNA–phosphate whereas the level of stable adducts was 0.1 μmol/mol 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 μmol 4-OHE ₂ -1(α,β)-N7Gua/molDNA–phosphate. When 4-OHE ₁ (E ₂ ) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87–440 μmol of 4-OHE ₁ (E ₂ )-1(α, β)-N7Gua was formed. After treatment with 4-OHE ₂ , rat mammary tissue contained 1.4 μmol of adduct/mol 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.

Estrogens induce tumors in laboratory animals and have been associated with breast and uterine cancers in humans. In relation to the role of estrogens in the induction of cancer, we examine formation of DNA adducts by reactive electrophilic estrogen metabolites, formation of reactive oxygen species by estrogens and the resulting indirect DNA damage by these oxidants, and, finally, genomic and gene mutations induced by estrogens. Quinone intermediates derived by oxidation of the catechol estrogens 4-hydroxyestradiol or 4-hydroxyestrone may react with purine bases of DNA to form depurinating adducts that generate highly mutagenic apurinic sites. In contrast, quinones of 2-hydroxylated estrogens produce less harmful, stable DNA adducts. The catechol estrogen metabolites may also generate potentially mutagenic oxygen radicals by metabolic redox cycling or other mechanisms. Several types of indirect DNA damage are caused by estrogen-induced oxidants, such as oxidized DNA bases, DNA strand breakage, and adduct formation by reactive aldehydes derived from lipid hydroperoxides. Estradiol and the synthetic estrogen diethylstilbestrol also induce numerical and structural chromosomal aberrations and several types of gene mutations in cells in culture and in vivo. In conclusion, estrogens, including the natural hormones estradiol and estrone, must be considered genotoxic carcinogens on the basis of the evidence outlined in this chapter.

Catechol estrogens and catecholamines are metabolized to quinones, and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogenic benzene can also be oxidized to its quinone. We report here that quinones obtained by enzymatic oxidation of catechol and dopamine with horseradish peroxidase, tyrosinase or phenobarbital-induced rat liver microsomes react with DNA by 1,4-Michael addition to form predominantly depurinating adducts at the N-7 of guanine and the N-3 of adenine. These adducts are analogous to the ones formed with DNA by enzymatically oxidized 4-catechol estrogens (Cavalieri,E.L., et al. (1997) Proc. Natl Acad. Sci., 94, 10937). The adducts were identified by comparison with standard adducts synthesized by reaction of catechol quinone or dopamine quinone with deoxyguanosine or adenine. We hypothesize that mutations induced by apurinic sites, generated by the depurinating adducts, may initiate cancer by benzene and estrogens, and some neurodegenerative diseases (e.g. Parkinson's disease) by dopamine. These data suggest that there is a unifying molecular mechanism, namely, formation of specific depurinating DNA adducts at the N-7 of guanine and N-3 of adenine, that could initiate many cancers and neurodegenerative diseases.

Studies of estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA can generate the critical mutations initiating breast, prostate and other cancers. The endogenous estrogens estrone (E ₁ ) and estradiol (E ₂ ) are oxidized to catechol estrogens (CE), 2- and 4-hydroxylated estrogens, which can be further oxidized to CE quinones. To determine possible DNA adducts of E ₁ (E ₂ )-3,4-quinones [E ₁ (E ₂ )-3,4-Q], we reported previously that the reaction of E ₁ (E ₂ )-3,4-Q with dG produces the depurinating adduct 4-hydroxyE ₁ (E ₂ )-1-N7Gua [4-OHE ₁ (E ₂ )-1-N7Gua] by 1,4-Michael addition (Stack et al ., Chem. Res. Toxicol ., 1996, 9, 851). We report here that reaction of E ₁ (E ₂ )-3,4-Q with Ade results in the formation of 4-OHE ₁ (E ₂ )-1-N3Ade by 1,4-Michael addition. The N7Gua and N3Ade depurinating adducts formed both in vitro and in rat mammary gland in vivo were analyzed by HPLC with electrochemical detection and, for some samples, by LC/MS/MS. When E ₂ -3,4-Q was reacted with DNA in vitro , the depurinating adducts 4-OHE ₁ (E ₂ )-1-N3Ade and 4-OHE ₁ (E ₂ )-1-N7Gua, which are rapidly lost from DNA by cleavage of the glycosyl bond, were formed (>99% of the total adducts), as well as traces of stable adducts, which remain in DNA unless removed by repair. Similar results were obtained when 4-OHE ₂ was oxidized by horseradish peroxidase, lactoperoxidase, tyrosinase or phenobarbital-induced rat liver microsomes in the presence of DNA. When 4-OHE ₂ or E ₂ -3,4-Q was injected into the mammary glands of female ACI rats in vivo and the mammary tissue was excised 1 h later, the depurinating adducts 4-OHE ₂ -1-N3Ade and 4-OHE ₂ -1-N7Gua constituted >99% of the total adducts formed. In addition, 4-OHE ₂ conjugates formed by reaction of E ₂ -3,4-Q with glutathione were also detected. These results demonstrate that the 4-CE are metabolized to CE-3,4-Q, which react with DNA to form primarily depurinating adducts. These adducts can generate the critical mutations that initiate cancer (Chakravarti et al ., Oncogene , 2001, 20, 7945; Chakravarti et al ., Proc. Am. Assoc. Cancer Res. , 2003, 44, 180).