Identification and quantification of stable DNA adducts formed from dibenzo[a,l]pyrene or its metabolites in vitro and in mouse skin and rat mammary gland.

The stable adducts of dibenzo[a,l]pyrene (DB[a,l]P) formed by rat liver microsomes in vitro were previously quantified, whereas the depurinating adducts were both identified and quantified [Li, et al. (1995) Biochemistry 34, 8043]. In this article, we report the identification and quantification of the stable DNA adducts obtained from DB[a,l]P and DB[a,l]P-11,12-dihydrodiol activated by rat liver microsomes and from reaction of (+/-)-anti-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) and (+/-)-syn-DB[a,l]PDE with DNA in vitro. In addition, the stable DNA adducts were identified and quantified following treatment of mouse skin with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (+/-)-anti-DB[a,l]PDE, or (+/-)-syn-DB[a,l]PDE in vivo and treatment of rat mammary gland with DB[a,l]P in vivo. The DNA adducts were analyzed by the (32)P-postlabeling method, and the major adducts were identified by comparison with standards. The six stable adducts of DB[a,l]P formed by rat liver microsomes in vitro were either guanine or adenine adducts of anti-DB[a,l]PDE or syn-DB[a,l]PDE. About 43% of the detected stable adducts from microsomes were with guanine and 44% were with adenine. The pattern of adducts formed from DB[a,l]P-11,12-dihydrodiol with microsomes was very similar to that from DB[a,l]P. Reaction of (+/-)-anti-DB[a,l]PDE with DNA in vitro formed higher levels of stable adducts (55% from guanine and 39% from adenine) than (+/-)-syn-DB[a,l]PDE did (about 44% with guanine and 47% with adenine). In mouse skin treated with DB[a,l]P, 1% of the total adducts detected were stable adducts, comprised of 51% guanine adducts and 46% from adenine; with DB[a,l]P-11,12-dihydrodiol, 54% of the total were stable adducts, with a pattern of adducts similar to those formed from DB[a,l]P. Treatment of mouse skin with (+/-)-syn-DB[a,l]PDE formed 68% stable adducts, mostly at guanine. With (+/-)-anti-DB[a,l]PDE, mouse skin contained almost exclusively (97%) stable adducts: 61% guanine adducts and 33% adenine adducts. In rat mammary gland treated with DB[a,l]P, 2% of the total adducts were stable, with 42% guanine adducts and 55% adenine adducts. Approximately equal to or greater amounts of stable guanine adducts were formed in all systems, except for rat mammary gland. In contrast, the majority of depurinating adducts were adenine adducts. The carcinogenic potencies of these compounds in mouse skin, published earlier, do not qualitatively or quantitatively correlate with stable adducts, but rather with depurinating adducts.

Tamoxifen versus aromatase inhibitors for breast cancer prevention.

Long-term exposure to estradiol is associated with an increased risk of breast cancer, but the mechanisms responsible are not firmly established. The prevailing theory postulates that estrogens increase the rate of cell proliferation by stimulating estrogen receptor (ER)-mediated transcription, thereby increasing the number of errors occurring during DNA replication. An alternative theory suggests that estradiol is metabolized to quinone derivatives, which directly remove base pairs from DNA through a process called depurination. Error-prone DNA repair then results in point mutations. We postulate that both processes act in an additive or synergistic fashion. If correct, aromatase inhibitors would block both processes, whereas antiestrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than antiestrogens. Our initial studies showed that catechol-estrogen metabolites are formed in MCF-7 human breast cancer cells in culture. We then used an animal model that allows dissociation of ER-mediated function from the effects of estradiol metabolites and showed formation of genotoxic estradiol metabolites. We also examined the incidence of tumors formed in these ERalpha knockout mice bearing the Wnt-1 transgene. The absence of estradiol markedly reduced the incidence of tumors and delayed their onset. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER-mediated mechanisms to induce breast cancer. These findings support the possibility that aromatase inhibitors might be more effective than antiestrogens in preventing breast cancer.

Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo.

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 (E1) and estradiol (E2) 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 E1(E2)-3,4-quinones [E1(E2)-3,4-Q], we reported previously that the reaction of E1(E2)-3,4-Q with dG produces the depurinating adduct 4-hydroxyE1(E2)-1-N7Gua [4-OHE1(E2)-1-N7Gua] by 1,4-Michael addition (Stack et al., Chem. Res. Toxicol., 1996, 9, 851). We report here that reaction of E1(E2)-3,4-Q with Ade results in the formation of 4-OHE1(E2)-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 E2-3,4-Q was reacted with DNA in vitro, the depurinating adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-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-OHE2 was oxidized by horseradish peroxidase, lactoperoxidase, tyrosinase or phenobarbital-induced rat liver microsomes in the presence of DNA. When 4-OHE2 or E2-3,4-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-OHE2-1-N3Ade and 4-OHE2-1-N7Gua constituted >99% of the total adducts formed. In addition, 4-OHE2 conjugates formed by reaction of E2-3,4-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).

Relative imbalances in estrogen metabolism and conjugation in breast tissue of women with carcinoma: potential biomarkers of susceptibility to cancer.

Exposure to estrogens has been associated with an increased risk of developing breast cancer. Breast biopsy tissues from 49 women without breast cancer (controls) and 28 with breast carcinoma (cases) were analyzed by HPLC with electrochemical detection for 31 estrogen metabolites and catechol estrogen quinone-glutathione conjugates. The levels of estrone and estradiol were higher in cases. More 2-catechol estrogen (CE) than 4-CE was observed in controls, but the 4-CE were three times higher than 2-CE in cases. In addition, the 4-CE were nearly four times higher in cases than in controls. Less O-methylation was observed for the CE in cases. The level of catechol estrogen quinone conjugates in cases was three times that in controls, suggesting in the cases a higher probability for the quinones to react with DNA and generate mutations that may initiate cancer. The levels of 4-CE and quinone conjugates were highly significant predictors of breast cancer. These results suggest that some catechol estrogen metabolites and conjugates could serve as biomarkers to predict risk of breast cancer.

Fluorobenzo[a]pyrenes as probes of the mechanism of cytochrome P450-catalyzed oxygen transfer in aromatic oxygenations.

Fluoro substitution of benzo[a]pyrene (BP) has been very useful in determining the mechanism of cytochrome P450-catalyzed oxygen transfer in the formation of 6-hydroxyBP (6-OHBP) and its resulting BP 1,6-, 3,6-, and 6,12-diones. We report here the metabolism of 1-FBP and 3-FBP, and PM3 calculations of charge densities and bond orders in the neutral molecules and radical cations of BP, 1-FBP, 3-FBP, and 6-FBP, to determine the mechanism of oxygen transfer for the formation of BP metabolites. 1-FBP and 3-FBP were metabolized by rat liver microsomes. The products were analyzed by HPLC and identified by NMR. Formation of BP 1,6-dione and BP 3,6-dione from 1-FBP and 3-FBP, respectively, can only occur by removal of the fluoro ion from C-1 and C-3, respectively, via one-electron oxidation of the substrate. The combined metabolic and theoretical studies reveal the mechanism of oxygen transfer in the P450-catalyzed formation of BP metabolites. Initial abstraction of a pi electron from BP by the [Fe(4+)=O](+)(*) of cytochrome P450 affords BP(+)(*). This is followed by oxygen transfer to the most electropositive carbon atoms, C-6, C-1, and C-3, with formation of 6-OHBP (and its quinones), 1-OHBP, and 3-OHBP, respectively, or the most electropositive 4,5-, 7,8-, and 9,10- double bonds, with formation of BP 4,5-, 7,8-, or 9,10-oxide.

Catechol ortho-quinones: the electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases.

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.

Catechol estrogen metabolites and conjugates in different regions of the prostate of Noble rats treated with 4-hydroxyestradiol: implications for estrogen-induced initiation of prostate cancer.

Prostate carcinomas arise in 100% of Noble rats treated with estradiol and testosterone. We hypothesize that estrogens initiate prostate cancer mainly by formation of 4-catechol estrogens (CE), followed by their oxidation to catechol estrogen-3,4-quinones (CE-3,4-Q), which can react with DNA. To avoid cancer initiation, CE can be detoxified by catechol-O-methyltransferase (COMT), and CE-3,4-Q by conjugation with glutathione (GSH) or by reduction to CE, catalyzed by quinone reductase and/or cytochrome P450 reductase. To investigate the prostatic metabolism of estrogens, Noble rats were treated with the CE 4-hydroxyestradiol (4-OHE2) or estradiol-3,4-quinone (E2-3,4-Q), and CE metabolites and conjugates were analyzed in the four regions of the prostate, which differ in susceptibility to carcinoma formation. Following treatment of rats with 4-OHE2 (6 micromol/100 g body weight in 200 microl of trioctanoin/dimethylsulfoxide (4:1) by intraperitoneal injection) for 90 min, the non-susceptible ventral (VP) and anterior (AP) prostate had higher levels of 4-methoxyCE and GSH conjugates than the susceptible dorsolateral prostate (DLP) and periurethral prostate (PUP). After treatment with the same molar amount of E2-3,4-Q, the VP and AP contained more GSH conjugates, 4-CE and 4-methoxyCE than the susceptible DLP and PUP. These results suggest that prostate areas susceptible to carcinoma induction have less protection by COMT, GSH, and quinone reductase and/or cytochrome P450 reductase, favoring reaction of CE-3,4-Q with DNA, presumably to initiate cancer.